CN105796581A - Application of paeoniflorin to preparation of medicine for regulating and controlling cholinergic anti-inflammatory pathway - Google Patents

Abstract

The invention relates to application of paeoniflorin to preparation of medicine for regulating and controlling a cholinergic anti-inflammatory pathway, and belongs to the field of medicine. The pharmacologic action mechanism of paeoniflorin on the cholinergic pathway is explored in vivo and in vitro, a myocardial ischemia reperfusion injury rat model is constructed, and the influence on expressions of signal molecules of the cholinergic pathway is monitored after paeoniflorin acts. Vascular endothelial cells and primarily cultured myocardial cells are selected as models in vitro, after anoxia/reaeration and hydrogen peroxide injuries, the protection function of paeoniflorin on the cells is observed, the influence on NF-kappa B in the cholinergic pathway is detected, therefore, it is revealed that paeoniflorin can be developed into the medicine for regulating and controlling myocardial ischemia reperfusion through the cholinergic anti-inflammatory pathway to achieve the protection function, and paeoniflorin has wide market prospects and high economic benefits when developed into the new medicine.

Description

Peoniflorin application in preparation regulation and control cholinergic anti-inflammatory pathway medicine

Technical field

The invention belongs to field of medicaments.

Background technology

Peoniflorin is got from Radix Paeoniae for 1963 first, the chemical entitled 5beta-of peoniflorin (Paeoniflorin) [(benzoyloxy)methyl]tetrahydro-5-hydroxy-2-methyl-2,5-methano-1h-3,4-dioxacy Clobuta [cd] pentalen-1alpha (2h)-yl-beta-d-glucopyranoside, molecular formula is C₂₃H₂₈O₁₁, molecule Amount: 480.45.For the amorphous chocolate brown powder of hygroscopicity (90% is off-white powder), fusing point: 196 DEG C.Peoniflorin is as Chinese herbaceous peony The main component of medicine total glycosides, is paid attention to by researcher the most, and current research is mainly in the following aspects:

1, the hypoglycemic activity of peoniflorin

Peoniflorin intravenous injection can substantially reduce the blood glucose of the diabetic mice of streptozotocin induction, and in blood plasma, sugar content is also It is gradually lowered along with the prolongation of administration time.After medication, during 25min, effect is the strongest, and peoniflorin reduces sugar content water in blood plasma Flat, act on and measure relevant (1～10mg/kg body weight), its hypoglycemic mechanism is unrelated with insulin.

2, to the heart, cerebrovascular system

Peoniflorin is to blood brain barrier during re perfusion after cerebral ischemia and cerebral ischemia neuropathologic change symptom, cerebral blood flow Impact.Find that peoniflorin has protective effect to cerebral ischemia associated with hydrocephalus, blood brain barrier, brain regional blood flow.Focal brain Ischemic animal application peoniflorin can substantially alleviate degree of cerebral edema, reduces Range of Cerebral Infarction, improves neuroethology symptom, blood Brain Barrier Permeability, increases brain regional blood flow.Display focal cerebral ischemic injury has significant protective effect, its protection Mechanism may be with following factors about suppression intracellular calcium overload, free radical resisting, the cerebrovascular that causes ischemia, anoxia The easypro dysfunction of contracting has good improvement result, has protective effect to blood brain barrier during re perfusion after cerebral ischemia, and can promote Enter the restitution of re perfusion cerebral blood flow in early days, but suppress the mechanism of intracellular calcium overload, to free radical resisting machine in cerebral tissue Which is shaped with, the most all need to study further to the effect of cerebrovascular function.

3, immunoregulation effect

It is certain immunoloregulation function that peoniflorin has, to rats in vitro splenocyte IL-2 with rat abdominal cavity is huge bites The IL 1 that cell produces, all has potentiation, and has inducing action to splenic lymphocytes, and can make AA rat Substantial amounts of low splenic lymphocytes is recovered and close to normal level.

4, the effect to tumor

Peoniflorin has certain inhibitory action to tumor cell, can suppress activity and the rising of ATP enzyme on tumor cell membrane Adenylic acid, cyclase (AC) activity.This medicine suppression tumor cell generates and Na, K-A TPase on suppression film to activate AC close Relevant.Result of study shows, peoniflorin 2mg/ml, 1mg/ml and 0.5mg/ml can suppress human liver cancer cell to a certain extent Breed to delay the process of hepatocarcinoma, and HepG can be induced₂Apoptosis, and can express by upregulation of apoptosis related gene P53 and bax, lower Apoptosis-related genes bcl-2 is expressed.

5, the effect to central nervous system

Peoniflorin has obvious protective function to PC12 cell in 6 kinds of ischemic brain dam age models, and its mechanism of action may Relevant to the intracellular calcium overload that the suppression damage later stage occurs.Equal to PC12 cell in 2 kinds of different types of calcium overloadinjury models There is obvious protective function.Peoniflorin can alleviate the rat radial mystery palace of Induced by scopoiamins and distinguish obstacle, and unilateral forebrain The rat four road radial maze of basal nuclei NBM wound inducement distinguishes obstacle；Activity of Cortical Neurons can be promoted, to astroglia The activity of cell has obvious inhibitory action, surviving the growth of neuron has facilitation.

