用sublytic C5b-9刺激上调的KLF5对大鼠肾小球系膜细胞合成IL-36α的影响

Effects of KLF5 expression on IL-36αproduction in the glomerular mesangial cell stimulated by sublytic C5b-9

目的:探讨转录因子Krüppel样因子5(Krüppel-like factor,KLF5)调控sublytic C5b-9刺激大鼠肾小球系膜细胞(glomerular mesangial cell,GMC)合成促炎因子白细胞介素(interleukin,IL)-36α的作用。方法:首先,培养大鼠GMC,体外用sublytic C5b-9刺激GMC后,不同时间点行反转录聚合酶链式反应(reverse transcription polymerase chain reaction,RT-PCR)和蛋白质印迹(Western blot)检查KLF5、IL-36αmRNA和蛋白水平的变化。接着,构建KLF5的过表达(pIRES2-KLF5)及发夹状小干扰RNA(shKLF5)质粒。将pIRES2-KLF5转染GMC或在shKLF5转染GMC后再用sublytic C5b-9刺激,行RT-PCR和Western blot检查过表达或沉默KLF5基因后对GMC合成IL-36α的影响。同时用荧光素酶报告实验检查过表达或沉默KLF5基因对IL-36α启动子活性的影响。结果:用sublytic C5b-9刺激GMC,能显著上调KLF5和IL-36α的mRNA和蛋白表达,且KLF5的表达时相早于IL-36α。过表达KLF5能上调IL-36α的生成,而沉默KLF5基因后再行sublytic C5b-9刺激,由GMC产生的IL-36α则显著下降。sublytic C5b-9刺激GMC或过表达KLF5基因均可提高IL-36α启动子的活性,而沉默KLF5基因则能明显减低sublytic C5b-9上调IL-36α启动子的活性。结论:KLF5的表达对sublytic C5b-9诱导GMC合成IL-36α有促进作用。

Objective:This study aims to explore the regulatory role of transcription factor Krüppel-like factor 5(KLF5)in the proinflammatory factor[i.e.interleukin(IL)-36α]production in rat glomerular mesangial cells(GMC)stimulated by sublytic C5 b-9.Methods:First,the changes of KLF5 or IL-36αmRNA and protein at different time were examined by reverse transcription PCR(RTPCR)and Western blot in the GMC stimulated by sublytic C5 b-9.Then,the plasmids of KLF5 overexpression(pIRES2-KLF5)and KLF5 short hairpin RNA(shKLF5)were generated and transfected rat GMC respectively,and the cells were treated with or without sublytic C5 b-9.The effects of overexpressing KLF5 gene or silencing KLF5 gene on IL-36αsynthesis in the GMC induced by sublytic C5 b-9 were detected by RT-PCR and Western blot.Meanwhile,the activity of IL-36αpromoter after KLF5 gene overexpression or knockdown in the GMC was measured by luciferase reporter assay.Results:The m RNA and protein expression of KLF5 and IL-36αwere obviously elevated in the GMC stimulated by sublytic C5 b-9,and the expression phase of KLF5 was earlier than that of IL-36α.KLF5 overexpression could promote IL-36αproduction,but KLF5 knockdown markedly decreased IL-36αexpression in the GMC upon sublytic C5 b-9 incubation.Sublytic C5 b-9 treatment or KLF5 overexpression could significantly up-regulate IL-36αpromoter activity,but KLF5 gene knockdown remarkably declined IL-36αpromoter activity stimulated by sublytic C5 b-9.Conclusion:The upregulation of KLF5 has a promoting role in IL-36αsynthesis in rat GMC stimulated by sublytic C5 b-9.