异牛肝菌素对人肝癌细胞增殖、凋亡影响及其与PI3K/Akt通路的关系

Effects of iso-suillin on proliferation and apoptosis of human hepatocellular carcinoma cells and its relationship with PI3K/Akt pathway

目的观察异牛肝菌素对人肝癌细胞增殖、凋亡的影响,并基于磷脂酸肌醇-3-激酶/蛋白激酶B(PI3K/Akt)通路探讨相关作用机制。方法取对数生长期的人肝癌细胞株HepG2,分为异牛肝菌素组、激活剂组、对照组。异牛肝菌素组加入50μg/mL的异牛肝菌素;激活剂组加入50μg/mL的异牛肝菌素和20μg/mL的PI3K/Akt通路激活剂FumonisinB1;对照组仅加入等容量生理盐水,不加任何药物。采用MTT法测算各组干预后24、48、72 h细胞的增殖抑制率,采用Hoechst 333258荧光染色法测算细胞凋亡率,Westernblotting法检测细胞中的凋亡相关蛋白(Bax、Bcl-2、Caspase-3)及PI3K/Akt通路相关蛋白(PI3K、pPI3K、pAkt)。结果干预后24、48、72 h异牛肝菌素组细胞OD值低于激活剂组和对照组,细胞增殖抑制率高于激活剂组和对照组(P均<0.05);随时间延长,异牛肝菌素组OD值呈降低趋势,细胞增殖抑制率呈升高趋势(P均<0.05)。异牛肝菌素组细胞凋亡率及Bax、Caspase-3蛋白表达高于激活剂组和对照组,Bcl-2、PI3K、pPI3K、pAkt蛋白表达低于激活剂组和对照组(P均<0.05)。结论异牛肝菌素可抑制HepG2细胞增殖并诱导其凋亡,作用机制可能与抑制PI3K/Akt通路有关。

Objective To observe the effects of iso-suillin on the proliferation and apoptosis of human hepatocellular carcinoma cells,and to explore the related mechanism based on the phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt)pathway.Methods Human hepatocellular carcinoma cell line HepG2 in the logarithmic growth phase was divid-ed into the iso-suillin group,activator group,and control group.Cells in the iso-suillin group were added with 50μg/mL iso-suillin,the activator group with 50μg/mL iso-suillin and 20μg/mL PI3K/Akt pathway activator Fumonisin B1,and the control group were only added with equal volume of normal saline without any drugs.MTT was used to measure the pro-liferation inhibition rate of each group at 24,48 and 72 h after intervention.Hoechst 333258 fluorescence staining method was used to measure the apoptosis rate.Apoptosis-related proteins(Bax,Bcl-2,Caspase-3)and PI3K/Akt pathway relat-ed proteins(PI3K,pPI3K,pAkt)were detected by Western blotting.Results OD values of the iso-suillin group were lower than those of the activator group and control group at 24,48 and 72 h after intervention,and inhibition rates of cell proliferation were higher than those of the activator group and control group(all P<0.05).The OD value of the iso-suillin group showed a decreasing trend,and the inhibition rate of cell proliferation showed an increasing trend over time(both P<0.05).The apoptosis rate and protein expression levels of Bax and Caspase-3 in the iso-suillin group were higher than those in the activator group and the control group,while the protein expression levels of Bcl-2,PI3K,pPI3K,and pAkt were lower than those in the activator group and the control group(all P<0.05).Conclusion Iso-suillin can inhibit the proliferation and induce apoptosis of HepG2 cells,and the mechanism may be related to the inhibition of PI3K/Akt path-way.