本文選題：戊二醛聚合豬血紅蛋白 + 失血性休克復(fù)蘇��； 參考：《西北大學(xué)》2015年博士論文

【摘要】：失血性休克是全世界范圍內(nèi)創(chuàng)傷患者死亡的主要原因,其治療策略主要采取控制出血,擴(kuò)張血容量,恢復(fù)組織灌注和氧供,維持血壓及體溫等對(duì)癥措施�；謴�(fù)組織灌注的復(fù)蘇液可采用如生理鹽水、乳酸林格液等晶體液,羥乙基淀粉、右旋糖酐等膠體液,或新鮮血漿、成分血以及幾種液體按比例聯(lián)合使用的方式。但采用生理鹽水、乳酸林格液等小分子晶體液復(fù)蘇時(shí),不僅難以維持血容量,還會(huì)造成細(xì)胞水腫、氧化應(yīng)激損傷和中性粒細(xì)胞的激活,進(jìn)而激活炎癥因子級(jí)聯(lián)反應(yīng),導(dǎo)致系統(tǒng)性炎癥反應(yīng)綜合征(SIRS),50%的患者會(huì)出現(xiàn)急性呼吸窘迫綜合癥(Acute Respiratory Distress Syndrome, ARDS),嚴(yán)重者發(fā)展為多器官功能障礙綜合癥(multiple organ dysfunction syndrome, MODs)。因此液體復(fù)蘇時(shí)也應(yīng)考慮抑制活性氧(ROS)產(chǎn)生和炎癥因子激活。在重度失血休克復(fù)蘇時(shí)除需擴(kuò)張血容量維持灌注外,通常需要輸血以改善組織缺氧狀態(tài),減輕系統(tǒng)性缺血再灌注損傷。由于潛在的感染風(fēng)險(xiǎn)、血液配型、保存方式和時(shí)間、老齡化社會(huì)的發(fā)展等因素,世界性的血液供給短缺還在不斷加劇,尋求合適血液替代用品的需求日益迫切�；谘t蛋白分子修飾的血紅蛋白氧載體HBOCs (Hemoglobin-based oxygen carriers)是一類(lèi)具有攜氧功能的聚合物,有望替代懸浮紅細(xì)胞(packed RBCs)用于失血性休克復(fù)蘇。課題組前期開(kāi)發(fā)了HBOCs產(chǎn)品戊二醛聚合豬血紅蛋白(Polymerized porcine hemoglobin, pPolyHb),發(fā)現(xiàn)大鼠靜脈注射會(huì)發(fā)生免疫耐受,繼而在100%換血實(shí)驗(yàn)中證實(shí)了其安全性和攜氧能力。因此本研究探討了pPolyHb在大鼠重度失血性休克復(fù)蘇中生理藥理應(yīng)用效果及其對(duì)血管內(nèi)皮細(xì)胞氧化應(yīng)激損傷及炎癥反應(yīng)影響和細(xì)胞信號(hào)轉(zhuǎn)導(dǎo)機(jī)制。首先,探討pPolyHb在大鼠重度失血性休克復(fù)蘇中對(duì)機(jī)體血壓、血液酸堿平衡、氧化應(yīng)激和炎癥反應(yīng)的影響以及不同液體復(fù)蘇組織病理?yè)p傷情況。試驗(yàn)建立大鼠重度失血性休克模型,同時(shí)控制血壓和血容量,控制血壓在35±5 mmHg,放血量60±5%(總血量按7%動(dòng)物體重計(jì))。休克90 min后,立即通過(guò)左側(cè)靜脈以0.5 mL/min速率注射復(fù)蘇液+2倍失血體積生理鹽水復(fù)蘇。受試動(dòng)物隨機(jī)分為5組進(jìn)行復(fù)蘇,①假手術(shù)組(Sham);②3倍失血體積生理鹽水復(fù)蘇組(NS組)；③2 g/kg pPolyHb復(fù)蘇組；④0.6%HES復(fù)蘇組(HES組)⑤自體血回輸組(以2 g/kg Hb計(jì),Blood組)。監(jiān)控大鼠血壓和動(dòng)脈血?dú)�。