自噬抑制剂3-甲基腺嘌呤对肝星状细胞增殖活化的影响

Effect of autophagy inhibitor 3-methyladenine on proliferation and activation of hepatic stellate cells

目的观察自噬抑制剂3-甲基腺嘌呤(3-MA)对大鼠肝星状细胞(HSC-T6)增殖活化的影响,并探讨其作用机制。方法体外培养大鼠肝星状细胞株HSC-T6,设立空白对照组和3-MA低剂量组(2.5 mmol/L)、中剂量组(5 mmol/L)、高剂量组(10 mmol/L)。RT-PCR分析不同浓度3-MA对HSC活化标志蛋白α-平滑肌肌动蛋白(α-SMA)以及Ⅰ型胶原蛋白基因表达的影响,Western blot法检测不同浓度3-MA对HSC中自噬水平标志蛋白LC3Ⅱ、α-SMA以及Ⅰ型胶原蛋白表达水平的影响;MTT法检测3-MA对HSC增殖的影响,流式细胞术分析3-MA对HSC细胞周期的影响。结果低、中、高剂量3-MA作用于HSC后,HSC-T6细胞自噬水平随3-MA浓度升高逐渐下降,α-SMA、Ⅰ型胶原蛋白mRNA相对表达量均明显低于对照组(P<0.05),LC3Ⅱ、α-SMA以及Ⅰ型胶原蛋白相对表达量均明显低于对照组(P<0.05)。与对照组相比,低、中、高剂量时3-MA对HSC增殖的影响均显著下降(P<0.05),G2期比例与对照组比较均明显增加(P<0.05)。结论 3-MA可下调大鼠HSC-T6细胞LC3Ⅱ的表达,抑制α-SMA及Ⅰ型胶原蛋白mRNA及蛋白的表达,使HSC-T6细胞周期停滞于G2期,抑制HSC增殖活化。

Objective To investigate the effect of autophagy inhibitor 3-methyladenine （3-MA） on proliferation and acti-vation of hepatic steUate cells （HSCs） and investigate the underlying molecular mechanism. Methods Cultured HSC-T6 cells were cultured in vitro and treated with different concentrations of 3-MA （ low-dose group, 2.5 mmol/L; middle-dose group, 5 mmol/L; high-dose group, 10 mmol/L; control group, 0 mmol/L）. The mRNA expressions of α-SMA and typeⅠ collagen were determined by RT-PCR; the protein expressions of LC3 Ⅱ, α-SMA and type Ⅰcollagen were detected by West-em blotting; cell proliferation was observed by MTT assay and cell cycle by flow cytometry. Results The autophagy of HSC- T6 cells decreased with the rising of the concentration of 3-MA. The mRNA expressions of α-SMA and type Ⅰ collagen in all three 3-MA-treated groups were significantly down-regulated, compared with those in control group （P 〈0.05）. Meanwhile, compared with the control group, the 3-MA-treated groups also showed significantly down-regulated protein expressions of LC311, α-SMA, and type Ⅰ collagen （P 〈 0.05）, significantly lower proliferation activity （P 〈0.05） and significantly higher numbers of HSCs in the G2 phase （ P 〈 0.05）. Conclusion The autophagy inhibitor 3-MA significantly down-regulated the expressions of LC311, α-SMA and type Ⅰ collagen in HSC-T6 and caused an arrest in the G2 phase of the cells, thus inhibiting the proliferation and activation of HSCs.