RIP1/RIP3/MLKL介导的细胞程序性坏死在免疫应激诱导的仔猪肠道损伤中的作用及其营养调控

Nutritional regulation is an important method in mitigating intestinal injury induced by immunological challenge in pigs. However, its efficacy is limited because the mechanism(s) by which immunological challenge induces intestinal injury are not completely understood. Necroptosis, a newly discovered form of cell death, plays an important role in tissue damages induced by several factors. This provides us a novel idea to explore the mechanism(s) of intestinal injury induced by immunological challenge. In the current study, firstly, we will investigate the effect of immunological challenge on necroptosis and its signaling pathway in small intestine of piglets. Secondly, we will further explore the effect of inhibiting the key regulatory components (RIP1, RIP3 and MLKL) of necroptosis on the injury of porcine intestine and IPEC-J2. Our objectives will be to elucidate whether necroptosis participates in the regulation of intestinal injury induced by immunological challenge. Thirdly, we will explore the downstream signals involved in RIP1/RIP3/MLKL mediated IPEC-J2 necroptosis. Our objectives will be to elucidate the molecular mechanism(s) of enterocytes necroptosis. Finally, we will investigate the modulatory effects of n-3 polyunsaturated fatty acid (PUFA) on necroptosis and its signaling pathway in small intestine of piglets. Our objectives are to investigate if n-3 PUFA can mitigate intestinal injury induced by immunological challenge via negative modulation of necroptosis. The results will be of great significance for us to elucidate the mechanism(s) of intestinal injury induced by immunological challenge and to explore the methods of nutritional regulation to mitigate the intestinal injury in piglets.

营养调控是缓解猪免疫应激诱导的肠道损伤的重要手段，但由于其损伤机制尚不很清楚，使得营养措施针对性不强。程序性坏死（necroptosis）是一种新鉴定的细胞死亡方式，在多种因素导致的组织损伤中发挥重要作用。这为探索免疫应激诱导肠道损伤的机制提供了崭新思路。本项目研究免疫应激对仔猪肠道程序性坏死及其信号通路的影响，并进一步研究抑制程序性坏死关键调控因子（RIP1/RIP3/MLKL）对仔猪肠道和IPEC1-J2细胞损伤的影响，以揭示程序性坏死是否参与肠道损伤的调控。其次，探讨RIP1/RIP3/MLKL介导IPEC1-J2程序性坏死的下游执行方式，以揭示肠细胞程序性坏死的分子机制。最后，研究n-3PUFA对程序性坏死及其信号通路的调控作用，以探索n-3PUFA是否可通过抑制程序性坏死从而缓解肠道损伤。本研究将为揭示猪免疫应激诱导肠道损伤的机制以及为通过营养调控来克服肠道损伤提供重要理论依据。

本项目探讨了程序性坏死在免疫应激导致的仔猪肠道损伤中的作用及n-3 多不饱和脂肪酸（PUFA）通过影响程序性坏死发挥对仔猪肠道损伤的保护作用。（1）采用脂多糖（LPS）刺激仔猪建立免疫应激模型，研究免疫应激对仔猪肠粘膜细胞程序性坏死及其信号通路关键调控因子的影响。结果表明，LPS刺激导致肠道结构、消化与屏障功能受损，引发肠道炎性因子表达升高，同时提高程序性坏死信号通路关键调控因子—RIP1、RIP3、MLKL、PGAM5、DRP1和HMGB1的蛋白表达量，激活程序性坏死信号通路。（2）通过动物试验和细胞试验，研究抑制程序性坏死关键调控因子对免疫应激诱导的仔猪肠道和细胞损伤的影响。首先探究Nec-1（RIP1抑制剂）、GSK’872（RIP3抑制剂）和GW806742X（MLKL抑制剂）对呕吐毒素（DON）诱导的IPEC-1细胞损伤的调控作用。结果表明，Nec-1、GSK’872和GW806742X可以通过阻断程序性坏死关键调控因子，抑制细胞程序性坏死的发生，进而缓解DON导致的细胞损伤，保护肠上皮细胞屏障功能的完整性。在细胞试验的基础上，采用Nec-1预处理仔猪，进一步探讨抑制程序性坏死对LPS诱导的仔猪肠道损伤的影响。结果表明，Nec-1可以通过抑制程序性坏死信号通路，缓解LPS诱导的肠道结构、消化和屏障功能损伤及肠道炎症。以上研究表明，程序性坏死介导免疫应激诱导的仔猪肠道损伤。（3）探究n-3 PUFA是否可以通过抑制程序性坏死从而缓解免疫应激导致的肠道损伤。首先探究二十碳五烯酸（EPA）和二十二碳六烯酸（DHA）对DON诱导的IPEC-1细胞损伤和程序性坏死的调控作用。结果表明，EPA和DHA显著缓解DON引起的RIP1、RIP3、PGAM5、DRP1和HMGB1蛋白表达量以及MLKL磷酸化的升高，缓解IPEC-1细胞损伤，从而保护肠上皮细胞屏障功能的完整性。随后研究日粮中添加5%的亚麻油（富含n-3 PUFA）对LPS刺激仔猪肠道形态结构与功能、程序性坏死信号通路相关基因表达的影响。结果表明，亚麻油缓解LPS引起的程序性坏死通路相关基因表达的升高，缓解LPS引起的肠道形态和屏障功能受损。以上研究表明，n-3 PUFA可通过抑制程序性坏死从而缓解肠道和肠细胞损伤。