CaSR信号通路在仔猪肠道炎症反应中的作用及机理

Activation of calcium sensor receptor(CaSR) in the intestinal epithelial system potentially may contribute to enhancement of gut health.CaSR is used to control the release of intracellular calcium and it can be allostericly activated by L-AAs and peptides. This suggests a potential role of CaSR activation in sensing availability of nutrient agonists or allosteric modifiers by which bioactivities of the gastrointestinal epithelial cells can be regulated. However, the functions of CaSR in modulating physiopathological conditions and enhancing GI health under inflammatory conditions have never been addressed. So, the objectives of this study will first clone and characterize porcine CaSR, and then investigate the tissue distribution of CaSR mRNA and protein in the gut by real-time PCR, Western Blot, and immunohistochemistry. Role of CaSR signalling in the inflammation will be investigated in virto using TNF-alpha-induced inflammatory IEC models.The protective and therapeutic anti-inflammatory activities of some functional peptides and amino acids will be also investigated in a vitro model and then their roles in the regulation of CaSR cell signalling will be checked.A porcine model of dextran sodium sulfate (DSS)-induced colitis and CaSR block reagents were used to in vivo evalutate the therapeutic efficacy of some functional peptides and amino acids and the role of CaSR signalling in this therapeutic efficacy. The main implication of this study will be improve the fundamental knowledge about how the CaSR agonistic dipeptide and amino acid -based nutritional interventions can modulate inflammation, and provide solid evidence to identify the cellular mechanisms underlying their therapeutic implication.

钙敏感受体（CaSR）位于细胞膜的表面，可以控制细胞内的钙流，能被一些肽和氨基酸激活，这提示我盟，CaSR能被营养物质调控，从而进一步调控胃肠道健康。然而，猪CaSR目前还没有被克隆鉴定，在肠道中的作用依然不是很清楚。本研究拟以先克隆和鉴定猪CaSR,然后利用real-time PCR、免疫印迹和免疫组化等技术来检测猪CaSR在胃肠道的分布及其在细胞内的定位。并通过建立TNF-alpha诱导的IEC细胞体外炎症模型和DSS诱导的猪肠道炎症模型，结合基因敲除技术和化学阻断技术，检测CaSR信号通路在细胞炎症中的作用和验证抗炎症的功能性肽和氨基酸是否通过改变CaSR信号通路而起作用。本研究结果将阐明CaSR信号通路在仔猪肠道肠炎中作用分子机理，这不仅可为养猪业开发新型的抗炎症营养性添加剂提供理论依据，也可为人类慢性肠炎的治疗提供参考。

项目根据任务书要求，项目开展了猪CaSR的克隆和鉴定；利用LPS 诱导的仔猪肠道炎症模型，通过日粮添加芳香族氨基酸L-Trp、L-Phe 和L-Tyr(TPT)，研究芳香族氨基酸在CaSR 信号通路和仔猪肠道炎症反应中的调控作用；利用LPS 诱导的IPEC-J2 细胞炎症模型，通过抑制和激动CaSR，研究CaSR 信号通路在芳香族氨基酸调控的炎症反应中的作用。主要科学发现有：日粮添加TPT 能够缓解LPS 诱导的组织病理学损伤，降低LPS 处理仔猪血清中促炎性细胞因子含量以及肠道组织中促炎性细胞因子mRNA 丰度，而显著提高抗炎性细胞因子水平；显著提高CaSR 和PLCβ2 的蛋白表达水平，降低LPS处理仔猪的p-NF-κB、IKKα/β和IκB 蛋白表达水平。提示日粮添加TPT 能激活CaSR 信号通路和抑制NF-κB 信号通路，缓解LPS 诱导的仔猪肠道炎症。在LPS 诱导的IPEC-J2 细胞炎症模型中，增加培养基TPT 浓度能提高CaSR蛋白表达水平而降低IκB 蛋白表达水平，抑制NF-κB 核转移，降低IL1β、IL6、GM-CSF 等促炎性因子mRNA 水平缓解LPS 诱导的猪肠上皮细胞IPEC-J2 炎症反应；NPS-2134 阻断CaSR 信号通路后，添加TPT 显著降低CaSR 蛋白表达水平而提高p-NF-κB 和IκB 蛋白水平，加剧LPS 诱导的猪肠上皮细胞IPEC-J2 炎症反应。以上结果说明，CaSR 信号通路在芳香族氨基酸TPT 缓解仔肠道炎症反应中发挥重要作用。结果可为养猪业开发新型的抗炎症营养性添加剂提供理论依据，也可为人类慢性肠炎的治疗提供参考。项目执行期间，基金资助发表论文6篇，其中SCI论文4篇；参加学术会议10次；培养研究生2名。