CN102580099A - Composition for resisting ischemia reperfusion injury and preparation method and application thereof - Google Patents

Composition for resisting ischemia reperfusion injury and preparation method and application thereof Download PDF

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CN102580099A
CN102580099A CN2011103212569A CN201110321256A CN102580099A CN 102580099 A CN102580099 A CN 102580099A CN 2011103212569 A CN2011103212569 A CN 2011103212569A CN 201110321256 A CN201110321256 A CN 201110321256A CN 102580099 A CN102580099 A CN 102580099A
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reperfusion injury
ischemia
liver
hepatic
ischemia resisting
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CN102580099B (en
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沈甫明
倪敏
赵婷
杨中伟
苏定冯
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Second Military Medical University SMMU
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Abstract

The invention relates to a medicinal composition for treating an ischemia reperfusion injury, particularly a hepatic ischemia reperfusion injury, and a preparation method and application thereof. The composition comprises an M-cholinergic receptor blocker and a cholinesterase inhibitor, has a simple and convenient preparation process, is safe to use, has an obvious curative effect, can be used for obviously improving the cell apoptosis caused by the ischemia reperfusion injury and overcomes the side effects caused by existing common medicaments. The invention expands novel medical application of an existing muscarinic receptor blocker and also provides a novel medicine intervention means for preventing and treating the ischemia reperfusion injury. The medicinal composition is suitable for large-scale production and commercial application in the industries of medicine, reagents and the like, has excellent application prospect and has obvious social benefits and economic benefits.

Description

A kind of ischemia resisting reperfusion injury composition and method of making the same and purposes
Technical field
The present invention relates to technical fields such as medicine, reagent; Specifically relate to a kind of pharmaceutical composition; More particularly relate to a kind of pharmaceutical composition that is used to treat ischemical reperfusion injury, particularly hepatic ischemia-reperfusion injury, specifically relate to the pharmaceutical composition of a kind of M of containing cholinergic receptor-blocking agent and cholinesterase inhibitor again.
Background technology
(1) research overview of ischemical reperfusion injury
1, general introduction
After the tissue of ischemia, organ regain blood supply; Tissue, organ dysfunction are recovered; Increased the weight of functional metabolism obstacle and structural damage on the contrary, this phenomenon is referred to as ischemical reperfusion injury (ischemia-reperfusion injury).
Clinically be common in the operation of liver severe trauma, liver neoplasm excision, liver transplantation etc.Human different reasons cause hepar damnification, even apoptosis is an important characteristic in the hepatic fibrosis process.Research is illustrated in the course of liver damage that ischemia-reperfusion causes and has hepatocellular apoptosis.
But the pathophysiological process of ischemical reperfusion injury is complicated, relates to approach such as apoptosis and necrosis, does not still have clear understanding so far, so the research of its mechanism has been become focus.
2, the mechanism of HIRI
The caused tissue injury of ischemia is the main cause of fatal disease, the myocardial infarction that causes such as coronary atherosclerosis, apoplexy etc.In ischemic diseases rescue and therapeutic process, physicians find that gradually only ischemia also is not enough to cause tissue injury; But when ischemia recovers blood supply (i.e. perfusion again) again suddenly after a period of time; Cause macrophage, neutrophilic granulocyte and platelet activation, cause a series of cell effects then, simultaneously because the infringement of vascular endothelial cell causes microcirculation disturbance; Further increase the weight of tissue injury, this damage is called as " tissue ischemia reperfusion injury ".
Hepatic ischemia-reperfusion injury (hepatic ischemia-reperfusion injury; Be called for short: the pathophysiological process that HIRI) is the complicacy of many factors participations; Good sending out in shock and multiple liver surgery is a multifactorial process (Banga NR, the Homer-Vanniasinkam S that influences liver function after liver transplantation, the liver leaf section excision; Graham A; Et al.Ischemic preconditioning in transplantation and major resection of the liver [J] .Br J Surg, 2005,92 (5): 528-538.).
Hepatic ischemia reperfusion can cause withered not formula cell (being the Kupffer cell) (Zeng Z; Huang HF; Et al.Heme oxygenase-1protects donor livers from ischemia/reperfusion injury:The role of Kupffer cells [J] .World J Gastroenterol; 2010Mar 14,16 (10): 1285-92.), neutrophilic granulocyte and platelet activation, cause a series of cell effects then; Simultaneously because the infringement of sinusoidal endothelial cell causes microcirculation disturbance; Further increase the weight of liver ischemia, anoxia: these Kupffer cells, neutrophilic granulocyte and monocytic activation can directly discharge a large amount of short scorching cytokines, like TNF-α, IL-1 β, IL-6; But also lipid release inflammatory mediator (is called for short: LTs), thromboxane A2 (is called for short: TXA2), platelet activating factor (is called for short: PAF) etc. like leukotriene.Inflammatory mediator can activate inflammatory cell, makes it synthetic and discharges multiple inflammatory factor; Inflammatory factor then both can also can cause hepatocyte injury through synergism to each other through single form in liver.
For example, TNF-α directly causes sinusoidal endothelial cell swelling; Interaction through neutrophilic granulocyte, endotheliocyte causes Liver Microcirculation (Zhou W; Zhang Y; Hosch MS.Subcellular site of superoxide dismutase expression differentially controls AP-1activity and injury in mouse liver following ischemia/reperfusion [J] .Hepatology; 2001,33 (4): 902-914; Tsuchiya Y; Suzuki S; Inaba K; Et al.Impact of endothelin-1on microcirculatory disturbance after partial hepatectomy under ischemia/reperfusion in thioacetamide-induced cirrhotic rats [J] .J Surg Res, 2003,111 (1): 100-8.); Can activate neutrophilic granulocyte release oxygen free radical; Stimulate mononuclear phagocyte and inflammatory factors such as other emiocytosis IL-1 β, IL-6 simultaneously.IL-1 β can induce the synthetic of IL-8 and increase cell adhesion molecule and select expression plain, that integration is plain, and these all strengthen sticking of neutrophilic granulocyte and endotheliocyte, further cause synthetic more cytokine.
Blood capillary that ischemic tissue causes when pouring into again and organa parenchymatosum's damage are mainly caused by reactive oxygen free radical, the proof that this has obtained in multiple organ.
Most scholars think that oxygen-derived free radicals plays a significant role when hepatic ischemia reperfusion, its generator is shaped on:
(1) the cell hypoxic-ischemic makes ATP generate minimizing, Ca 2+The entering mitochondrion increases, and makes mitochondrial function impaired, increases thereby oxygen-derived free radicals is generated;
(2) calcium overload can activate calcium ion dependence protein enzyme again, but latter's catalysis xanthine dehydrogenase is converted into xanthine oxidase., and xanthine oxidase produces a large amount of oxygen-derived free radicals when under aerobic conditions, can promote xanthine being decomposed into uric acid.Under the situation that a large amount of oxygen get into when pouring into again; Xanthine oxidase acts on hypoxanthine, produces a large amount of ultra-oxygen anion free radicals (Liu PG, He SQ; Wu Jian; Et al.Protective effects of apocynin and allopurinol on ischemia/reperfusion induced liver injury in mice [J] .World J Gastroenterol, 2008,14 (18): 2832-2837.; Liu JN; Zhang JX, Zhang XL, et al.The effect of oxidative stress in myocardial cell injury in mice exposed to chronic intermittent hypoxia [J] .Chin Med J (Engl); 2010,123 (1): 74-8.).Use SOD to remove radical pair ischemic reperfusion tissue injury protective effect is arranged;
When (3) pouring into again; Because anoxia, ATP reduce and calcium entering mitochondrion increases; Cytochrome oxidase functional disorder, the forfeiture of intracellular plastochondria membrane potential, respiratory chain dysfunction, electron transport chain are leaked electricity, and son increases, superoxide dismutase generates minimizing etc.; Can cause simultaneously a large amount of oxygen-derived free radicals to generate (Wang NT, Lin HI, et al.Effects ofthe antioxidants lycium barbarum and ascorbic acid on reperfusion liver injury in rats [J] .Transplant Proc; 2009,41 (10): 4110-3.);
(4) ischemic stage,, the histiocyte oxygen content reduced, and is not enough as the oxygen content of electron acceptor, perfusion back recovery organization oxygen supply again, make oxygen-derived free radicals at short notice burst increase.
The hepatocellular mechanism of oxygen free radical injury mainly contains:
(1) oxidation cell membrane, the flowability and the permeability of change film produce MDA, the coup injury cell;
(2) through directly acting on the attachment sites of sinusoidal endothelial cell film platelet increasing and neutrophilic granulocyte; Platelet increasing and neutrophilic granulocyte stick, assemble; Cause microcirculation disturbance (GlanemannM; VollmarB, NusslerAK, et al.Ischemic preconditioning protects from hepatic ischemia/reperfusion injury by preservation of microcirculation and mitochondria redox-state [J] .J Hepatol; 2003,38 (1): 59-66.);
(3) activate oxidation nuclease in the hepatic parenchymal cells nuclear, make the dna double chain interruption and cause the damage of liver structure and function;
(4) oxygen-derived free radicals suppresses mitochondrial oxidative phosphorylation, and energy matter is reduced.
Ischemia-reperfusion can cause the apoptotic fact to be reported to some extent.Apoptosis is after phalangeal cell receives some distinctive signals and stimulates, and by the cell death of the active of its internal mechanism regulation and control, belongs to the physiological activity of body self, is also referred to as programmed cell death and (is called for short: PCD).
PCD is one of important mechanisms that causes ischemical reperfusion injury; Be major reason (the Malhi H of liver failure; Gores GJ, Lemasters JJ, et al.Apoptosis and necrosis in the liver:a tale oftwo deaths [J] Hepatology; 2006,43 (2Suppl 1): 831-44.).
Apoptosis behind the ischemia-reperfusion is a complex process; But energy metabolism impairment, oxygen-derived free radicals, cytokine, intracellular calcium overload and Caspase family, Bcl-2 family be cell death inducing all; Wherein the formation of the burst of oxygen-derived free radicals is key factor; Free radical and metabolic center product thereof increase in the oxidative phosphorylation obstacle when ischemia-reperfusion, mitochondrion, and active decline of free radical defense system this moment; The DNA damage of free radical mediated can excite activation and the p53 accumulation of poly ADP ribose transferring enzyme, this two all relevant with apoptosis.
3, the pharmaceutical intervention of HIRI
But up to the present, still do not have sure Therapeutic Method and can change the whole course of disease.In the prior art, the method for treatment ischemical reperfusion injury is numerous, comprises that mainly ischemia adapts to pretreatment and the intervention of premedicant thing.
(1) ischemia adapts to pretreatment
Ischemia adapts to pretreatment and is meant that of short duration ischemia-reperfusion process render organ has more tolerance to subsequently more persistent ischemical reperfusion injury; It can reduce ATP degraded in the continuous ischemia process; The accumulation of sugar decomposition intermediate product and lactic acid product (Clavien PA; Yadav S, Sindram D, et al.Protective effects of ischemic preconditioning for liver resection performed under inflow occlusion in humans [J] Ann Surg; 2000,232 (12): 163-165.).
The medicine pretreatment is to utilize the direct or indirect pharmacological action of some active substance to reach the pretreated protective effect of similar ischemia, strengthens tissue or the cell toleration to ischemical reperfusion injury, thereby alleviates damage.
The medicine main principle that HIRI is had pretreating effect has:
1. protect biomembrane, microcirculation improvement and cellular energy metabolism.Ischemic tissue's zymolysis process strengthens, and therefore replenishes the glycolysis substrate and can effectively protect ischemic tissue, helps the recovery of biomembrane function.The metabolism of mitochondrion oxidative phosphorylation is obstructed during ischemia, Given this, can give exogenous ATP, phosphagen, cytochrome C etc.;
2. free radical scavenger: hang down the molecule free radical scavenger; For example vitamin A, vitamin C, vitamin E and glutathion reductase (are called for short: GTH-PX) (Zhang Houzhong; Han Xichun; Jiang Weidong. glutathion and ischemia pretreatment are to the influence [J] of rat liver ischemical reperfusion injury. Chinese gerontology magazine, 2006,26 (10): 91-93.); Enzyme type free radical scavenger, for example: catalase (is called for short: CAT), surpass the oxide dismutase and (be called for short: SOD), peroxidase;
3. reaction reduces inflammation: have the monoclonal antibody of the specific adhesion molecule of adhesive reaction between the glucocorticoid, blocking-up neutrophilic granulocyte and endotheliocyte of stable lysosome enzyme membrane effect etc.; All can obviously alleviate ischemical reperfusion injury (Ma Xingjiu; Geng Zhenhong, Li Dechun waits the protective action [J] of. Wu Sitating to hepatic ischemia-reperfusion injury. and the doctor studies magazine; 2005,28 (7): 22-28.);
4. alleviate the calcium channel blocker of calcium overload.
(2) the premedicant thing is intervened
The drug main of intervening before the art at present, will be main with plant extract.Chinese herbal medicine is big with safety range, toxic and side effects is little, effect advantages such as link is more, becomes the focus of research.
Some blood-activating and stasis-removing can be removed oxygen-derived free radicals; The internal organs ischemical reperfusion injury is had the certain protection effect, as Rhizoma Chuanxiong, Radix Salviae Miltiorrhizae, ginseng attach, Folium Ginkgo extract, emodin or the like (yellow rich, Chen Gaofei; Chen Guoyong; Deng. tea polyphenols is to the protective effect [J] of rat liver ischemia perfusion injury. gastroenterology and hepatopathy magazine, 2006,15 (5): 522-524.; Lin Shengzhang, Yu Yaojun, the trip great waves, etc. emodin is to the preventive effect [J] of rat liver ischemical reperfusion injury. Chinese combination of Chinese and Western medicine surgical magazine, 2006,12:136-138.; Zhao Ke. Folium Ginkgo extract is to the protective effect [J] of rat liver ischemical reperfusion injury. Central Plains doctor's periodical, 2007,34 (3): 5-6.).
Rhizoma Chuanxiong can suppress PMN and assemble adhesion, alleviate calcium overload, anti-TXA 2, synthetic, stop platelet aggregation, remove free radical, protective wire plastochondria; Radix Salviae Miltiorrhizae has the OFR of attenuating lipid peroxide contents, and SOD activity improving reduces calcium overload, promotes the Regeneration and Repair function.
In addition; There are some researches show that Radix Notoginseng total arasaponins, sodium ferulate (effective ingredient of Radix Angelicae Sinensis), green tea Pingpien Gingseng Rhizome thing (are called for short: GTE), Radix Astragali total flavones etc. all has protective effect (Ke QH to hepatic ischemia-reperfusion injury; Zheng SS; Liang TB; Et al.Pre-treatment of hypertonic saline can increase endogenous interleukin 10release to attenuate hepatic ischemia reperfusion injury [J] .Dig Dis Sc, 2006,51:2257-2263.).
Wherein many medicines have bigger toxic and side effects; Use the crude extract of plant clinically always; To its medicine property one-tenth be hard to tell, part not such as mechanism of action is not clear, quality is unstable, toxic and side effects is big, influenced their application clinically, be difficult to meet clinical needs.
In recent years, hypertonic saline (is called for short: HS) also begin to receive people's attention.Low dose of HS can not only improve the hemodynamic parameter of shock fast, improves myocardial contraction and CO, microcirculation improvement, and can also suppress the excessive inflammatory response under the stress state.Discover (Ke QH; Zheng SS; Liang TB; Et al.Pre-treatment ofhypertonic saline can increase endogenous interleukin 10release to attenuate hepatic ischemia reperfusion injury [J] .Dig Dis Sc, 2006,51:2257-2263.; Gonzalez EA; Kozar RA; Suliburk JW, et al.Conventional dose hypertonic saline provides optimal gut protection and limits remote organ injury after gut ischemia reperfusion [J] .J Trauma, 2006; 61 (1): 66-73.); HS can suppress the neutrophilic granulocyte excessive activation, suppresses the secretion of TNF-α, IL-6 and adhesion molecule, increases release and the expression of anti-inflammatory cytokines IL-10 simultaneously.The hypertonic saline pretreatment can obviously strengthen the mRNA and the protein expression of liver Heme oxygenase-1 behind the ischemia-reperfusion, obviously improves liver microcirculation, and hepatic ischemia-reperfusion injury is had the certain protection effect.
4, prospect
The control of hepatic ischemia-reperfusion injury has been poured into aspects such as condition, application cell protective agent and transfer body endogenous defence capability again and has been obtained great achievement in control.
Along with the research to the cholinergic anti-inflammatory pathway constantly is tending towards perfect, the application thinking and the practicable method that also are bound to produce more heterogeneous pass are served clinical treatment, for research control ischemical reperfusion injury provides one new strategy.Continuous progress in science and technology makes people deepen continuously to the Mechanism Study of ischemical reperfusion injury, and the method for disease preventing and treating also will be more and more effectively.
But; The mechanism of hepatic ischemia-reperfusion injury is complicated; Be mutual, the coefficient result of multiple factor, and single Study on Mechanism often can not reach satisfied effect, so comprehensive study number of mechanisms, that the multiple medicines thing is united utilization remain further to be carried out.
(2) research overview of M cholinergic receptor-blocking agent
1, general introduction
Various stimulations cause that being released in of endogenous acetylcholine brought out in the asthma and play an important role.Therefore the effect of M cholinergic receptor-blocking agent ability blockage of acetylcholine can be used for treating asthma.For example Atem (ipratropium) is mainly used in chronic asthmatic bronchitis: inhalation seldom absorbs; Therefore obvious expansion bronchus effect is arranged; Increase by first second forced volume,expiratory, and do not influence sputum secretion, do not have obvious general untoward reaction yet.
2, cholinoceptor and classification thereof
Cholinoceptor comprises two kinds:
(1) cholinoceptor
Cholinoceptor comprises two kinds:
1. (be called for short: m receptor), produce the parasympathetic nervous hormesis, both cardiomotility inhibition, bronchus gastrointestinal smooth muscle and detrusor of bladder shrink muscarinic receptor, digestive gland is secreted increase, contracted pupil etc.Atropine is the M-ChR blocker.
2. nicotine receptor (be called for short: n receptor), N 1Be positioned at the neuroganglion postsynaptic membrane, can cause that the postganglionic neuron of autonomic ganglion is excited; N 2Receptor is positioned at the skeletal muscle telolemma, can cause the motor end plate current potential, causes skeletal muscle excited.The main block N of six hydrocarbon quaternary amines 1Function of receptors, the tubocurarine block N 2Function of receptors.
(2) hypotype of M cholinoceptor and distribution
1. pharmacology's typing of M cholinoceptor
The recognition site of M cholinoceptor is quite conservative between each hypotype, though obtained multiplely to M cholinoceptor blocker selectively at present, finds the blocker that only a kind of receptor subtype is had high selectivity yet.
