Study Of Crosstalk Mechanisms Between CCL₇ And TGF-?₁ Induced Cardiac Fibrosis

Background:With the advent of the aging population and the prevalence of unhealthy lifestyles,the incidence of cardiovascular diseases is increasing year by year,and chronic heart failure is the common end stage performance of various cardiovascular diseases.Myocardial fibrosis?MF?is an important pathological basis for the development of chronic heart failure.With stimulated by hypertension,myocardial infarction and other pathological factors,Cardiac fibroblasts?CFs?are activated and transformed into myofibroblasts,which secrete excessive extracellular matrix?ECM?,leads to the imbalance of collagen ratio.Growth factorTGF-?1 is an important factor regulating myocardial fibrosis,which transduces signal through the intracellular protein smad 2/3 and ERKl/2.In the study of mouse epidermal fibrosis,both TGF-?1 and CCL₇ can promote fibrosis and have a cross-regulatory effect.The latest research has found that the amount of CCL₇ expression increases during aldosterone induced myocardial fibrosis.However,whether CCL₇ promotes the development of myocardial fibrosis and whether there is interaction with TGF-?1 remain unclear.Objective:The purpose of this study is to investigate the signal pathway of myocardial fibrosis induced by CCL₇,and to explore the interaction mechanism between CCL₇ and TGF-?1.Methods:Cardiac fibroblasts?CFs?were obtained by enzyme digestion and differential adherent method from 10 SD rats born 1-3 days after birth.The fibroblasts were identified by immune fluorescence with vimentin,FSP-1 and troponin antibodies.The best intervention concentration of TGF-?1 and CCL₇ was detected by Western Blot.Screening of viruses with down-regulation of CCL₇ or TGF-?1 by Elisa.The study includes the following groups:normal control group,TGF-?1 groups,TGF-?1+virus group,TGF-?1+CCL₇ siRNA virus group,CCL₇ group,CCL₇+virus group,CCL₇+TGF-?1 siRNA virus group.Each the group were transfected with corresponding virus,then given drug intervention.The mRNA expression of type ?and III collagen was detected by RT-PCR.the phosphorylation level of signal protein Smad2/3 and ERK1/2,type I collagen and type III collagen were detected by Western blot.was used to detect.Cell proliferation was detected by CCK8.Results:Cultured cardiac fibroblast cells were identified high purity of cardiac fibroblasts,with the expression of vimentin and FSP-1 is more than 95%,troponin T is less then 3%.Western Blot test showed the best intervention concentration of TGF-?1 was 4ng/ml,and CCL₇ was 200ng/ml.Elisa assay showed that TGF-?1-siRNA3 and CCL₇-siRNAl had the highest down-regulation efficiency.The phosphorylation levels of smad2/3 or ERK1/2,col ?,col ? mRNA expression and protein expression and the cell proliferation level were higher in TGF-?1 group than those in the control group?P<0.05?.However the phosphorylation levels of smad2/3 or ERK1/2,col ?,col? mRNA expression and protein expression,and the cell proliferation level were lower in TGF-?1+ CCL₇ siRNA group than those in the TGF-?1group?P<0.05?.The phosphorylation levels of smad2/3 or ERK1/2,col ?,col? mRNA expression and protein expression;and the cell proliferation level were higher in CCL₇ group than those in the control group?P<0.05?.But the phosphorylation levels of smad2/3 or ERKl/2,col ?,col? mRNA expression and protein expression,and the cell proliferation level were no significant diffierent between CCL₇+TGF-?1siRNA group and those in the CCL₇ group?P>0.05?.Conclusion:First,CCL₇ plays a key role in the signal pathway of TGF-?1 induced myocardial fibrosis.Down-regulation of CCL₇ can improve myocardial fibrosis induced by TGF-?1.Second,CCL₇ can cause myocardial fibrosis independently,and works through the intracellular signal protein smad 2/3 and ERK 1/2.