摘要
旨在初步阐明Sirt1在奶牛乳腺氧化应激中对紧密连接的影响及机制.以奶牛乳腺上皮细胞(MAC-T)为研究对象,使用H2O2诱导细胞氧化应激构建细胞模型,添加Sirt1激动剂SRT 2104,使用流式细胞术检测细胞中活性氧的含量;通过 RT-PCR 检测 Sirt1 基因的转录水平;用 Western blot 检测 Sirt1、ZO-1、Occludin、Cludin 3、p38MAPK、JNK、MLCK、MLC2的蛋白表达水平;通过细胞免疫荧光检测ZO-1、Occludin、Cludin 3的表达;同时使用试剂盒检测细胞的抗氧化能力.结果表明,H2O2刺激会显著抑制MAC-T细胞活性并抑制Sirt1基因的表达以及Sirt1、ZO-1、Occludin和Cludin 3的蛋白表达;SRT 2104可以有效改善H2O2刺激导致的ZO-1、Occludin和Cludin 3的蛋白表达下降;同时SRT 2104会增强细胞的抗氧化能力并抑制p38MAPK、JNK、MLCK蛋白的表达和MLC2的磷酸化.以上结果表明Sirt1通过抑制p38MAPK/JNK信号传导途径增强细胞的抗氧化能力和紧密连接蛋白表达.
Abstract
This study aims to investigate the influence and mechanism of Sirt1 on tight junctions in bovine mammary epithelial cells during oxidative stress.The bovine mammary epithelial cell line,MAC-T,was utilized to create an oxidative stress model induced by hydrogen peroxide,in combi-nation with the Sirt1 activator,SRT 2104.The study involved assessing intracellular levels of reac-tive oxygen species using flow cytometry,quantifying Sirt1 gene expression through real-time quantitative PCR,analyzing the protein expression of Sirtl,ZO-1,Occludin,and Claudin-3 via Western blot,and detecting the expression of ZO-1,Occludin,and Claudin-3 through cellular im-munofluorescence.Additionally,the antioxidant capacity of cells was measured using assay kits.The results indicated that hydrogen peroxide stimulation significantly reduced MAC-T cell viabili-ty and suppressed both the gene expression and protein expression of Sirt1,as well as the protein expression of ZO-1,Occludin,and Claudin-3.SRT 2104 effectively restored the decreased protein expression of ZO-1,Occludin,and Claudin-3 induced by H2O2 stimulation.SRT 2104 enhanced cell antioxidant capacity and inhibited the protein expression of p38MAPK,JNK,MLCK,and the phos-phorylation of MLC2.These findings suggest that Sirt1 enhances both cell antioxidant capacity and tight junction protein expression by inhibiting the p38MAPK/JNK signaling pathway.
基金项目
国家自然科学基金(32102624)
国家自然科学基金(32372961)
重庆市自然科学基金(CSTB2022NSCQ-MSX0920)
NETL
NSTL
万方数据