siRNA抑制饥饿素-O-乙酰基转移酶对肝细胞脂肪变性的影响及作用机制

摘要

目的 探讨siRNA抑制饥饿素-O-乙酰基转移酶(GOAT)对肝脂肪变性影响及作用机制.方法 游离脂肪酸(FFA)作用于人肝细胞系LO2细胞以诱导肝细胞脂肪变性.FFA与siRNA-GOAT单独或联合处理LO2细胞,分为4组,即空白对照(NC)组:仅用PBS处理;siRNA-GOAT组:仅以终浓度为10 nmol/L的siRNA-GOAT处理;FFA组:仅以终浓度为1 mmol/L的FFA处理;FFA+ siRNA-GOAT组:采用上述浓度FFA和siRNA-GOAT混合处理.肝细胞油红O染色检测脂肪滴形成情况;TG检测试剂盒检测LO2细胞脂质水平;免疫印迹法(Western Blot)、实时定量逆转录聚合酶链反应(RT-PCR)、免疫荧光染色、电子显微镜检测自噬活性;酶联免疫吸附测定法、RT-PCR检测TNFα、IL-6水平.Western Blot检测雷帕霉素(mTOR)、磷酸化mTOR(pmTOR)、AMP-活化蛋白激酶(AMPK)以及磷酸化AMPK(p-AMPK)的蛋白水平变化.计量资料多组间比较采用单因素方差分析,进一步两两比较采用LSD-t检验.结果 FFA+ siRNA-GOAT组脂肪滴形成较FFA组明显减轻,TG水平明显低于FFA组,差异有统计学意义(P ＜0.001).FFA+ siRNA-GOAT组TNFα和IL-6蛋白、mRNA表达水平较FFA组均明显下降,差异均有统计学意义(P值均＜0.005).siRNA-GOTA组LC3-Ⅱ、Beclin-1 mRNA表达水平明显高于NC组,差异均有统计学意义(P值均＜0.001).FFA+siRNA-GOAT组LC3-Ⅱ、Beclin-1 mRNA表达水平明显高于FFA组,差异均有统计学意义(P值均＜0.001).siRNA-GOAT组LC3-Ⅱ、Beclin-1蛋白水平明显高于NC组,差异均有统计学意义(P值均＜0.05).FFA+ siRNA-GOAT组LC3-Ⅱ、Beclin-1蛋白水平明显高于FFA组,差异均有统计学意义(P值均＜0.05).免疫荧光染色示FFA+ siRNA-GOAT组LO2细胞中内源性LC3-Ⅱ表达水平较FFA组、siRNA-GOAT组升高.电子显微镜观察结果示FFA+ siRNA-GOAT组自噬体表达较FFA组增加.自噬抑制剂siRNA-ATG5、3-MA或刺激剂雷帕霉素处理LO2细胞后,FFA+ siRNA-ATG5组、FFA+3-MA组TG水平分别与FFA+ siRNA-GOAT组、FFA+雷帕霉素组比较,差异均有统计学意义(P值均＜0.001).FFA+ siRNA-GOAT组、FFA+雷帕霉素组LC3-Ⅰ/Ⅱ水平明显高于FFA+ siRNA-ATG5组、FFA+3-MA组,差异均有统计学意义(P值均＜0.05).FFA+ siRNA-GOAT组p-AMPK蛋白表达水平明显高于于FFA组、p-mTOR蛋白表达水平明显低于FFA组,差异均有统计学意义(P值均＜0.05).结论 siRNA抑制GOAT可通过调节AMPK/mTOR通路,上调自噬活性,进而减轻肝细胞内脂肪变性.%Objective To investigate the effect of inhibition of ghrelin O-acyltransferase (GOAT) by small interfering RNA (siRNA) on hepatocyte fatty degeneration and related mechanism of action.Methods Human LO2 hepatocytes were treated with free fatty acid (FFA)to induce hepatocyte fatty degeneration.LO2 hepatocytes were treated with FFA and siRNA-GOAT alone or in combination and then divided into normal control (NC) group (treated with phosphate buffered saline alone),siRNA-GOAT group (treated with siRNA-GOAT at a final concentration of 10 nm),FFA group (treated with FFA at a final concentration of 1 mm),and FFA + siRNA-GOAT group (treated with FFA at a final concentration of 1 mm and siRNA-GOAT at a final concentration of 10 nm).Oil red O staining was performed for hepatocytes to identify lipid droplets;the triglyceride (TG) test kit was used to measure the lipid level in LO2 hepatocytes;Western blot,qRT-PCR,immunofluorescent staining,and electron microscopy were used to measure autophagy;ELISA and RT-PCR were used to measure the levels of tumor necrosis factor-α (TNFα) and interleukin-6 (IL-6);ELISA was used to measure the changes in the levels of mammalian target of rapamycin (mTOR),phosphorylated mTOR (p-mTOR),AMP-activated protein kinase (AMPK),and phosphorylated AMPK.A one-way analysis of variance was used for comparison between multiple groups,and the least significant difference t-test was used for further comparison between any two groups.Results Compared with the FFA group,the FFA + siRNA-GOAT group had a significant reduction in the formation of lipid droplets and a significantly lower TG level (P ＜0.001).Compared with the FFA group,the FFA + siRNA-GOAT group had significant reductions in the protein and mRNA expression of TNFα and IL-6 (all P ＜ 0.005).The siRNA + GOAT group had significantly higher mRNA expression of LC3-Ⅱ and Beclin-1 than the NC group (all P ＜0.001).The FFA + siRNA-GOAT group had significantly higher mRNA expression of LC3-Ⅱ and Beclin-1 than the FFA group (all P ＜0.001).The siRNA + GOAT group had significantly higher protein expression of LC3-Ⅱ and Beclin-1 than the NC group (all P ＜ 0.05).The FFA + siRNA-GOAT group had significantly higher protein expression of LC3-Ⅱ and Beclin-1 than the FFA group (all P ＜ 0.05).Immunofluorescent staining showed that compared with the FFA group and the siRNA-GOAT group,the FFA + siRNA-GOAT group had a significant increase in the expression of endogenous LC3-Ⅱ in LO2 hepatocytes.Electron microscopy showed that compared with the FFA group,the FFA + siRNA-GOAT group had a significant increase in the expression of autophagosome.After the LO2 hepatocytes were treated by autophagy inhibitors siRNA-ATG5 and 3-MA or an autophagy stimulant,rapamycin,there was a significant difference in TG level between the FFA + siRNA-ATG5 group and the FFA + siRNA-GOAT group (P ＜ 0.001),as well as between the FFA + 3-MA group and the FFA + rapamycin group (P ＜ 0.001).The FFA + siRNA-GOAT group had a significantly higher level of LC3-Ⅰ/Ⅱ than the FFA + siRNA-ATG5 group (P ＜0.05),and the FFA + rapamycin group had a significantly higher level of LC3-Ⅰ/Ⅱ than the FFA + 3-MA group (P ＜ 0.05).Compared with the FFA group,the FFA + siRNA-GOAT group had significantly higher protein expression of p-AMPK (P ＜ 0.05) and significantly lower protein expression of p-rmTOR (P ＜ 0.05).Conclusion GOAT inhibition by siRNA can upregnlate autophagy and alleviate hepatocyte fatty degeneration,possibly by regulating the AMPK/mTOR pathway.