6, the impact on smooth muscle

Peoniflorin may be by regulation cell membrane Ca²⁺/Na⁺Exchange carrys out diastole smooth muscle cell, it is also possible to direct regulation and control is put down Sliding myocyte's Kv passage, by open Kv passage, makes film hyperpolarization, suppresses Ca²⁺Interior stream, thus reduce [Ca²⁺] i, it is achieved that right The diastole effect of smooth muscle；Suppression parasympathetic nervous is excited, reduces smooth muscle tension and its motion of suppression.

7, the impact on blood

Peoniflorin significantly raise lonizing radiation hyperamization deficiency syndrome mouse peripheral blood quantity of leucocyte and bone marrow cfu-gm, BFU-E, CFU-E, CFU-mix quantity, raises bone marrow Epo and G-CSF gene expression.Bone marrow hematogenesis somatomedin Epo and G-CSF gene Express, be up radiating one of hyperamization deficiency syndrome mouse peripheral blood quantity of leucocyte, the mechanism promoting various proliferation of hematopoietic progenitors.

The meaning of peoniflorin Study on mechanism

China's Paeoniaceae plant resources enriches, and has 11 kinds and more than 10 kind of mutation, and extraction process of paeoniflorin is the most ripe, Purity is up to 98%.Peoniflorin has multiple biological activity, regulation blood glucose, cardiovascular, immunomodulating, central nervous system, The aspect such as smooth muscle, blood all has the pharmacological action of affirmative.In view of its extensively pharmacologically active of uniqueness and clinical practice, have into The value of one step research and the prospect of exploitation monomer medicine.Its mechanism of action is studied by application modern pharmacology method, can Think that the clinical efficacy of Paeoniaceae plant provides new theoretical foundation, developing clinical application range the most widely, it is possible to its For lead compound, it is carried out new drug development.

Summary of the invention

The present invention provides the application in preparation regulation and control cholinergic anti-inflammatory pathway medicine of a kind of peoniflorin.

The present invention adopts the technical scheme that: a kind of peoniflorin answering in preparation regulation and control cholinergic anti-inflammatory pathway medicine With.

Peoniflorin plays suppression adhesion factor and p38MAPK signal path, Jin Ergai by regulation and control cholinergic anti-inflammatory pathway Kind endothelial cell inflammation reaction, alleviates tissue injury, to HUVECs cells play protective effect.

When the present invention is used for preparing regulation and control cholinergic anti-inflammatory pathway medicine, it is administered orally or parenterally all safety , in the case of oral, it can be administered with any conventionally form, such as powder, granule, tablet, capsule, pill, solution, hangs Supernatant liquid, syrup, buccal tablets, sublingual lozenge etc.: when this drugs for parenteral delivery, any conventionally form can be taked, such as, note Penetrate agent: such as intravenous injection, ointment, suppository, percutaneous dosing, inhalant etc..

It is by effective ingredient monomer or effective ingredient and solid or liquid that the present invention prepares regulation and control cholinergic anti-inflammatory pathway medicine The excipient of body is constituted together, and the excipient of solid used herein or liquid is well known in the art, and names Several object lessons, powder is powder agent for oral administration, and its excipient has lactose, starch, paste essence, calcium carbonate, synthesis or sky So aluminum sulfate, magnesium oxide, magnesium stearate, sodium bicarbonate, dry yeast etc.；The excipient of solution have water, glycerol, 1,2-the third two Alcohol, simple syrup, ethanol, ethylene glycol, Polyethylene Glycol, Sorbitol etc.；The excipient of ointment can use fatty oil, aqueous Pilus Caprae seu Ovis Fat, vaseline, glycerol, Cera Flava, wood cured, white oil, resin, the senior water-repelling agent being combined into such as cured or hydrophilizing agent.

The present invention explores the peoniflorin pharmacological mechanism to cholinergic pathway in terms of internal, external two, builds cardiac muscle Ischemical reperfusion injury rat model, after monitoring peoniflorin effect, the expression on cholinergic pathway signaling molecule affects.External choosing Select vascular endothelial cell and Primary cultured myocardial cells as model, anoxia _ reoxygenation and Hydroperoxide injury after, observe Radix Paeoniae The glycosides protective effect to cell, and detect the impact of NF-κ B in cholinergic pathway, thus disclose peoniflorin and can be developed into By cholinergic anti-inflammatory pathway regulation and control myocardial ischemia-reperfusion, thus the medicine played a protective role.

