浒苔多糖通过NF-κB/MLCK通路缓解热应激肉鸡肠上皮细胞紧密连接损伤的分子机制

热应激（HS）导致肠上皮细胞紧密连接（TJs）损伤，危害肉鸡生产。浒苔多糖（EPS）具有免疫调节、抗氧化和抗炎等生物活性。申报人前期发现，EPS能改善HS肉鸡空肠TJs蛋白表达，降低NF-κB p65和MLCK蛋白表达量，提示EPS可通过NF-κB/MLCK通路缓解HS肉鸡肠道TJs损伤，但具体作用机制尚不明确。为此，本项目拟通过体内试验，利用qPCR、Western Blot、免疫荧光、免疫组化等实验手段，揭示EPS对HS肉鸡肠道NF-κB/MLCK通路相关信号分子及TJs的调控作用；通过体外试验，构建HS诱导肉鸡原代小肠上皮细胞TJs损伤模型，采用信号通路激活剂和抑制剂进行正反验证试验，结合qPCR、Western Blot、免疫荧光等实验技术，阐明EPS通过NF-κB/MLCK通路缓解HS肉鸡肠道TJs损伤的分子机制。研究结果将为EPS作为抗热应激活性物质在肉鸡上的应用奠定理论基础。

Heat stress (HS) results in the impairment of intestinal epithelial cells tight junctions (TJs), thus negatively affecting broilers production. Enteromorpha polysaccharides (EPS) has multiple biological activities, including immunomodulation, antioxidant and anti-inflammatory etc. We previously found that EPS could improve the protein expression of jejunal TJs, while reduce the protein expression levels of NF-κB p65 and MLCK in HS broilers, suggesting that EPS could alleviate the intestinal TJs injury of HS broilers through the NF-κB/MLCK pathway, however, the specific mechanism is not yet clear. Therefore, the present project aims to study the effect of dietary EPS on NF-κB/MLCK pathway-related signal molecules and TJs in HS broilers through in vivo experiment by using qPCR, Western Blot, immunofluorescence, and immunohistochemistry etc. Furthermore, based on the in vitro experiment, HS-induced primary intestinal epithelial cells TJs injury model of broilers will be constructed, using signaling pathway activators and inhibitors for positive and negative verification tests, and combine with the experimental techniques such as qPCR, Western Blot and immunofluorescence, to reveal that the molecular mechanism of EPS alleviates the intestinal TJs injury by regulating NF-κB/MLCK pathway in HS broilers. The present findings will provide a theoretical foundation for the application of EPS as an anti-HS active substance in broiler chickens.