【作者】 李俊；

【导师】 吴清和；

【作者基本信息】 广州中医药大学 ， 中药学， 2010， 博士

【摘要】 目的：在成功复制脓毒症动物模型基础之上,从分子细胞层面探讨脓毒症的发病机制,并采用安宫牛黄丸进行干预,观察安宫牛黄丸对脓毒症大鼠的保护作用,并在此基础上观察安宫牛黄丸对脓毒症早期炎症介质TNF-α、晚期炎症介质HMGB-1的mRNA表达的影响以及对JAK/STAT通路活化的影响,从分子水平探索安宫牛黄丸治疗脓毒症的机制和深层次靶点,为中医药治疗脓毒症的机理研究能够深入到分子水平、为丰富和发展中医温病学治法理论的科学内涵、阐述中医名方安宫牛黄丸治疗温热病危重症候(脓毒症)的现代药理机制、为进一步探索中西医结合治疗高发病率、高病死率重大疾病脓毒症的方案打下理论基础。方法：采用盲肠结扎穿孔法(CLP)复制脓毒症动物模型。SPF级大鼠90只,雌雄各半,随机分为5组,(1)正常对照组(n=10),麻醉后取血,处死取材待测；(2)模型组(n=20),于CLP前0.5h,CLP后6h,以10ml／kg体重灌胃蒸馏水,并于术后24h取血、处死取材待测；(3)安宫牛黄丸低、中、高剂量干预组各20只(n=60),于CLP前0.5h,CLP后6h,分别以1.0g／kg、2.0g／kg、3.0g／kg体重灌胃给药,并于术后24h取血、处死取材待测。同时观察各组动物24小时内死亡率及一般表现；动物尸体解剖腹腔感染程度；检测术后第6h、12h、24h动物肛温；血分析仪检测术后24小时动物白细胞计数；全自动生化分析仪测定血清ALT、AST、BUN、Crea、TB水平；酶学分光光度法测定肺组织髓过氧化物酶(MPO)含量；显色基质鲎试剂测定血浆内毒素含量；半定量RT-PCR法检测肝脏、肺脏、肾脏STAT1、STAT3、TNF-α、HMGB-1 mRNA的表达；EMSA法检测肝脏、肺脏STAT1、STAT3 DNA结合活性。结果：采用CLP法复制脓毒症动物模型,动物术后6小时后病态逐渐明显,并开始出现死亡,动物活动减少,不再合群取暖,嗜睡,竖毛,不饮水,有的出现呼吸急促,脓尿,腹泻,眼角渗出物等,24小时的累积死亡率达到了55%；从动物尸检发现腹腔感染程度比较严重；模型动物一直处于一个低温状态(中心体温<36℃)并出现了白细胞减少症(外周血白细胞计数或<4×109／L),并同时伴有主要脏器的严重损害(血清ALT、AST、TB水平显著高于正常对照组动物、肺脏MPO活性显著高于正常对照组)；脓毒症动物血浆内毒素水平要显著高于正常对照组；脓毒症大鼠各主要脏器STAT1、STAT3 DNA活性显著升高,JAK/STAT通路高度活化,同时早期炎症介质TNF-α、晚期炎症介质HMGB-1的mRNA表达显著上调。安宫牛黄丸能够改善脓毒症大鼠的一般表现,显著降低脓毒症大鼠的死亡率,提高脓毒症休克时大鼠的体温,提高脓毒症大鼠的白细胞计数,安宫牛黄丸具有降低脓毒症大鼠血清ALT、AST水平的趋势,能够显著降低脓毒症大鼠血清TB水平,降低脓毒症大鼠肺脏MPO活性,降低脓毒症大鼠血浆内毒素含量；安宫牛黄丸能够显著下调JAK/STAT通路中STAT1和STAT3的mRNA表达,能够显著减弱STATs RNA的结合活性,显著降低JAK/STAT通路的活化程度,同时还能显著下调TNF-α和HMG-1mRNA的表达。结论：采用CLP法能够成功复制脓毒症大鼠模型；大鼠脓毒症的发病机制可能与JAK/STAT通路高度活化以及由此通路激活的早期TNF-α、晚期炎症介质HMGB-1等炎症介质释放有关,并由此而导致了炎症反应失控,最终可能引起脏器的损害,引发MODS、脓毒症休克等。安宫牛黄丸能够降低脓毒症大鼠模型的死亡率,提高脓毒症休克时大鼠的体温,提高脓毒症大鼠的白细胞计数,对脓毒症大鼠整体有较好的保护作用。同时,安宫牛黄丸能够具有降低脓毒症大鼠血清ALT、AST水平的趋势,能够显著降低脓毒症大鼠血清TB水平,降低脓毒症大鼠肺脏MPO活性、血浆内毒素含量,对脓毒症大鼠主要脏器功能有明显的改善作用,这可能是安宫牛黄丸治疗脓毒症的机制之一。安宫牛黄丸能够显著下调脓毒症大鼠肝脏、肺脏、肾脏等主要脏器中早期炎症介质TNF-α和晚期炎症介质HMGB-1 mRNA表达；能够显著下调脓毒症大鼠肝脏、肺脏、肾脏等主要脏器中STAT1、STAT3 mRNA表达；还能显著降低JAK/STAT通路中STAT1和STAT3 DNA结合活性,降低JAK/STAT通路的活化程度。这从分子水平进一步阐明了安宫牛黄丸治疗脓毒症的作用机制,为安宫牛黄丸在危重病急救领域中的应用提供了理论基础。更多还原