The beneficial effects of the present invention is, the sickness rate of cardiovascular disease is in the trend risen year by year, and ischemia-reperfusion Damage is the important complication in the treatment of myocardial ischemia disease, is lacked as treatment by cholinergic anti-inflammatory pathway for peoniflorin Courageous and upright cardiopathic new drug development provides solid foundation, and the research and development turning out to be like product of this mechanism provide condition, adopt Take measure preventing and treating myocardial ischemia reperfusion injury significant to promotion myocyte survival, developed into new drug, then There are wide market prospect and higher economic benefit.

Accompanying drawing explanation

Fig. 1 peoniflorin impact (WB) figure on simulated ischemia Reperfu-sion endotheliocyte p-p38MAPK protein expression,

1: cell controls group, 2: model group, 3: peoniflorin 0.078mg/ml, 4: peoniflorin 0.156mg/ml；

Fig. 2 peoniflorin preconditioning on rat myocardial infarction area affect figure,

A. sham operated rats, b. model group, c. peoniflorin low dose group, d. peoniflorin high dose group；

The impact (HE light microscopic 200 ×) on myocardial ischemia-reperfusion rat heart muscle tissue leukocyte infiltration of Fig. 3 peoniflorin；

A. sham operated rats, b. model group, c. peoniflorin low dose group, d. peoniflorin high dose group；

The impact that myocardial ischemia-reperfusion rat heart muscle NF-κ B (A～D), ICAM-1 (E～H) are expressed by Fig. 4 peoniflorin (IHC 200×)；

A:Sham group, B:Model group, C:PF 30mg/kg, D:PF 60mg/kg, E:Sham group, F: Model group, G:PF 30mg/kg, H:PF 60mg/kg.

Detailed description of the invention

The application in preparation regulation and control cholinergic anti-inflammatory pathway medicine of a kind of peoniflorin.

Test example: peoniflorin passes through cholinergic anti-inflammatory pathway Ischemic myocardium reperfusion injury.

1, extraction and purification process: radix paeoniae rubra decoction pieces 1000g 70% alcohol reflux 3 times, each solvent load is respectively For 6000mL, 5000mL and 5000mL, return time is respectively 2.0h, 1.5h and 1.5h；United extraction liquid, filters, and filtrate exists Less than 60 DEG C are concentrated in vacuo to melicera extractum, there are 425.4g；Analyzing through HPLC, in extractum, the content of peoniflorin is 11.9%, the response rate of Effect of Alcohol Extracting Procedureto Total is 98.2%, the extractum 700mL water dissolution that leaching obtains, and first extracts with petroleum ether Take 2 times, each 700mL；Aqueous phase after extraction is extracted with ethyl acetate 2 times, each 700mL；Aqueous phase after ethyl acetate extraction With n-butanol extraction 3 times, each 700mL, merge the butanol solution of 3 extractions, below 60 DEG C, paste is concentrated in vacuo to obtain 152.3g；Analyzing through HPLC, in paste, the content of peoniflorin is 31.6%, and recovery of extraction is 95.3%.

Silica gel chloroform fully soaks, and after degassing, fills post by wet method, the paste 10mL of every gram of n-butanol extraction gained Eluant dissolve after upper prop, then with eluent (chloroform-methanol mixed solution), elution flow rate about 3mL/min, sub-bottle collection Effluent, qualitative with TLC, merge the part containing peoniflorin, be evaporated to do, alcohol-water solution reflux, extract, operation is preferably adopted With 70% alcohol reflux 3 times, the purity peoniflorin product more than 99.0% can be prepared by repeatedly column chromatography.

2, peoniflorin passes through cholinergic anti-inflammatory pathway Ischemic myocardium reperfusion injury

2.1 peoniflorins are to Human umbilical vein endothelial cells cholinergic anti-inflammatory pathway after simulated ischemia Reperfu-sion and correlation factor Impact

One, materials and methods

(1) experiment reagent peoniflorin, white powder, the good research center of Beijing University's generation provides, content 95%, lot number: 140420, test front normal saline and be made into desired concn；Trizol reagent, purchased from American I nvitrogen company；ICAM-1、 VCAM-1 test kit, purchased from R&D Systems company of the U.S.；P-p38MAPK sc-17852-R monoclonal antibody, purchased from the U.S. Santa Cruz company；P38MAPK 5F11 monoclonal antibody and Protein Extraction Reagent box are public purchased from U.S. Cell signaling Department；Goat anti-rabbit igg (HRP labelling), DAB staining kit, AEC staining kit are purchased from Wuhan doctor's moral.