The selective exclusion agent of M cholinoceptor hypotype commonly used is following:
M 1That the cholinoceptor affinity is the highest is MT17, and it is a kind of toxin of finding in recent years in the African green poisonous snake snake venom that is contained in.Other still has 4-DAMP, tripitramine, pirenzepine etc.
M 2That the cholinoceptor affinity is the highest is tripitramine.Other still have AFDX384, himbacine and 4-DAMP.
M 3That the cholinoceptor affinity is the highest is 4-DAMP.Other still have darifenacin.Exocrine gland, smooth muscle, blood vessel endothelium, brain and autonomic ganglion.
M 4That the cholinoceptor affinity is the highest is MT 3Other still have 4-DAMP, himbacine and AFDX384.
M 5That the cholinoceptor affinity is the highest is 4-DAMP.Other still have darifenacin.
2. the molecular biology typing of M cholinoceptor
Along with the development of molecular biology clone technology,, find to exist at least the m receptor hypotype of five kinds of different genes codings, respectively called after m through the m receptor cDNAs of being cloned into being transcribed into the analysis of different amino acid sequence 1, m 2, m 3, m 4, m 5Receptor comparatively is recognized that M 1, M 2, and M 3Three kinds of hypotypes.
3. M cholinoceptor subtype distribution
M 1Cholinoceptor mainly is distributed in parietal cell, neuroganglion and central nervous system;
M 2Cholinoceptor mainly is distributed in heart, brain, autonomic ganglion and smooth muscle;
M 3Cholinoceptor mainly is distributed in exocrine gland, smooth muscle, blood vessel endothelium, brain and autonomic ganglion;
(3) N cholinoceptor and distribution thereof
N receptor is divided into N according to the difference that distributes 1, N 2Two kinds.The both is a part door-control type cationic channel, when Ach with after n receptor combines, the n receptor space conformation changes, focal depolarization takes place in channel opener.When the depolarization level reached sodium channel opening threshold value, the sodium channel was open, causes action potential.Has N 2The Skeletal Muscle Cell of receptor shows as stream and intracellular Ca2+ release in the extracellular Ca2, muscle contraction; Has N 1The neuroganglion of receptor time one-level neuron shows as excited continuation transmission.
N 1Receptor is distributed in neuroganglion;
N 2Receptor is distributed in neuromuscular junction (Skeletal Muscle Cell film).
3, the classification of M cholinergic receptor-blocking agent
The M cholinergic receptor-blocking agent comprises three types:
1. the alkaloid of natural formation is like atropine and scopolamine; Atropine, scopolamine and Anisodamine all can be by extracting in the plant, and naturally occurring alkaloid is unsettled left-handed hyoscyamine, in leaching process, can obtain stable racemization hyoscyamine, is atropine.Scopolamine is a levo form, works than the strong manyfold of d-isomer.Atropine and analog basic structure thereof are the tertiary amine alkaloid fat of tropic acid.
2. the semi-synthetic derivant of natural alkaloid, its physiological disposition is different with parent drug with action time;
3. synthetic alkaloid, wherein some drugs has the selectivity antagonism to M cholinoceptor hypotype, like melyltropeine, tropicamide etc.
4, the effect of M cholinergic receptor-blocking agent and mechanism thereof
This type of drug effect is extensive, has the effect of lax visceral smooth muscle, releasing smooth muscle spasm; The secretion of salivary gland, sweat gland, gastrointestinal gland etc. can be suppressed, xerostomia and thirsty sense can be caused after the medication; Remove the inhibition of vagus nerve, make palpitating speed heart; Lax iris sphincter, the big pupil that looses, intraocular pressure raises; Excited respiratory center.
This type of medicine can combine with cholinoceptor but not produce or seldom produce the choline effect of intending; But can hinder acetylcholine (be called for short: ACh) or the cholinoceptor excitomotor combine with smooth muscle, cardiac muscle, gland cell, peripheral nervous joint and central nervous system's M cholinoceptor, thereby its plan choline effect of antagonism.The central nervous system, like bone marrow, cortex and subcortical center level, its cholinergic transmission all relates to the function of M cholinoceptor.
There is research data to show; The effect of M cholinergic receptor-blocking agent antiendotoxin shock relates to vagus nerve cholinergic anti-inflammatory pathway; And nicotine α 7 acetylcholinergic receptors (α 7-nicotinic acetylcholine receptor; Be called for short: α 7nAChR) participate in this process (Liu C, et al.Antishock effect of anisodamine involves a novel pathway for activating alpha7nicotinic acetylcholine receptor.Crit Care Med.2009; 37:634-641).
Recent pertinent literature report is pointed out; Nicotinic receptor agonists can alleviate hepatic ischemia-reperfusion injury (Crockett ET, et al.Protection of early phase hepatic ischemia-reperfusion injury by cholinergic agonists.BMC Clin Pathol.2006Feb 15; 6:3); After vagus nerve cuts off; The specific agonist of α 7nAChR can improve hepatocellular apoptosis (Hiramoto T, the et al.The hepatic vagus nerve attenuates fas-induced apoptosis in the mouse liver via alpha7nicotinic acetylcholine receptor.Gastroenterology.2008 of acute severe hepatitis due to the Fas albumen; 134:2122-2131).
But the relation of M cholinergic receptor-blocking agent and ischemical reperfusion injury is not appeared in the newspapers.
5, the purposes of M cholinergic receptor-blocking agent and application
M cholinergic receptor-blocking agent such as atropine are usually used in preanesthetic medication, can reduce the mucous secretion of anesthesia process mesobronchus, in case respiratory tract obstruction and aspiration pneumonitis; Can eliminate the inhibition of morphine, remove vagal excitation property, can prevent the laryngospasm of penthiobarbital breathing.To the septic shock due to toxic dysentery, the toxic pneumonitis etc.; Available heavy dose of M cholinergic receptor agonist such as atropine are treated; The microcirculation improvement to remove arteriolospasm; Make the blood pressure rise and improve symptom, routine dose of its treatment endotoxin shock is: common dose atropine 1～2mg, and every at a distance from intravenous injection in 15～20 minutes; Anisodamine 10～20mg, every at a distance from intravenous injection in 15～20 minutes; Scopolamine 0.01～0.03mg/kg, per 30 minutes quiet pushing away once; Its side effect mainly shows as xerostomia, flushing, slight platycoria, it is fuzzy etc. to look nearly thing, and individual patient has heart rate to accelerate and dysuria.
The M cholinergic receptor-blocking agent is clinical to be mainly used in:
1. separate the spasmic pain of gastrointestinal smooth muscle;
2. the slow preanesthetic medication that is used for reduces the mucous secretion of anesthesia process mesobronchus, in case respiratory tract obstruction and aspiration pneumonitis; Can eliminate the inhibition of morphine, remove vagal excitation property, can prevent the laryngospasm of penthiobarbital breathing;
3. be used for the infectious shock of poisoning.To the septic shock due to toxic dysentery, the toxic pneumonitis etc., available heavy dose of atropine treatment, the microcirculation improvement to remove arteriolospasm gos up blood pressure and improves symptom;
4. treat arrhythmia;
5. topical is used for iridocyclitis, makes platycoria, makes ciliary muscle obtain lax rest the (regulating paralysis), reaches antiinflammatory, aim of alleviating pain; And the big pupil that looses capable of using be used for checking fundus oculi disease;
6. save the organophosphorus compounds pesticide intoxication.
In recent years, through clinical observation, also find the new purposes of this type medicine:
1. treat the fulminant epidemic cerebrospinal meningitis;
2. treat children asthmatic brouchitis;
3. treat cold injury;
4. treat sciatica.
In addition, Anisodamine to children's's enteritis in autumn in winter, acute hemorrhagic necrotic enteritis, focal nerve levy, children's's " knot brain ", heavy infantile pneumonia, acute diffuse glomerulonephritis, acute appendicitis in infants and children, children epilepsy persistent state, hypertension, coronary heart disease, rheumatoid arthritis, ascariasis of biliary tract, cholecystitis, acute hemiplegia in infant and childhood disease etc. have certain curative effect.
(3) research overview of cholinesterase inhibitor
1, general introduction
Cholinesterase inhibitor is the same with ACh, can (be called for short: AChE) combines, but combine more firmly, hydrolysis is slower, is that the AChE activity is suppressed, thereby the ACh that causes the cholinergic nerve tip to discharge piles up, and produces the effect of plan choline with Acetylcholinesterase.
2, the effect of cholinesterase inhibitor
This type of medicine has the gastrointestinal smooth muscle of contraction, increases the effect of smooth muscle tension force; Stimulate sphincter pupillae and ciliary muscle contraction, regulate ciliary spasm; Suppress skeletal muscle neuromuscular junction AChE, relaxed muscle; Can suppress the secretion of salivary gland, sweat gland, gastrointestinal gland etc.; Act on the vasomotor center of oblongata, decreased heart rate, cardiac output are descended.
3, the purposes of cholinesterase inhibitor and application
Cholinesterase inhibitor such as neostigmine are commonly used to help gastrointestinal function recovery; Share with anesthetis, can be used for anaesthetizing back analgesia, need not continue medication to obtain good and persistent postoperative analgesic effect, causes unify side effect (the Hofer S. of digestive system of cardiovascular system but can cause that acetylcholine is accumulated; Eisenbach C., Lukic I.K., Schneider L., Bode K.; Brueckmann M., Mautner S., Wente M.N., Encke J.; Werner J., Dalpke A.H., Stremmel W., Nawroth P.P.; Martin E., Krammer P.H., Bierhaus A.; Weigand M.A., Pharmacologic cholinesterase inhibition improves survival in experimental sepsis.Crit.Care.Med.2008,36 (2): 404-408).
Be mainly used in clinically:
1. direct or indirect excited skeletal muscle, the treatment myasthenia gravis;
2. can excited intestinal smooth muscle and detrusor of bladder, promote aerofluxus and urinate, be usually used in postoperative urine retention;
3. treat glaucoma, can make contracted pupil behind the eye drip, intraocular pressure descends;
Detoxifcation when 4. competitive neuromuscular blocking drug is excessive.
The new method of domestic in recent years this medicine of use and new purposes:
1. increasing cholinesterase inhibitor by drugs approved by FDA be used for alzheimer disease (Alzheimer ' s disease, be called for short: treatment AD).AD patient's central position, especially cortex lower area, like nucleus basalis cholinergic neuron integrity defective, the method that therefore available cholinesterase inhibitor increases cholinergic neurotransmitter in the central nervous system is improved cognitive function, delays the progress of the AD state of an illness;
2. acupoint injection therapy helps restoration of gastrointestinal function;
3. share with anesthetis, be used to anaesthetize the back analgesia, need not continue medication to obtain good and persistent postoperative analgesic effect.
Therefore, it is still imperative to seek the little ischemia resisting reperfusion injury of novel, definite ingredients, determined curative effect, untoward reaction.But the medicine composite for curing ischemical reperfusion injury that M cholinergic receptor-blocking agent and cholinesterase inhibitor are united use does not appear in the newspapers as yet.
Through the document retrieval etc., up to the present, find as yet to have new ischemia resisting reperfusion injury product and its production and use etc. the report of aspect.
Summary of the invention
This finds that the technical problem of required solution is the pharmaceutical composition that discloses a kind of M of containing cholinergic receptor-blocking agent and cholinesterase inhibitor; Promptly this pharmaceutical composition has the new role of ischemia resisting reperfusion injury; Can be used in preparation ischemia resisting reperfusion injury product, to overcome the above-mentioned defective that prior art exists.
That is to say, the present invention is directed to the deficiency of prior art, through experimentation and theory study, one of purpose is intended to provide a kind of new pharmaceutical composition, and the drug regimen composition formula of a kind of M of containing cholinergic receptor-blocking agent and cholinesterase inhibitor promptly is provided;
Two of the object of the invention provides a kind of ischemia resisting reperfusion injury preparation of compositions method;
Three of the object of the invention provides a kind of purposes of ischemia resisting reperfusion injury compositions, promptly provides to contain the application of aforementioned pharmaceutical compositions as ischemia resisting reperfusion injury product aspect.
Ischemia resisting reperfusion injury product of the present invention is meant in the technical fields such as medicine, a kind of product that directly is used to prevent, diagnose, detect, protect, treat and study ischemical reperfusion injury and directly related disease thereof;
The preferred product that directly is used to prevent, diagnose, detect, protect, treat and study hepatic ischemia-reperfusion injury and directly related disease thereof, promptly anti-hepatic ischemia-reperfusion injury product.
Described ischemia resisting reperfusion injury product is to comprise in medicine, the reagent etc. one or more, preferred agents.
(1) technical conceive
Independent development research and development original new drug is a present urgent task of China, and the new combination of discovery novel drugs, existing medicine or new purposes etc. all are effectively quick approach, also are the advantage places of Chinese quick original new drug development.
The control of ischemical reperfusion injury is a research focus in recent years.When disturbance of blood circulation such as traumatic shock, surgical operation, organ transplantation, burn, cold injury and thrombosis, all postischemic reperfusion damage can appear.Histoorgan requires degree high more to oxygen, and ischemical reperfusion injury takes place more easily, after one's own heart, brain, liver, kidney etc.
But the mechanism of ischemical reperfusion injury is complicated, be mutual, the coefficient result of multiple factor, and single Study on Mechanism often can not reach satisfied effect, so comprehensive study number of mechanisms, that the multiple medicines thing is united utilization remains further to be carried out.
At present, ischemical reperfusion injury becomes one of medical measure key of success factors such as thrombolytic therapy, Coronary Artery Bypass, the replantation of amputated limb, organ transplantation.Therefore, the product, particularly medicine of aspects such as development control ischemical reperfusion injury, significant, and have remarkable social benefit and economic benefit.
Along with the research to the cholinergic anti-inflammatory pathway constantly is tending towards perfect, the application thinking and the practicable method that also are bound to produce more heterogeneous pass are served clinical treatment, also for research control ischemical reperfusion injury one new strategy are provided simultaneously.
The inventor is through carrying out the screening of system to the pharmaceutical composition that contains M cholinergic receptor-blocking agent and cholinesterase inhibitor; And infer that this pharmaceutical composition is in the drug effect of preventing and treating aspects such as ischemical reperfusion injury; Should mainly bring into play through the cholinergic anti-inflammatory pathway, result of study also proves and has confirmed that this pharmaceutical composition has the pharmacological action of significant ischemia resisting reperfusion injury.
According to this idea and thinking, the inventor passes through experimentation and analysis and theory study repeatedly, successfully obtains the result of study and the application product of expecting.
(2) ischemia resisting reperfusion injury composition and method of making the same
Described ischemia resisting reperfusion injury compositions is the pharmaceutical composition that comprises M cholinergic receptor-blocking agent and cholinesterase inhibitor;
The weight ratio of described M cholinergic receptor-blocking agent and cholinesterase inhibitor is 1: 0.005～50; Further preferred 1: 0.1～8; Most preferably be 1: 2.
Described M cholinergic receptor-blocking agent is the conventional M cholinergic receptor-blocking agent that uses in this area; Be to comprise atropine, Anisodamine, contain in plant extract, the scopolamine of Anisodamine, the plant extract that contains scopolamine, melyltropeine or the tropicamide etc. one or more; Preferred atropine, Anisodamine, the plant extract that contains Anisodamine, scopolamine or contain in the plant extract etc. of scopolamine one or more; In further preferred atropine, Anisodamine or the scopolamine etc. one or more, most preferably atropine;
Described atropinic structural formula is suc as formula shown in 1, and it is the esters that is formed by tropic acid and organic base, and structure and Anisodamine and scopolamine are similar;
Figure BDA0000100492130000101
Formula 1
The structural formula of described Anisodamine is suc as formula shown in 2:
Figure BDA0000100492130000111
Formula 2
The structural formula of described scopolamine is suc as formula shown in 3:
Formula 3
Anisodamine of the present invention and scopolamine can also extract from plant;
Described Anisodamine can extract from the plant Radix Anisodi Tangutici; But method for distilling list of references " Simultaneous analysis of hyoscyamine; Scopolamine; 6 β-hydroxyhyoscyamine and apoatropine in Solanaceous hairy roots by reversed-phase high-performance liquid chromatography " (Journal of Chromatography A, 2005,1091 (1-2): 32-39);
Described scopolamine can extract from plant of Solanaceae, but method for distilling list of references " Analysis of tropane and related alkaloids " (Journal ofChromatography A, 2002,978 (1-2): 1-35);
Described cholinesterase inhibitor is the conventional cholinesterase inhibitor that uses in this area, one or more in preferred neostigmine or the physostigmine etc., further preferred neostigmine;
The structural formula of described neostigmine is suc as formula shown in 4:
Figure BDA0000100492130000113
Formula 4
The structural formula of described physostigmine is suc as formula shown in 5:
Figure BDA0000100492130000121
Formula 5
Ischemia resisting reperfusion injury preparation of compositions method is following:
Described M cholinergic receptor-blocking agent, cholinesterase inhibitor are mixed in proportion, promptly get described ischemia resisting reperfusion injury compositions;
Preparation of drug combination method of the present invention can be with the existing commercially available above-claimed cpd that gets; Or the extract that contains above-claimed cpd that from plant, extracts; By proportioning, adopt the conventional method of this area to mix, promptly get described ischemia resisting reperfusion injury compositions.Each raw material addressed among the present invention or reagent are all commercially available to be got.
Certainly, in actual use or in emergency circumstances, can be directly M cholinergic receptor-blocking agent and cholinesterase inhibitor single variety be mixed earlier re-using, for example mode such as intravenous drip, intramuscular injection;
Also can no longer carry out prior mixing, but directly M cholinergic receptor-blocking agent and cholinesterase inhibitor single variety used respectively, and successively use continuously, for example mode such as intravenous drip, intramuscular injection.
(3) research of ischemia resisting reperfusion injury composite formula basis
1, experimentation
(1) checking liver organization express alpha 7nAChR
Get mouse liver, detect the expression of its α 7nAChR behind the extraction total protein, find that hepatic tissue has the expression of α 7nAChR.
(2) activate α 7nAChR and alleviate hepatic ischemia-reperfusion injury
Mice group, that is: Sham (sham-operation), I/R group (perfusion again behind the liver ischemia), PNU-282987 (α 7nAChR selective agonist)+I/R group, Atr.+Neo. (Atr., atropine, M cholinergic receptor-blocking agent; Neo., neostigmine, cholinesterase inhibitor)+the I/R group.A plurality of time points are set: promptly pour into several time points of back again, each time point is put to death animal, gets hepatic tissue and carries out pathological examination.
Pathological examination shows: selectedly pour into that 6h time point hepar damnification is the most serious, apoptosis hepatocyte quantity is maximum again.
