pPolyHb在大鼠重度失血性休克复苏中应用及其对内皮细胞氧化应激及炎症反应影响研究

发布时间：2018-05-03 01:11

本文选题：戊二醛聚合猪血红蛋白 + 失血性休克复苏　；参考：《西北大学》2015年博士论文

【摘要】：失血性休克是全世界范围内创伤患者死亡的主要原因,其治疗策略主要采取控制出血,扩张血容量,恢复组织灌注和氧供,维持血压及体温等对症措施。恢复组织灌注的复苏液可采用如生理盐水、乳酸林格液等晶体液,羟乙基淀粉、右旋糖酐等胶体液,或新鲜血浆、成分血以及几种液体按比例联合使用的方式。但采用生理盐水、乳酸林格液等小分子晶体液复苏时,不仅难以维持血容量,还会造成细胞水肿、氧化应激损伤和中性粒细胞的激活,进而激活炎症因子级联反应,导致系统性炎症反应综合征(SIRS),50%的患者会出现急性呼吸窘迫综合症(Acute Respiratory Distress Syndrome, ARDS),严重者发展为多器官功能障碍综合症(multiple organ dysfunction syndrome, MODs)。因此液体复苏时也应考虑抑制活性氧(ROS)产生和炎症因子激活。在重度失血休克复苏时除需扩张血容量维持灌注外,通常需要输血以改善组织缺氧状态,减轻系统性缺血再灌注损伤。由于潜在的感染风险、血液配型、保存方式和时间、老龄化社会的发展等因素,世界性的血液供给短缺还在不断加剧,寻求合适血液替代用品的需求日益迫切。基于血红蛋白分子修饰的血红蛋白氧载体HBOCs (Hemoglobin-based oxygen carriers)是一类具有携氧功能的聚合物,有望替代悬浮红细胞(packed RBCs)用于失血性休克复苏。课题组前期开发了HBOCs产品戊二醛聚合猪血红蛋白(Polymerized porcine hemoglobin, pPolyHb),发现大鼠静脉注射会发生免疫耐受,继而在100%换血实验中证实了其安全性和携氧能力。因此本研究探讨了pPolyHb在大鼠重度失血性休克复苏中生理药理应用效果及其对血管内皮细胞氧化应激损伤及炎症反应影响和细胞信号转导机制。首先,探讨pPolyHb在大鼠重度失血性休克复苏中对机体血压、血液酸碱平衡、氧化应激和炎症反应的影响以及不同液体复苏组织病理损伤情况。试验建立大鼠重度失血性休克模型,同时控制血压和血容量,控制血压在35±5 mmHg,放血量60±5%(总血量按7%动物体重计)。休克90 min后,立即通过左侧静脉以0.5 mL/min速率注射复苏液+2倍失血体积生理盐水复苏。受试动物随机分为5组进行复苏,①假手术组(Sham);②3倍失血体积生理盐水复苏组(NS组)；③2 g/kg pPolyHb复苏组；④0.6%HES复苏组(HES组)⑤自体血回输组(以2 g/kg Hb计,Blood组)。监控大鼠血压和动脉血气。监测基础、休克末、复苏1 h、复苏3 h及复苏6 h血清中TNF-α、IL-1β、 MPO、MDA含量。复苏6 h,处死动物检测心、肝、肺、肾组织中乳酸(Lac)、MPO、MDA及HMGB1含量,并通过HE染色和TUNEL法检测组织病理和细胞凋亡情况；检测血清胱抑素C (Cys C)、中性粒细胞明胶酶载脂蛋白(NGAL)及组织肾损伤分子-1(KIM-])含量,反映肾损伤情况。pPolyHb复苏对血压、血液酸碱平衡及血清中炎症因子含量和氧化应激水平的影响。pPolyHb组复苏过程血压维持在84±11 mmHg,比NS组(55±10mmHg)能更有效地维持血压；同时与NS组相比,复苏后pPolyHb组BE值(-6.3士5.7 vs-13.7土4.2)及代谢物Lac值(3.22±1.5 vs.6.0士2.2)得到较显著改善,有效地纠正了组织酸中毒状态,与Blood组复苏效果相似。pPolyHb复苏后血清中炎症因子TNF-a与IL-1β含量显著低于NS组(P0.05),也低于HES组(P0.05)；复苏6 h血清HMGB1,除NS与Sham相比,有显著升高,其余各组与Sham组无显著差异。以上表明,pPolyHb复苏能显著抑制休克复苏过程中血清炎症因子释放。血清MPO反映复苏液对循环中性粒细胞聚集的影响,NS组复苏过程中表现出持续上升趋势,pPolyHb组复苏6h, MPO水平显著低于NS组(P0.05),与HES组及Blood组无显著差异(JP0.05)。各复苏组血清中氧化应激产物MDA含量随时间延长不断增加,复苏6h时,各组间无显著差异。血清MPO和MDA含量表明,pPolyHb复苏对休克复苏引起的氧化应激有一定抑制作用。pPolyHb复苏重度失血性休克对不同组织炎症、氧化应激和病理损伤的影响。失血性休克复苏时,脏器如肾、肠、肺等易受到缺血／再灌注(Ischemia/Reperfusion, I/R)损伤。在肝、肾组织中,pPolyHb复苏组Lac含量与NS相比显著降低(P0.05),表明灌注效果较好。复苏后,各组肺组织中MPO含量显著升高(与Sham相比,P0.01),NS组升高最显著,pPolyHb与Blood组MPO含量无显著区别(P0.05)；在心、肝和肾组织中,pPolyHb组MPO水平高于Blood组,但无显著差异(P0.05)。复苏后各组织中脂质过氧化物MDA水平,NS组显著升高,pPolyHb组与NS组及Blood组均无显著差异,与血清结果一致。组织中炎症介质HMGB1表明,NS组显著升高,pPolyHb复苏与Blood相似,对肺组织晚期炎症有显著的抑制作用。从组织病理角度可见,NS复苏时心、肝、肺组织损伤较明显,pPolyHb与Blood复苏时损伤均能得到减轻。pPolyHb复苏重度失血性休克对肾脏损伤的影响。肾脏属于远端器官,在失血性休克复苏时I/R损伤比较严重。各复苏组肾组织中HMGB1含量升高,但各组间无显著差异。肾损伤生物标志物CysC及NGAL显示pPolyHb复苏损伤程度与NS组及Blood组相似,均显著低于HES组；四种复苏方式KIM-1均显著升高(P0.05)。HE染色病理分析发现Blood组损伤较轻,pPolyHb与NS组损伤相似。HES组TUNEL凋亡率最高,NS组与pPolyHb组水平一致,而Blood组细胞凋亡率最小,组织损伤与细胞凋亡相关。由以上结果可知,NS复苏后血清和组织中炎症因子和氧化应激水平明显增加,且复苏6h后组织仍处于缺氧状态,从病理角度看,肺组织炎症浸润严重,肾组织水化变性。而pPolyHb复苏显著抑制了血清和组织中炎症因子和中性粒细胞的浸润,肺脏损伤显著减轻,作为具有携氧功能的“氧治疗”剂对大鼠重度失血性休克复苏的益处大于不良反应。其次,研究了pPolyHb预处理对内皮细胞氧化应激影响及血红素加氧酶-1(HO-1)信号通路的作用。pPolyHb在携氧的同时有一定抑制氧化应激和炎症反应作用,在预处理抑制I/R损伤中显示广阔的前景。有研究报道HO-1在HBOCs抑制氧化应激损伤发生中起作用,而内皮系统损伤是I/R损伤发生的关键部位,H202又是细胞内ROS的主要存在形式。因此,试验建立人脐静脉内皮细胞(HUVECs)的H202损伤模型,MTT法检测细胞存活率,Westernblot检测细胞凋亡相关蛋白及HO-1的表达,对pPolyHb预处理产生的保护效应和信号通路进行探讨。