【作者】 全胜；

【导师】 姒健敏；

【作者基本信息】 浙江大学 ， 内科学， 2006， 硕士

【摘要】 全身炎症反应综合征（SIRS）是1991年由ACCP／SCCM（美国胸科医师协会和危重病医师协会）提出的一个新概念。SIRS是因为感染或非感染因素作用于机体而引起的一种全身性炎症反应临床综合征，具有以下各项中的2项或者2项以上者，SIRS诊断即可成立：①体温＞38℃或＜36℃；②心率＞90次／分；③呼吸频率＞20次／分或PaCO₂＜32mmHg（4.3kPa）；④白细胞计数＞12×10⁹／L或＜4.0×10⁹／L或幼稚粒细胞＞10％。2001年12月，ACCP等组织修改了诊断标准，纳入更广的症状和征象范畴，其中包括了寒战、尿量减少、皮肤灌注降低、血小板计数下降、高血糖以及无法解释的精神状态改变等。近期还提出SIRS是机体针对免疫触发而同时产生的过度炎症、凝血以及被削弱的纤溶三种过程之间复杂的相互作用的结果，这是SIRS病理生理学的核心。SIRS的发生主要与全身炎症系统功能紊乱，大量炎症因子的产生和释放，继而产生全身炎症瀑布反应有关，SIRS是MODS的发病基础，炎症因子的失控性释放也是SIRS向MODS转化的关键。因此，针对炎症因子的治疗具有较强的临床应用价值。国内外以往使用诸如清除内毒素、调节免疫功能、脏器功能的支持、改善营养代谢紊乱等方法防治SIRS，却始终无法得到满意的疗效。中药葛根素源自豆科植物野葛的干燥根，主要成分为8-β-D吡喃葡萄糖-4，7-二羟异黄酮甙。近年来，随着临床研究以及动物实验的不断深入，葛根素对于脑组织和心肌缺血再灌注后炎症因子的调节作用已经有了一定的报道。本实验旨在通过SIRS动物模型的建立，探讨葛根素对于SIRS的炎症因子（TNF-α；IL-6；IL-10）的影响，从而为寻找中医防治SIRS提供实验依据。目的：通过SIRS动物模型的建立，探讨葛根素对于SIRS的防治及相关作用机理研究。材料1．实验动物：SD大鼠：清洁级，5-7周龄，体重160-200g，雌雄各半，恒温22℃室内饲养，正常光照时间8：00a.m.-20.00p.m.，无饮食饮水限制。2．实验试剂：葛根素100mg／2ml／amp酵母多糖（zymosan A）液体石蜡TNF-αELISA试剂盒IL-6 ELISA试剂盒IL-10 ELISA试剂盒方法：1．模型制备将酵母多糖粉剂和液体石蜡混合，高频振荡15分钟，然后在100℃水浴80分钟灭菌，制成酵母多糖浓度为100mg／ml的混悬液备用，用时以40℃水浴，高频振荡15分钟后注药。制模实验分4组（n=48）：腹腔注射酵母多糖混悬液1000mg／kg组、腹腔注射酵母多糖混悬液750mg／kg组、腹腔注射酵母多糖混悬液500mg／kg组、正常对照组。本实验采用胡森提供的动物SIRS标准，以直肠温度较正常升高或者降低1℃和白细胞总数超过对照值的2倍或减少50％的作为大鼠SIRS选取标准。2．实验分组在成功制模的基础上，动物被随机分成五个组：A组（12只）为正常对照组；B1、B2组（每组12只）为1000mg／kg（体重）酵母多糖混悬液诱导的SIRS组；C1、C2组（每组12只）为750mg／kg（体重）酵母多糖混悬液诱导的SIRS组；其中B2、C2组尾静脉注射葛根素（100mg／2ml／amp）0.25ml／200g；A、B1、C1组尾静脉注射生理盐水0.25ml／200g。实验SD大鼠在实验前18h开始禁食（不禁水），上午8时先称重、测直肠温度，然后腹腔注射酵母多糖混悬液，立即尾静脉注射葛根素或者等量生理盐水，1次／日／只。3．观察项目大鼠腹膜炎诱导的SIRS模型的成模率。大鼠SIR模型死亡率的观察对比（B1和B2组）。大鼠SIR模型相关炎症因子IL-10、IL-6、TNF-α的检测对比（C1和C2组）。4．统计学方法统计学方法数据均以均数±标准差（X±S）表示。组间先行F检验，再行t检验，p＜0.05为差异有统计学意义，p＜0.01为差异有高度统计学意义。结果：1．成功的创建了大鼠腹膜炎诱导的SIRS模型体温和白细胞总数变化中符合SIRS标准的有两组（腹腔注射1000mg／kg组和750mg／kg组），成模率分别为83.3％和91.6％。鉴于腹腔注射1000mg／kg组大鼠快速向MODS甚至MOF发展，导致大鼠较高的死亡率，故本实验采用此组作为正式实验中死亡率对比组（B1、B2）的剂量。而腹腔注射酵母多糖750mg／kg组大鼠死亡率低，实验可行性高，作为正式试验中炎症因子对比组（C1、C2）的剂量。2．在葛根素的干预下，降低了SIRS组大鼠48h死亡率，与对照组比较有显著的统计学差异，但对SIRS组大鼠24小时死亡率影响与对照组无显著差异（附表3）。3．葛根素对SIRS炎症因子的调节作用：促炎因子TNF-α、IL-6分泌显著减少。（附表4）抗炎因子IL-10分泌显著增加（附表4）。结论：1．酵母多糖石蜡油混悬液腹腔注射建立大鼠SIRS模型成功。2．葛根素能有效的降低SIRS大鼠48h死亡率，具有保护作用。3．葛根素对于SIRS的保护作用可能通过对SIRS相关炎症因子的调节作用实现：抑制促炎因子TNF-α、IL-6分泌；促进抗炎因子IL-10分泌等。更多还原

【Abstract】 The SIRS was introduced by ACCP and SCCM since 1991.The definition of SIRS did not necessarily require the presence of bacterial infection. Clinical SIRS and MODS were diagnosed by meeting two or more of following criteria. ① Temperature >38℃ or<36℃; ② Heart rate >90 beats per minute;③ Respiratory rate >20 breaths per minute or PaCO₂<32 mmHg; ④ White blood cell count >12×10⁹/L or <4×10⁹/L or presence of >10% immature