CN102813650B - Application of zacopride in preparing pharmaceuticals for preventing ventricular remodeling - Google Patents
Application of zacopride in preparing pharmaceuticals for preventing ventricular remodeling Download PDFInfo
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Abstract
The invention relates to pharmaceuticals for treating ventricular remodeling the field of pharmaceuticals, in particular to application of zacopride in preparing pharmaceuticals for preventing ventricular remodeling, and aims to discover novel pharmaceuticals for preventing ventricular remodeling and prove that special agonist IK1 passage can prevent ventricular remodeling. Compared with the prior art, zacopride is applicable to researches of novel pharmaceuticals for preventing ventricular remodeling, and the fact that zacopride prevents ventricular remodeling through special agonist IK1 passage is discovered for the first time at present. Zacopride as pharmaceuticals for preventing ventricular remodeling has similar effect to captopril, and is stable in effect and large in safety range.
Description
Technical field
The present invention relates to field of medicaments, relate generally to the medicine for the treatment of remodeling ventricle, be specially Zacopride as the application of the anti-remodeling ventricle medicine of preparation.
Background technology
Remodeling ventricle refers to that ventricle increases in myocardial damage or load a series of variations such as size, shape, chamber wall thickness and organizational structure produce by ventricle, is the pathologic-physiological reaction process of the whole compensatory and secondary of pathological changes reparation and ventricle.Remodeling ventricle can damage cardiac function, and complication increases, and also obviously increase of mortality rate, and therefore anti-remodeling ventricle has become one of of paramount importance research contents in current cardiovascular field.
Hypertension, myocardial ischemia and myocardial infarction, drug intoxication, myocardial damage etc. all can cause remodeling ventricle, develop into the end stage eventually, will form heart failure.In recent years, although obtained very large progress in the Drug therapy of heart failure, the mortality rate of Patients with Cardiac Failure is still higher.Therefore disclosing the molecular mechanism that remodeling ventricle and heart failure produce, find effectively preventing measure, is the important topic of cardiovascular field research.
The paathogenic factor of remodeling ventricle can be divided into two classes:
1. mechanical factor: the pressure overload that hypertension, aortic stenosis etc. causes; The volume load that a moving venous fistula, chronic anaemia, atrial septal defect etc. cause is excessive.
2. humoral factor: norepinephrine, Angiotensin II (AngII), thyroxine, steroid material, vassopressin, Endothelin and multiple somatomedin are as the somatomedin of nerve growth factor, endothelial cell growth factor (ECGF), platelet source property, insulin like growth factor etc.
From cell and molecular level, myocardial remodelling comprises the change of two kinds of different structures: (1) myocardial cell: because phenotype changes, normal cell transforms to fetal type.This myocardial cell power consumption is low, and muscle maximal velocity of contraction and tension force development speed are slow, can not meet long-term efficiency operation needs, finally causes myocardium shrinkage function to decline.(2) non-myocardial cell composition (intercellular substance): due to interstitial cell hyperplasia, the content of collagen protein, type etc. change, myocardium hardness increases, and diastolic function is impaired.Current research shows, interrupts reconstruction progress and can delay or prevent that cardiac function from continuing to worsen, and improves survival rate.
Classification and the mechanism of action of current anti-remodeling ventricle medicine used:
1. central depressor
Methyldopa acts on maincenter alpha-2 receptor by its metabolite-normetadrenaline and produces expansion blood vessel and negative chronotropic effect, reduces total peripheral resistance and blood pressure lowering.Research shows, the methyldopa left ventricular hypertrophy (LVH) that can go down, clonidine does not have retrogradation to LVH in zoopery, but after hypertensive patient's medication in blood pressure drops, LVH is also compared with obviously going down before medication.
2. adrenergic receptor blocker
α-blockers can reverse LVH, and its incidence rate that reduces LVH is similar to calcium antagonist, diuretic and beta-Blocking agent.The go down effect of LVH of beta-Blocking agent may be that it reduces arterial pressure, reduces sympathetic nervous system tension force and reduces the coefficient results such as plasma renin activity.No matter be selectivity Beta 1 blocking agent atenolol and metoprolol, or nonselective timolol, the effect that reverses LVH all there is; The acebutolol with intrinsic sympathomimetic acitivity can make LVH go down too.
3. hypertensin 2 (AngII) receptor is according to anti-agent and angiotensin converting enzyme inhibitor (ACEI)
Blocking-up ACE or AT1 are subject to the effective prevention of physical ability even to reverse cardiac myocyte hypertrophy and Myocardial interstitial remodeling.Its mechanism is except reducing blood pressure, improve large artery trunks compliance and reduce outside afterload, can also alleviate LVH by suppressing the adrenergic secretion synthetic and angiotensin mediation of the myocardium protein that angiotensin relies on, lower than the AT1 receptor antagonist Losartan of hypotensive dose, just can suppress the fibrosis of myocardium interstitial.ACEI, as ramipril, lisinopril, captopril etc. all can make serious LVH alleviate.
