The Protective Effect And Mechanisms Of Yulangsan Flavonoids On Myocardial Ischemia Reperfusion Injury In Rats

Myocardial ischemia reperfusion injury(MIRI)refers to such a phenomenon that reperfusion to myocardial ischemic tissues aggravates the injuried myocardial structure and function.The research on MIRI becomes a focus in recent years.The natural resources of Chinese medicinal materials is very rich in China. It is of great significance to search for the safe and effective herbs for the prevention and treatment of MIRI,particularly for the active components in these herbs and the study of their pharmacodynamics and mechanisms on anti-MIRI,which can help to develop our country's herbal resources and to make the modernization of Traditional Chinese Medicine.The natural resources of Chinese medicinal materials is plentiful in Guangxi province.Yulangsan is a kind of the widely used folk herbs in Guangxi province.Research showed that crude extract of Yulangsan(YLS)could relieve MIRI.It has not been reported whether Yulangsan flavonoids(YF),one of the active components in YLS,has the effect on anti-MIRI.This study is designed to research the protective effect of YF and its mechanisms on MIRI at following levels,in vivo,cultured cardiocyte and molecular or genic level so as to provide the experimental and theoretical basis for developing YF to treat coronary artery disease clinically.Objective:1.To study effect of YF on cardiac hemodynamic in normal rats:To observe the effects of YF on heart rate(HR),left ventricular systolic pressure (LVSP),the maximum ascending rate in left ventricular pressure(+dp/dt_max)and action time in healthy rats with normal ECG.2.To study the protective effect and mechanisms of YF on MIRI in rats:To observe the effects of YF on the cardiac hemodynamic changes,pathological structure and cardiocyte ultrastructure,lipid peroxidation injury,myocardial infarct size,activity of nitric oxide synthase(NOS),gene expressions of ANT1 and Caspase-3,and expressions of Bcl-2 and Bax proteins in myocardial tissues in rats with MIRI.3.To study the mechanism of YF on anti-MIRI by using hypoxia/ reoxygenation(H/R)injury model in neonatal rat myocardial cells cultured in vitro.Methods:1.To study effect of YF on cardiac hemodynamic in normal rats:SD rats with normal ECG were picked out and randomly divided into 3 groups with 10 rats(half male and half female)in each group:the blank group,the normal saline group and the YF group.MS4000 biological signals record and analysis system(MS4000)was used to record HR,LVSP,and +dp/dt_maxby intubation in the carotid artery to the left ventricle.2.To study the protective effect and mechanisms of YF on MIRI in rats: SD rats with normal ECG were picked out and randomly divided into 7 groups with 10 rats(half male and half female)in each group:sham operation(SHAM) group,myocardial ischemia reperfusion injury(MIRI)group,normal saline(NS) group,YF low dose(YFL)group,YF medium dose(YFM)group,YF high (YFH)dose,and positive drug(DIL)group.Different doses of YF were administered into duodenum 30 min before ligation of the left anterior descending coronary artery(LAD)for 30 min and followed with reperfusion for 60 min to establish MIRI model.The following indexes were determined at the end of reperfusion for 60 min.2.1 MS 4000 was used to record HR,LVSP,+ dp/dt_max,the maximum descending rate of left ventricular pressure(-dp/dt_max),left ventricular diastolic pressure(LVDP),left ventricular end-diastolic pressure(LVDEP),and interval from beginning of left ventricular contraction to + dp/dt_max(t-dp/dt_max).2.2 Myocardial ischemic tissues were taken to make histological section and electron microscopic section S.A.for observing the changes of histopathology and ultrastructure of myocardium under optical microscope and transmission electron microscope respectively,and taking the pictures with camera simultaneously.2.3 The blood was collected via the abdominal aorta,centrifuged,and the upper plasma was taken for detecting activities of AST,CK,CK-MB,LDH, LDH1,SOD and GSH-Px,and content of MDA in each rat.2.4 The blood was taken via the abdominal aorta,and centrifuged.The upper plasma was collected for detecting the activity of nitric oxide synthase (NOS),and myocardial infarct size was assessed by TTC staining in each rat.2.5 The myocardial ischemic tissues were taken to extract RNA for detecting mRNA expressions of ANT1 and Caspase3 by RT-PCR.2.6 The myocardial ischemic tissues were taken for