【作者】 于征淼；

【导师】 钟嘉熙；

【作者基本信息】 广州中医药大学 ， 中医临床基础， 2009， 博士

【摘要】 研究目的:“炎症介质组”是指一个细胞或一个组织在单一时相点的全部炎症介质,在感染过程中常常被高度表达。之所以将这些炎症介质称为“组”,是因为它们由于功能上的紧密联系而构成了一个相对独立的有机整体。本研究从海量的蛋白质种类中挑选部分指标作为明确的研究靶点,由于研究范围的缩窄,指标的测量可以改用比较精确、直接的方法,而数据处理的难度也相对降低。这样可以克服目前整体生物学技术和数据处理技术的局限性,对于整体分析研究中医治疗机制具有重要意义。全身炎症反应综合征(SIRS)中的失控性炎症,实际上是一种介质病,主要由细胞因子链锁反应所致。但是至今为止,拮抗炎症介质治疗感染性休克未能取得确切疗效。研究失败的原因可能有二:第一,炎症介质种类很多,目前已知有30多种,仅针对其中个别的炎症介质治疗不能改变病情。第二,炎症介质本身可能在感染性休克的发展中对机体有一定的保护作用,抑制炎症介质的同时也促进了感染对机体的损伤。解决策略如下:针对第一种原因,解决策略是干预多种炎症介质。针对第二种原因,解决策略是精确干预。不是盲目地清除或拮抗炎症介质,而是在病程每个时相点对炎症介质采取正确和适度的治疗。同时对多种炎症介质进行精确干预是最理想的治疗策略,称之为“介质组调节”,中医药由于其整体作用特点而有可能具备这种“介质组调节”的治疗机制。清瘟败毒饮和醒脑静治疗温病疗效肯定,已经被证明可以影响多种炎症介质水平,故拟通过本研究探索清瘟败毒饮和醒脑静对SIRS实验动物模型炎症介质组的影响。研究方法:本研究是一项使用清瘟败毒饮和醒脑静治疗SIRS,并较大规模地平行检测多种血清炎症介质的实验,共包括4个部分。在“实验一”中,探索了不同剂量和不同次数注射脂多糖(LPS)制作新西兰兔温病实验动物模型的方法。在“实验二”中,通过间隔24h的2次LPS注射法(施瓦茨曼法)制作新西兰兔SIRS模型,使用含有不同剂量生石膏的清瘟败毒饮灌胃治疗。治疗后第1、2.5、5.5h测量肛温,观察神志、皮肤粘膜改变和其他症状;治疗后1、2.5h耳动脉采血,采用酶联免疫吸附法测定促炎介质IL-1、IL-6、IL-12、IL-18、TNF-α、IFN-γ和抑炎介质IL-4、IL-10、IL-13、TGF-β1;并进行病理检查。在“实验三”中,使用与实验二中相同的动物模型,使用醒脑静注射液耳缘静脉注射治疗SIRS。治疗后第1、2.5、5.5h测量肛温,观察神志、皮肤粘膜改变和其他症状;治疗后1、2.5h耳动脉采血,采用酶联免疫吸附法测定促炎介质IL-1、IL-6、IL-12、IL-18、TNF-α、IFN-γ和抑炎介质IL-4、IL-10、IL-13、TGF-β1;并进行病理检查。在“实验四”中,通过双变量相关分析、偏相关分析、聚类分析、因子分析、多元线性回归分析和Logistic回归分析等多元统计学方法研究实验一、二、三中49只新西兰兔的炎症介质检测指标和体温,探索炎症介质组的变化规律。研究结果:在不同剂量注射实验中,1次注射后实验动物出现发热、少动倦怠、呼吸增快等;2次注射后仍可出现发热、少动倦怠和呼吸增快,伴随耳缘静脉出血时间延长;随着注射脂多糖剂量增加,病情有加剧的趋势,大剂量注射后出现精神极度萎靡、耳缘冰冷等内毒素休克的表现,易导致死亡。在多次注射实验中,在7日中,等量或递增剂量的LPS每日1次注射均可引起发热。在验证实验中,第1次注射LPS后全部38只兔出现发热,其中2只于24h内死亡,再次注射LPS后,全部兔再次出现发热,继续观察48h,未再出现死亡。含有不同剂量生石膏的清瘟败毒饮治疗后1、2.5h,治疗组体温均低于对照组,差别有统计学意义。在治疗后1h,大剂量组的TNF-α水平低于对照组,TGF-β1水平高于对照组,差别有统计学意义,在全部炎症介质中,小剂量组的炎症介质水平常常介于对照组和大剂量组之间,而对照组的炎症介质水平从不会位于小、大剂量组之间。在治疗后2.5h,大剂量组的TNF-α水平低于对照组,差别有统计学意义,小、大剂量组的IL-4水平均高于对照组,差别接近有统计学意义(P=0.056),小剂量组的炎症介质水平同样常常介于对照组和大剂量组之间。醒脑静治疗后1、2.5h体温均低于对照组,差别有统计学意义。醒脑静治疗后1h醒脑静组的TNF-α水平低于对照组,IL-18水平高于对照组,差别有统计学意义。醒脑静治疗后2.5h各种炎症介质的组间差别均无统计学意义。各治疗组相对于对照组的炎症介质变化趋势的比较结果显示,炎症介质组的变化具有一定的规律性,特别是对于小、大剂量的清瘟败毒饮组来说,几乎各时限所有的炎症介质改变的方向都是相同的。积矩相关系数、等级相关系数和偏相关系数的计算结果显示,各种抑炎介质之间均呈正相关,各种促炎介质之间多呈正相关,而促炎介质与抑炎介质之间的相关关系则比较复杂,部分炎症介质之间相关系数较高。指标聚类的结果显示,第一步将IL-12与IL-13聚为一类,第二步将IL-12、IL-13与TGF-β1聚为一类,共进行9步,最后一步将TNF-α与其他炎症介质聚类。因子分析得到3个公因子:第一个公因子包括IL-12、IL-13、TGF-β1、IL-4共4种炎症介质,第二个公因子包括IFN-γ、TNF-α、IL-18、IL-10共4种炎症介质,第三个公因子包括IL-1、IL-6、IL-10共3种炎症介质。在全变量回归分析、逐步回归分析和二分类逐步logistic回归分析中,均得到了有统计学意义的回归方程模型,但决定系数较低,有统计学意义的变量较少。结论:LPS致热效果肯定,不同剂量、不同次数的LPS注射可以成功制作卫、气、营、血各阶段动物模型。卫气分模型宜用单次静脉注射法,营血分或气血两燔证模型用间隔24h的两次LPS注射法,长期发热的温病模型如留恋气分证或肝肾阴伤证可用多次注射法,内闭外脱证模型使用大剂量LPS注射。含有不同剂量生石膏的清瘟败毒饮均具有退热作用,其中以大剂量组的退热作用更为显著。清瘟败毒饮的作用机制可能包括抑制促炎介质TNF-α和提升抑炎介质TGF-β1,因而具有抗炎作用。全部炎症介质数据的分布表现出较明显的规律性,提示含有不同剂量生石膏的清瘟败毒饮可能致使介质水平向着相同的方向改变,大剂量组的改变更为显著。醒脑静具有退热作用。醒脑静能够降低促炎介质TNF-α水平而抗炎,而提升IL-18水平的意义不明。炎症介质变化方向的规律性提示各组药物对介质组的作用机制有相似之处;各种炎症介质之间具有比较密切的关系;指标聚类的结果提示,IL-12、IL-13与TGF-β1的数据相似程度最高,而TNF-α的数据特点与其他炎症介质有较大区别;因子分析结果中的第一个公因子主要反映抑炎介质,而第二、三个公因子主要反映不同的促炎介质,提示了炎症介质组的变化特点。更多还原

