氧化苦参碱改善棕榈酸诱导的HepG2细胞脂质沉积及氧化应激的研究

Effect of oxymatrine on lipid accumulation in Hepg2 cells exposed to palmitic acid

目的:观察氧化苦参碱对高脂诱导的HepG2细胞脂质沉积的干预作用及其机制。方法:高脂干预法培养HepG2细胞建立非酒精性脂肪性肝(nonalcoholic fatty liver disease,NAFLD)体外细胞模型。造模成功后随机分为高脂组(Pa组)、氧化苦参碱组(Omt组)和二甲双胍组(Met组),同时设立正常培养基对照组(N组)。药物干预48 h后收集细胞,利用相应试剂盒及2,7-二氯氢化荧光素二乙酸酯(2,7-dichlorofuorescin diacetate,DCFH-DA)荧光探针评估各组细胞内的脂质沉积水平和氧化应激状态;通过PCR和Western blot分别检测参与脂代谢关键分子的基因和蛋白表达。结果:与Pa组相比,氧化苦参碱及二甲双胍干预后可显著降低细胞内甘油三酯(triglyceride,TG)含量(POmt=0.023,PMet=0.043),上调肉碱脂酰转移酶-1(carnitine palmitoyl transferase-1,CPT-1)的m RNA表达(POmt=0.000,PMet=0.000),下调脂肪酸转位酶(fatty acid translocase,FAT/CD36)的m RNA(POmt=0.000,PMet=0.000)及蛋白表达水平(POmt=0.000,PMet=0.000);DCFH-DA荧光染色、总超氧化物歧化酶(total superoxide dismutase,T-SOD)、谷胱甘肽过氧化物酶(glutathion peroxidase,GSH-Px)及丙二醛(malondialdehyde,MDA)的测定结果显示药物干预可显著增强T-SOD(POmt=0.007,PMet=0.007)及GSH-Px(POmt=0.183,PMet=0.020)的活性,减少活性氧(reactive oxygen species,ROS)和MDA(POmt=0.016,PMet=0.001)的生成。结论:氧化苦参碱可改善高脂诱导的HepG2细胞细胞内脂质沉积,可能是通过改善脂代谢及细胞内氧化应激状态实现的。

Objective：To explore the effect of oxymatrine on lipid accumulation in HepG2 cells exposed to high fat intervention and to elucidate the molecular mechanism. Methods：Nonalcoholic fatty liver disease（NAFLD）in HepG2 cells was induced by cultured with medium containing 0.25 mmol/L palmitic acid（Pa）. The concentrations of triglyceride in HepG2 cells were measured by red oil O staining and triglyceride assay kit. After successful modeling,the Pa-medium group were randomly divided into 3 groups：Pa group（0.25 mmol/L）,metformin（Met）group（0.2 mg/m L）,oxymatrine（Omt）group（0.08 mg/m L）. The normal-medium（N）group was established at the same time. After 48 h of drug intervention,the cells were collected respectively. Triglyceride content and the related markers of oxygen stress were measured by kits and 2,7-dichlorofuorescin diacetate（DCFH-DA）staining. The expressions of the key mediators involved in fatty acid transportation and oxidation were examined by real time PCR（q RT-PCR）and Western blot.Results：Red oil O staining and measurement of triglyceride（TG）showed that there were lipid accumulation in HepG2 cells exposedto palmitic acid for 24 h,while the lipid accumulation ameliorated obviously after oxymatrine and metformin administration（POmt=0.023,PMet=0.043）. Compared with Pa group,oxymatrine and met formin administration increased the m RNA expression of carnitine palmitoyl transferase-1（CPT-1）（POmt=0.000,PMet=0.000）,and suppressed the m RNA（POmt=0.000,PMet=0.000）and protein（POmt=0.000,PMet=0.000）expression of CD36 simultaneously. After the treatment with oxymatrine and metformin,the generation of reactive oxygen species（ROS）and malondialdehyde（MDA）（POmt=0.016,PMet=0.001）reduced significantly,while the activities of total superoxide dismutase（T-SOD）（POmt=0.007,PMet=0.007）and glutathion peroxidase（GSH-Px）（POmt=0.183,PMet=0.020）increased. Conclusion：Oxymatrine could alleviate lipid accumulation in HepG2 cells exposed to palmitic acid through reducing the long-chain fatty acid uptake,increasing the β-oxydation and improving oxygen stress state.