研究发现,pPolyHb预处理增加了内皮细胞存活率,同时减弱细胞凋亡蛋白Bax的表达,增加细胞存活蛋白Bcl2的表达,降低两者比率,并减少CytC及Cleave-Caspase 3的释放,从而抑制线粒体途径引起的HUVECs细胞凋亡。进一步对细胞信号途径研究表明,H202损伤HUVECs激活JNK/p38 MAPK磷酸化增加,特异性抑制剂SB 203580及SP 600125能增加细胞存活率。pPolyHb预处理诱导了HO-1的表达,并随时间和浓度的增加而增加,同时抑制了H202损伤所引起的JNK/p38 MAPK磷酸化增加。HO-1抑制剂SnPP能够减弱pPolyHb对细胞存活的保护效应,同时部分抵消对JNK/p38 MAPK磷酸化的抑制效果。预处理还减弱了H202诱导的内皮细胞ROS水平。因此pPolyHb对内皮细胞的氧化应激损伤保护作用,可能是通过诱导HO-1表达,而抑制JNK/p38 MAPK磷酸化和细胞内ROS产生来发挥。最后,探讨了pPolyHb预处理对TNF-a诱导内皮细胞炎症因子表达影响以及HO-1对炎症信号通路影响。微血管中性粒细胞的过度浸润是相应器官发生MODs的标志,而内皮细胞对中性粒细胞粘附和激活是损伤发展的第一步,大量研究表明HO-1对炎症的发生也有抑制作用。为此采用10ng/mL TNF-α刺激内皮细胞制作炎症模型,不同浓度pPolyHb进行预处理,ELISA检测培养上清中MCP-1、sICAM-1及VCAM-1等炎症相关分子的释放,荧光实时定量PCR检测MCP-1、ICAM-1及HO-1mRNA相对表达量,Westemblot检测蛋白和信号分子表达,免疫荧光检测细胞内ICAM-1和HO-1含量,考察了pPolyHb预处理对炎症因子TNF-α刺激下相关炎症分子表达和释放的影响及其分子机制。研究表明,TNF-α刺激下HUVECs培养上清中MCP-1、sICAM-1及VCAM-1的含量增加,细胞中MCP-1、ICAM-1和MMP-9的表达增加,而pPolyHb预处理可抑制这些相关炎症因子的释放和表达。HO-1抑制剂SnPP减弱了pPolyHb预处理对炎症相关因子的抑制作用,说明预处理的保护效应与诱导HO-1产生相关。pPolyHb预处理时,细胞内GSH含量显著降低,pPolyHb Fe2+向pPolyHb Fe3+转化,因此HO-1的诱导表达可能与转化过程中ROS产生相关。进一步研究表明,pPolyHb预处理激活了p38MAPK/Nrf2信号途径促使Nrf2进入核内,进而诱导了HO-1的转录表达。研究还发现pPolyHb预处理能抑制主要的内皮细胞粘附分子表达途径NF-κB通路,且HO-1在一定程度上能抑制NF-κB核转位,两者之间存在交互作用。以上结果证实,pPolyHb预处理对内皮细胞炎症抑制作用可能是通过产生一定量ROS后,激活p38 MAPK/Nrf2途径诱导HO-1的表达,以及抑制NF-κB途径激活发挥作用的。引入抗氧化基团后的pPolyHb有显著的抑制中性粒细胞浸润和炎症因子释放作用,显著减弱肺脏损伤,可用于RBC替代治疗。而pPolyHb预处理可能通过p38 MAPK/Nrf2途径激活诱导HO-1,抑制内皮细胞氧化应激损伤和炎症反应。在移植器官保存、择期手术预处理、贫血及血管相关疾病治疗等方面有良好的应用前景。
[Abstract]:Hemorrhagic shock is the main cause of death in trauma patients worldwide. Its treatment strategies are mainly to control bleeding, dilate blood volume, restore tissue perfusion and oxygen supply, maintain blood pressure and body temperature. Recovery of resuscitation from tissue perfusion can be used as physiological saline, lactate linger solution, hydroxyethyl starch and dextran. It is not only difficult to maintain blood capacity, but also causes edema, oxidative stress damage and activation of neutrophils, and then activates the cascade reaction of inflammatory factors, and then activates the cascade reaction of inflammatory factors. For systemic inflammatory response syndrome (SIRS), 50% of patients have acute respiratory distress syndrome (Acute Respiratory Distress Syndrome, ARDS), and the serious people develop multiple organ dysfunction syndrome (multiple organ dysfunction syndrome, MODs). Therefore, the inhibition of reactive oxygen species (ROS) production and inflammatory factors should be considered in the resuscitation of liquid body. Activation. In the case of severe hemorrhagic shock and resuscitation, in addition to expanding blood volume to maintain perfusion, blood transfusion is usually needed to improve tissue hypoxia and reduce systemic ischemia reperfusion injury. Due to potential infection risk, blood distribution, preservation methods and time, and the development of aging society, the worldwide shortage of blood supply is still being added. The demand for suitable blood substitutes is increasingly urgent. Hemoglobin based hemoglobin