neutrophils.Altered organ function such that homeostasis needs to be maintained with intervention.SIRS is accompanied with a complex, unbalanced, fatal activation of the immune and inflammatory systems. Although the mechanisms are not yet completely explored, a variety of infectious and noninfectious conditions can lead to hyperactive inflammatory responses at the onset of SIRS, followed by immunoparalysis, compromised immune function and defective innate immune responses at a later stage.It was previously reported that both pro- and anti-inflammatory cytokines are elevated in SIRS. Cytokine mediated systemic neutrophil activation is a direct consequence of SIRS, and can lead to multiple organ dysfunction syndrome （MODS）. This prospective study assessed the risk of SIRS and MODS by measuring the circulating levels of inflammatory cytokines such as IL-6, TNF-a and IL-10 as well as the neutrophil functions as a marker of organ failure. Object:To study the prevention effect of Puerarin on SIRS and MODS induced by peritonitis in rats.Materials:1. AnimalsSD mice （5-7 weeks age） weighing 160-200 g, purchased from the Animal Care and Use Center （Breeding of Laboratory Animals, Zhejiang University, Hangzhou, China）, were kept in a room with controlled temperature （22 ℃） and lighting （lights on 8:00 a.m.-20:00 p.m.） and free access to food and water. All experiments were conducted between 8:00 a.m. and 4:00 p.m.2. Drugs and ReagentsPuerarin 100mg/2ml/ampZymosan A （Sigma）Mineral oilTNF-α ELISA KitIL-6 ELISA KitIL-10 ELISA KitMethods:1.The SIRS ModelZymosan A （Sigma） was suspended in mineral oil at a concentration of 100 mg/ml, and sterilized in a boiling water bath for 80 min. The zymosan in oil suspension was then sonicated in 5 ml aliquots for 20 s and frozen at-70℃ until used. Zymosan was injected intraperitoneally （ip）.The SIRS models of rat were diagnosed by meeting two offollowing criteria.① Compared with the normal group, the rectum temperature raisedor declined ≥1℃; ② Compared with the normal group, the total white cell amount exceeded 200% or reduced 50%.2. The Experiment GroupsBased on the successful manufacture of SIRS model in rats, SD mice were randomized （on experiment hour 0, time =0h） into five initial groups: Group A （Normal Control） received no zymosan and no puerarin. Group B1 （SIRS Control） received zymosan （1000mg/kg mouse BW t=0） and no puerarin. Group B2 （SIRS Treatment） receive zymosan （1000mg/kg mouse BW, t=0） and puerarin （62.5mg/kg mouse BW, t=0）. Group C1 （SIRS Control） receive zymosan （750mg/kg mouse BW t=0） and no puerarin. Group C2 （SIRS Treatment） receive zymosan （750mg/kg mouse