【作者】 刘鑫；

【导师】 郑江；

【作者基本信息】 第三军医大学 ， 临床检验诊断学， 2010， 博士

【摘要】 一、背景脓毒症(sepsis)是感染引起的全身性炎症反应综合征(systemic inflammatory response syndrome,SIRS),是严重烧伤、创伤、感染性疾病以及大手术后患者的常见并发症,脓毒症已成为导致临床危重症患者死亡的首要因素。但目前尚无可靠有效的防治药物用于临床治疗。研究表明,细菌是引发脓毒症最主要感染因素。临床上由细菌介导的脓毒症占95%以上,其中约50%的细菌脓毒症是直接由G-细菌引起,与此同时,在疾病状态下肠道G-菌群可发生移位,引起继发性G-细菌感染,也会导致脓毒症发病。因此拮抗G-细菌引发的脓毒症具有特殊的意义。G-细菌存在多种致病因子,但其外膜成分脂多糖/内毒素(lipopolysaccharide/endotoxin, LPS)和其细菌基因组DNA(bDNA/CpG DNA)在G-细菌普遍存在,分布最为广泛,同时毒力最强,因此是G-细菌诱发脓毒症最主要的致病因子/病原分子(pattern recognition molecular patterns, PAMPs)。LPS和CpG DNA分别与其模式识别受体(pattern recognition receptors, PRRs) TLR4(Toll like receptor 4,TLR4)和TLR9结合,诱导炎症反应细胞大量合成与释放多种炎症介质(如TNF-α、IL-1和IL-6等),引发机体免疫应答紊乱,最终导致脓毒症的发病。近来发现,LPS和CpG DNA在体内还可协同发挥作用,诱发更为剧烈的全身性炎症反应,导致更加严重的脓毒症。国内外针对脓毒症致病因子的拮抗策略和措施研究仍然主要围绕LPS进行,由于不能对另一个主要致病因子CpG DNA进行同步拮抗,因此往往疗效有限。基于上述认识,如能根据LPS和CpG DNA的特点与作用设计新颖的脓毒症拮抗策略,寻找同时针对LPS和CpG DNA的药物拮抗措施,脓毒症的防治就可能会取得突破性进展。中草药长期用于感染和炎症治疗。中草药制剂对于脓毒症具有一定疗效。但中药成分复杂,拮抗脓毒症的物质基础和作用机制不明,为解决该问题,在前期研究中我们将LPS和CpG DNA包被于生物传感器上,建立了以脓毒症主要病原分子为靶标的中药筛选和定向分离实验平台。先后从中药大黄和地骨皮中获得了具有同时结合和拮抗LPS和CpG DNA作用的活性组分。该结果为同时针对主要致病因子(LPS和CpG DNA)的脓毒症拮抗策略的实施奠定了基础。本研究采用生物传感器技术和色谱技术相结合的方法,从大黄和地骨皮活性组分中分离出具有同时拮抗LPS和CpG DNA活性的单体化合物,并通过体内外生物学活性评价方法对活性单体化合物拮抗脓毒症的效应和机制进行研究。二、目的建立以细菌主要致病因子LPS和CpG DNA为靶标的脓毒症防治策略,从中药活性组分中制备出对LPS和CpG DNA具有拮抗活性的单体化合物,并对其进行防治脓毒症的主要药效和作用机制研究。三、材料与方法1.应用标准物质高效液相色谱(high performance liquid chromatography, HPLC)比对的方法对前期获得的大黄活性组分(D组分,Fraction D)进行成分分析,检测D组分中含有的单体成分对LPS和CpG DNA的结合和拮抗活性;2.应用以LPS和CpG DNA为靶标的生物传感器技术平台,以前期得到的地骨皮活性粗组分(cortex lycii -4, CL-4)为研究对象,应用离子交换树脂和反相HPLC技术对CL-4组分进行纯化,对获得的各成分进行亲和力测定和拮抗LPS和CpG DNA的活性评价,最终对活性单体化合物进行纯度分析和结构鉴定。3.地骨皮活性单体化合物在体内外拮抗LPS和CpG DNA的活性及机制研究。3.1体外活性研究:①应用生物传感器技术平台,检测单体化合物与LPS或CpG DNA的结合作用并计算Kd值;②动态浊度法鲎试验测定单体化合物对LPS的体外中和能力;③激光共聚焦显微镜和流式细胞术观察/检测单体化合物对荧光素标记的LPS和CpG DNA与RAW 264.7细胞结合的影响;④real-time RT-PCR法检测单体化合物干预后,LPS和CpG DNA刺激炎症介质TNF-α、IL-6、iNOS和COX-2的mRNA在RAW264.7细胞的表达的变化;⑤ELISA方法检测单体化合物对LPS和CpG DNA刺激RAW264.7细胞活化(分泌细胞因子TNF-α)的影响(时效和量效关系);⑥观察单体化合物对在小鼠腹腔巨噬细胞(原代细胞)对LPS和CpG DNA的拮抗活性。3.2体内活性研究:采用尾静脉注射热灭活大肠埃希菌模拟LPS和CpG DNA的混合攻击,制备小鼠脓毒症模型:观察单体对致死剂量热灭活大肠埃希菌攻击小鼠的保护作用和对亚致死剂量热灭活大肠埃希菌攻击小鼠的治疗作用(造模后0、4、8、12、24、48和72 h采集血标本,动态浊度法鲎试验检测血LPS水平,ELISA法检测血清TNF-α水平);3.3机制研究:①单体化合物与LPS阳性拮抗剂PMB对LPS和CpG DNA的结合和抑制活性比较;②单体化合物对多个PAMPs的拮抗活性比较;③检测单体化合物不同加样方式对其拮抗LPS和CpG DNA活性的影响;④采用real-time RT-PCR检测单体对RAW264.7细胞TLR4和TLR9受体表达的影响;⑤westernblot检测单体化合物对LPS、CpG DNA、TNF-α和IL-1β刺激RAW264.7细胞信号分子IκB-α和p38活化影响;⑥ELISA法测定单体化合物对LPS和CpG DNA刺激RAW 264.7细胞转录因子NF-κB活化的影响。3.4单体化合物初步药物毒性评价实验:①MTT法检测单独给予25-800μM的KB对RAW 264.7细胞和小鼠腹腔巨噬细胞增殖活力的影响;②小鼠静脉注射60 mg/kg的单体化合物,观察对动物生存率和生存状态的影响,同时于静脉注射后24、48和72 h取主要的脏器组织,常规HE染色观察心、肺、肝、肾和肠组织学形态是否异常。四、结果1.中药大黄活性部位D组分经鉴定含有单体化合物大黄酸(rhein)和大黄素(emodin)。二者与LPS和CpG DNA具有高亲和活性,并可以单独或协同抑制LPS和CpG DNA刺激引起的TNF-α的释放。2.应用生物传感器平台和离子交换柱层析技术,从中药地骨皮活性部位CL-4中分离出三个组分CL-4a、CL-4b和CL-4c,其中CL-4b组分与LPS和CpG DNA具有高亲和力,可抑制LPS和CpG DNA刺激引起的TNF-α和IL-6的释放。在体外也可显著提高致死剂量热灭活大肠埃希菌攻击小鼠的存活率。对CL-4b采用反相HPLC技术纯化,制备得到两个单体化合物CL-4b1和CL-4b2。其中CL-4b2与LPS和CpG DNA具有高亲和活性,在体外能够中和LPS,抑制LPS和CpG DNA诱导RAW 264.7细胞释放TNF-α。经HPLC纯度分析显示,CL-4b2纯度大于99%,经过质谱和核磁共振分析,确定CL-4b2为苦柯胺B(kukoamine B, KB),分子式为C28H42N4O6。