背俞指针疗法对GERD大鼠内质网Ca2+-mTOR/Caspase-12介导ICC自噬与凋亡的影响

Gastroesophageal reflux disease (GERD) is a common disease. Combination of acid suppression, prokinetic and mucosal protective agents often gain poor efficacy and high recurrent rate. Previous researches confirmed the hypothesis of the" the intersectional imbalance of meridian qi of Ren and Du caused the imbalance of the motions of the spleen and stomach qi, this is the essence of the pathogenesis of GERD" and the "upper gastrointestinal motility disorder is closely related to GERD". Researches also confirmed the back-shu finger therapeutics is an effective treatment for this disease, its mechanism is related to its action and coordinating the esophageal ENS-ICC-SMC system. We further speculate that the back-shu finger therapeutics may modulate the autophagy of ICC cells for antagonizing apoptosis, maintain the ICC to play its pacing function, and to maintain the integrity of the esophagus ENS-ICC-SMC system. Thus, in the aims to reveal the molecular mechanism of the back-shu finger therapeutics, this project has culture ICC in vitro, then use confocal microscopy to detect the fluorescence changes of Ca2 +; use the electron microscope to observe the endoplasmic reticulum autophagosomes of ICC; use the Western Blot and other methods to detect the autophagy-apoptotic proteins; use the back-shu finger therapeutics as an intervention for GERD rat models; then observe the endoplasmic reticulum autophagosomes and the autophagy-apoptotic proteins; investigate the antagonizing apoptosis effects of Ca2+-mTOR/Caspase-12 pathway through mediating the autophagy of endoplasmic reticulum.

胃食管反流病(GERD)是常见多发病，抑酸、促动力与粘膜保护剂联合治疗时常疗效欠佳，病情易反复。前期临床和实验证实了“任督二脉经气升降交会失衡所致脾胃升降失衡是本病的病机本质”且与“上消化道动力障碍”密切相关的假说，也证实了背俞指针疗法治疗本病疗效肯定，其作用机制与协调食管ENS-ICC-SMC系统有关。我们进一步推测，背俞指针疗法可能调节ICC细胞的自噬而拮抗凋亡，维持ICC的生存而发挥起搏功能，保持食管ENS-ICC-SMC系统的完整性。因而，本项目通过体外ICC培养，共聚焦显微镜检测Ca2+荧光变化，电镜观察ICC内质网自噬体、Western Blot等方法检测自噬凋亡蛋白；并以背俞指针疗法干预GERD模型大鼠，观察内质网自噬体、检测自噬凋亡相关蛋白，探讨Ca2+-mTOR/Caspase-12通路介导内质网自噬而拮抗凋亡的效应，揭示该疗法作用的分子机制。

背景：目前，西药治疗GERD尚有不足。中医非药物疗法疗效确切，但其作用机理研究尚浅。近年来，我团队在临床运用背俞指针疗法治疗GERD效果显著，故通过本次研究进一步探讨背俞指针疗法对GERD的作用机制。主要内容：将235只SD大鼠随机分为正常组、模型组、西药组、指针组、空白指针组，采用贲门钢圈固定法进行造模，各组予相应的干预方法，疗程为14天。采用透射电镜观察各组食管下段组织ICC超微结构；采用免疫组化法和Western blot检测CaMKKβ、AMPK、mTOR、calpain、Caspase12、Bcl-2、Beclin1蛋白表达；采用RT-qPCR法检测mRNA的表达；采用Co-IP检测Bcl2/Belin1相互作用。体内实验结果：1.与空白组比较，模型组ICC超微结构模糊，细胞核固缩坏死、出现自噬体；与模型组比较，指针组ICC细胞形态结构改善。2.RT-qPCR法检测结果：与空白组相比，模型组Bcl-2、Caspase12、Beclin1 mRNA表达显著降低（P＜0.01）3.Western blot结果：模型组AMPK蛋白表达量较正常组低（P＜0.05）；指针组AMPK蛋白较正常组高，而BCL-2蛋白表达低（P＜0.05）；4.Co-IP结果：模型组Bcl-2/Beclin1相互作用较正常组水平升高（P＜0.01）；②指针组Bcl-2/Beclin1相互作用水平较模型组降低（P＜0.05）。体外实验结果：1.Western blot结果：与正常组比较，诱导组、抑制组中calpain蛋白表达均升高（P＜0.05），mTOR蛋白表达均降低（P＜0.05）。2.Co-IP：Bcl-2/beclin-1蛋白相互作用水平无统计学意义（P＞0.05）；3、共聚焦测Ca2+结果：抑制组、诱导组钙离子浓度均高于正常组（P＜0.01）；诱导组与抑制组比较，诱导组钙离子浓度高于抑制组（P＜0.01）。结论，GERD模型大鼠食管ICC存在“自噬-凋亡”现象，背俞指针疗法干预后ICC超微结构改善。其机制可能与背俞指针疗法影响Bcl-2/Beclin1之间蛋白相互作用水平及AMPK、BCL-2蛋白相关。L-谷氨酸诱导体外食管ICC细胞自噬及3-MA抑制体外食管ICC自噬与Ca2+浓度相关，同时，其可能通过影响自噬相关蛋白mTOR及凋亡相关蛋白calpain从而影响ICC的自噬。