【作者】 张新颖；

【导师】 王玉林；

【作者基本信息】 山东大学 ， 儿科学， 2011， 博士

【摘要】 研究背景和目的急性肺损伤(Acute lung injury, ALI)是指机体遭受心源性以外的各种肺内外因素(严重感染、创伤、休克、酸中毒及有害气体吸入等)打击后,出现弥漫性肺泡-毛细血管膜损伤所导致肺水肿和微肺不张等病理特征的临床综合征。ALI的临床表现为进行性呼吸窘迫和顽固性低氧血症,晚期常并发急性呼吸窘迫综合征(Acute respiratory distress syndrome, ARDS)及多脏器功能障碍综合征(Multiple organ dysfunction syndrome, MODS),发病急,病情凶险,死亡率高居不下,是现代危重医学中的难题。ALI的发病机理错综复杂,迄今尚未完全阐明。目前人们普遍认为ALI是全身炎症反应综合征(Systemic inflammatory response syndrom, SIRS)在肺部的表现,其本质是一种肺内过度性、失控性炎性反应。而ALI与一般炎症的区别在于它在某些环节上存在抗炎性反应和炎性反应的失衡,损伤与抗损伤的平衡机制遭到破坏,细胞因子在此过程中起到至关重要的作用。因此,细胞因子网络的平衡及其上游的调控研究,成为All发病机制的研究重点,对于揭示ALI的发生发展以及以此为靶因素的治疗措施的研究具有重要意义。细胞因子按其在炎性反应中的不同作用可分为促炎性细胞因子和抗炎性细胞因子。促炎性细胞因子中肿瘤坏死因子(Tumor necrosis factor, TNF)-α是释放最早、最重要的内源性介质,在炎症过程中具有启动和触发作用。白细胞介素(Interleukin, IL)-10则被认为是体内最重要的抗炎性细胞因子,可作用于炎症级联反应的多个环节,在全身性炎性反应过程中起保护作用。丝裂原活化蛋白激酶(Mitogen-aetivated protein kinase, MAPK)是真核细胞内的一类丝氨酸／苏氨酸蛋白激酶,通过磷酸化其他细胞蛋白,从胞浆移位至细胞核而调节转录因子的活性,启动相关细胞因子和炎性介质的基因表达。其中p38 MAPK信号通路与炎症调控反应机制密切相关,被认为是属于“应激诱导”的MAPK, p38MAPK在ALI肺组织早期炎症反应的内在调节机制中可能具有重要作用。乌司他丁(ulinastatin,UST)是近年来研制成功并广泛应用于临床的一种尿胰蛋白酶抑制剂(urinary trypsin inhibitor, UTI)。近年来有资料显示UTI可抑制炎症介质释放,预防细胞因子级联反应,抑制白细胞过度激活,阻断细胞因子与白细胞之间的恶性循环,在脓毒血症、DIC、多器官功能衰竭等全身性炎症疾病中具有肺保护作用,从而在早期阻断SIRS向MODS的发展中扮演了重要角色,但是其详细的作用机制还不十分明确,已有的研究基本停留在UTI对促炎性细胞因子的生成及产生作用的抑制效应上,对于细胞信号转导机制的研究甚少。UTI的肺保护作用是否和p38MAPK信号途径相关,国内外尚无相关报道,其机制尚有待研究。内毒素即脂多糖(Lipopolysaccharide, LPS)是G菌细胞壁上的主要致病成分。感染时LPS释放入血,可激活具有信号转导功能的Toll样受体4(Toll-like receptor, TLR4),并经过一系列的级联信号传递,激活细胞内p38MAPK信号转导通路,导致TNF-α、IL-1等炎症介质和各种黏附分子等基因的表达。本研究建立LPS诱导ALI的大鼠模型,静脉注射UTI进行干预,一方面通过观察肺组织病理、湿／干重比值及血清中细胞因子的变化揭示UTI对大鼠肺组织的保护作用,另一方面采用Real-time RT-PCR技术检测肺组织中TNF-αmRNA的水平,并分别应用免疫组织化学染色和Western blot方法观察肺组织中磷酸化的p38 MAPK的表达,揭示UTI抑制促炎性细胞因子TNF-α产生的可能的分子生物学机制,为UTI的临床应用提供一定的理论及实验依据。研究方法1.动物模型的建立及分组：选用健康雄性6-8周龄Wistar大鼠66只(清洁级)随机分成：对照组、模型组和乌司他丁干预组,对照组按1、3、6h分为3个亚组,模型组和干预组按0.5、1、3、6h分为4个亚组,共11组,每组6只。模型组大鼠给予LPS(5mg/kg)尾静脉注射,干预组大鼠在尾静脉注射LPS(5mg/kg)前30min给予UTI(50000单位/kg)尾静脉注射。对照组大鼠给予尾静脉注射等量的生理盐水(NS)。整个实验期间所有大鼠均喂标准饲料,实验前禁食12h,自由饮水。2.标本采集：各组动物在相应的不同时点分别给予3%戊巴比妥钠腹腔麻醉打开并暴露胸腔,肉眼观察肺组织大体病理改变。经左心室穿刺抽血,离心后留取血清置于-80℃冰箱保存。