【作者】 谢俊然；

【导师】 黄卫东；

【作者基本信息】 浙江大学 ， 临床医学， 2010， 博士

【摘要】 早期液体复苏是脓毒血症综合治疗手段中必不可少的一项治疗措施,但是关于脓毒血症的液体治疗一直存有争议,其中包括晶体与胶体之争、白蛋白与人工胶体之争,除此外,甚至面对市场上众多的晶体、胶体制剂,究竟选择何种具体制剂亦是临床医生感到困惑的问题。羟乙基淀粉(hydroxyethyl starch, HES)是目前临床上经常被使用的一种胶体,从玉米、高梁或者土豆中提炼制备而成,根据不同的分子量、摩尔取代度,HES分为多种剂型,其中HES 130/0.4属于新的第三代HES制剂,不仅过敏发生率低,对肾功能以及凝血功能的影响亦很小。前人有研究报道HES不仅有稳定血流动力学扩容作用,而且可能还有全身抗炎作用,例如可以下调全身的炎症介质,包括细胞因子、粘附分子、趋化因子等,同时又可以抑制白细胞粘附浸润减少毛细血管渗漏,但是关于HES抗炎以及堵漏的确切机制并不清楚。Toll样受体(Toll-like receptors, TLRs)／核因子(nuclear factor, NF)-kB信号转导通路在脓毒血症发生发展中占重要地位。TLRs是一类具有信号转导功能的跨膜蛋白,脓毒血症中,TLRs与相应配体,例如内毒素(lipopolysaccharide, LPS)、肽聚糖等结合后通过一系列跨膜信号转导激活细胞内的转录因子NF-kB,活化的NF-kB获自由从胞质移入胞核内从而促进一系列炎症介质的表达,包括多种细胞因子、粘附分子等,最终促进脓毒血症的发生。本实验欲应用大鼠盲肠结扎穿孔模型(cecal ligation and puncture, CLP)模拟脓毒血症观察第三代羟乙基淀粉制剂HES130/0.4的全身抗炎作用,并且试图从是否影响TLRs/NF-kB信号转导通路方面解释HES 130/0.4的可能抗炎机制。第一部分羟乙基淀粉(HES130/0.4)对脓毒血症大鼠血浆细胞因子的影响以及Toll样受体4／核因子-κB信号转导通路在其中的作用研究胶体HES 130/0.4是羟乙基淀粉的第三代制剂,目前在临床广为使用,除了扩容作用,前人研究提示HES可能还具备全身抗炎作用,但具体机制不清。本部分实验目的是观察HES 130/0.4对脓毒血症大鼠血浆细胞因子的影响以及探讨LPS-TLR4-NF-KB信号转导通路在其中所起的作用。方法是应用大鼠CLP模型模拟脓毒血症,CLP 3h后输注不同剂量的HES130/0.4(7.5ml/kg,15ml/kg及30ml/kg), CLP 5h后收集大鼠血浆、外周血单核细胞标本,采用鲎三肽偶氮显色法测定血浆LPS浓度；采用酶联免疫吸附方法检测大鼠血浆肿瘤坏死因子(tumor necrosis factor, TNF)-a,白介素(interleukin, IL)-6浓度；采用逆转录-聚合酶链方法检测外周血单核细胞TLR4 mRNA表达；采用免疫蛋白印迹方法检测外周血单核细胞TLR4蛋白相对浓度；采用凝胶电迁移方法检测外周血单核细胞转录因子NF-kB活化。实验结果表明CLP大鼠输注HES130/0.4后可以明显降低血浆TNF-αIL-6水平,15ml/kg剂量组降低细胞因子水平最明显；同时,HES 130/0.4可以显著抑制CLP大鼠外周血单核细胞TLR4的生成以及转录因子NF-kB的活化,与影响细胞因子一样,15ml/kg剂量表现最大抑制作用。上述实验结果表明HES 130/0.4在脓毒血症大鼠中具有下调全身炎症介质的作用,该作用可能与抑制TLR4/NF-KB信号转导通路有关。第二部分羟乙基淀粉(HES130／0.4)对脓毒血症大鼠肺毛细血管渗漏的影响以及Toll样受体／核因子-KB信号转导在其中的作用研究肺是脓毒血症发生时最易受累的脏器组织,根据严重程度可发展为急性肺损伤(acute lung injury, ALI)/急性呼吸窘迫综合征(acute respiratory distress syndrome, ARDS)。前人研究报道HES可能通过抗炎作用降低白细胞浸润粘附从而减轻毛细血管渗漏,但确切作用机制不清楚。本部分实验目的是观察HES130／0.4是否能够下调肺部炎症介质减少中性粒细胞浸润；是否能够减轻脓毒血症大鼠肺部毛细血管渗漏,并初步探讨TLRs (TLR2, TLR4)/NF-kB信号转导在其中可能所起的作用。方法是应用大鼠CLP模型模拟脓毒血症所致ALI, CLP 3h后输注15ml/kgHES130／0.4或者同剂量的琥珀酰明胶,CLP 5h后收集大鼠肺脏标本,采用酶联免疫吸附方法测定大鼠肺组织中的细胞因子TNF-α、IL-1β与粘附分子intercellular adhesion molecule(ICAM-1)-1浓度；采用逆转录-聚合酶链方法检测肺组织TLR2、TLR4 mRNA表达；采用免疫蛋白印迹方法检测肺组织TLR2、TLR4蛋白相对浓度；采用凝胶电迁移方法检测肺组织转录因子NF-kB活化。CLP 12h后测定大鼠肺脏湿干重比、毛细血管渗漏以及髓过氧化酶(myeloperoxidase, MPO)活性。实验结果表明15ml/kg HES 130/0.4可以明显减轻脓毒症大鼠的肺水肿以及肺毛细血管渗漏,同时HES130／0.4可以显著下调CLP大鼠肺部的炎症介质,包括细胞因子TNF-α、IL-1β与粘附分子ICAM-1;还可抑制CLP大鼠肺部的TLR2、TLR4生成以及转录因子NF-kB的活化。同剂量的琥珀酰明胶与HES130／0.4相比虽然也可减轻CLP大鼠肺毛细血管渗漏、降低炎症介质浓度,但作用没有HES130／0.4明显,且琥珀酰明胶对TLR2.TLR4以及NF-kB没有影响。上述实验结果表明HES130／0.4确实可以减轻脓毒血症大鼠的肺毛细血管渗漏,这可能与其下调肺部炎症介质、减少中性粒细胞浸润有关,另外实验结果提示HES130／0.4下调炎症介质发挥堵漏作用可能与抑制TLRs (TLR2, TLR4)/NF-kB信号转导有关。本实验主要欲观察第三代羟乙基淀粉制剂HES130／0.4在脓毒血症大鼠中的全身抗炎作用以及减轻毛细血管渗漏作用,并探讨与TLRs/NF-kB信号转导通路可能相关的分子机制。实验结果确实表明HES130／0.4可以下调脓毒血症大鼠全身的炎症介质,也可以减轻肺毛细血管渗漏,同时可以抑制外周血单核细胞与肺组织中的TLRs受体生成与转录因子NF-kB活化,提示HES130／0.4可能通过抑制TLRs/NF-kB信号转导下调全身炎症介质、减少中性粒细胞浸润以及减轻毛细血管渗漏。作为胶体制剂,HES130／0.4的主要作用仍然为扩容改善血流动力学作用,但是面对市场上众多的液体制剂,如果相比较,HES130／0.4还具有全身抗炎、减轻毛细血管渗漏作用,那么势必会增加其应用优势。正如实验第二部分结果显示HES130／0.4相比同剂量的胶体琥珀酰明胶能够更加明显的减轻脓毒症大鼠肺毛细血管渗漏,如果该实验成果能够得到临床进一步验证,那么临床医生对脓毒症患者进行液体治疗时便会考虑HES130／0.4的这一治疗优势,这便是该实验的最大临床意义。更多还原