(3) activate α 7nAChR and reduce Caspases family active in the hepatic ischemia reperfusion process
Mice group, that is: Sham group, I/R group, PNU-282987+I/R group, Atr.+Neo.+I/R group.A plurality of time points are set: promptly pour into several time points of back again, each time point is put to death animal.Get serum and liver organization and measure inflammatory factor; Get liver organization.This time point, with I/R group relatively, all can obviously alleviate the hepatic injury due to the ischemia-reperfusion after PNU-282987 and the Atr.+Neo. pretreatment; Caspase-3, Caspase-8, the active significantly decline of Caspase-9; But the inflammatory factor of hepatic tissue and serum does not all have significant change with each time point between each group.
(4) activate α 7nAChR and alleviate response to oxidative stress in the hepatic ischemia reperfusion process
Mice group, that is: Sham, I/R group, PNU-282987+I/R group, Atr.+Neo.+I/R group are poured into behind the ischemia again; Put to death animal; Get hepatic tissue and detect its MDA content and SOD activity, find: compare the active significantly rising of the growing amount of model group MDA and SOD with sham operated rats; The growing amount of the MDA of PNU-282987 and Atr.+Neo. pretreated group significantly is lower than model group, and the SOD activity is significantly higher than model group.
(5) effect of rami hepatici nervi vagi in hepatic ischemia-reperfusion injury
Mice group, that is: Sham, I/R group, Vago group (rami hepatici nervi vagi excision), Vago+I/R group, PNU-282987+Vago+I/R group, Atr.+Neo.+Vago+I/R group, Atr.+Neo.+I/R group.Get liver organization and measure Caspase-3.The result shows: compare with the Vago+I/R group, the Caspase-3 of PNU-282987+Vago+I/R group significantly reduces, and points out no matter whether complete vagus nerve is, activates α 7nAChR and can suppress apoptosis; Organize relatively with Vago+I/R, the Caspase-3 of Atr.+Neo.+Vago+I/R group does not have significance and changes, and prompting Atr.+Neo. inhibition apoptotic effect needs vagal integrity; Compare with the Atr.+Neo.+Vago+I/R group, the Caspase-3 of Atr.+Neo.+I/R group is active significantly to be reduced, and pointing out vagal preservation is the basis that Atr.+Neo. brings into play anti-apoptotic effect.
Above-mentioned experiment prompting α 7nAChR is relevant with ischemical reperfusion injury, and the M cholinergic receptor-blocking agent can pass through to strengthen indirectly the activation of vagus nerve mediator acetylcholine to α 7nAChR, thereby plays the effect that alleviates ischemical reperfusion injury.
Conclusion: α 7nAChR apoptosis damage due to antioxidation plays an important role in the hepatic ischemia-reperfusion injury process; Atropine can effectively improve hepatic ischemia-reperfusion injury; Preserving of vagus nerve function is beneficial to anti-hepatic ischemia-reperfusion injury.
2, theory study
The inventor discover hypertension the time cholinergic anti-inflammatory pathway function reduction, the rat heart, kidney and aortic tissue α 7nAChR expression reduce, α 7nAChR -/-Hypertension induced organ injury increases the weight of; And α 7nAChR selective agonist treatment spontaneous hypertensive rat can alleviate its organ injury (Li DJ; Evans RG; Yang ZW, Song SW, Wang P; Ma XJ, et al.Dysfunction of the cholinergic anti-inflammatory pathway mediates organ damage in hypertension.Hypertension.2011; 57:298-307).
Research confirms that also α 7nAChR influences the apoptosis and the birth process of cell; Exciting α 7nAChR can significantly suppress (Egleton RD such as apoptosis, induction of vascular new life and promotion cell proliferation; Brown KC, Dasgupta P.Nicotinic acetylcholine receptors in cancer:Multiple roles in proliferation and inhibition of apoptosis.Trends Pharmacol Sci.2008; 29:151-158; Ni M; Yang ZW; Li DJ, Li Q, Zhang SH; Su DE, et al.Apotential role of alpha-7nicotinic acetylcholine receptor in cardiac angiogenesis in a pressure-overload rat model.J Pharmacol Sci.2010; 114:311-319).
Hiramoto etc. discover; After vagus nerve cuts off; The specific agonist PNU-282987 of α 7nAChR can improve the hepatocellular apoptosis of acute severe hepatitis due to the Fas albumen, mechanism maybe with suppress cell and produce excessive active oxygen relevant (Hiramoto T, ChidaY; Sonoda J; Yoshihara K, Sudo N, Kubo C.The hepatic vagus nerve attenuates fas-induced apoptosis in the mouse liver via alpha7nicotinic acetylcholine receptor.Gastroenterology.2008; 134:2122-2131); Reports such as Parada; α 7nAChR on the exciting SH-SY5Y cell can suppress apoptosis (the Parada E due to the anoxia; Egea J, Romero A, del Barrio L; Garcia AG, Lopez MG.Poststress treatment with pnu282987can rescue sh-sy5y cells undergoing apoptosis via alpha7 nicotinic receptors linked to a jak2/akt/ho-1signaling pathway.Free Radic Biol Med.2010; 49:1815-1821).
But α 7nAChR does not appear in the newspapers to the influence of hepatic ischemia-reperfusion injury.Therefore, the inventor supposes that the activation of α 7nAChR can alleviate hepatic ischemia-reperfusion injury.
The effect of M cholinergic receptor-blocking agent such as Anisodamine antiendotoxin shock relates to vagus nerve cholinergic anti-inflammatory pathway, and warps such as M cholinergic receptor-blocking agent such as Anisodamine blocking-up macrophage m receptor can strengthen the activation of vagus nerve mediator acetylcholine to α 7nAChR indirectly.
Different with M cholinergic receptor-blocking agent such as Anisodamine is, atropine is usually used in administration before the surgical operation, and nearly all patient with operation can use.Therefore, can the atropine of preferably studying in the M cholinergic receptor-blocking agent be realized α 7nAChR is activated the anti-hepatic ischemia-reperfusion injury effect of performance through vagus nerve cholinergic anti-inflammatory pathway, has very important clinical meaning.
In order to avoid the side effect of postoperatives such as M cholinergic receptor-blocking agent such as atropine as much as possible; The inventor is giving M cholinergic receptor-blocking agent such as atropinic cholinesterase inhibitor such as the neostigmine etc. of giving simultaneously; The slow down hydrolysis rate of vagus nerve mediator acetylcholine is to obtain better to prevent the effect of reperfusion injury.
In view of this, inventor hypothesis: ischemical reperfusion injury is particularly in the hepatic ischemia-reperfusion injury process, and α 7nAChR apoptosis and inflammation damnification due to antioxidation play an important role; M cholinergic receptor-blocking agent such as atropine etc. can effectively improve hepatic ischemia-reperfusion injury, guarantee that the integrity of rami hepatici nervi vagi helps alleviating reperfusion injury.
(4) mechanism of action of ischemia resisting reperfusion injury compositions
Hepatic ischemia-reperfusion injury is a surgery of liver common pathological physiological process; All relate to this process (Banga NR like severe hepatic trauma, hepatectomy, liver transplantation etc. in the clinical position; Homer-Vanniasinkam S, Graham A, Al-Mukhtar A; White SA, Prasad KR.Ischaemic preconditioning in transplantation and major resection ofthe liver.Br J Surg.2005; 92:528-538).Ischemical reperfusion injury mechanism is extremely complicated, relates to approach such as apoptosis and necrosis.Human different reasons cause hepar damnification, even apoptosis is an important characteristic (AkazawaY, Gores GJ.Death receptor-mediated liver injury.Semin Liver Dis.2007 in the hepatic fibrosis process; 27:327-338).Research is illustrated in the course of liver damage that ischemia-reperfusion causes and has hepatocellular apoptosis (Neuma MG.Apotosis in disease ofthe liver.Crit Rev Clin Lab Sci.2001; 38:109-166).
1, cholinergic anti-inflammatory pathway
Inflammatory reaction is protection and the defense reaction that immune system produces when body receives infection and wound strike; The degree that body is regulated innate immune response through the endogenous mechanism of high conservative; Make and cause scorching and anti-inflammatory response tends to balance; Thereby stable (Nandakumar KS, the Holmdahl R.Therapeutic cleavage of igg:New avenues for treating inflammation.Trends Immunol.2008 of environment in keeping; 29:173-178).
Lack inflammatory reaction or excessive inflammatory response and all can cause the pathologic reaction; Lacking suitable inflammatory reaction meeting increases infection rate or causes secondary infection; And overreaction can cause that pro-inflammatory mediators such as IL-6, TNF-α discharge in a large number, forms systemic inflammatory reaction, causes serious pathologic complication such as sepsis and autoimmune disease, thereby causes the bigger damage of body.
Therefore intravital inflammatory reaction intensity must receive meticulous adjusting, and body controls inflammation reaction mainly through two mechanism: innate immunity mechanism and the brain source property immunomodulating path self controlled.The former discharges performance feedback regulation effect in inflammatory reaction such as IL-10, IL-4, TGF by activatory immunocyte; The latter has HPAA control glucocorticoid to discharge the regulation and control immunoreation.Discovered in recent years, nervous system be through the α 7nAChR of vagus nerve on can the activating macrophage film, and then suppress the release of TNF-α significantly, apace, alleviates systemic inflammatory reaction.This physiological mechanism is called " cholinergic anti-inflammatory pathway " (cholinergic anti-inflammatory pathway) (Fodale V, Santamaria LB.Cholinesterase inhibitors improe surival in experimental sepsis:A new way to activate the cholinergic anti-inflammatory pathway.Crit Care Med.2008; 36:622-623).
(α 7-nicotinic acetylcholine receptor, be called for short: α 7nAChR) become the research focus, people have begun the research to the effect of α 7nAChR in ischemical reperfusion injury along with nicotine α 7 cholinoceptors.
α 7nAChR extensively exists at body; Comprise (Lindstrom J such as neuronal cell, immunocyte, endotheliocyte and epithelial cell; Luo J, Kuryatov A.Myasthenia gravis and the tops and bottoms of achrs:Antigenic structure of the mir and specific immunosuppression of eamg using achr cytoplasmic domains.Ann N Y Acad Sci.2008; 1132:29-41); α 7nAChR is that an important inflammation is regulated receptor; Nerve-process of immune regulation in the mediation of cholinergic anti-inflammatory pathway has important effect (Wang H, Yu M, Ochani M; Amella CA; Tanovic M, Susarla S, et al.Nicotinic acetylcholine receptor alpha7subunit is an essential regulator of inflammation.Nature.2003; 421:384-388).
The core content of cholinergic anti-inflammatory pathway is: the main mediator acetylcholine that vagus nerve discharges can activating macrophage film α 7nAChR; Effectively reducing macrophage discharges proinflammatory cytokine and regulates anti-inflammatory cytokines content; Thereby reach the extent of reaction that controls inflammation, the damage of performance anti-inflammatory, infection and shock effect (Ulloa L.The vagus nerve and the nicotinic anti-inflammatory pathway.Nat Rev Drug Discov.2005; 4:673-684).
Research to α 7nAChR mainly concentrates on the nerve-immunomodulating path in the mediation of cholinergic anti-inflammatory pathway; The function that makes researcher comparatively pay close attention to is to be expressed in anti-inflammatory properties (the Wang DW that macrophage etc. produces the α 7nAChR mediation on the inflammatory factor cell membrane; Zhou RB; Yao YM.Role of cholinergic anti-inflammatory pathway in regulating host response and its interventional strategy for inflammatory diseases [J] .Chin J Traumatol; 2009Dec, 12 (6): 355-64.; Liu C; Shen FM, Su DF, et al.Antishock effect ofanisodamine involves a novel pathway for activating alpha7nicotinic acetylcholine receptor [J] .Crit Care Med; 2009,37 (2): 634-41.; Parrish WR; Rosas-Ballina M; Pavlov VA; Et al.Modulation of TNF release by choline requires alpha7subunit nicotinic acetylcholine receptor-mediated signaling [J] .Mol Med, 2008,14 (9-10): 567-74.).
People (Elahe T Crockett such as Elahe T Crockett; James J Galligan; Et al.Protection ofearly phase hepatic ischemia-reperfusion injury by cholinergic agonists [J] .BMC Clinical Pathology, 2006, Feb 15:6-3.) discover; Give the expression that alpha 7 nAChR agonists can reduce the cellular inflammation factor in early days at hepatic ischemia reperfusion, alleviate hepatic injury.This possibly be because the α 7nAChR on the Kuffer cell is activated, and has started α 7nAChR anti-inflammatory pathway, thereby has alleviated tissue injury.The acetylcholine that α 7nAChR anti-inflammatory pathway vagus nerve tip discharges is with after the α 7nAChR on the macrophage membrane combines; Adjustable inflammatory reaction degree (the Wang DW that causes by infection, shock etc.; Zhou RB; Yao YM.Role of cholinergic anti-inflammatory pathway in regulating host response and its interventional strategy for inflammatory diseases [J] .Chin J Traumatol; 2009Dec, 12 (6): 355-64.).
Up to now, α 7nAChR does not appear in the newspapers in the influence of hepatic ischemia-reperfusion injury.The inventor supposes that the activation of α 7nAChR can alleviate hepatic ischemia-reperfusion injury.
2, the antishock α 7nAChR mechanism of Anisodamine
Belladonna alkaloid is organic lipid of tropic acid and organic base be combined into; Comprise atropine, scopolamine, Anisodamine and Anisodine etc., belladonna alkaloid belongs to the M cholinergic receptor-blocking agent, its pharmacological action and strong and weak relevant with its chemical structural formula; Such medicine has pharmacological action widely; Comprise the inhibition glandular secretion, lax visceral smooth muscle, blood vessel dilating etc.Atropine can be removed smooth muscle spasm, and the microcirculation improvement obstacle is better to the early efficacy of shock.In addition, administration before the atropine Rhizoma Atractylodis Macrocephalae commonly used clinically.
Discover recently; Anisodamine can increase the combination rate of acetylcholine and N type cholinoceptor, thus reinforcing alpha 7nAChR antiinflammatory action (Liu C, Shen FM; Su DF; Et al.Antishock effect of anisodamine involves a novel pathway for activating alpha7nicotinic acetylcholine receptor [J] .Crit Care Med, 2009,37 (2): 634-41.; Parrish WR; Rosas-Ballina M; Pavlov VA; Et al.Modulation ofTNF release by choline requires alpha7subunit nicotinic acetylcholine receptor-mediated signaling [J] .Mol Med, 2008,14 (9-10): 567-74.).
Anisodamine can be removed vasospasm (especially blood capillary) as the M cholinergic receptor-blocking agent, and microcirculation improvement can make the hypertension of reduction under the prerequisite of replenishment of blood content.Also discovery of early stage research (Meng Xingkai, Shi Liubin, the refined window of Peng, etc. the experimentation of the anti-hepatic ischemia-reperfusion injury of Anisodamine [J]. Chinese emergency medicine, 2001,21 (1): 4-5; Huang Ren. anisodamine supplies the protective effect [J] of liver-heat ischemical reperfusion injury to rat. clinical and The Journal of Experimental Medicine; 2009,8 (1): 17-18.), Anisodamine has the effect that suppresses the Na-Ca exchange; Can prevent calcium overload, certain effect arranged preventing reperfusion injury.Anisodamine has certain cytoprotection, can be on cellular level the stabilising membrane structure, the destruction that prevents the radical pair plasma membrane.Use the treatment group hepatic necrosis of Anisodamine lighter, MDA content significantly lowers, and proves that Anisodamine has the effect of anti-law during ischemia damage.These researchs are the protective effect of α 7nAChR in ischemical reperfusion injury evidence are provided, and for the control of ischemical reperfusion injury new target spot are provided simultaneously.
Inventor's research in earlier stage shows; M cholinergic receptor-blocking agent Anisodamine can strengthen the activation of vagus nerve mediator acetylcholine to α 7nAChR indirectly through blocking-up macrophage m receptor, brings into play the effect of its antiendotoxin shock, and this mechanism of action relates to vagus nerve cholinergic anti-inflammatory pathway (Liu C; Shen FM; Le YY, Kong Y, Liu X; Cai GJ, et al.Antishock effect of anisodamine involves a novel pathway for activating alpha7nicotinic acetylcholine receptor.Crit Care Med.2009; 37:634-641).
Administration before different with Anisodamine is atropine the is usually used in surgical operation, nearly all patient with operation can use.Therefore, atropine has more near the probability of clinical practice, studies atropine accordingly and whether has anti-hepatic ischemia-reperfusion injury effect, has very important clinical meaning.In order to avoid the atropine postoperative as much as possible to the unify untoward reaction of digestive system of cardiovascular system; The inventor is giving the atropinic neostigmine that gives simultaneously; The hydrolysis rate of vagus nerve mediator acetylcholine slows down; In the hope of obtaining better to prevent the effect of reperfusion injury, this also meets clinical practice.
There is more side effect in atropinic action range when heavy dose of, common have xerostomia, dizzy, face or erubescence, a tachycardia etc., also can cause the unify inhibition of digestive system of cardiovascular system.Therefore, often unite clinically and give neostigmine, to alleviate atropinic side effect.Neostigmine is a cholinesterase inhibitor, can promote gastrointestinal secretion, alleviates atropine to the gastrointestinal inhibitory action.
In view of this, proof is attempted in this experiment: activate α 7nAChR and can alleviate hepatic ischemia-reperfusion injury; Atropine coupling neostigmine can alleviate hepatic ischemia-reperfusion injury, and its effect depends on vagal integrity.
The present invention is around α 7nAChR; Utilize mouse liver ischemia-reperfusion injury model, rami hepatici nervi vagi to cut off and combine the hepatic ischemia reperfusion model, share neostigmine with α 7nAChR selective agonist, atropine is that intervention means has been studied the influence of α 7nAChR activation to hepatic ischemia-reperfusion injury.
The completion of this experiment will provide new mechanism and prevention target spot for hepatic ischemia-reperfusion injury; The significance of prompting liver surgery process protection rami hepatici nervi vagi provides experimental basis for clinical atropine share neostigmine prevention hepatic ischemia-reperfusion injury.
The present invention utilizes the C57BL/6 mice, by mice part ischemia-reperfusion injury model, has at first studied the effect of α 7nAChR in the hepatic ischemia-reperfusion injury process.The main discovery comprises: pour into the 6h time point behind the ischemia again, hepar damnification is the most serious, and apoptosis is important characteristic change.PNU-282987 pretreatment of α 7nAChR selective agonist or Atr.+Neo. pretreatment can obviously alleviate ischemical reperfusion injury.Prompting thus: α 7nAChR activates has protective effect to hepatic ischemia-reperfusion injury, and the atropine pretreatment can alleviate hepatic ischemia-reperfusion injury.
Experimental result has confirmed content:
1. activate α 7nAChR and can alleviate hepatic ischemia-reperfusion injury;
2. atropine share neostigmine and can alleviate hepatic ischemia-reperfusion injury, and this effect depends on vagal integrity; Vagal preserve to be beneficial to alleviate hepatic ischemia-reperfusion injury;
3. suppressing apoptosis is to activate α 7nAChR to alleviate the main mechanism of hepatic ischemia-reperfusion injury effect.
