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.
Protective role of Sirtuin 1(Sirt1)in mitigating oxidative stress-induced damage to bovine mammary epithelial cells
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.
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