摘要
目的:探讨氧化苦参碱干预砷致肝星状细胞(HSC)活化过程中细胞自噬的变化。方法:本实验使用100 μmol/L亚砷酸钠作用于人胎肝细胞株LO2 24 h,收取培养上清。将染砷LO2细胞培养上清与正常培养液按照1 ∶ 4比例混合,用于培养人肝星状细胞株LX-2,24 h后采用低(0.25 g/L)和高(1 g/L)剂量氧化苦参碱干预培养24 h;采用全自动生化分析仪检测染砷LO2细胞培养上清中天冬氨酸氨基转移酶(AST)和丙氨酸氨基转移酶(ALT)水平;ELISA检测染砷LO2细胞培养上清中转化生长因子β1(TGF-β1)水平;MTT法检测LX-2细胞活力;Western blot检测α-平滑肌肌动蛋白(α-SMA)、自噬相关基因12(Atg12)、自噬相关基因5(Atg5)和微管相关蛋白1轻链3-Ⅱ(LC3-Ⅱ)蛋白表达水平变化;油红O染色光镜下观察LX-2细胞中脂滴。结果:(1)染砷LO2细胞培养上清AST和ALT水平显著增加( P <0.05),此上清加入LX-2细胞培养基中可明显增强LX-2细胞活力( P <0.05),与染砷LO2细胞培养上清共孵育的LX-2细胞内脂滴数量显著减少,且LX-2活化标志蛋白α-SMA表达水平显著上调( P <0.05);(2)与染砷LO2细胞培养上清共孵育可上调LX-2细胞Atg12、Atg5和LC3Ⅱ蛋白表达,较对照组差异具有显著性( P <0.05);(3)低、高剂量氧化苦参碱干预可抑制与染砷LO2细胞培养上清共孵育的LX-2细胞的活力( P <0.05),且使LX-2细胞α-SMA、Atg12、Atg5和LC3-Ⅱ蛋白表达水平显著下调( P <0.05),高剂量氧化苦参碱干预的LX-2细胞自噬标志蛋白LC3-Ⅱ表达水平降低较低剂量氧化苦参碱干预更为显著( P <0.05);(4)低、高剂量氧化苦参碱干预后LX-2细胞内脂滴数量较染砷LO2细胞培养上清共孵育的LX-2细胞均显著增多。结论:砷致肝纤维化过程中间接活化HSC与砷损伤肝细胞有关,细胞自噬参与这一过程,且HSC可能通过促进其内脂滴降解而促进自身的活化。氧化苦参碱可通过抑制细胞自噬,减少HSC活化实现其减轻肝纤维化�
AIM: To investigate the effect of oxymatrine (OM) on the autophagy of hepatic stellate cells (HSC) during HSC activation induced by arsenic (As). METHODS: Supernatant of human LO2 hepatocytes cultured with 100 μmol/L NaAsO 2 for 24 h was collected. Human HSC line LX-2 was cultured for 24 h before mingling culture supernatant of LO2 cells with arsenic exposure and normal culture media in a 1∶ 4 ratio, and then treated with low dose (0.25 g/L) and high dose (1 g/L) of OM. The levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in culture medium were measured by biochemical method. The level of transforming growth factor-β1 (TGF-β1) in the culture supernatant of LO2 cells with arsenic exposure was detected by ELISA. The cell viability was examined by MTT assay. The protein levels of α-smooth muscle actin (α-SMA), autophagy-related gene 12 (Atg12), autophagy-related gene 5 (Atg5) and microtubule-associated protein 1 light chain 3-Ⅱ(LC3-Ⅱ) were determined by Western blot. Lipid droplets were observed by oil red O staining under microscope. RESULTS: The levels of AST and ALT were obviously increased in the culture supernatant of LO2 cells with arsenic exposure ( P <0.05). The viability of LX-2 cells was obviously enhanced while adding supernatant of LO2 cells with arsenic exposure into the culture media of LX-2 cells ( P <0.05), the number of lipid drops decreased, and the expression of α-SMA was increased ( P <0.05). Co-incubation with supernatant of LO2 cells and arsenic exposure obviously increased the expression of Atg12, Atg5, LC3-Ⅱ at protein level in the LX-2 cells as compared with control group ( P <0.05). Low dose and high dose of OM inhibited the viability of LX-2 cells caused by co-incubation of the supernatant of LO2 cells with arsenic exposure ( P <0.05) and decreased the protein expression of α-SMA, Atg12, Atg5 and LC3-Ⅱ in the LX-2 cells ( P <0.05). High dose of OM was more effective in expression of LC3-Ⅱ than low dose ( P <0.05). The number of lipid droplets
作者
马子华
张景允
杨柳
田甜
汤雷
郑璐
蔡爽
韩冰
谢汝佳
杨婷
杨勤
MA Zi-hua;ZHANG Jing-yun;YANG Liu;TIAN Tian;TANG Lei;ZHENG Lu;CAI Shuang;HAN Bing;XIE Ru-jia;YANG Ting;YANG Qin(Department of Pathophysiology, Guizhou Provincial Key Laboratory of Pathogensis & Drug Research on Common Chronic Diseases, Guizhou Medical University, Guiyang 550000 , China)
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2019年第9期1662-1667,共6页
Chinese Journal of Pathophysiology
基金
国家自然科学基金资助项目(No.81460484)
关键词
肝星状细胞
氧化苦参碱
自噬
细胞活力
肝纤维化
Hepatic stellate cells
Oxymatrine
Autophagy
Cell viability
Hepatic fibrosis
