【作者】 苗健；

【导师】 陈海龙；

【作者基本信息】 大连医科大学 ， 中西医结合临床， 2009， 博士

【摘要】 背景及目的:脓毒症多继发于严重感染、创伤、休克和大手术后,是由于局部或全身感染等因素所导致的全身炎症反应综合症( systemic inflammatory response syndrome,SIRS)。发展到晚期的脓毒性休克和多器官功能障碍综合征(multiple organs dysfunction syndrom, MODS)是该症的主要死亡原因,也是临床上危重病中最常见的死亡原因。脓毒症病情进展迅速,是临床医学中一种难治的综合征。目前关于脓毒症的研究多集中于对某些脏器以及对不同时期各种炎性因子的研究。脓毒症在创伤和感染等应激状态下,肠道的屏障功能受损,肠道内大量的细菌和内毒素经门静脉及淋巴系统侵入血液循环,造成肠源性感染和毒性网络,引起全身多器官功能衰竭。急性肺损伤(acute lung injury,ALI)发生早、病情严重,常是病人死亡的直接原因。ALI是以通透性肺水肿为特征的一种临床综合征,其严重阶段即是急性呼吸窘迫综合征(ARDS);ARDS是多种原因引起的肺部失控性炎症反应的结果,而革兰氏阴性杆菌内毒素(LPS)介导炎症细胞产生释放大量细胞因子和炎性介质在ARDS的发病过程中起关键性作用,但ARDS发病机制尤其是肺水肿的形成机制仍未阐明清楚。肿瘤坏死因子-α(TNF-α)、白介素-1(IL-1)、白介素-6(IL-6)、细胞间粘附分子-1(intercellular adhesion molecule,ICAM-1)、水通道蛋白(AQPs)、丝裂原活化蛋白激酶(MAPK)等诸因素均可能与水肿的发生有关,但是其内在相互关系及调控并不完全清楚。肿瘤坏死因子(tumor necrosis factor,TNF),包括TNF-α和TNF-β。TNF-α又称为恶液质因子,是介导内毒素休克、SIRS、急性肺损伤(ALI)和多器官功能障碍综合征(MODS)的重要起始因子,可诱导其它多种因子的产生,这些促炎症细胞因子之间相互作用可形成许多正反馈环,导致所谓炎症“级联效应”的发生。细胞间粘附分子-1(intercellular adhesion molecule , ICAM-1)又名CD54。研究发现其参与许多炎症性疾病过程。其可能介导肺微血管内皮细胞-多形核粒细胞(PMVEC-PMN)的粘附,导致PMN肺微血管内的扣押,微血栓形成和血管内皮损伤,引起血管通透性增加,形成肺水肿,进而导致ALI。水通道蛋白(AQPs),其主要功能是介导自由水的跨细胞膜转运。肺组织中主要有4种水通道蛋白的表达,其中AQP-l主要定位于肺微血管内皮,对于维持血管与间质之间的水运动平衡意义重大。MAPK是蛋白激酶家族成员,其中p38通路与炎症反应的调控密切相关,在许多细胞因子如信号转导中起重要作用。研究显示p38可促进ICAM-1的表达。p38可由TNF-α、IL-1等诱导刺激而激活,而TNF-α、IL-1、IL-6以及IL-8又是p38依赖性的。脓毒症过程所引起的急性肺损伤(ALI)与上述基因表达调控的相关性研究较少,并且未见在同一感染模型下对上述基因表达调控的平行研究,亦未见复方清下汤的相关治疗性研究的报道。本课题拟以盲肠结扎穿孔导致大肠杆菌腹膜炎,进而诱发脓毒症肺损伤为模型,检测炎性反应时上述基因的调控变化,探讨肺水肿的形成机制,调控机制及复方清下汤、p38抑制剂对肺损伤的作用,以期为脓毒症以及多器官功能衰竭综合症(MODS)的防治提出可能的新途径。方法实验一:将健康SD大鼠随机分为4组,每组10只:假手术组(SHAM组), SHAM组只翻动盲肠,不做其他处理;脓毒症肺损伤组(模型组),以盲肠结扎穿孔诱发ALI模型;盲肠结扎穿孔+复方清下汤组(造模后立即灌胃给药,造模后8小时再次灌胃1次,剂量:10 ml/kg);盲肠结扎穿孔+头孢派酮舒巴坦组(抗生素舒普深)(造模后立即静脉注射1次,造模后8小时再次静脉注射1次,剂量:0.2g/kg)造模24h后收集标本。分别观察大鼠的一般状态,肺组织匀浆MPO的测定,留取下腔静脉血清进行TNF-ɑ、IL-1、IL-6的测定。镜下观察肺组织病理形态学改变,测量肺湿/干比值的变化。实验二:将健康SD大鼠随即分为4组,每组10只:(1)假手术组(SHAM组),SHAM组只翻动盲肠,不做其他处理;(2)脓毒症肺损伤组(模型组),以盲肠结扎穿孔诱发ALI模型;(3)盲肠结扎穿孔+复方清下汤组(造模后立即灌胃给药,造模后8小时再次灌胃1次,剂量:10 ml/kg);(4)盲肠结扎穿孔+头孢派酮舒巴坦(抗生素舒普深)(造模后立即静脉注射1次,造模后8小时再次静脉注射1次,剂量:0.2 g/kg)造模24h后收集标本。应用免疫组织化学和western blot方法检测肺组织中TNF-α、IL-1、IL-6、AQP-l、ICAM-1以及p38的表达,RT-PCR法检测肺组织上述蛋白mRNA表达。实验三:将健康SD大鼠随机分为4组,每组10只:(1)假手术组(SHAM组), SHAM组只翻动盲肠,不做其他处理; (2)脓毒症肺损伤组(模型组),以盲肠结扎穿孔诱发ALI模型; (3)盲肠结扎穿孔+复方清下汤组(造模后立即灌胃给药,造模后8小时再次灌胃1次,剂量:10 ml/kg);(4)p38抑制剂处理组,在动物模型组制备前30分钟灌胃给予SB203580( 12.5 mg/kg)。应用免疫组织化学和western blot法方法检测肺组织中TNF-α、IL-1、IL-6、ICAM-1的表达。采用RT-PCR法检测肺组织上述蛋白mRNA表达。结果1.实验一:与SHAM组比较,模型组MPO、TNF-α、IL-1、IL-6水平明显升高(P<0.01),肺间质和肺泡内水肿,伴大量红细胞渗出(出血)和纤维素沉积,肺泡间隔毛细血管内皮细胞高度肿胀。肺湿/干比值明显增加(P<0.01),抗生素及中药处理组与模型组比较, MPO、TNF-α、白介素-1(IL-1)、白介素-6(IL-6)水平明显降低(P<0.01),肺湿/干比值明显降低(P<0.01),肺组织镜下表现:抗生素处理组、中药处理组较模型组,肺泡间隔变窄,毛细血管内皮细胞肿胀减轻,出血明显减少,纤维素渗出相对减少。2.实验二:与SHAM组比较,模型组应用免疫组织化学及western blot法检测TNF-α、白介素-1(IL-1)、白介素-6(IL-6)、ICAM-1以及p38的表达均显著升高(P<0.01),而AQP-l则表达明显降低(P<0.01)、RT-PCR法检测mRNA转录水平与蛋白表达结果基本一致。抗生素及中药处理组与模型组比较,上述细胞因子除AQP-l外的表达明显降低(P<0.05),而AQPl表达上调(P<0.01),低于假手术组,抗生素及中药处理组两组检测数据相近。上述结果提示中药组可能通过抑制某种核心细胞因子的表达,继而抑制其他细胞因子的过度表达来减轻脓毒症肺损伤。3.