(2) experimental technique

The collection of 1.HUVECs cell uses primary huve cell culture method to gather cell with cultivating, and chooses health Anemia of pregnant woman's cesarean, distortionless, give a discount, (patient signs informed consent to the damaged fresh umbilical cord of neonate 20～25cm the most voluntarily Book), in repeatedly rinsing umbilical vein with D-Hank ' s liquid, residual blood and thrombosis are to blood to the greatest extent, liquid in umbilical vein to the greatest extent.Draw 0.1% glue Protoenzyme II Digestive system (37 DEG C of pre-temperature) about 10ml, injects umbilical vein, puts in the beaker filling D-Hank ' s liquid, and 37 DEG C of water-baths disappear Change 15min, the sucking-off Digestive system containing HUVECs cell, injects the centrifuge tube equipped with 10mlHUVECs cell culture fluid.Use endothelium After cell culture fluid cleans, piping and druming mixes, by HUVECs cell suspension inoculation in the 25cm being coated²In culture bottle, put incubator (37 DEG C, 5%CO₂) cultivate after 24 hours, carry out changing liquid.After cell covers with, with without Ca²⁺、Mg²⁺PBS, 0.125% After the of short duration digestion of pancreatin, by 1:2 Secondary Culture, take 3-5 generation growth logarithmic (log) phase HUVECs serum-free quiescent culture standby.

2. experiment packet, is divided into Normal group: use normal endothelial cell culture medium culturing；Model group: simulated ischemia Culture fluid changes normal culture fluid after cultivating 30min and is further cultured for 4h；Peoniflorin low dose group: add peoniflorin pretreatment 48h, the denseest Degree is for 0.078mg/ml, and rear simulated ischemia culture fluid cultivates 30min, then changes normal culture fluid and be further cultured for 4h；Dosage in peoniflorin Group: add peoniflorin pretreatment 48h, final concentration of 0.156mg/ml, rear simulated ischemia culture fluid is cultivated 30min, then is changed normal Culture fluid is further cultured for 4h；Peoniflorin high dose group: add peoniflorin pretreatment 48h, final concentration of 0.312mg/ml, rear simulation lacks Hemoculture liquid cultivates 30min, then changes normal culture fluid and be further cultured for 4h.

3.HUVECs cell simulation ischemia-reperfusion is cultivated to cultivate HUVECs cell and is being comprised 118mmol/L sodium chloride, 24mmol/L sodium bicarbonate, 1.0mmol/L sodium phosphate, 2.5mmol/L calcium chloride, 1.2mmol/L magnesium chloride, 20mmol/L lactic acid Sodium, 16mmol/L potassium chloride, 30min in " the ischemia liquid " of 10mmol/L 1,5-anhydroglucitol (pH is adjusted to 6.2), then it is changed to Reperfu-sion liquid i.e. normal incubation medium carries out Reperfu-sion 4h.

4. Enzyme-linked Immunosorbent Assay reaction method (ELISA) measures each group of ICAM-1, VCAM-1 soluble protein careful collection The supernatant of HUVECs, to take its supernatant again after 1000r/min centrifugation 5min, and cryopreservation is treated under the conditions of-80 DEG C Detection.ICAM-1, VCAM-1 detection kit, application double-antibody sandwich elisa is used to measure ICAM-1, VCAM-1 egg in specimen White expression.With substrate tetramethyl benzidine (TMB) colour developing after thoroughly washing.In the depth of color and sample ICAM-1, VCAM-1 content is proportionate.Measuring absorbance (OD value) by microplate reader, wavelength is 450nm, calculates sample concentration.

5.Western blot method detection p-p38MAPK protein expression is collected each group of cell and is extracted total egg to specifications In vain, protein concentration is measured with Bradford method；After Ding Liang, every swimming lane takes the sample corresponding to total protein 10 μ g volume and becomes Property, sample-adding, carry out SDS-PAGE electrophoresis with 10% acrylamide gel；Taking out separation gel, electrotransfer is to nitrocellulose filter Supreme, wash film after closing 1h with confining liquid at room temperature, under the conditions of 4 DEG C, hatch p-p38MAPK monoclonal antibody and conduct respectively The p38MAPK monoclonal antibody (working concentration 1:500) of internal reference overnight, and hatches goat anti-rabbit igg (HRP labelling) at room temperature 1h, often walks and all rinses 3 times with TBST liquid, each 10min；Finally, dyeing with AEC, treat that colour developing completes, distillation washing film dries, Sealed storage is at 4 DEG C of lucifuges.Use image analysis system scanning analysis, correct as internal reference using total p38MAPK, use p- P38MAPK and the relative expression's content representing phospho p38 MAPK with the ratio of pipe internal reference total p38MAPK intensity.

(3) statistical procedures data above represents with x ± s, and statistical method application SPSS13.0 software makees variance and phase Pass property is analyzed.

Two, result

(1) in peoniflorin produces the normal umbilical vein cultivated of impact of ICAM-1 to simulated ischemia Reperfu-sion endotheliocyte Chrotoplast can express ICAM-1, and the expression showed increased of ICAM-1 during simulated ischemia Reperfu-sion, after adding peoniflorin medicinal liquid The endotheliocyte that can make simulated ischemia Reperfu-sion is expressed the amount of ICAM-1 and is reduced.

Table 1 peoniflorin on simulated ischemia Reperfu-sion endotheliocyte produce ICAM-1 impact (N=10)

Note: " * * " represents compared with model group, p < 0.01.