3.对KB的体外活性研究显示:①KB对LPS和CpG DNA均具有高亲和活性,其Kd值分别为1.24和0.66μM;②KB在体外可剂量依赖地中和LPS,并且可阻断LPS和CpG DNA与RAW 264.7细胞的结合;③KB可抑制LPS和CpG DNA刺激RAW 264.7细胞初级和次级炎症介质TNF-α、IL-6、iNOS和COX-2的mRNA表达上调;④KB可以剂量依赖和时间依赖的方式抑制LPS和CpG DNA刺激RAW 264.7细胞释放TNF-α;⑤MTT检测显示KB协同LPS或CpG DNA加样均不影响RAW 264.7细胞的活力;⑥KB在小鼠原代巨噬细胞同样能够阻断LPS和CpG DNA与细胞的结合,抑制二者刺激引起的TNF-α的释放增加。对KB的体内活性研究结果显示,KB对热灭活大肠埃希菌攻击小鼠具有显著的保护作用,并且呈现剂量-效应关系,同时KB还可降低该模型小鼠的血浆LPS及血清TNF-α水平。对KB的作用机制研究显示:①KB对LPS和CpG DNA具有双重结合和拮抗活性,PMB仅对LPS结合和拮抗活性;②KB对LPS和CpG DNA具有选择性拮抗活性,对Pam3CSK4、Poly I:C及TNF-α和IL-1β的刺激无抑制作用;③KB与LPS或CpG DNA预孵育,其拮抗活性明显增强,KB与细胞预孵育,拮抗活性无明显变化,延迟给予KB,其抑制作用逐渐减弱,直至消失。④KB可抑制LPS和CpG DNA对RAW 264.7细胞TLR4和TLR9表达的上调作用;⑤KB可抑制LPS和CpG DNA对信号分子IκB-α和p38的活化,但不影响TNF-α和IL-1β引起的IκB-α和p38的活化;⑥KB对LPS和CpG DNA诱导的转录因子NF-κB p50和p65的转位活化具有抑制活性。对KB的初步毒性评价显示:KB对RAW 264.7细胞和小鼠腹腔巨噬细胞均无毒性,体内单独注射KB对小鼠的生存和重要脏器(心、肝、肺、肾和肠)的组织学形态无明显影响。五、结论本论文立足于脓毒症发病机制的最新认识,提出以细菌LPS和CpG DNA为双靶标的全新脓毒症防治策略,并基于此策略对中药大黄和地骨皮抗脓毒症有效成分进行了分离制备和活性研究。明确中药大黄中的单体化合物大黄酸和大黄素具有结合和协同拮抗LPS和CpG DNA的作用。随后从中药地骨皮中分离获得与LPS和CpG DNA均具有更高亲和活力和更强拮抗活性的化合物苦柯胺B(kukoamine B, KB),首次证实了KB通过同时针对LPS和CpG DNA的选择性结合和中和作用,阻断了二者与受体的结合,有效抑制了后续炎症反应,从而避免了脓毒症致死效应产生这一新颖的作用机制。KB药效可靠,作用机制明确,具有良好的成药前景,经后续开发有望为解决脓毒症问题提供全新的药物防治措施。更多还原

【Abstract】 Introduction Sepsis, a systemic inflammatory response syndrome caused by infection, is common complications for the severe burns, trauma, infectious diseases and major operations. Sepsis has become the leading cause of death for critical patients but no effective drugs are available currently.Bacteria is thought to be the major infectious factors for sepsis as over 95 % of sepsis cases in clinic are induced by bacterial infection. About 50% of the bacterial sepsis cases are triggered by G- bacterial infection directly. On the other hand, intestinal G-bacteria can enter circulation via translocation to trigger secondary G- bacterial infection. As a result, it is of vital importance to combat G- bacterial. There are two major pattern recognition molecular patterns (PAMPs) for the induction of G- bacterial sepsis, the out membrane lipopolysaccharide/endotoxin (LPS) and genome DNA (bDNA/CpG DNA). LPS and CpG DNA are widely distributed among all kinds of G- bacteria and act as major pathogenic factors fortors for sepsis. They bind with their pattern recognition receptors (PRRs) TLR4 and TLR9 to activate MyD88 dependent/independent signaling pathways, which lead to synthesis and release of large quantities of proinflammatory mediators (TNF-α, IL-1 and IL-6). The sequential reactions will cause the disorder of immune response and eventually cause sepsis. Recently, LPS and CpG DNA were also found to act simultaneously for the induction of sepsis more severely.Current anti sepsis study mainly focus on LPS. However, these measures are with limited effects as they are unable to fight against CpG DNA. However, there has been no report on the anti sepsis study targeting on both LPS and CpG DNA. So we believe bacterial sepsis will be prevented effectively if dual inhibitors of LPS and CpG DNA are found and developed based on a new anti sepsis