不同胶体液对失血性休克大鼠血流变和炎症反应的影响及CNP抗炎机制研究

发布时间：2018-01-15 13:39

本文关键词：不同胶体液对失血性休克大鼠血流变和炎症反应的影响及CNP抗炎机制研究　出处：《中国人民解放军军事医学科学院》2014年博士论文　论文类型：学位论文

【摘要】：研究背景和目的失血性休克是创伤导致死亡的主要原因。液体复苏是失血性休克救治的重要措施，用以恢复并维持有效循环血量，减轻机体损伤。然而，在失血性休克治疗中，通过复苏恢复正常血容量后，一部分患者仍然面临全身炎症反应甚至多器官功能障碍征发生的危险。研究表明，失血性休克复苏后氧化应激、炎症反应及微循环障碍是导致多器官功能障碍的主要原因。首先，失血性休克及其复苏过程中缺血再灌损伤会引发氧化应激和炎症反应，破坏肠道粘膜屏障功能，导致肠道内毒素入血，引发全身炎症反应综合征甚至多器官功能障碍综合征。其次，休克复苏会影响血液流变学特性，进一步影响组织微循环功能，与多器官功能障碍的发生相关。因此，液体复苏作为失血性休克早期治疗的重要手段，在恢复和维持正常循环血容量的同时，还应该减少机体氧化应激和炎症反应的产生，改善血流变特性和微循环功能。以往研究发现，复苏液的种类与失血性休克复苏后血流变特性、氧化应激及炎症损伤相关，直接影响失血性休克的救治效果。在前期临床调查中，我们发现，失血性休克救治中复苏液的选择仍然缺乏统一的标准。目前的研究中，临床常用的人工胶体复苏液羟乙基淀粉(HES)130/0.4、HES200/0.5以及琥珀酰明胶(GEL)对重度失血性休克复苏后氧化应激、炎症反应及血流变的差异性影响尚缺乏系统的比较研究。所以，在第一、二部分实验中，利用大鼠重度失血性休克模型，研究目前常用的人工胶体复苏液对重度失血性休克复苏后氧化应激、炎症反应及血流变特性的不同影响，为战场失血性休克的救治中复苏液的选择提供思路和实验依据。失血性休克救治中，在扩容治疗的同时，还需要利用抗休克药物抑制休克后炎症反应、改善微循环。目前，具有抑制炎症反应、改善微循环功能的抗休克药物研发是失血性休克救治研究中的热点。C型利尿钠肽(C-type natriuretic peptide,CNP)是利尿钠肽家族重要成员，其生物学作用广泛，具有利钠排尿、舒张血管等功能。失血性休克救治中，在充分扩容后，CNP能够发挥减轻组织水肿、改善微循环、保护肾功能等作用，是一种潜在的新型抗休克药物。以往研究表明，在血管球囊损伤动物模型中，CNP能够降低血管粘附分子的表达，显示其可能具有抗炎作用。目前，CNP的抗炎机制尚不明确。第三部分研究以失血性休克后炎症反应发生发展过程中关键环节-内皮细胞活化作为切入点，建立脂多糖诱导的内皮细胞活化模型，研究CNP是否通过减弱内皮细胞活化发挥抗炎作用，并从致炎信号通路入手探讨CNP抗炎的分子机制，为下一步体内研究提供理论依据和实验基础。第一部分：合成胶体复苏液在体外和失血性休克模型中对血流变特性的影响方法：在体外实验中，雄性SD大鼠经腹腔戊巴比妥钠麻醉后，分离股动脉并插管放血，肝素抗凝，离心调整红细胞压积到40%后分成四份，一份作为空白对照组，另外三份分别与6%的羟乙基淀粉130/0.4(hydroxyethyl starch130/0.4, HES130)、6%的羟乙基淀粉200/0.5(hydroxyethyl starch200/0.5, HES200)以及琥珀酰明胶(succinylated gelatin, GEL)以5:1和3:1(由临床报道的血药浓度确定)的比例混合。37°C孵育15分钟后，离心调节红细胞压积至40%，测量红细胞变形指数、聚集指数及聚集幅度，上层血浆用于检测血浆黏度。在体内实验中，将雄性SD大鼠随机分为假手术组(Sham组)、HES130组、HES200组及GEL组。本研究采用控量失血性休克模型，大鼠经戊巴比妥钠麻醉后，分离股动脉和股静脉并插管，除Sham组外，各组大鼠稳定10分钟后开始放血，首先，在30分钟内，经股动脉以0.26ml/min速率放血，放血量约为30%的总血量，接着，从第30分钟开始以0.13ml/min的速率放血，放血量约为20-25%的总血量，并维持休克状态到65分钟。当大鼠动脉酸碱剩余值在-9mmol/L-12mmol/L之间时，HES130、HES200以及GEL组分别输注一倍失血量的胶体液，复苏2h后，取动脉血测量红细胞变形指数(elongation index, EI)、血浆黏度、聚集指数(aggregation index, AI)及聚集幅度(aggregation amplitude,AMP)。结果：在体外实验中，HES130、HES200以及GEL组血浆黏度显著高于对照组。HES200组血浆黏度明显低于GEL组，另外，HES130和HES200组血浆黏度无显著差异。体内实验中，相对于假手术组，输注GEL显著提高血浆黏度，而输注HES130和HES200都明显降低血浆黏度。HES130和HES200组血浆黏度无显著差异。体外实验中，全血分别与三种复苏液以3:1混合后，各组100s-1剪切率下EI显著低于空白对照组。而在其他剪切率下，各组EI无显著性差异。当全血分别与三种复苏液以5:1混合后，各组EI无显著性差异。体内实验也显示各组EI无显著差异。体外实验中，混合比例为3:1条件下，相对于空白对照组，GEL能显著增加AI和AMP。HES130和HES200组AI显著低于GEL组。HES200组AMP显著低于GEL组。对于AI和AMP，HES130、HES200和空白对照组之间无显著差异。混合比例为5:1条件下，相对于空白对照组，GEL能显著增加AI和AMP。HES130和HES200组AI和AMP显著低于GEL组。