18α-甘草次酸对游离脂肪酸致HepG2细胞脂毒性的保护作用

Protective effect of 18α-glycyrrhetic acid on lipotoxicity in HepG2 cells induced by free fatty acids

目的探讨18α-甘草次酸对游离脂肪酸(FFAs)致HepG2细胞脂毒性的保护作用及经线粒体途径调节的分子机制。方法给予1 mmol/L FFAs,油酸(OA)–棕榈酸(PA)(2∶1)建立体外HepG2细胞脂肪变性模型,细胞分为对照组、模型组和18α-甘草次酸低、中、高剂量(10、20、40μmol/L)组,阳性药(凋亡抑制剂Z-VAD-FMK,10μmol/L)组,采用MTT法检测细胞活力,用Hoechst33258染色法观察细胞凋亡;免疫荧光染色法观察Bax蛋白激活情况;Western blotting检测细胞色素C在线粒体和胞浆的分布情况;Caspase-3酶活检测试剂盒测定细胞内Caspase-3活性。结果18α-甘草次酸能保护FFAs致HepG2细胞活力下降,减少HepG2细胞脂性凋亡数量;18α-甘草次酸能下调Bax蛋白激活,减少细胞色素C的释放,从而阻止Caspase-3活性的增高,且呈剂量相关性。结论18α-甘草次酸对FFAs致HepG2细胞的脂毒性中存在保护作用,其作用机制与抑制线粒体凋亡通路有关。

Objective To investigate the protective effect of 18α-glycyrrhetic acid on lipotoxicity in HepG2 cells induced by free fatty acids(FFAs)and its potential cellular pathway.Methods The HepG2 cell steatosis model in vitro were incubated with 1 mmol/L FFAs,oleic acid(OA)-palmitic acid(PA)(2:1).The HepG2 cells were randomly divided into control group,model group,18α-glycyrrhetic acid 10,20,and 40μmol/L groups,Z-VAD-FMK group.Cell viability was assessed by MTT method.Cell apoptosis was determined by Hoechst 33258.Bax activation was detected by immuno fluorescence staining.Mitochondrial dysfunction was analyzed by detecting cytochrome C release by Western blotting method.Caspase-3 enzyme activity was determined by Caspase-3 enzyme activity kit.Results 18α-Glycyrrhetic acid could protect the decrease of HepG2 cell activity and reduce the number of lipid apoptosis in HepG2.18α-Glycyrrhetic acid could reduce the activation of Bax protein and decrease the release of cytochrome C.Thus,18α-glycyrrhetic acid could decrease the Caspase-3 enzyme activity,and they were dose-dependent.Conclusion 18αGlycyrrhetic acid has the protection of lipotoxicity in HepG2 cells induced by FFAs,and its mechanism may be related to inhibition of mitochondrial apoptosis pathway.