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

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5 μg/L的TGF-β1作用于NRK-49F。不同浓度（0~5 μmol/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（5 μg/L）可促进NRK-49F细胞增殖，而MG-132（0.25~5 μmol/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（5 μg/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、2.50 μmol/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（2.5 μmol/L）+TGF-β1组及 MG-132（2.5 μmol/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。

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

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 pro-treated with MG-132 (0～5 μmol/L). The cell proliferation was measured with MTT method. Cell cycle and apoptosis were analyzed by flow cytometry. 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 (5 μg/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-β1 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 2.5 μmol/L MG-132 with or without 5 μg/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. Protoasome inhibitor may be a new strategy to treat renal interstitial fibrosis.