(5) the ischemia resisting reperfusion injury experiment and the result thereof of ischemia resisting reperfusion injury compositions
The apoptosis that the generation of free radical, oxidative stress etc. cause in the tissue behind the ischemia is the principal character of ischemical reperfusion injury.Apoptosis is the physiological pathological stimuli signal of cell to environment, and cell is under specific endogenous and external source signal induction, and its dead approach is activated, and issues life and death in the regulation and control of related gene and die.
Cellular oxidation phosphorylation obstacle when ischemia-reperfusion, (oxygen free radical is called for short: OFR) to produce a large amount of oxygen-derived free radicals; OFR can react with mitochondrion and intracellular other organelles, and interior free radical of mitochondrion and metabolic center product thereof are increased, and active decline of free radical defense system this moment; The DNA damage of free radical mediated can excite the activation and the p53 accumulation of poly ADP ribose transferring enzyme; Cause apoptosis (Uchiyama T, Otani H, Okada T; Ninomiya H; Kido M, Imamura H, et al.Nitric oxide induces caspase-dependent apoptosis and necrosis in neonatal rat cardiomyocytes.J Mol Cell Cardiol.2002; 34:1049-1061).OFR can react with cell membrane phospholipid; Product is the damaging cells film further, influences the permeability of mitochondrial inner membrane, causes apoptosis (Turrens JF; Beconi M; Barilla J, Chavez UB, McCord JM.Mitochondrial generation of oxygen radicals during reoxygenation ofischemic tissues.Free Radic Res Commun.1991; 12-13Pt 2:681-689).Therefore, in the hepatic injury that ischemia-reperfusion causes, apoptosis occupies critical role.
As effector composition important in the apoptosis mechanism; Caspase family participates in multiple physiology relevant with apoptosis and pathological process (Fauvel H; Marchetti P; Chopin C, Formstecher P, Neviere R.Differential effects of caspase inhibitors on endotoxin-induced myocardial dysfunction and heart apoptosis.Am J Physiol Heart Circ Physiol.2001; 280:H1608-1614).Under multiple incentive condition effect, multiple apoptosis signal transduction approach is activated in the cell, finally will assemble in this common mechanism of Caspase family protein enzyme.Caspase family is present in the cell with the pepsinogen form of non-activity usually.Essential through activation, form active Caspase and just can cause apoptosis.
Different according to initial activated Caspase, the activated approach of apoptosis is divided into 3 kinds: extrinsic pathway, outside approach and endoplasmic reticulum approach.Extrinsic pathway starts from the cell surface death receptor.Because the stimulation of various apoptosis signals, FasL (Fas ligand) and TNF-α act on corresponding receptors, form the apoptosis complex.Complex forms the back and combines in Caspase-8 proenzyme or Caspase-10 proenzyme.Making the activation of Caspase proenzyme is activated Caspase.The Caspase-3 in activatory Caspase-8 or 10 activation downstream, 6,7; Apoptosis (the Malhi H that finally causes cell; Gores GJ, Lemasters JJ Apoptosis and necrosis in the liver:Atale oftwo deaths? Hepatology.2006; 43:S31-44).The endogenous cell apoptosis pathway starts from mitochondrion.When receiving the apoptosis signal stimulus.Mitochondrion discharges cytochrome C to cytoplasm.The proteinase activated factor of a kind of apoptotic cell that exists in cytochrome C and the cell (is called for short: Apaf-1) combines, under the condition that dATP exists, raise the effect in territory, Apaf-1 and the combination of Caspase-9 proenzyme through cysteine proteinase.Make the activation of Caspase-9 proenzyme, activate the effect cysteine proteinase such as the Caspase-2,3,6,7,8,10 in downstream then, thereby start apoptosis (JozaN; Susin SA; Daugas E, Stanford WL, Cho SK; Li CY, et al.Essential role of the mitochondrial apoptosis-inducing factor in programmed cell death.Nature.2001; 410:549-554).
Given this, the inventor has studied the variation of apoptosis factor Csapase family active in the mouse liver ischemia reperfusion injury in the present invention.
The main discovery: compare the active significantly rising of each time point model group Caspases of perfusion back again with matched group; Compare with model group, pour into 6h and 24h time point PNU-282987 and Atr.+Neo. pretreated group again and can significantly reduce the Caspases activity.Prompting thus: PNU-282987 and Atr.+Neo. pretreatment can alleviate the apoptosis due to the hepatic ischemia reperfusion.
Can activate Kupffer cell, neutrophilic granulocyte and mononuclear cell behind the hepatic ischemia reperfusion; The activation of these cells; Can directly discharge short scorching cytokine (Kojima Y; Suzuki S, Tsuchiya Y, Konno H; Baba S, Nakamura S.Regulation of pro-inflammatory and anti-inflammatory cytokine responses by kupffer cells in endotoxin-enhanced reperfusion injury after total hepatic ischemia.Transpl Int.2003; 16:231-240), like TNF-α, IL-1 β, IL-6.Inflammatory mediator further activates inflammatory cell, makes it synthetic and discharges multiple inflammatory factor.Interaction through neutrophilic granulocyte, endotheliocyte causes Liver Microcirculation.Activated neutrophilic granulocyte release oxygen free radical stimulates mononuclear phagocyte and other emiocytosis inflammatory factor.
The inventor has measured the expression of serum inflammatory factor behind the mouse liver part ischemia-reperfusion.Find: after pouring into 3h again; The normal matched group of the expression of model group IL-1 β obviously raises; The expression of the IL-1 β of PNU-282987 pretreated group and Atr.+Neo. pretreated group obviously reduces than model group, other again infusion time put equal no significant difference; The expression of TNF-α is at the equal no significant difference of each time point.Because the expression of inflammatory factor is low excessively, the inventor thinks that it is all expressed in normal range, do not have pathological significance.Consider the organismic internal environment more complicated, the expression of inflammatory factor receives influence of various factors, and apoptosis mechanism also has many approach to participate in, so the inventor infers: in the hepar damnification process due to the ischemia-reperfusion, the inflammation infringement is not a chief reason.
In the liver ischemic stage, oxygen concentration sharply descends, and anerobic glycolysis has substituted aerobic metabolism energy is provided.Produce a large amount of oxygen-derived free radicals, oxygen-derived free radicals acts on lipid generation peroxidization, and the oxidation end-product is that malonaldehyde (is called for short: MDA), can cause the cross-linked polymeric of life macromolecules such as protein, nucleic acid, and have cytotoxicity.Flush phase again, the redox reaction of cell causes the chain type lipid peroxidation because oxygen-derived free radicals produces too much and the active decline of antioxidant reductase, and damaging cells film, organelle and even nucleus cause the necrocytosis apoptosis.Under the normal condition, there be complete antioxidase and the antioxidant system of a cover in the organism, can in time remove them, so to body and not damaged.Antioxidase comprises that superoxide dismutase (is called for short: SOD), glutathion peroxidase (is called for short: GSH-PX) with catalase (abbreviation: CAT).They are present in endochylema and the mitochondrion, and its important function is to reduce H 2O 2Concentration, the protection cell does not receive the damage of strong toxicity OFR.
In the present invention, the inventor has detected oxygen-derived free radicals generation peroxidization afterproduct MDA, and scavenging system SOD is active.Find: compare with matched group, the growing amount that pours into 3h and 6h time point model group MDA significantly raises, and PNU-282987 and Atr.+Neo. pretreatment can significantly reduce perfusion again and cause that MDA increases; Compare with model group, PNU-282987 and Atr.+Neo. pretreatment can further significantly SOD be active.Prompting: PNU-282987 and Atr.+Neo. pretreatment can reduce the MDA increase that ischemia-reperfusion causes, enhance SOD activity simultaneously.
Before address; Vagal main mediator ACh can suppress the macrophage proinflammatory cytokine through " cholinergic anti-inflammatory pathway " and discharge; The damage of performance anti-inflammatory, infection and shock effect (Straub RH; Gluck T; Zeuner M, Scholmerich J, Lang B.Association of pupillary parasympathetic hyperreflexia and systemic inflammation in patients with systemic lupus erythematosus.Br J Rheumatol.1998; 37:665-670; Espanol AJ, Sales ME.Parasympathetic modulation of local acute inflammation in murine submandibular glands.Inflammation.2003; 27:97-105).α 7nAChR is important adjusting target spot in this mechanism.
In addition; Activated another important effect of α 7nAChR is to suppress apoptosis (Parada E; Egea J, Romero A, del Barrio L; Garcia AG, Lopez MG.Poststress treatment with pnu282987can rescue sh-sy5y cells undergoing apoptosis via alpha7nicotinic receptors linked to a jak2/akt/ho-1signaling pathway.Free Radic Biol Med.2010; 49:1815-1821; Egleton RD; Brown KC, Dasgupta P.Nicotinic acetylcholine receptors in cancer:Multiple roles in proliferation and inhibition of apoptosis.Trends Pharmacol Sci.2008; 29:151-158; Ni M; Yang ZW; Li DJ, Li Q, Zhang SH; Su DF, et al.Apotential role of alpha-7nicotinic acetylcholine receptor in cardiac angiogenesis in a pressure-overload rat model.J Pharmacol Sci.2010; 114:311-319).The main mediator acetylcholine that vagus nerve discharges is a α 7nAChR endogenic ligand.Therefore, estimating vagus nerve, to avoid the rami hepatici nervi vagi damage in the effect of hepatic ischemia-reperfusion injury for the caution surgical operation be actively significant.
The inventor utilizes rami hepatici nervi vagi to cut off model this problem is studied.
Discover: no matter whether complete the vagus nerve preservation is, activate α 7nAChR and can both suppress apoptosis, and Atr.+Neo. inhibition apoptotic effect need depend on the vagus nerve integrity.Prompting thus: liver surgery process protection rami hepatici nervi vagi has the important clinical meaning.
1, ischemia resisting reperfusion injury compositions can be resisted ischemia-reperfusion and caused the apoptosis effect
This experiment is intended to observe the influence of ischemia resisting reperfusion injury compositions to hepatic ischemia reperfusion mouse model hepatocellular apoptosis.
With mice group; Sham operated rats is sewed up after opening abdomen immediately; Other organizes respectively, and intraperitoneal injection of saline solution is that model group, M cholinergic receptor-blocking agent and/or cholinesterase inhibitor divide some groups according to the experiment needs by ID; Row liver ischemia is handled behind injectable drug, and the ischemia some hrs begins to pour into again, pours into to get liver behind the some hrs and do the TUNEL apoptosis and detect again.
Experimental result confirms: ischemia resisting reperfusion injury compositions can obviously alleviate hepatic ischemia-reperfusion injury.Show that thus two medicines share hepatic ischemia-reperfusion injury model mice hepatocellular apoptosis quantity is significantly smaller than the application separately respectively of two medicines, the collaborative anti-hepatic ischemia-reperfusion injury effect of two kinds of medicines is strengthened greatly.And, the untoward reaction such as side effect that two types of drug combinations also obviously reduce these two types of prescriptions when solely using.
2, ischemia resisting reperfusion injury compositions can weaken hepatic ischemia reperfusion mouse model transaminase activity
This experiment is intended to observe the influence of ischemia resisting reperfusion injury compositions to transaminase in the hepatic ischemia reperfusion mouse model serum.
With mice group, sham operated rats is opened behind the abdomen and is sewed up immediately, and other organizes respectively that intraperitoneal injection of saline solution is model group, M cholinergic receptor-blocking agent and/or cholinesterase inhibitor; Row liver ischemia is handled behind injectable drug, and beginning perfusion again behind the ischemia some hrs is poured into and plucked eyeball behind the some hrs and get whole blood; After room temperature leaves standstill; Centrifugal, get supernatant, measure transaminase activity in the serum.
Ischemia resisting reperfusion injury compositions can reduce hepatic ischemia reperfusion mice serum glutamic oxaloacetic transaminase, GOT and gpt activity; Show thus; Two kinds of drug combinations are used separately respectively less than two medicines the liver dysfunction of hepatic ischemia-reperfusion injury model mice, and the collaborative anti-hepatic ischemia-reperfusion injury effect of two kinds of medicines is strengthened greatly.
The transaminase is a requisite enzyme in this normal operation process of human liver, is liver function, and hepatocyte is transaminase's main existence ground.When hepatocyte is inflamed, poisons, it is hepatocellular impaired to cause during necrosis etc., the transaminase just can be discharged in the blood, and serum transaminase is raise.The transaminase participates in amino acid whose decomposition and synthetic.
3, ischemia resisting reperfusion injury compositions can strengthen hepatic ischemia reperfusion mouse model liver organization SOD activity
This experiment is intended to observe ischemia resisting reperfusion injury compositions to the active influence of hepatic ischemia reperfusion mouse model hepatic tissue SOD, explores the mechanism that ischemia resisting reperfusion injury compositions suppresses lipid peroxidation.
With mice group, sham operated rats is opened behind the abdomen and is sewed up immediately, and other organizes respectively that intraperitoneal injection of saline solution is model group, M cholinergic receptor-blocking agent and/or cholinesterase inhibitor; Row liver ischemia is handled behind injectable drug, and beginning perfusion again behind the ischemia some hrs is opened abdomen after pouring into some hrs again; Hepatic portal vein is put pipe, and row liver perfusion art is got liver tissue homogenate; Get supernatant, measure superoxide dismutase in the liver organization and (be called for short: SOD) activity.
Superoxide dismutase plays crucial effects to the oxidation and the antioxidation balance of body.Ultra oxide anion too much in the body can cause lipid oxidation, and damaging cells is relevant with pathological processes such as multiple disease and agings.But the ultra oxide anion generation of this enzyme catalysis dismutation reaction makes it to be converted into hydrogen peroxide and oxygen, and the protection cell is avoided damage.
4, ischemia resisting reperfusion injury compositions can lower hepatic ischemia reperfusion mouse model liver organization MDA concentration
This experiment is intended to observe ischemia resisting reperfusion injury compositions hepatic tissue lipid peroxidation metabolite malonaldehyde (is called for short: the MDA) influence of content.
With mice group, sham operated rats is opened behind the abdomen and is sewed up immediately, and other organizes respectively that intraperitoneal injection of saline solution is model group, M cholinergic receptor-blocking agent and/or cholinesterase inhibitor; Row liver ischemia is handled behind injectable drug, and beginning perfusion again behind the ischemia some hrs is opened abdomen after pouring into some hrs again; Hepatic portal vein is put pipe, and row liver perfusion art is got liver tissue homogenate; Get supernatant, measure MDA activity in the liver organization.
The generation of the interior free radical of tissue, the cell injury that oxidative stress causes are behind the ischemia.The respiratory chain outburst and the oxidative stress that produce a large amount of free radicals behind the tissue ischemia and cause cause lipid peroxidation.Lipid peroxide be in biomembrane and the cell the contained polybasic unsaturated fatty acid of phosphorus matter by radical damage, oxidation and the peroxidating product that forms; Can cause reactions such as membrane damage, enzyme inhibition, lysosome release, protein-crosslinking, DNA and RNA structural deterioration; Thereby destroy the cell normal physiological function, finally can cause cell death.
(6) purposes of ischemia resisting reperfusion injury compositions
1, general introduction
The mechanism of drug action that the present invention's research relates to is relevant with the cholinergic anti-inflammatory pathway, and has carried out further animal experiment study and theory study.
The inventor through the latest find of research is: ischemia resisting reperfusion injury compositions can be resisted ischemia-reperfusion and caused the apoptosis effect; Weaken hepatic ischemia reperfusion mouse model transaminase activity, strengthen hepatic ischemia reperfusion mouse model liver organization superoxide dismutase activity and lower hepatic ischemia reperfusion mouse model liver organization concentration of malondialdehyde.
The inventor is research emphasis with the hepatic ischemia-reperfusion injury; Set forth the relation between M cholinergic receptor-blocking agent such as atropine etc. and the ischemical reperfusion injury in detail; That is: atropine etc. is through blocking-up M cholinoceptor, and indirect activation α 7nAChR starts the cholinergic path; The abnormal oxidation reduction reaction that inhibition is caused by ischemia-reperfusion, thus the ischemical reperfusion injury of liver alleviated.
But, when disturbance of blood circulation such as traumatic shock, surgical operation, organ transplantation, burn, cold injury and thrombosis, all postischemic reperfusion damage can appear.Histoorgan requires degree high more to oxygen, and ischemical reperfusion injury takes place more easily, after one's own heart, histoorgan that brain, liver, kidney etc. are relevant.And α 7nAChR extensively exists at body; Comprise (Lindstrom J such as neuronal cell, immunocyte, endotheliocyte and epithelial cell; Luo J, Kuryatov A.Myasthenia gravis and the tops and bottoms of achrs:Antigenic structure of the mir and specific immunosuppression of eamg using achr cytoplasmic domains.Ann N Y Acad Sci.2008; 1132:29-41).Therefore, ischemia resisting reperfusion injury compositions all has protective effect in the ischemical reperfusion injury process of all histoorgans that have α 7nAChR to express.
That is to say; Ischemia resisting reperfusion injury compositions can be used in preparation ischemia resisting reperfusion injury product; Can be used for the application such as protection, treatment of the ischemical reperfusion injury of relevant histoorgan such as the related heart such as traumatic shock, surgical operation, organ transplantation, burn, cold injury and thrombosis, brain, liver, kidney, most preferably anti-hepatic ischemia-reperfusion injury product.
Show the outer development that can significantly delay relevant disease of ischemia resisting reperfusion injury composition through experimentation.Completed acute toxicity testing proves; The mouse peritoneal drug administration by injection surpasses 1mg/kg to the maximum tolerated dose of this ischemia resisting reperfusion injury compositions; Be equivalent to more than 20 times of clinical recommended drug dosage; Show that this effective site is safe and reliable, solved such drug dose and used the problem in the taboo.
In sum; The inventor resists the ischemical reperfusion injury compositions and has carried out theory study; Comprise secular pharmacology test through a large amount of experimentatioies; Find that the ischemia resisting reperfusion injury compositions of being addressed has the activity of significant prevention, diagnosis, detection, protection, treatment and the reperfusion injury of research ischemia resisting,, scientific basis is provided for further developing the existing medicine of China for development ischemia resisting reperfusion injury medicine provides new source.
Therefore, the compositions of ischemia resisting reperfusion injury compositions especially pharmaceutical composition can be used for preparing ischemia resisting reperfusion injury product, is the medicine that feedstock production forms with ischemia resisting reperfusion injury compositions of the present invention preferably.
2, the method for using of ischemia resisting reperfusion injury compositions and requirement
Ischemia resisting reperfusion injury compositions of the present invention can be united use with other active component separately or further; Comprise that being used for preparation is used for prevention, diagnosis, detection, protection, treatment and research ischemia resisting reperfusion injury product; Comprise medicine or reagent etc., especially medicine.