4. calcium antagonist
With diltiazem, verapamil and nitrendipine short term therapy hypertensive patient, can significantly alleviate LVH.The mechanism of action of calcium antagonist comprises: (1) improves the early stage fast full function of diastole, and does not substantially affect isobaric diastolic function; (2) reduce afterload; (3) LVH reverses rear its contractile function does not affect, and even can also improve; (4) verapamil and diltiazem can also reduce ventricular arythmia.
5. statins
Patel etc. utilize simvastatin intervention mankind hypertrophic neuropathy model transgenic rabbits, and after 12 weeks, left ventricular mass decreased average 37%, interventricular septal thickness minimizing 21%, collagen volume fraction reduce 44%.Insulin resistant can increase the weight of remodeling ventricle, and statins can obviously improve insulin resistant, and its mechanism of action that improves remodeling ventricle may be: 1. improve vascular endothelial function, stop structural change; 2. reduce concentration and the activity of Angiotensin-Converting, reduce the generation of AngII; 3. regulate the expression of AngII receptor; 4. antioxidation; 5. improve insulin resistant.
6. aldosterone antagonists
Studies have shown that over nearly 10 years, in cardiac muscular tissue, external except existing AngII to be subject to. also have a large amount of aldosterone receptors.Aldosterone directly mediates myocardial remodelling (cardiac myocyte hypertrophy, extracellular Matrix collagen increase and fibrosis) by its receptor.RALES applies after low dose of spironolactone in treatment heart failure patient, observes its impact on left ventricular remodeling, finds that treatment has obtained useful effect.
7. endothelin-receptor antagonists
Endothelin 1 raises may be relevant with following factor with the change of Left Ventricular Structure: Endothelin 1 can stimulate Cardiac Fibroblasts I, III Collagen Type VI propagation, synthesize, promote myocardial interstitial collagen network remodeling, at myocardial fibrosis with in reinventing, play an important role: Endothelin 1 can be induced myocardial cell C-fos gene expression, promote myocardial cell hypertrophy, hypertrophy; Endothelin 1 can activate local renin-angiotensin-aldosterone system, and the releases such as the somatomedin of promotion platelet source property, transforming growth factor, promote myocardial cell hypertrophy indirectly, cause myocardial hypertrophy.Research discovery, endothelin receptor antagonists can stop uremia's Rat Myocardial Fibrosis and myocardial cell to ask thickening of small artery blood vessel wall, has confirmed that from another point of view Endothelin 1 and left ventricular remodeling are in close relations.Endothelin-receptor antagonists can produce resisting hypertension and improve the effect of remodeling ventricle.
During the anti-remodeling ventricle medicine of former studies, inward rectifyimg potassium channel (I
k1passage) be a unheeded target spot.I
k1be myocardium topmost background outward current, participate in maintaining and the multipole at myocardial action potential (Action potential, AP) 3 final ends of resting potential (Resting membrane potential, RMP).Its molecular basis is mainly inward rectifyimg potassium channel gene Kir2.x subfamily.In the past owing to lacking I
k1modality specificity agonist/blocker, has greatly limited the research of channel function.Our reported first an I
k1modality specificity agonist-Zacopride (zacopride), utilizes this pharmacological tool medicine, and we attempt by exciting/rise I
k1the expression of passage, brings into play anti-remodeling ventricle, improves the effect of cardiac function.Chloroquine is traditional antimalarial, and having bibliographical information 0.3 μ mol/L chloroquine is rat ventricular myocytes I
k1passage relative specificity blocker (command potential is-during 60mV, and I
k1passage is blocked 51% left and right).
Summary of the invention
Gastric motility promoter Zacopride (Zacopride) is domestic and international first found selectivity cardiac muscle I
k1channel agonist, has the new purposes that remodeling ventricle medicine is treated in anti-remodeling ventricle effect and preparation.
In fact, the present invention relates to Zacopride as the application of preparation treatment remodeling ventricle medicine.
Described medicine is capsule, microcapsule, liposome, granule, injection, tablet and oral liquid.
Zacopride (Zacopride), gastric motility promoter, is croak pyridine benzamide derivant, belongs to 5-HT
3receptor antagonist and 5-HT
4receptor stimulating agent, at present for promoting gastric motility and intestinal running, also as promoting brain metabolic cycles medicine treatment senile dementia.Our current research finds that Zacopride also can be used as anti-arrhythmic and treats some triggered arrhythmia.
We find that Zacopride (Zacopride) is I
k1channel selectivity agonist.This results suggest Zacopride (Zacopride) can be by exciting ventricular muscles I
k1passage, becomes a kind of new anti-remodeling ventricle medicine.