实验三:与SHAM组比较,模型组应用免疫组织化学方法、RT-PCR法及western blot法检测TNF-α、L-1、IL-6、ICAM-1的水平表达明显升高。而中药处理组及p38抑制剂组与模型组比较,上述细胞因子的表达则明显降低(P<0.05),此两组检测数据相近。结论脓毒症在创伤和感染等应激状态下,肠道的屏障功能受损,肠道内大量的细菌和内毒素经门静脉及淋巴系统侵入血液循环,造成肠源性感染和毒性网络,引起全身多器官功能衰竭,急性肺损伤(acute lung injury,ALI)发生早、病情严重,常是病人死亡的直接原因。ALI是以通透性肺水肿为特征的一种临床综合征,其严重阶段即是急性呼吸窘迫综合征(ARDS); ARDS是多种原因引起的肺部失控性炎症反应的结果,革兰氏阴性杆菌内毒素(LPS)介导炎症细胞释放大量细胞因子和炎性介质在ARDS的发病过程中起关键性作用。TNF-α是机体受到有害刺激后最初分泌的细胞因子,它可诱导其它多种因子的产生,这些促炎症细胞因子之间相互作用可形成许多正反馈环,导致所谓炎症“级联效应”的发生,但其内在相互关系及调控可能与某种核心细胞因子有关,它可联系上下游的靶基因。1.实验一证实脓毒症大鼠肺损伤时几种主要的炎性细胞因子过度表达的情况,并从病理学角度充分证实。证实了炎性介质的过度表达是造成脓毒症肺损伤的重要原因。经复方清下汤处理的动物模型肺损伤得以减轻。2.脓毒症诱发ALI的内在机制相当复杂,本实验通过脓毒症大鼠肺损伤模型的建立,应用免疫组织化学、RT-PCR以及western blot等手段,通过测定各组细胞因子灰度值,证实了脓毒血症大鼠肺损伤时几种主要的炎性细胞因子过度表达和某些蛋白表达的情况,并从分子生物学水平证实炎性介质TNF-α、IL-1、IL-6、ICAM-1的过度表达和p38通路的活化是造成脓毒症肺损伤的重要原因,而AQP-l的分泌表达则具有相反效果。经复方清下汤处理的动物模型肺损伤得以减轻,提示我们是否可以通过抑制某种介导多细胞因子过度表达的某种信号通路,为治疗脓毒血症大鼠肺损伤提供一个可能的新的手段。3.在实验一、二基础上,实验三通过脓毒症大鼠肺损伤模型的建立,应用免疫组织化学、RT-PCR以及western blot等手段,通过测定各组细胞因子灰度值,证实复方清下汤和p38抑制剂处理的动物模型肺损伤得以减轻,炎性介质TNF-α、IL-1、IL-6、ICAM-1的表达下调。提示脓毒症大鼠肺损伤时处于上下游中心环节的p38通路过度表达可能是导致某些炎性因子过度表达的主要原因之一。中药复方清下汤可能在某种水平上通过对p38的抑制,来抑制下游靶基因的表达从而达到减轻脓毒症肺损伤的效果更多还原

【Abstract】 Background and objective: The systemic inflammation response syndrome (SIRS) is defined as the host response to infection and other forms of tissue injury. When SIRS is attributed to an identifiable infectious process, it is termed sepsis. A wide range of stress such as trauma and infection may cause injury of bowel barrier. Bacteria and endotoxin thus enter blood circulation through portal vein and lymph system resulting in enterogenous infection and toxic network. The majority of patients with SIRS or sepsis who fail to survive may present with septic shock and multiple organ dysfunction syndrome (MODS), representing the leading cause of death in clinical emergency. The most common manifestation of MODS is acute lung injury (ALI), which may progress to acute respiratory distress syndrome (ARDS) in the subset of patients at the severe end of the spectrum. ARDS is characterized by“severe hypoxemia, diffuse bilateral pulmonary inflitrates, and decreased lung compliance.Polymorphonuclear leukocytes (PMNs) have been recognized as important contributors to the pathogenesis of ARDS. In respond to SIRS, leukocytes become activated in pulmonary microcirculation and migrate to pulmonary interstitium and finally in the alveolar space. The infiltration of PMNs results in diffuse alveolar damage and capillary leak and edema formation. These progress are mediated by early-response cytokines, cell surface adhension molecules, and chemotactic molecules, chemokines. It is now accepted that inlammatory mediators plays a key role in the pathogenesis of ARDS. These mediators include tumor necrosis factor (TNF)-α, interleukins (e.g. IL-1,6), adehension molecules (e.g. intercellular adhension molecule-1/ICAM-1), mitogen-activated protein kinases (MAPKs), aquaporin (AQPs), et al.TNF