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
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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

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R692.3

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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)


Sublytic C5b⁃9 induces IL⁃23 production in glomerular mesangial cells of rats with Thy⁃1 nephritis via up⁃regulated KLF5 expression
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1.Department of Microbiology and Immunology,School of Clinical Medicine,Jiangsu Health Vocational College,Nanjing 211800 ; 2.Department of Immunology,School of Basic Medicine,Nanjing Medical University,Nanjing 211166 ; 3.Department of General Practice,School of Clinical Medicine,Jiangsu Health Vocational College,Nanjing 211800 ,China

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    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(pGL3-IL-23-FL)转染 GMC,再给予sublytic C5b-9刺激,或将pGL3-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:①Thy-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。③Sublytic 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。

    Abstract:

    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-1 nephritis(Thy-1N). Methods:①A 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). ②The levels of mRNA and protein of KLF5 and IL -23 in the GMCs transfected with either a KLF5 overexpressing plasmid (pIRES2 - KLF5)or a KLF5 small interfering plasmid(shKLF5)were examined by RT - qPCR and WB. ③The full - length IL - 23 promoter luciferase reporter gene plasmid(pGL3-IL-23-FL)was transfected into GMCs,followed by stimulation with sublytic C5b-9.Alternatively,pGL3- 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. ④The 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:①The 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. ②Overexpression 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. ④IL-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 Thy-1N in rats,sublytic C5b-9 stimulates GMCs to promote the transcription and expression of the IL-23 gene by upregulating KLF5.

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刘玉,应帅,罗灿,李玉,荣灿,王迎伟,邱文. 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[J].南京医科大学学报(自然科学版),2024,(9):1198-1206

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  • 收稿日期:2024-06-05
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  • 在线发布日期: 2024-09-13
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