本文關(guān)鍵詞：不同膠體液對失血性休克大鼠血流變和炎癥反應(yīng)的影響及CNP抗炎機(jī)制研究　出處：《中國人民解放軍軍事醫(yī)學(xué)科學(xué)院》2014年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 失血性休克 液體復(fù)蘇 炎癥反應(yīng) 血流變 C-型利尿鈉肽

【摘要】：研究背景和目的 失血性休克是創(chuàng)傷導(dǎo)致死亡的主要原因。液體復(fù)蘇是失血性休克救治的重要措施，用以恢復(fù)并維持有效循環(huán)血量，減輕機(jī)體損傷。然而，在失血性休克治療中，通過復(fù)蘇恢復(fù)正常血容量后，一部分患者仍然面臨全身炎癥反應(yīng)甚至多器官功能障礙征發(fā)生的危險(xiǎn)。研究表明，失血性休克復(fù)蘇后氧化應(yīng)激、炎癥反應(yīng)及微循環(huán)障礙是導(dǎo)致多器官功能障礙的主要原因。首先，失血性休克及其復(fù)蘇過程中缺血再灌損傷會引發(fā)氧化應(yīng)激和炎癥反應(yīng)，破壞腸道粘膜屏障功能，導(dǎo)致腸道內(nèi)毒素入血，引發(fā)全身炎癥反應(yīng)綜合征甚至多器官功能障礙綜合征。其次，休克復(fù)蘇會影響血液流變學(xué)特性，進(jìn)一步影響組織微循環(huán)功能，與多器官功能障礙的發(fā)生相關(guān)。因此，液體復(fù)蘇作為失血性休克早期治療的重要手段，在恢復(fù)和維持正常循環(huán)血容量的同時，還應(yīng)該減少機(jī)體氧化應(yīng)激和炎癥反應(yīng)的產(chǎn)生，改善血流變特性和微循環(huán)功能。以往研究發(fā)現(xiàn)，復(fù)蘇液的種類與失血性休克復(fù)蘇后血流變特性、氧化應(yīng)激及炎癥損傷相關(guān)，直接影響失血性休克的救治效果。在前期臨床調(diào)查中，我們發(fā)現(xiàn)，失血性休克救治中復(fù)蘇液的選擇仍然缺乏統(tǒng)一的標(biāo)準(zhǔn)。目前的研究中，臨床常用的人工膠體復(fù)蘇液羥乙基淀粉(HES)130/0.4、HES200/0.5以及琥珀酰明膠(GEL)對重度失血性休克復(fù)蘇后氧化應(yīng)激、炎癥反應(yīng)及血流變的差異性影響尚缺乏系統(tǒng)的比較研究。所以，在第一、二部分實(shí)驗(yàn)中，利用大鼠重度失血性休克模型，研究目前常用的人工膠體復(fù)蘇液對重度失血性休克復(fù)蘇后氧化應(yīng)激、炎癥反應(yīng)及血流變特性的不同影響，為戰(zhàn)場失血性休克的救治中復(fù)蘇液的選擇提供思路和實(shí)驗(yàn)依據(jù)。 失血性休克救治中，在擴(kuò)容治療的同時，還需要利用抗休克藥物抑制休克后炎癥反應(yīng)、改善微循環(huán)。目前，具有抑制炎癥反應(yīng)、改善微循環(huán)功能的抗休克藥物研發(fā)是失血性休克救治研究中的熱點(diǎn)。C型利尿鈉肽(C-type natriuretic peptide,CNP)是利尿鈉肽家族重要成員，其生物學(xué)作用廣泛，具有利鈉排尿、舒張血管等功能。失血性休克救治中，在充分?jǐn)U容后，CNP能夠發(fā)揮減輕組織水腫、改善微循環(huán)、保護(hù)腎功能等作用，是一種潛在的新型抗休克藥物。以往研究表明，在血管球囊損傷動物模型中，CNP能夠降低血管粘附分子的表達(dá)，顯示其可能具有抗炎作用。目前，CNP的抗炎機(jī)制尚不明確。