The present invention adopts SD rat to test, by measure Zacopride (Zacopride) to levothyroxine sodium cause myocardial hypertrophy, isoproterenol causes myocardial hypertrophy, attenuates pressure-overload left causes the overweight and left anterior descending coronary artery ligation of pressure load and causes the effect of four kinds of remodeling ventricle models such as heart infarction, and applies I
k1passage relative specificity blocker chloroquine reverses the anti-remodeling ventricle effect of Zacopride, has confirmed above-mentioned enlightenment.Concrete confirmation thes contents are as follows:
A.5 within the scope of～50 μ g/kg, Zacopride (Zacopride) can reverse ventricular remodeling in rats by dose dependent, 15 μ g/kg are its ceiling effect dosage (P < 0.05), and the effect of anti-remodeling ventricle is similar to the anti-remodeling ventricle medicine captopril (Captoril) of generally acknowledging.
Effectively antagonism levothyroxine sodium causes myocardial hypertrophy to 15 μ g/kg Zacoprides, isoproterenol causes myocardial hypertrophy, and attenuates pressure-overload left causes the remodeling ventricle that the overweight and left anterior descending coronary artery ligation of pressure load causes four kinds of rat models such as heart infarction.Compare with normal rat, model (remodeling ventricle) rat heart volume increases, and left compartment muscle cell is obviously loose; Cardiac myocyte hypertrophy under light microscopic, degeneration necrosis; It is thick, tortuous, disorderly that Electronic Speculum shows that cardiac muscular tissue's sarcostyle increases, muscle segment structural fuzzy, and intercalated disc is fuzzy, broadening, and mitochondrion quantity showed increased, irregularly shaped is cavity shape and changes, and the concentric circular/myelin sample that is having changes; Immunohistochemical staining shows that apoptosis of cardiac muscle increases; Ultrasonic cardiography illustrates left chamber systole internal diameter (LVIDs), Dd (LVIDd), ejection fraction (EF) and left LVSF (FS) obviously to be reduced, and interventricular septal thickness is abnormal; Hemodynamics detects shows that rat left ventricular systolic pressure (LVSP), left ventricular pressure rise/fall maximum rate (± dp/dtmax) all obviously reduce, and left ventricular end diastolic presses (LVDP) to significantly improve, prompting ventricular hypertrophy and left systolic heart reduce.Rat myocardial cell injury after Zacopride (15 μ g/kg) is intervened obviously alleviates; downright bad and apoptosis reduces; form approaches normal; left chamber systole internal diameter, Dd, all compared with model group, obviously reduce; interventricular septal thickness approaches normal; EF, FS significantly raise, and heart function recovery is to normal or approach normal level.
B. chloroquine is rat ventricular myocytes I
k1passage relative specificity blocker, low dosage chloroquine (7.5 μ g/kg) and Zacopride use in conjunction can obviously suppress the anti-remodeling ventricle effect of Zacopride, and prompting Zacopride may pass through exciting myocardium I
k1passage and bring into play anti-remodeling ventricle effect.
The present invention compared with prior art, has the following advantages and effect:
1. Zacopride (Zacopride) pass through selectivity excitement I as what find first at present
k1reverse remodeling ventricle and also obviously improve the medicine of cardiac function, can be used for the research of anti-remodeling ventricle medicine.
2. Zacopride (Zacopride) is as the medicine for the treatment of remodeling ventricle, and its effect is similar to captopril, and effect is stable, and safety range is large, non-evident effect.
Accompanying drawing explanation
Fig. 1. levothyroxine rat model typical case ultrasoundcardiogram; In Fig. 1, A: Normal group; B: model group; C: Zacopride intervention group; D: Zacopride+chloroquine group;
Fig. 2. heart infarction rat model typical case ultrasoundcardiogram; In Fig. 2, A: pseudo-operation group; B: heart infarction group; C; Zacopride intervention group; D; Captopril group;
Fig. 3. each experimental group rat typical heart morphologic observation of isoproterenol prime model; In Fig. 3, A: normal rat; B: isoproterenol rat model; C: Zacopride is intervened rat;
Fig. 4. each experimental group Rat left ventricular myocardium cellular morphology of levothyroxine model is observed; In Fig. 4, A: normal rat; B: levothyroxine rat model; C: Zacopride is intervened rat;
Fig. 5. heart infarction rat model left compartment muscle ultrastructure; In Fig. 5, A: pseudo-operation group; B: heart infarction group; C; Zacopride intervention group; D; Captopril intervention group;
Fig. 6. heart infarction model is respectively organized rat pathology HE coloration result; In Fig. 6, A: pseudo-operation group; B: heart infarction group; C; Zacopride intervention group; D; Captopril intervention group;
Fig. 7. heart infarction model is respectively organized the comparison that rat caspase-3 expresses; In Fig. 7, A: pseudo-operation group; B: heart infarction group; C; Zacopride intervention group; D; Captopril intervention group;
The specific embodiment
Below with Zacopride (Zacopride) to levothyroxine sodium cause myocardial hypertrophy, isoproterenol causes myocardial hypertrophy, attenuates pressure-overload left causes the effect that the overweight and left anterior descending coronary artery ligation of pressure load causes four kinds of remodeling ventricle models such as heart infarction, illustrates that it is in the new purposes of pharmaceutical field.
1 materials and methods
1.1 laboratory animals:
Healthy SD rat, male, body weight 200～250g, purchased from Mountain Western Medicine S University's Experimental Animal Center.
