高酮血症抑制过渡期奶牛中性粒细胞胞外诱捕网形成的机制

Neutrophil extracellular traps (NETs) formation is the third defence function of neutrophils except for phagocytosis and degranulation. Dairy cows with ketosis displayed innate immune suppression, which may be associated with NETs, while the underlying mechanism is unknown. Therefore, neutrophils experiment are carried out both in dairy cows with hyperketonemia and in vitro. The effects of β-hydroxybutyrate (BHBA) and acetoacetic acid (AcAc) on the formation and release of NETs will be evaluated. The content, expression and activity of key NETs.formation and regulation molecules ROS, Nox2, and so on will be detected, which will reveal the inhibition effects of BHBA and AcAc on the NETs formation is dependent or independent of ROS. The effects of BHBA and AcAc on the upstream and downstream molecules of mTOR and NF-κB signaling pathway will be detected. Furthermore, the relationship between these signaling pathways and NETs formation.will be evaluated. The effects of BHBA and AcAc on the expression of HCAR2, and the effects of the activation and inhibition of HCAR2 on the mTOR and NF-κB signaling pathway will be detected, thereby evaluating the signaling mechanism of NETs formation medicated by HCAR2-induced mTOR and NF-κB pathways, which will provide valuable theoretical base to reveal a mechanism on NETs formation inhibited by hyperketocemia in ketotic dairy cows.

新发现中性粒细胞胞外诱捕网（NETs）是中性粒细胞除吞噬和脱颗粒作用之外的第三种防御机能。酮病奶牛存在的先天免疫抑制可能与高酮血症抑制NETs的形成有关，但其机制仍不清楚。本项目拟通过高酮血症病牛中性粒细胞体内和体外实验，观测酮血症主要成分β羟丁酸（BHBA）和乙酰乙酸（AcAc）对NETs形成和释放的影响，检测NETs形成及调节关键分子ROS和PLC等的含量、表达和活性，明确BHBA和AcAc抑制NETs形成是否依赖ROS；明确BHBA和AcAc对中性粒细胞mTOR和NF-κB通路的上游和下游信号分子的影响及其与NETs形成的关系；检验BHBA和AcAc对中性粒细胞BHBA受体HCAR2表达以及激活和抑制HCAR2对mTOR和NF-κB通路的影响，探讨HCAR2所调节的mTOR和NF-κB通路介导NETs形成的可能性，为揭示高酮血症抑制过渡期奶牛血液NETs形成的机制提供理论依据。

奶牛酮病是围产期奶牛高发的能量代谢障碍性疾病。其会导致机体免疫功能紊乱和系统性炎症，表现为酮病奶牛继发感染性疾病的发病率提高，如酮病奶牛乳房炎患病率提高3-5倍。中性粒细胞胞外诱捕网（NETs）是中性粒细胞除吞噬和脱颗粒作用之外的第三种防御机能。酮病奶牛存在的先天免疫抑制可能与高酮血症抑制NETs的形成有关，但其机制仍不清楚。. 本项目主要围绕探索酮病对奶牛外周血中性粒细胞NETs释放的影响及其内在的分子机制。本项目明确了酮病奶牛外周血中性粒细胞自噬积累和NETs释放增加，明确了酮病奶牛中性粒细胞NETs关键蛋白的表达增加。体外高浓度BHBA和ACAC可以促进NETs关键蛋白的表达和NETs的释放，并进一步证明了中性粒细胞释放NETs的过程需要GPR109A-mTOR-自噬和ROS/NF-κB信号通路的参与。此外，通过抑制自噬或激活mTOR可以阻断ACAC对NETs释放的促进作用；通过抑制GPR109A、自噬和ROS产生均可阻断BHBA对NETs释放的促进作用。. 以上结果说明酮病奶牛高BHBA和ACAC血症可以通过激活GPR109A-mTOR自噬途径和ROS/NF-κB途径介导的NETs释放，为阐明围产期奶牛酮病发生免疫抑制的机制提供了理论基础。