Study On The Material Base And Its Mechanism Of Shexiang Tongxin Dropping Pill On PI3K/Akt Signal Transduction Pathway To Myocardial Ischemia

Objective Shexiang Tongxin Dropping Pill?STP?is a well-known traditional Chinese medicine that is widely used for the treatment of ischemic heart disease?IHD?,although its mechanisms remain unclear.To observe the effects and active ingredients of Shexiang Tongxin Dropping Pill?STP?on PI3K/Akt signal transduction pathway in ischemic heart disease,so as to discuss the effect of STP on cardiomyocytes apoptosis,further to partly explain its mechanism and effective material basic in the treatment of myocardial ischemia.Methods1.Protective effects of Shexiang Tongxin Dropping Pill on pituitrin-induced acute myocardial ischemia in rats.SD rats were equally divided into five groups:control group,model group,positive group?Isosorbide mononitrate,4.0 m g/kg?,STP groups?42,21 mg crude drug/kg?dose group.After one week administering,pituitrin solution was injected into the abdominal cavity to induce myocardial ischemia in rats.Then the blood and myocardial tissue of rats were taken from each treatment group for the next using.ST-segment elevation,biochemical parameters creatine kinase-MB?CK-MB?and lactate dehydrogenase?LDH?activity were measured.Following heart excision,histological analysis by HE and TUNEL staining and levels of mRNA and protein expression of PI3K,Akt,Bax and Bcl-2 were determined by qRT-PCR and Westen Blot respectively.2.Protective effects of Shexiang Tongxin Dropping Pill against Na₂S₂O₄-induced Hypoxia-Reoxygenation injury in H9c2 cell.Ischemic myocardial cell injury was detected with MTT to optimize its best dose-effect and time-effect relationships of Na₂S₂O₄-induced myocardial apoptosis and to determine effective intervention time and dose of STP.H9c2 cells were randomly divided into control group,model group,STP group.After the pretreatment of STP for 12 h,all the groups were added 1 mmol/ml Na₂S₂O₄ solution for 4 h except control group.Then the cells were reoxygenated under normoxic conditions for 6 hours.The activity of cardiomyocytes was determined through MTT,cardiomyocytes apoptosis were detected by Hoechst staining and apotosis rates were measured by flow cytometry.The mRNA and protein expression levels of PI3K,Akt,Bax and Bcl-2 were measured by qRT-PCR and Western Blot.3.The pharmacodynamic chemical basis of STP was studied by using the pharmacochemistry method which was the coupling technique of HPLC-Q-TOF-MS/MS and GC-MS to analyze the effective substances in STP.First,the chromatographic method should be established and the main constituents in STP should be identified.Then,to study the active compounds in STP,cardiac myocytes in the Na₂S₂O₄-induced hypoxia/reoxygenation?H/R?injury were treated with some compounds of STP,and MTT assay was used to evaluate cell viability.Finaly,High-content cytometers were used to detected the cardiomyocytes apoptosis by Hoechst stainingResults1.Our results demonstrated that STP treatment protected against ST elevation and reduced the serum levels of CK-MB and LDH activity following acute myocardial infarction.In addition,STP treatment restored the histopathological change following PTT induced myocardial ischemia,and resulted in the down-regulated expression of Bax and up-regulated expression of Bcl-2,PI3K and Akt in myocardial tissue.The present study demonstrates the cardioprotective ability of STP in a rat model of myocardial ischemic injury,which can be attributable to its anti-apoptotic properties.2.The results indicated that STP pretreatment could significantly increased cells viability and inhibited morphological changes on H9c2 cardiomyocytes induced by Na₂S₂O₄.STP pretreatment could attenuate hypoxic damage and resulted in the up-regulated expression of Bax and up-regulated expression of Bcl-2,PI3K and Akt in H9c2 cell.Finding of the present study suggest that STP was cardioprotective in hypoxia-reoxygenation injury in H9c2 cell.The mechanism might involve protections of the cell membrance from hypoxic damage and of the cells via inhibition of cellular apoptosis.3.By optimizing chromatographic and mass spectrometer conditions of HPLC-Q-TOF-MS/MS and analyzing the obtained data,we identified 41 compounds including ginsenosides,bufadienolides,cholic acids and some other constituents in STP.By searching the data obtained from GC-MS in NIST 11 database,17 volatile compouds were identified.Though MTT and High-content cytometers assay,the result showed that ginsenoside Rgl,ginsenosides Rg3,borneol,isoborneol,muscone and so on could protect cardiac myocytes in the hypoxia/reoxygenation?H/R?injury.ConclusionShexiang Tongxin Dropping Pill can block up PI3K/Akt signal transduction pathway of inducing myocardial cell apoptosis,reduce apoptosis quantity of cardiomyocytes,improve morphosis of cardiomyocytes,inhibit postpone degeneration of cardiomyocytes,help cardiomyocytes to recover,which may worked by the active continuents,such as ginsenosides,bufadienolides,cholic acids and so on.The elucidation of the effective substances of STP provided helpful chemical information for further pharmacology and active mechanism research.