桂枝、荆芥挥发油抗炎效应的NLRP3炎症小体通路机制研究

Early studies have confirmed that the mechanisms of anti-inflammatory and antiviral effects of volatile oil of Guizhi and Jinjie were related to interfereing the signal transduction pathway of TLR2/4 and TLR7. Recent studies have shown that NLRP3 inflammasome is an important link between inflammation and immune response，that activation of NLRP3 inflammasome is key link of IL-1β maturation, and be considered as the new treatment target on infectious and inflammatory diseases. It also has close connection with TLR activation. Under the guidance of TCM theory, and based on the pathogenic microorganism infection, inflammation is closely related to the "Exterior syndrome ", and volatile oil are the main material basis of drugs for relieving Exterior Disorder that show "pungent flavour" characteristic，this study will further explain the mechanism of anti-inflammatory action of volatile oil of Guizhi and Jinjie from NLRP3 inflammasome pathway. In study, we will choose NLRP3 inflammasome as the pointcut, and make inflammatory cell/tissue - inflammatory cytokines - signal transduction as the main line, use the study mode of combining in vitro with in vivo experiments, to evaluate the expressions of the key target molecules or products in NLRP3 inflammasome activation pathways by using the methods of fluorescence quantitative PCR, Western blot, gene chip and cell fluorescence image analysis. The results will further explore the molecular mechanism of anti-inflammatory action of volatile oil of Guizhi and Jinjie, meanwhile rich the scientific connotation of "xinsan jiebiao" efficacy of drugs for relieving Exterior Disorder. …

前期研究证实，桂枝、荆芥挥发油抗炎、抗病毒效应的发挥与干预TLR2/4、TLR7信号转导通路有关。新近研究表明，NLRP3炎症小体作为连接炎症与免疫的重要纽带，该复合体的活化是IL-1β等促炎因子成熟的关键环节，被认为是感染性、炎症性疾病的治疗新靶点，且与胞膜TLR的激活存在密切联系。本研究在中医药理论指导下，基于病原微生物感染、炎症反应与"表证"密切相关，挥发油是解表药"辛味"的主要物质基础的基本事实，围绕桂枝、荆芥挥发油的抗炎目标效应，选择NLRP3炎症小体为切入点，以炎性细胞/组织-炎性细胞因子-信号转导为主线，采用体外与体内实验相结合研究模式，运用荧光定量PCR、Western blot、基因芯片、细胞荧光成像分析等方法技术，以NLRP3炎症小体激活通路途径的若干关键靶分子、产物为评价指标，进一步探索桂枝、荆芥挥发油的抗炎效应的分子机制，有利于丰富解表药"辛散解表"性效的科学内涵。

课题在中医药理论指导下，基于“表证”的临床病理特征，解表药桂枝、荆芥具有抗炎抗感染效应等事实，及其抗病毒作用与调控TLR受体通路相关的前期基础，本研究围绕抗炎目标效应，以桂枝、荆芥挥发油（RECO、EOST）及主要成分桂皮醛（CA）、胡薄荷酮为研究对象，将NLRP3炎症小体通路作为切入点，通过体外与体内研究，以炎性细胞/组织-炎性细胞因子-信号转导为主线，揭示药物抗炎免疫调节作用与调控NLRP3炎症小体通路的相关性。研究发现：① 以ATP和nigericin为NLRP3炎症小体激活物时，RECO、EOST、CA和胡薄荷酮能下调THP-1巨噬样细胞Pro-Caspase-1、p20、Pro-IL-1β和IL-1β蛋白及NLRP3、ASC、Caspase-1和IL-1β mRNA的表达，降低细胞中ROS表达，显著抑制ATP致细胞中NLRP3和ASC的共定位效应，减少CPPD诱导的细胞中Cathepsin B蛋白表达。RECO、EOST和胡薄荷酮显著降低细胞上清中Pro-IL-1β蛋白表达， CA、胡薄荷酮和EOST降低细胞上清中Caspase-1（p20）水平。② 给予4种药物后，能改善内毒素中毒C57BL/6J小鼠的精神状态和活动情况。显著抑制血清中IL-1β、IL-18、IL-5、IFN-γ、MIP-1β和M-CSF的表达，减少肺组织中嗜中性粒细胞数量和NO含量，下调肺组织中NLRP3、p20、Pro-IL-1β和P2X7R蛋白表达，下调肺中NLRP3、IL-1β、iNOS、IFN-β mRNA表达。此外，RECO、EOST能减少血清MCP-1含量和肺中STAT1 mRNA表达，CA、EOST和胡薄荷酮下调肺中Cathepsin B蛋白表达及升高COP-1蛋白水平，后两者亦减少血清TNF-α水平。③ 桂枝乙酸乙酯部位和桂皮酸能减少内毒素中毒KM模型小鼠血清中BUN、IL-18和TNF-α含量，下调肺中NLRP3和p20蛋白及NLRP3、Caspase-1和IL-1β mRNA的表达，减少肺中嗜中性粒细胞数量。乙酸乙酯部位能显著减少血清CRE和肝脏MDA含量，增加肝SOD活力和血清IFN-γ含量，下调Pro-IL-1β蛋白的表达。综上，荆芥、桂枝挥发油及桂皮醛、胡薄荷酮抗炎作用的发挥与抑制NLRP3炎症小体的激活有关，为其治疗“表证”的临床运用提供了更为扎实的科学依据