The Research On Wenxinkeli Inhibiting Cardiac Hypertrophy By Regulating Cardiomyocytes Autophagy

Objectives We aimed to elucidate the role of cardiomyocytes autophagy and its regulatory mechanisms by Wenxinkeli（WXKL） in subjected to hypertrophy.Methods We established H₉C₂ myocardial as experimental subjects. The first part of the experiment is divided into 8 groups, namely, Control group, EBSS group, AngII group,AngII + 1g / LWXKL group, AngII + 1g / LWXKL group, AngII + 5g / LWXKL group,AngII + 10 g / LWXKL group. Using immunofluorescence to detect cytoskeletal proteins,as well as self-expression of autophagy marker proteins. Using Western Blot technique to detect the expression of LC3- Ⅱ protein and mTOR protein. Transmission electron microscopy was applied to examine the ultrastructural changes. CellTiter-Glo? uminescent Cell Viability Assay was used to detect the content of ATP. Flow cytometry was used to detect apoptotic cardiomyocytes. Fluorescence microplate reader was used to detect myocardial cell fluorescence intensity. The second part of the experiment is divided into three groups, namely, Control group, overexpression CaN group, silence CaN group, in each group were added AngII, WXKL, Rapa, 3-MA drugs. Use the following methods to detect, first constructed cell lines overexpressing and silence; CellTiter-Glo? uminescent Cell Viability Assay was used to detect the content of ATP. Flow cytometry was used to detect apoptotic cardiomyocytes. Fluorescence microplate reader was used to detect myocardial cell fluorescence intensity.Results 1. Compared with the control group, AngⅡ group can increase the length and width of normal cells, while 3-MA group and the group of Wenxinkeli can be returned to normal cell morphology（P<0.05）. Ang Ⅱ group can make autophagy marker protein fluorescence enhancement than the control group, while the 3-MA group and the group of Wenxinkeli would decrease the intensity of fluorescence.2.Compared to the control cardiomyocytes, AngII-treated cardiomyocytes showed markedly increased the expression LC3II/I, but decreased the expression of mTOR. While the 3-MA and Wenxinkeli could downregulate the expression of the amount of LC3- Ⅱprotein AngⅡ induced upregulation and mTOR protein expression（P <0.05） the presence of autophagosome. And after Wenxinkeli intervention, cardiac muscle cells and mitochondrial filaments arranged neatly law, no autophagosome.4.Compared with control group, Ang Ⅱ group can make the content of ATP decreased, while Wenxinkeli group increased the content（n = 3, P <0.01）. Silence CaN significantly increased the content of ATP（n = 3, P <0.05）, over-expressedCaN can decrease significantly the content of ATP, and after adding Wenxinkeli to be that the content was increased（n = 3, P <0.05）.5.Compared with the control group, AngⅡgroup can make cardiomyocytes apoptosis rate increased, while Wenxinkeli can reduce AngⅡ induced apoptosis of myocardial（n =3, P<0.05）. Silencing CaN decreased the rate of apoptosis significantly, while over-expressCaN increased the rate of apoptosis. Wenxinkeli group can reduce the rate of apoptosis by each group.（n = 3, P <0.05）.6. Compared with the control group, AngⅡgroup increased autophagic fluorescence value and the number of autophagosome. The Wenxinkeli group could make it decrease（ n= 3, P <0.05）. Compared with the normal group,Silence CaN group dsecreased autophagic fluorescence value and the number of autophagosome.. OverexpressionCaN increased autophagic fluorescence value and the number of autophagosome. And after Wenxinkeli intervention, each group could decrease the autophagy value and the number of autophagosome.（n = 3, P <0.05）.Conclusions 1.Wenxinkeli reversal AngII induced cardiac hypertrophy may be correlated with the inhibiting mTOR activity, decreased LC3 II expression, improve cardiac autophagy,increased ATP production, inhibition of apoptosis of myocardial cells.2.Overexprese CaN increased the levels of autophagy, increased apoptosis. After Wenxinkeli intervention can reduce the level of apoptosis and autophagy, and its mechanism may be to some extent with the CaN-NFAT signaling pathway.