Study On The Intervention Effect And Mechanism Of Kanli Granule On Myocardial Remodeling Based On Runx2 Gene

Purpose:1.Establish a myocardial remodeling model of H9c2 and explore the intervention effect of Kanli particles on myocardial remodeling;2.Establish Runx2 gene knockout and overexpression H9c2 stable cell model,and explore the effect of Runx2 on myocardial remodeling and the effect of Kanli particles on Runx2 protein expression.Methods:1.Stimulate H9c2 cardiomyocytes with 100 n M Ang II to establish a myocardial remodeling cell model,and observed the morphology of H9c2 at 24 h,48h and 72 h.Analyzed the protein expression levels of ANP,BNP,?-MHC,MMP-1,MMP-9 and TIMP-1 in order to determine the optimal modeling time of myocardial remodeling.2.Through the cytotoxicity test,determined the non-toxic effective concentration range of Kanli Granule.And determined the low,medium and high concentrations of the medicinal particles in the effective range.And set up a blank control group,Ang II(model group),low concentration of Kanli Granule(Ang II+low concentration of Kanli Granule),medium concentration of Kanli Granule(Ang II+medium concentration in the granule),high concentration of Kanli Granule(Ang II+high concentration of Kanli Granule),in order to observe the intervention effect of Kanli particles on myocardial remodeling and the effect on Runx2,and to determine the optimal drug concentration of Kanli particles.3.Establish Runx2 gene knockout and its overexpressed stable cell line.Three cell models of normal H9c2 cells,Runx2 knockout stably transfected cell line and Runx2 gene overexpressing stable cell line are used as the research object.Three kinds of cell models were stimulated by Ang II to simulate myocardial remodeling.The optimal concentration of Kanli particles was used as an intervention to explore the effect of Runx2 on myocardial remodeling and the effect of Kanli particles on Runx2.Results:1.After Ang II stimulates cardiomyocytes,the cell volume became larger,and the expression of key indicators of myocardial remodeling ANP,BNP,?-MHC,MMP-1,MMP-9,TIMP-1 was increased,especially in 48 hours.At the same time,Ang II up-regulated the expression level of Runx2 protein,which was most obvious at 48 hours.2.The results of cytotoxicity experiments indicated that the non-toxic effective dose of the granules was between 0-100 ug/ml and the cell survival rate was above90%.3.Kanli particles reduced ANP,BNP,?-MHC,MMP-1,MMP-9,TIMP-1 protein levels,inhibited myocardial remodeling,and improved part of cardiac function.Moreover,the expression of Runx2 protein was significantly reduced,and the effect was most significant at a concentration of 25 ug/ml.4.Construction of Runx2 knockout stable cell line: DNA sequencing results showed that the DNA sequence of Runx2 was double-peaked after the target site repeat,and the Western blot showed that Runx2 had no protein expression,which proved the successful construction of Runx2 knockout stable cell line.5.The Runx2 gene overexpression stable cell line was constructed: RT-q PCR showed that the protein expression level of Runx2 was increased by more than 10 fold,and Western blotting showed that the expression of Runx2 protein was significantly increased,which proved that the stable cell line overexpressing Runx2 gene was successfully constructed.6.After the Runx2 gene knockout,the expression of BNP,MMP-1,MMP-9,TIMP-1 protein was not significantly decreased after intervention with Kanli granules;Runx2 knockout was compared with normal H9c2 cells.After that,the MMP-9/TIMP-1 ratio was decreased(P<0.05).7.After overexpression of Runx2 gene,compared with the model group,the expression of BNP,MMP-1,MMP-9,TIMP-1 protein was significantly decreased after the intervention of traditional Chinese medicine;The overexpression of Runx2 compared with normal H9c2 cells,the ratio of MMP-9/TIMP-1 was increased(P<0.05).Conclusion:1.Use Ang II stimulated H9c2 cells for 48 hours could establish the myocardial remodeling to a certain extent,and it up-regulates Runx2 protein expression,which plays a role in promoting myocardial remodeling.2.Kanli particles can alleviate the effect of Ang II on myocardial remodeling and reduce Runx2 protein expression.3.After the Runx2 gene knocked out,the degree of myocardial remodeling can be alleviated,and the intervention of the granules is not good.4.After overexpression of Runx2 gene,the degree of myocardial remodeling is aggravated,and the intervention of Kanli granule is significant.5.Kanli granules have a good effect on CHF.The mechanism may be to reduce myocardial remodeling and improve heart failure symptoms by reducing Runx2 expression in cardiomyocytes.