基于腹膜间皮细胞CD40/CD40L共刺激通路研究活血通腑方防治大鼠术后腹腔粘连作用机制

The interaction of CD40 with its ligand (CD40L) in peritoneal mesothelial cells (PMCs) mediates activation of some immune cytokines, which is the triggers for inflammation-fibrosis cascade reaction, playing a crucial role in the various pathological and physiological changes after peritoneal injury. More evidences demonstrate that PMCs exert the gate for adhesion formation mediated by the activation of CD40/CD40L co-stimulating signal. Based on our findings in long-time studies that effective clinical prescription Blood-activating and Organ-purging formula regulated PMCs function to prevent postoperative peritoneal adhesion at time-effect dependent manner after surgery, we suggested that these effects of this prescription may be associated with its regulation of PMCs CD40/40L co-stimulating signal pathway and inflammatory factor-induced the cascade reaction. Therefore, this project will investigate the mechanisms by which Blood-activating and Organ-purging formula prevents postoperative peritoneal adhesion mediated by PMCs CD40/CD40L co-stimulating signal pathway. To further understand this design, this project will investigate the effects of Blood-activating and Organ-purging formula focusing on CD40/CD40L co-stimulating signal pathway and inflammatory regulation factors in PMCs of rats after surgery, as well as in vitro model of PMCs by the methods of immunofluorescence histochemistry, Western blotting and Real-Time PCR, respectively. This projest will find the signal pathway mediated by Blood-activating and Organ-purging formula in this condition. These results from this study will reveal the molecular mechanisms of Blood-activating and Organ-purging formula and new targets of drug action, as well as provide the scientific evidences for clinical application.

腹膜间皮细胞(PMCs) CD40/CD40L相互作用激活介导产生免疫细胞因子是腹膜损伤后炎症-纤维化级联反应的触发点，在腹膜损伤后继发多种病理生理改变中起关键作用。有证据显示在CD40/CD40L信号激活介导下，PMCs呈现出对粘连形成的闸门效应。申请者在长期观察活血通腑方防治术后腹腔粘连时程效应基础上，提出该方效应机制可能与调控CD40/CD40L通路及刺激炎症因子所致级联状态进程有关，设计:基于CD40/CD40L共刺激通路开展该方防治大鼠术后腹腔粘连作用机制研究。拟围绕CD40/CD40L信号及炎症调控因子，用免疫荧光组化、Western blotting 和RT-PCR 等技术手段系统研究活血通腑方对整体动物术后及体外PMCs CD40/CD40L共刺激信号的干预作用，发现其介导效应的信号通路，阐明其防治术后腹腔粘连分子作用机制，明确药物新的作用靶点，为临床推广应用提供科学依据。

术后腹腔粘连是腹部外科临床难以避免的医源性并发症。受剂型、材料或副作用等诸多因素影响，当前临床缺乏针对性强的防治措施。加之腹腔粘连会继发引起肠梗阻、肠损伤、不孕不育以及腹痛等典型病症，术后腹腔粘连已带来较重的卫生经济与疾病负担。.手术创伤与素体虚弱导致的气血瘀结是术后腹腔粘连主要病机，理气活血、通腑散结是防治术后腹腔粘连主要治则。前期临床研究证实，活血通腑方防治术后腹腔粘连疗效显著，为探讨其疗效靶点及作用机制，项目在优化组方配伍、探讨适宜剂型、明确质量标准的基础上，通过整体动物与体外细胞研究，开展了活血通腑方防治术后腹腔粘连作用靶点与疗效机制研究。.项目建立了术后腹腔粘连动物模型，形成了腹膜间皮细胞原代培养及鉴定技术体系。探明了术后腹腔粘连形成的关键病理进程与有效干预时间窗。阐明了腹腔免疫失衡与炎症级联反应是粘连关键病理环节。运用网络药理学、生物信息学及分子生物学手段，证实CD40/CD40L相互作用激活介导产生免疫细胞因子失衡是腹膜损伤后炎症、纤维化级联反应的触发点，从组织、细胞和分子水平阐释活血通腑方多层次防治术后腹腔粘连的作用特点。项目明确了活血通腑方调控CD40/CD40L通路平衡免疫激活；调控IL-8、IL-10和TNF-α等炎性因子表达平衡炎性反应；调控腹膜组织型纤溶酶原激活物活性从而减少基质沉积；调控MCP-1、MMP等粘附分子及基质金属蛋白酶从而调节纤溶平衡减少胶原沉积，实现平衡CD40/CD40L表达，抑制炎症因子所致级联状态以及纤溶失衡所致胶原沉积，发挥防治术后腹腔粘连效应。.项目明确了术后腹腔粘连中医“气血瘀结”病机与炎症级联反应-纤溶失衡交互影响的生物学病理机制相关性，通过粘连动物模型制备及腹膜间皮细胞原代培养技术，阐明了活血通腑方作用靶点与药效机制，为术后腹腔粘连病理机制研究及中药新药创制提供了科学依据。