1.2 medicines and reagent:
Hydrochloric acid Zacopride (Zacopride hydrochloride) is purchased from TOCRIS.Euthyrox (levothyroxine sodium, L-thyroxine, L-Thy, specification is 50 μ g/ sheets) production of Merck KGaA company.Chloroquine, captopril (Captoril) are purchased from U.S. sigma company.Collagenase P is purchased from German Bochringer Mannhein company; taurine (taurine) Pidolidone (L-glutamicacid), HEPES (N-2-hudroxyethylpiperazine-N '-2-ethane-sulfonic acide) purchased from U.S. sigma company, all the other are domestic production analytical pure product.
1.3 key instruments:
1.3.1GE Vivid 7Pro color ultrasonic devices, 1OS probe, frequency probe 8.0MHz, is equipped with two-dimentional strain imaging software, Echo PAC work station
1.3.2Olympus FV 1000 laser confocal microscopes
1.3.3RM6240 multi-path physiology signal acquiring processing system (Chengdu Instruement Factory)
1.3.4HX-200 animal respirator (Chengdu TME Technology Co., Ltd.)
1.4 reagent preparation:
1.4.1 after Euthyrox tablet being ground, with normal saline, be mixed with the suspension of 1mg/ml, during use, by 1mg/kg/d, take.
1.4.2 Zacopride (Zacopride) stock solution: be 1mmol/L by deionized water dissolving Zacopride concentration, dilute on demand during use.
1.4.3 chloroquine (Chroxine) stock solution: be 1mmol/L by deionized water dissolved chlorine quinoline concentration, dilute on demand during use.
1.4.4 captopril (Captoril): dissolve captopril with tap water, every day, fresh preparation, took by 100mg/kg/d during use.
1.4.5 tyrode's solution (mmol/L): NaCl 140, and KCl 5.4, and NaH2PO4 0.33, and HEPES 5.0, and glucose 10, and MgCl2 1.0, and CaCl2 1.8, regulates PH to 7.38 with NaOH.
1.4.6 enzyme liquid (mmol/L): NaCl 125, KCl 5.4, CaCl275, MgCl2 1.0, and NaH2PO4 0.33,4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid (HEPES) 10, glucose 10, taurine 20, Collagenase P (Boehringe Mannheim, Germary) collagenase P 0.1～0.3mg/ml (3.5～5mg).
1.4.7KB liquid (mmol/L): KOH 85, Pidolidone 50, KCl 30, and taurine 20, and KH2PO4 30, MgCl21.0, HEPES 10, glucose 10, EGTA 0.5, with KOH, regulates PH to 7.4.
2 experimental techniques:
2.1 Zacoprides (Zacopride) cause the impact of remodeling ventricle model on levothyroxine sodium
2.1.1 experiment grouping:
Healthy SD rat is divided into group at random, i.e. Normal group, levothyroxine model group, Zacopride 5 μ g/kg, 15 μ g/kg, 50 μ g/kg intervention group, Zacopride 15 μ g/kg+ chloroquines (7.5 μ g/kg) group and captopril positive controls.
2.1.2 experimental technique:
SD rat is weighed.Except Normal group, all the other each treated animals are all with Euthyrox (1mg/kg/d) gavage of suspendible, and Normal group is with the distilled water gavage of equivalent.Zacopride various dose group is lumbar injection 5 μ g/kg, 15 μ g/kg, 50 μ g/kg Zacoprides respectively, Zacopride+chloroquine group is lumbar injection 15 μ g/kg Zacoprides and 7.5 μ g/kg chloroquines simultaneously, captopril positive controls by 100mg/kg/d drink water to captopril.Continuous 10 days.All rats are all raised with conventional solid feed and tap water, in drug withdrawal 24h overnight fasting, then observe corresponding index.
2.2 Zacoprides (Zacopride) cause the impact of remodeling ventricle model on isoproterenol
2.2.1 experiment grouping:
8 week age, the healthy SD rat of size was divided into 5 groups at random, i.e. Normal group, isoproterenol model group, Zacopride (Zacopride) 15 μ g/kg intervention group, Zacopride (15 μ g/kg)+chloroquine (7.5 μ g/kg) intervention group and captopril positive controls.
2.2.2 experimental technique:
SD rat is weighed.Except Normal group, the equal lumbar injection isoproterenol of all the other each treated animals (3mg/kg/d), the normal saline of Normal group lumbar injection equivalent.Zacopride intervention group lumbar injection 15 μ g/kg, Zacopride+chloroquine group is lumbar injection 15 μ g/kg Zacoprides and 7.5 μ g/kg chloroquines simultaneously, captopril positive controls by 100mg/kg/d drink water to captopril.Continuous 10 days.All rats are all raised with conventional solid feed and tap water, in drug withdrawal 24h overnight fasting, then observe corresponding index.
