OBJECTIVETo observe the effect of crocetin on autophagy in rat hepatocytes exposed to lipopolysaccharide (LPS) and D-galactosamine (D-gal) and explore the mechanism.

METHODSCultured rat hepatocytes were exposed to LPS (1 mg/L) and Dgal (60 mg/L) to induce cell injury and treated with crocetin, 3MA, or crocetin+3MA. Twelve hours after the treatments, the cells were examined for levels of ALT, AST and LDH in the supernatant using ELISA. LC3 fluorescence in the cells following immunofluorescence staining was observed using fluorescence microscopy. Autophagosomes in the cells were observed by transmission electron microscopy, and the cellular expressions of LC3, p62 and SIRT1 were detected using Western blotting.

RESULTSThe levels of ALT, AST and LDH in the hepatocytes were elevated after LPS- and D-gal-induced injury, reached the highest levels after 3MA treatment, but were decreased significantly by crocetin treatment. LC3 fluorescence increased obviously in the injured hepatoctyes, and the increment was the most obvious in crocetin-treated cells; LC3 fluorescence was decreased significantly after 3MA treatment. Cell injury induced obvious increase in autophagy in the hepatocytes, and the number of autophagosomes increased significantly after crocetin treatment but was reduced significantly after 3MA treatment. The cell injury caused an obvious up-regulation of LC3 and SIRT1 expression and down-regulated p62 expression. LC3 and SIRT1 expression levels were the highest and the expression of p62 was the lowest in cells with crocetin treatment. 3MA treatment significantly reduced the expression of LC3 and SIRT1 and increased the expression of p62 in the injured cells.

CONCLUSIONSAutophagy is increased in injured rat hepatocytes, and crocetin can promote autophagy in the injured cells to reduce further cell injury.