Research On Cyclovirobuxinum D's Mechanism Of Anti-myocardial Ischemia

Cyclovirobuxinum D(abbreviated as Cvb-D) is a new treating cardiovascular disease drug, is developed successfully in recent years in China. It have the function of promoting qi to activate blood and activating meridians to relieve pain, mainly used for the treatment with the syndrome such as thoracic obstruction, arrhythmia, coronary artery heart disease caused by stagnation of vital energy and blood stasis. This research applied the medicine myocardial ischemia animal model and the ligative myocardial ischemia animal model to study Cvb-D' s mechanism of anti-myocardial ischemia. The aim is developed to provide the experiment evidence of the clinical application and new medicine development.First section: Research of Documents I. The Cvb-D studies overviewStudies on the cardiovascular pharmacology of the Cvb-D in recent years is mainly concentrated as following several aspects: (1) strengthen the myocardial contraction force, (2)anti-myocardial ischemia function, (3) anti-arrhythmia function , (4)improvement hemorrheology and hemodynamics, (5)extend the blood vessel and improve microcirculation function, (6)effect on the blood pressure, (7)effect on the active experimental cerebral ischemia, (8) strengthen the ability of bearing the hypoxia, (9) pharmacokinetics research.II. The pathologic physiology foundation of myocardial ischemia?change on hemadynamics rcoronary artery circulation dynamics , active myocardial ischemia and relaxing period complaisance, myocardial consumption of oxygen closely related with blood pressure and cardiac rhythm, autoregulation when being in myocardial ischemia local coronary artery flow rate and cardiac muscle rhythmicitily contraction; ?narrow the coronary artery; ? coronary artery reconstitution; (4) The ability of myocard contraction lowers; ?Function of myocardial relaxation lowers;?changes on myocard electrophysiology; ? variety of blood platelets function and hemorrheology; ?influence of ischemia on myocard metabolism, ?change on myocard morphology, ?distribution and variation of myocardial ischemia. The impairment mechanisms of myocardial ischemia: CD lacks of energy; (2) calcium overload; ?The free radicals is born to increase. In addition, acid poisoning, the cell membrane permeability increment are also a important factor caused myocard damage. I II. Animal model of myocardial ischemiaThe relevant model of myocardial ischemia and myocardial infarction: (l)The electricity stimulation method;(2)The medicine method: ?medicine causes the coronary artery spasm; (2) medicine increase oxygen consumption;(3)Blocking coronary artery :coronary artery ligation method. Otherwise coronry artery saccus pneumaticus and occlusive selective coronary artery intubation can also be used; (4)isolated heart.Second section: Experiment ResearchMethods(1)Effeet on myocardial ischemia rats (caused by pituitr in) plasma SOD, MDA,LDH, CPK activityFifty SD rats were randomly divided into 5 groups: The model group, treatment group (0. 55 mg/ kg, 1.1 mg/kg, 2. 2 mg/kg ), positive control group(Isosorbide Dinitrate, 25 mg/kg).ig, for 30 days. When given medicine finally , the rat was anestheated by pentobarbital sodium and standard II lead electrocardiogram (ECG) was recorded. Pituitrin (1. 5u/kg, 0. lml/lOOg) was injected by vena caudal is in 15 seconds to caused coronary artery spasticity myocardial ischemia and record the ECG change in 15s, lmin, 3min,5min while measuring T wave peak value. When the myocardial ischemia model was replicated, superoxide dismutase (SOD), malonaldehyde (MDA), lactate dehydrogenase (LDH), phosphocreatine kinase (CPK) activity. (2)Effect on myocardial ischemia rats(caused by pituitrin) myocardium and serum NO,NOS, iNOS levelForty-eight SD rats were randomly divided into 6 groups :blank control group, model group, treatment group (0.55mg/kg, 1. lmg/kg, 2.2 mg/kg ), positive control group (the complex prescription DanShen tablet, abbreviated as DanShen). Pituitrin (20u/kg) was injected by intraperitoneal injection to induce myocardial ischemia. 20 minutes later , serum LDH and serum and myocardium tissue homogenate NO, NOS, iNOS level were determined. (3)Effect on myocardial ischemia rats (caused by isoprenaline) serum CPK LD, FFA and myocardium tissue SOD, MDA(pForty SD rats were randomly divided into 5 groups: model group, treatment group(0.55 mg/kg, 1.1 mg/kg, 2. 