The impact of 2.3Zacopride on rats with abdominal aorta coarctation remodeling ventricle
2.3.1 experiment grouping:
1. pseudo-operation group: intraperitoneal injection every day 2ml normal saline; 2. model group: intraperitoneal injection every day 2ml normal saline; 3. Zacopride intervention group: lumbar injection Zacopride every day (15 μ g/kg); 4. captopril positive controls: captopril dissolves in (100mg/kg/d) in drinking-water, freely drinks water.
2.3.2 experimental technique:
Get 80 of healthy male SD rats, body weight 220～250g.Choosing at random 20 rats is pseudo-operation group (sham group), makes attenuates pressure-overload left model for all the other 60.10% chloral hydrate 3ml/kg intraperitoneal anesthesia for rat after preserved skin.Xiphoid-process median incision of lower abdomen, minute layer open abdominal cavity, at the free ventral aorta of the above passivity of renal artery branch, by No. 7 parallel being placed on ventral aorta of syringe needle, with 4 trumpeter's art silk threads, by ventral aorta and together ligation of syringe needle, then slowly syringe needle is withdrawn from, close abdomen, layering is sewed up.Making rat aorta diameter constriction is 0.7mm.Sham operated rats will be performed the operation silk thread through ventral aorta after opening abdomen, but not ligation ventral aorta.60 rats with abdominal aorta coarctations are divided into model group at random, Zacopride intervention group and captopril positive controls (n=20).Postoperative three days conventional application penicillin prevention infection.Each is organized rat and within the 3rd day after surgery, starts administration, successive administration 8 weeks.After 8 weeks, detect corresponding index.
The impact of 2.4 Zacoprides (Zacopride) on ventricular remodeling in rats model after myocardial infarction
2.4.1 laboratory animal and grouping
Get 80 of healthy male SD rats, body weight 220～250g.Choosing at random 20 rats is pseudo-operation group (sham group), makes myocardial infarction model for all the other 60.60 Rat of Myocardial Infarction are divided into myocardial infarction group (MI) more at random, Zacopride intervention group and captopril positive controls (respectively organizing n=20).
2.4.2 experimental technique:
10% chloral hydrate 3ml/kg intraperitoneal anesthesia for rat after preserved skin.After anesthesia, tracheostomy tube connects artificial animal respirator and makes mechanical ventilation, 60 times/min of frequency, tidal volume 8ml, respiratory quotient 2: 1.Connect electrocardiograph, first record each treated animal normal ECG, after left breast routine disinfection, in left the 4th intercostal, open breast and expose heart, on the boundary and apex of the heart line of left auricle and pulmonary conus, wear 6-0 surgical thread following coronary artery occlusion left anterior descending branch, see that immediately the apex of the heart turns white, electrocardiogram demonstration ST section is raised, and is modeling success, and negative pressure is closed thoracic cavity, layer-by-layer suture muscle skin.Sham operated rats is only opened breast, exposes heart threading but following coronary artery occlusion not.Postoperative three days conventional application penicillin prevention infection.
Each is organized rat and within the 3rd day after surgery, starts administration, successive administration 8 weeks.1. pseudo-operation group: intraperitoneal injection every day 2ml normal saline.2. myocardial infarction group: following coronary artery occlusion left anterior descending branch (LAD), intraperitoneal injection every day 2ml normal saline.3. Zacopride intervention group: lumbar injection Zacopride every day (15 μ g/kg/d).4. captopril positive controls: intraperitoneal injection every day 2ml normal saline, captopril dissolves in (100mg/kg/d) in drinking-water.Each organizes rat in the postoperative detection of carrying out corresponding index for 8 weeks.
2.5 laboratory observation indexs:
2.5.1 ultrasoundcardiogram (ultrasound cardiograph, UCG) checks
1. rat is weighed, and 10% chloral hydrate (0.3ml/100g) is through intraperitoneal injection of anesthesia.By the rat supine position of having anaesthetized, on Mus plate, with cotton cord fixing limbs and head, chest is for hair.
2. with the 10s being equipped with in GE Vivid 7Pro Ultrasound Instrument, pop one's head in and be equipped with rodent pattern rat heart is carried out to scanning, detecting angle and the degree of depth tries one's best little of to improve frame rate, be generally 15 °-30 °, the degree of depth is 2-3cm, frame frequency > 250/Sec, the highest frame frequency can reach 400/Sec.On long axis view of the left heart, measure and record left ventricular interior diameter (LV), the interventricular septal thickness (IVS) of rat heart, by the ultrasonic acquisition Left Ventricular Ejection Fraction of M type (EF) and left LVSF (FS), all data are all measured 3 times and are got its meansigma methods.The error causing in order to reduce rat Individual Size difference, levothyroxine rat model adopts body surface area index to compare, LV index=LV/ body surface area wherein, IVS index=IVS/ body surface area.Rat body surface area computing formula: body surface area ((m
2)=0.09 * [weight (kg)]
2/3.