監(jiān)測(cè)基礎(chǔ)、休克末、復(fù)蘇1 h、復(fù)蘇3 h及復(fù)蘇6 h血清中TNF-α、IL-1β、 MPO、MDA含量。復(fù)蘇6 h,處死動(dòng)物檢測(cè)心、肝、肺、腎組織中乳酸(Lac)、MPO、MDA及HMGB1含量,并通過(guò)HE染色和TUNEL法檢測(cè)組織病理和細(xì)胞凋亡情況；檢測(cè)血清胱抑素C (Cys C)、中性粒細(xì)胞明膠酶載脂蛋白(NGAL)及組織腎損傷分子-1(KIM-])含量,反映腎損傷情況。pPolyHb復(fù)蘇對(duì)血壓、血液酸堿平衡及血清中炎癥因子含量和氧化應(yīng)激水平的影響。pPolyHb組復(fù)蘇過(guò)程血壓維持在84±11 mmHg,比NS組(55±10mmHg)能更有效地維持血壓；同時(shí)與NS組相比,復(fù)蘇后pPolyHb組BE值(-6.3士5.7 vs-13.7土4.2)及代謝物L(fēng)ac值(3.22±1.5 vs.6.0士2.2)得到較顯著改善,有效地糾正了組織酸中毒狀態(tài),與Blood組復(fù)蘇效果相似。pPolyHb復(fù)蘇后血清中炎癥因子TNF-a與IL-1β含量顯著低于NS組(P0.05),也低于HES組(P0.05)；復(fù)蘇6 h血清HMGB1,除NS與Sham相比,有顯著升高,其余各組與Sham組無(wú)顯著差異。以上表明,pPolyHb復(fù)蘇能顯著抑制休克復(fù)蘇過(guò)程中血清炎癥因子釋放。血清MPO反映復(fù)蘇液對(duì)循環(huán)中性粒細(xì)胞聚集的影響,NS組復(fù)蘇過(guò)程中表現(xiàn)出持續(xù)上升趨勢(shì),pPolyHb組復(fù)蘇6h, MPO水平顯著低于NS組(P0.05),與HES組及Blood組無(wú)顯著差異(JP0.05)。各復(fù)蘇組血清中氧化應(yīng)激產(chǎn)物MDA含量隨時(shí)間延長(zhǎng)不斷增加,復(fù)蘇6h時(shí),各組間無(wú)顯著差異。血清MPO和MDA含量表明,pPolyHb復(fù)蘇對(duì)休克復(fù)蘇引起的氧化應(yīng)激有一定抑制作用。pPolyHb復(fù)蘇重度失血性休克對(duì)不同組織炎癥、氧化應(yīng)激和病理?yè)p傷的影響。失血性休克復(fù)蘇時(shí),臟器如腎、腸、肺等易受到缺血／再灌注(Ischemia/Reperfusion, I/R)損傷。在肝、腎組織中,pPolyHb復(fù)蘇組Lac含量與NS相比顯著降低(P0.05),表明灌注效果較好。復(fù)蘇后,各組肺組織中MPO含量顯著升高(與Sham相比,P0.01),NS組升高最顯著,pPolyHb與Blood組MPO含量無(wú)顯著區(qū)別(P0.05)；在心、肝和腎組織中,pPolyHb組MPO水平高于Blood組,但無(wú)顯著差異(P0.05)。復(fù)蘇后各組織中脂質(zhì)過(guò)氧化物MDA水平,NS組顯著升高,pPolyHb組與NS組及Blood組均無(wú)顯著差異,與血清結(jié)果一致。組織中炎癥介質(zhì)HMGB1表明,NS組顯著升高,pPolyHb復(fù)蘇與Blood相似,對(duì)肺組織晚期炎癥有顯著的抑制作用。從組織病理角度可見(jiàn),NS復(fù)蘇時(shí)心、肝、肺組織損傷較明顯,pPolyHb與Blood復(fù)蘇時(shí)損傷均能得到減輕。pPolyHb復(fù)蘇重度失血性休克對(duì)腎臟損傷的影響。腎臟屬于遠(yuǎn)端器官,在失血性休克復(fù)蘇時(shí)I/R損傷比較嚴(yán)重。各復(fù)蘇組腎組織中HMGB1含量升高,但各組間無(wú)顯著差異。