TLR4介导可溶性尿酸诱导的肾小管上皮细胞自噬流异常  

Effect of TLR4 on regulation of autophagy in renal tubular epithelial cells by soluble uric acid

作　　者：易扬 陈缘 蒋远霞 杨海波[2] 谢恺庆[1] YI Yang;CHEN Yuan;JIANG Yuan-xia;YANG Hai-bo;XIE Kai-qing(Department of Nephrology,The Second Affiliated Hospital,Guangxi Medical University,Nanning 530021,China;Department of Microbiology,Guangxi Medical University,Nanning 530021,China)

机构地区：[1]广西医科大学第二附属医院肾内科,广西南宁530021 [2]广西医科大学微生物学教研室,广西南宁530021

出　　处：《中国病理生理杂志》2020年第8期1444-1449,共6页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.81360243);广西自然科学基金资助项目(No.2018GXNSFAA050057)。

摘　　要：目的:探寻Toll样受体4(TLR4)与可溶性尿酸诱导肾小管上皮细胞HK-2自噬的关系。方法:用可溶性尿酸或/和氯喹(CQ)刺激HK-2细胞,采用Western blot检测LC3-Ⅱ和P62的表达水平,通过透射电子显微镜观察细胞内自噬小体及自噬溶酶体形成。在可溶性尿酸刺激HK-2细胞的基础上加上TLR4抑制剂TAK242后采用RT-qPCR检测TLR4的mRNA表达水平,Western blot检测TLR4、LC3-Ⅱ和P62的蛋白水平,并通过自噬双标腺病毒(mRFP-GFP-LC3)标记观察细胞内自噬流的变化。结果:可溶性尿酸可诱导HK-2细胞LC3-Ⅱ和P62的表达上调,其与CQ共同处理后LC3-Ⅱ及P62的表达进一步增加,透射电子显微镜下观察到自噬小体增多,自噬溶酶体少见,提示自噬流受阻。Western blot结果显示,与正常对照组相比,可溶性尿酸组TLR4、LC3-Ⅱ和P62的蛋白水平增高,而TAK242则抑制可溶性尿酸诱导HK-2细胞的TLR4、LC3-Ⅱ和P62的表达水平。自噬双标腺病毒实验结果显示,可溶性尿酸组与对照组相比,自噬小体显著增多,且自噬溶酶体较少;而与可溶性尿酸组相比,TAK242+可溶性尿酸组自噬小体减少,自噬溶酶体相对增多,表明自噬小体与溶酶体融合现象增多,形成自噬溶酶体的进程更顺畅。结论:可溶性尿酸导致肾小管上皮细胞自噬流受阻;同时,可溶性尿酸可通过TLR4介导肾小管上皮细胞自噬流异常。AIM:To investigate the role of Toll-like receptor 4(TLR4)in autophagy induced by soluble uric acid in renal tubular epithelial HK-2 cells.METHODS:After HK-2 cells were co-stimulated with soluble uric acid or/and chloroquine(CQ),the protein expression of LC3-Ⅱ and P62 was determined by Western blot. Autophagosomes and autophagolysosomes were observed by transmission electron microscopy. Next,HK-2 cells were co-stimulated with soluble uric acid and TLR4 inhibitor TAK242. The mRNA expression of TLR4 was detected by RT-qPCR,the protein expression of TLR4,LC3-Ⅱ and P62 was determined by Western blot,and the autophagic flux was observed by the method of mRFPGFP-LC3.RESULTS:The expression of LC3-Ⅱand P62 in the HK-2 cells was up-regulated by soluble uric acid. The expression of LC3-Ⅱ and P62 was further increased after co-stimulated with uric acid and CQ,and the number of autophagic body was increased while the number of autophagolyososome was decreased as observed by TEM,indicating that the autophagic flux was blocked. The expression levels of TLR4,LC3-Ⅱ and P62 in soluble uric acid group were higher than those in control group. Meanwhile,TAK242 inhibited the up-regulation of TLR4,LC3-Ⅱ and P62 by soluble uric acid.The results of mRFP-GFP-LC3 experiment showed that the levels of autophagosomes were significantly higher in soluble uric acid group than that in control group,and the levels of autophagolysosomes were lower. Compared with soluble uric acid group,the level of autophagosomes was decreased,and autophagolysosomes was increased in TAK242+soluble uric acid group,indicating that the fusion of autophagosomes and lysosomes was increased,and the process of autophagolysosome formation was smoother.CONCLUSION:Soluble uric acid leads blocked autophagic flux in renal tubular epithelial cells. Soluble uric acid mediates abnormal tubular autophagic flux through TLR4.

关 键 词：TOLL样受体4 可溶性尿酸 肾小管上皮细胞 自噬 

分 类 号：R691.2[医药卫生—泌尿科学] R363.2[医药卫生—外科学]