| Background and objectiveMyocardial fibrosis?MF?is a common pathophysiological change at the end of the development of hypertension,ischemic heart disease,valvular heart disease and cardiomyopathy.The main pathological basis is the abnormal proliferation of cardiac fibroblasts?CFs?and excessive synthesis and deposition of extracellular matrix,leading to cardiac contraction and diastolic dysfunction,and eventually to severe heart failure,which brings heavy economic burden to patients and society.Therefore,it is of great significance to strengthen the research on the mechanism of myocardial fibrosis and to find effective intervention targets for the prevention and treatment of heart failure.Studies at home and abroad have shown that Wnt/?-Catenin signaling pathway is closely related to myocardial fibrosis.As a receptor of Wnt/beta-Catenin signaling pathway,Lgr5 can activate Wnt/?-Catenin signaling pathway by binding with R-spondin ligand.Therefore,this study proposes the following theoretical hypothesis:Lgr5 activates Wnt/?-Catenin signaling pathway by binding to R-Spondin ligand during the development of myocardial fibrosis,and?-Catenin accumulates in the nucleus and regulates target gene transcription by binding to TCF/LEF,thus participating in the regulation of myocardial fibrosis.In this study,we established an angiotensin II?Ang II?induced fibrosis model of CFs,transfected CFs with Lgr5-siRNA,observed the effects of Lgr5-siRNA on the proliferation,transdifferentiation and collagen synthesis of CFs,and detected the expression of Wnt/?-Catenin signaling pathway-related molecules to explore the role of Lgr5 in regulating Wnt/?-Catenin in myocardial fibrosis.The results of this study are expected to prevent and treat myocardial fibrosis.Diversification and heart failure provide new intervention targets.Methods:1.Primary isolation,culture and characterization of cardiac fibroblastsThe primary isolation and culture of mouse cardiac fibroblasts were performed by trypsin digestion combined with differential adherent method,and the purity of cardiac fibroblasts was identified by vimentin combined with immunofluorescence.The C57BL/6 neonatal mice were born on the 13 day,and the purity of the fibroblasts was determined by trypsin digestion and differential adhesion.2.Ang II-induced myocardial fibroblast cell model in vitroThe effects of different concentrations of Ang II on the proliferation of CFs were detected by CCK8 kit.The expression of collagen I/collagen III protein and mRNA was detected by Western blotting and real-time fluorescence quantitative PCR?RT-PCR?,and the optimal concentration of angiotensin II was found in the cell model of myocardial fibrosis.The experiment was divided into 5 groups:control group:Ang II(1×10-8mol/L)?Ang II(1×10-7mol/L)?Ang II(1×10-6mol/L)?Ang II(1×10-5mol/L).3.The effect of siRNAs Lgr5 on proliferation and transdifferentiation of cardiac fibroblasts induced by Ang?The effects of Lgr5-siRNA on the proliferation and transdifferentiation of cardiac fibroblasts induced by Ang?(1×10-6mol/L)were observed by CCK8 kit and immunofluorescence and Western blotting.The experiment was divided into four groups.:control group?Ang II?Ang II+Lgr5-scRNA?Ang II+Lgr5-siRNA.4.The effect of siRNA Lgr5 on the function of cardiac fibroblasts and the expression of signal molecules related to Wnt/?-Catenin pathway.The model of myocardial fibroblasts induced by Ang?was established in this experiment.Western blotting and real-time fluorescence quantitative PCR were used in the experiment.To observe the effect of Lgr5-siRNA on the secretion function of cardiac fibroblasts and the expression of signal molecules related to Wnt/?-Catenin pathway,The experiment was divided into four groups:control group?Ang II?Ang II+Lgr5-scRNA?Ang II+Lgr5-siRNA.Results:1.Primary isolation,culture and identification of myocardial fibroblastsThe morphology of CFs was irregular,fusiform or ellipsoid under microscope.After 34 days of culture,the cardiac fibroblasts proliferated and fused.Under microscope,the fibroblasts were long fusiform.The expression of Vimentin antigen in mouse cardiac fibroblasts was positive by immunofluorescence,and the positive rate was about 96%.The cultured cardiac fibroblasts have high purity and are suitable for experiment.2.Ang II-induced myocardial fibroblast cell model in vitroThe results of CCK8 showed that the proliferation ability of CFs increased with the increase of Ang?concentration,and the proliferation ability of cells increased at 1×10-6mol/L?1×10-5mol/L after CFs treatment with different concentrations of Ang?for 24hours.The difference was significant?P<0.05?.Real-time fluorescence quantitative PCR?RT-PCR?was used to detect the expression of collagen I/collagen III.Compared with the control group,the concentration of Ang II was significantly different in 1×10-6mol/L?1×10-5mol/L?P<0.05?.Western blotting,Compared with the control group,at1×10-6mol/L?1×10-5mol/L could significantly increase the expression of collagen I/collagen III protein.The difference was statistically significant?P<0.05?.3.The effect of siRNAs Lgr5 on proliferation and transdifferentiation of cardiac fibroblasts induced by Ang?The results of CCK8 showed that compared with the control group,CFs in Ang II group?1 X10-6mol/L?increased significantly in A450 value?P<0.05?.Compared with Ang II group,there was no significant difference in A450 value in Ang II+Lgr5-scRNA group?P>0.05?,but A45050 value in Ang II+Lgr5-siRNA group decreased significantly?P<0.05?.The results of immunofluorescence showed that?-SMA protein immunofluorescencestainingwasnegativeincontrolgroup,?-SMA immunofluorescence staining was positive in Ang?group and Ang?+negative siRNA group.There was no significant difference between the two groups?P>0.05?.Compared with Ang?group,the green fluorescence expression of?-SMA in Ang?+siRNA group was significantly lower?P<0.05?.The results of Western blotting analysis showed that the level of?-SMA protein was in phase with that of immunofluorescence.4.The effect of siRNA Lgr5 on the function of cardiac fibroblasts and the expression of signal molecules related to Wnt/?-Catenin pathway.The results of Western blotting and real-time fluorescence quantitative PCR showed that compared with the control group,Ang?could significantly increase the mRNA and protein expression of collagen I,collagen III,TIMPs,Lgr5,?-Catenin,and decrease the expression of MMPs mRNA and protein.The difference had statistical meaning?P<0.05?.After Lgr5-siRNA intervention,collagen I,compared with that of Ang II group.The expression of collagen I,collagen III,TIMPs,Lgr5,?-Catenin mRNA and protein significantly decreased,MMPs mRNA and increased protein surface.Conclusion:1.Lgr5-siRNA could significantly inhibit the proliferation of myocardial fibroblasts induced by Ang II and the transformation of cardiac fibroblasts into myofibroblasts.2.Lgr5-siRNA may inhibit myocardial fibrosis by down-regulating the Wnt/?-Catenin signal pathway,thereby inhibiting the secretion of collagen I,collagen III,MMPs,to alleviate myocardial fibrosis. |
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