【Abstract】 In the treatment of sepsis, early fluid therapy appears to be essential to optimize hemodynamics and obtain suitable tissue perfusion. However, much controversy still exists about the role of crystalloids and colloids in fluid therapy. Clinicians are often confused about which fluid fraction is most appropriate for septic patients.Hydroxyethyl starch (HES) is one of the most frequently used plasma substitutes and derived from maize, sorghum or potato. According to its in vitro molecular weight (MW) and molar substitution (MS), HES is classified into different starch preparations. HES130/0.4, the third generation of HES, was developed with lower MW (130) and MS (0.4) to enhance degradation and to minimize side effects.In addition to maintaining the stability of hemodynamics, previous studies have shown that HES may have anti-inflammatory effects, such as down-regulating systemic inflammatory mediators and reducing capillary permeability through inhibiting leukocyte adhesion and infiltration. However, the accurate mechanism of the anti-inflammatory effect of HES is still unclear.The Toll-like receptors (TLRs)/nuclear factor-kappa B (NF-кB) signaling pathway plays an important role in initiating the inflammatory response in sepsis. TLR family members are transmembrane proteins, which can specifically recognize pathogen-associated molecular patterns. Among 10 mammalian TRLs, TLR4 functions as a receptor for lipopolysaccharide (LPS) from Gram-negative bacteria, and TLR2 is involved in the recognition of multiple products of Gram-positive bacteria. TLR signals finally activate the transcription factor NF-kB and permit the transactivation of proinflammatory cytokine genes. Our experiments were designed to investigate the anti-inflammatory effect of HES 130/0.4 in septic rats induced by cecal ligation and puncture (CLP) and the possible mechanism related with TLRs/NF-kB signaling pathway.Section one:Effect of HES130/0.4 on plasma cytokines in septic rats and possible mechanism related with TLR4/NF-kB signaling pathwayIn addition to expanding blood volume, HES130/0.4, the third generation of HES colloidal solutions, has been reported to exert an anti-inflammatory effect. However, the accurate mechanism is still obscure. The aim of the experiment was to observe the effect of HES130/0.4 on plasma cytokines in septic rats and investigate the possible mechanism related with LPS-TLR4-NF-kB signaling pathway. Rats with sepsis induced by CLP were treated with HES 130/0.4 (7.5,15, or 30 ml/kg) intravenously, then, rat plasma and peripheral blood monocytes were isolated from blood at 5h after CLP. The plasma levels of LPS and cytokines (tumor necrosis factor [TNF]-a and interleukin [IL]-6), NF-kB activities and mRNA and protein levels of TLR4 in peripheral blood monocytes were determined by limulus amebocyte lysate test, enzyme-linked immunosorbent assay, electrophoretic mobility shift assay, reverse transcription-polymerase chain reaction and western blotting test, respectively. The results showed that HES 130/0.4 dose-dependently reduced the plasma levels of TNF-a and IL-6 in rats with sepsis. As compared with 7.5 and 30 ml/kg HES130/0.4,15 ml/kg caused greater reduction of plasma TNF-a and IL-6 concentrations. HES 130/0.4 also significantly inhibited NF-kB activation and TLR4 expression in monocytes. The results suggest that during sepsis HES 130/0.4 could down-regulate the inflammatory response, possibly through inhibiting the TLR4/NF-kB signaling pathway, and could be one more appropriate plasma substitute in sepsis.Section two:Effect of HES130/0.4 on pulmonary capillary permeability in septic rats and possible mechanism related with TLRs/NF-kB signaling pathwaySepsis frequently causes multiorgan dysfunction