Rab27b信号转导通路参与锦红汤调节脓毒症时血小板囊泡分泌的机制研究

Jinhong decoction, one prescription invented by the famous tranditional Chinese physician Gu Bohua from Shangha, consists of rhubarb, Sargentgloryvine Stem, dandelion and others. Jinhong decoction has been rewarded Science and Technology Progress Award of Shanghai for its inhibition of systemic inflammatory response and adjustment of inflammatory cytokines expression. It has been demonstrated that platelet play an important role not only in coagulation, but also in immunity. In the early stage of sepsis, a lot of inflammatory and coagulation factors are released from the platelet vesicles after platelet activation, which is one part of inflammatory cascade reaction. As small molecule GTP enzyme and a member of Rab protein family, Rab27b is mainly distributed in platelet, regulating platelet vesicle formation, transport, and secretion. We have found that in the early stage of sepsis platelet vesicle can secrete lots of inflammatory factors to promote coagulation disorder, and early application of Jinhong decoction can reduce the incidence of sepsis-induced coagulation dysfunction. Therefore, we prepare to study the mechanism of Jinhong Decoction regulating platelet vesicle secretion for inflammatory factors by the Rab27b signal transduction pathway in mouse Rab27b and NF-E2 knockout model, in which platelet aggregation rate test, platelet dense granule secretion test, platelet alpha granule secretion test, RT-PCR, Western blot, transmission electron microscope and flow cytometry were executed.

锦红汤是上海名中医顾伯华验方，功效清热解毒、行气通腑，可调正脓毒症炎症因子表达，抑制全身过度炎症反应。血小板囊泡含有大量炎症和凝血因子，脓毒症时会快速释放入血，促进脓毒症向多器官功能衰竭发展。Rab27b是Rab蛋白家族成员，分布在血小板及其内体膜表面，主要调控血小板内的囊泡形成、转运、分泌。基于锦红汤治疗脓毒症的临床疗效、Rab27b调控血小板囊泡分泌的信号转导机制在脓毒症发展中的重要作用，本课题在坚实工作基础（第六届全国重症医学大会三等奖）上，采用小鼠盲肠结扎模型，应用血小板聚集率测定、血小板致密颗粒分泌测定、血小板α颗粒分泌测定、RT-PCR、Western blot、透射电镜、流式细胞仪、免疫荧光法等方法，采用Rab27b基因敲除鼠模型，研究锦红汤通过调控脓毒症小鼠Rab27b的基因转录进而调节血小板囊泡分泌的具体机制。

锦红汤是上海名医顾伯华验方，功效清热解毒、行气通腑，可调正脓毒症炎症因子表达 ，抑制全身过度炎症反应。血小板囊泡含有大量炎症因子，会促进脓毒症发展。Rab27b分布在血小板膜表面，主要调控血小板内的囊泡分泌。本课题采用鼠盲肠结扎模型和Rab27b基因敲除鼠模型，设立假手术组、CLP组、JHT组、Rab27b(-/-)组、Rab27b(-/-)CLP组、Rab27b(-/-)CLPJHT组，分析JHT对血小板计数的影响，电镜法观察血小板形态，血小板聚集仪测定血小板聚集率，ELISA法检测5-HT，流式细胞仪法检测p选择素，激光共聚焦法检测Rab27b 细胞内分布，RT-PCR检测Rab27b mRNA，Western blot检测Rab27b 蛋白。主要结果发现：术后4h，CLP组和JHT组的PLT计数较假手术组降低；术后72h，JHT组较CLP组升高。PLT聚集率实验显示，术后4h时CLP组较假手术组升高，JHT组较CLP组降低；术后72h时CLP组较假手术组降低，JHT组较CLP组升高。术后4h和72h时，CLP组较假手术组升高，JHT组较CLP组降低。5-HT分泌试验结果显示，术后4h时 CLP组、Rab27b(-/-)+CLP组和Rab27b(-/-)CLPJHT组较假手术组降低。激光共聚焦实验显示，术后72h时CLP组的rab27b阳性率低于假手术组，JHT组高于CLP组。RT-PCR试验结果，术后4h和72h时，假手术组、 CLP组和JHT组的Rab27b mRNA水平无差异。Western blot试验结果，术后4h时，CLP组的Rab27b 蛋白水平低于假手术组，JHT组较CLP组升高；术后72h时，CLP组低于假手术组，JHT组与CLP组无差异。结论：锦红汤能够改善脓毒症时血小板聚集功能和调节P选择素的表达，并上调血小板计数，但该作用与Rab27b通路无关。