Fuzheng Huayu Recipe Regulates The Cellular And Molecular Mechanism Of MiR-29b-5p Targeting TGF-?/smads Pathway Against Myocardial Fibrosis

Objective:to explore the possible cellular and molecular mechanism of Fuzheng Huayu recipe against myocardial fibrosis from the point of view of miR-29b-5p targeting TGF-?/smads signaling pathway regulating CFs proliferation,apoptosis and ECM metabolism.Methods:(1)Neonatal rat cardiac fibroblasts were isolated from rats by differential adhesion method.The proliferation,apoptosis,ECM metabolism and the expression of miR-29b-5p and TGF-?/Smads in CFs stimulated by Ang ? were observed.And the intervention effect of Fuzheng Huayu recipe.The morphology of CFs cells was observed under inverted microscope,the proliferation of CFs was detected by CCK-8 assay,and the apoptosis of CFs was detected by flow cytometry.The protein andmRNA expression of type? collagen,type ? collagen,MMP2,MMP9,TIMP1 and TIMP2were observed by Elisa and qPCR,and the expression of miR-29b-5p was observed by qPCR method.The expression of TGF-?1,smad2,p-smad3,smad4 and smad7 was detected by Western blot.(2)MiR-29b-5p mimics/miR-29b-5p inhibitor was transfected into CFs,and the model of miR-29b-5p overexpression/expression suppression was established,and the miR-29b-5p inhibitor model was interfered with Fuzheng Huayu recipe.The expression of miR-29b-5p,TGF-?1,smad2,p-smad2,smad3,p-smad3,smad4 and smad7 and the intervention effect of Fuzheng Huayu recipe were observed.The detection method is the same as above.Results:(1)Compared with blank control group,the proliferation of CFs in DMSO groupwas no significant change;compared with blank control group,the proliferation of CFs in Ang? group was significantly increased(P<0.01);compared withAng? group,the proliferation of CFs in different concentrations of FZHY group was decreased significantly(P<0.05 or P<0.01)in a dose dependent manner.Compared with blank control group,the early apoptosis rate and total apoptosis rate in Ang ? group were decreased significantly(P<0.01);compared with Ang ? group,the early apoptosis rate and total apoptotic rate of FZHY group were increased(P<0.01).(2)Compared with blank control group,the contents and the mRNA expression of Col?,Col ?,MMP2,MMP9,TIMP1 and TIMP2 in Ang ? group were increased(P<0.05 or P<0.01),the ratio ofMMP2/TIMP2 and MMP9/TIMP1 had decreased tendency;compared with Ang? group,the contents and the mRNA expression of Col ?,Col ?,MMP2,MMP9,TIMP1,TIMP2 and the ratio of MMP2/TIMP2 were decreased(P<0.05 or P<0.01),and the ratio of MMP9/TIMP1 had decreased tendency.(3)Compared with blank control group,the expression of miR-29b-5p in Ang ? group was significantly decreased(P<0.05);compared with Ang ? group,the expression of miR-29b-5p in FZHY group was significantly increased(P<0.05).Compared with blank control group,the expression of TGF-?1,smad2,p-smad2,smad3,p-smad3,smad4 and smad7 in DMSO group had no significant change;compared with blank control group,the expression of TGF-?1,smad2,p-smad2,smad3,p-smad3,smad4 in Ang ? group was significantly increased,while the expression of smad7 in Ang ? group was significantly decreased(P<0.05 or P<0.01);compared with Ang? group,the expression of TGF-?1,p-smad2,smad3 and p-smad3 was significantly decreased(P<0.05 or P<0.01),the expression of smad2 and smad4 had decreased tendency,and the expression of smad7 had increased tendency.(4)Compared with blank control group,the proliferation of CFs inmimics negative control(mimics NC)group and inhibitor negative control(inhibitor NC)grouphad no significant change;compared with mimics NC group,the proliferation of CFs intransfected miR-29b-5p mimics(miR-29b-5p overexpression)group was significantly decreased(P<0.05);compared with inhibitor NC group,the proliferation of CFs intransfected miR-29b-5p inhibitor(miR-29b-5pexpression-inhibited)group was significantly increased(P<0.01).Compared with blank control group,there was no significant change in the expression of TGF-?1,smad2,p-smad2,smad3,p-smad3,smad4 and smad7 in mimics NC groupand inhibitor NC group;compared with mimics NC group,the expression of TGF-?1,smad2,p-smad2,smad3,p-smad3,smad4 in miR-29b-5p mimics group was significantly decreased(P<0.05 or P<0.01),and the expression of smad7 was significantly increased(P<0.01);compared with inhibitor NC group,the expression of TGF-?1,smad2,p-smad2,smad3,p-smad3 in miR-29b-5p inhibitor group was significantly increased(P<0.05 or P<0.01),the expression of smad7 was significantly decreased(P<0.01),and smad4 had increased tendency.(5)Compared with blank control group,the expression of TGF-?1,smad2,p-smad2,smad3,p-smad3,smad4 and smad7 in inhibitor NC group had no significant change;compared with inhibitor NC group,the expression of TGF-?1,smad2,p-smad2,smad3,p-smad3,smad4 in miR-29b-5p inhibitor group was significantly increased,but the expression of smad7 was significantly decreased(P<0.05 or P<0.01);compared with miR-29b-5p inhibitor group,the expression of TGF-?1,smad2,smad3,smad4 in FZHY group was significantly decreased,the expression of smad7 was significantly increased(P<0.05 or P<0.01),p-smad3 and p-smad2 had decreased tendency.Conclusion:The cellular and molecular mechanism of Fuzheng Huayu recipe against myocardial fibrosis may be related to its targeted miR-29b-5p regulation of TGF-?/smads signaling pathway mediated CFs proliferation,apoptosis and matrix metabolism.