3-甲基腺嘌呤对转化生长因子-β诱导大鼠肝脏星形细胞株HSC-T6活化及自噬的影响观察

Effects of 3-methyladenine on activation and autophagy of rat hepatic stellate cell line HSC-T6 induced by TGF-β

目的观察3-甲基腺嘌呤(3-MA)对转化生长因子-β(TGF-β)诱导的大鼠肝星形细胞株HSC-T6活化及自噬的影响。方法取对数生长期的HSC-T6细胞分为空白组、TGF-β+PBS组、TGF-β+3-MA组。TGF-β+3-MA组细胞加入浓度为2 ng/mL TGF-β培养72 h后加入3-MA(0.5 mg/mL)处理24 h,TGF-β+PBS组细胞加入浓度为2 ng/mL TGF-β培养72 h后加入等量PBS处理24 h,空白组加入等量PBS处理,采用实时荧光定量PCR法检测各组细胞活化标志物α-SMA、TGF-βmRNA和自噬标志物LC3、Beclin-1、Atg5 mRNA,采用Western blotting法检测各组细胞活化标志物α-SMA、TGF-β蛋白和自噬标志物LC3、Beclin-1、Atg5蛋白。结果TGF-β+PBS组、TGF-β+3-MA组细胞活化标志物α-SMA、TGF-βmRNA和蛋白均高于空白组,且TGF-β+3-MA组均低于TGF-β+PBS组(P均<0.05)。TGF-β+PBS组、TGF-β+3-MA组细胞自噬标志物LC3、Beclin-1、Atg5 mRNA和蛋白均高于空白组,且TGF-β+3-MA组均低于TGF-β+PBS组(P均<0.05)。结论3-MA可抑制TGF-β诱导的大鼠肝星形细胞株HSC-T6活化及自噬。

Objective To investigate the effects of 3-methyladenine(3-MA)on activation and autophagy of rat he-patic stellate cell line HSC-T6 induced by transforming growth faction-β(TGF-β).Methods HSC-T6 cells in the loga-rithmic growth phase were divided into the blank group,TGF-β+PBS group,and TGF-β+3-MA group.Cells of TGF-β+3-MA group were cultured with 2 ng/mL TGF-βfor 72 h and treated with 3-MA(0.5 mg/mL)for 24 h.Cells of TGF-β+PBS group were cultured with 2 ng/mL TGF-βfor 72 h and treated with the same volume of PBS for 24 h.Cells in the blank group were treated with equal volume of PBS,and the mRNA levels of cell activation markersα-SMA and TGF-βand au-tophagy markers LC3,Beclin-1 and Atg5 in each group were detected by real-time fluorescence quantitative PCR.Cell ac-tivation markersα-SMA,TGF-βproteins and autophagy markers LC3,Beclin-1,Atg5 proteins were detected by Western blotting.Results The mRNA and protein levels of cell activation markersα-SMA and TGF-βin the TGF-β+PBS group and TGF-β+3-MA group were higher than those in the blank group,and those were lower in the TGF-β+3-MA group than in the TGF-β+PBS group(all P<0.05).The mRNA and protein levels of autophagy markers LC3,Beclin-1 and Atg5 in the TGF-β+PBS group and TGF-β+3-MA group were higher than those in the blank group,and those were lower in the TGF-β+3-MA group than in the TGF-β+PBS group(all P<0.05).Conclusion 3-MA can inhibit TGF-β-induced HSC-T6 activation and autophagy in rat hepatic astrocytes.