对于AI和AMP，HES130、HES200和空白对照组之间无显著差异。另外，各组在两种不同混合比例下的AI之间无显著性差异。混合比例3:1条件下，HES130和HES200组AMP显著高于混合比例5:1条件下AMP。体内实验中，各组AI无显著性差异。相对于假手术组，GEL能够显著提高AMP。HES130和HES200组AMP显著低于GEL组。对于AMP，HES130、HES200和假手术组之间无显著差异。结论： HES200/0.5和HES130/0.4对红细胞聚集特性没有明显影响；而GEL能够促进红细胞聚集并显著提高血浆黏度。第二部分：合成胶体复苏液对失血性休克后氧化应激和炎症反应的影响比较方法：将雄性SD大鼠随机分为假手术组(Sham组)、HES130组、HES200组、GEL组。采用控量失血性休克模型，大鼠经戊巴比妥钠麻醉后，分离股动脉和股静脉并插管，除Sham组外，各组大鼠稳定10分钟后开始放血，首先，在30分钟内，经股动脉以0.26ml/min速率放血，放血量约为大鼠30%的总血量，接着，从第30分钟开始以0.13ml/min的速率放血，放血量约为总血量20-25%，并维持休克状态到65分钟。当大鼠动脉酸碱剩余值在-9mmol/L-12mmol/L之间时，分别以一倍失血量的胶体液进行输注，复苏2h后处死。实验过程中，检测放血前、休克后及复苏后2h的动脉血气。取动物组织检测肝、脑、肺和小肠中髓过氧化物酶(myeloperoxidase, MPO)活性和丙二醛(malondialdehyde, MDA)水平。并检测小肠IL-6和TNF-α水平。结果：实验结果表明，各组大鼠的基础pH，pCO2，pO2以及酸碱剩余值(base excess,BE)无统计学差异。失血后，各组大鼠的pH，pCO2，pO2以及BE值同样无统计学差异。相较于GEL组，HES130组大鼠肝、肺、小肠和脑组织中MDA水平都显著降低。相较于HES200，HES130显著抑制肝、小肠和脑中MDA水平，但是两组肺组织中MDA水平无统计学差异。另外，在所有组织中，GEL和HES200组之间MDA水平没有显著性差异。HES130组大鼠肝、肺、小肠和脑组织中MPO活性显著低于HES200组。在四种组织中，HES130组MPO活性都显著低于GEL组。而对于GEL组和HES200组，各个组织中MPO活性无显著性差异。相对于HES200，HES130输注显著降低小肠TNF-α水平。HES130组小肠TNF-α水平也显著低于GEL组。对于HES200和GEL组，小肠TNF-α水平无统计差异。结论：在大鼠失血性休克模型中，相较于HES200/0.5和GEL，HES130/0.4输注降低失血性休克复苏后各组织中氧化应激和炎症反应。在33ml/kg输注剂量下，HES200/0.5或GEL组中氧化应激和炎症反应水平无显著差异。第三部分：C型利尿钠肽抑制内毒素诱导的内皮细胞活化及机制研究方法：采用MTT比色法检测CNP对LPS刺激下细胞活力的影响；应用Westernblot及实时荧光定量PCR检测不同浓度CNP对LPS刺激下脐静脉内皮细胞(HUVECs)粘附分子表达；用Western blot和ELISA方法检测NF-κB p65磷酸化水平和NF-κB活化；利用Western blot检测信号分子磷酸化水平；进一步应用荧光探针检测CNP对LPS刺激下HUVECs内活性氧水平的影响。结果：研究发现，LPS处理24h能够显著降低HUVECs活性，0.01、0.1及1μMCNP处理没有改变LPS刺激下的内皮细胞活性。1μM CNP预处理能够显著抑制LPS诱导的内皮细胞粘附分子-1(VCAM-1)、细胞间粘附分子-1(ICAM-1)、P-selectin和E-selectin的表达。LPS处理能够显著增强NF-κB p65的磷酸化水平和DNA结合活性，而CNP能够显著抑制NF-κB p65的的磷酸化水平和DNA结合活性。LPS处理能够在短时间内引起内皮细胞中ERK1/2、p38MAPK及JNK的活化。CNP分别在15和30分钟时都显著抑制ERK1/2和p38MAPK的磷酸化。而CNP在检测的各个时间点都没有影响JNK的活化水平。单独抑制p38MAPK或联合抑制p38MAPK和ERK1/2磷酸化都能够显著减弱LPS引起的ICAM-1和VCAM-1的mRNA和蛋白表达水平升高。而单独抑制ERK1/2不能降低LPS诱导的ICAM-1和VCAM-1的mRNA和蛋白表达水平。LPS处理能够迅速引起细胞内Akt磷酸化。与LPS组相比，CNP处理在15分钟时增强Akt的磷酸化，在30分钟时，CNP处理能够维持Akt的磷酸化。抑制PI3K/Akt信号通路活化能够逆转CNP的抑炎作用。另外，CNP能够显著增强HO-1的mRNA和蛋白表达水平。LPS处理1和3h能显著增加ROS水平。而CNP处理能够显著抑制LPS引起的ROS水平升高。结论：CNP可以通过抑制p38和NF-κB通路，清除ROS及激活PI3K/Akt/HO-1通路减弱LPS诱导的内皮细胞活化。在失血性休克救治中，选择合适的复苏液对患者的救治至关重要。本研究结果提示，，琥珀酰明胶能够提高血浆黏度，改善血流变特性，可以应用于失血性休克早期血浆黏度严重下降的患者救治。HES130/0.4抑制失血性休克复苏后组织氧化应激和炎症反应水平，可以应用于具有全身炎症反应发生倾向的失血性休克患者治疗。另外，CNP能够通过多个通路发挥抗炎作用，提示CNP有望成为一种新型抗休克药物，当然，这些还需要临床试验和动物实验的验证。本研究为失血性休克救治中复苏液的选择提供重要的实验依据，并且为新型抗休克药物的研制提供线索。
[Abstract]:Background and purpose of research
