RANKL在小鼠肝纤维化形成中抑制肝星状细胞的活化

RANKL inhibits activation of hepatic stellate cells during hepatic fibrosis in mice

目的 探讨核因子-κB受体活化因子配体(receptor activator of nuclear factor kappa-B ligand,RANKL)在小鼠肝星状细胞(hepatic stellate cells,HSCs)活化及肝纤维化进程中的生物学作用。方法 将26只6~8周龄雄性C57BL/6鼠随机分为(n=13):肝纤维化造模组[按照5μL/g腹腔注射体积分数10%的四氯化碳(carbon tetracholoride,CCL_(4))油剂溶液,2次/周诱导小鼠肝纤维化]和对照组(同时注射等体积的无菌油剂)。6周后通过RT-qPCR、IHC和Western blot等方法检测肝组织、原代肝细胞(hepatocyte,HC)和HSCs中的RANKL,α-平滑肌动蛋白(α-SMA)和Ⅰ型胶原(Collagen-Ⅰ)的mRNA及蛋白表达水平。用不同浓度外源性重组RANKL细胞因子培养原代HSCs,采用RT-qPCR和IF分别检测各组RANKL、α-SMA和Collagen-ⅠmRNA及蛋白表达水平。结果 造模组小鼠肝组织中RANKL的mRNA和蛋白表达水平显著增高(P<0.05),且蛋白表达呈弥散分布;对照组小鼠体内,RT-qPCR和Western blot结果均显示HC的RANKL mRNA和蛋白表达水平高于在HSCs中的表达水平(P<0.001),且在CCL_(4)诱导小鼠肝纤维化后,HC的RANKL表达水平进一步升高,而HSCs的RANKL表达水平出现下降;在体外刺激原代HSCs活化时加入不同浓度的RANKL重组细胞因子后,与对照组相比,加入重组RANKL后的HSCs在活化过程中RANKL、α-SMA和Collagen-ⅠmRNA和蛋白表达水平均降低(P<0.05或P<0.01或P<0.001),且有剂量依赖性。结论 在小鼠肝纤维化的进程中,HC表达大量RANKL,而RANKL可抑制HSCs的活化。

Objective To explore the biological role of receptor activator of nuclear factor kappa-B ligand(RANKL) in the activation of hepatic stellate cells(HSCs) and the process of hepatic fibrosis.Methods Twenty-six male C57BL/6 mice(6~8 weeks old) were randomly assigned to control group and liver fibrosis group(n=13).The mice in the latter group were injected intraperitoneally with 5 μL/g 10% carbon tetracholoride(CCL_(4),twice/week) to induce liver fibrosis,while those of the former group were given same volume of sterile oil solution.In 6 weeks later,the expression of RANKL,α-SMA and Collagen-Ⅰ at mRNA and protein levels in liver tissue,primary hepatocytes(HCs) and HSCs was detected by quantitative real-time PCR(RT-qPCR),immunohistochemical assay and Western blotting,respectively.After primary HSCs were treated with different concentrations of exogenous recombinant RANKL,the mRNA and protein levels of above molecules were measured with RT-qPCR and immunofluorescence assay.Results The mRNA and protein levels of RANKL were significantly increased in the liver tissue from the model group than the control group(P<0.05),and the protein was in a diffuse distribution.In the control mice,the mRNA and protein levels of RANKL was obviously higher in HCs than HSCs(P<0.001),while CCL_(4) treatment induced liver fibrosis resulted in the expression level of RANKL was further increased in HCs,while decreased in HSCs.After different concentrations of recombinant RANKL were added to stimulate the activation of primary HSCs,the expression of RANKL,α-SMA and Collagen-Ⅰat mRNA and protein levels in HSCs activated by recombinant RANKL was decreased in a dose-dependent manner(P<0.05,P<0.01 or P<0.001).Conclusion In the process of liver fibrosis in mice,HCs express a large amount of RANKL,and RANKL can inhibit the activation of HSCs.