Role Of Rac1b In AngⅡ-induced Atrial Fibrosis In Rats

Objective:To investigate the effect of Rac1b overexpression on atrial fibrosis under AngⅡ stimulation by constructing a rat model of Raclb gene overexpression SM22α-Rac1b-TG,and to explore new ideas for clinical treatment of AFMethods:The research ideas were divided into in vivo and in vitro parts of rats1,Vivo partThe AngⅡ pump was implanted subcutaneously on the back of 8-week-old wild-type(WT)male SD rats and SM22a-Rac1b-TG rats to construct an AngⅡ-induced rat atrial fibrillation model and set up a corresponding control group.Masson staining of rat left atrium tissue was used to evaluate the collagen fiber content of left atrium tissue.The expression of Rac1b gene in left atrium tissue of rats in each group was evaluated by real-time fluorescence quantitative PCR.Rac1b and Rac1b in rat left atrium were evaluated by Western blotting.Expression of type I collagen fibers and α-SMA protein to evaluate atrial fibrosis in rats2.Vitro part1.Isolation and culture of fibroblasts from primary wild-type(WT)and SM22α-Rac1b-TG rat suckling rats in vitro,which were divided into two groups:control group and AngⅡ intervention group.Western blot and real-time quantitative PCR were used.Evaluation The expression of Rac1b,type I collagen fibers,and α-SMA protein in rat cardiac fibroblasts was evaluated2.Isolate WT suckling mouse heart fibroblasts and divide them into three groups of transfected virus:no-load virus group,V12 virus group,Rac1b virus group,and then evaluate the apoptosis of the three groups of cells by flow cytometry,and pass CCK-8 experimental analysis of the three groups of cell proliferation3.Isolate cardiac fibroblasts from wild type(WT)and SM22α-Rac1b-TG rat suckling rats,and analyze the effect of Rac1b on the migration ability of cardiac fibroblasts through cell scratch testResults:1.The area of atrial collagen fibers in the WT AngⅡ group was significantly higher than that in the control group,with a statistical difference(P<0.01)The area of atrial collagen fibers in SM22α-Rac1b-TG AngⅡ group was significantly higher than that in WT AngⅡ group(P<0.01)2.Compared with the control group,the expression of Raclb protein was increased in the WT AngⅡ group in the atrial tissue(P<0.001),and the expression of fibrosis-related protein CollagenI and α-SMA was significantly increased(P<0.01)Compared with WT mice,SM22α-Rac1b-TG and WT mice showed higher expressions of chemically-related mRNAs(P<0.01)3.At the cell level,the expression of Rac1b protein was increased in the WT AngⅡgroup compared with the control group(P<0.001),and the expression of fibrosis-related proteins CollagenI and α-SMA was significantly increased(P<0.01).The expression of related mRNA was increased.Compared with WT mice,SM22α-Rac1b-TG and WT mice showed higher expression of CollagenI and α-SMA mRNA(P<0.01)4.Flow cytometry experiments showed that the percentage of apoptosis in the Raclb virus group was less than that in the no-load virus group(P<0.001);CCK-8 experiments showed that the Rac1b virus group had a higher proliferation ability than the no-load virus group(P<0.001)5.Cell scratch test showed that SM22α-Rac1b-TG mouse heart fibroblasts had higher migration ability than WT mice(P<0.001)Conclusion:Rac1b promotes AngⅡ-induced atrial fibrosis in rats.