Traumatic hemorrhagic shock is the main cause of death. Fluid resuscitation is an important measure of hemorrhagic shock, to restore and maintain effective blood circulation, reduce the damage to the body. However, in the treatment of hemorrhagic shock, restore normal blood volume through the recovery, some patients still face the risk of systemic inflammatory response and multiple organ dysfunction syndrome occurred. The study shows that the resuscitation of hemorrhagic shock after oxidative stress, inflammation and microcirculation disorder is the main cause of multiple organ dysfunction. Firstly, hemorrhagic shock and resuscitation in the process of ischemia reperfusion injury can cause oxidative stress and inflammation, damage of intestinal mucosal barrier function, cause the intestinal endotoxin into the blood, cause systemic inflammatory response syndrome and multiple organ dysfunction syndrome. Secondly, shock resuscitation will affect the hemorheology, further influence group The fabric microcirculation function, associated with the occurrence of multiple organ dysfunction. Therefore, as an important means of early resuscitation of hemorrhagic shock treatment, to restore and maintain normal blood volume at the same time, should also reduce oxidative stress and inflammation, improve blood rheology and microcirculation function. Previous studies have found that the type of resuscitation fluid the blood loss and blood rheology of resuscitation, oxidative stress and inflammation related, directly affect the treatment effect of hemorrhagic shock. In the early stage of clinical investigation, we found that in the treatment of hemorrhagic shock resuscitation fluid selection is still a lack of unified standards. In the current study, the clinical commonly used artificial colloid fluid resuscitation hydroxyethyl starch (HES) 130/0.4, HES200/0.5 and succinylated gelatin (GEL) on severe hemorrhagic shock after resuscitation, oxidative stress, inflammatory reaction and the differences of the effects of blood rheology is A comparative study of the lack of system. So, in the first, second part of the experiment, the rat model of severe hemorrhagic shock, the current study used artificial colloid solution on the recovery of severe hemorrhagic shock and resuscitation after oxidative stress, different rheological properties of inflammatory reaction and blood, to provide ideas and experimental basis for the treatment of hemorrhagic shock in the battlefield Su liquid.