include TNF-αand TNF-β. TNF-αis also called“cachectin”, which represents as an improtant initial factor mediating septic shock, SIRS, ALI and MODS. It can induce formation of downstream proinflammatory cytokines that produce many cascade feedback..ICAM-1, also called CD54, involves in inflmmatory process via mediating adhesion of PMVEC-PMN which results in accumulation of PMN in the pulmonary microcirculation, formation of microthrombus and injury of vascular endothelium. These process lead to increase in vessel permeability and pulmonary edema and ALI develops.AQPs involve in transmembrane transport of free water. There are four types of AQPs in lung tissue, among which AQP-1 is located in the microvessel endothelium and plays an important role in maintaining water balance between vessel and interstitium.MAPK is one member of protein kinase family and is composed of three major groups: the extracellular regulated kinases (ERKs), the C-Jun N-terminal Kinases (JNKs) and the p38 MAPKs. The p38 MAPK kinase pathway shows close relationship with inflammatory reaction. Research reveal that p38 can promote expression of ICAM-1. It is activated in response to cytokines such as TNF-α, IL-1 and so on. Simultaneously, TNF-αand IL-1, 6, 8 are p38-dependent.Although more attention are paid to the pathophysiology of these mediators, the interaction among them and their gene modulation in the development of ALI due to sepsis still need to be elucidated. This research uses ALI animal model due to sepsis, which was established by intestinal perforation-induced peritonitis after cecum ligation. The pathogenesis and gene modulation mechanisms of lung edema were investigated via measuring the upmentioned gene expression in the inflammatory process. Furthermore, there is no research about the role of traditional Chinese medicine FU FANG QING XIA TANG (FFQX) in the pathophysiology of ALI. The mechenisms of FFQX as well as inhibitor of p38 were explored to reveal a new method for the preventation and treatment of sepsis and MODS. MethodsExperiment 1. SD rats were divided into four groups: SHAM group (n=10) : the rats didn’t undergo any operation; ALI group (n=10): the cecum were ligated and produec intestinal perforation and induce ALI; FFQX group (n=10): FFQX (10mg/kg) were administrated by lavage immediately and 8 hours after formation of ALI respectively; antibiotics group (ALI+ Cefoperazone-Sulbactam, pfizer, n=10): Cefoperazone-Sulbactam (0.2g/Kg) were administrated by intravenous injection immediately and 8 hours after formation of ALI respectively. Specimen were collected 24 hours after ALI. Evaluate common condition of rats. Myeloperoxidase (MPO) were detected in homogenate from lung tissue. Serum TNF-α, IL-1 and IL-6 were measured from blood sample of inferior vena cava. Observe pathologic changes in lung tissue. Calculate the ratio of lung wet weight to dry weight (W/D).Experiment 2: Expression of