Objective: To observe the effect of crocetin on autophagy in rat hepatocytes exposed to lipopolysaccharide (LPS) and D-galactosamine (D-gal) and explore the mechanism.
Methods: Cultured 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.
Results: The 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.
Conclusions: Autophagy is increased in injured rat hepatocytes, and crocetin can promote autophagy in the injured cells to reduce further cell injury.
目的: 研究西红花酸对肝细胞损伤中细胞自噬的影响及其机制。
方法: 体外分离和培养大鼠肝细胞，调整细胞密度为1×108/L，接种于24孔板中，分为对照组、损伤组、西红花酸组、3甲基腺嘌呤（3MA）组和西红花酸+3MA组，肝细胞培养24 h后，加入脂多糖1 mg/L+D-氨基半乳糖胺60mg/L进行细胞损伤处理，西红花酸组和3MA组分别加入西红花酸和3MA进行干预，西红花酸+3MA组则加入西红花酸与3MA进行干预，继续培养12 h。采用ELISA检测各组上清液中的ALT、AST和LDH；细胞经免疫荧光染色后，荧光显微镜观察染色细胞LC3荧光；细胞经染色切片后，透射电子显微镜观察自噬体；Western blot检测肝细胞中LC3、p62、SIRT1的表达。
结果: ALT、AST和LDH水平在肝细胞损伤后升高，3MA处理后达最高，而西红花酸处理后明显降低。肝细胞损伤后LC3荧光增多，西红花酸处理后LC3荧光最多，3MA处理后LC3荧光明显减少。肝细胞损伤后自噬体形成增加，西红花酸处理后自噬体增加明显，3MA处理后自噬体形成明显抑制。肝细胞损伤后LC3和SIRT1表达上调、p62表达下调，西红花酸处理后LC3和SIRT1表达最高、p62表达最低，3MA处理后LC3和SIRT1表达最低、p62表达最高。
结论: 肝细胞损伤后细胞自噬增加，西红花酸可促进肝细胞损伤的自噬、减少肝细胞进一步损伤。
Keywords: autophagy; crocetin; liver cell injury.