【作者】 伏建峰；

【导师】 郑江；

【作者基本信息】 第三军医大学 ， 临床检验诊断学， 2008， 博士

【摘要】 脓毒症是感染因素介导的全身炎症反应综合征(systemic inflammatory response syndrome, SIRS),是烧伤、创伤和感染性疾病患者的常见并发症。流行病学资料显示,每年全球有超过1800万的脓毒症病例,且患者数目每年以1.0%速度递增。重症脓毒症和脓毒性休克是重症监护病房(intensive care units, ICU)病人死亡的首要因素,死亡率高达50%-60%。内毒素(endotoxin/Lipopolysaccharide, LPS)是G-菌外膜的主要成分,是介导细菌脓毒症的重要病原体相关分子模式(pathogen-associated molecular patterns,PAMPs)。LPS进入机体内后,首先与内毒素结合蛋白(lipopolysaccharide-binding protein,LBP)结合,在LBP的作用下,LPS与单核/巨噬细胞膜上的受体CD14结合形成LPS-CD14复合物,复合物中的LPS与跨膜受体TLR4(Toll like receptor 4,TLR4)结合,将信号转入胞内,导致靶细胞的活化,释放TNF-α、IL-6等炎症介质,介导脓毒症的发生。尽管对脓毒症的病理生理机制及其防治策略的研究较为深入,但至今仍无特殊有效的药物供临床治疗。中草药在脓毒症中的应用具有悠久的历史,已证实多种中草药具有较好的拮抗内毒素作用,但由于中药成分复杂,加之缺乏有效的分离检测手段,限制了中草药在脓毒症防治中的应用与发展。在前期的研究中,我们将LPS的活性中心Lipid A包被于生物传感器上,建立了以Lipid A为靶点的筛选拮抗LPS药物的实验平台。基于此技术平台,本研究采用大孔吸附树脂、超滤膜分离和高效液相色谱(high performance liquid chromatogram, HPLC)技术,对中药水提液进行多级分离纯化,以期从天然药物中获取具有拮抗LPS活性的单体化合物,为脓毒症防治药物的研发提供新的思路和方法。方法1.应用以Lipid A为靶点的生物传感器技术平台,检测60种中药水煎液与Lipid A的结合活性;对明确具有与Lipid A结合活性的中药,采用大孔吸附树脂分离技术、超滤膜技术和HPLC技术进行分离提取,应用生物传感器进行活性追踪监测,收集能与Lipid A结合的组分,并对其进行针对LPS的体内外生物学活性评估。对活性明确的有效组分,用HPLC作进一步的分离纯化,选择与Lipid A具有较高结合活性的HPLC产物作进一步的研究,对所得单体化合物进行纯度分析和结构解析。2.对得到的单体化合物进行针对细菌脓毒症的体内外生物学活性评价,包括:①鲎试剂法检测单体对LPS的体内外中和作用;②ELISA法检测单体对LPS刺激巨噬细胞产生细胞因子(TNF-α和IL-6)的抑制作用;③RT-PCR法检测单体对LPS诱导巨噬细胞TLR4和TNF-αmRNA表达的影响;④观察单体化合物对脓毒症模型小鼠的保护作用。结果1.应用生物传感器技术平台,从60种中药中筛选出黄芩、胡连、赤芍、五倍子、诃子等5种具有与Lipid A较高结合活性的中药(RU>200 arc seconds)。以黄芩为研究对象,从黄芩水提液中分离出与Lipid A具有结合活性的组分SbG-4;SbG-4 (40mg/kg)能显著提高热灭活大肠埃希氏菌攻击小鼠72h的存活率;物质属性鉴定SbG-4为黄酮类物质。应用膜分离技术从SbG-4中分离出分子量小于5000Da的5KL,5KL经HPLC分离获得5个HPLC组分(5KL-1～5);在5个组分中,以5KL-1与Lipid A的结合活性最高;在体外5KL-1对LPS具有直接中和作用、可显著抑制LPS诱导的RAW264.7细胞释放TNF-α,体内评价显示5KL-1对脓毒症模型小鼠具有显著的保护作用;5KL-1经HPLC进一步分离后,得到5KL-1A、5KL-1B和5KL-1C三个产物;其中仅5KL-1B是单一物质并与Lipid A的结合活性最高,纯度分析显示,5KL-1B的纯度因子为999.685;经质谱、核磁、红外等波谱分析,对5KL-1B的结构指认为2’, 5, 6’, 7-四羟基二氢黄酮醇(2’, 5, 6’, 7-tetrahydroxy-flavanonol, THF),分子式为:C15H12O7。2.对THF的活性研究显示:①、THF在体内外对LPS均具有直接中和作用;②、THF能够显著抑制LPS诱导RAW264.7细胞释放TNF-α和IL-6,并具有明显的量效关系;③、THF对LPS诱导的RAW264.7细胞TLR4和TNF-α在mRNA水平的表达均具有显著的抑制作用;④、THF对致死剂量热灭活大肠埃希氏菌攻击的小鼠具有显著的保护作用。结论应用现代生物技术从黄芩中定向分离出2’, 5, 6’, 7-四羟基二氢黄酮醇;2’, 5, 6’, 7-四羟基二氢黄酮醇在体内外对LPS均具有拮抗作用,对脓毒症模型小鼠具有保护作用。更多还原