(2) in peoniflorin produces the normal umbilical vein cultivated of impact of VCAM-1 to simulated ischemia Reperfu-sion endotheliocyte Chrotoplast can express a certain amount of VCAM-1, and during simulated ischemia Reperfu-sion, the expression of VCAM-1 increases, and adds variable concentrations Peoniflorin medicinal liquid after can make simulated ischemia Reperfu-sion endotheliocyte express VCAM-1 amount reduce, with model group comparing difference Statistically significant.

Table 2 peoniflorin on simulated ischemia Reperfu-sion endotheliocyte produce VCAM-1 impact (N=10)

Note: " * " represents compared with model group, p < 0.05, " * * " represents compared with model group, p < 0.01.

(3) result that affects that simulated ischemia Reperfu-sion endotheliocyte p-p38MAPK is expressed by peoniflorin shows, model group P-p38MAPK protein expression substantially increases, and peoniflorin is respectively organized and all can be significantly reduced its expression.As shown in table 3, Fig. 1.

Impact that simulated ischemia Reperfu-sion endotheliocyte p-p38MAPK is expressed by table 3 peoniflorin (n=3,)

Note: compare with cell controls group, △ △ P < 0.01；Compare with model group, * P < 0.05.

Three, discuss

Inflammatory reaction is to cause the important mechanisms of serious pathological damage after ischemia-reperfusion, and research shows, in ischemia/reperfusion In note pathological process, the generation of inflammatory mediator and oxygen-derived free radicals promotes the expression of inflammatory factor, acute phase reactive protein, and then Transcribing and synthesizing of regulation and control adhesion factor agent genes, induces the expression of adhesion factor, shape in neutrophilic granulocyte and endotheliocyte Become positive feedback effect, then increase the weight of the damage of affected cells.

Adhesion molecule is present among cell surface and substrate more, and its effect is to promote cell and cellular matrix or other are thin Born of the same parents are sticked.At present research is thought, adhesion molecule wide participation inflammatory reaction, neoplasm metastasis, immunne response, wound recover, The physiology such as blood coagulation, pathological process.Wherein, inflammatory reaction is one of important mechanisms of mediate ischemia reperfusion damage, is lacking In blood reperfusion injury generating process, its local vascular endotheliocyte is first subjected to stimulate, and promotes that ICAM-1, VCAM-1 etc. stick Molecule high expressed.The intercellular adhesion molecule of endothelial cell surface can be mutual with the CD11/CD18 molecule of neutrophils surface Effect, thus promote neutrophil adhesion in Surface of Vascular Endothelial Cells, improve its migration and go out blood vessel wall entrance surrounding tissue Probability, in turn result in tissue injury.Having research to confirm, ginkalide B can reduce intercellular adhesion molecule expresses, thus to little The little cerebral ischemia reperfusion injury of Mus plays protective effect.Tian XF etc. are by the research to intestinal ischemia-reperfusion injury in rats Showing, there is significant leukocyte infiltration for rear damaged tissue local again in blood, increases with Expression of Cell Adhesion Molecules, becomes For the key factor causing ischemical reperfusion injury to occur.Meanwhile, at myocardial ischemia reperfusion injury, chronic congestion heart failure Exhaust, the research of the disease such as postcardiac surgery endothelial injury shows the most successively, in ischemical reperfusion injury generating process, and cell adhesion The expression of molecule is significantly higher than normally.Illustrate that ICAM-1, VCAM-1 participate in the ischemical reperfusion injury process of Various Tissues.

In addition to adhesion factor, mitogen activated protein kinase (MAPK) is as signal transduction pathway system important in organism One of system, also may participate in the regulation and control of inflammatory reaction.Have it is demonstrated experimentally that the p38MAPK signal path belonging to MAPK family can be many Planting and play a role on cell, mediating inflammatory reacts；Activation on HUVECs cell is the phosphoric acid caused by AT1 receptor pathway Change realization.Therefore, medicine anti-inflammatory response can be inquired into by p-p38MAPK protein expression level on detection HUVECs cell The mechanism of effect.

In this experiment, in model group simulated ischemia Reperfu-sion cell, ICAM-1, VCAM-1, p-p38MAPK protein expression is obvious Higher than matched group (p < 0.01), consistent with the studies above result.Experimental data shows simultaneously, and basic, normal, high dosage peoniflorin is located in advance Reason group ICAM-1, VCAM-1 express and are all substantially less than model group (p < 0.01), dosage peoniflorin pretreated group p-low, middle P38MAPK protein expression is also substantially less than model group (p < 0.05), it was demonstrated that it effectively inhibits glutinous in Ischemia-Reperfusion Injury Attached molecule and the expression of phosphorylation, thus by suppression adhesion molecule respectively and the inflammatory reaction of p38MAPK signal path mediation, Effectively play the protective effect to vascular endothelial cell, and be proportionate with concentration.

