Effect of ghrelin O-acyltransferase inhibition by small interfering RNA on hepatocyte fatty degeneration and related mechanism of action

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 ( m TOR) , phosphorylated m TOR ( p-m TOR) , 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-m TOR ( P < 0. 05) . Conclusion GOAT inhibition by siRNA can upregulate autophagy and alleviate hepatocyte fatty degeneration, possibly by regulating the AMPK/m TOR pathway.