棕榈酸诱发的肝细胞脂质沉积和炎症机制中AMPKα2的作用研究

Research the role of AMPKα2 in palmitic acid-induced lipid deposition and inflammation in hepatocyte

目的:探讨AMP活化蛋白激酶(AMPK)α2在棕榈酸(PA)诱发的肝细胞脂质沉积和脂多糖(LPS)诱导的炎症中的作用和机制。方法:分离小鼠原代肝细胞,将一部分细胞分为非特异性小干扰RNA(siRNA)对照组(siNR)和敲除AMPKα2(siAMPKα2)组,每组细胞再分别用10%牛血清白蛋白(BSA)、200μmol/L PA、磷酸盐缓冲液(PBS)、10μg/mL LPS孵育16 h或6 h。油红O染色检测细胞中的脂质,Western印迹检测核因子κB抑制蛋白(IκB)激酶(IKK)和激活c-Jun氨基末端激酶(JNK)的磷酸化。将另一部分肝细胞分为转染对照腺病毒组(Ad-GPF)和AMPKα2腺病毒组(Ad-AMPKα2),每组再分别用10%BSA、200μmol/L PA、PBS、10μg/mL LPS孵育16 h或6 h,Western印迹检测JNK和IKKα/β的磷酸化。结果:与对照组相比,siAMPKα2组PA诱导的细胞内脂质积累增加(t=2.133,P<0.05),PA和LPS磷酸化JNK(1.51±0.08,t=2.701,P<0.05)和IKKα/β(1.35±0.03,t=1.657,P<0.05)的作用加强,而AMPKα2腺病毒组PA和LPS升高JNK和IKKα/β磷酸化的作用显著受抑制。结论:AMPKα2可能通过抑制JNK及核因子-κB信号通路,改善PA诱导的肝细胞脂质沉积以及和PA和LPS诱导的肝细胞炎症。

Objective:To investigate the role and mechanism of AMP-activated kinase(AMPK)α2 in palmitic acid-induced lipid deposition and lipopolysaccharide(LPS)induced inflammation in hepatocytes.Methods:Primary mouse hepatocytes were isolated and divided into non-specific small interfering RNA(siRNA)control group(siNR)and AMPKα2 knock down group(siAMPKα2).The hepatocytes were incubated with 10%bovine serum albumin(BSA),200μmol/L palmitic acid,PBS,10μg/mL LPS for 16 h or 6 h to induce lipid deposition.The lipid in cells were detected by oil red O staining,and the phosphorylation of IKK and JNK were detected by Western blotting.Another part of hepatocytes were divided into control adenovirus group(Ad-GPF)and AMPKα2 adenovirus group(Ad-AMPKα2),and each group was incubated with 10%BSA,200μmol/L PA,PBS and 10μg/ml LPS for 16 h or 6 h respectively.The phosphorylation of JNK and IKKα/βwere detected by Western blotting.Results:Compared with the control group,in siAMPKα2 group PA induced intracellular lipid accumu lation was increased(t=2.133,P<0.05),the phosphorylation of JNK(1.51±0.08,t=2.701,P<0.05)and IKKα/β(1.35±0.03,t=1.657,P<0.05)by PA and LPS were enhanced,while the increased phosphorylation of JNK and IKKα/βby PA and LPS was significantly inhibited in AMPKα2 adenovirus group.Conclusion:AMPKα2 may improve PA induced lipid deposition as well as PA and LPS induced inflammation in hepatocytes by inhibiting JNK and NF-κB signaling pathway.