非酯化脂肪酸对隐性乳腺炎奶牛中性粒细胞跨内皮迁移的影响机制

Negative energy balance during the lactation period of dairy cattles leads to fat mobilization which results in elevated levels of nonesterified fatty acid (NEFA) in blood. The negative energy balance makes high incidence of subclinical mastitis. A large number of neutrophils migrate into the milk and release peroxides and enzymes which lead to decline in milk quality and serious economic loss. On the basis of that the tight junction between the vascular endothelial cells plays a critical role in neutrophil trans-endothelial migration and disbands in time according to tissue infection or injury. We will investigate the mechanism of neutrophil trans-endothelial migration under the condition of negative energy balance and elevated levels of NEFA during the lactation period of dairy cattles. In this proposal, we will culture vascular endothelial cells and identify which kinds of NEFA affect the trans-endothelial migration of neutrophil. Furthermore, we will clarify the mechanism of the effects of NEFA on the opening and closing of tight junction by detecting the effects of NEFA on the expressions of adhesion molecules and the accumulation of lipid rafts. To reveal the molecular targets of the effects of NEFA on tight junction, we will detect the effects of NEFA on AMP-activated protein kinase signaling pathway. By clarifying the mechanism of the effects of NEFA on the opening and closing of tight junction between the vascular endothelial cells, this project will provide a valuable theoretical base to develop functional additives and prevention and treatment drugs for subclinical mastitis during the lactation period of dairy cattles under negative energy balance.

本项目针对奶牛能量代谢负平衡体脂动员导致高非酯化脂肪酸血症时，隐性乳腺炎发病率升高，大量中性粒细胞迁移至乳中，裂解释放过氧化物和活性酶类，导致原乳品质严重下降或废弃，给奶牛养殖造成巨大经济损失这一生产实践问题，从血管内皮细胞紧密连接对中性粒细胞跨内皮迁移的门控作用，需要根据组织损伤或感染情况及时打开或关闭出发，探讨非酯化脂肪酸对紧密连接开合的影响，以揭示奶牛隐性乳腺炎发病率升高的原因和机制。项目拟通过分离培养血管内皮细胞，评价不同非酯化脂肪酸对中性粒细胞迁移的影响，甄别促进或抑制中性粒细胞迁移的非酯化脂肪酸；分析内皮细胞黏附分子表达及脂筏聚集、检测腺苷酸活化蛋白激酶信号通路，以阐明非酯化脂肪酸影响内皮细胞紧密连接开合的机制，从而探寻如何调控中性粒细胞迁移与乳腺组织感染或损伤相协调，为功能性饲料添加剂和防治药物研发提供思路，以解决能量代谢负平衡状态下奶牛隐性乳腺炎发病率升高这一生产实践问题。

本项目针对奶牛能量代谢负平衡体脂动员导致高非酯化脂肪酸血症时，隐性乳腺炎发病率升高，大量中性粒细胞迁移至乳中，裂解释放过氧化物和活性酶类，导致原乳品质严重下降或废弃，给奶牛养殖造成巨大经济损失这一生产实践问题，以血管内皮细胞紧密连接对中性粒细胞跨内皮迁移的门控作用需要根据组织损伤或感染情况及时打开或关闭为依据，探讨了非酯化脂肪酸NEFA对紧密连接开合的影响，阐述奶牛隐性乳腺炎发病率升高的原因和机制。.项目通过观察NEFA对中性粒细胞迁移的影响；分析内皮细胞黏附分子表达及脂筏聚集、检测腺苷酸活化蛋白激酶信号通路，阐明这些非酯化脂肪酸影响内皮细胞紧密连接开合的机制；从而探寻如何调控中性粒细胞根据乳腺组织感染或损伤及时迁移的思路或方法。.体外研究结果表明油酸、亚油酸对PMN的迁移率没有影响。棕榈酸、硬脂酸可以增加PMN的迁移率；油酸、亚油酸对ICAM，JAM和Claudin的表达没有影响。棕榈酸、硬脂酸可以上调ICAM，JAM和Claudin的表达；油酸、亚油酸对脂筏在血管内皮细胞中的分布没有影响。棕榈酸、硬脂酸可以扰乱脂筏在血管内皮细胞中的分布。NEFA对血管内皮细胞AMPK信号通路影响的检测结果表明油酸、亚油酸对在血管内皮细胞PPAR-a和SREBP1表达没有影响。棕榈酸、硬脂酸可以上调血管内皮细胞PPAR-a和SREBP1表达；阻断AMPK，棕榈酸、硬脂酸上调PPAR-a和SREBP1表达受到抑制。体内研究结果表明添加棕榈酸和硬脂酸后，乳中体细胞数升高；棕榈酸、硬脂酸可以上调ICAM，JAM和Claudin的表达，以及上调RRAR和SREBP的表达。.以上研究结果可为功能性饲料添加剂和防治药物的研发提供实验依据，为生产实践中有效降低隐性乳腺炎发病率提供理论依据和指导。