strategy targeting on both LPS and CpG DNA.Traditional Chinese herbs have been traditionally applied in treating infectious and inflammatory diseases. Many herbal formulas are effective for less severe sepsis. Nevertheless, herbs are composed with complicated components which make it difficult to discover contributing compounds and elucidate the innate mechanisms. To solve the above problems, affinity biosensor was introduced in our previous study to establish traditional Chinese herbs screening and separation via LPS and CpG DNA based double targets. 140 herbs were screened and active fractions were separated from radix et rhizoma rhei and cortex lycii. To find the contributing compound inside the active fractions, biosensor technique and chromatography were coupled for the isolation of active monomers. Bioactivity assessments in vitro and in vivo were also applied to investigate the effects and mechanisms of their anti LPS and anti CpG DNA activity.Objectives To establish anti sepsis strategy targeting on both LPS and CpG DNA, isolate natural compounds with botj anti LPS and anti CpG DNA activities and investigate their effectiveness and mechanism of their activities.Methods First, HPLC technique was utilized for determination of standard monomers in Fraction D of radix et rhizoma rhei. Both binding affinities for LPS and CpG DNA and anti LPS/CpG DNA activities of the containing monomers were detected in vitro. Second, biosensor technique was coulped with chromatography to isolate active fractions and monomers with high affinity for LPS and CpG DNA from fraction CL-4 of coetex lyciii. These fractions and monomers were analyzed via bioactivity assessment and the active monomer underwent purity analysis and structure determination.Third, effects and mechanism of anti LPS and CpG DNA activity of active monomer isolated from cortex lycii were studied. The binding affinity and dissociation constant (Kd value) for LPS and CpG DNA was first detected by biosensor. Then in vitro LPS neutralizing activity of the monomer was assayed via limulus amebocyte lysate test (LAL) tests and the binding of FITC-LPS and 5-FAM-CpG DNA to RAW 264.7 cells was analyzed by flow cytometry and laser scanning confocal microscopy (LSM). Effects of the monomer onmRNA expressions of TNF-α, IL-6, iNOS and COX-2 in RAW 264.7 cells were measured by real-time RT-PCR and the releases of cytokines were detected with ELISA. Lastly, the anti LPS and CpG DNA activity of the monomer was also observed in murine primary macrophages. In vivo, anti LPS and CpG DNA activity study of monomer was studied by observing effects of the monomer on survival of mice challenged intravenously with lethal dose of heat inactivated E. coli and detecting plasma LPS and serum TNF-αin mice injected with sublethal dose of E. coli. For mechanism study, binding and inhibitory activity of the monomer and PMB was studied and compared. Then inhibitory activity of monomer for multiple PAMPs was observed. Real-time RT-PCR was applied for the detection of TLR4 and TLR9 expression. Western