基于MLCK信号通路探讨手法干预脾虚FD家兔频率-效应-补泻的相关性

The previous study of our project had confirmed that Pushing Manipulation with One Finger(PMOF) and Rubbing Manipulation(RM) could improve / worsen the related symptoms of spleen-deficient rabbits respectively in the frequency range of 101-150,201-250 (beats / min). This project aims to explore that whether the effect of TUINA manipulation has the same differences on molecular protein level and find the targets of the specific protein signaling pathways. This project explore the mechanism from MLCK Signaling Pathway in two parts:”PMOF part and RM part”.Spleen-deficiency FD model rabbits would be randomly divided into Blank control group, Model control group, Low frequency group, High frequency group, Inhibitor group 1 and Inhibitor group 2. We carry out the experiment under the design idea “two massage manipulations, two frequency bands and independent observation in every part”.We will observe and compare the changes of gastric emptying rate,small intestine advance rate,MLCK protein and its mRNA expressions in the gastric tissue in order to verify the hypothesis:"Massage manipulation can intervene the contraction and relaxation of gastrointestinal smooth muscle of spleen-deficient FD rabbits by regulating the MLCK signaling pathway.Between frequency and manipulation effect, there is a correlation that can be named as Frequency - Effect - Reinforcing and Reducing Axis Network ".The gastric emptying rate and small intestine advance rate were detected by charcoal gavage.The MLCK protein and the mRNA expressions were measured by ELISA,immunohistochemistry,Western blot and RT-PCR.The results can basically clarify the scientific connotation of slow-rapid reinforcing-reducing from the perspective of frequency in TUINA manipulation,which is of positive significance for enhancing the scientificity of TUINA and promoting its transformation from empirical medicine to modern science.

前期研究证实一指禅推和摩法在101-150、201-250（次/min）频率段可分别改善/恶化脾虚家兔症状，本项目旨在探究在分子蛋白层面是否具有同样的差异性及其效应靶点。研究以MLCK信号通路为切入点，将脾虚FD家兔模型分为空白组、模型组、低频组、高频组、抑制剂组1和抑制剂组2，实验分一指禅推和摩法两次完成，在“两种手法、两个频率段、独立观察”的研究思路下，采用炭末灌胃法检测胃内残留率和小肠推进率，采用免疫组化、Western blot和RT-qPCR法检测胃组织MLCK通路蛋白及mRNA表达量，以验证“手法可通过调节MLCK通路干预脾虚FD家兔胃肠道平滑肌的收缩和舒张，频率高低与效应相关，推拿治疗存在频率-效应-补泻疗效轴”的科学假说。成果可从频率角度基本阐明手法缓急补泻的科学内涵，为手法操作提供量化标准，对提升推拿的科学性、促进其从经验医学向现代科学方向转化具有积极意义。

本项目前期分别研究证实“手法在101-150次/min频率段对改善脾虚家兔行为学症状、生化指标及胃肠道病理结构等方面优于其它段，在201-250次/min段则不但无效，甚至表现为相关指标恶化”基础上，为了解决“上述两个频率段的手法能否被界定为推拿学中的补法和泻法、前期研究及预实验结果所显示的差异性在更微观的分子蛋白层面是否具有同样的差异性、在分子蛋白层面手法干预产生这种差异性的效应靶点在哪儿？”等问题，并验证“手法可以通过调节MLCK信号通路干预脾虚型FD家兔胃肠道平滑肌的收缩和舒张，频率高低与手法效应密切相关，推拿治疗存在“频率-效应-补泻”的疗效轴”的科学假说，开展了本次研究。. 主要研究内容是用上述两个频率段分别用一指禅推法和摩法干预脾虚家兔，设置抑制剂组，从信号通路上下游，多水平、多层次地系统观察不同频率段推拿手法对脾虚证FD的MLCK通路的调节作用。观察造模及手法治疗前后家兔的行为学症状、手法影响胃排空和改善小肠传输功能情况、检测胃肠细胞Ca2+含量观察FD家兔MLCK通路上游因子的变化情况，荧光实时定量RT-qPCR方法、Western-blot法、免疫组化法检测家兔胃、小肠中CaM、MLCK和MLCP的mRNA和蛋白的表达， 综合上述研究结果，系统评价不同频率段手法干预脾虚FD家兔MLCK信号传导通路的变化，观察手法干预的信号靶点。项目除胃肠平滑肌细胞Ca2+由于标本处理失误导致部分数据缺失外，其余实验数据均按照计划收集完毕（附件15-49）。. 本项目研究的科学意义在于：1、结合前期研究成果，从频率角度基本阐明手法缓急补泻的科学内涵；2、为手法补泻操作提供量化依据，提升了推拿疗法的科学性，为临床治疗、技能考核、学术交流和课堂教学等提供量化了标准。3、拓宽推拿实验的研究思路，对促进推拿学从经验医学向现代科学方向转化具有积极意义。