【作者】 黄新莉；

【导师】 凌亦凌；

【作者基本信息】 河北医科大学 ， 病理学与病理生理学， 2004， 博士

【摘要】 革兰氏阴性细菌内毒素的主要活性成分脂多糖(lipopolysaccharide,LPS)是导致内毒素休克(endotoxic shock,ES)的主要因素，ES是临床常见的危重病症，死亡率极高，肺脏是ES时最易受损的靶器官之一。肺动脉高压是ES发病早期的特征性病理变化，肺动脉压增高的程度及其维持的时间是休克并发急性肺损伤(acute lung injury,ALI)的重要因素。ES时，肺动脉平滑肌细胞(pulmonary artery smooth muscle cell,PASMC)在维持肺动脉的反应性中发挥重要作用。研究显示促炎症细胞因子、氧自由基和一氧化氮(nitric oxide,NO)等多种致炎物质在ES时肺损伤的发生中起主要作用，针对氧化性损伤，组织可产生多种抗氧化物质，如血红素氧合酶-1(heme oxygenase,HO-1)和胆囊收缩素(cholecystokinin,CCK)等。HO-1是一种新发现的应激蛋白，可被许多能引起氧化损伤的因素诱生。我室周君琳等的研究发现，HO-1及其代谢产物一氧化碳(carbon monoxide,CO)在肢体缺血／再灌注肺损伤中发挥保护作用。CCK是一种脑肠肽，参与了多种生理及病理过程。八肽胆囊收缩素(cholecystokinin octapeptide,CCK-8)为内源性CCK的重要功能片段。CCK的作用主要由两种受体介导，即CCK-A受体(CCK-A receptor,CCK-AR)和CCK-B受体(CCK-B receptor,CCK-BR)。以往对CCK的研究多偏重于其在消化系统、神经系统及内分泌系统中的调节作用。CCK-8在肺脏也有分布，然而CCK-8在肺脏特别是在ES时肺脏病理变化中的作用报道较少。我室系列研究发现CCK-8具有显著的抗ES作用，可部分逆转肺动脉压增高并可改善LPS所致的肺损伤。以往研究结果提示HO-1及其产物CO与CCK-8改善ALI之间可能存在着内在联系。 丝裂原激活蛋白激酶(mitogen-activated protein kinase,MAPK)是多种细胞外刺激激活各种信号途径的交汇点，具有调节细胞基本生命活动的功能。迄今已证明，在真核细胞中该家族有4个成员，即：①细胞 中文摘要外信号调节激酶(extraeellular一signal:egulated kinase，E双)，②e一Jun氨基末端激酶(e一Jun NHZ一terminal kinase，刃冈K)，③p38MApK，④E既5/B MK，(big mitogen一aetivated protein kinase，BMK:)。引闪K又被称为应激激活蛋白激酶(stress aetivated protein kinase，s妙K)，它可被多种应激原、促炎细胞因子和炎症介质等激活，激活的JNK在氧化应激反应中发挥重要作用。众所周知，LPS可激活MAPK信号转导通路，以往研究证实在胰腺腺泡细胞CCK也可激活MAPK通路。转录因子是MAPK的重要目标，活化蛋冬l(aetivator protein一1，Ap一l)是第一个被发现的转录因子，在机体抵抗炎症反应的过程中发挥重要作用。大量的实验结果表明，HO一l的表达可能受AP一1的调节。AP一1不是一个单一的蛋白质，它是由含有亮氨酸拉链区的多种蛋白质构成的二聚体，在其组成成员中，c一Jun的作用是最强的，只有c一Jun持续表达并同时被磷酸化才足以诱导HO一1表达。已证实JNK是c一Jun磷酸化调节的一个重要因素，是MAPK家族中唯一能够有效磷酸化c一Jun的Ser一63和Ser一73位点的蛋白激酶‘由此我们推测JNKJc一Jun通路参与了LPS诱导的HO一1基因表达。 CCK一8对LPS诱导的大鼠肺组织中HO一1表达有何作用，它是否通过调控HO一l/CO来发挥细胞保护作用，以及刃阿K/c一Jun通路是否参与CCK一8对LPS诱导的HO一l表达的调控均未见报道。本研究在我室以往系列研究的基础上，从整体、细胞及分子水平，观察CCK一8对LPS所致的ALI及PASMC损伤的改善作用，CCK受体(CCK reeeptor，CCK-R)在PASMC中的分布及表达变化，以及CCK一8对LPS诱导的HO-1/CO的影响及JN下了c一Jun通路在此过程中的作用，以探讨内源性CO在CCK一8抗LPS所致的ALI中的作用，为临床防治ES时的ALI提供新的对策。1内源性CO在CCK一8减轻内毒素所致的急性肺损伤中的作用 本实验通过舌静脉注入LPS复制AU模型，观察内源性CO在CCK一8抗LPS所致的ALI中的作用。将84只雄性SD大鼠随机分为7组(每组12只):①对照组:注入等量生理盐水(0 .5 ml瓜g);②LPS组:注入LpS(5 mg/kg，1 0 mg/ml);③LpS+Znpp(HO一l特异性抑制剂)组:注入LPs前一0 min注入znPP(1 0 mg服g，xo mg/ml):④ 中文摘要LPS+Hm(Hemin，CO供体)组:注入LpS前1 0 min注入Hm(1 0 mg/Kg，10m留ml);⑤CCK一8+LpS组:注入LpS前10 min注入CCK-8 (40”眺g，0.05 mg/ml);⑥CCK一8+Lps+znPP组:注入LPs前20min先注入ZnPP，其后1 0 min注入CCK一8;⑦CCK一8组:注入CCK-8。各组给药后Zh，6h，12h进行观察:每组6只动物用于支气管肺泡灌洗(bronchoalveolar lavage，BAL)，检测支气管肺泡灌洗液 (bronchoalveolar lavage fluid，BALF)中中性粒细胞 (polymo甲honuelear neutrophil，pMN)数目;分离各组另外6只未进行BAL动物的右侧颈静脉插管至右心房，分离左颈动脉插管至主动脉根部，采集血液分别代表入肺血(in一flowing pulmona汀blood，IPB)和出肺血(out一going pulmona仃blood，OpB)，检测血液中碳氧血红蛋白 (earbo材hemoglobin，COHb)水平代表CO更多还原