TNF-α, IL-1 and IL-6, AQP-1, ICAM-1 and p38 in lung tissue were measured via immunohistochemistry and Western blotting, respectively. RT-PCR were performed to measure mRNA of these mediators.Experiment 3: p38I group (p38 inhibitor administration group, n=10): p38 inhibitor SB203580 (12.5 mg/Kg) were administrated by lavage 30minutes before the preparation of ALI animal model.Results1. In Experiment 1, the level of serum TNF-α, IL-1 and IL-6 and MPO in homogenate were significantly increased in ALI group compared with SHAM group (p<0.01). Pulmonary interstitial and intra-alveolar edema with exudate of RBCs and deposit of fibrin were seen. Endothelial cell of alveolar septum capillaries were obviously swollen. The ratio of W/D in ALI group is higher than that in SHAM group (p<0.01). Compared with ALI group, the level of serum TNF-α, IL-1 and IL-6 and MPO in homogenate were significantly decreased and the ratio of W/D is lower in FFQX group and antibiotics group (p<0.01 each). Narrowed alveolar septum, slight endothelial cell swelling and decreased bleeding and exudate can be seen in both FFQX group and antibiotics group.2. In Experiment 2, immunohistochemistry and Western blotting revealed the increased expression of TNF-α, IL-1 and IL-6, AQP-1, ICAM-1 and p38 in ALI group compared with SHAM group (p<0.01). The expression of TNF-α, IL-1 and IL-6, AQP-1, ICAM-1 and p38 in mRNA transcription level showed coincident results with protein level. The protein and mRNA expression of these upmentioned cytokines droped significantly in FFQX group and antibiotics group compared with ALI group (p<0.01 each) although there were no obvious difference between FFQX group and antibiotics group. The results suggest FFQX may inhibit expression of certain key cytokines to relieve lung injury due to sepsis.3. In Experiment 3, the protein and mRNA expression of these upmentioned cytokines droped significantly in FFQX group and p38I group compared with ALI group (p<0.01 each) and there were no significant difference between FFQX group and p38I group. The results suggest FFQX may inhibit certain procedure of p38 pathway to relieve lung injury due to sepsis.ConclusionsOur research revealed that overexpression of several major inflammatory factors such as TNF-α, IL-1 and IL-6 and activation of p38 MAPK kinase pathway in sepsis-induced ALI rat model may be the result of sepsis-induced ALI. However, AQP-1 had different effects from TNF-α, IL-1 and IL-6 in pathogenesis of ALI.FFQX can decrease the secretion of the several cytokines,also can alleviate the extent of sepsis-induced acute lung injury . And the relief of ALI after administration of FFQX and inhibitor of p38 suggested that specific inhibitors of p38 pathway may alleviate sepsis-induced lung injury. FFQX may inhibit p38 pathway which mediated overexpression of certain inflammatory factors. The use of FFQX can provide a new method for treating this disease.更多还原