miR-211-5p靶向TLR4通路减轻自身免疫性肝炎小鼠肝损害

Exogenous hydrogen sulfide miR-211-5p targeting TLR4 pathway mitigates liver damage in autoimmune hepatitis mice

目的 探讨微小核糖核酸-211-5p(miR-211-5p)靶向Toll样受体4(TLR4)通路对自身免疫性肝炎小鼠肝损害的影响。方法 将通过尾静脉注射刀豆蛋白A(Con A)建立的自身免疫性肝炎小鼠随机分为模型组、对照组、沉默组、过表达组,分别经尾静脉注射生理盐水、miR-NC、miR-211-5p antagomir、miR-211-5p agomir。检测各组小鼠肝功能及炎症因子水平;苏木素-伊红(HE)染色观察肝组织病理改变;实时荧光定量聚合酶链反应(RT-qPCR)检测脾脏组织TLR4、肝组织miR-211-5p、TLR4、核因子-κB(NF-κB p65)、NOD样受体热蛋白结构域相关蛋白3(NLRP3)信使核糖核酸(mRNA)表达;Western blot法检测肝组织TLR4、NF-κB p65、NLRP3蛋白表达及磷酸化NF-κB p65(p-NF-κB p65)水平;荧光素酶活性实验验证miR-211-5p是否可靶向调控TLR4。结果 与模型组和对照组比,沉默组ALT、AST、TNF-α和IL-1β水平升高、IL-10水平降低(P<0.05),肝组织有严重炎症细胞浸润,过表达组ALT、AST、TNF-α和IL-1β水平降低、IL-10水平升高(P<0.05),肝组织有轻度炎性细胞浸润。与模型组和对照组比,沉默组脾脏组织TLR4 mRNA表达、肝组织TLR4、NF-κB p65、NLRP3 mRNA及蛋白表达及p-NF-κB p65水平升高,miR-211-5p表达降低(P<0.05),过表达组脾脏组织TLR4 mRNA表达、肝组织TLR4、NF-κB p65、NLRP3 mRNA及蛋白表达及p-NF-κB p65水平降低,miR-211-5p表达升高(P<0.05);TLR4是miR-211-5p的靶基因。结论 miR-211-5p可通过靶向抑制TLR4通路减轻自身免疫性肝炎小鼠肝损害。

This study was designed to investigate the effect of microRNA-211-5p(miR-211-5p) targeting Toll-like receptor 4(TLR4) TLR4 pathway on liver damage in mice with autoimmune hepatitis. Firstly, the autoimmune hepatitis mice established by tail vein injection of concanavalin A(Con A) were randomly divided into model group, control group, silence group and overexpression group, which were injected with normal saline, miRNC and miR-211-5p antagomir, miR-211-5p agomir through tail vein respectively. Then the liver function and inflammatory factor levels of mice in each group were detected. Hematoxylin-eosin(HE) staining was used to observe the pathological changes of liver tissue;RT-qPCR was used to detect TLR4 in spleen tissues, miR-211-5p,TLR4, nuclear factor-κB(NF-κB p65), NOD-like receptor pyrridine domain-related protein 3(NLRP3) mRNA expression in liver tissue;Western blot was used to detect the protein expression of TLR4, NF-κB p65, NLRP3 and the levels of phosphorylated NF-κB p65(p-NF-κB p65) in liver tissue;luciferase activity assay was applied to verify whether miR-211-5p can target and regulate TLR4. Data showed that compared with the model group and the control group, the levels of ALT, AST, TNF-α and IL-1β in the silence group were increased, while the level ofIL-10 was decreased(P<0.05), and there was severeinflammatory cell infiltration in the liver tissue. The levels of ALT, AST, TNF-α and IL-1β in the overexpression group were decreased, while the level of IL-10 wasincreased(P<0.05), and there was mild inflammatory cell infiltration in the liver tissue. Compared with the modelgroup and the control group, the expression of TLR4 mRNA in spleen tissue, the mRNA and protein expressions ofTLR4, NF-κB p65, NLRP3 and p-NF-κB p65 in liver tissue were increased, and the expression of miR-211-5pwere decreased in the silence group(P<0.05), while the overexpression group demonstrated contrary changes(P<0.05). TLR4 is a target gene of miR-211-5p. In conclusion, miR-211-5p can alleviate liver damage in autoimmunehepatitis mice by targeting TLR4 pathway.