Hemorrhagic shock, the expansion of treatment at the same time, but also need to use anti shock drug to inhibit the inflammatory reaction after shock microcirculation. At present, it can inhibit inflammation, improve microcirculation function of anti shock drug development is hot blood loss.C type natriuretic peptide in the treatment of septic shock (C-type natriuretic, peptide, CNP) is an important member of natriuretic peptide family, which has extensive biological effects, diuresis and natriuresis, vasodilation function. Hemorrhagic shock, in full expansion, CNP can play edema, improve microcirculation, protect the renal function, is a new type of anti shock drug potential. Previous studies have shown that and in the vascular balloon injury in animal models, CNP can reduce the expression of vascular adhesion molecules, suggesting its anti-inflammatory effect. At present, the anti-inflammatory mechanism of CNP is not clear. The third part of the study on blood loss Shock inflammation after the key link in the development of endothelial cell activation as a starting point, establish the model of LPS induced activation of endothelial cells, to study whether CNP by decreasing endothelial cell activation exerts anti-inflammatory effects, and inflammatory signal pathway from the start to explore the molecular mechanism of CNP anti inflammation, provide theoretical and experimental basis for the study of in vivo the next step.
Part one: the effect of synthetic colloid resuscitation on blood rheological properties in vitro and hemorrhagic shock models
Methods: in vitro experiment, male SD rats by intraperitoneal injection of pentobarbital sodium anesthesia after separation of femoral artery was bleeding, heparin, centrifugal adjusted hematocrit to 40% into four, as a blank control group, the other three respectively and 6% hydroxyethyl starch 130/ (hydroxyethyl starch130/0.4, 0.4 HES130), hydroxyethyl starch 200/0.5 6% (hydroxyethyl starch200/0.5, HES200) and succinylated gelatin (succinylated gelatin, GEL) with 5:1 and 3:1 (determined by the blood concentration of clinical reports) ratio of.37 ~ C after 15 min of incubation, centrifugal adjusted hematocrit to 40%, measuring the erythrocyte deformation index, aggregation index and aggregation rate, the upper plasma is used to detect the plasma viscosity.