【Abstract】 Introduction Sepsis is defined as suspected or proven infection plus a systemic inflammatory response syndrome. It is the most common complication of burn, trauma and infectious diseases. An epidemiological study found that there were approximately 18 million cases of sepsis annually worldwide and the incidence was expected to increase 1% per year. Septic shock and multiorgan dysfunction resulting from sepsis are the leading cause of death in intensive care units, with the mortality rates between 50 and 60%.Lipopolysaccharide (LPS; known also as endotoxin) is an outer membrane component of gram-negative bacteria, and thought to be a major PAMP of sepsis. In the presence of lipopolysaccharide-binding protein (LBP), LPS interacts with monocyte/macrophage cells via CD14 and the two form the LPS-CD14 complex. Then the complex binds to TLR4 and triggers the TLR4-mediated signal transduction to induce the release of pro-inflammatory cytokines, including TNF-αand IL-6. Over expression of cytokines will trigger systemic inflammatory response syndrome, as a result, sepsis and septic shock take place. Although great attentions have been paid in studies of the pathophysiology and treatment of sepsis, there is currently no effective anti-sepsis drug in clinical use.Application of traditional Chinese medicines in septic therapy has a long history. Recent clinical trials and studies also demonstrated that a lot of herbs possess the properties of anti-LPS. However, herbs usually have a large number of complex constituents and thus extremely difficult to identify the anti-LPS ones, which limit their clinical uses. In previous studies, we successfully established an effective method to screen anti-LPS compound from traditional Chinese herbs using affinity biosensor technology. In this study, based on this technology platform, the aqueous extracts from traditional Chinese herbs were isolated by using macroporous adsorptive resins technology, ultra filtration membrane technology and high performance liquid chromatography (HPLC) technology in order to obtain anti-LPS monomer to be used for sepsis therapy.Methods First, the aqueous extracts from sixty traditional Chinese herbs were tested for detecting the binding activity to Lipid A using the affinity biosensor technology. Herb has high Lipid A-binding activity in the screening test was isolated by using macroporous adsorptive resins technology, ultra filtration membrane technology and HPLC technology. In the isolation proceedings of aqueous extract from the herb, the biosensor affinity technology has been used to detect Lipid A-binding activity for various kinds of elutes in order to screen the active site from them. Furthermore, we evaluated the anti-LPS activity of the elute fraction which has the highest binding activity to Lipid A among them in vitro and in vivo, As a result, the active compound against LPS was attained.Second, the active compound was purified by HPLC and the purified monomer with high Lipid A-binding activity was analyzed by infrared, mass spectrometry and NMR (nuclear magnetic resonance) in order to identify its structure.Finally, anti-sepsis activity of the monomer was studied in vitro and in vivo. In detail, the neutralization of the monomer to LPS was detected by kinetic turbidimetric limulus test. Meanwhile, ELISA and RT-PCR was used respectively to measure the release of cytokine (TNF-αand IL-6) and detect the expression of TLR4 mRNA in RAW264.7 cells exposure to LPS with or without pretreatment of the monomer. Furthermore, the protective effect on mice subjected to lethal dose of heat-killed E.coli challenge with or without treatment of the monomer was observed.Result Five herbs were found to possess high Lipid A-binding activity in the screening test of aqueous extracts from sixty traditional Chinese herbs (RU>200 arc second). They were Radix Paeoniae Rubra, Rhizoma Picrorhizae, Galla Chinensis, Scutellaria baicalensis Georgi and Fructus Chebulae. To weigh the merits and demerits, we selected Scutellaria baicalensis Georgi for further study. Subsequently, SbG-4 was isolated from the herb, which had high Lipid A-binding activity. Moreover, SbG-4 could significantly increase the survival rate of sepsis mice induced by heat-killed E.coli. Interestingly, the Chemical compound assessment of SbG-4 indicated it is a flavonoid compound. So, we selected SbG-4 further to isolate using ultra filtration membrane technology and HPLC technology. In this step, we collected five HPLC fractions and named from 5KL-1 to 5. Among them, 5KL-1 had the highest Lipid A-binding activity and significantly neutralizing LPS in vitro. In RAW264.7 cells, the supernatant TNF-αlevel was increased after the cells exposure to LPS, however, this effect was attenuated by the pretreatment with 5KL-1. Likewise, 5KL-1 could protect mice from heat-killed E.coli challenge. Collectively, 5KL-1 has anti-LPS activity and deserves further study.After purified by HPLC, 5KL-1 yielded three products and named from 5KL-1A to C. among them, 5KL-1B had the highest binding activity to Lipid A. So, we selected it for further study. HPLC analysis to show that 5KL-1B is a single peak and purity factor is 999.685. After the structure was analyzed by mass spectrometry, infrared and NMR, 5KL-1B was confirmed as 2’,5 ,6’,7-tetrahydroxy-flavanonol (THF), molecular formula is C15H12O7.A series of experiments were used to evaluate the anti-LPS activity of THF. In detail, THF could neutralize LPS and decrease the release of TNF-αand IL-6 in LPS-induced RAW264.7 cells in a dose-dependent manner. Furthermore, THF reduced the expression of TLR4 and TNF-αmRNA in RAW264.7 cells induced by LPS in a dose-dependent manner too. In vivo experiments, Escherichia coli 35218 (E.coli 35218) was used to induce sepsis in an animal model. We also found that THF could protect mice against a lethal challenge with heat-killed E.coli in a dose-dependent manner.Conclusion An active monomer, 2’, 5, 6’, 7-tetrahydroxy-flavanonol (THF) was attained from Scutellaria baicalensis Georgi. In vitro, THF could neutralize LPS and suppress the activating of RAW264.7 cells induced by LPS. In vivo, THF could significantly decrease the plasme LPS level in endotoxemia mice and protect mice against a lethal challenge with heat-killed E.coli. Collectively, THF has anti-sepsis activity.更多还原