二氢姜黄素对油酸诱导的非酒精性脂肪肝体外模型的预防作用与机制研究

Preventive effect and underlying mechanism of dihydrocumin on NAFLD in oleic acid-treated HepG2 cells

目的研究二氢姜黄素对非酒精性脂肪肝病(NAFLD)的预防作用与机制。方法 HepG2细胞经0.5 mmol/L油酸(OA)与二氢姜黄素(0、5、10、20μmol/L)同时处理24 h后,检测细胞内三酰甘油(TG)、活性氧自由基(ROS)、一氧化氮(NO)含量以及细胞对荧光D-葡萄糖同系物(2-NBDG)的摄取能力;采用RT-qPCR和Western blotting方法检测细胞中糖脂代谢与氧化应激相关基因固醇调节元件结合蛋白1c(SREBP-1C)、Patatin样磷酯酶结构域蛋3(PNPLA3)、过氧化物酶体增殖物激活受体α(PPARα)、磷脂酰肌醇-3-激酶(PI3K)、磷酸化蛋白激酶B(pAKT)、蛋白激酶B(AKT)和核转录因子Nrf2(Nrf2)的mRNA与蛋白表达量。结果与对照组相比,OA处理组细胞内TG、ROS及NO含量明显上升,对2-NBDG的摄取能力降低,SREBP-1C和PNPLA3的mRNA及蛋白表达上调,PPARα、PI3K、pAKT/AKT和Nrf2的蛋白表达下调;与OA处理组相比,OA+二氢姜黄素组细胞内TG与NO含量降低,对2-NBDG的摄取能力升高,SREBP-1C和PNPLA3的mRNA与蛋白表达下调,PPARα、PI3K和Nrf2的蛋白表达及pAKT/AKT上调。结论二氢姜黄素对体外NAFLD细胞模型的预防作用机制包括:通过抑制脂合成关键基因(SREBP-1C和PNPLA3)及诱导脂氧化关键基因PPARα的表达,预防肝细胞脂堆积;通过上调PI3K的表达及pAKT/AKT水平,缓解胰岛素抵抗,促进肝细胞对葡萄糖的摄取;通过上调Nrf2的表达量,降低细胞内NO含量,缓解肝细胞的炎症反应及氧化损伤。

Objective To evaluate the preventive effect of dihydrocumin（DHC） on an in vitro model of nonalcoholic fatty liver disease（NAFLD） and investigate the signal transduction pathways underlying DHC treatment. Methods Oleic acid（OA, 0.5 mmol/L） induced hepatic steatosis was established in HepG2 cells as in vitro model of NAFLD. After cells were co-treated by OA and DHC（0, 5, 10, and 20 μmol/L） for 24 h, the cellular contents of triglyceride（TG）, reactive oxygen species（ROS） and NO were determined by cellular biochemical assays. Signaling pathways involved in glucolipid metabolism and oxidative stress including SREBP-1 C, PNPLA3, PPARα, PI3 K, the phosphorylation of AKT（pAKT）, AKT, and Nrf2 on the mRNA and protein levels were determined by RT-qPCR and Western blotting. The glucose uptake was determined by fluorospectrophotometry using 2-NBDG as a fluorescence probe. Results Compared with the control group, the content of TG, ROS and NO was significantly increased, the uptake of 2-NBDG was decreased. and the expression of SREBP-1 C and PNPLA3 on the mRNA and protein levels were up-regulated and the protein expression levels for PPARα, PI3 K and Nrf2, as well as the ratio of pAKT to AKT were down-regulated in OA-induced cells. Compared with OA treated group, DHC decreased the levels of cellular TG and NO, as well as the mRNA and protein expression levels of SREBP-1 C and PNPLA3, and increased the uptake of 2-NBDG, while at the same time increasing the cellular glucose uptake and the protein expression levels of PPARα, PI3 K, pAKT/AKT, and Nrf2 in OA-induced HepG2 cells. Conclusion DHC protected OA-induced hepatic steatosis by inhibiting lipid accumulation and oxidative/nitrative stress and increasing hepatic insulin sensitivity. Furthermore, the effect of DHC is likely associated with its role in the regulation of SREBP-1 C, PNPLA3, PPARα, Nrf2, PI3 K and AKT signaling pathways. DHC may have the effects on relieving the resistance of insulin and promoting the uptake of glucose in the hepatocytes by upregulating PI3 K expression and pAKT/AKT level. Through increasing the expression of Nrf2 and reducing the content of NO in the cells, DHC could alleviate the inflammatory reaction and oxidative damage of liver cells.