Experimentation proves, peoniflorin can be subject to by regulation and control M-cholinergic receptors signal path, regulation M-cholinergic receptors and M Body-G-protein-K_ATPPassage plays the protective effect to inflammatory cell.Separately having research display, cholinergic anti-inflammatory pathway can reduce The expression of adhesion factor, additionally, its mechanisms of anti-inflammatory and p38MAPK activity decrease also have dependency, it can pass through α 7-cigarette Alkaline acetylcholinergic receptor (α 7nAChR) suppression p38MAPK signal pathway, blocks NF-κ B and c-Myc path, alleviates local scorching Disease is reacted, thus produces protective effect.Therefore peoniflorin by regulation and control cholinergic anti-inflammatory pathway play suppression adhesion factor and P38MAPK signal path, and then improve endothelial cell inflammation reaction, alleviate tissue injury, to HUVECs cells play protectiveness Effect.

2.2 peoniflorins are to the protective effect of myocardial ischemia-reperfusion injury and the tune to nuclear factor kappa B expression Joint

One, materials and methods

(1) trial drug peoniflorin, purchased from Nat'l Pharmaceutical & Biological Products Control Institute, mass fraction is 99.0%.Used time with Normal saline becomes the peoniflorin medicinal liquid of 3.0mg/ml.

(2) animal SD rat, male and female half and half, body weight: 270～300g, by Institute of Experimental Animals, Chinese Academy of Medical Sciences Breeding field provides, the quality certification number: SCXK (capital) 2013-0003.

(3) reagent NBT (Nitrotetrazolium Blue chloride, NBT), purchased from the U.S. Sigma-aldrich company；Concentrated type NF-κ B rat Ig-G monoclonal antibody, sheep ICAM-1 polyclonal antibody, win purchased from Beijing Ao Sen Bioisystech Co., Ltd；SABC staining kit, DAB developer, SPES bonding die agent etc., biological purchased from Wuhan Boster Engineering Co., Ltd.AST (AST) test kit, lactic acid dehydrogenase (LDH) test kit, creatine kinase (CK) Test kit, CK-MB (CK-MB) test kit, build up Bioengineering Research Institute purchased from Nanjing.

(4) instrument HX-300S type toy respirator；BIOPAC polygraph；AE100 electronic analytical balance； Shimadzu ultra-violet and visible spectrophotometer；Leica paraffin slicing machine；CIAS-1000 cell image analyzes system；OLMPUS is micro- Mirror and Digit camera.

(5) method

1. model is prepared after rat adaptability raises 1 week, anaesthetizes with pentobarbital sodium (45mg/kg), and dorsal position is fixed.Will BIOPAC polygraph cardiac diagnosis lead electrode is connected with rat extremity, records normal II lead electrocardiogram.Cervical region does gas Cannula, connects toy respirator (60 times/min of respiratory frequency, ventilation 20ml/kg).Cut along left border of sternum 3～4 intercostal Open thoracic wall and expose heart, ligature ramus descendens anterior arteriae coronariae sinistrae (LAD) at 2mm level in declining away from left auricle, and under ligature After placing one section of thin latex tubing (diameter 1.5mm), ligature is tightened up, cause myocardial ischemia.Extract latex tubing after 30min out to lead to again Reperfu-sion 2h.Using " II lead electrocardiogram ST section substantially raises or T wave height is alarmmed " as the successful standard of coronary ligation；So that " II leads Connection reduction in ST segment depression more than 50% or T ripple declines " as the successful standard of Reperfu-sion^[7].Meet this standard person selected real Test.

2. experiment packet takes into mould rat and is randomly divided into model group, peoniflorin low dosage (30mg/kg) group, peoniflorin height agent Amount (60mg/kg) group, often organizes each 20.Separately take rat 20, connect respirator open chest surgery, but do not ligature arteria coronaria, as vacation Operation group.Each group before preoperative 25h, 1h and Reperfu-sion, 20min carries out sublingual vein respectively or tail vein injection is administered, sham-operation Group and model group give equal-volume normal saline.Often in group 10 carry out myocardial infarction area mensuration, other carry out cardiac muscular tissue HE dyeing and immunohistochemical experiment, all take the indexs such as Virus monitory AST, LDH.

3. Electrocardiography uses BIOPAC polygraph that each group of rat is carried out continuous ECG record, analyzes Before being administered after its ligation arteria coronaria, be administered after ligation arteria coronaria after after 5min, 10min, 20min, Reperfu-sion when 30min, 1h, 2h The change of ECG T wave change absolute value.

4. myocardial infarction area measures and uses NBT dyeing display infarcted region and ischemic region.Put to death rat, core dirty rapidly It is placed on ice platform, rinses well with PBS and be stained with filter paper dry.By equal for crosscutting for ventricle one-tenth thickness 5 from the apex of the heart, Being placed in 0.1%NBT (with the 0.2mol/L Tris liquid preparation of pH 7.4-7.8) solution, 37 DEG C of water-bath 15min, it is unnecessary to wash away Dyestuff, infarcted region is not colored, non-infarcted region by NBT dye for hyacinthine.Taking a picture infarcted myocardium, application image processes software meter Calculate infarcted myocardium area and account for the percentage ratio of the ventricle gross area.