腎損傷生物標(biāo)志物CysC及NGAL顯示pPolyHb復(fù)蘇損傷程度與NS組及Blood組相似,均顯著低于HES組；四種復(fù)蘇方式KIM-1均顯著升高(P0.05)。HE染色病理分析發(fā)現(xiàn)Blood組損傷較輕,pPolyHb與NS組損傷相似。HES組TUNEL凋亡率最高,NS組與pPolyHb組水平一致,而B(niǎo)lood組細(xì)胞凋亡率最小,組織損傷與細(xì)胞凋亡相關(guān)。由以上結(jié)果可知,NS復(fù)蘇后血清和組織中炎癥因子和氧化應(yīng)激水平明顯增加,且復(fù)蘇6h后組織仍處于缺氧狀態(tài),從病理角度看,肺組織炎癥浸潤(rùn)嚴(yán)重,腎組織水化變性。而pPolyHb復(fù)蘇顯著抑制了血清和組織中炎癥因子和中性粒細(xì)胞的浸潤(rùn),肺臟損傷顯著減輕,作為具有攜氧功能的“氧治療”劑對(duì)大鼠重度失血性休克復(fù)蘇的益處大于不良反應(yīng)。其次,研究了pPolyHb預(yù)處理對(duì)內(nèi)皮細(xì)胞氧化應(yīng)激影響及血紅素加氧酶-1(HO-1)信號(hào)通路的作用。pPolyHb在攜氧的同時(shí)有一定抑制氧化應(yīng)激和炎癥反應(yīng)作用,在預(yù)處理抑制I/R損傷中顯示廣闊的前景。有研究報(bào)道HO-1在HBOCs抑制氧化應(yīng)激損傷發(fā)生中起作用,而內(nèi)皮系統(tǒng)損傷是I/R損傷發(fā)生的關(guān)鍵部位,H202又是細(xì)胞內(nèi)ROS的主要存在形式。因此,試驗(yàn)建立人臍靜脈內(nèi)皮細(xì)胞(HUVECs)的H202損傷模型,MTT法檢測(cè)細(xì)胞存活率,Westernblot檢測(cè)細(xì)胞凋亡相關(guān)蛋白及HO-1的表達(dá),對(duì)pPolyHb預(yù)處理產(chǎn)生的保護(hù)效應(yīng)和信號(hào)通路進(jìn)行探討。研究發(fā)現(xiàn),pPolyHb預(yù)處理增加了內(nèi)皮細(xì)胞存活率,同時(shí)減弱細(xì)胞凋亡蛋白Bax的表達(dá),增加細(xì)胞存活蛋白Bcl2的表達(dá),降低兩者比率,并減少CytC及Cleave-Caspase 3的釋放,從而抑制線(xiàn)粒體途徑引起的HUVECs細(xì)胞凋亡。進(jìn)一步對(duì)細(xì)胞信號(hào)途徑研究表明,H202損傷HUVECs激活JNK/p38 MAPK磷酸化增加,特異性抑制劑SB 203580及SP 600125能增加細(xì)胞存活率。pPolyHb預(yù)處理誘導(dǎo)了HO-1的表達(dá),并隨時(shí)間和濃度的增加而增加,同時(shí)抑制了H202損傷所引起的JNK/p38 MAPK磷酸化增加。HO-1抑制劑SnPP能夠減弱pPolyHb對(duì)細(xì)胞存活的保護(hù)效應(yīng),同時(shí)部分抵消對(duì)JNK/p38 MAPK磷酸化的抑制效果。預(yù)處理還減弱了H202誘導(dǎo)的內(nèi)皮細(xì)胞ROS水平。因此pPolyHb對(duì)內(nèi)皮細(xì)胞的氧化應(yīng)激損傷保護(hù)作用,可能是通過(guò)誘導(dǎo)HO-1表達(dá),而抑制JNK/p38 MAPK磷酸化和細(xì)胞內(nèi)ROS產(chǎn)生來(lái)發(fā)揮。最后,探討了pPolyHb預(yù)處理對(duì)TNF-a誘導(dǎo)內(nèi)皮細(xì)胞炎癥因子表達(dá)影響以及HO-1對(duì)炎癥信號(hào)通路影響。