基于Nrf2/ARE与ChTLR15/MyD88/NFκB/MAPK-NLRP3信号通路串话探讨乳酸菌抑制鸡球虫感染所致肠道炎性损伤的分子机制

禽类特有ChTLR15在肠道病原感染中作用重要，但其配体及信号通路尚不明确。前期工作表明，球虫子孢子(EtSp)特异激活ChTLR15/NFκB/MAPK-NLRP3(C/N/M/N)通路释放IL-1β，提示该通路激活与炎性损伤及EtSp入侵相关。研究显示，乳酸菌可激活Nrf2抗氧化通路，降低球虫感染所致肠道病变。氧化应激与炎症关系密切，本研究提出乳酸菌活化Nrf2串话ChTLR15/NLRP3，负向调控C/N/M/N通路，抑制EtSp入侵，减缓炎症损伤是乳酸菌抗球虫作用的新机制。研究拟采用siRNA，过表达及通路抑制剂，借助激光共聚焦，QRT-PCR，流式细胞仪等技术从转录/蛋白/细胞水平解析C/N/M/N通路信号转导机制及其与EtSp入侵关系；在此基础上，通过体外/内实验从整体水平阐明乳酸菌活化Nrf2调控C/N/M/N通路并抑制炎症损伤的分子机制。研究为新一代抗球虫制剂研制提供参考。

Avian-specific toll-like receptor 15 (ChTLR15) play an vital role during infection of intestinal pathogen, but the ligand of ChTLR15 and its signal pathway were not clearly reported untill now. Our previous research showed that the signal pathway way of ChTLR15/NFκB/MAPK-NLRP3(C/N/M/N) in DF1 cells was specifically activated by E. tenella sporozoites (EtSp) and IL-1β was released, which suggested that the activation of the C/N/M/N pathway was strictly related to inflammatory injury and EtSp invasion into cells. Several researches displayed that Nrf2 antioxidant pathway was activated by lactic acid bacteria, and the intestinal pathogenic changes caused by Eimeria infection was alleviated by lactic acid bacteria. Considering the oxidation stress and inflammatory injury are closely related, the novel mechanism for lactic acid bacteria on anticoccidial effects was proposed in which the activation of Nrf2 pathway and the crosstalk between Nrf2 and ChTLR15/NLRP3 negatively regulate C/N/M/N pathway and then inhibit invasion of EtSp into cells, and thus decrease inflammatory injury. In this study, the signal trancduction of C/N/M/N pathway and its relation to EtSp invasion of cells were analyzed at the transcription level, protein level and cell level by using small interfering RNA (siRNA), over expression technology and pathway inhibitor, by applying confocal laser scanning, real-time PCR and flow cytometer etc. On the basis of above research, the molecular mechanism for lactic acid bacteria on inhibiting inflammatory injury through activating Nrf2 and regulating C/N/M/N pathway was elucidated at the whole level based on experiment in virto and vivo. This research provide reference for preparing new generation anticoccidial agents.