【Abstract】 Objective:"Inflammatory Mediatorome",usually highly expressed during inflammation, were all mediators of inflammation in a cell or tissue in one time-phase.As an organic whole,mediators of inflammation,closely linked in function,were called "mediatorome" In this research,several kinds of protein were selected to study from a great deal kinds of protein.Because studying target was limited in a small circumscription,experimental detection could be finished by precise technique and data processing would be easy.This strategy was helpful to resolve problems both in experimental techniques and data processing and very important in TCM research by holistic cellular biology techniques.Uncontrolled inflammation in systemic inflammatory response syndrome(SIRS) was actually a mediator disease,mainly caused by chain reaction of cytokine.But up to now,no anti-mediator therapy had made sure effect in treating septic shock.There were two possible reasons.First,there were so many kinds of mediator of inflammation that pathogenetic condition could not be improved by treating only a few mediators.Second,mediators of inflammation could protect the body in septic shock and anti-mediator therapy could destroy the protection.For the first reason,many kinds of mediators of inflammation should be treated together。For the second reason,every mediator in every time-phase should be treated correctly and moderately instead of being simply cleaned up.Exactly treating many kinds of mediators together was a ideal strategy, which was called "mediatorome regulating".TCM could regulate the mediatorome because of its integrity characteristics.Decoction for clearing away pestilent factors and detoxification(DCPD) and Xingnaojing injection(XNJI) had sure therapeutic effect to exogenous febrile disease and could affect mediators.In this research,SIRS experimental animals were treated by DCPD and XNJI while the change of mediatorome was observed and analyzed.Method:This research,in which SIRS experimental animals were treated by DCPD and XNJI while many kinds of mediator of inflammation were detected,included 4 experiments.In "experiment 1",New Zealand rabbits received LPS injection of different times and different doses to indue different exogenous febrile disease model.In "experiment 2",New Zealand rabbits SIRS models were induced by twice(with a 24-hour interval) lipopolysaccharide(LPS) infusion through vein.SIRS rabbits received intragastric administration of DCPD containing different dose of gypsum.Symptoms and objective signs,such as rectal temperature,consciousness,skin and mucosa,were observed at 1,2.5 and 5.5 hour after dosage and blood samples were collected at 1 and 2.5 hour after dosage.Then,10 kinds of cytokines,including interleukin1(IL-1), interleukin4(IL-4),interleukin6(IL-6),interleukin10(IL-10), interleukin12(IL-12),interleukin13(IL-13),interleukin18(IL-18),tumor necrosis factor-α(TNF-α),interferon-γ(INF-γ) and transforming growth factor(TGF-β1),were tested by enzyme linked immunosorbent assay.Samples of lung and liver were got for pathological section at the end of experiment.In "experiment 3",SIRS models,indueed in the same way,received XNJI intravenous injection in ear vein.Symptoms and objective signs,such as rectal temperature,consciousness,skin