肝郁脾虚证IBS-D内脏高敏感性中枢敏化机制及中药干预研究

Visceral hyperalgesia (VHL) was the core pathophysiology of diarrehea-irritable bowel syndrome（IBS-D）. Central sensitization was the sign of the formation of VHL，and TRPV1、ERK and microglia was the critical path in IBS central sensitization. Liver stagnation and spleen deficiency was the core of TCM pathogenesis.Our previous study demonstrated：Shuganjianpi therapy was effective in treating IBS-D and could improve the VHL，the mechanism connected with the activation of brain -gut pathway (CRH pathway)，but the central sensitization mechanism in subsequent gut-brain pathway was no reports. Accordingly，the project would to observed the change of TRPV1 expression in colonic and L6S1 spinal cord、the change of pERK1/2 expression in L6S1 spinal cord 、the change of activation of microglia (OX42)、the change of expression of p38MAPK and P2X4 receptors in microglia in IBS-D rats，after these rats were treated by Tongxie Yaofang；at the same time, the ERK1/2 inhibitor PD98059 and p38MAPK inhibitor SB203580 were used to observe the changes of VHL. We intended to further explore the role of liver stagnation and spleen deficiency in the pathogenesis of IBS-D VHL central sensitization and the mechanism of soothing liver and invigorating spleen treating IBS-D，which would open up a new idea for the research of the dysfunction of liver and spleen in IBS-D.

内脏高敏感性(VHL)是腹泻型肠易激综合征（IBS-D）的核心病理生理机制，中枢敏化是VHL形成的标志，TRPV1、ERK和小胶质细胞是中枢敏化的关键通路。肝郁脾虚是IBS-D的中医核心病机。我们前期研究证实：疏肝健脾法治疗IBS-D疗效确切且可改善其VHL，与脑→肠通路(CRH通路)激活有关，而其后续肠→脑过程中枢敏化机制研究尚无报道。据此，本项目拟观察疏肝健脾法对IBS-D内脏高敏感性大鼠结肠和L6S1段脊髓背角TRPV1表达、L6S1段脊髓背角中pERK1/2表达、L6S1段脊髓背角中小胶质细胞的活化状况（OX42）、L6S1段脊髓背角中小胶质细胞p38MAPK和P2X4受体表达的影响，并同时给予ERK1/2抑制剂PD98059、p38MAPK阻断剂SB203580来观察大鼠内脏敏感性变化，探讨肝郁脾虚IBS-D的VHL中枢敏化作用机制，以全新的角度探讨IBS-D肝脾失调的发生机制。

本项目的科学假说为：TRPV1-ERK/小胶质细胞是中枢敏化和肠-脑轴的关键环节，也是肝郁脾虚证IBS-D的关键病理机制，疏肝健脾法通过调节TRPV1-ERK/小胶质细胞通路抑制中枢敏化调节VHL达到治疗IBS-D的目的。.本研究运用母子分离+慢性束缚+番泻叶灌胃复制肝郁脾虚证IBS-D大鼠模型，并通过评价AWR、AEMG及血清5-HT等指标明确VHL的形成。同时，在造模后8h、3d、7d、14d及21d的6个时间点分别进行模型评价，确定造模后14天内为指标检测和药物干预的最佳时间窗。据此结果，开展了疏肝健脾法治疗肝郁脾虚证IBS-D的效应研究，明确了疏肝健脾法通过调节大鼠VHL治疗IBS-D的作用。在此基础上，以中枢敏化通路TRPV1-ERK/小胶质细胞为切入点，采用ERK1/2抑制剂PD98059和p38MAPK（小胶质细胞功能性受体）阻断剂SB203580髓鞘内干预，并运用免疫印迹、免疫荧光及免疫组化等实验方法，观察并比较不同干预组大鼠结肠和L6S1段脊髓背角TRPV1的表达，L6S1段脊髓背角中pERK1/2的表达、小胶质细胞活化状态（OX42）及其特异性受体P2X4和p38MAPK的表达情况，进一步探讨疏肝健脾法调节IBS-D大鼠中枢神经敏化的的分子机制和作用靶点。结果表明，肝郁脾虚证IBS-D大鼠结肠及L6S1段脊髓背角的TRPV1表达显著升高、L6S1段脊髓背角中pERK1/2的表达增加、小胶质细胞活化，表现为小胶质细胞标记物OX42表达增加、特异性受体P2X4及p38MAPK的表达升高。疏肝健脾中药干预能显著逆转上述蛋白的表达变化；PD98059及SB203580干预对上述蛋白的表达均具有抑制作用，且PD98059和SB203580联合及与中药联合的干预作用与单用中药无显著差异。.综上，本研究探讨了脑-肠轴功能紊乱与肝郁脾虚证IBS-D间的关系；发现了肝郁脾虚证IBS-D大鼠VHL及中枢敏化机制与TRPV1诱导ERK和小胶质细胞活化密切相关；明确了疏肝健脾法通过调节中枢敏化关键通路TRPV1-ERK/小胶质细胞治疗肝郁脾虚证IBS-D的效应机制；初步揭示了疏肝健脾中药治疗IBS-D可能的作用靶点及分子机制。本研究结果将为下一步深入开展疏肝健脾中药治疗IBS-D的详细效应靶点及后续的临床转化研究提供数据基础。项目资助发表论文4篇。培养硕士4名，博士1名