2.5.2 hemodynamics detects
After modeling eight weeks, claim rat body weight, 10% chloral hydrate 3ml/kg intraperitoneal anesthesia.After anesthesia, be fixed on operating-table.Longitudinal incision skin of neck 1.5～2cm, isolates right carotid.Ligation, away from the common carotid artery of heart end, is clamped the common carotid artery of proximal part with bulldog clamp.Make common carotid artery full, by artery scissors one osculum, insert 1mm polyethylene catheter, in conduit, be full of in advance heparin saline.Utilize RM6240B biological function experimental system to measure the left ventricular systolic pressure (LVSP) of rat, left ventricular end diastolic pressure (LVEDP), left ventricular pressure rise/fall maximum rate (± dp/dtmax).After inspection, experimental rat carotid artery blood sampling is standby, opens rapidly breast and takes out heart, takes weight and left ventricular mass whole-heartedly.Get left chamber part cardiac muscular tissue and immerse in 10% formalin, remaining tissue-70 ℃ refrigerator is frozen standby.
2.5.3 after the mensuration drug withdrawal 24h of myocardial hypertrophy index, weigh (BW).Open breast chamber, take out fast heart and be placed in the normal saline of 4 ℃ of pre-coolings, by aorta, drive in the wrong direction and rinse after heart blood, with double-deck filter paper suck dry moisture, electronic balance accurately weighs wet quality (HW) whole-heartedly, remove atrium and valvular tissue, along interventricular septum, cut off left ventricle (comprising interventricular septum) and right ventricle, remove trunk and pericardial tissue, weigh left ventricle (comprising interventricular septum) quality (LVW), finally calculate quality/weight (HW/BW whole-heartedly, mg/g) ratio, left ventricular mass/weight (LVW/BW, mg/g) ratio, be designated as respectively plump index whole-heartedly, left ventricular hypertrophy index.
2.5.4 separated single left compartment muscle cell to measure cell surface long-pending
Modeling success is after 10 days, each experimental group animal is got respectively 3,15 minutes abdominal vein injecting heparins (1000U/kg) in the preoperative, pentobarbital sodium (65mg/kg, ip) anesthesia rear neck artery blood-letting, open rapidly breast and take out heart, be placed in 4 ℃ with saturated the pruning without calcium tyrode's solution of 100% oxygen, then heart is suspended on Langendorff perfusion device through the retrograde perfusion of aorta.First use without calcium Zinciodati Comp solution perfusion 8-10 minute, then use enzyme liquid circulation perfusion 15～20 minutes.In perfusing course, keep 37 ℃ of constant temperature, perfusion pressure 70cmH
2o, and continue to pass to 100% oxygen.After becoming large, deliquescing, ventricular muscles tissue chooses left ventricle, being placed in KB liquid shreds, piping and druming obtains the left compartment muscle cell disperseing gently, after the strainer filtering in 150 μ m apertures, be stored in KB liquid, room temperature is placed after 2 hours and is used the multiple calcium of calcic tyrode gradient, and Laser Scanning Confocal Microscope carries out form observation.Each heart is chosen at random 100 ventricular muscle cells and is measured length and width, and reference area, gets its meansigma methods as the numerical value of this animal individual corresponding index.
2.5.5 heart tissue pathological examination
Heart infarction rat model, gets infarction cardiac muscular tissue around, is cut into 1mm
3size was built in 2.5% glutaraldehyde fixing at 1 minute, transmission electron microscope observing.All the other rats are got part left ventricular tissues, and to be placed in 10% formalin fixing, does specimens paraffin embedding slices, does the expression of HE dyeing and Immunohistochemistry apoptotic proteins enzyme (caspase)-3.
2.6 date processing:
Data represent with mean ± standard deviation (Mean ± SD), adopt SPSS15.0 statistical software to carry out one factor analysis of variance, and P < 0.05 is for having significant difference.
3 experimental results
3.1 conventional Ultrasound kinetocardiogram results
3.1.1 levothyroxine model
Compare with Normal group, levothyroxine model group rat left chamber systole internal diameter (LVIDs), Dd (LVIDd), interventricular septal thickness (IVS) all significantly increase (P < 0.01), ejection fraction (EF) and left LVSF (FS) obviously reduce, and prompting ventricular hypertrophy and left heart function reduce.Rat all obviously reduces (P < 0.01) compared with model group through Zacopride (15 μ g/kg) preventive administration rear left chamber systole internal diameter, Dd, interventricular septal thickness; EF, FS significantly raise (P < 0.01), and heart function recovery is to normal or approach normal level.Application low dosage chloroquine (7.5 μ g/kg, I
k1passage relative specificity blocker) blocking-up Zacopride is to I
k1the stirring effect of passage, can obviously suppress the anti-remodeling ventricle effect of Zacopride, and prompting Zacopride may pass through exciting myocardium I
k1passage and bring into play anti-remodeling ventricle effect.The results are shown in Table 1, Fig. 1.
Table 1. levothyroxine rat model echocardiographic parameters changes (mean ± standard deviation)
LVIDd: left ventricular internal dimension diastole; LVIDs: left ventricular internal dimension systole; IVS: interventricular septal thickness; EF: Left Ventricular Ejection Fraction (%); The left LVSF of FS (%). compare with matched group
*p < 0.01,
*p < 0.05; Compare with model group
Δ Δp < 0.01; Compare with Zacopride group
##p < 0.01.