and mucosa,were observed at 1,2.5 and 5.5 hour after dosage and blood samples were collected at 1 and 2.5 hour after dosage.Then,10 kinds of cytokines,including IL-1,IL-4,IL-6,IL-10,IL-12, IL-13,IL-18,TNF-α,INF-γand TGF-β1,were tested by enzyme linked immunosorbent assay.Samples of lung and liver were also got for pathological section.In "experiment 4",the level of inflammatory mediators and rectal temperature of 49 rabbits in other three experiments were analyzed through bivariate correlate,partial correlate,hierarchical cluster,factor date reduction,linear regression and Logistic regression to explore the variation rule of inflammatory mediatorome.Results:In the different-dose injecting experiment,the first LPS injection caused fever,less activity,lassitude and tachypnea in rabbits,then similar symptoms accompanied with prolonged venous hemorrhage time were found after the second injection.As the LPS dose increasing,pathogenetic condition intensified.Large-dose LPS injection caused endotoxin shock symptoms such as unconsciousness and ice cold ears,possibly causing death.In the multiple injecting experiment,injection with equal dose or progressively increasing dose could induce fever every time during 7 days.In the validity proving experiment,all the 38 rabbits had fever after first injection and 2 rabbits died in 24 hours,then all the remained 36 rabbits had fever again after second injection and no rabbit died in next 48 hours.The DCPD treated groups received intragastric administration of DCPD containing different dose of gypsum.Rectal temperature of both the little-dose and large-dose DCPD group was lower than the controlled group at 1h and 2.5h after dose and there was statistical significance in the difference. At 1h after dose,TNF-αlever of the large-dose DCPD group was lower than the controlled group while TGF-β1 lever was higher,the difference was statistically significant.Lever of inflammatory mediators in little-dose DCPD group was usually between the controlled group and large-dose DCPD group, while lever of inflammatory mediators in the controlled group was never between the little-dose and large-dose DCPD group.At 2.5h after dose,TNF-αlever of large-dose DCPD group was significantly lower than the controlled group, while IL-4 lever of both the little-dose and large-dose DCPD group was higher than the controlled group,the difference is nearly significant(P= 0.056).Lever of inflammatory mediators in little-dose DCPD group was usually between the other 2 groups.Rectal temperature of the XNJI treated group was significantly lower than the controlled group at 1h and 2.5h after dose.At 1h after dose,TNF-αlever of the XNJI treated group was lower than the controlled group and IL-18 lever was higher,the difference was statistically significant.At 2.5h after dose, no statistical significance was obtained in all the inflammatory mediators between 2 groups.Variation tendency of inflammatory mediators in all the treated groups was compared to the controlled group and variation of inflammatory mediatorome showed some