蛋白酶体抑制剂对肾间质成纤维细胞增殖、凋亡及其相关蛋白的影响

Effects of proteasome inhibitor on proliferation, apoptosis and related proteins in renal interstitial fibroblasts

目的探讨蛋白酶体抑制剂MG-132对转化生长因子β1（TGF-β1）诱导下的大鼠肾间质成纤维细胞（NRK-49F）增殖、凋亡及其相关蛋白的影响。方法用5μg／L的TGF-β1作用于NRK-49F。不同浓度（0-5μmol／L）MG-132预处理NRK-49F细胞后，MTT法检测细胞增殖；应用流式细胞仪检测细胞周期和细胞凋亡率；DNA凝胶电泳法观察细胞的凋亡情况；Western印迹检测p53、p27、p21、caspase-3、Bcl-2和Bax蛋白的变化。结果TGF-β1（5μg／L）可促进NRK-49F细胞增殖，而MG-132（0．25-5μmol／L）呈剂量依赖性地抑制这种效应，使细胞生长停滞在G1期。TGF-β1（5μg／L）单独不能诱导NRK-49F的凋亡（3．880％±0．365％比4．723％±1．582％），而MG-132（0．10、0．25、0．50、0．75、1．00、2．50μmol／L）预处理可呈剂量依赖性地促进细胞凋亡（调亡率分别为8．8％、18．7％、22．8％、31．3％、37．7％、38．9％）。MG-132（2．5μmol／L）＋TGF-β1组及MG-132（2．5μmol／L）组在DNA电泳中呈典型的梯状条带现象。Western印迹结果显示，MG-132可剂量依赖性地激活TGF-β1诱导的细胞周期及凋亡相关蛋白p53蛋白表达，促进caspase-3的活性片段产生，Bcl-2表达下调，并能使Bax和p21表达上调，但对p27没有明显的影响。结论MG-132可抑制TGF-β1诱导的肾间质成纤维细胞增殖，并促其凋亡。该作用可能与MG-132对细胞周期及凋亡相关蛋白p53、p21、caspase-3、Bcl-2和Bax的调节有关，提示泛素蛋白酶体抑制剂可能成为将来防治。肾间质纤维化的一个潜在的治疗手段。

Objective To investigate the role of MG-132, a specific dipeptide proteasome inhibitor, on the proliferation, apoptosis and the related proteins in renal interstitial fibroblasts. Methods Renal interstitial fibroblasts （NRK-49F） were induced by transforming growth factor β1 （TGF-β1, 5 μg/L） and pre-treated with MG-132 （0-5 μmol/L）. The cell proliferation was measured with MTT method. Cell cycle and apoptosis were analyzed by flow eytometry. The apoptosis was also analyzed by Annexin V/PI staining and DNA ladder. Expression of p53, p27, p21, caspase-3, Bcl-2 and Bax protein was examined by Western blot. Results TGF-β1 （5μg/L） could stimulate the proliferation of NRK-49F. MG-132 （0.25-5 μmol/L） could inhibit TGF-β1-induced proliferation in a dose-dependent manner through G1-arrest. TGF-151 alone could not induce apoptosis （3.880%±0.365% vs 4.723%±1.582%）. But pretreatment of MG-132 （0.1-2.5 μmol/L） could significantly induce apoptosis of TGF-β1-stimulated NRK-49F in a dose-dependent manner. Typical DNA ladder was also confirmed in these two groups in the DNA fragments analysis after being incubated with 2.5μmol/L MG-132 with or without 5μg/L TGF-β1. Western blot showed that MG-132 could activate the cell-cycle and apoptosis-related proteins such as p53, p21, caspase-3, Bax and inhibit Bcl-2 in a dose-dependent manner, while expression of p27 remained unchanged. Conclusions Proteasome inhibitor MG-132 can inhibit proliferation and induce the cell apoptosis in renal interstitial fibroblasts stimulated by TGF-β1. The mechanism may be associated to the mediation of p53, p21, caspase-3, Bcl-2 and bax pathways. Proteasome inhibitor may be a new strategy to treat renal interstitial fibrosis.