背俞指针疗法对GERD大鼠SCF-c-kit-ICC/PLC-Ca2+-PKC/CaM信号通路与任督二脉穴位皮温的影响

In recent years, the incidence of Gastroesophageal reflux disease（GERD）is rising and the Western medical treatment alone still show a lot of confusion. Beishu Acupoint Finger Acupuncture Therapy(BAFAT) of traditional Chinese medicine is effective, but the mechanism is unknown. we have concluded that "Functional unbalance of hoisting and delivering in Ren-Du meridian should be the pathogenic essence, as the spleen and stomach's hoisting functional unbalance caused GERD", and the close relativity between the unbalance of Ren-Du meridian and the dyskinesis of upper gastrointestinal has been proved. Recent study has proven that SCF/c-kit-ICC through Ca2+-CaM-MLCK pathways and PLC-Ca2+-PKC pathway regulating the stomach esophagus during contraction and relaxation of isolated smooth muscle function. So, we consider that this therapy could improve the unbalance of Ren-Du meridian, further influencing PLC-Ca2+-PKC and SCF/c-kit-ICC pacemaker and enhance Ca2+-CaM-MLCK pathways during contraction and relaxation of isolated smooth muscle of the function. In order to deeply study the essence about the meridian-molecular related pathogenesis, to further prove the scientific of the previous hypothesis, and also to provide the theoretical basis of the methods applying in other gastrointestinal dynamic disorders, we will investigate the main acupoints' skin temperature in Ren-Du meridian, PLC-Ca2+-PKC signaling pathways associated with the ICC and their relationship by BAFAT on GERD model rats.

胃食管反流病(GERD)发病率呈上升趋势，单纯西医药治疗尚有不少困惑。中医背俞指针疗法疗效肯定，但机制不明。我们提出了"任督二脉经气升降交会失衡是脾胃升降失衡致病的病机本质"的假说，且已证明任督二脉经气升降交会失衡与上消化道动力障碍密切相关。新近发现SCF/c-kit-ICC可通过Ca2+-CaM-MLCK通路与PLC-Ca2+-PKC通路调控胃食管平滑肌舒缩功能。因而，我们认为本疗法很可能通过改善任督二脉经气交会失衡，进而影响PLC-Ca2+-PKC与SCF/c-kit-ICC起搏并增强Ca2+-CaM-MLCK通路调控平滑肌的舒缩功能。本项目采用背俞指针疗法干预GERD模型大鼠，探讨其对任督二脉主要穴位皮温、PLC-Ca2+-PKC与ICC相关信号通路的影响及其相关性，深入研究GERD经络-分子相关病机之本质，进一步证明前期假说的科学性，为该疗法在消化道动力障碍疾病的应用提供科学依据。

背景：迄今为止，单纯西药治疗GERD尚存在许多困惑。而中医非药物疗法疗效虽然确切，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，但作用机制研究仍不够深入，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120只SD大鼠随机分为空白组、模型组、西药组、指针组，采用经食管支架植入术进行造模，各组予相应的干预方法，疗程为14天。采用红外热成像技术观察各组治疗前后任督二脉主要穴位皮温变化的特点；采用光镜观察大鼠食管中下段组织的变化；采用电镜观察ICCs超微结构；采用免疫组化法和Western blot法检测β-actin、PLC、DG、IP3、PKC、c-kit、SCF、CaM、MLCK、及MLC20磷酸化蛋白表达；采用RT-qPCR法检测mRNA的表达。重要结果：1.干预前，指针组及西药组与模型组穴位皮温无差异，经干预后，两者均高于模型组。2.光镜下观察的病理结果示：与模型组相比，指针组食管粘膜结构基本恢复正常，鳞状上皮修复完整，黏膜固有层未见明显的炎症细胞浸润。3.RT-qPCR法检测结果表明：①西药组：与空白组相比，其c-kit、CaM的mRNA表达有所升高，SCF、MLCK的mRNA表达则有所降低。相较模型组，其c-kit mRNA表达显著升高。②指针组：与空白组相比，其SCF、MLCK的mRNA表达有所降低，CaM mRNA则显著升高。相较模型组，其c-kit mRNA表达显著降低，CaM、MLCK的mRNA表达则有所升高。4.Western blot结果示：PLC、DG在模型组的蛋白表达较空白组有所下降，而与模型组相比，其西药组与指针组的蛋白表达均有所升高。5.免疫组化法观察的结果表明：①各因子指针组及西药组ICCs计数较模型组显著增加。②除IP3、SCF、c-kit外，其余因子指针组蛋白表达水平较模型组显著升高。③除SCF外，其余因子西药组蛋白表达水平较模型组显著升高。6.电镜下观察ICCs超微结构结果示：指针组较模型组的ICCs数目及结构改善显著。以上结果表明，任督二脉皮温的变化与GERD的发病相关，背俞指针疗法治疗GERD的有效机制可能与改善任督二脉经气、修复ICCs并增加其数目，调节PLC-DG-IP3-Ca2+-PKC，SCF/c-kit-ICC及Ca2+-CaM-MLCK信号通路上的因子相关。