牛乳腺炎无乳链球菌β溶血素CylE基因对牛巨噬细胞炎性细胞因子的转录和表达及对信号通路MAPK的影响机制研究

Effects of the β hemolysin CylE gene of Streptococcus agalactiae on transcription and expression of inflammatory cytokines in bovine macrophages and its mechanism in the MAPK signaling pathway

目的探讨牛乳腺炎无乳链球菌β溶血素CylE基因对牛巨噬细胞吞噬功能的免疫调理分子机制。方法利用牛乳腺炎无乳链球菌临床分离野生菌株及β溶血素CylE基因缺失突变株对牛巨噬细胞进行侵染,提取侵染细胞的总mRNA;利用qPCR检测细胞因子Il-8、Il-10、Il-12、TNFα及NOS2的转录水平,ELISA检测相应表达水平。在此基础上,利用免疫印迹实验鉴定无乳链球菌β溶血CylE基因对MAPK信号通路下游接头分子p-38、p44/42的表达及其磷酸化水平的影响,以明确其调控该信号通路的分子机制。结果巨噬细胞-GBS^(WI)组与巨噬细胞-GBS^(ΔcyIE)组相比,IL-10、TNF-α细胞因子上调,而IL-8、IL-12、NOS2细胞因子表达水平降低,表明无乳链球菌β溶血素CylE对细胞因子IL-10、TNF-α的影响结果一致,相对转录水平均上调,同时抑制细胞因子IL-8、IL-12及NOS2的转录。巨噬细胞-GBS;组与巨噬细胞-GBS;组相比,IL-10及TNF-α的相对表达量增加,而IL-8及IL-12的相对表达量下调,差异均具有统计学意义(均P>0.05),NOS2的相对表达量降低。对MAPK信号通路下游分子磷酸化检测结果显示,无乳链球菌β溶血素CylE基因可激活其下游接头分子p38磷酸化为p-p38,不能激活下游分子p44/42。结论根据上述结果,p38 MAPK是无乳链球菌溶血素CylE基因发挥免疫调控的识别途径,可能成为对其进行识别钝化,实现信号通路封闭,提高对牛乳腺炎预防与治疗有效性的新靶位,为有效防控该病原菌的危害,保障乳源安全提供新的思路和有效途径。

Objective To explore the molecular mechanism of immune regulation by the β hemolysin gene of Streptococcus agalactiae(which causes dairy mastitis) in the phagocytosis of bovine macrophages. Methods The wild strain and the strain of S. agalactiae with a mutant β hemolysin gene were used to infect bovine macrophages. Total mRNA was extracted from the infected cells. The levels of IL-8, IL-10, IL-12, TNF-α, and NOS2 transcription were detected using qPCR and the levels of expression were detected using ELISA. The effects of the β hemolysin CylE gene of S. agalactiae on the expression and phosphorylation of the downstream junction molecules p38 and p44/42 in the MAPK signaling pathway were determined using Western blotting. Results Compared to the macrophage-GBS;group, the cytokines IL-10 and TNF-α were up-regulated in the macrophage-GBS;group while the levels of expression of the cytokines IL-8, IL-12, and NOS2 decreased. The β hemolysin CylE gene of S. agalactiae had similar effects on the levels of the cytokines IL-10 and TNF, and the relative levels of transcription were both up-regulated. The β hemolysin CylE gene of S. agalactiae inhibited the relative transcription of the cytokines IL-8 and IL-12. There were significant differences in the levels of transcription(P<0.05), and the relative level of NOS2 expression decreased. Detection of molecular phosphorylation downstream of the MAPK signaling pathway indicated that the β hemolysin CylE gene of S. agalactiae activated phosphorylation of the downstream junction molecule p38 to p-p38 but it did not activate the p-44/42 molecule. Conclusion Together, these results suggest that p38 MAPK was the pathway by which the β hemolysin CylE gene of S. agalactiae regulated immunity. It can be identified and purified, and it will serve as a new target to block the signaling pathway and to improve the effectiveness of prevention and treatment of bovine mastitis. It will provide new insight into the effective prevention and control of this harmful bacterium and ensure the safety of milk sources.