摘要
目的:我们发现Toll样受体9(TLR9)在嘌呤霉素氨基核苷(PAN)处理的足细胞中上调。本研究试图确定TLR9是否参与PAN对足细胞的损伤作用。方法:(1)用PAN诱导体外培养的足细胞损伤模型,以定量逆转录PCR(qRT-PCR)和Western Blot检测足细胞TLR9及该通路其他组分的变化。同时,利用流式细胞术检查足细胞凋亡情况。用PAN诱导大鼠足细胞损伤,以qRT-PCR和免疫组化法检测大鼠肾小球中TLR9表达变化。(2)将TLR9干扰RNA(siRNA)表达质粒和对照质粒分别转染足细胞,用Western Blot检测比较核因子κB(NF-κB)p65和p38丝裂原活化蛋白激酶(p38 MAPK)磷酸化、qRT-PCR比较白细胞介素12(IL-12)的mRNA变化,流式细胞仪比较足细胞凋亡情况。结果:(1)与对照组相比,经PAN刺激的足细胞,其TLR9、丝氨酸/苏氨酸蛋白激酶1(IRAK1)、肿瘤坏死因子受体相关因子6(TRAF6)、IL-12的mRNA水平明显上调,磷酸化p65和磷酸化p38显著增加,细胞凋亡增加。PAN大鼠模型的肾小球中TLR9 mRNA表达上调,免疫组化显示TLR9蛋白显著增加。(2)足细胞转染TLR9干扰质粒后,TLR9的mRNA和蛋白表达水平降低;而经PAN处理后,该细胞与转染对照siRNA的细胞相比,其磷酸化p65和磷酸化p38的水平降低,IL-12 mRNA表达降低,足细胞凋亡减少。结论:TLR9信号通路参与了PAN诱导的足细胞损伤过程,其机制可能是促进炎症因子产生及p38 MAPK依赖的细胞凋亡。
Objective:We have found that Toll-like receptor 9 (TLR9) was upregulated in the podocytes treated with puromycin aminonucleoside (PAN).This study aims to determine whether TLR9 mediates PAN-induced podocyte injury.Methodology:(1) PAN was used to induce injury in cultured podocy()s,and the expression or activation of TLR9 and the components of the pathway was examined by qRT-PCR or Western blotting.Podocyte apoptosis was assayed by Annexin V staining followed by flow cytometry.PAN-induced podocyte injury model of rat was generated and TLR9 expression in the glomeruli was examined by immunohistochemistry and qRT-PCR.(2) To explore the role of TLR9 signaling in podocyte injury,we transfected podocytes with TLR9 siRNA expresssing plasmid and control plasmid,respectively,followed by PAN treatment.We then compared the p65 and p38 phosphorylation by Western blotting,and IL12 mRNA levels by qRT-PCR,and cell apoptosis by Annexin V-flow cytometry between the cells transfected with TLR9 or control siRNA plasmid.Results:(1) The mRNA levels of TLR9,IRAK1,TRAF6 and ILl2,as well as the phosphorylation of p65 and p38,and apoptosis in the podocytes was increased by PAN.Both TLR9 mRNA and protein were upregulated in the podocytes of rats treated with PAN.(2) Compared with control plasmid,the transfection with TLR9 siRNA plasmid decreased TLR9 mRNA and protein levels,p65 and p38 phosphorylation,as well as podocyte apoptosis; in addition,mRNA levels of IL12 was also downregulated.Conclusion:TLR9 signaling was involved in the podocyte injury induced by PAN.This finding may provide a novel mechanism underlying podocyte apoptosis in glomerular diseases.
出处
《肾脏病与透析肾移植杂志》
CAS
CSCD
北大核心
2014年第3期207-215,共9页
Chinese Journal of Nephrology,Dialysis & Transplantation
基金
国家自然科学基金(81370827)
国家重点基础研究发展计划(973计划)[2012CB517606]
江苏省自然科学基金(BK20131324)