Chinese Medicine Extractum Huaiqihuang Inhibits Proliferation Of Rat Mesangial Cells And Connective Tissue Growth Factor Expression Via Tgf-β–smad2 Pathway

Aim Mesangial cells play a key role in the maintenance of glomerular structure and function,mesangial cell proliferation and cell - matrix remodeling may lead to glomerulosclerosis.CTGF is an important fibrotic mediator downstream of TGF-β1.This study is aimed to construct the model of mesangial cell proliferation induced by TGF-β1, and investigate whether Huaiqihuang (HQH) could inhibit TGF-β1- induced proliferation of rat glomerular mesangial cells (GMC) and CTGF expression,and the involvement of Smad and ERK signaling pathways.Method 1.GMC was cultured and pre-incubated for 12h with different concentrations of Huai Qi Huang (0ng/ml, 6ng/ml, 12ng/ml, 18ng/ml),and then exposed to 2ng/ml TGF-β1 for 24h,the cell proliferation was evaluated by MTT assay and cell cycle phases were measured by flow cytometry.2.GMC was exposed to 2ng/ml TGF-β1 for different time(0,1,2,3,6,9,12,24,48h,the protein expression of CTGF was detected through westernblot. 3.GMC was pre-incubated with different concentrations of Huai Qi Huang (0ng/ml, 6ng/ml, 12ng/ml, 18ng/ml) for 12h,and then exposed to 2ng/ml TGF-β1 for 2h,the effect of HQH on TGF-β1-induced protein expression of CTGF was detected through westernblot.4.The signal transduction pathways involved in HQH inhibition of GMC proliferation and CTGF expression were studied through nuclear translocation and westernblot analysis.Result 1.MTT results showed that HQH could significantly inhibit GMC proliferation by 20% at a concentration of 6ng/ml,and the inhibitory rate increased with the increment of HQH concentration, the highest inhibitory rate 60%,was obtained at a concentration of 18ng/ml,which demonstrated that HQH inhibits GMC proliferation on a concentration-dependent manner(P<0.05).2.The percentage of GMC at G0/G1 phase was significantly higher and that at S and G2/M phases were significantly lower after HQH treatment,when compared with the control group(P<0.05).3.Westernblot showed that HQH reduced CTGF protein expression on a concentration-dependent manner.4.TGF- 1 increased phosphorylation of smad2 in 1 hour,which was maintained for 6 hours and then gradually decreased,the total Smad2/3 protein showed no significant change.Smad2 phosphorylation could be blocked by HQH pretreatment.4.TGF- 1 induced ERK1/2 phosphorylation,which was peaked at 2h,without change of total ERK1/2 protein.HQH showed little effect on the phosphorylation of ERK1/2 induced by TGF- 1.5.TGF- 1 increased the nuclear level of phosphorylated Smad2,total Smad2 /3,Smad4,which were blocked by pretreatment with HQH.Conclusion These findings suggest that HQH inhibits messangial cell proliferation and CTGF expression on concentration-dependent manner via reducing Smad2 phosphorylation and nuclear translocation of smad2/3,smad4.