表没食子儿茶素没食子酸酯对脂多糖诱导的奶牛乳腺上皮细胞炎性损伤和凋亡的干预作用

Effects of EGCG on Inflammatory Injury and Apoptosis of BMECs Induced by LPS

【目的】探究表没食子儿茶素没食子酸酯(EGCG)对脂多糖(LPS)诱导的奶牛乳腺上皮细胞(BMECs)炎症和凋亡的影响及其潜在的保护机制。【方法】利用LPS诱导BMECs构建细胞炎症模型。通过CCK-8检测细胞活力,筛选EGCG最佳浓度。将BMECs分为对照组、LPS处理组和EGCG+LPS共处理组,利用实时荧光定量PCR检测细胞中炎性因子、凋亡因子以及抗氧化相关基因mRNA表达水平;利用试剂盒检测BMECs的线粒体膜电位和活性氧(ROS)水平;通过流式细胞术检测BMECs凋亡情况。【结果】使用LPS处理BMECs后,与0 h相比,各时间段细胞中炎性因子白细胞介素-6(IL-6)、IL-8和IL-1β基因mRNA表达量均极显著或显著升高(P<0.01;P<0.05),成功构建BMECs细胞炎性损伤模型。CCK-8检测细胞活力结果显示,EGCG最佳作用浓度为5μmol/L。实时荧光定量PCR结果显示,与对照组相比,LPS诱导的BMECs中IL-6、IL-8、丙二醛(MDA)、Bcl-2相关X蛋白(Bax)和半胱氨酸天门冬氨酸特异性蛋白酶-3(Caspase-3)基因mRNA表达量均极显著升高(P<0.01),谷胱甘肽过氧化物酶(GSH-Px)、B细胞淋巴瘤蛋白-2(Bcl-2)基因mRNA表达量极显著降低(P<0.01);与LPS组相比,EGCG+LPS组细胞中IL-6、IL-8、IL-1β、MDA、Bax和Caspase-3基因mRNA表达量均显著或极显著降低(P<0.05;P<0.01),GSH-Px、超氧化物歧化酶(SOD)、Bcl-2基因mRNA表达量均显著或极显著升高(P<0.05;P<0.01)。线粒体膜电位检测结果显示,与对照组相比,LPS组BMECs红色荧光明显减弱,绿色荧光有所增强,而EGCG和LPS共孵育后则对BMECs红色荧光没有明显影响,绿色荧光相对减弱;ROS水平检测结果显示,与对照组相比,LPS组细胞绿色荧光明显增强,而EGCG和LPS共孵育后,细胞绿色荧光有所减弱。流式细胞术检测结果显示,与对照组相比,LPS处理后死亡细胞和凋亡细胞明显增多,细胞凋亡率极显著升高(P<0.01),而EGCG和LPS共孵育后凋亡细胞则明显减少,细胞凋亡率显著降低(P<0.05)。【结论】EGCG可以通过抑制LPS诱导BMECs中ROS释放和缓解线粒体损伤等综合作用,缓解炎性反应并抑制细胞凋亡,研究结果为EGCG预防和治疗奶牛乳腺炎提供理论基础。

【Objective】The aim of this experiment was to investigate the effects of epigallocatechin gallate(EGCG)on lipopolysaccharide(LPS)induced inflammation and apoptosis in bovine mammary epithelial cells(BMECs)and its potential protective mechanisms.【Method】The cell inflammation model was constructed by LPS-induced BMECs.The cell viability was detected by CCK-8 and the optimal concentration of EGCG was screened.BMECs were divided into control group,LPS treatment group and EGCG+LPS co-treatment group.Real-time quantitative PCR was used to detect the mRNA expression levels of inflammatory factors,apoptosis factors and anti-oxidation-related genes.Mitochondrial membrane potential and reactive oxygen species(ROS)levels of BMECs were detected by kit.The apoptosis of BMECs was detected by flow cytometry.【Result】After LPS-induced BMECs,the mRNA expressions of inflammatory factors interleukin-6(IL-6),IL-8 and IL-1βgenes were extremely significantly or significantly increased at all time periods compared with 0 h(P<0.01 or P<0.05),the cell inflammatory damage model of BMECs was successfully constructed.CCK-8 assay showed that the optimal concentration of EGCG was 5μmol/L.Real-time quantitative PCR results showed that,compared with control group,the mRNA expressions of IL-6,IL-8,MDA,Bax and Caspase-3 genes in LPS-induced BMECs were extremely significantly increased(P<0.01),while the mRNA expressions of GSH-Px and Bcl-2 genes were extremely significantly decreased(P<0.01).Compared with LPS group,mRNA expressions of IL-6,IL-8,IL-1β,MDA,Bax and Caspase-3 genes were significantly or extremely significantly decreased in EGCG+LPS group(P<0.05 or P<0.01),and the mRNA expressions of GSH-Px,SOD and Bcl-2 gene were significantly or extremely significantly increased(P<0.05 or P<0.01).The results of mitochondrial membrane potential showed that compared with control group,LPS significantly weakened the red fluorescence and enhanced the green fluorescence of BMECs,while the co-incubation of EGCG and LPS had no significant effect on the red fluorescence of BMECs,and the green fluorescence was relatively weakened.ROS level detection results showed that compared with control group,the green fluorescence of cells in LPS group was significantly enhanced,while the green fluorescence of cells was weakened after co-incubation of EGCG and LPS.Flow cytometry showed that compared with control group,death cells and apoptotic cells increased significantly after LPS treatment,the apoptosis rate was extremely significantly increased(P<0.01).After co-incubation with EGCG and LPS,the apoptotic cells were significantly decreased,and the apoptotic rate was significantly decreased(P<0.05).【Conclusion】EGCG could alleviate inflammatory response and inhibit cell apoptosis by inhibiting ROS release induced by LPS in BMECs and alleviating mitochondrial damage.The results provide a theoretical basis for the prevention and treatment of mastitis in dairy cows with EGCG.