基于Ca2+-CaM-MLCK通路研究黄连“健胃-败胃”量效双向调节机制

Coptis has the saying that"small doses strengthen stomach and large doses spoil stomach". The project aims at discussing the bidirectional dose-effect mechanism of "strengthen stomach-spoil stomach" of different doses of Coptis. The studying team found previously that "strengthen stomach-spoil stomach" of Coptis was related to regulating gastrointestinal motility.Combining with some relative researches, the hypothesis that "different doses of Coptis regulate Ca2+-CaM-MLCK signal pathway differently,influence gastrointestinal smooth muscle contraction and gastrointestinal motility correspondingly shown as the bidirectional effect of 'strengthen stomach-spoil stomach'".The project is to cut in Ca2+-CaM-MLCK signal pathway generally accepted in regulating smooth muscle contraction and relaxation,to adopt body states of normal and spleen-deficiency model animal,and to combine with research modes of in vitro and vivo to discuss the bidirectional regulation mechanism of "strengthen stomach- spoil stomach" in Coptis. The expression of CaM、MLCK protein and gene and Ca2+ concentration regulated by different doses of Coptis is to be detected by RT-PCR, Western blot and laser scanning confocal microscope. Research results would be beneficial to reveal the amount-effect essence of "strengthen stomach"、"spoil stomach",to provide experimental basis for reasonable application of Coptis clinically and for development of new drugs,and to provide reference for studying the scientific connotation of the nature-efficiency expression in bitter-cold herbs.

黄连有"小剂量健胃，大剂量败胃"之说。本项目旨在探讨不同剂量黄连"健胃-败胃"的双向量效机制。课题组前期研究发现黄连"健胃-败胃"与其对调节胃肠动力的调节密切相关。结合有关研究，本项目提出工作假说："不同剂量黄连对Ca2+-CaM-MLCK通路的调控存在差异，进而影响胃肠平滑肌细胞收缩与胃肠运动，表现为'健胃-败胃'的双向效应"。本项目切入现今公认的调节胃肠平滑肌舒缩的经典机制Ca2+-CaM-MLCK信号通路，拟采用正常、脾虚模型动物两种机体状态，结合体内外实验研究，探讨黄连"健胃-败胃"的双向调节机制。综合运用荧光定量PCR、Western blot和激光共聚焦显微镜等技术，检测不同剂量黄连对CaM、MLCK蛋白、基因表达及Ca2+浓度的调控。研究结果将揭示黄连"健胃"、"败胃"的量效本质，为黄连的临床合理运用与新药开发提供实验依据。并可为苦寒药性效表达的科学内涵研究提供参考。"

黄连有“小剂量健胃，大剂量败胃”之说。本项目以调节胃肠平滑肌舒缩的Ca2+-CaM-MLCK信号通路为切入点，采用正常、脾虚模型动物两种机体状态，结合体内外实验研究，综合运用荧光定量PCR、Western blot等技术，检测不同剂量黄连对CaM、MLCK蛋白、基因表达的调控。结果显示：正常黄连各给药组大鼠胃残留率均明显高于正常组，正常黄连中剂量组小肠推进率明显低于正常组；脾虚模型组大鼠胃残留率和小肠推进率均明显高于正常组；脾虚黄连高、中剂量组大鼠胃残留率和小肠推进率均明显低于脾虚模型组。脾虚黄连高、中剂量组胃窦平滑肌细胞收缩率均明显低于脾虚模型组，脾虚黄连低剂量组回肠平滑肌细胞收缩率明显高于脾虚模型组。脾虚模型组胃粘膜萎缩变薄，皱襞低平，出现胃粘膜中断，水肿明显，脂肪及胶原纤维组织增生。黄连高、中、低剂量组胃粘膜中段、水肿等病理现象均不同程度减轻，脂肪及胶原纤维组织增生也减少。与空白对照组比较，模型组胃窦平滑肌组织CaM和MLCK mRNA含量均显著下降；与模型组比较，黄连高、中、低剂量组CaM mRNA含量均显著升高；与模型组比较，多潘立酮组、黄连高、中剂量组MLCK mRNA含量均显著升高。以上研究结果表明一定剂量黄连水煎液通过改善脾虚状态大鼠的胃肠功能紊乱情况，保护胃黏膜，增加胃粘膜组织CaM/MLCK mRNA的表达调控“Ca2+-CaM-MLCK”实现“健胃”作用的。研究结果将揭示黄连“健胃”、“败胃”的量效本质，为黄连的临床合理运用与新药开发提供实验依据。并可为苦寒药性效表达的科学内涵研究提供参考。”