Aspect concrete use, ischemia resisting reperfusion injury compositions of the present invention can directly be used separately, can also use with other many chemical substances.These chemical substances biologically active or have the function of treatment disease whether no matter; Comprise miscellaneous function like collaborative amplification, antagonism or alleviate the side effect etc. of ischemia resisting reperfusion injury compositions, these chemical substances are to comprise in pharmaceutically acceptable carrier, natural product, chemical synthetic drug or the human medication etc. one or more; Preferably include in pharmaceutically acceptable carrier etc. one or more.
" the pharmaceutically acceptable carrier " that this paper uses comprises one or more in any He all physiology suitable solvent, disperse medium, afterbirth, antibacterial and antifungal, isotonic agent or the absorption delay agent etc.The example of pharmaceutically acceptable carrier comprises one or more water, saline, PBS, glucose, glycerol or ethanol etc. and in the compositions one or more thereof.In many cases, in said composition, preferably include isotonic agent, for example, sugar, such as in the polyhydric alcohol of mannitol, sorbitol, sorbitol or the sodium chloride etc. one or more.Pharmaceutically acceptable carrier can also comprise a spot of auxiliary substance, one or more in wetting agent or emulsifying agent, antiseptic or the buffer etc. for example, and they have strengthened the effect duration or the effectiveness of this ischemia resisting reperfusion injury compositions.
See that from concrete classification said pharmaceutically acceptable carrier is meant the pharmaceutical carrier that the medicine and pharmacology field is conventional, comprises lubricant, like in Pulvis Talci, Polyethylene Glycol or the magnesium stearate etc. one or more; Disintegrating agent is like in microcrystalline Cellulose, sodium carboxymethyl cellulose or the low-substituted hydroxypropyl cellulose etc. one or more; Filler is like in starch, dextrin or the lactose etc. one or more; Binding agent is like in pregelatinized Starch, cellulose derivative, alginate, gelatin, polyvinylpyrrolidone, polyvinylpyrrolidone or the hydroxypropyl cellulose etc. one or more; Osmotic pressure regulator is like in sodium chloride, glucose, sucrose, sorbitol or the mannitol etc. one or more; The pH regulator agent, one or more in acid such as example hydrochloric acid, sodium hydroxide or the alkali; Solvent, as in water, buffer, ethanol or the propylene glycol etc. one or more etc.; Antioxidant and chelating agent are like among sodium sulfite, the EDTA etc. one or more; Surfactant is like quaternary ammonium compound, hexadecanol etc.; Absorption carrier is like in Kaolin or the soap clay etc. one or more; The macromolecular scaffold agent is like in cyclodextrin, Polyethylene Glycol, the poloxamer etc. one or more; In diluent such as starch, Icing Sugar, dextrin, microcrystalline Cellulose, mannitol, lactose and the Semen sojae atricolor wet goods one or more; In stabilizing agent such as sodium carboxymethyl cellulose or the cyclodextrin etc. one or more; In antiseptic such as ethylparaben or the sodium benzoate etc. one or more.In addition, can also in this pharmaceutical composition, add other adjuvant, like in flavouring agent and/or sweeting agent such as sucrose, fructose and the aspartame etc. one or more.
For example; With ischemia resisting reperfusion injury composition dissolves, suspendible or (for example be emulsifiable in the suitable aqueous solvent; In distilled water, normal saline or the Green's solution etc. one or more) or in the oil-based solvent (for example; Vegetable oil is one or more in olive oil, Oleum sesami, Oleum Gossypii semen, Semen Maydis oil or the propylene glycol etc. for example) in; Can make ejection preparation; Wherein can contain solubilizing agent (for example, one or more in polyoxyethylene sorbitan monoleate, polyoxyethylene hydrogenated Oleum Ricini, polyvidone, cyclodextrin, poloxamer, Polyethylene Glycol, benzyl alcohol, chlorobutanol or the phenol etc.), osmotic pressure regulator (for example, one or more in sodium chloride, glycerol, D9-mannose, D-sorbitol or the glucose etc.) in the solvent.In this case, if necessary, can add additive, for example stabilizing agent (for example, human serum albumin etc.), analgesic (for example, one or more in procaine hydrochloride or the lignocaine etc.) etc.
According to the invention and ischemia resisting reperfusion injury compositions can also unite use, particularly with other chemical substance such as medicine animal especially mammal is comprised that people or other animals treat compositions for use or similar compositions.Said mammal; Comprise in people, mice, rat, sheep, monkey, cattle, pig, horse, rabbit, dog, chimpanzee, baboon, Adeps seu carnis Rhiopithecus roxellanae, macaque or the Rhesus Macacus etc. one or more; In preferred people, mice, rat, monkey, pig, rabbit or the dog etc. one or more, one or more in further preferred people, rat or the monkey etc.For example, can with ischemia resisting reperfusion injury compositions of the present invention add be suitable for to curee's Pharmaceutical composition in.Usually, this Pharmaceutical composition comprises ischemia resisting reperfusion injury compositions of the present invention and pharmaceutically acceptable carrier.
The compositions of ischemia resisting reperfusion injury compositions particularly pharmaceutical composition can have various forms, comprises in the dosage forms such as liquid, semisolid and solid for example one or more; Wherein said pharmaceutical composition comprises that the ischemia resisting reperfusion injury compositions of treating effective dose is an active component, and one or more pharmaceutically acceptable carriers.
The compositions of ischemia resisting reperfusion injury compositions of the present invention especially pharmaceutical composition can adopt conventional production method well known in the art to process any dosage form that is suitable for testing, study or uses clinically; Comprise solid preparation such as capsule, tablet, granular preparation etc., liquid preparation such as oral liquid or injection etc.
Active component is mixed with one or more carriers, be made into required dosage form then.Described dosage form comprises one or more in tablet, capsule, granule, suspensoid, Emulsion, solution, syrup or the injection etc., takes one or more route of administration in oral or injection (comprise in intravenous injection, intravenous drip, intramuscular injection or the subcutaneous injection etc. one or more), the mucosa dialysis etc. to carry out prevention, diagnosis, detection, protection, treatment or the scientific research of ischemia resisting reperfusion injury.
The compositions of ischemia resisting reperfusion injury compositions especially pharmaceutical composition generally must be aseptic and stable under the production condition of storage.Can said composition be mixed with solution, microemulsion, dispersion liquid, liposome or other is suitable for the ordered structure of high drug level.Through with a kind of of this ischemia resisting reperfusion injury compositions of aequum and required mentioned component or combine to add in the appropriate solvent and then carry out aseptic filtration and prepare aseptic parenteral solution.Generally speaking, prepare dispersion liquid through this ischemia resisting reperfusion injury compositions being added in the aseptic solvent that contains basic disperse medium and required above-mentioned other composition.Under the situation of the sterile powder that is used to prepare aseptic parenteral solution, the method for preparing of recommendation is vacuum drying and lyophilized preparation.For example, through passing through to keep required granular size such as the coating of lecithin, under the situation of dispersion liquid and, can keeping the adequate liquidity of solution through using surfactant.
Can comprise the medicament that postpones absorption in the said composition, for example Monostearate or gelatin absorb with the prolongation that reaches injectable composition; Can comprise the high molecular polymer carrier,, discharge with the prolongation that reaches Orally administered composition like hydroxypropyl methylcellulose or polyoxyethylene.
When being used for the patient; Ischemia resisting reperfusion injury composition dosage of the present invention is 0.005～0.05mg/kgd; Can use one or more times, this dosage or consumption decide according to the age of patient or user and the situation of body weight and health or patient's symptom usually.
The compositions of ischemia resisting reperfusion injury compositions of the present invention especially pharmaceutical composition can comprise the ischemia resisting reperfusion injury compositions of the present invention of " treatment effective dose " or " prevention effective dose "." treatment effective dose " is meant at the dosage of necessity and effectively reaches the amount of required therapeutic effect under the time.The treatment effective dose of ischemia resisting reperfusion injury compositions can cause that at this individuality the factors such as ability of required reaction change according to the patient's condition, age, sex and body weight and this ischemia resisting reperfusion injury compositions such as individuality.The treatment effective dose also refers to that the useful therapeutic effect of this ischemia resisting reperfusion injury compositions surpasses the amount of its any toxicity or harmful effect.
" prevention effective dose " is meant the amount that under necessary dosage and time, effectively reaches required preventive effect.Because preventive dose is used for the ill preceding or early stage curee of disease, the prevention effective dose is usually less than the treatment effective dose.The typical non-limiting scope of the treatment of ischemia resisting reperfusion injury compositions of the present invention or prevention effective dose is 0.005～0.05mg/kg, more preferably 0.01～0.02mg/kg.Should note; Dose value will change according to disease type of desiring to alleviate and seriousness; That is to say when being used for the patient; Ischemia resisting reperfusion injury composition dosage of the present invention or consumption decide according to the age of patient or user and the situation of body weight and health or patient's symptom usually.
In addition; Should understand; For any specific curee; Should along with the time according to individual need and give with or supervision give the professional judgement adjustment given dose system with the people of said compositions, and the dosage range that this paper sets is merely illustrative, the scope or the practice of compositions that can't the requirement for restriction protection.
That is to say, need be according to object, route of administration, institute's disease of treating and the situation etc. of treatment, change ischemia resisting reperfusion injury compositions of the present invention at every turn and/or dosage or the consumption of every day.For example, give mammal through vein, adult (like body weight 60kg) especially, the single dose of said ischemia resisting reperfusion injury compositions is about 0.3～3mg, preferably about 0.5mg, preferred administration every day 1 time.Can adjust dosage unit, to propose the best required reaction of arch (for example, treatment or prevention are replied).
For example, can single heavy dose of administration can give several divided doses or reduce or increase dosage in proportion according to the urgency of treatment situation in a period of time.The non-intestinal compositions that preparation is easy to the unified dosage unit form of administration and dosage is especially favourable.The dosage unit form that this paper uses refers to be suitable for the physical separation unit of dosage unit of the mammalian subject of desire treatment; The calculating that each unit contains scheduled volume is used for together producing with required pharmaceutical carrier the active matter ischemia resisting reperfusion injury compositions of required therapeutic effect.The specification of dosage unit form of the present invention; Mixing the interior of this technology that is used for treating individual sensitivity ischemia resisting reperfusion injury compositions by the following specific characteristic of confirming and directly depending on following (a) this ischemia resisting reperfusion injury compositions and the particular treatment of desiring to reach or preventive effect with (b) in restriction.
3, the pharmaceutical dosage form of ischemia resisting reperfusion injury compositions and route of administration
The compositions of ischemia resisting reperfusion injury compositions of the present invention is being used for prevention, diagnosis, detecting of preparation of pharmaceutical compositions, protection, treatment and research ischemia resisting reperfusion injury product especially, and wherein the product according to the requirement preparation of reagent technical field can be used in diagnosis, detection, studies the ischemia resisting reperfusion injury; Can be used in patient's treatment, diagnosis, prevention or research according to the product of the requirement of medical technical field preparation; Can either directly be used to prepare the medicine of treatment, prevention or research separately; Also can mix with many chemical substances or make up, directly or indirectly be used to prepare the medicine of treatment, prevention or research.Chemical substance described here identical with described in this joint preceding text.
In the present invention, required material comprises raw material of the present invention, above-mentioned matching used chemical substance etc., all should adopt the material of SILVER REAGENT or pharmaceutical grade according to practical situation and needs.
The compositions of ischemia resisting reperfusion injury compositions of the present invention is pharmaceutical composition especially, can use the whole bag of tricks administration known in the art, although route of administration/administering mode of in many therapeutic use, recommending is spray or oral administration.But the technical staff will appreciate that route of administration/administering mode changes with required result.In some practical implementation, this reactive compound can together prepare for example controlled release preparation with the carrier of protecting this chemical compound to avoid rapid release, comprises that graft transmission system, transdermal paste one or more in transmission system or the microcapsule transmission system etc.In addition, can also use biodegradable, biocompatible polymer, for example one or more in ethylene-ethyl acetate, polyanhydride, polyglycolic acid, collagen protein, polyorthoesters or the polylactic acid etc.Prepare the equal patent applied for of many methods of this preparation or be generally those skilled in the art and know (Sustained and Controlled Release Drug Delivery Systems for example; J.R.Robinson edits, Marcel Dekker, Inc.; New York, 1978).
The compositions of ischemia resisting reperfusion injury compositions of the present invention especially in the pharmaceutical composition, can contain pharmaceutically acceptable carrier well known in the art and other optional member.Carrier comprises carboxymethyl starch, starch, cellulose, gelatin, sodium bicarbonate, propylene glycol or Tween 80 etc.Optional member for example is coloring agent, sweeting agent, antioxidant etc.
The compositions of ischemia resisting reperfusion injury compositions of the present invention is pharmaceutical composition especially, and one or more modes in administered through oral, rectum or the parenteral etc. are applied to the patient who needs this treatment usually.
The compositions of ischemia resisting reperfusion injury compositions of the present invention especially pharmaceutical composition can be processed any dosage form that is suitable for using clinically; Comprise solid preparation; Like capsule, tablet, granular preparation etc.; Semi-solid preparation such as ointment etc., liquid preparation such as oral liquid, suspensoid, Emulsion etc., perhaps injection.Take one or more route of administration in oral or injection (comprise in intravenous injection, intravenous drip, intramuscular injection or the subcutaneous injection etc. one or more), the mucosa dialysis etc. to carry out prevention, diagnosis, detection, protection, treatment or the scientific research of ischemia resisting reperfusion injury.
Be used for when oral, can be made into conventional solid preparation such as in tablet, powder, granule or the capsule etc. one or more.When implementing, ischemia resisting reperfusion injury compositions of the present invention can be together oral with for example inert diluent or assimilable edible carrier.This ischemia resisting reperfusion injury compositions (with other composition, if desired) can also be wrapped in hard or soft shell gelatin capsules, is pressed into tablet or directly adds in curee's the meals.About oral therapeutic administration, can said ischemia resisting reperfusion injury compositions be added with excipient and uses with one or more forms in edible tablet, buccal tablet agent, lozenge, capsule, suspension, syrup or wafer or the like.
For to give ischemia resisting reperfusion injury compositions of the present invention outside the parenterai administration, possibly together give to this ischemia resisting reperfusion injury compositions coating or with this ischemia resisting reperfusion injury compositions with the material that prevents its inactivation.Can also the reactive compound that replenish be added in the said composition.Other medicine that in the specific implementation, ischemia resisting reperfusion injury compositions of the present invention and one or more can be used to treat disease is prepared altogether and/or is given altogether.Thisly unite use, can utilize this medicine that gives primely, therefore avoid possible toxicity or the complication relevant with various monotherapies than low dosage.
Process in liquid preparation such as water preparation, oil-suspending agent or other liquid preparation one or more, like in syrup, tincture or the elixir etc. one or more; When being used for parenteral, can be made in solution, water preparation or the oiliness suspending agent etc. of injection one or more.
Above medicine or pharmaceutical composition can use various approach; In described type of service; Preferred form is that oral formulations (like in tablet, coated tablet, capsule, solution or the suspension etc. one or more), non-intestinal give one or more in the dosage form (like in injection, ointment or the patch etc. one or more) etc.; Further one or more in preferred tablet, capsule or the injection etc., a kind of in special preferred tablet or the injection.
In sum, the compositions of ischemia resisting reperfusion injury compositions of the present invention especially pharmaceutical composition can be used for preventing, diagnoses, detects, protects, treats and studies ischemia resisting reperfusion injury product, preferred agents and food, further preferred agents.
(7) technological speciality
The present invention provides a kind of new medicament sources and application forms for prevention, diagnosis, detection, protection, treatment and the reperfusion injury of research ischemia resisting; Thereby to existing ischemia resisting reperfusion injury product systems particularly medicine carried out improvement, improved, thereby the application of having expanded existing medicine.
The present invention is safe and effective, and practicality is stronger, and is inexpensive; Convenient and swift; Easy, the easy operation of its preparation technology, easy to use, evident in efficacy, can be used for preventing, diagnose, detect, protect, treat and studying all kinds of ischemical reperfusion injuries and directly related treatment of diseases and prevention.
The present invention studies ischemical reperfusion injury targetedly, has found a kind of new ischemia resisting reperfusion injury compositions, has made beyond thought achievement; Simultaneously, the present invention also studies the activity of ischemia resisting reperfusion injury compositions targetedly, and its pharmacological action is stronger, and is safe in utilization, brought into play effect to greatest extent.The present invention resists the ischemical reperfusion injury compositions and has expanded new medical usage, also for prevention, diagnosis, detection, protection, treatment and the reperfusion injury of research ischemia resisting a kind of new medicament sources is provided.
This pharmaceutical composition is used to treat ischemical reperfusion injury, particularly hepatic ischemia-reperfusion injury; Can obviously improve the apoptosis that ischemical reperfusion injury causes; Therapeutic effect is remarkable, and toxic and side effects is little, has overcome the side effect that existing common drug causes.The present invention has expanded the new medical usage of existing M-ChR blocker, also for the control ischemical reperfusion injury a kind of new pharmaceutical intervention means is provided.
Ischemia resisting reperfusion injury compositions of the present invention pharmacological action is stronger, and effect is obvious, and its raw material sources are abundant, inexpensive; Stable in properties; Preparation technology is simple, and convenient quality control is more suitable for the large-scale production and the commercial application of industry such as medicine, reagent and industry; The scope of application is wide especially, therefore applies easily, can have a tremendous social and economic benefits in the short period of time.
In a word; Active adaption of the present invention modern medical service and the job demand of scientific research field and the needs of human nature service; For researching and developing new ischemia resisting reperfusion injury product new medicine and preparation source is provided; Having important value to developing the existing medicine of China, is the safe raw material that is used to prevent, diagnose, detect, protect, treat and study aspect such as ischemical reperfusion injury, has important value to improving and improving existing medical level.
Description of drawings
Fig. 1 is the sketch map of the murine liver tissue α 7nAChR expression of the embodiment of the invention 1;
Fig. 2 is the sketch map that the mouse liver ischemia-reperfusion process liver blood flow of the embodiment of the invention 2 changes situation;
Fig. 3 is the sketch map of the various dose Atr.+Neo. therapeutic alliance of the embodiment of the invention 3 to the liver organization Caspases family active change of ischemical reperfusion injury; *P＜0.01, * *P＜0.001 vs Sham; #P＜0.05, ##P＜0.01, ###P＜0.001vs I/R
Fig. 4 alleviates the HE dyeing sketch map of hepatic ischemia-reperfusion injury for the Atr.+Neo. therapeutic alliance of the embodiment of the invention 4;
The transmissioning electric mirror checking that Fig. 5 alleviates hepatic ischemia-reperfusion injury for the Atr.+Neo. therapeutic alliance of the embodiment of the invention 4 is sketch map as a result;
Fig. 6 is the sketch map of the Atr.+Neo. therapeutic alliance of the embodiment of the invention 5 to the influence of serum proinflammatory cytokine expression; *P＜0.05 vs Sham; ##P＜0.01 vs I/R
Fig. 7 is that the Atr.+Neo. therapeutic alliance of the embodiment of the invention 6 is to the difference sketch map that changes of the liver organization Caspases family active of infusion time point damage again; *P＜0.05; *P＜0.01 vs Sham; #P＜0.05, ##P＜0.01 vs I/R
Fig. 8 is the sketch map of the Atr.+Neo. therapeutic alliance of the embodiment of the invention 7 to hepatic tissue MDA growing amount and the active influence of SOD; *P＜0.05; *P＜0.01 vs Sham; #P＜0.05, ##P＜0.01 vs I/R
Fig. 9 be the embodiment of the invention 8 Atr.+Neo. therapeutic alliance rami hepatici nervi vagi excision to hepatic ischemia-reperfusion injury the sketch map of liver organization Caspase-3 activity change influence; *P＜0.01 vs other black bar, #P＜0.05 vs other blank bar, §P＜0.05 vs vago+I/R+Atr.+Neo..