BW t=0） and puerarin （62.5mg/kg mouse BW, t=0）. Mice were sacrificed when they were preterminal （clinical signs of shaking, shivering, or paralysis） or on Experiment Hour 48 （survivor）.At the termination of the experiment, mice were anesthetized with Phenobarbital and diethyl ether, and blood was obtained for blood routine assays by severing the carotid artery and collecting the blood in eppendorf syringes （EDTA-K2 500ul）. The blood was immediately centrifliged, and plasma was rapidly separated for the ELISA test of IL-10, IL-6, and TNF-α.3. The Observing Aim① The success rate of SIRS model induced by peritonitis in rats; ② The mortality in the model of SIRS were compared betweenGroup B1 and Group B2;③ The inflammatory cytokines such as IL-10, IL-6 and TNF-αwere tested and compared between Group C1 and Group C2. Both of them were compared to the control group injected i.p with normal saline. RESULT1. The SIRS models induced by peritonitis in rats were made successfully.There are two groups meeting two criteria （Group 1000mg/kg and Group 750mg/kg）. （TAB1 and TAB2）TAB1: The temperature and the total white cell amount of model groupsMost rats in group 1000mg/kg were dead in 48 hours TAB2: The Mortality of model groups2. Compared with untreated mice （Group B1）, puerarin-treated mice （Group B2） had significantly lower level of mortality （48h） .When control mice （Group A） were compared to Group Bl, the zymosan-exposed mice had higher morbidity scores, shorter length of survival. Functionally, the zymosan-treated mice demonstrated evidence of multiple organ system dysfunction.Mortality: Treatment of mice （Group B2） showing clinical evidence of MODS with puerarin produced a significant reduction in mortality when compared to similar mice （Group B1）, which did not receive puerarin. （TAB3） TAB3: The Mortality of SIRS group mice3. Compared with untreated mice （Group C1）, puerarin-treated mice（Group C2） had significantly higher level of IL-10 and lower level of IL-6 and TNF-α（P<0.01）.（TAB4）TAB4: Effect of puerarin on inflammatory factors system in peritonitis-induced SIRS in rat （48h）CONCLUSION1. Intraperitoneal zymosan in a mineral oil carrier induces SIRS model in rats successfully; 2. Puerarin administration reduces mortality in the model of SIRS significantly;3. Puerarin administration inhibits the release of pro-inflammatory cytokines such as TNF-a（P<0.01）, IL-6（P<0.01）, promotes the release of anti-inflammatory cytokines such as IL-10（P<0.01）,which may be the mechanism of puerarin’s protection.Understanding the mechanism of puerarin’s effects in SIRS and MODS may lead to therapeutic benefits.更多还原