自噬抑制剂对乙醇诱导的肝星状细胞活化作用的影响

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

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(5mmol/L 3-MA+100 mmol/L乙醇)、高剂量组(10 mmol/L 3-MA+100 mmol/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。

Objective To observe the effect of autophagy inhibitor on the activation of alcohol induced hepatic stel-late cells, and the mechanisms thereof. Methods HSC-T6 cells were cultured in vitro and divided into four groups, includ-ing blank control group, alcohol group, 5 mmol/L 3-MA＋alcohol group （low alcohol group） and 10 mmol/L 3-MA＋alcohol group （high alcohol group）. RT-PCR was used to detect the expression levels ofα-smooth muscle actin （α-SMA） and typeⅠcollagen. The levels of LC3Ⅱ,α-SMA and typeⅠcollagen were detected by Western blot assay. The cell viability of HSC-T6 was detected by MTT assay. Results The mRNA expressions ofα-SMA, typeⅠcollagen and the protein of expressionsα-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 10 mmol/L 3-MA＋alcohol group （P〈0.01）. The mRNA and protein levels ofα-SMA and typeⅠcollagen were significantly decreased in two 3-MA-treated groups compared with those in alcohol group （P〈0.05）. Meanwhile, compared with the 5 mmol/L 3-MA＋alcohol group,the protein expressions ofα-SMA, typeⅠcollagen and LC3Ⅱwere significantly decreased in10 mmol/L 3-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-T6 cells, and inhibit the proliferation of HSC cells.