铁超载对非酒精性脂肪肝病HepG2细胞模型中Hepcidin和Fpn-1的影响  

Impact of iron overload on Hepcidin and Fpn-1 in nonalcoholic fatty liver disease model of HepG2 cells

作　　者：沈洁[1] 蔡静明 孙梦云[1] 曹玥[1] 赵艳[1] SHEN Jie;CAI Jingming;SUN Mengyun;CAO Yue;ZHAO Yan(Department of Nutrition and Food Hygiene, School of Public Health, Harbin Medical University Harbin 150081, Heilongjiang, China)

机构地区：[1]哈尔滨医科大学公共卫生学院营养与食品卫生学教研室,黑龙江哈尔滨150081

出　　处：《癌变．畸变．突变》2017年第3期184-188,共5页Carcinogenesis,Teratogenesis & Mutagenesis

基　　金：国家自然科学基金(81273062;81573136)

摘　　要：目的:探讨铁超载对非酒精性脂肪肝病(NAFLD)HepG2细胞模型中铁代谢指标Hepcidin和Fpn-1的影响。方法:采用四甲基噻唑蓝(MTT)法分别检测浓度均为0.062 5、0.125、0.25、0.5、1.0、2.0 mmol/L的油酸(OA)和硫酸亚铁铵[Fe(NH4)2·(SO 4)2·6H 2O,下称Fe]对HepG2细胞活力的影响,确定OA和Fe联合给药浓度。采用0.5 mmol/L OA联合不同浓度(0、0.125、0.25、0.5 mmol/L)的Fe诱导HepG2细胞建立NAFLD细胞模型,并设阴性对照组(未经药物处理的细胞)和0.5 mmol/L Fe单独处理组为对照,测定细胞内甘油三酯(TG)和铁蛋白(Fn)含量。采用实时荧光定量PCR(qPCR)法和Western blot法测定细胞中铁调素(Hepcidin)、膜铁转运蛋白(Fpn-1)m RNA和蛋白的表达。结果:与阴性对照组相比,0.5 mmol/L的OA,0.125、0.25、0.5 mmol/L的Fe对细胞存活率无明显影响(P>0.05)。与对照组和0.5 mmol/L OA组相比,0.5 mmol/L OA与各浓度Fe联合作用时,随着铁浓度增大,细胞内的TG和铁蛋白(Fn)含量逐渐增加,差异具有统计学意义(P<0.05)。与对照组和0.5 mmol/L OA组相比,0.5 mmol/L OA联合不同浓度Fe处理后Hepcidin的m RNA和蛋白表达均明显下降(P<0.05);0.5 mmol/L OA联合不同浓度Fe处理后Fpn-1 m RNA的表达均明显上调(P<0.05);与对照组相比,0.5 mmol/L Fe组、0.5 mmol/L OA联合0.25、0.5 mmol/L Fe组Fpn-1蛋白水平明显降低(P<0.05)。结论:0.5 mmol/L OA联合不同浓度Fe处理时可引起细胞发生脂质沉积,使体内正常铁代谢发生紊乱,Hepcidin的m RNA和蛋白表达均明显下降,Fpn-1 m RNA表达上调而蛋白表达下降,进一步加重NAFLD的进展。OBJECTIVE:To explore the impact of iron overload on iron metabolism index of Hepcidin and Fpn-1in HepG2cells,a model of nonalcoholic fatty liver disease(NAFLD).METHODS:Cell viability was determined using the MTT assay at different concentrations(0.0625,0.125,0.25,0.5,1.0,2.0mmol/L,respectively)of oleic acid(OA)and iron and to determine the combined drug concentration.The model of NAFLD was established by using0.5mmol/L OA in combination with different concentrations(0,0.125,0.25,0.5mmol/L)of the Fe induced in HepG2cells.The contents of intracellular triglyceride(TG)and ferritin(Fn)were determined.The mRNA levels of Hepcidin and Ferroportin1(Fpn-1)were determined by quantitative real-time PCR and the protein expression of Hepcidin and Fpn-1were determined by Western blot.A negative control group(no drug treatment of cells)and a0.5mmol/L Fe group were used as controls.RESULTS:Compared with the negative control group,0.5mmol/L OA,0.125,0.25,0.5mmol/L Fe had no obvious effect on cell viability(P>0.05).Compared with the control and the0.5mmol/L OA groups,in groups of0.5mmol/L OA joint with various concentrations of Fe and with increased of the concentration of iron,intracellular TG and ferritin(Fn)content increased gradually and significantly(P<0.05).Compared with the control and the0.5mmol/L OA group,the joint effect of0.5mmol/L OA with various concentrations of Fe resulted in the decrease of mRNA and protein expression of Hepcidin(P<0.05),resulted in the increase of mRNA of Fpn-1(P<0.05).Compared with the control,0.5mmol/L Fe group,0.5mmol/L OA combined with0.25and0.5mmol/L Fe resulted in the decrease of protein expression ofFpn-1(P<0.05).CONCLUSION:The joint effect of0.5mmol/L OA with various concentrations of Fe aggravated the pathogenesis of NAFLD through lipid deposition and iron dysfunction.

关 键 词：铁超载 铁蛋白 铁代谢 非酒精性脂肪肝病 HEPCIDIN Fpn-1 

分 类 号：R994.4[医药卫生—毒理学]