| Background: Salvia Miltiorrhiza Bunge(SM)is commonly used botanical medicine for benefiting myocardial ischemia(AMI).However,the therapeutic mechanisms of SM extract on AMI remain unclear.Recent studies have shown that the changes in cardiac metabolite could affect the host metabolism and contribute to progression of AMI.Objective: This study first explored the protective effect and potential therapeutic mechanism of SM extract on myocardial injury induced by isoproterenol(ISO)through metabolomics.In order to further clarify the effective substance basis and the "multi target multi pathway" mechanism of action of SM extract,liquid chromatography-mass spectrometry and bioinformatics analysis experiments were designed.Using the above experimental results as a starting point,in vitro experiments were conducted to establish rat H9c2 myocardial cell injury,which was validated at the cellular level,providing a scientific basis for the study of Mongolian medicine resources with SM extract as the pharmacological ingredient.Methods:(1)First,SD rats were given different doses of SM extract(0.18,0.9,1.8g/kg)by preventive gavage for 4 weeks,and then ISO(4 mg/kg/d,2 days)was injected intraperitoneally to establish acute myocardial ischemia(AMI)model.Observe the ECG and cardiac tissue pathological changes,and detect the serum myocardial enzymes,myocardial tissue oxidative stress and inflammation-related indicators to evaluate the efficacy of SM extract on ISO induced cardiac injury in rats.At the same time,a cardiac metabolomics method based on UPLC-Q-TOF/MS was designed to clarify the changes of cardiac metabolites and the effects of SM extract on metabolic pathways in rats.(2)The components of SM extract were identified and determined by HPLC-MS/MS to determine its chemical components and lay a foundation for the subsequent potential effective components;Use network pharmacology and molecular docking to screen the potential active components,key targets,and signal pathways of SM extract against MI.The interaction between the main active ingredients and the core target was further verified by molecular docking.(3)The ischemia model was induced by ISO-induced H9c2 myocardial cell injury,and the mechanism of anti-MI effect of SM extract was verified from m RNA level using CCK-8,ELISA,RT-q PCR,and other methods.Results:(1)In vivo experiments showed that SM extract pretreatment could alleviate the myocardial injury caused by ISO,specifically manifested by reducing the myocardial ischemic area,changing the ECG,histopathology and myocardial enzyme abnormalities(CK-MB,CK,LDH,AST(p<0.05)),and reducing the level of pro-inflammatory cytokines(TNF-α、 IL-1 β、 IL-6(p<0.05),inhibit oxidative stress(CAT,GSH-Px,SOD(p<0.05)),especially in the 1.8 g/kg dose group.The results of metabolomics analysis showed that the level of 24 differential metabolites after SM extract treatment was close to that of the normal group.Through the analysis of metabolic pathway,the differential metabolites mainly involved the metabolism of histidine,alanine,aspartic acid and glutamic acid,glycerol phospholipid and glycine,serine,and threonine.Correlation analysis showed that the level of SM extract’s regulatory effect on inflammation and oxidative stress was related to the change of endogenous metabolites.(2)In this study,the chemical components of SM extract were identified.A total of 140 chemical components were identified.Among them,salvianolic acid B has the highest relative content,and there are 18 species with a relative content higher than 1%.Based on the analysis of 18 compounds based on network pharmacology,a total of 613 potential targets were screened,246 disease targets were obtained,and 62 anti-MI targets of SM extract were obtained.Through the construction of protein-protein interaction network and enrichment analysis,SM extract may regulate PI3K-Akt signal pathway,JAK-STAT signal pathway through STAT3、AKT1、PIK3CA、IL6,etc.to affect the occurrence of cellular inflammation and oxidative stress,and then treat MI,showing the characteristics of "multi-component and multi-target",and play a therapeutic role through the interaction between various targets.(3).In vitro experimental results showed that SM extract can reduce the activity of CK,CK-MB,CTnl,and LDH in H9c2 cells treated with ISO.By verifying key targets in key signaling pathways,it was found that SM extract upregulated EGFR,PIK3 CA,AKT1,and Bcl2 in PI3K/Akt and JAK-STAT signaling pathways,and downregulated the expression of JAK2 and STAT3(p<0.05),indicating that SM extract exerts anti AMI effects by regulating PI3 K Akt and JAK-STAT signaling pathways.Conclusion: This study aims to investigate the protective effect and mechanism of SM extract on ISO induced cardiac tissue and H9c2 cell damage in rats through in vitro and in vivo studies.In summary,this study clarified the chemical composition of SM extract and confirmed its clear preventive effect on ISO induced rat AMI model.Its potential effective components include salvianolic acid A,luteolin,salvianolic acid B,and caffeic acid.After entering the body,it regulates metabolic pathways such as histidine metabolism,alanine,aspartic acid,and glutamate metabolism,glycerol phospholipid metabolism,and glycine,serine,and threonine metabolism.Furthermore,it regulates the PI3K/Akt and JAK2-STAT3 signaling pathways to exert cardioprotective effects. |
