姜黄素对非酒精性脂肪肝细胞模型保护作用以及机制研究

目的 探讨姜黄素对非酒精性脂肪肝病(NAFLD)细胞模型的保护作用以及保护机制。方法 体外用0.6 mmol/L 油酸诱导HepG2 细胞脂质沉积,建立NAFLD细胞模型。 将HepG2细胞分为对照组(Con)、模型组(OA)、姜黄素对照组和姜黄素干预组。采用Bodipy493/503荧光染色观察各组细胞内脂滴分布,使用透射电镜观察细胞线粒体超微结构变化,使用试剂盒检测培养上清液肿瘤坏死因子α和白介素-6(IL-6),采用DCFH-DA法检测HepG2细胞活性氧(ROS)生成量,采用Hoechst 33258染色检测HepG2细胞凋亡情况,采用Western blot法检测凋亡和炎症相关蛋白Bcl-2、Bax、NF-κB、Caspase-3/9和线粒体内细胞色素C(mCytc)。结果 与 Con组比,OA组 HepG2细胞脂质沉积明显增加,而姜黄素干预组细胞脂质沉积明显减少;OA组细胞线粒体明显损伤,而姜黄素干预组细胞线粒体损伤明显减轻;与Con组比,OA组 HepG2细胞上清液TNF-α和IL-6显著升高,而姜黄素干预组细胞上清TNF-α和IL-6明显降低;OA组HepG2细胞绿色荧光强度为(52.24±5.11)%,显著强于Con组【(6.71±2.31)%, P<0.05],而姜黄素干预的HepG2细胞绿色荧光强度降低; OA组 HepG2细胞凋亡率为(12.12±0.72)%,显著增高Con组【(2.04±0.57)%,P<0.05 ],而黄素干预组细胞凋亡率显著降低【(5.71±0.61)%,P<0.05】;与Con组比,OA组细胞Bax、NF-κB和cleaved-Caspase-3/9蛋白表达增强,而Bcl-2和mCytc表达减弱(P均<0.05),而姜黄素干预组细胞Bax、NF-κB和Caspase-3/9蛋白表达减弱,而mCytc和Bcl-2表达增强(P均<0.05)。结论 姜黄素可缓解NAFLD细胞脂肪变性,其作用机制可能与减轻炎症反应、抑制氧化应激损伤和细胞凋亡有关。

Effect and underlying mechanism of curcumin on oleic acid-induced steatosis of HepG2 cells in vitro

Objective The aim of this experiment was to explore the effect and underlying mechanism of curcumin on oleic acid-induced steatosis of HepG2 cells in vitro.Methods HepG2 cells were treated with or without 1 mmol/L oleic acid(OA)to establish nonalcoholic fatty liver disease(NAFLD)cell model.The HepG2 cells were divided into four groups,e.g.control(con),steatosis model(OA),curcumin control and curcumin-intervened groups.Bodipy493/503 staining was used to detect the distribution of lipid droplets in the HepG2 cells.The ultrastructure of mitochondria was examined by transmission electron microscopy.Reactive oxygen species(ROS)levels were detected by DCFH-DA.The TNF-αand IL-6 levels in the supernatants were measured by a commercial kit.The apoptosis was determined by Hoechst 33258 staining.Western blott was applied to determine the expression of Bcl-2,Bax,mCytc,NF-κB,and Caspase-3/9 proteins.Results Compared with in the control cells,the cells treated with OA showed significantly increased lipid droplets accumulation,while the cells treated with curcumin showed reduced lipid droplets accumulation;the mitochondrial damage including mitochondrial swelling and vesiculation in OA group was more obvious than that in control group,while the mitochondrial damage treated by curcumin was significantly improved;the TNF-αand IL-6 levels in OA group were much higher than that in the control group[(210.11±12.36)pg/ml vs.(80.43±6.87)pg/ml and(14.74±0.78)ng/l vs.(4.03±0.31)ng/l,respectively,all P<0.05],while they decreased greatly in curcumin-inervened group[(125.22±11.54)pg/ml nd(6.47±0.34)ng/l,respectively,both P<0.05];the ROS levels was(52.24±5.11)%in OA group,significantly higher than(6.71±2.31)%in the control group,while it decreased to(37.44±7.21)%in curcumin-treated group(P<0.05);the apoptosis rate in OA group was(12.12±0.72)%,significantly higher than(2.04±0.57)%in the control group,while it decreased significantly in curcumin-treated group[(5.71±0.61)%,P<0.05];the expressions of Bax,NF-κB and cleaved-Caspase-3/9 intensified,and Bcl-2 and mCytc decreased greatly in OA group as compare to those in the control,while the expressions of Bax,NF-κB and Caspase-3/9 decreased,and Cytc and Bcl-2 increased(P<0.05)in curcumin-intervened group.Conclusion Curcumin effectively prevent oleic acid-induced steatosis in HepG2 cells,which might be related to the alleviation of inflammatory reaction,oxidative stress and inhibition of apoptosis.