天津医药

• 细胞与分子生物学 • 上一篇    下一篇

自噬抑制剂对乙醇诱导的肝星状细胞活化作用的影响

何月1,贾宝辉1,刘曼2,罗文2,张吉翔3   

  1. 1. 南昌大学第四附属医院
    2. 南昌大学第二附属医院
    3. 南昌大学第二附医院消化内科
  • 收稿日期:2013-11-05 修回日期:2014-03-06 出版日期:2014-07-15 发布日期:2014-07-15
  • 通讯作者: 张吉翔

The Effects of Autophagy Inhibitor on Activation of Alcohol induced Hepatic Stellate Cells

  • Received:2013-11-05 Revised:2014-03-06 Published:2014-07-15 Online:2014-07-15

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摘要： 【摘要】目的 观察自噬抑制剂3-甲基腺嘌呤（3-MA）对乙醇诱导的肝星状细胞（HSC）活化作用，并探讨其作用机制。方法 体外培养大鼠肝星状细胞株HSC-T6，设立空白对照组、阳性对照组（乙醇刺激组）、低剂量组（5 mmol/L3-MA+100mmol/L乙醇）、高剂量组（10mmol/L3-MA+100mmol/L乙醇）。RT-PCR分析各组HSC活化标志蛋白α-平滑肌肌动蛋白(α-SMA)及Ⅰ型胶原基因表达，Western blot法检测各组HSC中自噬水平标志蛋白LC3Ⅱ、α- SMA、Ⅰ型胶原表达；MTT法检测3-MA对乙醇诱导的HSC增殖的影响。结果与空白对照组比较，阳性对照组中α- SMA、Ⅰ型胶原mRNA和蛋白表达及LC3Ⅱ表达量明显增加（P＜0.05），高剂量组却显著减少（P＜0.01）；与阳性对照组比较，低剂量组和高剂量组中α-SMA、Ⅰ型胶原mRNA和蛋白表达及LC3Ⅱ表达量逐渐减少（P＜0.05）；与低剂量组比较，高剂量组中α-SMA、Ⅰ型胶原mRNA和蛋白表达及LC3Ⅱ表达减少（P＜0.05）。与阳性对照组比较，加入3- MA处理后的HSC增殖显著减少（P＜0.05）。结论3-MA可抑制乙醇诱导的HSC-T6细胞中LC3Ⅱ蛋白的表达、α- SMA、Ⅰ型胶原mRNA及蛋白的表达，抑制HSC增殖，且高剂量的作用更明显。

关键词: 自噬, 酒精, 3-甲基腺嘌呤, 肝星状细胞

Abstract: [Abstract] Objective To observe the effect of autophagy inhibitor on the activation of alcohol induced hepatic stel? late cells, and the mechanisms thereof. Methods HSC-T6cells were cultured in vitro and divided into four groups, includ? ing blank control group, alcohol group, 5mmol/L3-MA+alcohol group (low alcohol group) and10mmol/L3-MA+alcohol group (high alcohol group). RT-PCR was used to detect the expression levels ofα-smooth muscle actin (α-SMA) and typeⅠ collagen. The levels of LC3Ⅱ, α-SMA and typeⅠcollagen were detected by Western blot assay. The cell viability of HSC T6was detected by MTT assay.Results The mRNA expressions ofα-SMA, typeⅠcollagen and the protein of expressions α-SMA, typeⅠcollagen and LC3Ⅱwere significantly up-regulated in alcohol group compared with those of control group (P<0.05), while the expressions of those parameters were significantly down- regulated in 10mmol/L3-MA+alcohol group (P<0.01). The mRNA and protein levels of α-SMA and typeⅠcollagen were significantly decreased in two3-MA-treated groups compared with those in alcohol group (P<0.05). Meanwhile, compared with the 5mmol/L3-MA+ alcohol group，the protein expressions ofα-SMA, typeⅠcollagen and LC3Ⅱwere significantly decreased in10mmol/L3-MA+alcohol group (P<0.05). Compared with the alcohol group，there was significantly lower proliferation activity in all two 3-MA-treated groups (P<0.05). Conclusion 3-MA can inhibit the protein expression of LC3Ⅱ, α-SMA and typeⅠcollagen induced by alcohol in HSC-T6cells, and inhibit the proliferation of HSC cells.

Key words: autophagy, alcohol, 3-methyladenine, hepatic stellate cells