基于PKCγ/ERK1/2/MAPK信号通路探讨安肠汤缓解腹泻型肠易激综合征腹痛研究

In the early days reasearch has proved Anchang decoction has obvious curative effect in treating IBS, and this mechanism was to reduce visceral hypersensitivity, which mainly related to peripheral sensitization and central sensitization of pain signal transduction. So invesitigation the key target of peripheral sensitization and central sensitization of visceral pain signal transduction pathways become an important problem. Some research shows that PKCγ,ERK1/2,mGluR5, NR2BR targets play important roles in PKCγ/ERK1/2/MAPK signal pathway to activate, transduce and regulate visceral hypersensitivity. Based on these findings, the project group intended to build a Rat Model of Irritable Bowel Syndrome of liver depression and spleen deficiency. Using Western bloting technique, calcium imaging technique and electrophysiological technique to test the protein expression of PKCγ,ERK1/2,mGluR5, NR2BR, the change of intracellular calcium ion concentration and the muscular tension of large intestine smooth muscle in vitro in Different levels neuron tissues intervened by Anchang decoction between pre and post treatment, respectively. Through the above measures, we want to clarify the key link between visceral hypersensitivity and visceral pain signal transduction pathways sensitization, and illustrate the main target of IBS intervened by Anchang decoction in molecular level.

前期研究证实安肠汤对IBS有明显的临床疗效，其主要通过降低内脏高敏感性而发挥效应，而内脏高敏感性主要与疼痛信号转导的外周敏化和中枢敏化有关，因此探讨内脏疼痛信号转导通路中外周敏化和中枢敏化关键靶点十分重要。研究表明，PKCγ/ERK1/2/MAPK信号通路中PKCγ,ERK1/2,mGluR5, NR2BR靶点在IBS的内脏疼痛信号激活、转导、调控中据有关键位置，与外周敏化和中枢敏化关系密切。项目拟通过建造肝郁脾虚IBS-D的大鼠模型，以利用Western bloting，钙离子成像和电生理技术观察安肠汤治疗前后不同级别神经元组织的PKCγ,ERK1/2,mGluR5, NR2BR蛋白表达，细胞内钙离子浓度的改变，大肠离体平滑肌肌张力的变化，阐明内脏高敏感性和内脏疼痛信息转导通路敏化的关键环节，从分子水平上探讨中药安肠汤干预IBS的主要靶点。

在前期研究中课题组已经证实安肠汤可以通过降低TRPV1在IBS大鼠脑肠轴中蛋白的表达，达到降低内脏高敏感性，减轻腹痛症状的作用，提示TRPV1是内脏疼痛外周初级伤害感受器，其信息可能汇合于PKCγ/ERK1/2/MAPK信号通路而向中枢转导，然而疼痛信号的转导与外周敏化和中枢敏化有关，因此本课题主要探讨内脏疼痛信号转导通路中外周敏化和中枢敏化的关键靶点。通过束缚+辣素刺激法建造肝郁脾虚型IBS-D大鼠模型，利用Western b loting，钙离子成像和电生理技术，以安肠汤干预IBS-D动物模型，观察对比安肠汤治疗前后模型脊髓段PKCγ,ERK1,mGluR5,NR2BR受体基因和蛋白表达，细胞内钙离子浓度的改变，大肠离体平滑肌肌张力的变化，阐述安肠汤缓解IBS-D腹痛症状的科学依据。动物实验研究，安肠汤干预IBS-D大鼠模型，研究安肠汤对模型大鼠腰段脊髓背角PKCγ,ERK1,mGluR5,NR2BR蛋白和基因表达，结果：脊髓背角PKCγ,ERK1,mGluR5,NR2BR蛋白和基因表达，模型组明显增高，与治疗组对比有统计学意义，说明PKCγ,ERK1,mGluR5,NR2BR的基因参与IBS－D大鼠疼痛信号中枢敏化机制，安肠汤可以有效调控PKCγ,ERK1,mGluR5,NR2BR蛋白和基因在脊髓背角的表达，从而达到缓解腹痛的临床效应机制。安肠汤干预IBS-D大鼠模型，研究背根神经节中PKCγ,ERK1,mGluR5,NR2BR在IBS-D大鼠中的表达，结果：安肠汤治疗组IBS-D模型大鼠背根神经节中PKCγ,ERK1,mGluR5,NR2BR的基因表达明显下降，与模型组比较，差异有统计学意义，说明PKCγ,ERK1,mGluR5,NR2BR的基因参与IBS－D大鼠疼痛信号外周敏化机制，安肠汤能有效降低IBS-D大鼠背根神经节中PKCγ,ERK1,mGluR5,NR2BR的基因表达，缓解腹痛症状。本课题实验证实了PKCγ,ERK1/2,mGluR5, NR2BR靶点在IBS的内脏疼痛信号激活、转导、调控中占据有关键位置，与外周敏化和中枢敏化关系密切。阐明了安肠汤通过干预背根神经节和脊髓背角PKCγ，ERK1，mGluR5，NR2BR的基因表达，调控PKC/ERK1/2/MAPK信号通路，缓解腹痛的重要作用机制。该项目为安肠汤治疗IBS-D提供了重要的科学依据