5. cardiac muscular tissue HE dyeing Reperfu-sion is cut rapidly heart and is placed on ice platform after terminating, with wash buffer, carefully Separate left ventricle antetheca ischemic region and also cut 3～5mm tissues, be placed in 10% neutral formalin and fix 12h, dehydration, paraffin embedding, cut Sheet, dewax, dye and neutral gum mounting, Microscopic observation cardiac muscular tissue form.

6. SABC detection tissue of being drawn materials aforementioned left ventricle antetheca ischemic region is placed in 4% paraformaldehyde fixing, de- Water, routine paraffin wax embeds, serial section, thick 4 μm of sheet, and every rat cuts 20 sections, and wherein 10 are used for nuclear factor Kappa B (NF-κ B) detects, and remaining detects for ICAM-1 (ICAM-1).The most conventional dewaxing is to water, with 3% Hydrogen peroxide treatment, high temperature reparation, serum is closed, and drips the one of 1:100 respectively and resists overnight, uses PBS to replace an anti-work simultaneously For negative control, 4 DEG C of overnight incubation；The next day drip two anti-continuation respectively and hatch 20min, DAB colour developing then, Microscopic observation develops the color Background.All to use 0.01mol/LPBS to rinse between last step 3 times, each 5min.After with haematoxylin redyeing, rush with distilled water Washing, gradient alcohol dehydration, dimethylbenzene is transparent, neutral gum mounting, tissues observed cell dyeing situation under light microscopic.

7., after Serum Indexes mensuration Reperfu-sion 2h terminates, ventral aorta is taken a blood sample, and under the conditions of 4 DEG C, 3500r/min is centrifuged 10min Separate serum.Microplate reader is used to measure Serum LDH and AST level；Use ultraviolet-uisible spectrophotometer respectively at 660nm wavelength Lower mensuration absorbance, to measure CK and CK-MB level (operating by test kit description).

8. statistical procedures data above represents with x ± s, and statistical method application SPSS13.0 software is made variance and is correlated with Property analyze.

Two, result

(1) the peoniflorin pretreatment impact on myocardial infarction area

From table 4, Fig. 2, after myocardial ischemia-reperfusion, b. model group myocardial infarction area significantly increases.With model group Relatively, d. peoniflorin high dose and c. low dose group myocardial infarction area the most substantially reduce, and have aobvious through statistical procedures difference Work property (P < 0.01)

Table 4 peoniflorin on the impact of myocardial ischemia-reperfusion rat myocardial infarction model area (n=10,)

Note: compare with model group, * * P < 0.01

(2) the peoniflorin pretreatment impact on cardiac muscular tissue's form

As shown in HE stained photo in Fig. 3, a. rats in sham-operated group cardiac muscle fiber marshalling, clear in structure, shape State is complete, even dyeing, has no the substantially pathological change such as cell hydropic degeneration, cell infiltration；B. model group cardiac muscle fiber row Row disorder, many places visible cell cavity sample degeneration, dissolve fracture, band disappears, part nucleus swelling, corrode, depigmentation, uneven Even eosin stains strengthens, and interstice is the most broadening, vasodilation, hyperemia, hemorrhage, with a large amount of neutrophil infiltration；Chinese herbaceous peony It is light that c, d myocardial cell injury relatively model group is respectively organized in medicine glycosides pretreatment, and has concentration dependence.Prompting peoniflorin pretreatment can There is the effect of protection cardiac muscle.

(3) impact that myocardial ischemia-reperfusion rat heart muscle NF-κ B, ICAM-1 are expressed by peoniflorin

Immunohistochemical staining result shows, after modeling, rat heart muscle tissue NF-κ B, ICAM-1 expression substantially rises High.Peoniflorin 60mg/kg and 30mg/kg group can obviously reduce the integral optical density of NF-κ B, the expression area of ICAM-1 and integration Optical density, difference has statistical significance.The results are shown in Table 5, Fig. 4.

Table 5 peoniflorin on the impact of myocardial ischemia-reperfusion rat NF-κ B and ICAM-1 protein expression (n=10,)

Note: compare with sham operated rats, △ △ P < 0.01；Compare with model group, * P < 0.05, * * P < 0.01.

(4) peoniflorin pretreatment is on serum CK, the impact of CK-MB, AST, LDH

As shown in table 6, comparing with sham operated rats, model group can significantly raise AST, LDH, CK, CK-MB value, through statistics Process difference and there is significance (P < 0.01)；Peoniflorin high dose group can significantly reduce CK, CK-MB, through statistical procedures difference There is significance (P < 0.05)；And each administration group all shows the trend reducing myocardium four enzymes.