2 mg/kg ), positive control group(DanShen). Medicine was given for 21 days. Recorded normal standard limbs lead ECG. After that all rats were injected isoprenaline (Iso) by intravenous(1 mg/kg) injection and ECG in all time point (1, 3, 5, 10, 20, 30, 60 min)were recorded. Taking total J point descending mv number (2 J) and the time of recovering to normal as indexes to compare the impairment degree of myocardium. ?Forty-eight rats were randomly divided into 6 groups: blank control group, treatment group(0. 55mg/kg, 1. lmg/kg, 2. 2mg/kg ),positive controlgroup(DanShen). Medicine was given for 21 days.20 minutes later after givenmedicine for the last time all rats were injected Iso (3 mg/kg). Repeatingit in 24h. lh later , determine free fatty acid(FFA), CPK, LDH activity ofserum.?Activity determination of MDA, SOD in serum and myocardium tissue.Experiment program was the same as experiment?, lh later after givenisoprenaline for the second time, determine MDA, SOD activity in serum andmyocardium tissue homogenate.(4)Effect on myocardial ischemia rats(caused by isoprenaIine)myocardiumce11 apoptos i sForty-eight SD rats were randomly divided into 6 groups: blank control group,model group, treatment group(0. 55mg/kg, 1. lmg/kg, 2.2 mg/kg ), positivecontrol group(DanShen). ig. 21d. 30min later after the last time drug wasgiven, iso (3mg/kg) was injected by subcutaneous injection, repeating itin the next day. lh later heart was taken out, stained by hematoxylinand eosin (HE).It was observed by light microscope. End labelling TUNELmethod was used to determined cell apoptosis. The results was expressed byaverage percentage of apoptosis cell to cell sum total.(5) Effect on myocardial ischemia rats' (caused by coronary artery I i gat ion)myocardium SOD, MDA, CPKForty-eight SD rats were randomly divided into 6 groups: model controlgroup, sham operation group, treatment group(0.55 mg/kg,1. 1 mg/kg,2.2mg/kg ), positive control group(DanShen). ig, total 30do 60mins after thelast time medicine was given the rats was was anestheated by 20% urethanethrough intraperitoneal injection. The left anterior descend(LAD)coronaryartery was ligated except for the sham operation group. Two hours before andafter operation the chest lead were recored respctively and to measurethe change about S~T segment. Two hours after operation the heart wastaken out and the cardiac apex tissue was stained by 0. 1% nitrobluetetrazolium(NBT) to compute the myocardial infarction percentage anddetermine CPK, SOD, MDA level by preparing myocardium tissue homogenate.(6)Effect on myocardial ischemia rats ' (caused by coronary arteryI i gat i on) p I asma endothe I i n (ET), ang i otens i n II (Ang II), MDA, PG12, TXA2Forty-eight SD rats were randomly divided into 6 groups: model controlgroup, sham operation group, drug treatment group(0.55 mg/kg, 1. 1 mg/kg, 2. 2mg/kg ), positive control group(DanShen). ig, total 30do Two hours after thelast time drug was given the rats was anestheated by urethane throughintraperitoneal injection. The left anterior descend(LAD)coronary arterywas ligated except for the sham operation group. 2h after coronaryartery ligation, the plasma ET, Ang II, PGI2, TXA2 and its' metabolite6-Keto-PGFia and TXB2 level were determined.(7)Effect on myocardial ischemia rat's (caused by coronary ligation)myocardium cell apoptosisForty-eight SD rats were randomly divided into 6 groups: model controlgroup, sham operation, treatment group (0. 55mg/kg, 1.1mg/kg, 2.2 mg/kg),positive control group (DanShen). ig, total 30d. The rats were anestheated byurethane. LAD coronary artery was ligated excepted by sham operation. Thecardiac apex tissue was taken out and was stained by HE. End labelling TUNELmethod to measure cell apoptosis. The results was expressed by averagepercentage of positive apoptosis cells to cell sum total.(8) Statistical treatment of experimental dataAll data was deal with SPSS11.0 software package, expressed with*±s,every index was compared with intergroup t-test.Results(DEffect on myocardial ischemia rats (caused by pituitrin) plasmaSOD,MDA, LDH, CPK activity?The animal number that ischemia change took place was as follow: therewere 10, 3, 5, 3, 3 rats in model groups, Isosorbide Dinitrate group,0.55rag/kg, 1. lmg/kg, 2.2mg/kg dosage Cvb~D groups respectively. The difference by compared Isosorbide Dinitrate group and drug treatment group with model group via x2 test was notable, and P< 0. 01 or P <0. 05. In the fifteenth second time point of model group, electrocardiogram T wave raised notably (P <0.05). But when in 1, 3, 5 min time point, T wave remarkable lower than normal (P <0. 