高NEFA和高BHBA血症奶牛血液中性粒细胞免疫机能低下的信号转导机制

The innate immunity suppression is an important biological feature for the transition period of dairy cows. It is known that transition period is a crucial period for incidence of negative energy balance and infectious diseases. Dairy cows with negative energy balance(NEB) are susceptible to infectious disease. Fat mobilization reaction to negative energy balance induces the high blood concentration of nonesterified fatty acid (NEFA) and/ or β-hydroxybutyric acid (BHBA). Therefore，we presumes that high blood concentration of NEFA and BHBA might be the main pathogenic factor for the dysfunction of neutrophilic granulocyte. High blood concentration of NEFA and BHBA might affect the signaling pathway of reactive oxygen species (ROS) and nitric oxide (NO) production and interfere with the signaling pathway of inflammatory reaction in neutrophilic granulocyte. In vivo and in vitro, the effects of high NEFA and / or BHBA on the immune function of neutrophils will be evaluated. And the key molecules changes of neutrophil respiratory burst signaling pathways which are responsible for phagocytic and bactericidal function, and tolls receptor signaling pathway involved in cell inflammatory reaction are determined in the transition period of healthy and diseased dairy cows, and in cultured neutrophils under a modeling high NEFA and / or BHBA. Therefore, the effects of high blood NEFA and BHBA on the immune function of neutrophil could be evaluated, and the mechanism of innate immunosuppression in transition period of dairy cows with NEB could be revealed. This study will provide a valuable theoretical base to elevate the innate immunity function and reduce the incidence of infectious diseases in the transition period of dairy cows.

过渡期奶牛存在先天免疫抑制，且其能量负平衡性疾病和感染性疾病高发，能量负平衡可增加感染性疾病的发生。因此，揭示能量负平衡抑制奶牛先天免疫机能的成因，是提高奶牛免疫机能和防治感染性疾病的关键。据此，本项目提出能量负平衡诱发的高非酯化脂肪酸（NEFA）和高β-羟丁酸（BHBA）有可能是奶牛中性粒细胞免疫功能低下的主要原因，高NEFA和/或高BHBA干扰中性粒细胞活性氧（ROS）和一氧化氮（NO）产生并激活炎性反应的信号转导可能是过渡期奶牛先天免疫抑制的重要环节。通过体内和体外实验评价高NEFA和/或高BHBA血症对中性粒细胞免疫机能的影响，检测中性粒细胞最迅速、最有效的防御机制-ROS和NO产生的信号通路及炎性反应Tolls样受体信号通路关键分子的变化。以确证高NEFA和高BHBA血症导致奶牛中性粒细胞免疫机能低下的信号转导机制，为提高过渡期奶牛先天免疫机能，降低感染性疾病的发生提供理论依据。

本项目运用细胞生物学和分子生物学技术，重点研究酮病奶牛中性粒细胞通过Toller样受体介导的炎症信号通路导致免疫机能低下的调控作用。获得如下研究成果：（1）NEFAs可以激活奶牛中性粒细胞TLR2/4-NF-κB信号通路，导致促炎因子IL-1β、IL-6和TNF-α释放。（2）NEFAs通过TLR2/4-NF-κB信号通路诱导PMN凋亡率的检测结果表明，NEFAs不能通过TLR2/4-NF-κB信号通路调节PMN的细胞凋亡。（3）NEFAs抑制PMN细胞内ROS的产生，导致PMN杀灭微生物能力下降。（4）NEFAs对PMN的脂毒性增加，可能是导致持续性炎症功能紊乱和细胞凋亡的主要原因。（5）BHBA可以增强ROS的产生，诱导中性粒细胞氧化应激。（6）BHBA可以通过氧化应激诱导中性粒细胞产生炎症应答。（7）IGF-1缓解高酮血症奶牛中性粒细胞免疫抑制的机制。以上研究新发现，不仅从基因水平明确了酮病奶牛中性粒细胞免疫机能抑制的机制，同时也揭示了围产期酮病奶牛感染性疾病高发的主要原因，从而为制订针对抗氧化应激、缓解凋亡和炎性损伤为目的的防治措施奠定了理论基础。