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

doi:10.3969/j.issn.1004-616x.2017.03.005

癌变·畸变·突变 ›› 2017, Vol. 29 ›› Issue (3): 184-188.doi: 10.3969/j.issn.1004-616x.2017.03.005

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

沈洁, 蔡静明, 孙梦云, 曹玥, 赵艳

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

收稿日期:2017-03-20 修回日期:2017-04-28 出版日期:2017-05-31 发布日期:2017-05-31
通讯作者: 赵艳,E-mail:amyzhaosb@163.com E-mail:amyzhaosb@163.com
作者简介:沈洁,E-mail:1367439434@qq.com。
基金资助:
国家自然科学基金（81273062，81573136）

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

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

Received:2017-03-20 Revised:2017-04-28 Online:2017-05-31 Published:2017-05-31

摘要/Abstract

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·（SO₄）₂·6H₂O，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） mRNA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mRNA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 mRNA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mRNA和蛋白表达均明显下降，Fpn-1 mRNA表达上调而蛋白表达下降，进一步加重NAFLD的进展。

关键词: 铁超载, 铁蛋白, 铁代谢, 非酒精性脂肪肝病, Hepcidin, Fpn

Abstract: OBJECTIVE:To explore the impact of iron overload on iron metabolism index of Hepcidin and Fpn-1 in HepG2 cells,a model of nonalcoholic fatty liver disease (NAFLD). METHODS:Cell viability was determined using the MTT assay at different concentrations (0.062 5,0.125,0.25,0.5,1.0,2.0 mmol/L,respectively) of oleic acid (OA) and iron and to determine the combined drug concentration. The model of NAFLD was established by using 0.5 mmol/L OA in combination with different concentrations (0,0.125,0.25,0.5 mmol/L) of the Fe induced in HepG2 cells. 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-1 were determined by Western blot. A negative control group (no drug treatment of cells) and a 0.5 mmol/L Fe group were used as controls. RESULTS:Compared with the negative control group,0.5 mmol/L OA,0.125,0.25,0.5 mmol/L Fe had no obvious effect on cell viability (P > 0.05). Compared with the control and the 0.5 mmol/L OA groups,in groups of 0.5 mmol/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 the 0.5 mmol/L OA group,the joint effect of 0.5 mmol/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,0.5 mmol/L Fe group,0.5 mmol/L OA combined with 0.25 and 0.5 mmol/L Fe resulted in the decrease of protein expression of Fpn-1 (P < 0.05). CONCLUSION:The joint effect of 0.5 mmol/L OA with various concentrations of Fe aggravated the pathogenesis of NAFLD through lipid deposition and iron dysfunction.

Key words: iron overload, ferritin, iron metabolism, nonalcoholic fatty liver disease, Hepcidin, Fpn

中图分类号:

R994.4

沈洁, 蔡静明, 孙梦云, 曹玥, 赵艳. 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[J]. 癌变·畸变·突变, 2017, 29(3): 184-188.

SHEN Jie, CAI Jingming, SUN Mengyun, CAO Yue, ZHAO Yan. Impact of iron overload on Hepcidin and Fpn-1 in nonalcoholic fatty liver disease model of HepG2 cells[J]. Carcinogenesis, Teratogenesis & Mutagenesis, 2017, 29(3): 184-188.

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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

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