肌肽抑制脂多糖诱导的小胶质细胞炎症小体活化和细胞焦亡
Carnosine inhibits LPS-induced inflammasome activation and pyroptosis in microglia
沈佳红 1温雨欣 1徐佳雯 1孙建良1
作者信息
- 1. 浙江大学医学院附属杭州市第一人民医院麻醉科,杭州 310006
- 折叠
摘要
目的:探讨肌肽对脂多糖(LPS)诱导的小胶质细胞炎症小体活化和细胞焦亡的影响,初步阐明其作用机制.方法:建立LPS(10 ng/ml)诱导的小胶质细胞活化模型,CCK-8法检测不同浓度(0.2、1、5、20、50 mmol/L)肌肽干预小胶质细胞6 h后的细胞活性,以及不同浓度肌肽预处理小胶质细胞0.5 h后再加LPS刺激6 h后的细胞活性,筛选药物浓度.筛选获得适宜的肌肽浓度后,将小胶质细胞分为对照组、肌肽组(5 mmol/L)、LPS模型组、LPS+肌肽组进行研究:倒置相差显微镜下观察各组细胞形态变化;ELISA检测各组小胶质细胞培养上清中IL-1β、TNF-α和IL-6水平;碘化丙啶(PI)染色法检测各组细胞焦亡情况;免疫荧光法检测各组小胶质细胞中NOD样受体热蛋白结构域3(NLRP3)蛋白表达情况.结果:与对照组比较,LPS组小胶质细胞存活率明显降低(P<0.01),形态多呈"阿米巴状"激活态,小胶质细胞培养上清中IL-1β、TNF-α、IL-6含量明显升高(P<0.01),PI阳性率和NLRP3阳性细胞数均明显增加(P<0.01);与LPS组比较,LPS+肌肽组小胶质细胞存活率明显升高(P<0.01),"阿米巴状"激活态小胶质细胞数减少,培养上清中IL-1β、TNF-α含量明显降低(P<0.01),IL-6含量降低(P<0.05),PI阳性率和NLRP3阳性细胞数均明显减少(P<0.01).结论:肌肽可抑制LPS诱导的小胶质细胞激活和炎症小体活化,进而抑制细胞焦亡和炎症因子释放.
Abstract
Objective:To investigate the effects of carnosine on lipopolysaccharide(LPS)-induced inflammasome activation and pyroptosis in microglia,and to clarify its mechanism.Methods:Activation model of microglia was established by LPS(10 ng/ml).CCK-8 assay was used to detect cell activity of microglia treated with different concentrations of carnosine(0.2,1,5,20,50 mmol/L)for 6 h,and the cell activity of microglia pretreated with different concentrations of carnosine for 0.5 h and then stimulated with LPS for 6 h,to screen a suitable concentration.Then microglia were divided into control group,carnosine group(5 mmol/L),LPS group,and LPS+carnosine group:cell morphological changes in each group were observed under an inverted phase contrast microscope;levels of IL-1β,TNF-α and IL-6 in microglial culture medium were measured by ELISA;propidium iodide(PI)staining was used to detect py-roptotic cells;immunofluorescence was used to observe protein expression of Nod-like receptor protein 3(NLRP3).Results:Com-pared with control group,cell viability of microglia in LPS group was significantly decreased(P<0.01),the shape of microglia was mostly"amoeboid",levels of IL-1β,TNF-α and IL-6 in microglial culture medium were significantly increased(P<0.01),the posi-tive rate of PI and the number of NLRP3 positive cells were significantly increased(P<0.01).Compared with LPS group,cell viability of microglia in LPS+carnosine group was significantly increased(P<0.01),the number of"amoeboid"microglia was decreased,levels of IL-1β,TNF-α in microglial culture medium were significantly decreased(P<0.01),and the level of IL-6 was decreased(P<0.05),the positive rate of PI and the number of NLRP3 positive cells were both significantly decreased(P<0.01).Conclusion:Carnosine can inhibit LPS-induced microglia activation and inflammasome activation,thereby inhibiting cell pyroptosis and the release of inflammato-ry factors.
关键词
肌肽/脂多糖/小胶质细胞/炎症小体/细胞焦亡Key words
Carnosine/Lipopolysaccharide/Microglia/Inflammasome/Pyroptosis引用本文复制引用
基金项目
国家自然科学基金青年科学基金(81901087)
浙大市一预研基金(YYJJ2019Z03)
杭州市医学重点学科建设项目(OO20200484)
出版年
2024
国家科技期刊平台
NSTL
万方数据