Klotho与自噬在脓毒症急性肾损伤细胞中的表达趋势  

The Changes of Klotho and Autophagy in HK-2 Cells Induced by LPS

作　　者：陈宇[1] 陈若如[1] 黄蔚霞[1] CHEN Yu;CHEN Ruoru;HUANG Weixia(Department of Nephrology,Wenzhou Traditional Chinese Medicine Hospital,Wenzhou,325000)

机构地区：[1]温州市中医院肾内科,温州325000

出　　处：《中国中西医结合肾病杂志》2023年第10期862-866,I0002,共6页Chinese Journal of Integrated Traditional and Western Nephrology

基　　金：浙江省自然科学基金资助项目(No.LY16H150007)。

摘　　要：目的:研究脂多糖(LPS)诱导的人近曲肾小管上皮细胞(HK-2)中Klotho与自噬的变化及其关系。方法:HK-2细胞分别在浓度为0、1、5、10、50、100μg/ml的LPS干预下孵育3 h,明确最佳的LPS诱导浓度。继而分别在LPS干预下孵育3 h、6 h、12 h、24 h,明确LPS诱导的最佳时间点。从而建立脓毒症急性肾损伤的细胞模型,Westernblot检测HK-2细胞中Klotho蛋白、LC3蛋白和P62蛋白的表达,电镜观察自噬体的形成,免疫荧光观察细胞自噬激活情况。SPSS 23.0软件采用独立样本资料的t检验进行统计学分析。结果:HK-2细胞在不同浓度梯度LPS诱导3 h后,Klotho蛋白在LPS 10μg/ml浓度时表达最弱。自噬相关蛋白包括LC3和P62,在LPS 10μg/ml浓度时LC3-Ⅱ表达最显著,P62表达最弱。HK-2细胞在不同时间梯度LPS诱导(10μg/ml浓度)下,Klotho蛋白在LPS作用24 h时表达最弱,而LC3-Ⅱ在该时间点表达最显著,P62表达最弱。电镜显示HK-2细胞在LPS 10μg/ml浓度诱导24 h时可发现自噬小体和吞噬泡现象。免疫荧光也同样显示细胞在LPS 10μg/ml浓度诱导24 h时自噬激活。结论:LPS诱导的HK-2细胞中Klotho减少,自噬增强;Klotho和自噬在脓毒症急性肾损伤细胞中的变化具有剂量和时间依赖性,在LPS 10μg/ml的浓度预处理24 h最显著。Objective:Investigate the changes and relationship between Klotho and autophagy in immortalized proximal tubular epithelial human cell line HK-2 cells which were induced by lipopolysaccharide(LPS).Methods:Cell model of sepsis-induced AKI was established by HK-2 cells that were exposed to LPS.HK-2 cells were incubated with 0,1,5,10,50,or 100μg/ml LPS for 3,6,12,or 24 hours to determin the best LPS incubation concentration and time.The protein of Klotho,LC3 and P62 were detected by Westernblot,and autophagosomes were observed by electron microscope and fluorescence microscope.Statistical analyses were performed using Student’s t-test by SPSS 23.0.Results:Cell model of sepsis-induced AKI was successfully established by HK-2 cells that were exposed to LPS.The activation of autophagy was indicated by the expression of LC3-Ⅱ/L3-Ⅰand p62.Klotho reduction and autophagy activation reached the maximum in HK-2 cells 24 hours after LPS incubation on the concentration of 10μg/ml.Also autophagosomes could be seen by electron microscope and fluorescence microscope on this concentration and time.Conclusion:It was a process of Klotho protein reduction and autophagy protein intension in HK-2 cells induced by LPS,the expression of Klotho and autophagy behaved dose and time dependence in septic AKI cell models,particularly 24 hours after LPS incubation on the concentration of 10μg/ml.

关 键 词：LPS HK-2 脓毒症 急性肾损伤 KLOTHO 自噬 LC3-Ⅱ P62 

分 类 号：R459.7[医药卫生—急诊医学] R692[医药卫生—治疗学]