益气健脾中药通过多胺和钙离子调控作用于小肠上皮细胞迁移的研究

The effect on gastrointestinal mucosal repair after wounding is one of the mechanisms of Yi-qi-jian-pi herbs. Migration of epithelial cells is important for the repair of gastrointestinal mucosal injuries. Our previous two researches indicated that Yi-qi-jian-pi herbs could promote cell migration, of which the mechanism is related to its effect on polyamine-mediated signal ways. Yi-qi-jian-pi herbs could improve cytosolic free calcium concentration ([Ca2+] cyt), by increasing Ca2+ release from intracellular stores and extracellular Ca2+ influx through Ca2+-permeable ion channels, which play an important role in the stimulation of cell migration after wounding, whereas extracellular Ca2+ influx is more important. The promoting effects of Yi-qi-jian-pi herbs attenuated apparently in absence of extracellular Ca2+, which suggested regulatory effects of Ca2+ are key points. This study will be undertaken to further investigate the mechanism of promoting effect of Yi-qi-jian-pi herbs on intestinal epithelial cell migration, focusing on calcium regulation in polyamines-dependent signal way. Key targets such as Ca2+ influx driving force, regulating effect of protein complexes (STIM1/TRPC1, STIM1/STIM2, Rac1/PLC-r1, RhoA/TRPC1, Cav1/TRPC1) and influence of specific protein inhibitor will be determined to study the mechanism of Yi-qi-jian-pi herbs protecting gastrointestinal mucosa, which will elucidate the scientific connotation of the therapeutic effects of Yi-qi-jian-pi herbs on Spleen and Stomach disease and the conception “spleen as the defense of five-zang viscera and six-fu organs” and also provide foundation to the researches of innovative drug of yi-qi-jian-pi herbs.

胃肠粘膜损伤修复是益气健脾中药作用机制之一，上皮细胞迁移是胃肠粘膜损伤修复的重要环节；申请人前两课题发现益气健脾中药能促进小肠上皮细胞迁移，作用机制与其影响多胺调控信号通路有关，可通过促进胞内钙库释放Ca2+和胞外Ca2+内流两途径（后者作用更明显），提高细胞Ca2+水平而促进细胞迁移；但若无Ca2+内流则益气健脾中药作用明显减弱，表明对Ca2+调控是关键指标，本课题进一步围绕多胺信号通路Ca2+调控过程，观察益气健脾中药促进细胞迁移的作用机制：对Ca2+内流驱动力、蛋白复合体调控（STIM1/TRPC1、STIM1/STIM2、Rac1/PLC-r1、RhoA/TRPC1、Cav1/TRPC1）、特定蛋白抑制后的影响等，以多胺和Ca2+调控为关键指标，研究益气健脾中药胃肠粘膜保护机制，对阐述益气健脾中药治疗脾胃病和“脾为之卫”的科学内涵有重要意义，也为益气健脾创新药物研究提供基础。

益气健脾中药胃肠黏膜损伤修复作用与其促进上皮细胞迁移有关，其中对多胺信号通路Ca2+的调节是关键；本课题围绕Ca2+调节蛋白及其复合体等机制开展研究。.1、益气健脾中药多糖提取物对IEC-6细胞迁移Ca2+调节影响的研究：以能促进小肠上皮细胞（IEC-6）迁移的黄芪多糖或人参多糖或四君子汤多糖为受试药，观察其对细胞迁移多胺信号通路Ca2+调节的影响。①对Ca2+调节蛋白及复合体的影响：促进STIM1向胞膜移位并提高其表达，降低STIM2表达；提高STIM1/TRPC1表达；调节STIM1/STIM2表达；提高RhoA、Rac1、Cav1、TRPC1、PLC-γ1和RhoA/TRPC1、Cav1/TRPC1、Rac1/PLC-γ1表达；逆转DFMO（多胺合成抑制剂）对上述指标的作用。②对Ca2+内流驱动力的影响：含钙培养时能促进胞外Ca2+内流从而提高Ca2+；含钙培养+CPA耗竭胞内Ca2+时能促进胞外Ca2+内流以维持Ca2+水平；无钙培养+CPA时可短暂升高细胞Ca2+水平。③对特定负荷的影响：能逆转si-Cav1所致的Cav1表达降低、Ca2+降低和细胞迁移抑制；逆转RhoA、Rac1抑制剂所致的Ca2+降低和细胞迁移抑制。.2、益气健脾中药提取物防治大鼠胃肠黏膜损伤的研究：①四君子汤水提物或多糖能防治吲哚美辛所致大鼠胃或小肠黏膜损伤，提高胃或小肠黏膜多胺（精脒）、Ca2+及紧密连接蛋白（ZO-1、Occludin、Claudin-3）和黏附连接蛋白（E-cadherin、α-catenin、β-catenin）表达；降低血浆D-乳酸。②人参多糖能防治吲哚美辛所致大鼠小肠粘膜损伤，提高小肠粘膜Ca2+，促进小肠（十二指肠）上皮细胞迁移。③黄芪、人参水提物能防治吲哚美辛所致大鼠小肠粘膜损伤。.3、益气健脾中药多糖提取物药效物质研究：在黄芪多糖或四君子汤多糖样品中，检测了HPLC、红外光谱、1H-NMR和13C NMR；计算分子量；水解并检测单糖比例。.本课题研究表明，多糖是益气健脾中药促进细胞迁移有效组分之一，作用机制与其影响多胺信号通路有关，尤其Ca2+调节蛋白及其复合体的作用是关键之一，可通过促进胞内钙库释放和增强胞外Ca2+内流而提高细胞Ca2+且后者作用更明显，结合动物实验结果为探讨益气健脾中药胃肠黏膜损伤修复机制提供了参考。