白花丹醌对人肝星状细胞凋亡及相关蛋白表达的影响

Effects of plumbagin on apoptosis and expression of apoptosis-related proteins in human hepatic stellate cells

目的:研究白花丹醌对瘦素(Leptin)刺激体外培养人肝星状细胞株HSC-LX2凋亡及相关蛋白表达的影响,以探讨其抗肝纤维化作用的机制.方法:体外培养HSC-LX2,将其分为以下几组:空白对照组、Leptin对照组、秋水仙碱组、2、8、16μmol/L白花丹醌组.瘦素刺激24h,药物与细胞共孵育24h后,应用流式细胞仪Annexin/PI双染方法检测细胞凋亡,透射电镜观察HSC-LX2凋亡形态学变化,免疫组织化学法检测HSC-LX2凋亡基因p53、Bax、Bcl-2的蛋白表达.结果:细胞流式术结果显示,白花丹醌作用HSC-LX224h,HSC凋亡率明显增加,白花丹醌8、16μmol/L组和秋水仙碱组HSC凋亡率均显著高于空白对照组和Leptin对照组(5.21±0.41,8.10±0.63,10.1±1.08vs1.40±0.13,2.85±0.21,均P<0.01).透射电镜结果,空白对照组细胞形态清晰,细胞膜、核膜完整,胞质内细胞器清晰,染色质均匀,细胞表面微绒毛;药物组可见少量坏死细胞和许多不同程度的凋亡细胞,体积变小,核膜消失,胞质浓缩,胞质内出现大量空泡,细胞核染色质固缩,可见核碎裂,细胞表面微绒毛消失.免疫组织化学结果显示,受Leptin刺激后HSC-LX2胞质内表达少量的Bax、Bcl-2、P53,可见少量的棕黄色颗粒,经白花丹醌2、8、16μmol/L干预后,HSC-LX2促凋亡基因Bax、P53蛋白表达明显升高,抗凋亡基因Bcl-2蛋白表达显著降低,与Leptin刺激组相比,差异均有统计学意义(Bax:85.24±1.08,86.35±1.12,91.13±1.13vs56.63±0.94;P53:25.32±0.6,38.14±0.71,41.19±0.72vs19.25±0.46;Bcl-2:32.12±0.43,27.71±0.38,21.46±0.46vs44.51±0.56,P<0.05或0.01).结论:白花丹醌能够促进活化的HSC-LX2凋亡,其促HSC-LX2凋亡机制可能与上调P53、Bax蛋白表达和下调Bcl-2蛋白有关.

AIM：To investigate the effects of plumbagin on leptin-induced apoptosis and expression of apoptosis-related protein in human hepatic satellite cells（HSC-LX2） and to explore the anti-fibrotic mechanism of plumbagin.METHODS：After HSC-LX2 cells were cultured in vitro,stimulated with leptin for 24 h,and treated with different concentrations of plumbagin for 24 h,cell apoptosis was detected by flow cytometry;cell ultrastructure was observed by transmission electron microscopy;and the protein expression of P53,Bax,and Bcl-2 was determined by immunocytochemistry.RESULTS：HSC-LX2 cells were divided into 6 groups：untreated cells（blank control group）,those treated with 100 μg/L leptin（leptin control group）,those treated with both leptin and colchicin（colchicin group）,those treated with both leptin and 2,8 or 16 μmol/L plumbagin（2,8,16 μmol/L plumbagin group）.The apoptosis rate of HSC-LX2 cells was signif icantly increased in plumbagin groups.The apoptosis rates of cells treated with 8 or 16 μmol/L plumbagin or colchicine were signif icantly higher than those of the blank control group and leptin group（5.21 ± 0.41,8.10 ± 0.63,10.1 ± 1.08 vs 1.40 ± 0.13,2.85 ± 0.21,all P 0.01）.Transmission electron microscopy revealed varying degrees of apoptosis in the leptin group or plumbagin groups.Immunocytochemistry analysis showed that the protein expression levels of P53 and Bax were higher and that of Bcl-2 was lower in plumbagin groups than in the leptin group（Bax：85.24 ± 1.08,86.35 ± 1.12,91.13 ± 1.13 vs 56.63 ± 0.94;P53：25.32 ± 0.6,38.14 ± 0.71,41.19 ± 0.72 vs 19.25 ± 0.46;Bcl-2：32.12 ± 0.43,27.71 ± 0.38,21.46 ± 0.46 vs 44.51 ± 0.56,all P 0.05 or 0.01）.CONCLUSION：Plumbagin can significantly accelerate leptin-induced apoptosis of HSC-LX2 cells possibly by up-regulating P53 and Bax expression and down-regulating Bcl-2 expression.