The specific embodiment
The present invention has studied existing ischemical reperfusion injury technology, and a kind of prescription of new ischemia resisting reperfusion injury product is provided, and is convenient to the safe handling of industries such as medical treatment, reagent.
The present invention finally need be prepared into ischemia resisting reperfusion injury product and use, and will enumerate embodiment below and further specify.If any problem, can contact directly 15900897066 with the inventor.Provide several kinds of film coating prescriptions and method for using and some experimental study contents by aforementioned summary of the invention in above-mentioned some experimental datas that provide and the following example; But the research contents that list in the place that should be appreciated that the present invention is not limited to this; Should also be appreciated that term as used herein only is used to describe certain embodiments, and be not qualification of the present invention.
The method for preparing of the common drug preparation of ischemia resisting reperfusion injury compositions and compositions thereof
The present invention prepares injectable powder and generally adopts conventional freeze-drying, as solvent, the steps include: to get ischemia resisting reperfusion injury compositions with water, adds excipient; Be dissolved in water, regulate pH, add active carbon, filtration sterilization; Plug is partly rolled in fill, and lyophilization, tamponade are rolled lid and got final product.Used excipient is selected from one or more in mannitol, gelatin hydrolysate, glucose, lactose, dextran, albumin, the pH regulator agent etc.Every bottle contains ischemia resisting reperfusion injury compositions 0.1～4mg.
The present invention prepares injectable powder also can adopt spray drying method, as solvent, the steps include: to get ischemia resisting reperfusion injury compositions with water, adds or do not add excipient (excipient is the same); Be dissolved in water, add active carbon, filtration sterilization; Spray drying, aseptic subpackaged, tamponade is rolled lid and is got final product.Every bottle contains ischemia resisting reperfusion injury compositions 0.1～4mg.
When the present invention prepares small-volume injection; Preparation gets final product as solvent with water for injection; Also can add appropriate amount of auxiliary materials, adjuvant is selected from one or more in ethanol, propylene glycol, glycerol, Polyethylene Glycol, benzoic acid, dimethyl acetylamide, pH regulator agent, surfactant, cyclodextrin, antioxidant, complexing of metal ion agent, the antibacterial.Injection can be mixed with solution, microemulsion, emulsion, liposome, microsphere, microcapsule or other is suitable for the ordered structure of high drug level; Wherein can comprise the medicament that postpones absorption; For example Monostearate, gelatin, ethylene-ethyl acetate, polyanhydride, polyglycolic acid, collagen protein, polyorthoesters or polylactic acid etc. absorb with the prolongation that reaches injectable composition.Every contains ischemia resisting reperfusion injury compositions 0.1～4mg.
The present invention prepares glucose infusion liquid or sodium chloride transfusion; With water for injection as solvent; Adding the preparation of an amount of glucose or sodium chloride gets final product; Also can add appropriate amount of auxiliary materials, adjuvant is selected from one or more in ethanol, propylene glycol, glycerol, Polyethylene Glycol, benzoic acid, dimethyl acetylamide, pH regulator agent, surfactant, antioxidant, cyclodextrin, complexing of metal ion agent, the antibacterial.Every bottle contains ischemia resisting reperfusion injury compositions 0.1～4mg.
The present invention prepares oral formulations such as tablet, capsule, granule, oral liquid, and adjuvant can be lactose, starch, dextrin, stearate etc., presses the routine techniques preparation.Can comprise the high molecular polymer carrier,, discharge with the prolongation that reaches Orally administered composition like hydroxypropyl methylcellulose or polyoxyethylene etc.
The existing just effect of ischemia resisting reperfusion injury of the present invention is an example with the hepatic ischemia-reperfusion injury, the concrete elaboration as follows:
Each pharmaceutical composition puts on the C57BL/6 mice that needs this treatment among the embodiment involved in the present invention with the lumbar injection form.The general dosage that imposes on the C57BL/6 mice that needs treatment is 0.1～25mg/kg for the M cholinergic receptor-blocking agent, and cholinesterase inhibitor is 0.2～8mg/kg; Preferred dosage M cholinergic receptor-blocking agent is 0.2～6.25mg/kg, and cholinesterase inhibitor is 0.4～1mg/kg; Further preferred dosage is atropine 0.2mg/kg, neostigmine 0.4mg/kg.
(1) checking liver organization express alpha 7nAChR
Get male C57BL/6 mouse liver, detect the expression of its α 7nAChR behind the extraction total protein, find that hepatic tissue has the expression of α 7nAChR.
(2) activate α 7nAChR and alleviate hepatic ischemia-reperfusion injury
Male C57BL/6 mice is divided 4 groups, that is: Sham (sham-operation), I/R group (perfusion again behind the liver 70% ischemia 60min), PNU-282987 (α 7nAChR selective agonist)+I/R group, Atr.+Neo. (Atr., atropine, M cholinergic receptor-blocking agent; Neo., neostigmine, cholinesterase inhibitor)+the I/R group.3 time points are set: promptly pour into back 3h, 6h, 24h time point again, each time point is put to death animal, gets hepatic tissue and carries out pathological examination.Pathological examination shows: pour between selected 3 hours that 6h time point hepar damnification is the most serious, apoptosis hepatocyte quantity is maximum again.
(3) activate α 7nAChR and reduce Caspases family active in the hepatic ischemia reperfusion process
Male C57BL/6 mice is divided 4 groups, that is: Sham group, I/R group, PNU-282987+I/R group, Atr.+Neo.+I/R group.3 time points are set: promptly pour into back 3h, 6h, 24h time point again, each time point is put to death animal.Get serum and liver organization and measure inflammatory factor (TNF-α and IL-1 β); Get liver organization.This time point, with I/R group relatively, all can obviously alleviate the hepatic injury due to the ischemia-reperfusion after PNU-282987 and the Atr.+Neo. pretreatment; Caspase-3, Caspase-8, the active significantly decline of Caspase-9; But the inflammatory factor of hepatic tissue and serum does not all have significant change with each time point between each group.
(4) activate α 7nAChR and alleviate response to oxidative stress in the hepatic ischemia reperfusion process
Male C57BL/6 mice is divided 4 groups; That is: Sham, I/R group, PNU-282987+I/R group, Atr.+Neo.+I/R group are poured into 6h again and are put to death animal behind the ischemia 60min, and it is active to get hepatic tissue its MDA content of detection and SOD; Find: compare with sham operated rats; The growing amount of model group MDA and SOD are active significantly to raise, and the growing amount of the MDA of PNU-282987 and Atr.+Neo. pretreated group significantly is lower than model group, and the SOD activity is significantly higher than model group.
(5) effect of rami hepatici nervi vagi in hepatic ischemia-reperfusion injury
Male C57BL/6 mice is divided 7 groups, that is: Sham, I/R group, Vago group (rami hepatici nervi vagi excision), Vago+I/R group, PNU-282987+Vago+I/R group, Atr.+Neo.+Vago+I/R group, Atr.+Neo.+I/R group.Get liver organization and measure Caspase-3.The result shows: compare with the Vago+I/R group, the Caspase-3 of PNU-282987+Vago+I/R group significantly reduces, and points out no matter whether complete vagus nerve is, activates α 7nAChR and can suppress apoptosis; Organize relatively with Vago+I/R, the Caspase-3 of Atr.+Neo.+Vago+I/R group does not have significance and changes, and prompting Atr.+Neo. inhibition apoptotic effect needs vagal integrity; Compare with the Atr.+Neo.+Vago+I/R group, the Caspase-3 of Atr.+Neo.+I/R group is active significantly to be reduced, and pointing out vagal preservation is the basis that Atr.+Neo. brings into play anti-apoptotic effect.
Conclusion: α 7nAChR apoptosis damage due to antioxidation plays an important role in the hepatic ischemia-reperfusion injury process; Atropine can effectively improve hepatic ischemia-reperfusion injury; Preserving of vagus nerve function is beneficial to anti-hepatic ischemia-reperfusion injury.
Below the used relevant experimental technique of the present invention just, set forth as follows:
1, the histopathology of liver detects
It is clean with normal saline flushing that each organizes mouse liver, the filter paper wipe dry.Get one in left middle lobe tissue, fix 24 hours with 10% neutral formalin liquid buffer
(1) pathological section is made
1. dehydration: gradient alcohol dehydration (every grade of 45min), 50%, 70%, 80%, 90%, 95%, 100% ethanol
2. on alcohol burner with the dewaxing of wax cup, boil wax, and stir with glass rod.Put into 55～60 ℃ of calorstats again, about 30～60min
3. transparent: 1/2 xylene+1/2 ethanol 30min, three grades of xylene, each 20min
4. waxdip: piece of tissue drops into the wax cup, earlier 1/2 xylene+1/2 paraffin 30min; Soak pure wax again three times, each 90min.
In calorstat, carry out, be higher than 2 ℃ of fusing points, keep dissolving
5. embedding: behind pure wax for the third time,, in the wax box, pour a small amount of wax into earlier, again piece of tissue is dropped into the wax box, pour more paraffin into to the piece of tissue label
6. drop into and make it be frozen into wax stone in the cold water
7. repair piece: wax stone is cut into trapezoidal, is fixed on the wooden unit
8. section: the wax stone that fixes is contained on the microtome, and the angle of adjustment microtome knife begins section, thickness 5 μ m at 15 °
9. paster: with evenly coating albumen glycerol on the clean microscope slide.Wax disk(-sc) provoked gently being laid on the constant temperature water surface with brush pen, launch the back fully and picks up with microscope slide
10. place 40 ℃ of calorstat 12h, wax disk(-sc) is pasted firmly
(2) HE dyeing
1. xylene dewaxing 110min
2. xylene dewaxing 115min
3. dehydrated alcohol I, each 1min of II flush away xylene
4. 95% ethanol 1min
5. 90% ethanol 1min
6. 85% ethanol 1min
7. wash 2min from the beginning
8. haematoxylin dyeing 1～5min (om observation control Color)
9. wash 1min from the beginning
10. the differentiation of 1% acidic alcohol is 20 seconds
(11) 1% weak ammonia return blue 30 seconds, tap water or distillation washing 1min
(12) 20 seconds to 5 minutes (om observation control Color) of Yihong dyeing
(13) wash 30 seconds from the beginning
(14) 85% dehydration of alcohols 20 seconds
(15) 90% ethanol 30 seconds
(16) 95% ethanol I, each 1min of II
(17) dehydrated alcohol I, each 2min of II
(18) xylene I, each 2min of II
(19) neutral gum mounting
(20) optical microscope X 40,100, and 200,400 times observation Colors: nucleus is blue, and cytoplasm, muscle, connective tissue, erythrocyte and eosinophil granule are redness in various degree
2, the histopathology ultrastructure of liver detects
Get the liver organization BIAO and BEN, pre-fix, vertically cut, form the 1mm3 small tissue blocks with 2.5% glutaraldehyde phosphate buffer; 0.2M after the rinsing of sucrose phosphate buffer, 1% starves the acid back fixes, the dyeing of saturated acetic acid uranium, and serial acetone dehydration, epoxy resin 618 embeddings, citron lead plumbate dyeing behind the ultrathin section, the JEM-2000EX transmission electron microscope observing is also taken pictures
3, hepatocellular apoptosis detects
Apoptosis (programmed cell death; PCD), claiming apoptosis (apoptosis) again, is the cell death pattern that normal structure is removed a kind of uniqueness of some cell; Extensively exist in vivo, in the organism growing development process, have crucial meaning.In apoptosis process, genomic DNA can rupture and produce two strands, low-molecular-weight dna fragmentation and the high-molecular weight single stranded DNA broken ends of fractured bone (breach), and these DNA chain breach can utilize fluorescent labeling nucleotide 3 ' terminal method to discern.TUNEL (TdT-mediated Dutp nick end labeling, the Brdurd of TdT mediation lacks 13 end labellings) is a kind of basic skills that is used in situ detection apoptosis situation.
1. 3%H2O2: incubated at room 15min is used in section, PBS flushing 3 times
2. 2% E.C. 3.4.21.64 incubated at room 20min, PBS flushing 3 times
3. labelling: PBS flushing is 2 times, dries sample water on every side, the TUNNEL reaction of Dropwise 50 μ l
Mix, solution (1: 9) is hatched 60min for 37 ℃ in wet box, PBS flushing 3 times
4. 5% bovine serum albumin seals 30min
5. add 50 μ l transforming agent-POD, in wet box, hatch 40min for 37 ℃, PBS flushing 3 times
6. add 100 μ l DRB substrate solutions, incubated at room 10min, PBS flushing 3 times, haematoxylin is redyed 4min, hydrochloride alcohol color separation 30 seconds, dehydration, transparent, mounting
7. under light microscopic, observe the apoptosis of cardiac muscle situation then.TUNEL dyeing apoptotic nucleus is pale brown color to yellow, and negative cells nuclear is redyed through haematoxylin and is blue
4, expressing quantity detects
(1) histone extracts
Get a certain amount of lysate and face with preceding adding 1mM DTT, 1mM PMSF, 0.5mM EDTA and protease inhibitor take by weighing the about 200mg/ml lysate of tissue weight, place on ice after the homogenate, and every pipe adds 6 μ l NP-40, places 15min behind the mixing on ice.4 ℃, centrifugal 14000rpm * 10min collects supernatant, after once centrifugal again, get supernatant.Detect protein content in the supernatant.