取血结束后立即留取肺部标本,取左肺计算肺组织湿／干重比值(W/D)；取右肺上叶以10%中性甲醛固定以备HE染色和免疫组化染色。取右肺中、下叶放入液氮中冻存,-80℃保存,以备Western blot及Real Time RT-PCR检查。3.常规HE染色并计算肺组织病理损伤评分。4. ELISA法检测血清中TNF-α和IL-10的浓度。5. Real Time RT-PCR法检测肺组织中TNF-αmRNA的相对表达量。6.免疫组化法检测磷酸化的p38 MAPK蛋白在肺组织中的表达。7. Western blot法检测肺组织中磷酸化的p38 MAPK的表达。8.统计学分析：采用SPSS 13.0统计软件包分析。计量资料用means±SD表示,各组之间的差异采用单因素方差分析(one-way ANOVA),其后两两之间比较采用LSD检验,p<0.05视为差异有统计学意义。研究结果1.一般情况：对照组大鼠呼吸平稳,活动无明显变化,全部存活,抓取时迅速逃避,解剖后肺外观色泽粉红,无明显异常改变；模型组大鼠各时点均出现竖毛、呼吸急促(>100次／分),口唇发绀,精神萎靡,少动少食,抓取时无力逃避,3h组和6h组各有1只大鼠死亡,濒死前出现抽搐,大小便失禁,深大呼吸,四肢僵硬,解剖后肉眼见肺组织体积增大,脏层胸膜张力较高,表面色泽暗红,可见包膜下点状、片状出血,切面疏松,有黄色或淡红色液体溢出,尤以6h组最为明显；干预组各时点表现相对较轻,所有动物均存活,抓取时能够逃避,解剖后肺有类似上述改变,但范围小,程度轻,体积增大不明显,表面呈红色,可见少量出血点。2.肺组织湿／干重比值(W/D)的变化：模型组大鼠肺组织的W/D在3h和6h明显高于对照组(p<0.01),干预组大鼠的W/D在上述两个时点均较模型组降低(p<0.05)。对照组及1h的模型组和干预组大鼠W/D无明显变化。3.肺病理学检查3.1 HE染色：光镜下,对照组肺组织结构完整,肺泡腔清晰,无渗出物,肺泡壁无增厚,肺泡间质无炎性细胞浸润,肺泡隔均匀一致。模型组肺组织的结构破坏明显,肺泡腔内可见出血、炎症细胞及液体渗出；血管周围间隙增宽,大量淡粉色网状结构;肺泡间质内有大量炎症细胞浸润,以中性粒细胞、单核巨噬细胞、浆细胞为主,可见少量淋巴细胞,部分肺间隔变薄、断裂,有肺气肿形成。与模型组相比,干预组大鼠肺组织的损伤程度减轻,表现为肺间隔轻度增宽、无明显出血、少量炎性细胞浸润。3.2病理损伤评分结果：模型组大鼠肺组织的病理损伤评分(5.90±0.48)明显高于对照组(1.60±0.28),应用UTI干预后,病理损伤评分降低(3.62±0.46),差异有统计学意义(p均<0.01)。4.血清中细胞因子浓度的变化4.1血清TNF-α浓度的变化：模型组大鼠血清中TNF-α的浓度在各个时点均明显高于对照组,3h为其表达高峰,6h浓度开始下降,UTI干预后大鼠血清中TNF-α的浓度在各个时点均低于模型组(p均<0.01)。4.2血清IL-10浓度的变化：对照组大鼠血清中IL-10的浓度低于检测水平,模型组大鼠注射LPS后,IL-10的浓度逐渐升高,6h最高,各时点间均有明显差异,UTI干预后血清中IL-10的浓度在各个时点均高于模型组(p均<0.01)。5.肺组织TNF-αmRNA的相对表达量：模型组肺组织中TNF-α的mRNA表达水平在各个时点均高于对照组,以1h时最明显,为对照组的128倍,UTI干预后,TNF-α的mRNA表达水平下降,最高时为对照组的58倍。与模型组相比,干预组TNF-α的mRNA表达水平在各个时点均降低,差异有统计学意义(0.5h、1hp<0.01,3h p<0.05)。6.肺组织磷酸化p38 MAPK蛋白的表达6.1免疫组化染色结果：对照组肺组织中p-p38MAPK呈阴性表达,模型组肺组织p-p38MAPK呈强阳性表达,表现为胞核内(或合并胞浆内)棕黄色颗粒,阳性细胞主要是肺间质内中性粒细胞、巨噬细胞和肺泡上皮细胞及血管内皮细胞,UTI干预组肺组织中p-p38 MAPK阳性细胞较模型组明显减少,呈低表达。6.2 Western blot检测结果：对照组中p-p38MAPK的表达很弱,注射LPS后,p-p38MAPK在模型组中的表达明显增多,并于1h达到高峰,随后开始下降,各时间点与对照组比较均有明显差异(p＜0.01)。UTI干预后p-p38MAPK在各个时点的表达均低于模型组(p＜0.01)。研究结论1.内毒素性ALI大鼠早期血清中促炎和抗炎性细胞因子的浓度均升高,相对于促炎性细胞因子,抗炎性细胞因子的浓度低,且时间滞后,使机体在ALI早期即存在促炎-抗炎反应的不平衡。2.静脉注射UTI干预内毒素性ALI,可减轻大鼠肺组织的病理损伤,降低肺水肿程度,具有肺保护作用。3.UTI通过抑制促炎性细胞因子和升高抗炎性细胞因子的浓度,改善机体促炎介质／抗炎介质的失衡状态发挥其肺保护作用。4. p38 MAPK信号通路参与了LPS诱导的ALI过程。5.UTI在转录水平抑制了促炎性细胞因子TNF-αmRNA的产生。6.UTI可能是通过抑制p38 MAPK信号通路减少TNF-α的mRNA的产生。创新及意义1.本研究阐述了UTI的肺保护作用,为ALI的临床治疗提供新的思路。2.本研究提示UTI抑制TNF-αmRNA的产生是通过p38 MAPK信号通路,为UTI的临床应用提供了理论和实验依据。更多还原