第三部分研究以失血性休克后炎癥反應(yīng)發(fā)生發(fā)展過程中關(guān)鍵環(huán)節(jié)-內(nèi)皮細(xì)胞活化作為切入點(diǎn)，建立脂多糖誘導(dǎo)的內(nèi)皮細(xì)胞活化模型，研究CNP是否通過減弱內(nèi)皮細(xì)胞活化發(fā)揮抗炎作用，并從致炎信號通路入手探討CNP抗炎的分子機(jī)制，為下一步體內(nèi)研究提供理論依據(jù)和實(shí)驗(yàn)基礎(chǔ)。 第一部分：合成膠體復(fù)蘇液在體外和失血性休克模型中對血流變特性的影響 方法：在體外實(shí)驗(yàn)中，雄性SD大鼠經(jīng)腹腔戊巴比妥鈉麻醉后，分離股動脈并插管放血，肝素抗凝，離心調(diào)整紅細(xì)胞壓積到40%后分成四份，一份作為空白對照組，另外三份分別與6%的羥乙基淀粉130/0.4(hydroxyethyl starch130/0.4, HES130)、6%的羥乙基淀粉200/0.5(hydroxyethyl starch200/0.5, HES200)以及琥珀酰明膠(succinylated gelatin, GEL)以5:1和3:1(由臨床報(bào)道的血藥濃度確定)的比例混合。37°C孵育15分鐘后，離心調(diào)節(jié)紅細(xì)胞壓積至40%，測量紅細(xì)胞變形指數(shù)、聚集指數(shù)及聚集幅度，上層血漿用于檢測血漿黏度。 在體內(nèi)實(shí)驗(yàn)中，將雄性SD大鼠隨機(jī)分為假手術(shù)組(Sham組)、HES130組、HES200組及GEL組。本研究采用控量失血性休克模型，大鼠經(jīng)戊巴比妥鈉麻醉后，分離股動脈和股靜脈并插管，除Sham組外，各組大鼠穩(wěn)定10分鐘后開始放血，首先，在30分鐘內(nèi)，經(jīng)股動脈以0.26ml/min速率放血，放血量約為30%的總血量，接著，從第30分鐘開始以0.13ml/min的速率放血，放血量約為20-25%的總血量，并維持休克狀態(tài)到65分鐘。當(dāng)大鼠動脈酸堿剩余值在-9mmol/L-12mmol/L之間時，HES130、HES200以及GEL組分別輸注一倍失血量的膠體液，復(fù)蘇2h后，取動脈血測量紅細(xì)胞變形指數(shù)(elongation index, EI)、血漿黏度、聚集指數(shù)(aggregation index, AI)及聚集幅度(aggregation amplitude,AMP)。 結(jié)果：在體外實(shí)驗(yàn)中，HES130、HES200以及GEL組血漿黏度顯著高于對照組。HES200組血漿黏度明顯低于GEL組，另外，HES130和HES200組血漿黏度無顯著差異。體內(nèi)實(shí)驗(yàn)中，相對于假手術(shù)組，輸注GEL顯著提高血漿黏度，而輸注HES130和HES200都明顯降低血漿黏度。HES130和HES200組血漿黏度無顯著差異。 體外實(shí)驗(yàn)中，全血分別與三種復(fù)蘇液以3:1混合后，各組100s-1剪切率下EI顯著低于空白對照組。而在其他剪切率下，各組EI無顯著性差異。當(dāng)全血分別與三種復(fù)蘇液以5:1混合后，各組EI無顯著性差異。體內(nèi)實(shí)驗(yàn)也顯示各組EI無顯著差異。 體外實(shí)驗(yàn)中，混合比例為3:1條件下，相對于空白對照組，GEL能顯著增加AI和AMP。HES130和HES200組AI顯著低于GEL組。HES200組AMP顯著低于GEL組。對于AI和AMP，HES130、HES200和空白對照組之間無顯著差異。混合比例為5:1條件下，相對于空白對照組，GEL能顯著增加AI和AMP。