(2) protein quantification
Determination of protein concentration adopts BCA quantification of protein kit method
1. (be called for short: BSA) standard solution (0.5mg/ml) joins respectively in 96 orifice plates, adds PBS and supplies 20l with 0,1,2,4,8,12,16,20 μ l bovine serum albumin
2. the sample with proper volume joins in the test tube, and supplies 20 μ l with PBS
3. in each test tube, add 200 μ l BCA work dye liquor, mixing is placed 30min for 37 ℃
4. measure the light absorption value at 570nm place with ELIASA, and the record reading; With the absorbance value of the sample that do not contain BSA as blank
5. drawing standard curve, the protein concentration in the calculation sample.If resulting protein concentration redeterminates behind the dilute sample not in standard curve range
6. press protein content and add 4 * sample-loading buffer and lysate, adjusting every tubulin amount concentration is 3 μ g/ μ l, and 95～100 ℃ of degeneration 10min deposit for-80 ℃ after the packing
(3) the protein PAGE (is called for short: SDS-PAGE)
1. the albumen after quantitative is pressed protein content and is added 4 * sample-loading buffer and lysate, and adjusting every tubulin amount concentration is 3 μ g/ μ l, and 95～100 ℃ of degeneration 10min deposit for-80 ℃ after the packing
2. glue: prepare 2 lower floor's glue (15ml altogether).After lower floor's gel solution shaken up, add to immediately between the glass plate of glue groove, respectively add 300 μ l isopropyl alcohol sealings then; After treating to separate out between gel and the isopropyl alcohol water layer (agglomerative sign, about 15～30min), wash down isopropyl alcohol with distilled water; Then with between the glass plate of upper strata glue (2 are total to 4ml) adding glue groove (noting: can not add bubble); Insert comb, more remaining upper strata glue is added, the liquid level upper limb of glue is concordant with the comb peritreme approximately; After about 5～10min treats gelling, carefully extract comb
3. go up appearance: the glue groove is unloaded from gum-making rack, be contained on the iontophoretic electrode, put into electrophoresis tank then, in the electrophoresis inside groove, fill it up with earlier electrophoresis liquid (be prone to see clearly the comb hole from side this moment), sample is added in the swimming lane
4. electrophoresis: with 80V voltage electrophoresis sample, when in sample gets into gel, becoming horizontal wire, strengthen voltage to 130～150V, continue electrophoresis, stop electrophoresis when treating bromophenol blue near the electrophoresis tank bottom
5. change film: after electrophoresis finishes; Unload the glue groove immediately; Take out gel, press following order and prepare electricity commentaries on classics film plastic clip: blackboard (negative pole)-ischemical reperfusion injury-filter paper-gel-NC film-filter paper-ischemical reperfusion injury-blank (positive pole) whenever adds one deck and all need catch up with most bubble; To change the film plastic clip then and insert in the commentaries on classics film groove, the black side of changeing the film plastic clip is to the black negative electrode.Voltage 100V, 1h
6. dyeing: on the NC film, add Ponceaux dyeing number min, visible protein band clearly if protein band is clear and do not have swimming lane or band disappearance then to be regarded as changeing film respond well, is promptly used 1 * PBS flushing
7. seal the nonspecific immune reaction binding site: take out the NC film, place in the culture dish, add the 1 * PBS solution 80ml that contains 5% defatted milk powder, room temperature jolting 3～4h on decolorization swinging table
8. adding one resists: 1 * PBST washes 3 times * 5min of film, the NC film is faced up to put into hatch box, adds freshly prepared anti-Kir6.2 antibody (dilution ratio: 1: 100) and anti-GAPDH (dilution ratio: 1: 5000) antibody-solutions, incubated at room 2h
9. add two anti-: from hatch take out the NC film in the box after, wash film 5min totally 3 times with 1 * PBST; Film is reentered into hatches in the box, add 1: 5000 goat-anti rabbit/mice IgG solution, incubated at room 1h
10. sweep film: from hatch in the box take out the NC film after, wash film 4 times with 1 * PBST, at every turn 5min; Obtain and memory image in Odyssey Infrared Imaging System
5, inflammatory factor detection of expression
1. the serum inflammatory factor detects: pluck eyeball and get blood, blood sample is placed 4 ℃ of refrigerator overnight, then in 4 ℃, the centrifugal 10min of 3000rpm gets the upper serum sample ,-20 ℃ of preservations
The detection of the tissue inflammation factor: quantitative albumen is sample
2. sample is diluted to debita spissitudo (preliminary experiment is confirmed diluted sample concentration) with sample diluting liquid
3. the ELISA test kit uses forward horizontal stand to room temperature, and every hole adds 50 μ l earlier and measures diluent RD1W, adds BIAO and BEN that 50 μ l standard substance, 50 μ l have diluted then in the respective reaction hole
4. mixing 1min gently covers with adhesive tape then, and room temperature is placed 2h
5. wash plate: get rid of liquid in the most plate as far as possible,, and remove water droplet (doing) at thick filter paper arsis with cleaning mixture washing reaction plate (every hole adds 400 μ l cleaning mixture); Cyclic washing five times
6. every hole adds the solution that 100 μ l contain anti-inflammatory factor antibody to be detected, covers with new adhesive tape then, and room temperature is placed 2h
7. wash plate: get rid of liquid in the most plate as far as possible,, and remove water droplet (doing) at thick filter paper arsis with cleaning mixture washing reaction plate (every hole adds 400 μ l cleaning mixture); Cyclic washing five times
8. every hole adds 100 μ l colour developing liquid, and room temperature is placed 30min, lucifuge
9. every hole adds 100 μ l stop buffers, and mixing reads the OD value in the 30min under the 450nm wavelength gently, and tuning wavelength is 570nm
10. utilize standard substance OD value drawing standard curve.According to standard curve and sample OD value, calculate sample
Factor concentration to be detected (pg/ml)
Tissue inflammation factor calculation method: factor concentration/protein content to be detected (pg/mg) in the sample
6, organize the active detection of Caspases
1. get a certain amount of lysate, take by weighing the about 200mg/ml lysate of tissue weight, place 15min after the homogenate on ice.4 ℃, centrifugal 12000rpm * 10min collects supernatant, after once centrifugal again, get supernatant.Detect protein content in the supernatant
2. prepare working solution, with the DTT of the reactant liquor of 2X dilution in 99: 1 1M, subsequent use
3. sample is diluted to debita spissitudo (preliminary experiment is confirmed diluted sample concentration) with sample diluting liquid
4. in 96 orifice plates, every hole adds 50 μ l working solutions earlier, adds BIAO and BEN that 50 μ l have diluted then in the respective reaction hole
5. in reacting hole, add 5 μ l reaction substrates.Mixing gently
6. measure the OD value at the 405nm place, be designated as ODX
7. use the adhesive tape shrouding, hatch 4h for 37 ℃, measure the OD value at the 405nm place, be designated as ODY
8. sample OD value is ODY-ODX
9. behind the equal difference dilution standard article, in 96 orifice plates, add the standard substance of 100 each concentration of μ l, measure the OD value at the 405nm place, meter drawing standard curve
10. active according to Caspases in the standard curve computation organization
7, organize the MDA content detection
1. go after the hepatic portal vein intubate, with PBS lavation liver, after liver bleaches, get liver right middle lobe, left middle lobe, lobus sinister ,-80 ℃ of preservations
2. get a certain amount of antioxidation lysate (1 * BHT), take by weighing the about 200mg/ml lysate of tissue weight, place 15min after the homogenate on ice.4 ℃, centrifugal 12000rpm * 10min collects supernatant, after the centrifugal again supernatant of once getting.Detect protein content in the supernatant
3. sample is diluted to debita spissitudo (preliminary experiment is confirmed diluted sample concentration) with sample diluting liquid
4. in the EP pipe, add 100 μ l samples, standard substance, in the EP pipe, add 100 μ lSDS lysates again, fully behind the mixing, room temperature is placed 5min
5. add 250 μ lTBA reaction substrates, shut EP pipe lid, 95 ℃ of heating in water bath 60min
6. ice bath 5～10min behind room temperature cooling 5min
7. 4 ℃, 3000rpm * 15min gets supernatant
8. in supernatant, add 300 μ l n-butyl alcohol, acutely rock 1～2min, 4 ℃, 10000g * 5min gets supernatant, and is subsequent use
9. in 96 orifice plates, add the supernatant 200 μ l of step 8, measure the OD value in the 532nm place
10. according to the MDA content in the standard curve computation organization
8, tissue SOD is active detects
1. go after the hepatic portal vein intubate, with PBS lavation liver, after liver bleaches, get liver right middle lobe, left middle lobe, lobus sinister ,-80 ℃ of preservations
2. take by weighing the about 200mg/ml lysate of tissue weight, place 15min after the homogenate on ice.4 ℃, 12000rpm * 10min, centrifugal, collect supernatant, after once centrifugal again, get supernatant.Detect protein content in the supernatant
3. sample is diluted to debita spissitudo (preliminary experiment is confirmed diluted sample concentration) with sample diluting liquid
4. in 96 orifice plates, add the BIAO and BEN that 20 μ l standard substance, 20 μ l have diluted successively, xanthine solution 5 μ l, selectivity chromophoric group solution 5 μ l, 10 * SOD reactant liquor, 10 μ l, deionized water 50 μ l
5. add 10 μ l xanthine oxidases at last, mixing
6. use the adhesive tape shrouding, hatch 1h for 37 ℃, measure the OD value at the 490nm place
7. according to the activity of the SOD in the standard curve computation organization
9, mice part ischemia-reperfusion injury model
Utilize the modeling of C57BL/6 mice; Anaesthetize (40mg/kg) along the capable abdominal of ventrimeson through pentobarbital sodium; Separate the blood vessel (tremulous pulse, portal vein, bile duct) of domination right middle lobe, left middle lobe, lobus sinister and clamp tremulous pulse, portal vein and bile duct, keep the blood flow of lobus dexter, caudate lobe, mastoid process, unclamp bulldog clamp behind ischemia (the blocking about 70% liver blood stream) 60min with bulldog clamp; Beginning is perfusion again, replenishes the liquid (300 μ l) that runs off with physiological saline solution when closing abdomen.
10, mice rami hepatici nervi vagi excision
With the C57BL/6 mouse anesthesia, open the abdominal cavity along its ventrimeson after, find rami hepatici nervi vagi by operating microscope along esophagus, separate rami hepatici and with its cut-out with the glass minute hand.
11, statistical procedures
When carrying out hemodynamics mensuration, ELISA, pathology detection, the observer adopt single blind method to detect.Continuous data is with mean ± standard error (mean ± SEM) expression.Relatively employing non-paired t test between two groups; Relatively select one factor analysis of variance (ANONA) for use between many groups; Relatively select the Dunnett-t check for use between a plurality of experimental grouies and a matched group; Relatively with the q check, P＜0.05 thinks that statistical analysis has significant difference to a plurality of sample averages in twos.Adopt the SAS13.1 statistical software to handle.
In the present invention, the embodiment of the above-described specific embodiment and the following stated all is in order to set forth the present invention better, is not to be used for limiting scope of invention.
Through embodiment the present invention is described in detail below.
The relevant explanation of embodiment
1, laboratory animal
The C57BL/6 mice, male, 22 ± 2g (Shanghai SIPPR-BK laboratory animal company limited provides).
Experimental session keeps the Animal House room temperature about 22 ± 2 ℃, relative humidity about 70%, early 8 extremely 8 illuminations automatically in evening.The animal ad lib is freely drunk water.The use of laboratory animal obtains the agreement of the care of animal mechanism of The 2nd Army Medical College, meets relevant management guideline, and the experimentation animal all obtains treating of hommization.
2, key instrument
Microcomputer (the Tianlin 11106C/1.2G of association)
Paraffin, freezing dual-purpose microtome (German Lecia company)
Perfusion tee T (French Plastimed company)
PE10 (internal diameter 0.30mm, external diameter 0.50mm) (French Biotrol company)
PE50 (internal diameter 0.58mm, external diameter 0.96mm) (French Biotrol company)
1ml syringe (BD company)
JA2003 electronic balance (Shanghai balance equipment factory)
Refiner (Ningbo Xin Zhi Instr Ltd.)
Shaking table (Shen, Shanghai ability lottery industry biotech firm)
Pipettor (Eppendorf company)
Baking sheet machine HI1220 type (German Leica company)
Optical microscope (German Leica company)
Multiple tracks cell harvestor (Shanghai medical apparatus factory)
Protein electrophoresis appearance Bio-Rad company (Bio-Rad company)
Albumen changes film appearance (Bio-Rad company)
Enzyme micro-plate reader (Finland Labsystems Dragon company)
Superclean bench (SuZhou Antai Air Tech Co., Ltd.)
Inverted phase contrast microscope (Japanese Olympus company)
Water bath with thermostatic control shaking table (going up the grand instrument plant of Nereid)
Low-temperature and high-speed desk centrifuge 5417R (Eppendorf company)
MP3 protein electrophoresis groove (Bio-Rad company)
PowerRasic electrophresis apparatus (Bio-Rad company)
Scotsman ice machine (Fisher company)
Image analysis system (sky, Shanghai ability Science and Technology Ltd.)
UV-2802 ultraviolet spectrophotometer (UNICO company)
Fluorescence microscope (Japanese Olympus company)
Odyssey far infrared image analyzers (U.S. LI-COR company)
MLS-3020 disinfection with high pressure steam pot (SANYO GS electrical equipment company)
6 orifice plates, 96 orifice plates (U.S. Costar company)
JEM-2000EX transmission electron microscope (JEOL company)
3, main agents and source thereof
Atropine (Sigma company)
Neostigmine (Sigma company)
PNU-282987 (Sigma company)
TUNEL-POD test kit (Roche company)
TNF-α ELISAkits (U.S. R&D company)
IL-1 β ELISAkits (U.S. R&D company)
Caspase-3kits (Japanese MBL company)
Caspase-8kits (Japanese MBL company)
Caspase-8kits (Japanese MBL company)
MDAkits (U.S. Cell Biolabs company)
SOD kits (U.S. Cell Biolabs company)
BCA protein quantification test kit (the green skies, Haimen City biotech company)
Mice source α 7nAChR monoclonal antibody (Millipore company)
Infred Dye 800CW sheep anti rabbit igg (Rockland company)
Infred Dye 800CW goat anti-mouse IgG (Rockland company)
The mice anti-GAPDH monoclonal antibody (go up Haikang become biotechnology company) of originating
Dodecyl sodium sulfate (SDS) (CalBiochem company)
Bromophenol blue (Shanghai ancient cooking vessel state company)
Protease Inhibitor Cocktail Set (CalBiochem company)
Tris alkali (vast Tyke, Shanghai biotech firm)
Glycine (photo bio scientific & technical corporation is won in Shanghai)
Ammonium persulfate. (is called for short: AP) (photo bio scientific & technical corporation is won in Shanghai)
Bovine serum albumin (is called for short: BSA) (photo bio scientific & technical corporation is won in Shanghai)
Ponceaux (photo bio scientific & technical corporation is won in Shanghai)
Dimethyl sulfoxide (is called for short: DMSO) (photo bio scientific & technical corporation is won in Shanghai)
DTT (photo bio scientific & technical corporation is won in Shanghai)
Triton-x-100 (photo bio scientific & technical corporation is won in Shanghai)
Ethylenediaminetetraacetic acid (is called for short: EDTA) (photo bio scientific & technical corporation is won in Shanghai)
Acrylamide (photo bio scientific & technical corporation is won in Shanghai)
Phenylmethanesulfonyl fluoride (is called for short: PMSF) (AMRESCO company)
Tetramethylethylenediamine stock solution (is called for short: TEMED) (Bio-Rad company)
Isopropyl alcohol (Shanghai traditional Chinese medicines group)
Glycerol (Shanghai traditional Chinese medicines group)
Chloroform (Shanghai traditional Chinese medicines group)
Pentobarbital sodium (Shanghai traditional Chinese medicines group)
40% formaldehyde (Shanghai traditional Chinese medicines group)
N-butyl alcohol (Shanghai traditional Chinese medicines group)
Coomassie brilliant blue dye liquor (giving birth to worker's biological engineering)
HAEMATOXYLIN (Hematoxylin) (Fluk company)
According to red Y (EosinY) (Shanghai reagent three factories)
Acid fuchsin (Acid Fuchsin) (Shanghai reagent three factories)
4, the preparation of main agents
(1) the required reagent of zoopery
1. atropine solution preparation
Adopt deionized water preparation atropine solution (containing atropine 50 μ g/ml)
2. neostigmine solution preparation:
Adopt deionized water preparation neostigmine solution (containing neostigmine 100 μ g/ml)
3. PNU-282987 solution preparation:
The 5mgPNU-282987 powder is dissolved among the 500 μ l DMSO, is made into storing solution, and-20 ℃ of preservations are diluted to 5 μ g/ml with deionized water during use
(2) the required reagent of histopathology
1. neutral formalin fixative
Measure 40% formalin solution 100ml, the PBS standardize solution causes 1L, and final concentration is 10%, and transferring PH is 7.2～7.6, and room temperature is preserved
2. 1% hydrochloride alcohol breaks up liquid
70% ethanol 99ml+ concentrated hydrochloric acid 1ml
3. ammonia returns blue liquid
Distilled water 99ml+ strong aqua ammonia 1rnl
4. the preparation of Ovum Gallus domesticus album glycerite
Fresh albumen and glycerite add a little sodium azide antiseptic in 1: 1 ratio mixing, and it is subsequent use to put into 4 ℃ of refrigerators after fully stirring
5. hematoxylin solution
Take by weighing hematoxylin 1g, distilled water 200ml, HgO 0.5g, dehydrated alcohol 10ml, Alumen 20g, be dissolved in the dehydrated alcohol hematoxylin subsequent use earlier.The Alumen heating for dissolving in distilled water, is added subsequent use hematoxylin again, boil 2min, glass rod adds HgO while stirring.Move to immediately in the frozen water after the dissolving fully, quicken its cooling, the hold over night after-filtration.Add glacial acetic acid with preceding ratio with 5%.This liquid can be placed 3 months to half a year
6. Yihong solution
Take by weighing Yihong 2.5～5g, be dissolved in the 500ml distilled water, add concentrated hydrochloric acid 10ml, stir the back hold over night.Filter, deposition is used the distilled water flushing secondary, refilters.It is dry that precipitate is put into baking oven together with filter paper, and reuse 95% dissolve with ethanol solution is made into saturated solution.Press 1: 1～2 times dilutions with 95% ethanol before using, and add 1～2% glacial acetic acid
7. PBS liquid
NaCl 8.0g, KCl 0.2g, Na 2HPO 412H 2O 3.48g, KH 2PO 40.2g deionization is water-soluble, separates to 1000ml 15 pounds of pressure sterilization 15min after the packing
(3) expressing quantity detects required reagent
1. protein extraction lysate
(1) cultured cell in vitro protein extraction lysate
2 * sds gel sample loading buffer (100mM TrisCl (pH6.8), 200mM DTT, 4.0%SDS, 0.2% bromophenol blue, 20% glycerol)
(2) animal tissue's protein extraction lysate
20mM?HEPES，420mM?NaCl，1.5mM?MgCl 2，1mM?DTT，1mM?PMSF，0.5mM?EDTA
2. 4 * sample-loading buffer
1M Tris-HCl (pH6.8) 5ml, SDS 0.8g, glycerol 2ml, bromjophenol blue 0.0012g, beta-mercaptoethanol 0.4ml, deionized water is settled to 10ml
③1.5M?Tris-HCl(pH8.8)
Take by weighing Tris 18.671g, be dissolved in the 100ml distilled water, with concentrated hydrochloric acid adjust pH to 8.8
④1M?Tris-HCl(pH6.8)
Take by weighing Tris 12.114g, be dissolved in the 100ml distilled water, with concentrated hydrochloric acid adjust pH to 6.8
5. 10% Ammonium persulfate. (AP)
Ammonium persulfate. powder 0.1g, distilled water is settled to 1ml, 4 ℃ of preservations
6. 5 * electrophoretic buffer
Tris alkali 15.1g, glycine 94g, 10%SDS 50ml, distilled water is settled to 1000ml.Time spent is got 200ml, and distilled water is settled to 1000ml.With before getting 200ml, adding distil water is settled to 1000ml, is 1 * electrophoretic buffer
7. 10 * change the film buffer
Tris alkali 30.3g, Gly 151.1g, distilled water is settled to 1000ml.Time spent is got 100ml, adds 200ml methanol, and distilled water is settled to 1000ml
⑧10×TBS(pH7.6)
Tris alkali 24.2g, NaCl 80g transfers pH to 7.6 with concentrated hydrochloric acid, and distilled water is settled to 1000ml
⑨1×TBST
10 * TBS 100ml, Tween-201ml, distilled water is settled to 1000ml
10. antibody diluent
Take by weighing BSA 1g, be dissolved among 100ml 1 * TBST, final concentration is 1%BSA, deposits for 4 ℃
Used animal, instrument and equipment, reagent and preparation thereof etc. all are from above-mentioned description or meet above-mentioned requirement in the experiment of following examples.
Embodiment 1, murine liver tissue α 7nAChR expression
Utilize the western-blotting method to detect the expression of the α 7nAChR of male C57BL/6 murine liver tissue; Take off cervical vertebra and put to death mice; Get hepatic homogenate; Total protein is organized in extraction, detects the expression of the α 7nAChR of hepatic tissue, finds that the normal mouse liver organization has α 7nAChR to express (see figure 1).
Embodiment 2, mouse liver ischemia-reperfusion process liver blood flow change situation
Select the C57BL/6 mice for use; Utilize doppler flowmeter in part ischemia-reperfusion operation process, monitor liver left middle lobe blood flow (hepatic blood flow HBF) in real time, find to clamp blood vessel moment with bulldog clamp; Blood flow is down to 32% ± 0.049; Blood flow is stabilized in 17% ± 0.032 of normal blood flow amount behind the ischemia 10min, removes bulldog clamp moment blood flow and returns to 66% ± 0.112, and blood flow returns to the normal level (see figure 2) fully behind the 10min.
Embodiment 3, various dose Atr.+Neo. therapeutic alliance are to the effect of ischemical reperfusion injury
With mouse liver part ischemia-reperfusion is model, and male C57BL/6 mice (22 ± 2.0g) each 35, be divided into five groups at random, open behind the abdomen for one group and sew up immediately.Distinguish intraperitoneal injection of saline solution (10ml/kg), PNU-282987 (40 μ g/kg), Atr. (0.2mg/kg)+Neo. (0.4mg/kg), Atr. (0.1mg/kg)+Neo. (0.2mg/kg) for other four groups; Row liver ischemia is handled behind the injectable drug 30min; Ischemia 60min begins to pour into again; Pour into 6h again and get liver organization, utilize Caspase-3,8, the 9 active (see figure 3)s of the liver of each treated animal of ELISA (ELISA) technology for detection.
The result finds: with the normal control group relatively, model group Caspase-3,8,9 actively significantly raise ( *P＜0.01, * *P＜0.001; N=7), PNU-282987 and Atr. (the 0.2mg/kg)+Caspase3,8 of Neo. (0.4mg/kg) pretreated group, 9 activity all significantly are lower than model group (#P＜0.05, ##P＜0.01; ###P＜0.001; N=7), the Caspase-8 of Atr. (0.1mg/kg)+Neo. (0.2mg/kg) pretreated group, 9 active and model group there was no significant differences.