oxygen carrier HBOCs (Hemoglobin-based oxygen carriers) is a class of polymers with oxygen carrying functions. It is expected to replace suspended red cells (packed RBCs) for the recovery of hemorrhagic shock. The research group has developed the HBOCs product amyl two in the early stage. Aldehyde polymerized Polymerized porcine hemoglobin (pPolyHb), it was found that the immune tolerance occurred in the rats by intravenous injection, and then confirmed the safety and oxygen carrying capacity in the 100% blood exchange experiment. Therefore, this study explored the physiological and pharmacological effects of pPolyHb in the resuscitation of severe hemorrhagic shock in rats and its effect on vascular endothelial cells. The effects of oxidative stress injury and inflammatory response and cell signal transduction mechanism. First, the effects of pPolyHb on the body blood pressure, blood acid base balance, oxidative stress and inflammatory response and the pathological damage of different fluid resuscitation tissues during severe hemorrhagic shock resuscitation in rats were investigated. The blood pressure and blood volume were controlled, the blood pressure was 35 + 5 mmHg, the blood volume was 60 + 5% (the total blood volume was 7% animal weight). After the shock of 90 min, the left vein was immediately injected with the +2 times of the resuscitation of the resuscitation fluid at 0.5 mL/min rate to recover the physiological saline. The subjects were randomly divided into 5 groups to carry out the resuscitation, (1) the sham operation group (Sham); and (2) 3 times the volume physiological salt of the volume of blood loss. Water resuscitation group (group NS); (3) 2 g/kg pPolyHb resuscitation group; (4) 0.6%HES resuscitation group (group HES) (group HES) of autologous blood transfusion (2 g/kg Hb, Blood group). Monitoring the blood pressure and arterial blood gas of rats. Monitoring foundation, shock end, recovery 1 h, recovery 3 h and resuscitation 6 h serum. The contents of lactic acid (Lac), MPO, MDA and HMGB1 were weave, and the histopathology and cell apoptosis were detected by HE staining and TUNEL method, serum cystatin C (Cys C), neutrophil gelatinase apolipoprotein (NGAL) and molecular -1 (KIM-]) content of tissue renal injury were detected, reflecting the blood pressure, blood acid base balance and serum in the renal injury. The effect of inflammatory factor content and oxidative stress level on the blood pressure in the.PPolyHb group was 84 + 11 mmHg, and the blood pressure was maintained more effectively than the NS group (55 + 10mmHg). At the same time, compared with the NS group, the BE value of the pPolyHb group (-6.3, 5.7 vs-13.7 4.2) and the metabolite Lac value (3.22 + 1.5 vs.6.0 man 2.2) were significantly improved and effectively corrected. The state of tissue acidosis was similar to that of the Blood group. The content of serum inflammatory factors TNF-a and IL-1 beta in serum was significantly lower than that of group NS (P0.05) and HES group (P0.05) after.PPolyHb resuscitation, and the recovery of 6 h serum HMGB1, except NS and Sham, was significantly higher, and the other groups were not significantly different from those of the group. Serum MPO reflected the release of serum inflammatory factors during the resuscitation of shock. The effect of resuscitation on circulating neutrophils was reflected in serum, and in group NS, there was a continuous upward trend in the process of resuscitation, 6h in group pPolyHb was resuscitation, MPO was significantly lower than that in group NS (P0.05), and there was no significant difference between group HES and Blood group (JP0.05). There was no significant difference in the content of 6h. The serum MPO and MDA content showed that the pPolyHb resuscitation had a certain inhibitory effect on the oxidative stress caused by shock and resuscitation, and the effect of.PPolyHb resuscitation on severe hemorrhagic shock on different tissues, oxidative stress and pathological damage. In the liver and kidney tissue, the content of Lac in the pPolyHb resuscitation group was significantly lower than that of NS (P0.05) in the liver and kidney tissues (P0.05), indicating that the perfusion effect was better. After the recovery, the MPO content in the lung tissue was significantly increased (compared with Sham, P0.01), and the NS group increased most significantly, pPolyHb and Blood group content was not. There was significant difference (P0.05). In heart, liver and kidney tissue, the level of MPO in group pPolyHb was higher than that in group Blood, but there was no significant difference (P0.05). The level of lipid peroxide MDA in each tissue after resuscitation was significantly higher, and there was no significant difference between the group of pPolyHb and the NS group and the Blood group. The inflammatory mediators in the tissue HMGB1 showed that the NS group was significantly elevated. LyHb resuscitation is similar to Blood and has a significant inhibitory effect on advanced inflammation of the lung tissue. From the histopathological point of view, the damage of the heart, liver and lung tissue in the resuscitation of NS is obvious. Both pPolyHb and Blood resuscitation can reduce the effect of severe hemorrhagic shock of.PPolyHb resuscitation on renal injury. The kidney belongs to the distal organ, and in the hemorrhagic shock. The I/R injury in the resuscitation group was more serious. There was no significant difference in the content of HMGB1 in the renal tissue of the resuscitation groups, but there was no significant difference between the groups of renal injury biomarkers CysC and NGAL. The damage degree of pPolyHb resuscitation was similar to that of the NS group and the Blood group, all of which were significantly lower than that of the HES group; the four resuscitation methods were significantly increased (P0.05).HE staining pathological analysis of Blood group The apoptosis rate of TUNEL was the highest in the group of pPolyHb and the NS group, and the NS group was the same