蛋白酶体抑制剂抑制肾间质成纤维细胞细胞外基质表达及其机制

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

目的:本研究旨在观察蛋白酶体抑制剂MG132对转化生长因子β1(TGF-β1)刺激下的大鼠肾间质成纤维细胞的细胞外基质表达的影响并初步探讨其机制。方法:体外培养大鼠肾间质成纤维细胞(NRK/49F),利用实时荧光定量PCR技术(Real-time PCR)分别观察MG132不同浓度直接刺激细胞和对TGF-β1(5ng/ml)诱导下产生的结缔组织生长因子(CTGF)、α平滑肌肌动蛋白(α-SMA)、纤维连结蛋白(FN)和III型胶原(ColIII)mRNA表达的影响。应用相同浓度的MG132(2.5μmol/L)预处理后,再加以TGF-β1分别刺激不同时间(0h,6h,12h,24h),同样用Realtime PCR检测上述因子的mRNA水平。利用Western blot观察MG132对TGF-β1诱导的FN和Smads蛋白表达的影响。ELISA方法观察MG132对TGF-β1诱导的FN蛋白分泌的影响。结果:MG132直接作用NRK/49F细胞即可明显降低CTGF、α-SMA和FN、ColIII的mRNA表达水平,随着MG132浓度(0.5、1、2.5、5μmol/L)增大,CTGF的mRNA表达均下降,分别为对照组的65%、46%、17%和20%;α-SMA的mRNA表达均下降,分别为对照组的10%、7%、4%和3%;FN的mRNA表达均下降,分别为对照组的54%、42%、25%和24%;ColIII的mRNA表达均下降,分别为对照组的30%、12%、5%和1%(均为P<0.05)。5ng/mlTGF-β1分别使CTGF、α-SMA和FN、ColIII mRNA表达增加为对照组的6.91、2.11、1.38和3.60倍(P<0.05),用MG132不同浓度(0.5、1、2.5、5μmol/L)预处理后,CTGF mRNA均下降,分别为对照组的3.30、2.84、1.06和0.74倍,α-SMA mRNA均下降,分别为对照组的0.50、0.31、0.28和0.19倍,FN mRNA均下降,分别为对照组的0.80、0.67、0.55和0.37倍,ColIII mRNA均下降,分别为对照组的0.57、0.35、0.28和0.05倍。MG132在各时间点均能使TGF-β1所引起的纤维化相关因子的mRNA表达水平下调。5ng/mlTGF-β1以时间依赖方式诱导p-Smad2、p-Smad3磷酸化增加,1h达到高峰。MG132预处理组p-Smad2、p-Smad 3及FN蛋白表达量与TGF-β1刺激组相比显著下降,各组Smad2/3蛋白表达无显著变化。MG132预处理组FN蛋白分泌量与TGF-β1刺激组相比显著下降。结论:MG132可下调肾间质成纤维细胞的纤维化相关因子的mRNA表达,它能够通过抑制Smad信号转导途径抑制TGF-β1介导的炎症反应,提示MG132在肾脏炎症中具有抑制间质纤维化的潜在作用。

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（2.5μmol/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 reahime 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 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 （P 〈0.05）. 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 IIl mRNA decreased by 0.57, 0.35, 0.28, 0.05 times （P 〈0.05） in MG132 （0. 5, 1, 2.5 and 5 μmol/L） pretreated groups. MG132 could decrease the mRNA level of fibro- sis-related factors which were induced by TGF-β1 at each time point. TGF-β 15ng/ml could significantly increase the phos- phorylation 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 fi- brosis-related factors. It could inhibit fibrosis induced by TGF-β1 via Smad signaling pathway. It has a potent effect on counteracting the renal fibrosis.