摘要
目的探讨乙型肝炎病毒X蛋白(hepatitis B virus X protein,HBx)在肾小球足细胞免疫紊乱中的作用及其调控机制。方法选取14只6周龄雄性乙型肝炎病毒(hepatitis B virus,HBV)转基因(HBV transgenic,HBV-Tg)小鼠,以相同周龄野生型(wild type,WT)小鼠作为对照,饲养至不同周龄,检测小鼠24 h尿蛋白量、血生化、肾脏病理以及电镜下足细胞改变;免疫组化法观察HBV-Tg小鼠肾组织HBx蛋白表达及免疫细胞浸润情况。应用pcDNA3.1/myc-HBx质粒转染人足细胞,免疫荧光法检测HBx、足细胞标志物Nephrin在足细胞中的定位;流式细胞术检测足细胞表面主要组织相容性复合体Ⅱ(major histocompatibility complexⅡ,MHC-Ⅱ)、共刺激分子CD40的表达;酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELISA)测定细胞培养上清中多种细胞因子的含量;转录组测序(Transcriptome sequencing,RNA-seq)筛选HBx调控的下游基因,实时定量PCR法验证其表达。利用过表达质粒或短发夹RNA(short hairpin RNA,shRNA)过表达或沉默Notch1基因后,观察其对足细胞表面免疫分子表达及细胞因子分泌的影响。使用Notch受体抑制剂N-[N-(3,5-二氟苯乙酰基-L-丙氨酰基)]-(s)-苯基甘氨酸叔丁酯{N-[N-(3,5-difluorophenyl-L-alanyl)]-(s)-phenylglycine tert-butyl ester,DAPT}阻断HBV-Tg小鼠体内Notch1信号通路后,观察小鼠血生化、肾脏病理改变以及肾组织免疫细胞浸润情况。结果与WT组小鼠相比,HBV-Tg组小鼠尿蛋白量显著增多(P<0.05),血肌酐、尿素氮水平显著升高(均P<0.05),肾脏病理损伤加重,肾组织HBx蛋白异常表达以及免疫细胞浸润明显。HBx转染人足细胞后其表面免疫分子MHC-Ⅱ、CD40表达上调(均P<0.05),培养上清中单核细胞趋化蛋白1(monocyte chemotactic protein-1,MCP-1)、肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)、白细胞介素(interleukin,IL)-1β含量增加(均P<0.05),IL-4/干扰素γ(interferonγ,IFN-γ)分泌失衡。RNA-seq筛选出HBx下游基因,如Notch1、PLA2R、TLR4等,进一步证实HBx可促进Notch1 mRNA和蛋白表达上调(均P<0.05)。将Notch1基因过表达后,HBx诱导的足细胞表面MHC-Ⅱ、CD40表达显著上调(均P<0.05),其上清中MCP-1、TNF-α及IL-1β的含量明显增加(均P<0.05),IL-4/IFN-γ失衡加重;将Notch1基因沉默后,上述结果则呈相反变化。体内实验结果显示,与HBV-Tg+溶剂组相比,HBV-Tg+Notch受体抑制剂(DAPT)组小鼠血肌酐水平显著下降(P<0.05),肾脏病理损伤及免疫细胞浸润明显改善。结论HBx蛋白可促进足细胞Notch1表达上调;Notch1进一步促进足细胞表面免疫分子表达,并调控细胞因子失衡,从而导致肾小球损伤和免疫微环境紊乱。
Objective To investigate the role of hepatitis B virus X protein(HBx)in glomerular podocyte immune disorder and its regulatory mechanism.Methods Fourteen 6-week-old male hepatitis B virus(HBV)transgenic(HBV⁃Tg)mice were selected,and age-matched wild type(WT)mice were as controls.They were fed to different weeks,and 24 h urinary protein,blood biochemistry,renal pathology and podocyte changes under electron microscope were detected.The expression of HBx and the infiltration of immune cells in kidney tissue of HBV⁃Tg mice were observed by immunohistochemistry.Human podocyte cell line was transfected with pcDNA3.1/myc-HBx plasmid,and the localization of HBx and Nephrin in podocytes was detected by immunofluorescence.The expression of major histocompatibility complexⅡ(MHC-Ⅱ)and co-stimulatory molecule CD40 on the cell surface was detected by flow cytometry.The contents of multiple cytokines in cell culture supernatants were determined by enzyme-linked immunosorbent assay.Transcriptome sequencing(RNA-seq)was used to screen the downstream related genes regulated by HBx,and real-time quantitative PCR was used to verify their expressions.After overexpression or silencing of Notch1 gene with overexpressed plasmids or short hairpin RNA(shRNA)in podocytes,the effects on the expression of immune molecules and cytokines secretion was observed.The Notch receptor inhibitor N-[N-(3,5-difluorophenyl-l-alanyl)]-(s)-phenylglycine tert-butyl ester(DAPT)was used to block Notch1 signaling pathway in HBV⁃Tg mice,and then blood biochemistry,renal pathological changes and infiltration of immune cells in kidney tissue were observed.Results Twenty-four-hour urine protein,serum creatinine and urea nitrogen levels were markedly increased(all P<0.05)and renal pathological injury was significantly aggravated in HBV⁃Tg mice than those in WT mice.Also,HBx was up-regulated and immune cells infiltrated in the glomerulus of HBV⁃Tg mice.After transfection with HBx in podocytes,the expression of MHC-Ⅱand CD40 on the cellular surface was up-regulated(all P<0.05),the contents of monocyte chemotactic protein⁃1(MCP-1),tumor necrosis factor⁃α(TNF-α)and interleukin(IL)-1βin the supernatants were increased(all P<0.05),and the secretion of IL-4 and interferonγ(IFN-γ)was unbalanced.RNA-seq screened downstream genes of HBx,such as Notch1,PLA2R,TLR4,etc;and further confirmed that HBx could promote the up-regulation of Notch1 mRNA and protein(all P<0.05).After over-expression of Notch1 gene,HBx-induced expression of MHC-Ⅱand CD40 on the cellular surface was significantly up-regulated(all P<0.05),and the contents of MCP-1,TNF-αand IL-1βin the supernatants were obviously increased(all P<0.05),and the imbalance of IL-4/IFN-γwas further aggravated.After Notch1 gene silencing,the above results showed the opposite changes.In vivo,the results indicated that serum creatinine levels were obviously decreased(all P<0.05),renal pathological injury and immune cell infiltration were significantly alleviated in HBV⁃Tg+DAPT group than those in HBV⁃Tg+DMSO group.Conclusions HBx protein can promote the up-regulation of Notch1 signaling pathway in podocytes.And Notch1 signaling pathway promotes the expression of immune molecules on the surface of podocytes and regulates the imbalance of cytokines,then causes glomerular injury and dysfunction of immune microenvironment.
作者
杨以通
牛余超
张树俭
邵乐平
袁伟杰
Yang Yitong;Niu Yuchao;Zhang Shujian;Shao Leping;Yuan Weijie(Department of Nephrology,Qingdao Municipal Hospital,the Third Clinical Medical School of Qingdao University Medical College,Qingdao 266071,China;Department of Oncology,Qingdao Central Hospital,University of Health and Rehabilitation Sciences,Qingdao 266042,China;Department of Nephrology,Jintan Affiliated Hospital of Jiangsu University,Changzhou 213200,China)
出处
《中华肾脏病杂志》
CAS
CSCD
北大核心
2024年第11期882-893,共12页
Chinese Journal of Nephrology
基金
国家自然科学基金(82100759,82170729)。