and the lung is the most vulnerable organ that is often affected. According to different severities, lung injury is classed into acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). Previous studies indicated that HES could inhibit leukocyte adhesion and infiltration due to its anti-inflammatory effect, and then reduce capillary permeability. However, the accurate mechanism is obscure. The aim of the experiment was to observe the effect of HES 130/0.4 on pulmonary inflammatory mediators, leukocyte infiltration and pulmonary capillary permeability and further investigate the mechanism related with TLRs (TLR2, TLR4)/NF-kB signaling pathway. Rats with sepsis induced by CLP were treated with HES130/0.4 or succinylated gelatin (15 ml/kg, intravenously), then, rat lungs were isolated from blood at 5 h after later. The pulmonary levels of inflammatory mediators (TNF-α, IL-1βand intercellular adhesion molecule [ICAM]-1), NF-kB activities and mRNA and protein levels of TLRs (TLR2 and TLR4) were determined by enzyme-linked immunosorbent assay, electrophoretic mobility shift assay, reverse transcription-polymerase chain reaction and western blotting test, respectively. At 12 h after CLP, the rat lungs were collected for determination of capillary permeability and myeloperoxidase (MPO) activities. The results showed that HES130/0.4 significantly reduced pulmonary edema and capillary permeability, down-regulated pulmonary inflammatory mediators including TNF-a, IL-1βand ICAM-1, and inhibited pulmonary NF-kB activation and TLRs (TLR2 and TLR4) expressions. Compared with HES130/0.4, gelatin could also decrease pulmonary levels of inflammatory mediators and reduce capillary permeability, but not impact NF-kB activation and TLRs expression. The results of the experiment indicated that HES 130/0.4 could reduce pulmonary capillary permeability possibly through down-regulating inflammatory mediators and inhibiting TLRs (TLR2, TLR4)/NF-kB signaling pathway in septic rats.ConclusionThe aim of the experiments was to observe the effect of HES 130/0.4 on systemic inflammatory response and pulmonary capillary permeability in septic rats and further investigate the possible mechanism related with TLRs/NF-kB signaling pathway. The results definitely demonstrated that HES 130/0.4 could down-regulate the septic rat systemic inflammatory mediators, reduce pulmonary capillary permeability and inhibit TLRs expression and NF-kB activation in peripheral monocytes and lung tissue, which suggests that the mechanism on anti-inflammatory effect of HES 130/0.4 in sepsis might be related with inhibiting TLRs/NF-kB signaling pathway.As one colloidal solution, the main pharmaceutical effect of HES is still to expand blood volume and improve hemodynamic parameters. However, compared with other many solutions in pharmaceutical market, the effects of anti-inflammatory and reducing capillary permeability would strengthen the competitive capacity of HES. As the results of the second experiment showed, HES 130/0.4, compared with the same dose of succinylated gelatin, could more significantly reduce pulmonary capillary permeability in septic rats. If the conclusion of our animal experiments was further conformed using clinical trials, the clinicians would consider the above pharmaceutical advantages of HES 130/0.4, which is the utmost clinical significance of our experiments.更多还原