determining Bcl-2 and Bax protein expressions by immunohistochemical method.3.The protective mechanisms of YF againist MIRI was studied in rat H/R injury model:Primary cardiac myocytes were cultured in vitro.Cardiocytes with beating well were picked out and divided randomly into following seven groups: control group(Control),positive drug verapamil group(Verapamil),YF high dose medicated serum group(YFH),YF medium dose medicated serum group (YFM),YF low dose medicated serum group(YFL),blank serum group (Blank),and H/R injury model group(Model).H/R injury model was established in each group except control group.YF medicated serum and blank serum were made by injecting YF and NS respectively into duodenum in SD rats with normal ECG.Activity of cardiocytes was detected by MTT after H/R,and the activities of LDH and NOS in culture solution before and after H/R were detected by the test kit,the activities of Na⁺-K⁺-ATPase and Ca²⁺-Mg²⁺-ATPase in cardiocytes after H/R were detected by the test kit,and the morphological changes and apoptosis rate of the cardiocytes were detected by acridine orange staining and Annexin V-FITC/PI double staining respectively after H/R.Results:1.The effect of YF on cardiac hemodynamic in normal rats:Compared with NS group,YF group could decrease HR,LVSP and +dp/dt_maxsignificantly (P＜0.05)at 40 min,50 min,and 60 min after treatment with YF.2.The protective effect and mechanisms of YF on MIRI in rats:2.1 Compared with MIRI group,HR,LVSP,+dp/dt_max,and -dp/dt_maxin YFH,YFM and DIL group increased significantly(P＜0.05),while the LVDP, LVDEP,and t-dp/dt_maxdecreased significantly(P＜0.05).2.2 Myocardial fibers were in disorder condition under optical microscope in MIRI group,in which cellular edema,break or necrosis,infiltration of inflammatory cells,or obvious leakage of red blood cells could be observed.The above injuries could be significantly alleviated in YFH and YFM group.The obvious damages of myocardial fibers,mitochondria and intercalated disks could be observed in MIRI group under electronic microscope,but thses damages were attenuated significantly in YFH and YFM group.2.3 Compared with MIRI group,the myocardial enzyme activity and the content of MDA in the plasma in YFH and YFM groups decreased significantly (P＜0.01),and the activities of SOD and GSH-Px in YFH and YFM groups increased significantly(P＜0.01).2.4 Compared with MIRI group,the myocardial infarct sizes were significantly smaller in YFH and YFM group(P＜0.01),the activities of tNOS and cNOS increased significantly(P＜0.01),and the activity of iNOS decreased significantly(P＜0.01).2.5 Compared with MIRI group,expression of ANT1mRNA increased and expression of Caspase 3 mRNA decreased in YFH significantly(P＜0.05).2.6 Compared with the MIRI group,expression of Bcl-2 protein had no difference in each group(P＞0.05),the expresion of Bax protein and the ratio of Bax protein to Bcl-2 decreased significantly in YFH and YFM(P＜0.05).3.Compared with the model group,the activities of cardiocytes increased significantly in YFM and YFH after H/R.The activities of LDH,tNOS,iNOS, and cNOS had no significant difference among all groups before H/R(P＞0.05); However,compared with Model,the activities of LDH and iNOS decreased obviously(P＜0.05),and the activities of tNOS and cNOS increased obviously (P＜0.05),the activities of Na⁺-K⁺-ATPase and Ca²⁺-Mg²⁺-ATPase increased significantly in YFM and YFH(P＜0.05)after H/R.The cytoplasms of cardiocytes were in red fluorescence,the nuclei in green in YFH and YFM after H/R,cardiocytes in YFH and YFM were denser than that in Model,but inferior to Control.Flow cytometry tests showed that the mortality rate and apoptosis rate of cardiocytes in YFH,YFM and YFL group after H/R decreased significantly compared with those of model group.Conclusion:1.YF,one active component of YLS,has negative chronotropic property and inotropic effect,obviously at about 50 min after treatment with YF.2.YF can protect the heart function and myocardial cellular structure against MIRI,and show a certain dose-effect relationship.Its protective mechanisms may be related to its negative chronotropic property and inotropic effect,anti-oxidative stress injury,increase in activity of cNOS,decrease in activity of iNOS,up-regulation of ANT1 mRNA expression,protect the activities of Na⁺-K⁺-ATPase and Ca²⁺-Mg²⁺-ATPase,and inhibition of cardiocyte apoptosis by down-regulation of Caspase 3 mRNA expression,Bax protein expression and the ratio of Bax/Bcl-2.