【Abstract】 Lipopolysaccharide (LPS) . the main component of Gram-negative bacterial endotoxin, is the main factor to induce endotoxin shock (ES). ES is a common and severe pathological process with high mortality in clinical practice. Lung is one of the target organs primarily impaired in the early stage of ES. Pulmonary artery hypertension (PAH) is the typical pathological change in the early phase of ES. It was reported that the degree and duration of PAH were important factors of ES accompanied by acute lung injury (ALI). Pulmonary artery smooth muscle cell (PASMC) played an important role in maintaining the tone of pulmonary artery during ES. Previous studies demonstrated that many inflammatory substances such as inflammatory cytokines, oxygen free radicals (OFR) and nitric oxide (NO) and so on played major roles in the process of ALI during ES. There was a rapid increase in anti-oxidative substances such as heme oxygenase-1 (HO-1) and cholecystokinin (CCK) that provided protection against oxidative injury during ES. HO-1 is a novel stress protein. It can be induced by many factors that could induce oxidative injury. It was demonstrated by the studies of Zhou JL et al in our laboratory that HO-1 and its metabolic product-carbon monoxide (CO) had protective effects during ALI induced by ischemia/refusion (I/R) of limb. It has been well documented that CCK, a kind of brain-gut peptide, is involved in many physiological and pathophysiological processes. Sulfated cholecystokinin-octapeptide (CCK-8) is the minimum sequence for biological activity. It was well known that the actions of CCK are mainly mediated by two distinct receptors, CCK-A receptor (CCK-AR) and CCK-B receptor (CCK-BR). Particular emphasis had been laid on its regulatory actions in nervous system, digestive system, and endocrine system in the previous studies of CCK. CCK is also demonstrated to be located in thelung. However, reports about the effects of CCK-8 on the lung especially on the pathophysical changes of the lung during ES were seldom seen. The serial studies of our laboratory demonstrated that CCK-8 had protective actions against ES, and could partly reverse the increase of pulmonary artery pressure as well as attenuate the lung injury induced by LPS. The results of previous studies showed that there maybe exist internal relationship among the above-mentioned protective actions of CCK-8, HO-1 and its product-carbon monoxide (CO).The mitogen-activated protein kinase (MAPK) family is the cross point of discrete signaling cascades for diverse extracellular stimuli, and function to regulate fundamental cellular processes. Four distinct subfamilies have been described in enkaryocyte: extracellular-signal regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK), p38MAPK and ERK5/BMK, (big mitogen-activated protein kinase). JNK, also called as stress-activated protein kinase (SAPK), which can be activated by various stressors, proinflammatory cytokines and inflammatory mediators, played an important role in oxidative stress response when it was activated. It was well known that LPS could activate MAPK signal pathway. CCK-8 has been demonstrated to have the similar role in pancreas acinus cell. Transcription factors are the important targets of MAPK. Activator protein-1(AP-1), the first identified transcription factor, played an important role in anti-inflammation response. Many experiments showed that AP-1 was very important in HO-1 regulation. AP-1 is not a single protein, but a dimmer consisted of many kinds protein with leucine zippers domain. Among its components, c-Jun is the one with the strongest action. Only when c-Jun was sustained expressed and phosphorylated meanwhile, could the expression of HO-1 be induced. It was verified that JNK was an important factor to regulate the phosphorylation of c-Jun as well as the only protein kinase to effectively phosphorylate Ser-63 and Ser-73 of c-Jun in MAPK family. From the above, we supposed that JNK/c-Jun signal pathway was invovled in the expression of HO-1 induced by LPS.12However, it hadn’t bee更多还原