【Abstract】 Objective:based on successful replication of sepsis animals, the paper studies on the pathogenesis of sepsis from the level of molecular cell. Using the intervene treat of Angong Niu Huang Wan, the paper will observes how the pill protects the septic rats. On the basis of that, it also will check the pills’mRNA expression of early sepsis inflammatory media TNF-aand later media HMGB-1, also its effect of Janus kinase/signal transducer and activators of transcription (JAK/STAT). The pape r will offer the theoretical foundation for the application of Angong Niu Huang Wa n in injury severity from exploring the mechanism of how Angong Niu Huang Wan treating the sepsis on the basis of molecular mechanism, and deep level target.Methods:adopting CLP to replicate the sepsis animal model; 90 SPF rats, hal f male and half female, divided into five groups, (1) the control (n=10), get the blo od after anesthesia then euthanize them for the test; (2) the model (n=20), get the blood 24h later after treating the rats with distilled water (10ml/kg) 0.5h before CL P and 6h after CLP, then euthanize them for the test; (3) Angong Niu Huang Wan low, middle and high, each 20 rats, get the blood 24 hours after gastric perfusion (1.0g/kg,2.0g/kg,3.0g/kg),0.5h before CLP and 6h after CLP, then euthanize them for the test; observe each groups and check the death rate and normal behaviors d uring the 24h; the degree of animal autopsy intraperitoneal infection; the rats rectal temperatures 6h,12h and 24h after operation; the WBC of the rats 24h after operati on measured by blood analyzer; the ALT, AST, BUN, Crea, TB levels of the Seru m measured by automatic biochemistry analyzer; the MPO content in the lung meas ured by enzymatic determination; the toxin content in the plasma by limulus polyph emus; the expression of STAT1, STAT3, TNF-αand HMGB-1 of liver, lung and kidney measured by semi-quantitative RT-PCR; the DNA binding activity of STAT1 and STAT2 measured by EMSA.Results:based on replication of sepsis animal models by CLP, the ill conditio ns of the rats gradually increased and some rats would even die; the activities of ra ts were induced and they no longer gather together to keep warm; they has the feat ures of sleepiness with pio-erection and do not drink water; some rats even suffer t he shortness of breath, pyuria, diarrhea and eye exudate. The cumulative rate of dea th reaches 55% in 24h. Post mortem examination reveals the serious degree of intra peritoneal infection; the models’body temperatures keep in a low condition (core te mperature less then 36℃), get leukopenia (WBC maybe less than 4×109/L) and the ir main organs are seriously damaged (ALT, AST and TB levels of serum and lung MPO activity are obviously higher than the control group); the toxin level in the s epsis rats’serum is very higher than the control group; the DNA activity of STAT1 and STAT 3 of the sepsis rats’main organs also rises obviously, JAK/STAT is al so highly activated in the pathway and meanwhile, the expression of early inflamma tory media TNF-αand later media HMGB-1 is also highly raised.Angong Niu Huang Wan can improve the normal behaviros of the sepsis rat s, obviouly reduce their death rate, raise their tempeatures during their shock and in crease their WBC count. The pills can lower the ALT and AST levels in the sepsis rats’serum, higly decreased their TB level in the serum, their lungs’MPO activity and the toxin content in their plasma. The pills can reduce JAK obviously. STAT1 and STAT3’s mRNA expression in the STAT activation pathway can reduce STAT s’RNA bending activity, the pathway’s activation degree of JAK/STAT and meanw hile the Mrna expression of TNF-αand HMGB-1.Conclusion:the CLP method can successfully replicate the sepsis rate models; the pathogenesis of the septic rats might be related to the higher pathway activatio n of JAK/STAT and the release of early inflammatory media TNF-αand later med ia HMGB-1 caused by the activatio pathway so as to cause the inflammatory effect s out of control and finally lead to the organs damage, MODS and septic shock. A ngong Niu Huang Wan can well protect the sepsis. The possible molecular mechani sms treatment of the pills for sepsis is to reduce the STAT1 and STAT3 expression in the JAK/STAT activation pathway so as to reduce the JAK and STAT’s activati on pathway degree, thus reduce the express of related cytokine such as TNF-α, H MGB-1, from JAK/STAT activation pathway, regulate the inflammatory infection tha t is out of control and reduce the inflection damage to organs so as to protect orga nism.更多还原