blot and ELISA were used to detect activation of signaling molecules p38 and IκBαand transcription factor NF-κB p50 and p65. Finally, effects of various administration methods of the monomer on its anti LPS and CpG DNA activity. For preliminary drug safety analysis, effects of the monomer on RAW 264.7 cells and murine peritoneal cells in vitro and on mice survival and histomorphology of heart, lung, liver, kidney and intestine were studied..Results First, rhein and emodin were identified to be components of fraction D. they were shown with high affinity for LPS and CpG DNA and acted separately and synergistically to inhibited TNF-αrelease induced by LPS and CpG DNA.Second, three fractions CL-4a、CL-4b and CL-4c were separated from CL-4 of which CL-4b was identified to possess anti LPS and anti CpG DNA activity in vitro and in vivo. Two monomers were purified from CL-4b and the one with anti LPS and anti CpG DNA activity was identified to be kukoamine B (KB).Third, KB was identified with high affinity for LPS and CpG DNA (Kd value at 1.24 and 0.66μM each). It neutralized LPS in vitro in a well dose dependent manner and interfered LPS and CpG DNA from binding and internalization in RAW 264.7 cells. Kb was also shown to inhibit mRNA expression of TNF-α, IL-6, iNOS and COX-2 and suppressed the release of TNF-αtime and dose dependently in RAW 264.7 cells. Similar inhibitory activity of KB was also observed in murine peritoneal macrophages.In vivo,KB protected mice from challenged with heat inactivated E.coli probably via reducing circulatory LPS and CpG DNA levels. In mechanism study, KB was shown to be a dual binder and dual inhibitor for LPS and CpG DNA, compared to the fact that PMB only bind and inhibited LPS. KB could only inhibit LPS and CpG DNA but did not affect the effects of other stimuli. Additionally, preincubation of KB with LPS or CpG DNA significantly increased its inhibitory effects. KB attenuated the upregulation of TLR4 and TLR9 and thus inhibited sustained activation by LPS and CpG DNA. KB was demonstrated to selectively inhibited LPS and CpG DNA induced signaling transduction and cytokines (TNF-αand IL-6). KB was also shown to be nontoxic as it did not affect the cell viability and the survival or histomorphology of major organs in mice in prelimimary studies.Conclusions Dual targets (LPS and CpG DNA) targeted anti sepsis strategy was set up and active anti sepsis monomers purified from radix et rhizoma rhei and cortex lycii were obtaind based on this strategy. Rhein and emodin from radix et rhizoma rhei were found with binding and simultaneous anti LPS and CpG DNA activity for the first time. Then kukoamine B (KB) from cortex lycii was identified with stronger binding and inhibitory activity for LPS and CpG DNA, which was demonstrated to be attributed directly to its selective and specific binding and neutralizing activity for the two PAMPs. KB is an active compound. It will be a promising anti sepsis drug candidate with which the treatment of sepsis may be dramatically improved.更多还原