Abstract:

Objective To discuss the therapeutic effect of astilbin （AST） in the mice with myocardial infarction（MI）， and to clarify its mechanism. Methods The MI model was established by ligating the left anterior descending artery（LAD） in the male C57BL/6 mice. A total of 50 mice were randomly divided into model group， low dose of AST group（AST-L group） （12.5 mg·kg^-1）， high dose of AST group （AST-H group） （50 mg·kg^-1）， enalapril group （2 mg·kg^-1）， and sham operation group （only thoracotomy without ligation）， and there were 10 mice in each group. Echocardiography was used to detect the left ventricular ejection fraction （LVEF） and left ventricular fractional shortening （LVFS） of the mice in various groups to evaluate cardiac function； Masson trichrome staining， wheat germ agglutinin （WGA） staining and TUNEL staining were used to detect the MI areas， cardiomyocyte sizes and cardiomyocyte apoptosis in the mice in various groups， respectively. In vitro， cobalt chloride （CoCl₂） was used to induce the H9C2 cells to establish the hypoxia injury model. The cells were divided into control group， CoCl₂ group， CoCl₂+low dose of AST（AST-L） group， CoCl₂+medium dose of AST（AST-M） group， and CoCl₂+high dose of AST（AST-H） group. Cell counting kit-8（CCK-8） method was used to detect the survival rates of the cells in various groups； Western blotting method was used to detect the expression levels of apoptosis-related proteins in the cells in various group； immunofluorescence method was used to detect the levels of reactive oxygen species （ROS） in the cells in various groups. In addition， databases including SwissTargetPrediction and PharmMapper were used to predict the targets related to AST and MI； protein-protein interaction （PPI） network was constructed to screen the core targets； Gene Ontology （GO） functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes （KEGG） signaling pathway enrichment analysis were performed； and molecular docking validation was conducted using Autodock software. Results In vivo， compared with sham operation group， the LVEF and LVFS of the mice in model group were decreased （P<0.001）， indicating impaired cardiac function； the Masson staining results showed severe myocardial fibrosis in the mice in model group； the cross sectional area of cardiomyocytes was increased （P<0.05）， the number of TUNEL staining positive cells was increased. Compared with model group， the LVEF and LVFS of the mice in AST-L group and AST-H group were increased （P<0.05 or P<0.001）； the myocardial fibrosis areas of the mice were decreased （P<0.05 or P<0.001）， and the cardiomyocyte cross-sectional areas were decreased （P<0.001）. In vitro， compared with control group， the survival rate of the cells in CoCl₂ group was decreased （P<0.01）， the expression level of B cell lymphoma 2 （Bcl-2） was decreased （P<0.01）， while the expression levels of Bcl-2-associated X protein （Bax） and cleaved cysteinyl aspartate-specific proteinase 3（Cleaved-Caspase-3） proteins were increased （P<0.05 or P<0.01）， and the ROS level was increased （P<0.001）. Compared with CoCl₂ group， the expression levels of Bax and Cleaved-Caspase-3 in the cells in CoCl₂+AST-L， CoCl₂+AST-M and CoCl₂+AST-H groups were decreased （P<0.05 or P<0.01）， the expression levels of Bcl-2 were increased （P<0.05 or P<0.01）， and the ROS levels were decreased （P<0.001）. The network pharmacology analysis results showed that AST had 357 potential targets； after intersecting with MI disease targets， core targets were screened by Degree value， among which epidermal growth factor receptor （EGFR） showed good molecular docking score with AST （binding energy <-8.0 kcal·mol^-1）. The KEGG pathway enrichment analysis results showed that the common targets were mainly enriched in phosphatidylinositol 3-kinase （PI3K）-protein kinase B （AKT） signaling pathway and EGFR signaling pathway， etc. The Western blotting results showed that compared with control group， the expression levels of EFGR， phosphorylated PI3K （p-PI3K） and phosphorylated AKT （p-AKT） proteins in the cells in CoCl₂ group were decreased （P<0.05 or P<0.01）； compared with CoCl₂ group， the expression levels of EGFR， p-AKT， and p-PI3K proteins in the cells in CoCl₂+AST-H group were increased （P<0.01）. Conclusion AST can exert anti-oxidative and anti-apoptotic effects through regulating EGFR/PI3K/AKT signaling pathway， improve cardiac function and myocardial pathological injury in MI mice， and its mechanism is related to regulating this signaling pathway.

Key words: Astilbin, Myocardial infarction, Cobalt chloride, Network pharmacology, Epidermal growth factor receptor signaling pathway