Department of Nephrology, Ruijin Hospital, Shanghai Jiao Tong University, Shanghai, 200025, P.R. China.
Pharmacol Rep. 2013;65(5):1357-65. doi: 10.1016/s1734-1140(13)71494-4.
The ubiquitin proteasome pathway plays a pivotal role in controlling cell proliferation, apoptosis and differentiation in a variety of normal and tumor cells. This study aimed to investigate the role of a proteasome inhibitor on proliferation, apoptosis and related proteins in renal interstitial fibroblasts (NRK-49F).
NRK-49F cells were induced using transforming growth factor-β1 (TGF-β1) and pretreated with the proteasome inhibitor MG-132. Cell proliferation was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). The cell cycle and apoptosis were analyzed using flow cytometry. Apoptosis was also analyzed using a DNA ladder. The protein expression of p53, p27, p21, caspase-3, Bcl-2 and Bax was examined using western blots.
The results showed that TGF-β1 (5 ng/ml) can stimulate the proliferation of NRK-49F cells.MG-132 (0.25-5 μM) inhibited TGF-β1-induced proliferation in a dose-dependent manner through G1-arrest; TGF-β1 alone did not induce apoptosis (3.8 ± 0.4% vs. 4.7 ± 1.6%). However, pretreatment with MG-132 significantly induced apoptosis in TGF-b1-stimulated NRK-49F cells in a dose-dependent manner. A typical DNA ladder was also confirmed in these two groups. Western blot analysis showed that MG-132 activated p53, p21, caspase-3 and Bax, and inhibited Bcl-2 in a dose-dependent manner, while p27 expression remained unchanged.
A proteasome inhibitor inhibited proliferation and induced apoptosis in renal interstitial fibroblasts stimulated by TGF-β1. The mechanism may relate to the p53, p21, caspase-3, Bcl-2 and Bax pathways. Our results suggest that a proteasome inhibitor could be a new strategy to treat renal interstitial fibrosis.
泛素蛋白酶体途径在多种正常和肿瘤细胞中控制细胞增殖、凋亡和分化中起着关键作用。本研究旨在探讨蛋白酶体抑制剂对转化生长因子-β1（TGF-β1）诱导的肾间质成纤维细胞（NRK-49F）增殖、凋亡及相关蛋白的作用。
用转化生长因子-β1（TGF-β1）诱导 NRK-49F 细胞，并预先用蛋白酶体抑制剂 MG-132 处理。用 3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐（MTT）测定细胞增殖。用流式细胞术分析细胞周期和凋亡。用 DNA 梯带分析凋亡。用 Western blot 检测 p53、p27、p21、caspase-3、Bcl-2 和 Bax 的蛋白表达。
结果表明，TGF-β1（5ng/ml）可刺激 NRK-49F 细胞增殖。MG-132（0.25-5μM）通过 G1 期阻滞呈剂量依赖性抑制 TGF-β1 诱导的增殖；TGF-β1 本身不诱导凋亡（3.8±0.4%比 4.7±1.6%）。然而，MG-132 预处理可显著诱导 TGF-β1 刺激的 NRK-49F 细胞凋亡呈剂量依赖性。这两组也证实了典型的 DNA 梯带。Western blot 分析显示，MG-132 呈剂量依赖性激活 p53、p21、caspase-3 和 Bax，抑制 Bcl-2，而 p27 表达保持不变。
蛋白酶体抑制剂抑制 TGF-β1 刺激的肾间质成纤维细胞增殖并诱导其凋亡。其机制可能与 p53、p21、caspase-3、Bcl-2 和 Bax 途径有关。我们的结果表明，蛋白酶体抑制剂可能是治疗肾间质纤维化的新策略。
