TLR4介导自噬流在脂多糖诱导肾小管上皮细胞炎症反应中的作用研究

The role of TLR4-mediated autophagic flux in lipopolysaccharide-induced inflammatory response in tubular epithelial cells

目的:探讨自噬流与脂多糖(LPS)诱导肾小管上皮细胞(HK-2细胞)炎症反应的关系,检测Toll样受体(TLR4)是否参与其中。方法:实验分为以下5组:阴性对照组、LPS(100μg/mL)组、LPS(100μg/mL)+雷帕霉素(RAP)(10μmol/L)组、LPS(100μg/mL)+氯喹(CQ)(10μmol/L)组、LPS(100μg/mL)+TAK242(TLR4拮抗剂)(2μmol/L)组,刺激HK-2细胞,通过实时荧光定量聚合酶链反应(qPCR)检测白细胞介素-1β(IL-1β)、TLR4、选择性自噬接头蛋白(P62)和自噬微管相关蛋白1轻链3(LC3)mRNA表达和Western blotting检测IL-1β、TLR4、P62和LC3蛋白表达;采用自噬双标腺病毒(mRFP-GFP-LC3)转染HK-2细胞检测自噬流,在共聚焦显微镜下观察自噬体与自噬溶酶体数量变化以判断自噬流表达情况。结果:与阴性对照组比较,LPS可诱导HK-2细胞IL-1β、TLR4和P62 mRNA和蛋白表达上调(P<0.05),LC3 mRNA和LC3-Ⅱ蛋白表达上调(P<0.05),共聚焦显微镜图示自噬体数明显增多、自噬溶酶体很少,表明自噬流受阻;与LPS组比较,LPS+RAP组IL-1β、TLR4 mRNA和蛋白表达下调(P<0.05),LC3 mRNA及LC3-Ⅱ蛋白表达上调(P<0.05),P62蛋白表达下调(P<0.05),共聚焦显微镜图示自噬溶酶体数量明显增多,表明自噬流得到改善;LPS+CQ组IL-1β、P62、TLR4 mRNA和蛋白表达上调(均P<0.05),共聚焦显微镜图示自噬体明显增多、基本无自噬溶酶体,表明自噬流受阻;LPS+TAK242组IL-1β、TLR4 mRNA和蛋白表达下调(P<0.05),LC3 mRNA及LC3-Ⅱ蛋白表达上调(P<0.05),P62蛋白表达下调(P<0.05),共聚焦显微镜图示自噬溶酶体数量明显增多,表明自噬流得到改善。结论:LPS诱导肾小管上皮细胞炎症反应,存在自噬流阻断;自噬流负调控LPS诱导肾小管上皮细胞炎症反应,TLR4参与其中。

Objective: To investigate the relationship between autophagy flow and lipopolysaccharide(LPS)-induced inflammatory response in renal tubular epithelial cells(HK-2 cells), and to detect whether toll-like receptor4(TLR4) was involved in the inflammatory response. Methods:The experiment was divided into negative control group, LPS(100 μg/mL) group, LPS(100 μg/mL) + rapamycin(RAP)(10 μmol/L) group, LPS(100 μg/mL)+ chloroquine(CQ)(10 μmol/L) group, LPS(100 μg/mL) + TAK242(TLR4 antagonist)(2 μmol/L)group.HK-2 cells were stimulated. the mRNA expressions ofIL-1β, TLR4, P62, and LC3 were detected by fluorescence quantitative(qPCR) and theproteinexpressionsof IL-1β, TLR4, P62, and LC3 were detected by western blotting. Autophagy flow was detected in HK-2 cells that transfected with autophagy double-labeled adenovirus(mRFP-GFPLC3).The changesin the number of autophagosomes and autophagy lysosomeswere observed by confocal microscopy, and the autophagy flowwas evaluated.Results: Compared with the negative control group,LPS could inducetheup-regulation of mRNA and protein expression of IL-1β, TLR4, and P62 in HK-2 cells(P<0.05), and the expression of LC3 mRNA and LC3-II protein were also up-regulated in HK-2 cells(P<0.05). Confocal microscopy showed a significant increase of autophagosomes and a small number of autophagy lysosomes,indicated that autophagic fluxwas blocked. Compared with the LPS group, in LPS +RAP group, the expression of IL-1β, TLR4 mRNA and protein were down-regulated(P<0.05), LC3 mRNA and LC3-IIprotein were up-regulated(P<0.05), and P62 protein was down-regulated(P<0.05). Confocal microscopy showed that the number of autophagy lysosomes wasincreased significantly and higher than that of autophagosomes, indicated that autophagic flux was improved. In LPS +CQ group, the expression of IL-1β, P62, and TLR4 mRNA and protein were up-regulated(P<0.05). Confocal microscopy showed a significant increase in autophagosomes and basically no autophagy lysosomes, indicated that autophagic flux was blocked. In LPS + TAK242 group, the expression of IL-1β, TLR4 mRNA and protein were down-regulated(P<0.05), LC3 mRNA and LC3-II protein were up-regulated(P<0.05), and P62 protein was down-regulated(P<0.05). Confocal microscopy showed that the number of autophagy lysosomes was significantly higher than that of autophagosomes, indicated that autophagic flux was improved. Conclusion: When LPS induce the inflammatory reaction of renal tubular epithelial cells, autophagic flux isblocked, autophagic flux negatively regulates LPS-inducedrenal tubular epithelial cells inflammation, in which TLR4 is involved.