In vivo experiment, male SD rats were randomly divided into sham operation group (Sham group), HES130 group, HES200 group and GEL group. This research adopts the volume control models of hemorrhagic shock in rats by sodium pentobarbital anesthesia, isolated femoral artery and vein and intubation, except group Sham, each group rats were stable 10 minutes after the start of bloodletting, first of all, in 30 minutes, through the femoral artery to 0.26ml/min rate was about bleeding bleeding, 30% of the total blood volume, then, from the start in thirtieth minutes at a rate of 0.13ml/min blood, blood volume is about 20-25% of the total blood volume, and maintain a state of shock when large to 65 minutes. Arterial acid-base residual value between -9mmol/L-12mmol/L, HES130, HES200 and GEL were colloid infusion twice the amount of blood loss, recovery after 2h, arterial blood measurement of erythrocyte deformation index (elongation, index, EI), plasma viscosity, aggregation index (aggregation, index, AI) and AGG (amplitude aggregation Regation amplitude, AMP).
Results: HES130 in vitro, HES200 and GEL group, plasma viscosity was significantly higher than that of control group, plasma viscosity in.HES200 group was significantly lower than that of group GEL, in addition, there was no significant difference between HES130 and HES200 group. The plasma viscosity in vivo, compared with sham operation group, GEL infusion significantly increased the viscosity of blood plasma, and the infusion of HES130 and HES200 decreased plasma viscosity and plasma viscosity.HES130 HES200 group had no significant difference.
In vitro experiments, respectively, and three kinds of blood resuscitation fluid mixed with 3:1, each 100s-1 EI shear rate was significantly lower than the control group. While in other shear rate, there is no significant difference between groups EI and three respectively. When the blood resuscitation fluid mixed with 5:1, there is no significant difference between groups in EI. The experiment also showed no significant difference between the groups of EI.
In vitro experiments, mixing ratio is 3:1, compared with the blank control group, GEL significantly increased AI and AMP.HES130 and AI in HES200 group was significantly lower than that of GEL group.HES200 group AMP was significantly lower than GEL group. For AI and AMP, HES130, no significant difference between HES200 and control group. The mixing proportion is 5:1. Compared with the blank control group, GEL significantly increased AI and AMP.HES130 and HES200 group AI and AMP were significantly lower than GEL group. For AI and AMP, HES130, no significant difference between HES200 and control group. In addition, there were no significant differences between two different mixing ratio of the mixed proportion of 3:1 under the condition of AI. HES130, AMP and HES200 group was significantly higher than the proportion of 5:1 under the condition of AMP. in vivo, there is no significant difference between groups AI. Compared with the sham operation group, GEL can significantly improve the AMP.HES130 and HES200 in group AMP was significantly lower than GEL group. For AMP, HES130, HES200 and hand There was no significant difference between the groups.
Conclusion: HES200/0.5 and HES130/0.4 have no significant effect on the characteristics of erythrocyte aggregation, while GEL can promote erythrocyte aggregation and significantly increase plasma viscosity.
The second part: the comparison of the effects of synthetic colloid resuscitation on oxidative stress and inflammatory response after hemorrhagic shock
Methods: male SD rats were randomly divided into sham operation group (Sham group), HES130 group, HES200 group, GEL group. The control model of hemorrhagic shock rats, anesthetized by pentobarbital sodium, isolated femoral artery and vein and intubation, except group Sham, rats in each group were stable for 10 minutes after the start of bloodletting, first of all, in 30 minutes, through the femoral artery to 0.26ml/min rate was about bleeding bleeding, total blood volume, 30% rats and thirtieth minutes from the start at a rate of 0.13ml/min blood, blood volume of about 20-25% total blood volume, and maintain a state of shock to 65 minutes. When the rat arterial pH surplus values in between -9mmol/L-12mmol/L, respectively in a times of blood loss amount of colloid solution infusion, recovery after 2H death. During the experiment, the detection of arterial blood gas before bleeding, 2h after shock and resuscitation. The detection of liver, animal brain, spinal cord and antioxidant enzymes in lung and intestine (myeloperoxidase, MPO) Activity and the level of malondialdehyde (MDA). And the levels of IL-6 and TNF- alpha in the small intestine were detected.