as that in the pPolyHb group. The apoptosis rate of the Blood group was the smallest and the tissue damage was related to the apoptosis. It was found that the serum and tissue inflammatory factors and oxidative stress levels increased obviously after NS resuscitation, and the tissue was still in the hypoxia after the recovery of 6h. From the pathological point of view, the inflammatory infiltration of the lung tissue is serious and the renal tissue is hydrated. The pPolyHb resuscitation significantly inhibits the infiltration of inflammatory factors and neutrophils in the serum and tissues, and the lung injury is significantly reduced. As a "oxygen therapy" agent with oxygen carrying function, the benefit of the recovery of severe hemorrhagic shock in rats is greater than that of adverse reactions. Secondly, the effect of pPolyHb pretreatment on oxidative stress of endothelial cells and the role of heme oxygenase -1 (HO-1) signaling pathway,.PPolyHb has a certain inhibitory effect on oxidative stress and inflammatory reaction at the same time as oxygen carrying, and it has a broad prospect in the preconditioning inhibition of I/R damage. A study has reported that HO-1 inhibits oxidative stress in HBOCs. It plays an important role, and endothelial damage is the key part of I/R damage, and H202 is the main form of intracellular ROS. Therefore, the experiment established the H202 damage model of human umbilical vein endothelial cells (HUVECs), MTT method to detect cell survival rate, Westernblot detection of apoptosis related protein and HO-1 expression, produced by pPolyHb preprocessing. The protective effect and signal pathway are discussed. It is found that pPolyHb preconditioning increases the survival rate of endothelial cells, decreases the expression of apoptotic protein Bax, increases the expression of cell survivin Bcl2, reduces the ratio of both, and reduces the release of CytC and Cleave-Caspase 3, thus inhibiting the apoptosis of HUVECs cells caused by mitochondrial pathway. Further study on cell signaling pathway showed that H202 damage HUVECs activation JNK/p38 MAPK phosphorylation increased, specific inhibitor SB 203580 and SP 600125 could increase cell survival rate.PPolyHb preconditioning induced HO-1 expression, and increased with time and concentration, and inhibited JNK/p38 MAPK phosphorylation caused by H202 damage. Adding.HO-1 inhibitor SnPP can weaken the protective effect of pPolyHb on cell survival and partially counteract the inhibition effect on JNK/p38 MAPK phosphorylation. Preconditioning also weakens the ROS level of H202 induced endothelial cells. Therefore, the protective effect of pPolyHb on oxidative stress damage to endothelial cells can be mediated by the induction of HO-1 expression, and the inhibition of JNK/p38 MAP. K phosphorylation and intracellular ROS production. Finally, the effect of pPolyHb preconditioning on the expression of inflammatory factors in endothelial cells induced by TNF-a and the effect of HO-1 on the inflammatory