基于TRP通道介导的钙信号调节研究温里药“温中止痛”的作用机制

《ShenNongBenCaoJing》reords " treating the cold with hot-nature drugs",interior-warming drugs expell cold and relieve pain by warming Zhongjiao,which can significantly improve abdominal cold pain, vomiting, diarrhea of cold-syndrome, modern studies explain these only with antiulcer and anti-diarrhea or anti-inflammatory and analgesic effects, failed to clarify the "natures-effects" intrinsic correlation, but how to integrate many effects to clarify the overall regulation effect of Chinese medicine is still a difficult problem in modern research.TRP channels,cold and hot sensory receptors, which found in recent years not only mediate temperature feeling also pain feeling,and research in TCM systems biology indicate that the regulatory factors theory of TRP superfamily is consistent with natures theory of Chinese medicine. Calcium and its signaling pathway mediated by TRP channels are important regulatory factors in cold-hot reaction of Chinese herbs and digestive diseases. Preliminary studies showed that interior-warming drugs inhibitted Ca2+ influx, intestinal contraction and pain.Thus we suppose the effects of "warming Zhongjiao and relieve pain" may be associated with recovery steady-state of calcium signaling pathways through regulation of TRP channels, which bring about "warm Zhongjiao to expell cold " "antispasm to relieve pain"in overall regulation. The project plans to study the action law and mechanisms of interior-warming drugs on cold sensory TRP channels and calcium signaling regulation factors(e.g IP3,PKC,cAMP),from three levels such as animals,tissue and cell. To provide the basis for scientific explanation with "warming Zhongjiao and relieve pain" and study in cold-hot natures of Chinese medicine.

《本经》云"疗寒以热药"，温里药温中散寒止痛，能明显改善里寒证的脘腹冷痛、呕吐泄泻，现代研究仅以抗溃疡抗腹泻或抗炎镇痛作用解释，未能阐明其"性-效"的本质关联,而如何综合多个作用阐明中药的整体调节效应是中药现代化研究的难点。寒热感受TRP通道是近年发现的温度、疼痛感受器，中医系统生物学研究提示TRP超家族的调控因子理论与药性理论一致。由TRP介导的钙信号及其通路在中药寒热反应、消化系统疾病中均是重要调节因子。前期研究表明温里药拮抗Ca2+流，抑制肠收缩、疼痛。基于此推测温里药"温中止痛"可能与经TRP通道调控钙信号通路恢复稳态有关，从而实现"温中散寒""解痉止痛"的综合调节作用。项目拟从整体动物、离体组织和细胞水平三个层次，观察温里药对冷感觉TRP通道亚型和相关的钙信号调节因素（如IP3、PKC、cAMP）的作用规律和作用机制。为中药的寒热属性研究与温里药"温中止痛"的科学阐释提供依据。

温里药温中散寒止痛，能明显改善里寒证的脘腹冷痛、呕吐泄泻，但其性-效本质关联未能阐明。近年发现瞬时感受器电位通道蛋白(TRP)作为温度感觉器，与中医寒热反应密切相关，为此本研究从整体动物和细胞水平，观察温里药对冷感受器TRPA1和TRPM8及其相关的钙信号调节因素的作用规律。采用整体动物和离体组织试验等优选受试温里药；采用脾胃虚寒肠易激综合征大鼠模型研究温里药对整体动物的冷感受器TRPA1和TRPM8基因及蛋白表达的影响；体外实验分别采用TRPA1-HEK293T细胞和MCF-7细胞研究温里药对TRPA1和TRPM8及其相关的钙信号调节因素的作用规律。研究结果表明，高良姜、吴茱萸、干姜、肉桂等温里药有不同程度的温中止痛作用；高良姜、吴茱萸能明显改善脾胃虚寒肠易激综合征大鼠模型的一般症状和指标，调节冷感受器TRPA1和TRPM8基因及蛋白表达；课题组成功构建了TRPA1稳转HEK-293T细胞，并发现高良姜、吴茱萸、干姜、肉桂、花椒、荜澄茄含药血清对TRPA1-HEK293T细胞的TRPA1表达作用不同，已证明高良姜含药血清能明显调控TRPA1基因和蛋白表达、细胞内钙离子浓度和cAMP水平等；高良姜、吴茱萸、干姜、肉桂、花椒、荜澄茄含药血清对体外细胞的TRPM8 表达及其介导的钙信号通路也有明显的调控作用。此外，课题组还基于药物对TRPA1的调控作用研究开展了高良姜的有效部位分离提取和活性筛选，也进行了高良姜的组方应用研究。温里药是中医临床常用的一类药物，研究首次表明其温中止痛的作用与调节病理状态下冷感受器TRPA1和TRPM8的异常表达及其介导的钙信号通路有关，为温里药温中止痛的科学阐释提供了依据，同时也对中药药性理论的发展发挥着积极的推动作用。