Sublytic C5b-9上调KLF5促进Thy-1肾炎大鼠肾小球系膜细胞生成IL-23的作用  

Sublytic C5b-9 induces IL-23 production in glomerular mesangial cells of rats with Thy-1nephritis via up-regulated KLF5 expression

作　　者：刘玉 应帅 罗灿 李玉 荣灿 王迎伟 邱文 LIU Yu;YING Shuai;LUO Can;LI Yu;RONG Can;WANG Yingwei;QIU Wen(Department of Microbiology and Immunology,School of Clinical Medicine,Jiangsu Health Vocational College,Nanjing 211800;Department of Immunology,School of Basic Medicine,Nanjing Medical University,Nanjing 211166;Department of General Practice,School of Clinical Medicine,Jiangsu Health Vocational College,Nanjing 211800,China)

机构地区：[1]江苏卫生健康职业学院临床医学院微生物与免疫学教研室,江苏南京211800 [2]南京医科大学基础医学院免疫学系,江苏南京211166 [3]江苏卫生健康职业学院临床医学院全科教研室,江苏南京211800

出　　处：《南京医科大学学报（自然科学版）》2024年第9期1198-1206,共9页Journal of Nanjing Medical University(Natural Sciences)

基　　金：国家自然科学基金(82171740);江苏省高等学校基础科学(自然科学)研究项目资助(23KJD310001)。

摘　　要：目的:研究亚溶解型C5b-9(sublytic C5b-9)上调转录因子Krüppel样因子5(Krüppel-like factor 5,KLF5)促进Thy-1肾炎(Thy-1 nephritis,Thy-1N)大鼠肾小球系膜细胞(glomerular mesangial cell,GMC)产生炎症因子白细胞介素(interleukin,IL)-23的作用。方法:(1)建立大鼠Thy-1N模型和体外培养大鼠GMC,用Western blot(WB)检查Thy-1N大鼠肾组织和受sublytic C5b-9刺激的GMC中KLF5和IL-23的表达。(2)分别将KLF5过表达质粒(pIRES2-KLF5)或KLF5小干扰质粒(shKLF5)转染GMC,通过实时荧光定量PCR和WB检测KLF5和IL-23的mRNA和蛋白水平。(3)将IL-23全长启动子荧光素酶报告基因质粒(p GL3-IL-23-FL)转染GMC,再给予sublytic C5b-9刺激,或将p GL3-IL-23-FL与pIRES2-KLF5或shKLF5共转染GMC,用荧光素酶报告基因实验检测IL-23启动子活性的变化。(4)将慢病毒(lentivirus,LV)包装的LV-shKLF5和LV-shCTR行肾动脉灌注术导入大鼠肾组织,经小动物脏器可见光三维成像和冰冻切片观察GFP表达,证实LV-shCTR在肾组织中富集效率。之后再复制大鼠Thy-1N,用WB检查肾组织中KLF5和IL-23的蛋白表达。结果:(1)Thy-1N大鼠的肾组织和sublytic C5b-9刺激的GMC中,KLF5和IL-23的表达均显著升高,且KLF5的表达高峰稍早于IL-23。(2)在GMC中过表达或敲低KLF5能分别引起IL-23表达的升高或降低。(3)Sublytic C5b-9刺激或KLF5过表达均可增加GMC中IL-23启动子的活性,但敲低KLF5后可明显下调由sublytic C5b-9刺激GMC诱导的IL-23启动子活性。(4)敲低Thy-1N大鼠肾组织中KLF5的表达后,其肾组织中IL-23的表达水平明显降低。结论:大鼠Thy-1N发病早期,sublytic C5b-9刺激GMC后可通过上调KLF5促进IL-23基因的转录与表达。Objective:To explore the role of sublytic C5b-9 upregulating the transcription factor Krüppel-like factor 5(KLF5)in promoting the production of the inflammatory cytokine interleukin-23(IL-23)in glomerular mesangial cells(GMCs)of rats with Thy-1nephritis(Thy-1N).Methods:(1)A rat model of Thy-1N was established and rat GMCs were cultured in vitro.The expression levels of KLF5 and IL-23 in the renal tissues of Thy-1N rats and in GMCs stimulated by sublytic C5b-9 were detected by using Western blot(WB).(2)The levels of mRNA and protein of KLF5 and IL-23 in the GMCs transfected with either a KLF5 overexpressing plasmid(p IRES2-KLF5)or a KLF5 small interfering plasmid(shKLF5)were examined by RT-qPCR and WB.(3) The full-length IL-23promoter luciferase reporter gene plasmid(pGL3-IL-23-FL)was transfected into GMCs,followed by stimulation with sublytic C5b-9.Alternatively,p GL3-IL-23-FL was co-transfected with either pIRES2-KLF5 or shKLF5 into GMCs,and changes in IL-23 promoter activity were measured using a luciferase reporter gene assay.(4)The LV-shKLF5 and LV-shCTR lentivirus vectors were respectively perfused into rat renal tissues via the artery perfusion.After confirming that LV-shCTR could enrich in rat kidney through animal imaging system and frozen section,the Thy-1N was reproduced,and the KLF5 and IL-23 expression in the renal tissues were measured by WB.Results:(1)The expressions of KLF5 and IL-23 were significantly increased in the renal tissues of Thy-1N rats and in GMCs stimulated by sublytic C5b-9,with KLF5 expression peaking slightly earlier than IL-23.(2)Overexpression or knockdown of KLF5 in GMCs led to an increase or decrease in IL-23 expression,respectively.(3) Sublytic C5b-9 stimulation or KLF5 overexpression upregulated the activity of IL-23 promoter,while KLF5 knockdown markedly reduced the IL-23 promoter activity induced by sublytic C5b-9.(4)IL-23 expression in the renal tissues of the rats treated by knocking down of renal KLF5 gene was significantly downregulated.Conclusion:In the early stage of

关 键 词：THY-1肾炎 亚溶解型C5b-9 肾小球系膜细胞 krüppel样因子5 白细胞介素-23 

分 类 号：R692.3[医药卫生—泌尿科学]