01), which indicated the cardial ischemia syndrome took place. Isosorbide Dinitrate group and drug treatment group all can inhibit the T wave' s ascending in the fifteenth second time point and T wave' s redounding and at the time of 3 min and 5 min. There were no significance difference on the aspect that improved T wave changes between medicine treatment group. ?Medicine treatment group and Isosorbide Dinitrate group all can improve activity of plasma SOD and reduce MDA content (P<0. 01 or P <0. 05). ?Medicine treatment group and and Isosorbide Dinitrate group all can reduce plasma LDH, CPK content notably (P <0. 01 or P <0.05).(2)Effect on myocardial ischemia rats(caused by pituitrin) myocardium and serum NO,NOS, iNOS level?Twenty minutes after the myocardial ischemia model was replicated successfully by hypophysin, LDH in model group raised significantly (P<0.05), compared with blank control group; LDH in medicine treatment groups cut down obviously(P<0.05 and P<0.05), compared with model group. Serum NO level was reduced obviously (P <0. 01), compared with blank control group, 2.2 mg/kg dosage Cvb-D group' s serum NO content obviously rises (P<0. 05), compared with the model group. (2) Myocardium NO content in model group was reduced obviously compared with blank group (P<0.01), Myocardium NO content in 1. lmg/kg and 2.2 mg/kg group rise obviously, compared with model group (P<0.01). (3)NOS and iNOS content in model group was obviously reduced compared with blank group (P <0. 01), NOS and iNOS content in 1.lmg/kg and 2.2 mg/kg dosage Cvb~D group obviously rised ,compared with the model group (p<0.01 or P <0.05). ?NOS and iNOS content in Model group was obviously reduced compared with blank group (P<0. 01), NOS and iNOS content in 1. lmg/kg and 2. 2 mg/kg dosage Cvb-D group obviously rised compared with model group p <0.01 or P<0. 05.(3)Effect on myocardial ischemia rats(caused by isoprenaline)serum CPK, LD, FFA and myocardium tissue SOD,MDA(DSerum FAA content of rats in all cardial ischemia group was higher than blank group. By compared the model group with blank group, P< 0. 01; the range of raising FFA in 1. 1 and 2. 2mg/kg Cvb-D group to reduce notably. ?Activity of serum CPK, LDH in model group was higher than the normal group notably. The rising of CPK and LDH activity in 1.1 and 2.2mg/kg Cvb-D group is suppressed. Except that CPK and LDH activity in 0. 55mg/kg group was higher than normal group notably, other all groups of activity has already been closed to the normal group's level. ?Rats' myocardium SOD activity in model group was lower notably than the blank Group, but MDA content was improved notably (P<0. 05); descending of myocardium SOD activity and rising of MDA content in 1.1 and 2. 2mg/kg Cvb-D group was suppressed notably, compared with model group (P<0. 05 or P <0.01).(4)Effect on myocardial ischemia rats (caused by isoprenaline)myocardium ce11 apoptos i s(T)HE stain: Model group: irregular dissolving necrosis accompanying with inflammatory cell infiltration under the left ventricle endomembrane myocardium .Some were medium-sized dissolving necrosis, or see the special mess dissolve necrosis. The myocardium close to epicardiumhad not been seen unusually. The blank group: The myocardium fibre shape is normal, had not seen obvious necrosis. The cell nucleus is intact, have not seen inflammatory cell infiltrate. Drug treatment group and DanShen group: The degree of pathological change of every sample myocardial cell necrosis or inflammatory cell infiltrate was not as serous as model group. ?Endlabeling stain(TUNEL method)-.There were few apoptosis cell in blank control group. Apoptosis cell of ischemia left ventricular wall in model group obviously exceeded blank group. There had been seen sporadic apoptosis cell in left ventricular wall among the samples of 1. lmg/kg, 2.2mg/kg dosage Cvb-D group and DanShen group. (5) Effect on myocardial ischemia rats' (caused by coronary artery I i gat ion) myocard i um SOD, MDA, CPK(T)ECG S-T segment in model group ascended obviously. The infarct size was 24.76%. P<0.01 by comparing with sham operation group. ECG S~T segment in 1. lmg/kg, 2.2mg/kg dosage Cvb-D group and DanShen group descended obviously and infarct size decreased with P<0.01 or P<0.05. But these index in 0.5mg/kg dosage Cvb~D group had no significant difference .compared with model group. ?The myocardium SOD activity of the model Group decreased notably and MDA content increased notably, compared with the sham operation group(P<0.01). SOD activity increased and MDA content increased in 1. lmg/kg, 2. 2mg/kg dosage Cvb-D group and DanShen group, compared with the model group(P<0.05 or P<0.01). (3) Myocardium CPK activity of the model group is reduced markedly, compared with the sham operation group (P<0.01). CPK activity increased in 1. lmg/kg, 2. 