3.1.2 attenuates pressure-overload left model
Ultrasonic cardiography diagram, compares with puppet operation group, and LVIDd, the LVIDs of model group rat obviously increase, LVEF, and FS obviously reduces.Compare with model group, Zacopride and Captopril group LVIDd, LVIDs significantly reduce; Left ventricle EF, FS significantly raises, and has significant difference (P < 0.05) with model group.The results are shown in Table 2.
The comparison (mean ± standard deviation) of table 2. attenuates pressure-overload left rat model ultrasoundcardiogram indices
With model group comparison,
*p < 0.05,
*p < 0.01; With the comparison of puppet operation group,
#p < 0.05,
##p < 0.01
3.1.3 heart infarction model
Ultrasonic cardiography diagram, compares with puppet operation group, and LVIDd, the LVIDs of heart infarction group rat obviously increase, left chamber EF, and FS obviously reduces, the obvious attenuation of IVSd.Compare with heart infarction group, Zacopride group and Captopril group LVIDd, LVIDs have reduction trend, but with heart infarction group no significant difference; IVSd is significantly higher than heart infarction group (P < 0.05); Left ventricle EF, FS significantly raises, and has significant difference (P < 0.05) with heart infarction group, and prompting cardiac function obviously improves.The results are shown in Figure 2, table 3.
Table 3. heart infarction model is respectively organized the comparison (mean ± standard deviation) of rat ultrasoundcardiogram indices
With the comparison of heart infarction group,
*p < 0.05,
*p < 0.01; With the comparison of puppet operation group,
#p < 0.05,
##p < 0.01
3.2 hemodynamics detect
3.2.1 attenuates pressure-overload left model
Compare with puppet operation group, model group left ventricular systolic pressure (LVSP) and significantly improving, left ventricular pressure rise/fall maximum rate (± dp/dtmax) all significantly reduces (P < 0.05), and left ventricular end diastolic is pressed (LVDP) significantly rising (P < 0.05); Compare with model group, Zacopride group and Captopril group LVSP, + dP/dtmax and-dP/dtmax all significantly raises (P < 0.05), and LVDP significantly reduces (P < 0.05), prompting rat heart contractile function obviously improves.Each indicator difference not statistically significant of Zacopride group and Captopril group (P > 0.05).In Table 4.
Table 4. attenuates pressure-overload left model is respectively organized the comparison (mean ± standard deviation) of hemodynamics index
With model group comparison,
*p < 0.05,
*p < 0.01; With the comparison of puppet operation group,
#p < 0.05,
##p < 0.01
3.2.2 heart infarction model
Compare with puppet operation group, heart infarction group LVSP ,+dP/dtmax and-dP/dtmax all significantly reduces (P < 0.05), LVDP significantly raise (P < 0.05); Compare with heart infarction group, Zacopride group and Captopril group LVSP, + dP/dtmax and-dP/dtmax all significantly raise (P < 0.05), and LVDP significantly reduces (P < 0.05), prompting rat heart contractile function and diastolic function obviously improve.Each indicator difference not statistically significant of Zacopride group and Captopril group (P > 0.05).In Table 5.
Table 5. is respectively organized the comparison (mean ± standard deviation) of hemodynamics index
With the comparison of heart infarction group,
*p < 0.05,
*p < 0.01; With the comparison of puppet operation group,
#p < 0.05,
##p < 0.01
The impact of 3.3 Zacoprides on different rat model myocardial hypertrophies
3.3.1 levothyroxine model
Rat oral gavage gives levothyroxine, after continuous 10 days, plump index (P < 0.01), left ventricular hypertrophy index (P < 0.01) compared with normal matched group obviously increase whole-heartedly, and the success of myocardial hypertrophy model copy is described.5～50 μ g/kg Zacoprides can dose dependent antagonism myocardial hypertrophy, and plump index, left ventricular hypertrophy index all reduce compared with model group whole-heartedly for they.5 μ g/kg are Zacopride ceiling effect dosage (P < 0.01), and its effect is compared there was no significant difference (P > 0.05) with Captopril group; And apply low dosage chloroquine (7.5 μ g/kg, I simultaneously
k1passage relative specificity blocker) can obviously suppress the anti-remodeling ventricle effect of Zacopride, prompting Zacopride may pass through exciting myocardium I
k1passage and bring into play anti-remodeling ventricle effect.The results are shown in Table 6.
Table 6. Zacopride on rat due to levothyroxine sodium whole-heartedly, the impact (mean ± standard deviation) of left ventricular hypertrophy index
Compare with matched group
*p < 0.01; Compare with model group
Δ Δp < 0.01,
Δp < 0.05; Compare with Zacopride 15 μ g/kg groups
##p < 0.01,
3.3.2 isoproterenol prime model
15 μ g/kg Zacopride reversible cardiac hypertrophy induced by isoprenaline, heart size is reduced, plump index, left ventricular hypertrophy index all reduce (P < 0.01) compared with model group whole-heartedly, and its effect has even surpassed Captopril group (P < 0.05); And apply low dosage chloroquine (7.5 μ g/kg, I simultaneously
k1relative specificity blocker) can obviously suppress the anti-remodeling ventricle effect of Zacopride, prompting Zacopride may pass through exciting myocardium I
k1passage and bring into play anti-remodeling ventricle effect.The results are shown in Table 7, Fig. 3.
