蛋白酶体抑制剂对肾间质成纤维细胞凋亡和增殖的影响

Proteasome inhibitor on proliferation and apoptosis of renal interstitial fibroblasts

目的：探讨泛素蛋白酶体抑制剂MG-132对大鼠肾间质成纤维细胞（NRK-49F）及其在转化生长因子β1（TGF-β1）诱导下的细胞凋亡和增殖情况，并初步探索其机制。方法：采用5ng／ml的TGF-β1作用于NRK-49F，不同浓度（0～5μM）的蛋白酶体抑制剂MG-132预处理NRK-49F细胞，应用MTT法检测其增殖情况，LDH法检测细胞毒性，应用流式细胞仪检测细胞周期和细胞凋亡率，Hoechst 33258染色观察细胞凋亡形态学变化，Westernblot检测p-IKβ1IKB蛋白的变化。结果：TGF-Bl（5ng／m1）能促进NRK-49F的增殖（0．661±0．04猫0．495±0．06），MG-132（O．25—5μM）呈剂量依赖型抑制这种效应；MG-132（0．1—5μM）对NRK-49F有一定的细胞毒性作用，在0．5μM时达高峰；TGF-β1单独不能促使NRK-49F的凋亡[（3．8±0．4）％摊（4．7±1．6）％]，但加用MG．132（0．1—2．5μM）干预后呈剂量依赖关系促进细胞凋亡，MG-132单独也有促进细胞凋亡作用；MG-132（0～1斗M）作用于TGF-β1诱导的NRK-49F后，其S期比例呈剂量依赖关系显著减少，G1期细胞比例增加；Hoechst33258染色显示MG．132（0．5μM）＋TGF-β1组及MG-132组细胞核均出现核固缩、核碎裂、凋亡小体等凋亡特异形态学改变；Westernblot结果显示，TGF-β1及MG-132（2．5μM）＋TGF-β1均能以时间依赖方式诱导p-IKB、IKB蛋白表达增加，且MG-132能上调TGF-β1诱导下的p-Iκβ, IκB蛋白表达。结论：泛素蛋白酶体抑制剂MG-132对正常及病理状态下的NRK-49F都有促凋亡作用，并可能通过G1期阻滞过程来抑制其增殖作用。其机制可能与MG-132阻断IKB蛋白的泛素化降解有关。

Objective：To investigate the role and mechanism of proteasome inhibitor on the proliferation and apoptosis in cultured rat renal interstitial fibroblasts（RIF） induced by TGF-β1. Methodology：RIF（NRK-49F） were induced by TGF-β1 （5 ng/ml） and pre-treated with MG-132（0 -5 μM）. The cell proliferation was measured with MTT method. The cell cytotoxicity was tested by LDH method. The cell cycle and apoptosis was analyzed by flow cytometry. The apoptotic nuclear morphology was detected by Hoechst 33 258 staining. The p-IκB and IκB protein expression was examined by Western blot. Results： TGF-β1 （5 ng/ml ） could stimulate the proliferation of NRK-49F （0. 661 ±0. 04 vs 0. 495 ±O. 06）. MG-132 （0. 25 -5 μM） could dose-dependently inhibit the effect caused by TGF-β1, and had cytotoxicity on NRK-49F with the optimal concentration course at O. 5 p.M. TGF-β1 alone could not induce apoptosis （3.8% ±0. 4% vs 4. 7% ±1.6% ）. But MG-132（0. 1 -2. 5 μM）could significantly induce apoptosis of TGF-β1-stimulated NRK-49F in a dose-dependent manner, and also had such effect alone. After being incubated with MG-132 （0- 1 μM） and TGF-β1 （5 ng/ml）, the percentage of Sstage of NRK-49F was decreasing in a dose-dependent manner, while the G1 stage percentage increased. Hoechst 33 258 staining showed condensed or fragmented nuclei, apoptotic body after being incubated with 2.5 μM MG-132 with or without 5 ng/ral TGF-β1. Western blot showed that 5 ng/ral TGF-β1 with or without 2. 5 μM MG-132 both significantly up-regulated p-IκB and IκB protein expression time-dependently, and MG-β2 could enhance the level of p-IκB and IκB protein expression induced by TGF-β1. Conclusion：Proteasome inhibitor MG-β2 could induce apoptosis in cultured normal and pathologic RIF and inhibit the cellular proliferation by Gl-arrest. The mechanism may contribute to the blockade of ubiquitin-proteasome-mediated degradation of IκB protein.