铁调素通过抑制肝细胞凋亡在肝纤维化中的作用机制研究

Effects of hepcidin on liver fibrosis by inhibiting hepatocytes apoptosis.

目的明确在小鼠肝纤维化发生、发展过程中铁调素(hepcidin)的调控作用。方法将野生型C57BL/6(WT)小鼠和hepcidin基因敲除(HAMP-/-)小鼠按随机数字表法分为正常对照组(n=10)和四氯化碳(CCl 4)组(n=10)。正常对照组小鼠给予橄榄油灌胃,CCl 4组小鼠给予25%CCl 4(CCl 4︰橄榄油=1︰3)灌胃,每周2次,共6周。通过酶联免疫吸附试验(ELISA)检测CCl 4灌胃1周、4周和6周小鼠血清中hepcidin的水平;通过Masson染色和苏木精-伊红(HE)染色来分别观察CCl 4所诱导的小鼠肝脏纤维化严重程度,以及肝内的炎症水平;通过实时荧光定量PCR(qPCR)检测小鼠肝脏内纤维化相关基因Ⅰ型胶原(CollagenⅠ)、Ⅲ型胶原(CollagenⅢ)和转化生长因子β(TGF-β)的表达,炎症相关基因白细胞介素-1β(IL-1β)、白细胞介素-6(IL-6)和肿瘤坏死因子-α(TNF-α)的表达,以及凋亡相关基因Bax、Fas、Bcl-xl和Bcl 2的表达;最后采用流式细胞术检测hepcidin多肽对CCl 4诱导的小鼠肝细胞系NCTC1469细胞凋亡的调控作用。结果ELISA结果表明,随着肝纤维化进展,小鼠血清中hepcidin水平呈现出逐渐升高的趋势,在6周时最高,与正常对照组小鼠相比,升高约1.9倍,差异有统计学意义(P<0.01)。肝组织病理学结果显示,与WT型CCl 4组小鼠相比,HAMP-/-CCl 4组小鼠肝纤维化程度和肝脏炎症细胞浸润更严重。qPCR结果显示,与WT型CCl 4组小鼠相比,HAMP-/-CCl 4组小鼠其肝脏CollagenⅠ、CollagenⅢ和TGF-β的表达水平显著升高,差异均有统计学意义(P<0.05);促炎性细胞因子IL-1β、TNF-α和IL-6表达水平升高,差异均有统计学意义(P<0.05);促凋亡基因Bax和Fas的表达也呈现出上调趋势,凋亡抑制基因Bcl-xl和Bcl 2的表达呈下调趋势,差异均有统计学意义(P<0.05)。流式细胞仪分析结果显示,hepcidin可显著抑制CCl 4诱导的小鼠肝细胞系NCTC1469细胞凋亡。结论Hepcidin可以通过抑制肝脏细胞的凋亡进而抑制肝脏炎症和肝纤维化。

Objective To investigate the regulatory effect of hepcidin on mice liver fibrosis.Methods Wild-type(WT)and hepcidin-knockout(HAMP-/-)mice were randomly divided into control group(n=10)and carbon tetrachloride(CCl 4)group(n=10).The normal control group mice were administered olive oil by gavage,and the CCl 4 group mice were administered 25%CCl 4(CCl 4︰olive oil=1︰3)by gavage twice a week for 6 weeks.Enzyme-linked immunosorbent assay(ELISA)was utilized to detect the level of hepcidin in the serum of mice with liver fibrosis at 0,1,4 and 6 weeks.Masson and Hematoxylin-eosin staining were used to observe mice liver fibrosis and inflammation changes.Quantitative real-time PCR(qPCR)was used to detect the expression of CollagenⅠ,CollagenⅢ,transforming growth factor-β(TGF-β),interleukin-1β(IL-1β),interleukin-6(IL-6),tumor necrosis factor-α(TNF-α),apoptosis related genes Bax,Fas,Bcl-xl and Bcl 2.Finally,flow cytometry was used to confirm the hepcidin regulatory effect on CCl 4-induced mice hepatic cell line NCTC1469 apoptosis.Results Serum ELISA result showed that hepcidin was increased gradually.Compared with control mice,the serum hepcidin level in fibrotic mice after 6 weeks CCl 4 gavage increased by 1.9 times,the difference was statistically significant(P<0.01).Masson and HE staining showed that the HAMP-/-mice had a more severe collagen deposition and liver inflammatory cells infiltration.QPCR results showed that compared with WT mice after CCl 4 administration,the HAMP-/-mice after CCl 4 administration had higher mRNA expression levels of CollagenⅠ,CollagenⅢ,TGF-β,IL-1β,TNF-αand IL-6,the differences were statistically significant(P<0.05).Compared with WT mice after CCl 4 administration,the mRNA level of apoptosis-related genes Bax and Fas expression were higher but Bcl-xl and Bcl 2 expression were lower in those of HAMP-/-mice,the differences were statistically significant(P<0.05).Flow cytometry result showed that hepcidin can inhibit CCl 4-induced mice hepatic cell line NCTC 1469 apoptosis in vitro.Conclusion Hepcidin can suppress liver inflammation and fibrosis through inhibiting hepatocytes apoptosis.