褪黑素调节LPS诱导奶牛乳腺上皮细胞炎性因子的表达及其机制

为探索褪黑素降低奶牛乳腺上皮细胞炎性损伤的作用机制,分别用不同浓度的脂多糖(LPS)诱导奶牛乳腺上皮细胞(MAC-T)产生炎症反应,采用MTT法确定LPS的最佳致炎剂量,建立体外MAC-T炎症模型;采用ELISA检测褪黑素对LPS诱导MAC-T分泌TNF-α和IL-6、IL-8的影响;采用荧光定量PCR检测褪黑素对LPS诱导MAC-T Toll样受体信号通路关键因子,TLR4、NF-κB p50、NF-κB p65 mRNA表达量的影响。结果显示,5 mg/L LPS是诱导MAC-T炎症模型的最佳剂量,可极显著提高MAC-T培养液上清中TNF-α和IL-6、IL-8的含量(P<0.05),而50μmol/L褪黑素可显著下调LPS诱导的MAC-T炎症因子TNF-α和IL-6、IL-8的蛋白表达(P<0.05)。同时,TLR4/NF-κB信号通路关键因子NF-κB p50 mRNA的表达量也显著下调。表明褪黑素可能通过调节TLR4/NF-κB信号通路,影响LPS诱导的MAC-T炎症损伤反应。

Melatonin Regulates LPS-induced Expression of Inflammatory Cytokines in Bovine Mammary Epithelial Cells and Its Mechanism

To explore the anti-inflammatory effect of melatonin on bovine mammary epithelial cells,MAC-T were induced by LPS at concentration of 0.00,0.1,1.00,5.00,10.00 and 20.00 mg/L,and the activities of MAC-T were detected by MTT assay.Furthermore,MAC-T cells cultured with melatonin for 12 h were induced by LPS at concentration of 5.00 mg/L,and TNF-α,IL-6,IL-8 in the culture supernatant of MAC-T were separately detected by ELISA,also expressions of TLR4,NF-κB p65 and NF-κB p50 were detected by real-time fluorescence quantitative PCR.The results showed that the cell growth inhibition rate in 5.00 mg/L LPS treated group was 7.91%,and the contents of TLR4,IL-6 and IL-8 in the cell culture supernatant were significantly increased(P<0.01).While the expressions of TLR4,IL-6 and IL-8 of MAC-T induced by LPS were significantly down-regulated by MLT(P<0.01).Meanwhile,the expressions of NF-κB p50 which including in the TLR4 signal transduction were also down-regulated in MLT treated group.These results showed that MLT might reduce the inflammatory injury by regulating the TLR4/NF-κB signaling pathway.