HSP72/IRAK-M抑制奶牛乳腺上皮细胞炎症TLR4信号转导机制

Mastitis in dairy cow is in a high incidence all over the world and its prevention and treatment have been becoming an increasingly hotspot of international research. It has been already proved that both HSP72 and IRAK-M increases their expressions in the inflammation, which in turn inhibit inflammatory process. HSP72 influences IRAK-M expression indirectly. However, the signal transmission mechanism of HSP72 and IRAK-M inhibiting inflammatory process is not clear. We hypothesize that HSP72 and IRAK-M mutual regulate and control each other, which negative feedback regulate TLR4 signal transmission mechanism. The research aims to explore the mechanism of HSP72 and IRAK-M in the inhibition of TLR4 inflammation signal transmission. We will study the mutual regulating relationship between HSP72 and IRAK-M both in the inflammation tissue of cow mammary gland (in vivo) and in the inflammation model of epithelial cells（in vitro）using advanced technologies in modern molecular biology and cytobiology, as well as the dynamic changes of the key molecules (IRAK-4/1, TRAF6, NF-κB, p38 and JNK) expression in the TLR4 signal pathway and inflammatory factor (IL-1β, IL-6, IL-8 and TNFα) in the inflammation model of epithelial cell. The correlations between these measured indicators and HSP72/IRAK-M inhibiting TLR4 signal transmission mechanism in both mammary gland and in the inflammation model of epithelial cells will be analyzed. The results will provide a theoretic and experimental base for finding drug target in treating clinical mastitis of dairy cow.

奶牛乳腺炎世界范围内高发，其防治是国际研究的热点。已证实在炎症反应中，HSP72和IRAK-M表达均升高并抑制炎症，而HSP72又间接影响IRAK-M的表达，但HSP72和IRAK-M抑制炎症信号转导机制尚不清楚。本研究拟以HSP72与IRAK-M存在调控关系，且负反馈调控TLR4信号转导为理论假说。从HSP72和IRAK-M抑制TLR4炎症信号转导为切入点，采用分子生物学和细胞生物学等研究手段，观测奶牛乳腺炎症组织（体内）和乳腺上皮细胞炎症模型（体外）中HSP72和IRAK-M相互调控关系，检测乳腺上皮细胞炎症模型中TLR4信号通路中关键因子（IRAK-4/1、TRAF6、NF-κB、p38和JNK）和炎症因子（IL-1β、IL-6、IL-8和TNFα）的变化。分析HSP72/IRAK-M抑制奶牛乳腺上皮细胞炎症TLR4信号转导机制，为寻找治疗奶牛乳腺炎药物靶点提供理论与实验依据。

已证实在炎症反应中，HSP72和IRAK-M表达均升高并抑制炎症，但HSP72和IRAKM抑制炎症信号转导机制尚不清楚。项目以奶牛乳腺上皮细胞炎症模型为研究对象，采用构建HSP72过表达载体、建立稳定高表达HSP72蛋白的牛乳腺上皮细胞系，采用现代生物学技术和方法，检测了HSP72、IRAK-M、IRAK-4/1、TRAF6、P65、p-P65、I-κB、TNFα、IL-1β、IL-6、IL-8等在炎症模型中的变化。项目最终阐明在炎症模型中，HSP72基因通过调控IRAK-M的表达可以提高Tollip、TRAF6的表达以及抑制IRAK-1/4、A20的表达来抑制牛乳腺上皮细胞炎症。此外，项目还研究了中药单体葛根素和黄芩苷以及谷胱甘肽抗炎的调控机制，葛根素和黄芩苷通过降低LPS诱导的奶牛乳腺上皮细胞中p-IκB-α/IκB-α、p-p65/p65蛋白的比值，以及TNF-α、IL-1β、caspase-3、NF-κB和IκB的表达量，减弱LPS引起的损伤，两种药物很可能是通过调控HSP72和NF-κB达到保护细胞的作用。谷胱甘肽可通过诱导HSP72高表达使IRAK-M、Tollip表达升高，降低A20、JNK、P38和p-P38的表达，炎性因子IL-1β、IL-6、IL-8、IL-10、IL-13和IL-17的产生，减轻由LPS诱导的炎性反应，起到保护奶牛乳腺上皮细胞的作用。研究成果将为深入研究奶牛乳腺炎疾病信号转导机制，为寻找治疗奶牛乳腺炎药物靶点提供理论与实验依据。在项目执行过程发表研究论文22篇，获得授权专利5项，中培养了6名研究生。