HES130和HES200組AI和AMP顯著低于GEL組。對于AI和AMP，HES130、HES200和空白對照組之間無顯著差異。另外，各組在兩種不同混合比例下的AI之間無顯著性差異�；旌媳壤�3:1條件下，HES130和HES200組AMP顯著高于混合比例5:1條件下AMP。體內(nèi)實(shí)驗(yàn)中，各組AI無顯著性差異。相對于假手術(shù)組，GEL能夠顯著提高AMP。HES130和HES200組AMP顯著低于GEL組。對于AMP，HES130、HES200和假手術(shù)組之間無顯著差異。 結(jié)論： HES200/0.5和HES130/0.4對紅細(xì)胞聚集特性沒有明顯影響；而GEL能夠促進(jìn)紅細(xì)胞聚集并顯著提高血漿黏度。 第二部分：合成膠體復(fù)蘇液對失血性休克后氧化應(yīng)激和炎癥反應(yīng)的影響比較 方法：將雄性SD大鼠隨機(jī)分為假手術(shù)組(Sham組)、HES130組、HES200組、GEL組。采用控量失血性休克模型，大鼠經(jīng)戊巴比妥鈉麻醉后，分離股動脈和股靜脈并插管，除Sham組外，各組大鼠穩(wěn)定10分鐘后開始放血，首先，在30分鐘內(nèi)，經(jīng)股動脈以0.26ml/min速率放血，放血量約為大鼠30%的總血量，接著，從第30分鐘開始以0.13ml/min的速率放血，放血量約為總血量20-25%，并維持休克狀態(tài)到65分鐘。當(dāng)大鼠動脈酸堿剩余值在-9mmol/L-12mmol/L之間時，分別以一倍失血量的膠體液進(jìn)行輸注，復(fù)蘇2h后處死。實(shí)驗(yàn)過程中，檢測放血前、休克后及復(fù)蘇后2h的動脈血?dú)�。取動物組織檢測肝、腦、肺和小腸中髓過氧化物酶(myeloperoxidase, MPO)活性和丙二醛(malondialdehyde, MDA)水平。并檢測小腸IL-6和TNF-α水平。 結(jié)果：實(shí)驗(yàn)結(jié)果表明，各組大鼠的基礎(chǔ)pH，pCO2，pO2以及酸堿剩余值(base excess,BE)無統(tǒng)計(jì)學(xué)差異。失血后，各組大鼠的pH，pCO2，pO2以及BE值同樣無統(tǒng)計(jì)學(xué)差異。相較于GEL組，HES130組大鼠肝、肺、小腸和腦組織中MDA水平都顯著降低。相較于HES200，HES130顯著抑制肝、小腸和腦中MDA水平，但是兩組肺組織中MDA水平無統(tǒng)計(jì)學(xué)差異。另外，在所有組織中，GEL和HES200組之間MDA水平?jīng)]有顯著性差異。HES130組大鼠肝、肺、小腸和腦組織中MPO活性顯著低于HES200組。在四種組織中，HES130組MPO活性都顯著低于GEL組。而對于GEL組和HES200組，各個組織中MPO活性無顯著性差異。相對于HES200，HES130輸注顯著降低小腸TNF-α水平。HES130組小腸TNF-α水平也顯著低于GEL組。對于HES200和GEL組，小腸TNF-α水平無統(tǒng)計(jì)差異。 結(jié)論：在大鼠失血性休克模型中，相較于HES200/0.5和GEL，HES130/0.4輸注降低失血性休克復(fù)蘇后各組織中氧化應(yīng)激和炎癥反應(yīng)。在33ml/kg輸注劑量下，HES200/0.5或GEL組中氧化應(yīng)激和炎癥反應(yīng)水平無顯著差異。 