Astragaloside Ⅳ Reduces Myocardial Fibrosis In Rat Through TGF-β1 Smad/NF-κB Signaling

Objective To study the effect of Astragaloside IV(ASIV)on myocardial fibrosis in rats and to observe the changes of TGF-β1 Smad / NF-κB signaling pathway on myocardial fibrosis in rats.Methods In vivo study,forty Sprague-Dawley(SD)rats were randomly divided into four groups,each group has 10 rats: sham group,ISO(isoproterenol)group(ISO 10 mg?kg-1?d-1,i.p.),PRO group(ISO 10 mg?kg-1?d-1,i.p.;PRO 40 mg?kg-1?d-1,i.g.),ASIV group(ISO 10 mg?kg-1?d-1,i.p.;ASIV 40 mg?kg-1?d-1,i.g.).Rats injected ISO for 30 days,while the rats of PRO group and ASIV group received PRO and ASIV simultaneously.Measured heart mass index(HMI)and left ventricle mass index(LVMI)of rats.The collagen deposition in rat heart was observed by Sirius red staining.The expression of TGF-β1 and TNF-α in rat heart was detected by immunohistochemistry assay.procollagen I carboxyterminal propeptide(PICP)and its inhibitor carboxyterminal telopeptide of type I collagen(ICTP)were detected by ELISA.The protein expression of TGF-β1,Smad2 / 3,Smad4,Smad7,TNF-α,p65 and TLR4 in rat heart was detected by western blot.The RNA expression ofα-Smooth muscle actin(α-SMA),type I collagen and type III collagen was determined by PCR assay.In vitro study,primary fibroblasts of neonatal SD rats were isolated by enzymatic digestion.To be passed to 3 generations later,cardiac fibroblasts were randomly divided into 4 groups: control group,TGF-β1 group(10 μg/L),PRO group(TGF-β1 10 μg/L;PRO 2 μM),ASIV group(TGF-β1 10 μg/L;ASIV 100 μM).fibroblasts were treated with or without ASIV for 2 h followed by incubation of TGF-β1 for 24 h.The activity of myocardial fibroblasts was detected by MTT assay.The protein expression of Smad2 / 3,Smad4,Smad7,TNF-α,p65 and TLR4 was detected by western blot.Results In vivo study,compared with the sham group,after ISO administration,(1)The HMI and LVMI of rat heart were increased;(2)The concentration of collagen fibers in the heart tissue of rats was increased;(3)The contents of PICP and ICTP in rats serum were increased;(4)The protein expressions of TGF-β1,Smad2 / 3 and Smad4,TNF-α,p65,TLR4 were increased in the heart tissue,while the protein expression of Smad7 was decreased;(5)The RNA expression of α-SMA,type I collagen and type III collagen in the heart tissue were increased,and the increase of type III collagen was faster than that of type I collagen.The ASIV group compared with the ISO group,(1)The HMI and LVMI of rat heart were decreased;(2)The concentration of collagen fibers in the heart tissue of rats was decreased;(3)The contents of PICP and ICTP in rats serum were decreased.(4)The protein expressions of TGF-β1,Smad2 / 3 and Smad4 TNF-α,p65,TLR4 were decreased in the heart tissue,while the protein expression of Smad7 was increased;(5)The RNA expression of α-SMA,type I collagen and type III collagen in the heart tissue were decreased,and the ratio of type I collagen to type III collagen increased.In vitro study,compared with the contol group,after TGF-β1 administration,(1)The proliferation of neonatal rats myocardial fibroblasts was increased;(2)The protein expressions of Smad2 / 3 and Smad4,TNF-α,p65,TLR4 were increased in the neonatal rats myocardial fibroblasts,while the protein expression of Smad7 was decreased.The ASIV group compared with the TGF-β1 group,(1)The proliferation of neonatal rats myocardial fibroblasts was decreased;(2)The protein expressions of Smad2 / 3 and Smad4,TNF-α,p65,TLR4 were decreased in the neonatal rats myocardial fibroblasts,while the protein expression of Smad7 was increased.Conclusions The results showed that ASIV had the inhibitory effect on myocardial fibrosis,which may be related to down-regulation of TGF-β1 Smad/ NF-κB signaling.