牛磺鹅去氧胆酸对大鼠肺泡巨噬细胞中TGR5受体介导的信号通路的影响

The early researches of applicant revealed that Taurochenodexyc acid (TCDCA), anactive substance, had effects of significant anti-inflammatory and immunoregulation. The aim of the research project is to investigate the molecular mechanisms of anti-inflammatory and immunoregulation effects of TCDCA and the influence of TCDCA on signaling pathway mediated by TGR5 in depth.The rat alveolar macrophage cell line NR8383 with the methods of modern molecular technology such as fluorescence polarization, western blot, real time PCR and etc. will be used to reveal: ①the interaction of TCDCA and TGR5; ②the influence of TCDCA on the signaling pathway during the relative immunologic factors synthesis process in the rat alveolar macrophage NR8383; ③the influence of TCDCA on synthesis and secretion of the relative immunologic factors in the rat alveolar macrophage NR8383; ④the influence of TCDCA and TGR5 agonist on the alteration of capacitance of cell membrane and [Ca2+]i. Through this study, it's able to provide an experimental basis for studying new target of immune-inflammatory drugs; and it has practical significance to accelerate TCDCA into pharmaceutical products, to improve the added value and utilization of animal bile, and to promote the development of medicine and animal husbandry.

申请者在前期研究中，揭示了体内活性物质牛磺鹅去氧胆酸（TCDCA）作为胆汁酸(BAs)的主要有效成分之一,对炎症与免疫功能均具有显著的调节作用。本课题旨在深入探讨TCDCA的抗炎与免疫调节作用的分子机制，探明TCDCA对胆汁酸受体TGR5介导的信号转导通路的影响。拟以NR8383大鼠肺泡巨噬细胞系为研究对象，采用荧光偏振、Western blot和荧光定量PCR等现代分子生物学技术揭示①TCDCA与TGR5的相互作用；②TCDCA对NR8383合成免疫相关因子过程中的细胞内信号转导通路的影响；③TCDCA对NR8383合成分泌免疫相关因子的影响；④TCDCA对TGR5介导的细胞膜电流及胞内[Ca2+]i的变化。通过该研究为研究抗炎免疫药物新的作用靶点提供实验依据。本研究对加快TCDCA转化成医药产品，提高动物胆汁的附加值和利用率，促进医药和畜牧业的发展均具有重要的现实意义。

为揭示牛磺鹅去氧胆酸（TCDCA）的抗炎免疫调节机制，本研究以NR8383细胞为研究对象，探讨了TCDCA对TGR5介导的AC-cAMP-PKA及IP3-CaM信号通路的影响。.主要研究内容：①用免疫荧光、q-PCR及Western Blot( WB )鉴定了TGR5在293T细胞中表达。结果：TGR5真核表达载体构建成功且在239T中表达。用荧光显微镜观察了293T细胞受体内陷，ELISA测定了293T细胞cAMP含量，用免疫荧光检测293T细胞荧光素酶报告基因活性。结果：TCDCA能够引起转染TGR5受体293T细胞发生内陷，细胞内cAMP和萤光素酶含量显著升高且呈现剂量依赖关系。②用ELISA检测了NR8383细胞阻断TGR5基因、AC和PDE后的cAMP含量。结果：TCDCA能够诱导NR8383细胞升高cAMP。③用q-PCR和WB检测了NR8383细胞中PKA和CREB活性。结果：TCDCA能够极显著增加NR8383细胞中PKA和CREB活性。④分别用q-PCR和ELISA法检测了NR8383细胞中TNF-α、IL-1β、IL-6、IL-8、IL-12、IκBα和COX-2的基因表达量和蛋白含量，WB检测了IκBα和COX-2的活性。结果：TCDCA能够显著降低NR8383细胞中TNF-α、IL-1β、IL-6、IL-8、IL-12、 IκBα和COX-2表达量。⑤用ELISA检测了NR8383细胞中IP3浓度， q-PCR和WB检测了NR8383细胞中CaM表达。结果：TCDCA显著提高NR8383细胞中IP3浓度及CaM表达。⑥酶标法检测NR8383细胞Ca2+浓度表明，TCDCA能增加NR8383细胞Ca2+浓度且存在浓度依赖性关系。.结论：①TCDCA能与TGR5发生相互结合。②TCDCA诱导细胞NR8383中cAMP、PKA、CREB升高与其激动TGR5有关。③TCDCA可抑制IκBα降解而降低NF-κB结合活性。④TCDCA对NR8383细胞中TNF-α、IL-1β、IL-6、IL-8 、IL-12和COX-2表达均具有显著抑制作用。⑤TCDCA能提高NR8383细胞中IP3和Ca2+浓度。⑥TCDCA能促进NR8383细胞中CaM表达。⑦TCDCA抗炎免疫调节作用与激活TGR5受体介导的AC-cAMP-PKA及IP3-CaM信号通路有关。