Effect of ghrelin O-acyltransferase inhibition by small interfering RNA on hepatocyte fatty degeneration and related mechanism of action / 临床肝胆病杂志

ABSTRACT

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.