signaling pathway. The excessive infiltration of microvascular neutrophils is a marker of MODs in the corresponding organs, while endothelial cells are damaging to the adhesion and activation of neutrophils. The first step, a large number of studies have shown that HO-1 has an inhibitory effect on the occurrence of inflammation. To this end, 10ng/mL TNF- alpha was used to stimulate endothelial cells to produce inflammatory models, different concentrations of pPolyHb were pretreated, ELISA was used to detect the release of MCP-1, sICAM-1 and VCAM-1 related molecules in the culture supernatant and real-time quantitative PCR detection of MCP-1, ICAM-1 and HO-1mRNA. The expression of the relative expression, the expression of Westemblot protein and signal molecules, the content of ICAM-1 and HO-1 in cells by immunofluorescence, and the effect of pPolyHb preconditioning on the expression and release of inflammatory molecules associated with inflammatory factor TNF- alpha and its molecular mechanism. The study showed that TNF- alpha stimulated MCP-1, sICAM-1 and VCAM-1 in the supernatant of HUVECs culture. The expression of MCP-1, ICAM-1 and MMP-9 increased in the cells, while pPolyHb preconditioning inhibited the release of these related inflammatory factors and the expression of.HO-1 inhibitor SnPP that weakened the inhibitory effect of pPolyHb preconditioning on inflammation related factors, indicating that the protective effect of preconditioning and the induction of HO-1 production associated.PPolyHb preprocessing, the intracellular GSH contains As the amount of pPolyHb Fe2+ was reduced to pPolyHb Fe3+, the induced expression of HO-1 may be associated with ROS in the transformation process. Further studies show that pPolyHb preconditioning activates the p38MAPK/Nrf2 signal pathway to induce Nrf2 into the nucleus and induces the transcriptional expression of HO-1. The study also found that pPolyHb pretreatment could inhibit the main endothelium. Cell adhesion molecules express the pathway of NF- kappa B, and HO-1 can inhibit the transposition of NF- kappa B nucleus to some extent, and there is a interaction between them. The above results confirm that the inhibitory effect of pPolyHb preconditioning on endothelial cell inflammation may be induced by the activation of p38 MAPK/ Nrf2 pathway to induce HO-1 expression and the inhibition of NF- kappa pathway. Activation plays a role. PPolyHb after the introduction of antioxidant groups significantly inhibits neutrophil infiltration and inflammatory factor release, significantly attenuated lung injury and can be used for RBC replacement therapy. PPolyHb preconditioning may activate HO-1 through p38 MAPK/Nrf2 pathway, inhibit oxidative stress injury and inflammatory reaction in the inner skin cells. There are good prospects for organ preservation, elective surgery, anemia and vascular related diseases.

【学位授予单位】：西北大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R459.7

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