2mg/kg dosage Cvb-D group and Danshen group (P <0. 05 or P <0. 01). (6)Effect on myocardial ischemia rats' (caused by coronary artery I i gat i on) p t asma endothe I i n (ET), ang i otens i n II(Angll), MDA, PGI2,TXA2 (T)Plasma ET content of the model group is obviously higher than the sham operation group (P <0. 01), which Indicated that when rats were in acute cardial ischemia impairment ET level increased. ET of plasma of acute cardial ischemia rat was reduced obviously, compared with the model group (P<0. 01). ?Angll content of the model is obviously higher than the sham operation group (P<0. 01), which indicated that plasma AngII level increase when the rats was of acute cardial ischemia state. AngII of plasma in1. lmg/kg, 2.2mg/kg dosage Cvb-D group and DanShen group was reduced obviously. There was significant difference compared with model group (P <0.01). (3) Plasma TXA2 level was higher than normal in myocardial ischemia model group. Plasma PGI2 level and PGI2/TXA2 ratio are obviously lower than sham operation group( P<0.05 or P<0.01), which indicated there existed the dysequilibrium of vascular endothelial cell when the rat was of myocardial ischemia state. 1. 1 mg/kg, 2. 2 mg/kg Cvb-D Group the plasma TXA2 level was obviously reduced, and plasma PGI2 level and PGI2/TXA2 ratio increased in 1.lmg/kg, 2.2mg/kg dosage Cvb-D group and DanShen group, compard with the model group(P <0. 05 or P<0. 01).(7)Effect on myocardial ischemia rat's (caused by coronary Ii gat i on)myocard i urn ce11 apoptos i s(D HE stain: There was extensively extremely coagulability necrosis under epicardium (surround nearly about left and right cardiac ventricle or involve by bulk left ventricle ), or medium-sized coagulability necrosis (involve by 1/3-2/3 of left ventricles), or special mess coagulability necrosis in ligature model group. There was myocardium nterstitial blood vessel broaden and congestion in all sample that appeared infarct. The blank control group: The myocardium fibre shape is normal, had not seen obvious necrosis, the cell nucleus was intact and the cardiac muscle had not been seen unusual. Degree of pathological change about myocardial cell necrosis or inflammatory infiltration of every sample in medicine treatment group and DanShen group was less than model group than model inflammation soaking into pathology. (2) The end labeling stain (TUNEL method): There was a extremely small quantity apoptosis cell in blank group , model group lack left room wall of blood can see more apoptosis cell than blank group in left ventricular wall of model group (P<0.01). Sporadic apoptosis cell can be seen in left ventricular wall in medicine treatment group and DanShen group which was obviously less than model group (P<0. 01).ConclusionThis paper applied the medicine myocardial ischemia animal model (caused byisoprenaline or pituitrin) and the ligative myocardials ischemia animalmodel to study Cvb~D' s mechanism of anti-myocardial ischemia. The aim isdeveloped to provide the experiment and theoretical foundation of theclinical application of anti-myocardial ischemia.(DCvb-D can cause the inhibition of T-wave change of rats of cardial ischemiamodel caused by pituitrin, improve model rats' plasma SOD activation, reduceplasma MDA, LDH, CPK content of model rats notably.(2)Cvb-D can cause the inhibition release of the myocardium enzyme (serumenzyme LDH) of cardial ischemia model rats caused by pituitrin, raise themodel rats' serum and tissue NO, NOS and iNOS content notably.@Cvb~D can reduce myocardial ischemia model (caused by isoprenaline) rats'SJ of ECG, shorten ECG resume time , reduce serum free fatty acid (FFA )content, serum CPK, LDH activation, reduce cardiac tissue MDA content, raisethe cardiac tissue SOD activation(4) Cvb-D can lighten myocardial ischemia model (caused by isoprenaline) rats' degree of pathological change .turbulence of myocardial fibre, plasmolysis of putrescence cell, myocardial cell necrosis or inflammatory infiltration. Cvb-D can reduce myocardial ischemia model (caused by isoprenaline) rats' apoptosis myocardial cell notably.(5) Cvb-D can cause the inhibition of the rise of section S-T ECG of myocardial ischemia model (caused by ligature coronary artery) rats notably, reduce rats' area of myocardium infarct of model rats notably, raise cardiac tissue SOD activation , reduce cardiac tissue MDA content, CPK activation notably.(6) Cvb-D can reduce plasma ET, Angll level of myocardial ischaemia model rats (caused by ligature coronary artery) notably, reduce the plasma TXA2 level,increase the plasma PGI2 level and PGI2/TXA2 ratio obviously, improve the...