Table 6 peoniflorin on the impact of myocardial ischemia-reperfusion rat heart muscle enzyme (n=10,Unit: U/L)

Note: compare with sham operated rats,^△△P<0.01；Compare with model group,^*P<0.05.

Three, discuss

Myocardial ischemic preconditioning (myocardial ischemia preconditioning, MIP) refers to Repeated Brief Myocardial ischemia can produce protective effect to cardiac muscle, so that the toleration of longer time ischemia is strengthened by cardiac muscle.But, as one Planting damaging preventive measure, based on ethics and methodology factor, MIP is difficult to the most effectively use.Research in recent years finds, By giving some drugs in advance, also can produce or directly simulate Endogenous protective substances by excitating organism, strengthening myocardium resistance to ischemia Hypoxia ability, thus under the conditions of non-damaging, realize the protective effect to cardiac muscle, i.e. " Pharmic preconditioning ".This experiment is by greatly Intravenous injection variable concentrations paeoniflorin preparations before Mus myocardial ischemia and before Reperfu-sion, studies it and damages rat myocardial ischemia and reperfusion The protective effect of wound.Myocardial infarction area is to evaluate medicine to myocardial ischemia reperfusion injury protectiveness with the ratio of the gross area The leading indicator of effect, this result of study shows, peoniflorin can obviously reduce myocardial infarction area, compares with model group, and it is poor Different have significance (P < 0.01).

When cardiac muscle occurs ischemical reperfusion injury, owing to intracellular calcium overload and oxygen-derived free radicals cause biomembrane lipid The organelles such as peroxidization, causes the 26S Proteasome Structure and Function of cell membrane to be destroyed, mitochondrion produce pathologic and change, so that Myocardial damage aggravation under ischemic state.Owing to now membrane permeability increases, intracellular enzyme leaks outside entrance blood circulation, Necessarily cause a series of serum enzyme to change, and present positive correlation with myocardial necrosis degree.Therefore, to AST in serum, LDH, The activity of CK, CK-MB detects, can be as myocardial ischemia reperfusion injury degree and the index of curative effect of medication.This experiment is tied Fruit display, compared with model group, peoniflorin high and low dose group all shows the trend reducing myocardium four enzymes, wherein peoniflorin The high dose group reduction effect notable (P < 0.05) to serum CK, CK-MB, points out it may be with increasing to the protective effect of cardiac muscle The stability of heart tonifying muscle cell membrane is relevant.

Inflammatory reaction is another important mechanisms causing the serious pathological of myocardial ischemia reperfusion injury to damage.Research table Bright, in ischemia/reperfusion pathological process, inflammatory mediator and oxygen-derived free radicals generate, and cause the activation of NF-κ B, thus promote Entered the expression of inflammatory factor, acute phase reactive protein, and between regulating cell adhesion factor ICAM-1 agent genes transcribe and Synthesis, induction of the expression of ICAM-1 in neutrophilic granulocyte and endotheliocyte, forms positive feedback effect, then increases the weight of myocardial cell Damage.Have research to point out, ICAM-1 to neutrophil adhesion during ischemical reperfusion injury, assemble and the process that activates plays Pivotal role.In this research, HE Coloration experiment result shows, has a large amount of neutrophil infiltration between model group myocardial cell, and Chinese herbaceous peony In medicine glycosides each dosage group, neutrophil infiltration situation all makes moderate progress, and has concentration dependence.Immunohistochemical experiment result Display, compared with model group, peoniflorin high dose group can obviously reduce the integral optical density of NF-κ B, ICAM-1, and difference has system Meaning (P < 0.05 or P < 0.01) learned by meter.

In sum, this result of study display peoniflorin preconditioning on myocardial ischemic reperfusion injury of rats has protectiveness Effect, its mechanism of action with by scavenging activated oxygen thus suppress Cell membrane lipids peroxidating, and by suppression NF-κ B egg The downstream inflammatory reaction approach that white expression and then impact are regulated and controled by NF-κ B is relevant, thus discloses peoniflorin and can be developed into leading to Cross the medicine of cholinergic anti-inflammatory pathway regulation and control myocardial ischemia-reperfusion, thus play a protective role.

Prepare the embodiment of medicament:

Peoniflorin 200.0g, medical starch is appropriate, and the two is sufficiently mixed, encapsulated, makes 1000 seed lac capsules, every weight 0.25g, containing peoniflorin 0.2g.

Claims (2)

1. peoniflorin application in preparation regulation and control cholinergic anti-inflammatory pathway medicine.

The peoniflorin the most according to claim 1 application in preparation regulation and control cholinergic anti-inflammatory pathway medicine, peoniflorin leads to Cross regulation and control cholinergic anti-inflammatory pathway and play suppression adhesion factor and p38MAPK signal path, and then it is anti-to improve endothelial cell inflammation Should, alleviate tissue injury, to HUVECs cells play protective effect.