脂联素抑制AKT信号拮抗砷诱导肝细胞脂质沉积

Adiponectin inhibits AKT signaling and antagonizes arsenic-induced lipid deposition in hepatocytes

目的探讨重组人脂联素(adiponectin,APN)对砷诱导肝HepG-2细胞脂质沉积的干预效应及其分子机制。方法实验分为对照组、亚砷酸钠(SA)染毒组、rAPN干预+SA染毒组、蛋白激酶B(AKT)抑制剂(GSK690693)干预+SA染毒组,分别对体外培养的HepG-2细胞进行处理,通过细胞涂片油红O染色观察细胞脂质沉积特征,应用生物化学及酶联免疫吸附试验(ELISA)检测线粒体膜电位(ΔΨm)、线粒体肉碱棕榈酰转移酶1(CPT-1)活性、游离脂肪酸(FFAs)和APN含量,免疫印迹检测磷酸化AKT(p-AKT)、线粒体谷胱甘肽S转移酶K1(GSTK1)和炎症因子半胱天冬酶1(Caspase-1)水平。结果相比对照组,SA染毒组显示细胞内脂质沉积,ΔΨm、CPT-1活性、GSTK1和APN水平下降,而FFAs、p-AKT和Caspase-1水平上调。相比SA染毒组,重组人APN干预后显示细胞脂质沉积程度减轻,ΔΨm、CPT-1活性、GSTK1和APN水平升高,而FFAs、p-AKT和Caspase-1水平下调。类似地,GSK690693干预后亦显示细胞脂质沉积程度减轻,ΔΨm、CPT-1活性、GSTK1水平升高,p-AKT和Caspase-1水平下调。结论砷诱导HepG-2细胞脂质沉积与AKT信号激活有关,脂联素可通过抑制AKT信号拮抗砷诱导肝细胞脂质代谢障碍及脂质沉积发生。

Objective To investigate the effects and molecular mechanism of recombinant human adiponectin(APN)on arsenic induced lipid deposition in hepatic HepG-2 cells.Methods The experiments were divided into control group,sodium arsenite(SA)ex­posure group,rAPN intervention+SA exposure group,and protein kinase B(AKT)inhibitor(gsk690693)intervention+SA exposure group.After cultured HepG-2 cells in vitro were treated respectively,the characteristics of cellular lipid deposition were observed by Oil Red O staining of cells smears,and the methods of biochemical or enzyme-linked immunosorbent assay(ELISA)were applied to determine the levels of intracellular mitochondrial membrane potential(ΔΨm),mitochondrial carnitine palmitoyltransferase-1(CPT-1)activity,free fatty acids(FFAs)and APN.Western-blotting(WB)was applied to detect the levels of phosphorylated AKT(p-AKT),mitochondrial Glutathione S-transferase K1(GSTK1)and inflammatory factor Caspase-1.Results Compared with control group,SA exposed group showed intracellular lipid deposition and decreased levels of mitochondriaΔΨm,CPT1 activity,GSTK1 or APN,while the levels of FFAs,p-AKT and Caspase-1 were increased.Compared with SA exposed group,recombinant human APN(rAPN)in­tervention led to the alleviated lipid deposition in hepatocytes and increased levels ofΔΨm,CPT1 activity,GSTK1 or APN,while the levels of FFAs,p-AKT and Caspase-1 were decreased.Similarly,the intervention of AKT inhibitor showed also the reduced cellular lipid deposition and the upregulated levels ofΔΨm,CPT1 or GSTK1,while the levels of p-AKT and caspase-1 were downregulated.Conclusion Arsenic induced lipid deposition in HepG-2 cells is associated with AKT signaling,and APN can antagonize arsenic-in­duced lipid metabolism disorder and lipid deposition in hepatocytes via inhibiting AKT signaling.