断奶仔猪小肠上皮屏障损伤的CHOP介导内质网应激细胞凋亡机制及壳寡糖保护效应研究

Disruption of intestinal epithelial barrier is a commonly encountered disease of early weaned piglets in practice, however, the underlying molecular mechanism of this disease is unclear. Transcription factor CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP) is a critical factor in endoplasmic reticulum (ER) stress-induced apoptosis, which has been recently revealed to play an important role in the pathogenesis of colitis. It is not clear whether CHOP and CHOP-mediated apoptosis involved in the apoptosis and injury of intestinal epithelial cells in early weaned piglets. In the present project, the gut cell apoptosis will be detected by morphological observation, terminal-deoxynucleoitidy transferase mediated nick end labeling (TUNEL) in vivo and in vitro. The expression profile of CHOP, the upstream unfolded protein response moleculars, as well as the downstream apoptosis proteins will be determined in intestinal epithelial cells of early weaned piglets by multiple technologies, such as real-time PCR, Western blotting, immunohistochemical staining and siRNA. One the basis, the effect of chitosan oligosaccharide to prevent intestinal epithelial barrier disruption will be investigated in vitro. Moreover, its underlying mechanism will be elucidated. The results will be important for us to identify new targets to improve intestine health status with special nutritional intervention in pigs.

小肠上皮屏障损伤是早期断奶仔猪面临的普遍问题，但损伤发生的分子机制尚不清楚。CHOP是内质网应激细胞凋亡途径的关键凋亡信号分子，其与人类肠炎发生密切相关。然而，断奶仔猪小肠上皮细胞中CHOP及介导的凋亡相关蛋白表达规律和与肠上皮细胞损伤的关系未见报道。本项目拟采用体内外相结合的方法，通过形态学检测、原位缺口末端标记和流式细胞分析等研究早期断奶仔猪肠上皮细胞凋亡的发生发展规律，运用免疫组化、小RNA干扰、RT-PCR及Western blotting技术，考察小肠上皮细胞中CHOP，及其上游内质网应激信号蛋白和下游细胞凋亡相关蛋白的表达规律，揭示CHOP介导的细胞凋亡与断奶仔猪小肠上皮损伤的关系及作用的分子机制。在此基础上，探索壳寡糖对肠上皮屏障的保护效应及可能作用途径。研究结果将为阐明小肠上皮屏障损伤的内质网应激细胞凋亡机制及通过营养手段调控仔猪肠道健康提供重要理论依据。

小肠上皮屏障损伤是早期断奶仔猪面临的普遍问题，但损伤发生的分子机制尚不清楚。CHOP是内质网应激细胞凋亡途径的关键凋亡信号分子，其与人类肠炎发生密切相关。然而。断奶仔猪小肠上皮细胞中CHOP及介导的凋亡相关蛋白表达规律和与肠上皮细胞损伤的关系未见报道。本项目采用体内外相关结合的方法，通过形态学检测、原味缺口末端标记和流式细胞分析等研究了早期断奶仔猪肠上皮细胞凋亡的发生规律，运用免疫组化、RT-PCR及western blotting技术，考察了小肠上皮细胞中CHOP及内质网应激信号蛋白和细胞凋亡相关蛋白的表达规律。结果发现，断奶应激导致仔猪空肠细胞凋亡显著增加、肠道屏障受损、通透性增加、养分转运功能紊乱，抑制仔猪生长；断奶后整体养分摄入的进一步限制，将显著加重断奶应激的危害，加重肠细胞凋亡和上皮屏障的损伤，影响肠道健康；断奶应激导致仔猪空肠细胞周期、免疫和代谢等功能的变化，断奶后7d空肠细胞功能逐渐恢复；断奶后整体养分摄入的进一步限制，对肠道代谢中脂肪代谢的影响更大；CHOP介导的内质网应激凋亡是导致仔猪断奶后肠道损伤的主要途径之一，死亡受体途径参与调控，但线粒体途径无变化；空肠上皮的糖基化过程被扰乱是CHOP介导的内质网应激凋亡导致肠上皮损伤的关键点。在此基础上，从天外和体内分别探索了不同条件下添加COS壳寡糖（COS）对肠上皮屏障的保护效应及可能作用途径。结果发现，断奶早期外源高剂量短期补充COS可显著缓减断奶应激对仔猪生产性能的抑制，显著降低空肠细胞的凋亡率，改善空肠上皮的通透性，且显著影响CHOP介导内质网应激凋亡途径关键分子基因表达，同时有效缓解肠道氧化应激损伤； 在diquat诱导氧化应激条件下，饲粮COS的添加显著缓减氧化应激导致的肠上皮损伤，保护和修复肠上皮糖基化过程，下调CHOP介导的内质网应激细胞凋亡通路信号分子的表达。研究结果为阐明小肠上皮屏障损伤的内质网应激细胞凋亡机制及通过营养手段手段调控仔猪肠道健康提供了重要理论依据。