辛二酰苯胺异羟肟酸诱导大鼠原代肝星状细胞凋亡

Suberoylanilide hydroxamic acid induces apoptosis of rat hepatic stellate cells in vitro

目的:通过研究辛二酰苯胺异羟肟酸(SAHA)对大鼠原代肝星状细胞(HSCs)凋亡及相关蛋白表达的影响,探讨SAHA诱导HSCs凋亡的作用机制。方法:采用Opti Prep梯度离心法分离大鼠原代HSCs;通过实时细胞分析技术检测SAHA对HSCs增殖的影响;倒置显微镜观察不同浓度SAHA处理HSCs后的形态变化;荧光显微镜及流式细胞术Annexin V-FITC/PI法检测细胞凋亡率;Western blotting法检测α-平滑肌肌动蛋白(α-SMA)、Ⅰ型胶原、金属蛋白酶组织抑制物1(TIMP1)、葡萄糖调节蛋白78(GRP78)和组蛋白去乙酰化酶6(HDAC6)的蛋白表达;免疫共沉淀法检测GRP78蛋白与HDAC6蛋白是否形成复合物。结果:成功分离的HSCs连续培养14 d,HSCs逐渐由静止状态变为激活状态。SAHA可显著抑制HSCs增殖,且呈剂量时间依赖性(P<0.05);SAHA对HSCs的促凋亡作用具有时间依赖性(P<0.05)。SAHA处理HSCs后,α-SMA、Ⅰ型胶原、HDAC6和TIMP1的蛋白表达水平明显降低(P<0.05),GRP78的蛋白表达水平明显升高(P<0.05)。与激活型的HSCs相比,SAHA处理后的HSCs中免疫共沉淀下的蛋白复合物中GRP78与总的乙酰化赖氨酸蛋白显著增多,而HDAC6蛋白显著降低,同时证明GRP78与HDAC6形成复合物。结论:SAHA抗肝纤维化的机制可能是,SAHA下调HDAC6蛋白表达水平,上调乙酰化GRP78蛋白表达水平,诱导HSCs内质网应激,促进肝星状细胞凋亡,从而起到抗肝纤维化的作用。

AIM： To study the effects of suberoylanilide hydroxamic acid （SAHA） on the apoptosis of hepatic stellate cells （HSCs） and expression of associated proteins, and to investigate the mechanisms of SAHA to induce apoptosis.METHODS： The rat HSCs were isolated by OptiPrep gradient centrifugation method. The effect of SAHA on HSC proliferation was detected by real-time cell analyzer. The morphological changes of HSCs treated with SAHA at different concentrations were observed under inverted microscope. The apoptotic rates of HSCs were analyzed by flow cytometry with Annexin V-FITC/PI staining and fluorescence microscopy. The protein expression of α-smooth muscle actin （α-SMA）, collagen I, tissue inhibitor of metalloproteinase 1 （TIMP1）, glucose-regulated protein 78 （GRP78） and histone deacetylase 6 （HDAC6） was detected by Western blotting. The interaction of GRP78 with HDAC6 in the HSCs was determined by co-immunoprecipitation.RESULTS： HSCs were successfully isolated and cultured for 14 d, during which the HSCs changed gradually from rest state to active state. SAHA significantly inhibited the proliferation of HSCs in a time-and dose-dependent manner （P〈0.05）. The results of Western blotting showed that the protein expression levels of α-SMA, TIMP1, collagen-I and HDAC6 were significantly decreased （P〈0.05）, while GRP78 was significantly increased （P〈0.05）. Compared with activated HSCs, GRP78 and total acetyl-lysine protein were significantly increased in the co-immunoprecipitated HSCs treated with SAHA, while HDAC6 protein was significantly decreased, indicting that GRP78 formed a complex with HDAC6.CONCLUSION： The anti-hepatic fibrosis effect of SAHA may be related to down-regulation of HDAC6 and up-regulation of acetylated GRP78, thus inducing endoplasmic reticulum stress of HSCs and promoting the apoptosis of HSCs.