regularity.For the 2 DCPD treated groups,the direction of variation of all the inflammatory mediators at different time-phase was almost same.According to Pearson coefficient correlation,Spearman coefficient correlation and partial coefficient correlation,positive correlation was found between pro-inflammatory mediators and between anti-inflammatory mediators,but correlation between pro-inflammatory mediators and antiinflammatory mediators was complicated.The coefficient correlation was high between several mediators.In a 9-step clustering analysis,IL-12 and IL-13 were clustered at the first step,IL-12,IL-13 and TGF-β1 were clustered at the second step,while TNF-αwas clustered with other inflammatory mediators at the last step.3 common factors were obtained in factor analysis.The first common factor included IL-12、IL-13、TGF-β1 and IL-4,the second common factor included IFN-γ、TNF-α、IL-18 and IL-10,while the last common factor consisted IL-1、IL-6 and IL-10.Regression equations obtained by linear regression and logistic regression were statistically significant,but coefficients of determination were poor and only a few variables had statistical significance.Conclusion:LPS injection could surely cause fever.LPS injection with different times and different doses could indue different exogenous febrile disease models of Weifen,qifen,yingfen and xuefen syndrome.Weifen and qifen syndrome could be endued by LPS infusion once,yingfen and xuefen syndrome could be endued by twice(with a 24-hour interval) LPS infusion,persistent qifen syndrome or deficiency of liver-yin and kidney-yin syndrome by multiple times infusion and unconsciousness and collapse syndrome could be caused by large-dose LPS injection.The results showed that DCPD containing different dose of gypsum gave antipyretic effect on SIRS rabbits and DCPD containing large dose of gypsum gave more obvious antipyretic effect.DCPD could antagonise inflammation possibly by restraining TNF-αand promoting TGF-β.All the data of inflammatory mediators showed regularity.DCPD with different dose of gypsum caused inflammatory mediators changing in the same direction and the effect of DCPD with large dose of gypsum was more obvious.The results showed that XNJZ also gave antipyretic effect on SIRS rabbits and antagonised inflammation possibly by restraining TNF-α,but the reason about promoting IL-18 was unknowed.Some variation characteristic of inflammatory mediatorome was revealed through several multivariate statistical analyzing.The regularity of variation of inflammatory mediators suggested that action mechanism of different treating drugs could be similar.Correlation analysis demonstrated that inflammatory mediators had close relationship.Clustering analysis showed that numerical data of IL-12,IL-13 and TGF-β1 was very similar and data characteristic of TNF-αwas quite different.3 common factors were obtained in factor analysis.The first common factor mainly reflected anti-inflammatory mediators,while the second and third common factors probably reflected different pro-inflammatory mediators.更多还原