Effect of proteasome inhibitor on TGF-β1-induced extracellular matrix expression in renal interstitial fibroblasts

Objective:To investigate the effect and its mechanism of proteasome inhibitor on TGF-β1-induced expression of extracellular matrix in rat renal interstitial fibroblasts. Methodology:The rat renal interstitial fibroblasts (NRK/49F cells) were cultured in vitro. The expression of connective tissue growth factor(CTGF),α-smooth muscle actin (α-SMA) and fibronectin (FN),collagen type III (Col III) mRNA were measured by realtime PCR in the NRK/49F treated by MG132,a proteasome inhibitor,in different concentrations and with or without TGF-β1 (5 ng/ml). The cells were also pretreated by MG132(2.5 μmol/L),then were stimulated by TGF-β1 for different time (0 h,6 h,12 h,24 h),the mRNA level of fibrosis-related factors were observed by realtime PCR. The effects of MG132 on the level of FN and signal transduction factors (smads protein) expression induced by TGF-β1 were measured by Western blot. Results:MG132 could directly decrease the mRNA expression of CTGF,α-SMA,FN and Col III. Compared with the control group,as the concentration of MG132 in 0.5,1,2.5 and 5 μmol/L was increased,the mRNA level of CTGF,α-SMA,FN and Col III were decreased. In addition,the expression of CTGF,α-SMA,FN and Col III mRNA was increased by 6.91,2.11,1.39 and 3.60 times after stimulation of 5ng/ml TGF-β1 (P0.05). And compared with 5 ng/ml TGF-β1 group,CTGF mRNA decreased by 3.30,2.84,1.06,0.74 times,α-SMA mRNA decreased by 0.50,0.31,0.28,0.19 times,FN mRNA decreased by 0.80,0.67,0.55,0.37 times,and Col III mRNA decreased by 0.57,0.35,0.28,0.05 times (P0.05) in MG132 (0.5,1,2.5 and 5 μmol/L) pretreated groups. MG132 could decrease the mRNA level of fibrosis-related factors which were induced by TGF-β1 at each time point. TGF-β 15ng/ml could significantly increase the phosphorylation of Smad 2,Smad 3 protein in a time-dependent manner,with the optimal time course at one hour. MG132 could downregulate the p-Smad 2,p-Smad 3,and FN protein expression compared with TGF-β1 group. No changes in the Smad 2/3 protein expression were observed in all groups. Conclusion:MG132 could reduce the mRNA expression of fibrosis-related factors. It could inhibit fibrosis induced by TGF-β1 via Smad signaling pathway. It has a potent effect on counteracting the renal fibrosis.