维生素D及其受体上调IGF-1抑制TGF-β1/SMAD2/3信号延缓CCl4诱导肝纤维化的进展OA
Mechanistic study of Vitamin D/Vitamin D receptor upregulation of IGF-1 in inhibiting TGF-β1/SMAD2/3 signaling to delay CCl4-induced liver fibrosis progression in mice
目的 探讨维生素D(VD3)及其受体(VDR)信号轴通过上调胰岛素样生长因子1(IGF-1)的表达拮抗肝纤维化的保护作用机制.方法 70只7～8周龄雄性C57BL/6小鼠随机分为对照组;模型组,建立四氯化碳(CCl4)-诱导肝纤维化小鼠模型;模型+VD3组,口服管饲法给予模型小鼠10 IU/(kg·d)VD3;模型+Lenti-VDR shRNA组,通过尾静脉注射Lenti-VDR shRNA慢病毒颗粒实现敲低VDR;模型+Lenti-VDR shRNA+VD3 组;模型+Lenti-shRNA NT 组;模型+Lenti-shRNA NT+VD3 组.人肝星状细胞 LX2 细胞体外实验分组为细胞对照组,LPS组,LPS+Lenti-VDR shRNA组;LPS+Lenti-shRNA NT组,LPS+Lenti-VDR OE组和LPS+Lenti-vector组.qPCR法测定小鼠肝脏组织VDR、IGF-1、Ⅰ型胶原α1(Col1α1)、丝氨酸蛋白酶抑制剂家族E成员1(Serpine 1)、基质金属蛋白酶抑制剂1(Timp1)和纤连蛋白1(Fn1)mRNA相对表达水平.免疫荧光染色测定小鼠肝组织切片中星状细胞活化相关标志物Collagen Ⅰ和内皮细胞相关标志物CD31的表达水平.Western blot法测定小鼠肝脏组织或LX2细胞内VDR、α-平滑肌肌动蛋白(α-SMA)、转化生长因子β1(TGF-β1)、磷酸化-SMAD家族成员2(p-SMAD2)、SMAD2、磷酸化-SMAD 家族成员 3(p-SMAD3)、SMAD3、核因子 κB(NF-κB)亚基 p65、磷酸化-p65、ERK 1/2、磷酸化-ERK 1/2的相对表达水平.ELISA法测定小鼠静脉血清IGF-1、丙氨酸氨基转移酶(ALT)、肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)和CC趋化因子配体2(CCl2)的水平.结果 与对照组相比,模型组小鼠肝组织IGF-1 mRNA和血清IGF-1水平升高(P＜0.05);血清1,25-(OH)2D3水平降低(P＜0.05);ALT水平升高(P＜0.05);小鼠血清TNF-α、IL-6和CCl2水平升高(P＜0.05);肝组织中肝纤维化标志物Col1 α1、Serpine 1、Timp1和Fn1 mRNA水平升高(P＜0.05);肝组织切片Collage Ⅰ和CD31免疫荧光染色相对荧光强度增强(P＜0.05);肝组织中TGF-β1、p-SMAD2/SMAD2、p-SMAD3/SMAD3相对表达水平升高(P＜0.05);p-p65/p65和p-ERK 1/2/ERK 1/2相对表达水平升高(P＜0.05).与模型组相比,模型+VD3组部分逆转了模型组小鼠的上述指标值.与模型+VD3组相比,模型+Lenti-VDR shRNA组恶化了模型组小鼠的上述指标值.与细胞对照组相比,LPS组肝星状细胞活化标志物α-SMA,以及TGF-β1、p-SMAD2和p-SMAD3相对表达水平升高(P＜0.05);与LPS组相比,LPS+Lenti-VDR shRNA组上述蛋白因子相对表达水平升高(P＜0.05),LPS+Lenti-VDR OE组降低(P＜0.05).结论 敲低VDR的表达将通过抑制IGF-1的表达进一步激活TGF-β1/SMAD2/3信号通路和肝星状细胞,恶化CCl4-诱导肝纤维化小鼠模型的疾病进展.
Objective To investigate the protective mechanism of the vitamin D(VD3)/vitamin D receptor(VDR)signaling axis in liver fibrosis,focusing on its ability to upregulate insulin-like growth factor-1(IGF-1)and antagonize the TGF-β1/SMAD2/3 signaling pathway.Methods Seventy male C57BL/6 mice(7-8 weeks old)were randomly assigned to seven groups:control,CCl4-induced fibrosis model,model+VD3(10 IU/kg/day orally),model+Lenti-VDR shRNA,model+Lenti-VDR shRNA+VD3,model+Lenti-shRNA NT,and model+Lenti-shRNA NT+VD3.Human hepatic stellate LX2 cells were also treated in vitro with LPS,Lenti-VDR shRNA,Lenti-VDR over-expression(OE),or controls.qPCR was used to quantify hepatic mRNA levels of VDR,IGF-1,Col1 α1,Serpine1,Timp1,and Fn1.Immunofluorescence detected Collagen Ⅰ and CD31.Western blot measured VDR,α-SMA,TGF-β1,p-SMAD2/3,NF-κB p65,p-p65,ERK1/2,and p-ERK1/2.ELISA quantified serum IGF-1,ALT,TNF-α,IL-6,and CCL2.Results Compared with controls,CCl4-treated mice showed decreased serum 1,25(OH)2D3,elevated ALT,TNF-α,IL-6,CCL2,Col1 α1,Serpine1,Timp1,Fn1,Collagen Ⅰ,CD31,TGF-β1,p-SMAD2/3,p-p65/p65,and p-ERK1/2/ERK1/2(P＜0.05).VD3 administration partially reversed these effects.VDR knockdown via Lenti-VDR shRNA aggravated fibrosis markers and signaling activation.In LX2 cells,VDR knockdown enhanced,whereas VDR overexpression reduced,LPS-induced α-SMA,TGF-β1,p-SMAD2,and p-SMAD3 lev-els(P＜0.05).Conclusion VDR knockdown exacerbates CCl4-induced liver fibrosis by inhibiting IGF-1 expres-sion,thereby activating the TGF-β1/SMAD2/3 pathway and hepatic stellate cell activation.VD3/VDR upregulation of IGF-1 represents a potential therapeutic strategy to delay liver fibrosis progression.
王翠翠;李倩;范旻;姚俊英
新疆维吾尔自治区人民医院临床营养研究所(新疆乌鲁木齐 830001)新疆维吾尔自治区人民医院临床营养研究所(新疆乌鲁木齐 830001)新疆维吾尔自治区人民医院临床营养研究所(新疆乌鲁木齐 830001)新疆维吾尔自治区人民医院临床营养研究所(新疆乌鲁木齐 830001)
医药卫生
肝纤维化维生素D胰岛素样生长因子-1肝星状细胞TGF-β1/SMAD2/3信号通路
liver fibrosisvitamin Dinsulin like growth factor 1hepatic stellate cellsTGF-β1/SMAD2/3 signaling pathway
《广东医学》 2026 (4)
496-507,12
自治区卫生健康青年医学科技人才专项科研项目(WJWY-202324)
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