Results: the experimental results show that based on pH, the rats of pCO2, pO2 and acid-base residual value (base excess, BE). There was no significant difference in blood loss, the rats of pH, pCO2, pO2 and BE also showed no significant difference. Compared with GEL group, HES130 group of rat liver, lung, MDA the level of small intestine and brain tissues were significantly decreased. Compared with HES200, HES130 significantly inhibited the level of MDA in liver, intestine and brain, but there was no significant difference in the levels of MDA in lung tissues in two groups. In addition, in all tissues, no significant difference between.HES130 group of rat liver, between GEL and HES200 MDA level of lung the activity of MPO, intestine and brain tissues was significantly lower than that of HES200 group. In four tissues, the activity of MPO in group HES130 were significantly lower than those of GEL group. In GEL group and HES200 group, there was no significant difference in MPO activity in various tissues. Compared with HES200, HES130 infusion significantly decreased intestinal TNF- alpha level of.HES130 group TNF- in the small intestine The level of alpha was significantly lower than that in the GEL group. There was no statistical difference in the level of TNF- alpha in the small intestine for HES200 and GEL groups.
Conclusion: the model of hemorrhagic shock rats, compared with HES200/0.5 and GEL, HES130/0.4 infusion reduced oxidative stress and inflammation after hemorrhagic shock resuscitation in various tissue reaction. In 33ml/kg infusion at the dose of HES200/0.5 or GEL group, oxidative stress and inflammatory response had no significant difference.
The third part: C type diuretic peptide inhibits endotoxin induced endothelial cell activation and its mechanism
Methods: MTT assay was used to study the effect of CNP on LPS induced cell viability; application of Westernblot and real-time fluorescence quantitative PCR to detect different concentrations of CNP on LPS induced human umbilical vein endothelial cells (HUVECs) adhesion molecule expression; blot activation with Western and ELISA method to detect NF- kappa B phosphorylation of p65 and NF- K B; by testing the signaling molecule Western phosphorylation of blot; the further application of fluorescent probe to detect CNP on LPS stimulation effect of active oxygen levels within the HUVECs.
Results: the study found that LPS 24h can significantly reduce the activity of HUVECs, 0.01,0.1 and 1 MCNP LPS treatment did not change under the stimulation of pretreatment of endothelial cells.1 M CNP can inhibit endothelial cell adhesion molecules induced by LPS -1 (VCAM-1), intercellular adhesion molecule -1 (ICAM-1), P-selectin and E-selectin expression.LPS treatment can significantly enhance the NF- kappa B p65 phosphorylation and DNA binding activity, while CNP can significantly inhibit NF- kappa B p65 phosphorylation and DNA binding activity of.LPS can induce ERK1/2 in endothelial cells in a short period of time, the activation of.CNP p38MAPK and JNK respectively in 15 and 30 minutes were significantly the inhibition of ERK1/2 and phosphorylation of p38MAPK and CNP. At each time point detection did not influence the level of JNK. The activation or inhibition of p38MAPK alone and ERK1/2 combined inhibition of p38MAPK phosphorylation can significantly attenuated LPS induced ICAM-1 Increased expression of mRNA and protein and VCAM-1. Inhibition of ERK1/2 alone could not decrease mRNA and protein induced by LPS ICAM-1 and VCAM-1.LPS expression level could cause Akt phosphorylation in cells rapidly. Compared with the LPS group, CNP treatment enhanced the phosphorylation of Akt in 15 minutes, 30 minutes, CNP to maintain the phosphorylation of Akt. Inhibition of the activation of PI3K/Akt pathway in inflammation inhibition can be reversed by CNP. In addition, CNP can significantly enhance mRNA HO-1 and protein expression level of.LPS 1 and 3H significantly increased the level of ROS. CNP treatment can inhibit the LPS induced increase in ROS levels.
Conclusion: CNP can inhibit the activation of endothelial cells induced by LPS by inhibiting the p38 and NF- kappa B pathway, eliminating ROS and activating PI3K/Akt/HO-1 pathway.
In hemorrhagic shock, resuscitation liquid suitable for treatment of patients is essential. The results of this study suggest that succinylated gelatin can improve blood viscosity, improve blood rheology, can be used in treatment of patients with.HES130/0.4 in early hemorrhagic shock plasma viscosity decrease seriously inhibit oxidative stress and inflammation tissue of hemorrhagic shock resuscitation reaction

【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R459.7

【参考文献】