Table 7. Zacopride on rat due to isoproterenol whole-heartedly, the impact (mean ± standard deviation) of left ventricular hypertrophy index
Compare with matched group
*p < 0.01; Compare with model group
Δ Δp < 0.01; Compare with Zacopride group,
##p < 0.01,
3.2.3 attenuates pressure-overload left model
Result is shown: model group and the comparison of pseudo-operation group, plump index, left ventricular hypertrophy index obviously increase (P < 0.01) whole-heartedly.Compare with model group, Zacopride and Captopril group whole-heartedly plump index, left ventricular hypertrophy index significantly reduce, Zacopride and Captopril group there was no significant difference (P > 0.05).In Table 8.
Table 8. Zacopride to heart infarction rat whole-heartedly, the comparison (mean ± standard deviation) of left ventricular hypertrophy index
With model group comparison,
*p < 0.05,
*p < 0.01; With the comparison of puppet operation group,
#p < 0.05,
##p < 0.01
3.2.4 heart infarction model
Result is shown: heart infarction group and the comparison of pseudo-operation group, plump index (P < 0.01), left ventricular hypertrophy index (P < 0.05) obviously increase whole-heartedly, heart infarction heart hypertrophy in rats is described, hints model copies successfully.Zacopride and Captopril group whole-heartedly, the decrease to some degree of left ventricular hypertrophy index, relatively there is significant difference (P < 0.01) with heart infarction group, Zacopride and Captopril group there was no significant difference (P > 0.05).In Table 9.
Table 9. Zacopride to rats with abdominal aorta coarctation whole-heartedly, the comparison (mean ± standard deviation) of left ventricular hypertrophy index
With the comparison of heart infarction group,
*p < 0.05,
*p < 0.01; With the comparison of puppet operation group group,
#p < 0.05,
##p < 0.01
The impact of 3.4 Zacoprides on levothyroxine rat model left compartment muscle cellular morphology
Under mirror, visible levothyroxine rat model left compartment muscle cell length, width obviously increase; cell volume increases (P < 0.01), and after 15 μ g/kg Zacopride interventions, cellular morphology returns to normally or approach normal level (P < 0.01).The results are shown in Table 10, Fig. 4.
Each experimental group left compartment muscle cellular morphology of table 10. changes (mean ± standard deviation)
Compare with matched group
*p < 0.01; Compare with model group
Δ Δp < 0.01
3.5 heart tissue pathological examinations
3.5.1 heart infarction rat model left compartment muscle Ultrastructural observation
Electronic Speculum result shows, the sarcostyle thickness of pseudo-operation group cardiac muscular tissue relatively evenly, marshalling, light and dark, the clear in structure such as intercalated disc, mitochondrion are visible; Heart infarction group cardiac muscular tissue sarcostyle increases thick, tortuous, disorderly, muscle segment structural fuzzy, and intercalated disc is fuzzy, broadening, and some fractures are discontinuous, and mitochondrion quantity showed increased, irregularly shaped is cavity shape and changes, and the concentric circular/myelin sample that is having changes; Zacopride group and Captopril group ultrastructure have clear improvement compared with model group.See Fig. 5.
3.5.2 heart infarction rat model left ventricle HE coloration result
Pseudo-operation group rat myocardial cell marshalling, form is normal, endochylema clean mark; Heart infarction group cardiac myocyte hypertrophy, degeneration necrosis; Zacopride and Captopril group myocardial cell Mild edema, form normal.See Fig. 6.
3.5.3 heart infarction rat model left ventricle ImmunohistochemistryResults Results
The quantity showed increased of heart infarction group caspase-3 positive cell, prompting apoptosis of cardiac muscle increases; And Zacopride group and Captopril group caspase-3 express obviously reduction, prompting Zacopride and the protective effect of captopril to myocardial cell.See Fig. 7.
Should be understood that, for those of ordinary skills, can be improved according to the above description or convert, and all these improvement and conversion all should belong to the protection domain of claims of the present invention.
Claims (2)
1. Zacopride (Zacopride) is as the application of preparation treatment remodeling ventricle medicine.
2. Zacopride according to claim 1, as the application of preparation treatment remodeling ventricle medicine, is characterized in that described medicine is capsule, microcapsule, liposome, granule, injection, tablet and oral liquid.
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| 李勋 等.自发性高血压大鼠左心室肌细胞动作电位延长的离子机制.《高血压杂志》.2000,第8卷(第1期),第74页摘要. |
| 自发性高血压大鼠左心室肌细胞动作电位延长的离子机制;李勋 等;《高血压杂志》;20000331;第8卷(第1期);第74页摘要 * |
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