第三部分：C型利尿鈉肽抑制內(nèi)毒素誘導(dǎo)的內(nèi)皮細(xì)胞活化及機(jī)制研究 方法：采用MTT比色法檢測CNP對LPS刺激下細(xì)胞活力的影響；應(yīng)用Westernblot及實(shí)時熒光定量PCR檢測不同濃度CNP對LPS刺激下臍靜脈內(nèi)皮細(xì)胞(HUVECs)粘附分子表達(dá)；用Western blot和ELISA方法檢測NF-κB p65磷酸化水平和NF-κB活化；利用Western blot檢測信號分子磷酸化水平；進(jìn)一步應(yīng)用熒光探針檢測CNP對LPS刺激下HUVECs內(nèi)活性氧水平的影響。 結(jié)果：研究發(fā)現(xiàn)，LPS處理24h能夠顯著降低HUVECs活性，0.01、0.1及1μMCNP處理沒有改變LPS刺激下的內(nèi)皮細(xì)胞活性。1μM CNP預(yù)處理能夠顯著抑制LPS誘導(dǎo)的內(nèi)皮細(xì)胞粘附分子-1(VCAM-1)、細(xì)胞間粘附分子-1(ICAM-1)、P-selectin和E-selectin的表達(dá)。LPS處理能夠顯著增強(qiáng)NF-κB p65的磷酸化水平和DNA結(jié)合活性，而CNP能夠顯著抑制NF-κB p65的的磷酸化水平和DNA結(jié)合活性。LPS處理能夠在短時間內(nèi)引起內(nèi)皮細(xì)胞中ERK1/2、p38MAPK及JNK的活化。CNP分別在15和30分鐘時都顯著抑制ERK1/2和p38MAPK的磷酸化。而CNP在檢測的各個時間點(diǎn)都沒有影響JNK的活化水平。單獨(dú)抑制p38MAPK或聯(lián)合抑制p38MAPK和ERK1/2磷酸化都能夠顯著減弱LPS引起的ICAM-1和VCAM-1的mRNA和蛋白表達(dá)水平升高。而單獨(dú)抑制ERK1/2不能降低LPS誘導(dǎo)的ICAM-1和VCAM-1的mRNA和蛋白表達(dá)水平。LPS處理能夠迅速引起細(xì)胞內(nèi)Akt磷酸化。與LPS組相比，CNP處理在15分鐘時增強(qiáng)Akt的磷酸化，在30分鐘時，CNP處理能夠維持Akt的磷酸化。抑制PI3K/Akt信號通路活化能夠逆轉(zhuǎn)CNP的抑炎作用。另外，CNP能夠顯著增強(qiáng)HO-1的mRNA和蛋白表達(dá)水平。LPS處理1和3h能顯著增加ROS水平。而CNP處理能夠顯著抑制LPS引起的ROS水平升高。 結(jié)論：CNP可以通過抑制p38和NF-κB通路，清除ROS及激活PI3K/Akt/HO-1通路減弱LPS誘導(dǎo)的內(nèi)皮細(xì)胞活化。 在失血性休克救治中，選擇合適的復(fù)蘇液對患者的救治至關(guān)重要。本研究結(jié)果提示，，琥珀酰明膠能夠提高血漿黏度，改善血流變特性，可以應(yīng)用于失血性休克早期血漿黏度嚴(yán)重下降的患者救治。HES130/0.4抑制失血性休克復(fù)蘇后組織氧化應(yīng)激和炎癥反應(yīng)水平，可以應(yīng)用于具有全身炎癥反應(yīng)發(fā)生傾向的失血性休克患者治療。另外，CNP能夠通過多個通路發(fā)揮抗炎作用，提示CNP有望成為一種新型抗休克藥物，當(dāng)然，這些還需要臨床試驗(yàn)和動物實(shí)驗(yàn)的驗(yàn)證。本研究為失血性休克救治中復(fù)蘇液的選擇提供重要的實(shí)驗(yàn)依據(jù)，并且為新型抗休克藥物的研制提供線索。
[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

【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R459.7

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