Results suggest: Atr.+Neo. therapeutic alliance effect becomes dose dependent within the specific limits.
Embodiment 4, activation α 7nAChR alleviate hepatic ischemia-reperfusion injury
Male C57BL/6 mice (22 ± 2.0g) 40, be divided into four groups at random, open behind the abdomen for one group and sew up immediately, get liver and do histopathology and ultrastructure detection.Distinguish intraperitoneal injection of saline solution (10ml/kg), PNU-282987 (40 μ g/kg), Atr. (0.2mg/kg)+Neo. (0.4mg/kg) for other three groups; The capable liver ischemia of 30min is handled behind injectable drug; Beginning perfusion again behind the ischemia 60min is poured into and is got liver behind 3h, 6h and the 24h and do histopathology; Pour into again and get that liver organization carries out Electronic Speculum and apoptosis detects behind the 6h.
(1) liver organization light microscopic HE chromoscopy result
Liver tissue slices optical microscope after HE dyeing is observed down.
Find: control group mice hepatic tissue structure is normal basically, the expansion of part venous tributary, congestion; The large stretch of coagulation necrosis of I/R model group hepatic tissue, downright bad is the center with the central vein, visible remaining portal area, downright bad periphery is steatosis significantly; The obvious degeneration of PNU-282987 pretreated group hepatic tissue, endochylema is light to be dyed, visible lamellar coagulation necrosis, downright bad is the center with the central vein, visible remaining portal area; Atr.+Neo. the obvious degeneration of pretreated group hepatic tissue, endochylema is light to be dyed, visible small pieces coagulation necrosis, downright bad is the center with the central vein, the hepatocyte structure is normal basically around the portal area.
Light microscopic inspection down finds that all there are damage in perfusion back 3h, 6h and 24h liver organization again, the most serious (see figure 4) of performance during with 6h.
(2) hepatocellular apoptosis changes
The TUNEL method detects the liver cell apoptosis, and optical microscope is observed down.
Find: the accidental apoptotic cell of matched group; The I/R model group is found large stretch of apoptotic cell; Compare with model group, PNU-282987 and Atr.+Neo. pretreated group apoptotic cell obviously reduce (see figure 4).
(3) liver transmissioning electric mirror checking result
Transmission electron microscope observing is found: chromatin uniform distribution in the matched group hepatocyte nucleus, and the mitochondrion ridge is high-visible, and chromatin is evenly distributed in the Kupffer clear-cut, nucleus; Model group hepatocyte nuclei dyeing chromaticness is piled up, and there are a large amount of cavitys in the cell and cell membrane place, and endoplasmic reticulum is seriously expanded, mitochondrial swelling, and the mitochondrion ridge is invisible; Kupffer membranolysis, endochylema leak outside and endochylema is interior a large amount of cavitys occur and engulfs granule; The hepatocyte nuclei dyeing chromaticness of PNU-282987 and Atr.+Neo. pretreated group is slightly assembled, and endoplasmic reticulum is slightly expanded, and mitochondrial swelling is not obvious, and the mitochondrion ridge is faintly visible, chromatin small pile-up (see figure 5) in the Kupffer clear-cut, nucleus.
Embodiment 5, activate the influence of α 7nAChR to the serum proinflammatory cytokine
With mouse liver part ischemia-reperfusion is model, and male C57BL/6 mice (22 ± 2.0g) 120, be divided into four groups at random, open behind the abdomen for one group and sew up immediately.Distinguish intraperitoneal injection of saline solution (10ml/kg), PNU-282987 (40 μ g/kg), Atr. (0.2mg/kg)+Neo. (0.4mg/kg) for other three groups; Row liver ischemia is handled behind injectable drug 30min; Ischemia 60min begins to pour into again, after pouring into 3h, 6h, 24h again, gets mice serum; Utilize ELISA (to be called for short: ELISA) serum il of each treated animal of technology for detection-1 β and TNF-alpha expression amount; The normal matched group of the expression of model group IL-1 β obviously raises, and the expression of the IL-1 β of PNU-282987 pretreated group and Atr.+Neo. pretreated group obviously reduces than model group, other again infusion time put equal no significant difference; But these expressions are all lower, should not have pathological significance.The expression of TNF-α is in the also equal no significant difference (see figure 6) of each time point.
Results suggest: the damage process of the less participation ischemia-reperfusion of inflammatory reaction.
Embodiment 6, activation α 7nAChR are to the active influence of liver organization Caspases
With mouse liver part ischemia-reperfusion is model, and male C57BL/6 mice (22 ± 2.0g) 120, be divided into four groups at random, open behind the abdomen for one group and sew up immediately.Distinguish intraperitoneal injection of saline solution (10ml/kg), PNU-282987 (40 μ g/kg), Atr. (0.2mg/kg)+Neo. (0.4mg/kg) for other three groups; Row liver ischemia is handled behind injectable drug 30min; Ischemia 60min begins to pour into again; After pouring into 3h, 6h, 24h again, get ischemia part mouse liver, utilize elisa technique to detect the liver Caspases family active of each treated animal.
Find: compare perfusion back 3h, 6h, each time point model group Caspase-8 of 24h, 9 active significantly risings again, perfusion back 6h, the active significantly rising of 24h time point model group Caspase-3 again with matched group; Compare with model group; Pour into 6h time point PNU-282987 and Atr.+Neo. pretreated group again and all significantly reduce Caspase-3,8,9 activity; Pour into 24h time point PNU-282987 pretreated group more only Caspase-8 is active and significantly descend, pour into 24h time point Atr.+Neo pretreated group Caspase-3,8,9 the activity (see figure 7) that all significantly descends again.
Embodiment 7, activation α 7nAChR are to hepatic tissue MDA growing amount and the active influence of SOD
With mouse liver part ischemia-reperfusion is model, and male C57BL/6 mice (22 ± 2.0g) 120, be divided into four groups at random, open behind the abdomen for one group and sew up immediately.Distinguish intraperitoneal injection of saline (10ml/kg), PNU-282987 (40 μ g/kg), Atr. (0.2mg/kg)+Neo. (0.4mg/kg) for other three groups; Row liver ischemia is handled behind administration 30min; Ischemia 60min begins to pour into again; Pouring into back 3h, 6h, 24h time point again, get ischemia part mouse liver, it is active to detect MDA growing amount and SOD.
The result shows: compare with matched group, the growing amount that pours into 3h and 6h time point model group MDA significantly raises, and PNU-282987 and Atr.+Neo. pretreatment can significantly reduce the MDA growing amount; Compare with matched group, again the active significantly rising of perfusion back 3h, 6h, 24h time point SOD; Compare with model group, PNU-282987 and Atr.+Neo. pretreatment can further remarkable enhance SOD activity (see figure 8)s.
Embodiment 8, rami hepatici nervi vagi excise the influence to hepatic ischemia-reperfusion injury
Utilize the rami hepatici nervi vagi excision (to be called for short: vago) and the ischemia-reperfusion operation; Be divided into 7 groups, that is: sham operated rats, vago group, I/R model group, vago+I/R group, PNU-282987 (40 μ g/kg)+vago+I/R group, Atr. (0.2mg/kg)+Neo. (0.4mg/kg)+vago+I/R group, Atr. (0.2mg/kg)+Neo. (0.4mg/kg)+I/R group.Get liver organization after the operation and measure the Caspase-3 activity.
The result shows: the vago+I/R group is than the active there was no significant difference of I/R group caspase-3, and the I/R+Atr.+Neo. group is than the active significantly decline (see figure 9) of the caspase-3 of vago+I/R+Atr.+Neo. group.Prompting: rami hepatici nervi vagi excision weakens the Atr.+Neo. therapeutic effect, and the preserving to be beneficial to of rami hepatici nervi vagi alleviates hepatic ischemia-reperfusion injury.
Embodiment 9, ischemia resisting reperfusion injury compositions can be resisted ischemia-reperfusion and caused the apoptosis effect
Male C57BL/6 mice (22 ± 2.0g); Random packet; Sham operated rats is sewed up after opening abdomen immediately, and other group intraperitoneal injection of saline solution (10ml/kg) respectively is that model group, M cholinergic receptor-blocking agent and/or cholinesterase inhibitor divide some groups according to the experiment needs by ID, and the capable liver ischemia of 30min is handled behind injectable drug; Beginning perfusion again behind the ischemia 60min is poured into and is got liver behind the 6h and do the TUNEL apoptosis and detect.
Experimental result is seen table 1:
The pharmaceutical composition opposing ischemia-reperfusion of table 1, M cholinergic receptor-blocking agent and cholinesterase inhibitor causes the apoptosis effect
Figure BDA0000100492130000421
Figure BDA0000100492130000431
*P＜0.05vs model group; *P＜0.01vs model group; * *P＜0.001vs model group;
From the experimental result of last table, can find that ischemia resisting reperfusion injury compositions can obviously alleviate hepatic ischemia-reperfusion injury.
Show that thus two medicines share hepatic ischemia-reperfusion injury model mice hepatocellular apoptosis apoptosis quantity is significantly smaller than the application separately respectively of two medicines, the collaborative anti-hepatic ischemia-reperfusion injury effect of two kinds of medicines is strengthened greatly.And, the untoward reaction such as side effect that two types of drug combinations also obviously reduce these two types of prescriptions when solely using.
Embodiment 10, ischemia resisting reperfusion injury compositions can weaken hepatic ischemia reperfusion mouse model transaminase activity
Male C57BL/6 mice (22 ± 2.0g), sew up immediately after opening abdomen by random packet, sham operated rats; Other group respectively intraperitoneal injection of saline solution (10ml/kg) be model group, M cholinergic receptor-blocking agent and (or) cholinesterase inhibitor, the capable liver ischemia of 30min is handled behind injectable drug, beginning perfusion again behind the ischemia 60min; Pour into again and pluck eyeball behind the 6h and get whole blood, after room temperature leaves standstill 2h, 3000 rev/mins of centrifugal 10min; Get supernatant, the ELASE method is measured transaminase activity in the serum
Experimental result is seen table 2:
The pharmaceutical composition of table 2, M cholinergic receptor-blocking agent and cholinesterase inhibitor
Weaken hepatic ischemia reperfusion mouse model transaminase activity
Figure 3
Figure BDA0000100492130000451
*P＜0.05vs model group; *P＜0.01vs model group; * *P＜0.001vs model group;
Ischemia resisting reperfusion injury compositions can reduce hepatic ischemia reperfusion mice serum glutamic oxaloacetic transaminase, GOT and gpt activity; Show thus; Two kinds of drug combinations are used separately respectively less than two medicines the liver dysfunction of hepatic ischemia-reperfusion injury model mice, and the collaborative anti-hepatic ischemia-reperfusion injury effect of two kinds of medicines is strengthened greatly.
Embodiment 11, ischemia resisting reperfusion injury compositions can strengthen hepatic ischemia reperfusion mouse model liver organization superoxide dismutase and (be called for short: SOD) activity
Male C57BL/6 mice (22 ± 2.0g), sew up immediately after opening abdomen by random packet, sham operated rats; Other group respectively intraperitoneal injection of saline solution (10ml/kg) be model group, M cholinergic receptor-blocking agent and (or) cholinesterase inhibitor, the capable liver ischemia of 30min is handled behind injectable drug, beginning perfusion again behind the ischemia 60min; Open abdomen after pouring into 6h again, hepatic portal vein is put pipe, with the capable liver perfusion of phosphate buffer art; The 50mg hepatic tissue adds the homogenate of 1ml phosphate buffer; 12000 rev/mins of 10min get supernatant, measure SOD activity in the liver organization with the nitrite method.
Experimental result is seen table 3:
The pharmaceutical composition of table 3, M cholinergic receptor-blocking agent and cholinesterase inhibitor
It is active to strengthen hepatic ischemia reperfusion mouse model liver organization SOD
Figure 2
Figure BDA0000100492130000461
*P＜0.05vs model group; *P＜0.01vs model group;
It is active that ischemia resisting reperfusion injury compositions can be able to strengthen hepatic ischemia reperfusion mouse model liver organization SOD.Show that thus the SOD activity of two kinds of drug combination enhancing hepatic ischemia-reperfusion injury model mices is superior to two medicines to be used separately respectively, the collaborative anti-hepatic ischemia-reperfusion injury effect of two kinds of medicines is strengthened greatly.
Embodiment 12, ischemia resisting reperfusion injury compositions can lower hepatic ischemia reperfusion mouse model liver organization malonaldehyde and (be called for short: MDA) concentration
Male C57BL/6 mice (22 ± 2.0g), sew up immediately after opening abdomen by random packet, sham operated rats; Other group intraperitoneal injection of saline solution (10ml/kg) respectively is model group, M cholinergic receptor-blocking agent and/or cholinesterase inhibitor, and the capable liver ischemia of 30min is handled behind injectable drug, beginning perfusion again behind the ischemia 60min; Open abdomen after pouring into 6h again, hepatic portal vein is put pipe, with the capable liver perfusion of phosphate buffer art; The 50mg hepatic tissue adds the homogenate of 1ml phosphate buffer; 12000 rev/mins of 10min get supernatant, measure MDA activity in the liver organization with the thiobarbituricacid method.
Experimental result is seen table 4:
The pharmaceutical composition of table 4, M cholinergic receptor-blocking agent and cholinesterase inhibitor
Lower hepatic ischemia reperfusion mouse model liver organization MDA concentration
Figure 20111032125691000021
Figure BDA0000100492130000481
*P＜0.05vs model group; *P＜0.01vs model group;
Ischemia resisting reperfusion injury compositions can reduce hepatic ischemia reperfusion mouse model liver organization MDA growing amount.Show that thus the MDA growing amount of two kinds of drug combination minimizing hepatic ischemia-reperfusion injury model mices is superior to two medicines to be used separately respectively, the collaborative anti-hepatic ischemia-reperfusion injury effect of two kinds of medicines is strengthened greatly.

Claims (17)

1. an ischemia resisting reperfusion injury compositions is characterized in that, described this ischemia resisting reperfusion injury compositions comprises following component: M cholinergic receptor-blocking agent and cholinesterase inhibitor.
2. ischemia resisting reperfusion injury compositions according to claim 1 is characterized in that the weight ratio of described M cholinergic receptor-blocking agent and cholinesterase inhibitor is 1: 0.005～50.
3. ischemia resisting reperfusion injury compositions according to claim 2 is characterized in that the weight ratio of described M cholinergic receptor-blocking agent and cholinesterase inhibitor is 1: 0.1～8.
4. ischemia resisting reperfusion injury compositions according to claim 3 is characterized in that the weight ratio of described M cholinergic receptor-blocking agent and cholinesterase inhibitor is 1: 2.
5. ischemia resisting reperfusion injury compositions according to claim 1 is characterized in that, described M cholinergic receptor-blocking agent is the conventional M cholinergic receptor-blocking agent that uses in this area.
6. ischemia resisting reperfusion injury compositions according to claim 5; It is characterized in that described M cholinergic receptor-blocking agent is to comprise atropine, Anisodamine, contain in plant extract, the scopolamine of Anisodamine, the plant extract that contains scopolamine, melyltropeine or the tropicamide one or more.
7. ischemia resisting reperfusion injury compositions according to claim 6 is characterized in that, described M cholinergic receptor-blocking agent is to comprise in atropine, Anisodamine or the scopolamine one or more.
8. ischemia resisting reperfusion injury compositions according to claim 7 is characterized in that described M cholinergic receptor-blocking agent is an atropine.
9. ischemia resisting reperfusion injury compositions according to claim 1 is characterized in that, described cholinesterase inhibitor is the conventional cholinesterase inhibitor that uses in this area.
10. ischemia resisting reperfusion injury compositions according to claim 9 is characterized in that, described cholinesterase inhibitor is to comprise in neostigmine or the physostigmine one or more.
11. ischemia resisting reperfusion injury compositions according to claim 10 is characterized in that described cholinesterase inhibitor is a neostigmine.
12., it is characterized in that the method for preparing of described said composition is following according to each described ischemia resisting reperfusion injury compositions of claim 1～11:
Described M cholinergic receptor-blocking agent, cholinesterase inhibitor are mixed in proportion, promptly get described ischemia resisting reperfusion injury compositions.
13., it is characterized in that the method for using of described said composition is following according to each described ischemia resisting reperfusion injury compositions of claim 1～11:
Do not carry out prior mixing, but directly M cholinergic receptor-blocking agent and cholinesterase inhibitor single variety are used respectively, and successively use continuously.
14. be used to prepare ischemia resisting reperfusion injury product according to each described ischemia resisting reperfusion injury compositions of claim 1～11.
15. ischemia resisting reperfusion injury compositions according to claim 14 is used to prepare anti-hepatic ischemia-reperfusion injury product.
16. the compositions according to each described ischemia resisting reperfusion injury compositions of claim 1～11 is used to prepare ischemia resisting reperfusion injury product.
17. the compositions of ischemia resisting reperfusion injury compositions according to claim 16 is used to prepare anti-hepatic ischemia-reperfusion injury product.
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CN104288772A (en) * 2014-09-11 2015-01-21 顾万清 Combined application of cholinesterase inhibitor and muscarinic receptor blocker
CN104940934A (en) * 2015-07-01 2015-09-30 顾万清 Pharmaceutical composition used for promoting skin healing and application thereof
CN105943537A (en) * 2016-06-28 2016-09-21 顾万清 Compound anisodamine and neostigmine sustained-release tablet and preparation method thereof
WO2021092275A1 (en) * 2019-11-06 2021-05-14 Children’S National Medical Center Methods and compositions for the prevention and treatment of ischemia reperfusion injury and infection

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102836157A (en) * 2012-09-12 2012-12-26 高尔医药科技(上海)有限公司 Application of neostigmine hyoscyamine in preparation of medicine for treating acute cerebral ischemic injury
CN104094896A (en) * 2013-07-19 2014-10-15 刘俊乐 Ischemic preconditioning small animal centrifuge
CN104094896B (en) * 2013-07-19 2017-03-22 刘俊乐 Ischemic preconditioning small animal centrifuge
CN104288772A (en) * 2014-09-11 2015-01-21 顾万清 Combined application of cholinesterase inhibitor and muscarinic receptor blocker
CN104288772B (en) * 2014-09-11 2017-09-01 顾万清 The use in conjunction of anticholinesterase and muscarinic acceptor blocker
CN104940934A (en) * 2015-07-01 2015-09-30 顾万清 Pharmaceutical composition used for promoting skin healing and application thereof
CN104940934B (en) * 2015-07-01 2018-02-09 顾万清 A kind of pharmaceutical composition and its application for being used to promote skin healing
CN105943537A (en) * 2016-06-28 2016-09-21 顾万清 Compound anisodamine and neostigmine sustained-release tablet and preparation method thereof
WO2021092275A1 (en) * 2019-11-06 2021-05-14 Children’S National Medical Center Methods and compositions for the prevention and treatment of ischemia reperfusion injury and infection

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