基于PI3K/PDK1和SCF/c-kit通路研究疏肝理气法调控胃Cajal间质细胞自噬与分化促胃动力的机制

Gastrointestinal Cajal interstitial cells (ICC) are the gastrointestinal activities pacemaker and moderator, thus insufficient ICC autophagy and differentiation is the key to gastrointestinal movement disorder. The method of Soothing Liver Regulating Qi is an effective therapy on gastrointestinal movement disorders. Does it mean this method can regulate ICC autophagy and differentiation? Based on the series of our previous research, in this study, we will discuss the mechanism of correlative signal transduction of the method of Soothing Liver Regulating Qi on mediating ICC autophagy and differentiation to promote gastric dynamics. According to the key link of PI3K/PDK1 pathway mediating ICC autophagy and SCF/c - kit promoting ICC differentiation, Functional Dyspepsia (FD) animal models were copied by the method of clipping tail to irater and stimulate. Gastric emptying of radionuclide was observed, and ICC ultra structures like morphology and autophagy were observed by laser cdonfocal microscopy and transmission electron microscope. Separation of ICC and subculture were done through enzymolysis approach, meanwhile cell self-macrophages was detected by PI staining flow cytometry. Changes of Ca2+ were tested by whole-cell patch clamp technique and Fluo-3 fluorescence, and gastric tissues, expressions of ICC cell, such as SCF, c-kit, PI3, PI3K, Beclin1, Bcl-2, Vps34, CaM, and MLCK etc. were detected respectively by immunohistochemistry and Western Blot as well. This study is to explore the mechanism of the method of Soothing Liver Regulating Qi on promoting gastric dynamics by regulate autophagy and differentiation of ICC. It will lay the foundation for reveal the deep mechanism of the method of Soothing Liver Regulating Qi on promoting gastric dynamics.

胃肠道Cajal间质细胞(ICC)是胃肠道活动起搏者和调节者，其自噬与分化不足是胃肠动力障碍的关键，疏肝理气法是胃动力障碍性疾病的有效治法，是否意味着疏肝理气法能够调控ICC自噬与分化？基于前期系列研究的基础上，我们拟从PI3K/PDK1通路介导ICC自噬与SCF/c-kit促进ICC分化的关键环节出发，夹尾激惹刺激法复制FD动物模型，观察核素胃排空，激光共聚焦显微镜、透射电镜观察ICC形态、自噬体等超微结构；酶解法分离ICC并传代培养，PI染色流式细胞术检测细胞自噬，全细胞膜片钳技术、Fluo-3荧光检测Ca2+变化；免疫组化及Western Blot检测胃组织、ICC细胞SCF、c-kit、PI3、PI3K、Beclin1、Bcl-2,Vps34,CaM、MLCK等表达，探讨疏肝理气法调控ICC自噬与分化促胃动力的相关信号转导机制,为揭示疏肝理气法促胃动力的深层机制奠定基础。

背景：功能性消化不良（FD）是胃肠动力障碍性疾病之一，临床上多见肝胃不和之证，常以疏肝理气法进行治疗，疗效较好，但是相关的机理尚不清晰。胃Cajal间质细胞对胃肠运动的作用极为重要，FD的发病机理可能与胃Cajal细胞自噬于分化不足相关，而疏肝理气法发挥疗效的机理可能与介导为Cajal细胞自噬于分化的PI3K/PDK1通路的干预有关。. 研究内容：本课题按照研究目的分别从动物实验、细胞实验进行研究，证实疏肝理气法促胃动力作用，深入探讨疏肝理气法对胃肠道Cajal间质细胞（ICC）自噬及分化的PI3K/PDK1和SCF/c-kit信号途径相关环节的影响。. 关键数据：与正常对照组比较，模型组胃排空率显著降低（P<0.05）。与模型组比较，柴胡疏肝散低、中、高剂量组及枳实低、中、高剂量组胃排空率显著升高（均P<0.05）。与正常对照组比较，模型组SCF、c-kit的荧光强度显著减弱（P<0.05）。与模型组比较，柴胡疏肝散低、中、高剂量组及枳实低、中、高剂量组SCF、c-kit的荧光强度均增强（均P<0.05）。与正常对照组比较，模型组胃ICC内Beclin1、LC3B蛋白MFI及mRNA IOD升高（P<0.05）；与模型组比较，柴胡疏肝散高剂量组、柴胡疏肝散中剂量组ICC内Beclin1蛋白MFI及mRNA IOD下降（P<0.05）；与正常组比较，模型组胃SMC中Bcl-2较正常组显著减少，Bax、NF-κB蛋白的表达明显升高（P<0.05）。柴胡疏肝散低、中、高剂量组中Bcl-2的表达较模型组均显著升高，Bax、NF-κB表达较模型组均显著降低（均P<0.05）。. 重要结果及科学意义：FD大鼠模型成功建立，对于构建FD大鼠模型有一定的推广意义。并且证实了柴胡疏肝散、枳实均有促进FD大鼠胃动力作用，具有一定的临床和科研意义；柴胡疏肝散可能通过上调SCF表达，促进ICC的增殖分化及表型的维持，从而促进胃动力；FD大鼠胃肠动力障碍的机制之一可能是胃ICC过度自噬，柴胡疏肝散能抑制ICC的过度自噬从而促进胃动力；柴胡疏肝散降低FD大鼠胃SMC的凋亡率，其机制可能是通过上调Bcl-2、抑制Bax的表达来调节线粒体途径的细胞凋亡，从而发挥促胃动力作用，同时也可能与抑制NF-κB凋亡信号通路异常激活导致的胃平滑肌的损伤有关。