微血管中性粒細(xì)胞的過(guò)度浸潤(rùn)是相應(yīng)器官發(fā)生MODs的標(biāo)志,而內(nèi)皮細(xì)胞對(duì)中性粒細(xì)胞粘附和激活是損傷發(fā)展的第一步,大量研究表明HO-1對(duì)炎癥的發(fā)生也有抑制作用。為此采用10ng/mL TNF-α刺激內(nèi)皮細(xì)胞制作炎癥模型,不同濃度pPolyHb進(jìn)行預(yù)處理,ELISA檢測(cè)培養(yǎng)上清中MCP-1、sICAM-1及VCAM-1等炎癥相關(guān)分子的釋放,熒光實(shí)時(shí)定量PCR檢測(cè)MCP-1、ICAM-1及HO-1mRNA相對(duì)表達(dá)量,Westemblot檢測(cè)蛋白和信號(hào)分子表達(dá),免疫熒光檢測(cè)細(xì)胞內(nèi)ICAM-1和HO-1含量,考察了pPolyHb預(yù)處理對(duì)炎癥因子TNF-α刺激下相關(guān)炎癥分子表達(dá)和釋放的影響及其分子機(jī)制。研究表明,TNF-α刺激下HUVECs培養(yǎng)上清中MCP-1、sICAM-1及VCAM-1的含量增加,細(xì)胞中MCP-1、ICAM-1和MMP-9的表達(dá)增加,而pPolyHb預(yù)處理可抑制這些相關(guān)炎癥因子的釋放和表達(dá)。HO-1抑制劑SnPP減弱了pPolyHb預(yù)處理對(duì)炎癥相關(guān)因子的抑制作用,說(shuō)明預(yù)處理的保護(hù)效應(yīng)與誘導(dǎo)HO-1產(chǎn)生相關(guān)。pPolyHb預(yù)處理時(shí),細(xì)胞內(nèi)GSH含量顯著降低,pPolyHb Fe2+向pPolyHb Fe3+轉(zhuǎn)化,因此HO-1的誘導(dǎo)表達(dá)可能與轉(zhuǎn)化過(guò)程中ROS產(chǎn)生相關(guān)。進(jìn)一步研究表明,pPolyHb預(yù)處理激活了p38MAPK/Nrf2信號(hào)途徑促使Nrf2進(jìn)入核內(nèi),進(jìn)而誘導(dǎo)了HO-1的轉(zhuǎn)錄表達(dá)。研究還發(fā)現(xiàn)pPolyHb預(yù)處理能抑制主要的內(nèi)皮細(xì)胞粘附分子表達(dá)途徑NF-κB通路,且HO-1在一定程度上能抑制NF-κB核轉(zhuǎn)位,兩者之間存在交互作用。以上結(jié)果證實(shí),pPolyHb預(yù)處理對(duì)內(nèi)皮細(xì)胞炎癥抑制作用可能是通過(guò)產(chǎn)生一定量ROS后,激活p38 MAPK/Nrf2途徑誘導(dǎo)HO-1的表達(dá),以及抑制NF-κB途徑激活發(fā)揮作用的。引入抗氧化基團(tuán)后的pPolyHb有顯著的抑制中性粒細(xì)胞浸潤(rùn)和炎癥因子釋放作用,顯著減弱肺臟損傷,可用于RBC替代治療。而pPolyHb預(yù)處理可能通過(guò)p38 MAPK/Nrf2途徑激活誘導(dǎo)HO-1,抑制內(nèi)皮細(xì)胞氧化應(yīng)激損傷和炎癥反應(yīng)。在移植器官保存、擇期手術(shù)預(yù)處理、貧血及血管相關(guān)疾病治療等方面有良好的應(yīng)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.

【學(xué)位授予單位】：西北大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：R459.7

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