金黄色葡萄球菌侵染对奶牛乳腺上皮细胞Nrf2和NF-κB信号通路转导的影响

Effects of Sta ph ylococcus aureus infection on Nrf2 and NF-κB signaling pathway in bovine mammary epithelial cells

金黄色葡萄球菌(S.aureus)是奶牛乳腺炎的主要致病菌之一,严重影响奶牛的生理健康和生产性能。本试验使用S.aureus侵染奶牛乳腺上皮细胞(BMECs),观察S.aureus侵袭对BMECs的NF-κB和Nrf2信号通路转导,及炎性因子TNF-α、IL-1β和IL-8表达的影响。采用S.aureus按MOI=1∶1侵染BMECs,在不同的时间点分别使用荧光定量PCR检测Nrf2通路关键基因Nrf2、Keap1、NQO-1与炎性因子TNF-α、IL-1β、IL-8的mRNA表达水平;Western blot检测Nrf2和NF-κB信号通路关键蛋白Nrf2、p65、P-p65、IκBα、P-IκBα的表达。结果显示:Nrf2和NQO-1基因mRNA表达量在感染后2,4 h显著或极显著升高(P<0.05或P<0.01),且Nrf2在感染后6,8 h时极显著下降(P<0.01);Keap1基因mRNA表达量在感染后2,4 h时显著或极显著下降(P<0.05或P<0.01),且在感染后8 h时显著升高(P<0.05)。炎性因子TNF-α、IL-1β和IL-8基因mRNA表达在感染后1,2,3,4,6,8 h时均显著或极显著升高(P<0.05或P<0.01)。Western blot结果显示:Nrf2蛋白表达量在感染后2 h时显著升高(P<0.05),且在感染后6,8 h时显著或极显著下降(P<0.05或P<0.01);p65蛋白磷酸化水平在感染后0.5,1,2,3,4,5,6 h时显著或极显著升高(P<0.05或P<0.01);IκBα蛋白磷酸化水平在感染后1,2,3,4 h时显著或极显著升高(P<0.05或P<0.01)。结果表明,S.aureus侵染BMECs后,通过激活Nrf2和NF-κB信号通路,促进炎性细胞因子表达,参与S.aureus侵袭BMECs的进程,为金黄色葡萄球菌性乳腺炎的防治提供新的思路。

Staphylococcus aureus(S.aureus) is one of the main pathogens causing mastitis in dairy cows,which seriously affects the physiological health and production performance of cows.In this study,S.aureus was used to infect the bovine mammary epithelial cells(BMECs).The effects of S.aureus invasion on the nuclear factor-erythroid 2-related factor 2(Nrf2),the nuclear factor kappa-B(NF-κB) signaling pathway and the mRNA expressions of TNF-α,IL-1β and IL-8 were investigated.S.aureus was used to infect BMECs according to MOI = 1:1.qRT-PCR was used to detect the mRNA expressions of Nrf2,Keap1,NQO-1,TNF-α,IL-1β,and IL-8 at different time.The expressions of Nrf2,p65,P-p65,IκBα,P-IκBαwere detected by Western blot at different time.The results showed that the mRNA expressions of Nrf2 and NQO-1 were significantly increased(P<0.05 or P<0.01)at 2 hand 4 hafter infection,and the mRNA expression of Nrf2 was significantly decreased(P<0.01)at 6 hand 8 hafter infection.The mRNA expression of Keap1 was significantly decreased(P<0.05 or P<0.01)at 2 hand 4 hafter infection,and increased(P<0.05)at8 hafter infection.Additionally,the mRNA expressions of TNF-α,IL-1βand IL-8 were significantly increased(P<0.05 or P<0.01)at 1,2,3,4,6,8 hafter infection.The result of Western blot showed that the expression level of Nrf2 was significantly increased(P<0.05)at 2 hafter infection,and decreased(P<0.05 or P<0.01)at 6 hand 8 hafter infection.The phosphorylation level of p65 was significantly increased(P<0.05 or P<0.01)at 0.5,1,2,3,4,5 and 6 hafter infection.The phosphorylation level of IκBαwas significantly increased(P<0.05 or P<0.01)at 1,2,3 and4 hafter infection.Incondusion when S.aureus infects BMECs,Nrf2 and NF-κB signaling pathways are activated,meanwhile,inflammatory cytokines are expressed,indicating that Nrf2,and NF-κB signaling pathways may participate in the damage of BMECs induced by S.aureus.