PKA依赖cAMP信号介导TRPV1敏化调控内脏敏感性探讨疏肝健脾法防治肠易激综合征研究

Previous studies have reported that Soothing liver and invigorating spleen decoction exerts an effect on reducing visceral hypersensitivity and relieving symptoms of IBS.The regulatory targets maybe relevant with changes of mast cells and Cajal cells and TRPV1 expression. What is the connection between TRPV1 expression and visceral sensitivity?PKA-dependent cAMP signaling mediated TRPV1 sensitization, non-selective cation channel opening, Ca2+ influx, increase the release of gastrointestinal peptides, leading to visceral hypersensitivity and gastrointestinal motility disorders, which participated in the pathogenesis of IBS. We hypothesized that soothing liver and invigorating spleen decoction may interfere with PKA-dependent cAMP signaling regulates TRPV1 sensitization, reduced visceral hypersensitivity, relieve IBS. In vivo, our research through animal experiment to observe the indexes correlated with PKA-dependent cAMP signal mediated TRPV1 sensitization before and after use of soothing liver and invigorating spleen decoction treatment of IBS. In vitro study, we assay soothing liver and invigorating spleen decoction on regulating effect of isolated colonic smooth muscle tension, intracellular calcium concentration and DRG cell's TRPV1 channel-opening . This study clarifies soothing liver and invigorating spleen decoction prevention and treatment of IBS may have an important significance.

前期证实疏肝健脾类方药具有降低内脏高敏感性，缓解IBS 效用，其靶点可能与调控肥大细胞、cajal细胞及TRPV1 表达有关。TRPV1表达与内脏敏感性有何联系？ PKA 依赖cAMP 信号介导TRPV1 敏化，非选择性阳离子通道开放，Ca2+内流，胃肠肽类物质释放增加，导致内脏高敏感性和胃肠运动功能紊乱，参与IBS发病。推论疏肝健脾类方药可能通过干预PKA 依赖cAMP 信号调控TRPV1敏化，降低内脏高敏感性，缓解IBS效用。项目拟通过动物实验观察疏肝健脾方药治疗前后PKA 依赖cAMP 信号介导TRPV1 敏化调控内脏敏感性中相关指标的改变，离体观察疏肝健脾方药对结肠离体平滑肌肌张力、细胞内钙离子浓度变化以及DRG细胞TRPV1通道开放的调节作用，对于阐明疏肝健脾类方药防治IBS 的可能科学内涵有重要意义。

目的：基于PKA依赖cAMP信号介导TRPV1敏化探讨疏肝健脾方降低内脏高敏感性，缓解肠易激综合征(IBS)机制。方法：⑴在体实验：选择72只SD大鼠，按体重随机分为正常组，模型组，治疗组（低、中、高剂量疏肝健脾方）、对照组（得舒特），除正常组外，均采用束缚应激联合辣素灌胃制备 IBS 模型，将疏肝健脾类方药及得舒特分别作用于上述模型。应用光镜及电镜检测结肠组织学及超微结构、电生理评估大鼠内脏敏感性、以及WB、RT-PCR检测结肠PKA、cAMP、P-TRPV1、SP、CGRP 表达。⑵离体实验：截取大鼠结肠平滑肌肌条，应用电生理技术观察疏肝健脾方对结肠平滑肌张力影响；分离培养大鼠结肠平滑肌细胞，应用激光共聚焦显微镜及流式细胞仪检测Ca2+含量；分离原代培养DRG细胞，应用膜片钳检测 DRG细胞 TRPV1 通道，应用WB及RT- PCR检测 DRG 中PKA、cAMP、P-TRPV1、SP、CGRP 表达。结果：⑴在体实验:光镜下大鼠结肠腺体结构、排列正常，符合IBS改变；电镜下模型组大鼠结肠紧密连接增宽；模型组大鼠腹部抬高和背部拱起的容量阈值均显著低于治疗组，提示内脏高敏感性；光镜下见结肠肥大细胞活化数及PKA、P-TRPV1、cAMP、CGRP、SP表达升高，与治疗组比较，有统计学差异（P＜0.05）。⑵离体实验：模型组大鼠平滑肌条给药前收缩值比正常组显著增高；而给药后收缩值发现两组无统计学意义。应用MTT法确定最佳给药浓度为1.56%，0.78%，0.39%，按照上述浓度给药进行Ca2+内流含量检测，激光共聚焦结果提示各组之间无明显差异；而流式细胞仪检测发现治疗组具有降低结肠平滑肌细胞内Ca2+浓度的效用。膜片钳结果提示：疏肝健脾方虽然无法直接激活DRG细胞中的TRPV1通道，却可以正向调控Capsaicin激动的TRPV1电流。DRG细胞模型组P-TRPV1、PKA、CGRP 、cAMP表达高于治疗组，SP表达量均低于治疗组。结论：疏肝健脾方通过下调PKA、cAMP，调控P-TRPV1，减少SP及CGRP表达，降低内脏敏感性发挥防治IBS效用。