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 of proteasome inhibitor MG132 on the expression of extracellular matrix of rat renal interstitial fibroblasts stimulated by transforming growth factor-β1 (TGF-β1) and its possible mechanism. Methods: Rat renal interstitial fibroblasts (NRK / 49F) were cultured in vitro. Real-time quantitative PCR (Real-time PCR) was used to observe the effects of MG132 cells directly stimulated with different concentrations of TGF-β1 (5ng / ml) (CTGF), alpha-smooth muscle actin (α-SMA), fibronectin (FN) and type III collagen (ColIII) After pretreatment with the same concentration of MG132 (2.5μmol / L), TGF-β1 and TGF-β1 were respectively stimulated for different time (0h, 6h, 12h and 24h). The effect of MG132 on TGF-β1-induced FN and Smads protein expression was observed by Western blot. The effect of MG132 on TGF-β1-induced FN protein secretion was observed by ELISA. Results: MG132 directly induced the mRNA expression of CTGF, α-SMA and FN, ColIII in NRK / 49F cells. With the increase of MG132 concentration (0.5, 1, 2.5 and 5μmol / L) , Which were respectively 65%, 46%, 17% and 20% of the control group. The mRNA expression of α-SMA decreased by 10%, 7%, 4% and 3% , Which were 54%, 42%, 25% and 24% of the control group respectively. The mRNA expression of ColIII decreased by 30%, 12%, 5% and 1% of the control group (all P <0.05). The mRNA expression of CTGF, α-SMA, FN and ColIII in 5ng / ml TGF-β1 group increased 6.91,2.11,1.38 and 3.60 times higher than those in control group (P <0.05) L) pretreatment, CTGF mRNA decreased 3.30, 2.84, 1.06 and 0.74 folds in the control group, while the α-SMA mRNA decreased in the control group (0.50, 0.31, 0.28 and 0.19 times, respectively) , Respectively, 0.80,0.67,0.55 and 0.37 times of the control group, ColIII mRNA decreased, respectively, the control group 0.57,0.35,0.28 and 0.05 times. At each time point, MG132 down-regulated the mRNA expression of fibrosis-related factors induced by TGF-β1. 5ng / ml TGF-β1 induced p-Smad2 in a time-dependent manner, phosphorylation of p-Smad3 increased and peaked at 1h. The expression of p-Smad2, p-Smad 3 and FN in MG132 pretreatment group was significantly lower than that in TGF-β1 -stimulated group, while the expression of Smad2 / 3 protein in each group did not change significantly. MG132 pretreatment group FN protein secretion was significantly decreased compared with TGF-β1 stimulation group. CONCLUSION: MG132 can down-regulate the mRNA expression of fibrosis-related factors in renal interstitial fibroblasts. It can inhibit the TGF-β1-mediated inflammatory response by inhibiting the Smad signaling pathway, suggesting that MG132 has the ability of inhibiting interstitial fibrosis Potential role.