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

doi:10.3969/j.issn.1672-5069.2020.03.006

摘要/Abstract

摘要： 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。

关键词: HepG2细胞, 非酒精性脂肪性肝病, 姜黄素, 氧化应激, 细胞凋亡 , ,

Abstract: 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-α and 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-α and IL-6 levels in OA group were much higher than that in the control group , while they decreased greatly in curcumin-inervened group ; 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 ; 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.

Key words: HepG2 cells, Nonalcoholic fatty liver disease, Curcumin, Apoptosis, Oxidative stress

吴鹏波,宋琪,俞媛洁,郭一天,饶倩,柴红,谭诗云. 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[J]. 实用肝脏病杂志, 2020, 23(3): 324-327.

Wu Pengbo, Song Qi, Yu Yuanjie, et al.. Effect and underlying mechanism of curcumin on oleic acid-induced steatosis of HepG2 cells in vitro[J]. Journal of Practical Hepatology, 2020, 23(3): 324-327.

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