乙型肝炎病毒X蛋白通过下调微RNA⁃223靶向NLRP3炎症小体促进乙型肝炎病毒相关性肾炎足细胞焦亡

Hepatitis B virus X protein promotes podocyte pyroptosis in hepatitis B virus‐associated glomerulonephritis by down‐regulating microRNA‐223 targeting NLRP3 inflammasome

目的探讨微RNA(microRNA,miRNA)⁃223在乙型肝炎病毒(hepatitis B virus,HBV)X蛋白(HBV X protein,HBx)诱导的HBV相关性肾炎(HBV⁃associated glomerulonephritis,HBV⁃GN)足细胞焦亡中的潜在功能及相关机制。方法采用人肾足细胞中过表达HBx基因来模拟HBV⁃GN的发病机制。实时荧光定量PCR和Western印迹分别检测焦亡相关蛋白[核苷酸结合寡聚化结构域样受体蛋白3(nucleotide⁃binding oligomerization domain⁃like receptor protein 3,NLRP3)、胱天蛋白酶1(Caspase⁃1)、凋亡相关斑点样蛋白(apoptosis⁃associated speck⁃like protein containing a CARD,ASC)]及炎性因子[白细胞介素1β、白细胞介素18]mRNA和蛋白的表达水平;双荧光素酶报告基因实验验证miRNA⁃223的下游靶标;TUNEL染色和流式细胞术检测细胞焦亡情况;免疫荧光检测足细胞损伤标志物Desmin和Nephrin的表达;Hoechst 33342染色观察足细胞细胞核的形态和数量变化;酶联免疫吸附测定检测Caspase⁃1活性。将足细胞分为以下9组:对照组(不予特殊处理)、空质粒组(转染空质粒)、HBx过表达组(转染HBx过表达慢病毒)、HBx过表达+miRNA⁃223 mimic组(共转染HBx过表达慢病毒和miRNA⁃223模拟物)、HBx过表达+miRNA⁃223 inhibitor组(共转染HBx过表达慢病毒和miRNA⁃223抑制剂)、HBx过表达+miRNA⁃223 mimic+NLRP3组(共转染HBx过表达慢病毒、miRNA⁃223模拟物和NLRP3过表达质粒)、HBx过表达+miRNA⁃223 mimic+NLRP3 siRNA组(共转染HBx过表达慢病毒、miRNA⁃223模拟物和NLRP3 siRNA)、HBx过表达+miRNA⁃223 inhibitor+NLRP3组(共转染HBx过表达慢病毒、miRNA⁃223抑制剂和NLRP3过表达质粒)、HBx过表达+miRNA⁃223 inhibitor+NLRP3 siRNA组(共转染HBx过表达慢病毒、miRNA⁃223抑制剂和NLRP3 siRNA)。结果与对照组相比,HBx过表达组miRNA⁃223表达较低(P<0.05)。TUNEL染色和免疫荧光结果显示,敲低NLRP3减弱HBx过表达引起的足细胞损伤和焦亡(P<0.05)。双荧光素酶报告基因实验证明NLRP3是miRNA⁃223的下游靶点之一。功能回复实验证明,NLRP3过表达削弱了miRNA⁃223对足细胞损伤的保护作用(P<0.05)。miRNA⁃223 mimic和NLRP3 siRNA的共同加入使HBx过表达诱导升高的NLRP3炎症小体及炎性因子表达下降,焦亡细胞数量减少(均P<0.05);而同时引入miRNA⁃223 inhibitor和NLRP3过表达质粒则使足细胞中NLRP3炎症小体和炎性因子的表达上调,Caspase⁃1活性升高,焦亡细胞数量增加(均P<0.05)。结论HBx可能通过下调miRNA⁃223靶向NLRP3炎症小体促进HBV⁃GN足细胞焦亡。miRNA⁃223有望成为治疗HBV⁃GN的潜在靶点。

Objective To investigate the potential function and related mechanism of microRNA‐223(miRNA‐223)in the podocyte pyroptosis of hepatitis B virus(HBV)‐associated glomerulonephritis induced by HBV X protein(HBx).Methods HBx‐overexpressing lentivirus was transfected into human renal podocytes to mimic the pathogenesis of HBV‐GN.Real‐time fluorescence quantitative PCR and Western blotting experiments were used to detect the mRNA and protein expression of pyroptosis‐related proteins[nucleotide‐binding oligomerization domain‐like receptor protein 3(NLRP3),apoptosis‐associated speck‐like protein containing a CARD(ASC)and caspase‐1],and inflammatory factors(interleukin‐1βand interleukin‐18),respectively.TUNEL staining and flow cytometry were used to detect the number of pyroptosis cells.Immunofluorescence staining was used to detect the expression of podocytes biomarkers desmin and nephrin;Hoechst 33342 staining was used to observe the morphological and quantitative changes of podocyte nuclei.Enzyme‐linked immunosorbent assay was used to measure caspase‐1 activity.The dual luciferase reporter gene assay was used to verify the downstream target of miRNA‐223.Podocytes were divided into the following nine groups:control group(no special treatment),empty plasmid group(transfected with empty plasmid),HBx overexpression group(transfected with HBx overexpression lentivirus),HBx overexpression+miRNA‐223 mimic group(transfected with HBx overexpression lentivirus and miRNA‐223 mimic),HBx overexpression+miRNA‐223 inhibitor group(transfected with HBx overexpression lentivirus and miRNA‐223 inhibitor),HBx overexpression+miRNA‐223 mimic+NLRP3 group(transfected with HBx overexpression lentivirus,miRNA‐223 mimic and NLRP3 overexpression plasmid),HBx overexpression+miRNA‐223 mimic+NLRP3 siRNA group(transfected with HBx overexpression lentivirus,miRNA‐223 mimic and NLRP3 siRNA),HBx overexpression+miRNA‐223 inhibitor+NLRP3 group(transfected with HBx overexpression lentivirus,miRNA‐223 inhibitor and NLRP3 overexpression plasmid),HBx overexpression+miRNA‐223 inhibitor+NLRP3 siRNA group(transfected with HBx overexpression lentivirus,miRNA‐223 inhibitor and NLRP3 siRNA).Results miRNA‐223 was down‐regulated in HBx overexpression group compared with the control group(P<0.05).TUNEL and immunofluorescence staining showed that NLRP3 knockdown attenuated podocyte injury and pyroptosis induced by HBx overexpression(P<0.05).Dual luciferase reporter gene assay demonstrated that NLRP3 was one of the downstream targets of miRNA‐223.Rescue experiments revealed that NLRP3 overexpression weakened the protective effect of miRNA‐223 in podocyte injury(P<0.05).The addition of miRNA‐223 mimic and NLRP3 siRNA decreased the expression of NLRP3 inflammasome and cytokines,and reduced the number of pyroptosis cells induced by HBx overexpression(all P<0.05);The addition of miRNA‐223 inhibitor and NLRP3 overexpression plasmid significantly increased the expression of NLRP3 inflammasome and cytokines,caspase‐1 activity,and the number of pyroptosis cells(all P<0.05).Conclusion HBx may promote podocyte pyroptosis of HBV-GN via downregulating miRNA‐223 targeting NLRP3 inflammasome,suggesting that miRNA‐223 is expected to be a potential target for the treatment of HBV‐GN.