Ginsenoside Rgl Regulates TLR4 And Inhibits Myocardial Apoptosis And Fibrosis

Objective:To investigate the therapeutic effects of ginsenoside Rgl on apoptosis and myocardial fibrosis using a mouse model of myocardial fibrosis;H9C2 cardiomyocytes were selected for in vitro studies to observe the effect mechanism of ginsenoside Rgl on inhibition of apoptosis and myocardial fibrosis in relation to the regulation of TLR4 expression.Methods:1.To enrich human cardiac transcriptome sequencing data from the GTEx database according to TLR4 expression grouping using GSEA enrichment analysis method.2.Animal experiment:Selected 30 male C57BL/6 mice randomly divided into control group,isoproterenol(ISO)group and Chinese ginsenoside Rg1 intervention group.The establishment of a myocardial fibrosis model induced by ISO and the effect of ginsenoside Rgl on myocardial histopathology and fibrosis evaluated by HE,Masson,Sirius Red,and immunohistochemistry after group intervention.Apoptotic cell death was evaluated by TUNEL staining;the transcription levels of mRNAs such as TLR4,Bax and Bcl-2 in myocardial tissues were detected by qRT-PCR,and the expression levels of TLR4,Bax and Bcl-2 proteins in myocardial tissues were detected by Western blotting.3.These cells were divided into control group,ISO group,ISO+TAK-242(TLR4 signaling pathway inhibitor)group and ISO+ginsenoside Rg1 intervention group.After the grouping intervention,apoptosis levels were detected by flow cytometry,mRNA transcript levels of TLR4,Bax and Bcl-2 were detected by qRT-PCR,and protein expression levels of TLR4,Bax and Bcl-2 were detected by Western blotting in cardiomyocytes.Results:1.The enrichment analysis of human heart tissue RNA-seq data GSEA indicated that high expression of TLR4 was closely associated with apoptosis as well as the classical pathway of fibrosis,TGF-β.2.Result of HE,Masson,Sirius Red and immunohistochemistry of myocardial tissue showed that myocardial fibrosis was increased in mice of ISO group compared with control group(P<0.05);myocardial fibrosis was significantly decreased in mice of ginsenoside Rg1 intervention group compared with ISO group(P<0.05).Results of TUNEL staining revealed that the content of apoptotic cells in myocardial tissue was significantly higher in the ISO group of mice compared with the control group(P<0.05).In comparison with the ISO group,the content of apoptotic cells in the myocardial tissue of mice in the ginsenoside Rg1 group was significantly lower(P<0.05).After the qRT-PCR results,the mRNA expression of TLR4,Bax,COL-1,and α-SMA was decreased(P<0.05)and that of Bcl-2 was increased(P<0.05)in mice with myocardial fibrosis model after ginsenoside Rg1 intervention.The expression of TLR4,Bax,COL-1 and α-SMA was increased and Bcl-2 was decreased in the myocardial tissue of mice in the ISO group compared with the control group as revealed by Western blotting.Expression of TLR4,Bax,COL-1,and α-SMA proteins in myocardial tissue of mice in the ISO+Rg1 group was significantly lower and Bcl-2 expression was significantly higher in the ISO group compared with the ISO group(P<0.05).3.The results of flow cytometry showed that the level of apoptosis in H9C2 cardiomyocytes was remarkably increased in the ISO group compared with the control group.The apoptotic level of H9C2 cardiomyocytes was significantly decreased in the ISO+Rg1 group compared with the ISO group.The results of qRT-PCR showed that the mRNA expression levels of TLR4 and Bax were increased and Bcl-2 was decreased in H9C2 cardiomyocytes of the ISO group compared with the control group.In the ISO+TAK-242 group,the mRNA expression levels of Bax were decreased and Bcl-2 was increased in H9C2 cardiomyocytes compared with the ISO group(P<0.05),while the mRNA expression levels of TLR4 and Bax were decreased(P<0.05)and Bcl-2 was increased(P<0.05)in H9C2 cardiomyocytes in the ISO+Rg1 group.In H9C2 cells,western blotting showed that TLR4 and Bax expression was elevated and Bcl-2 expression was decreased in the ISO group compared with the control group;Comparing with the ISO group,H9C2 cardiomyocytes in the ISO+TAK-242 group showed reduced protein expression levels of Bax and significantly elevated Bcl-2(P<0.05),and H9C2 cardiomyocytes in the ISO+Rg1 group showed significantly lower protein expression of TLR4 and Bax and elevated Bcl-2 in the ISO group(P<0.05).Conclusion:There is the therapeutic effect of ginsenoside Rg1 to inhibit ISO-induced cardiomyocyte apoptosis and myocardial fibrosis;The effect mechanism of ginsenoside Rg1 to inhibit cardiomyocyte apoptosis and myocardial fibrosis was related to the regulation of TLR4 expression.