【Abstract】 Backgroud and objectivesAcute lung injury(ALI) refers to clinical manifestations of respiratory distress and refractory hypoxemia.sufferring from severe infection, the wounds, shock, acidosis and harmful gases inhalation factors.Its pathologic characteristics are pulmonary edema and micro atelectasis caused by diffusive alveolar-capillary membrane damage. Common complications of ALI are acute respiratory distress syndrome(ARDS) and multiple organ dysfunction syndrome(MODS).ALI has the high mortality rate and is a difficult problem of modern critical medical.The pathogenesis of ALI has not yet been fully clarified. It is now widespread thought that ALI is a performance in the lungs of systemic inflammatory response syndrome (SIRS), its essence is a kind of incontrollable and excessive inflammation reaction. The difference between ALI and general inflammation is there is imbalance of proinflammatory reaction and anti-inflammatory reaction in ALI.Cytokines play a crucial role in this process.Therefore, research on the balance of cytokine network and the regulation of upstream become focus.It has important significance to reveal the development of ALI as well as the treatment.Cytokines can be divided into proinflammatory and anti-inflammatory cytokines according to their role in inflammatory reaction.Tumor necrosis factor alpha(TNF-a) is the earliest release and most important endogenous medium among proinflammatoty cytokines.It plays a trigger role during inflammation.Interleukin 10 is considered the most important factor of anti-inflammatory cytokines.It acts on the multiple links of inflammation cascade in systemic inflammatory reaction process and plays a protective role.Mitogen activated protein kinase(MAPK) is a kind of serine/threonine protein kinase within the eukaryotic cells.It can phosphorylated other proteins to shift them from the cytoplasm to nuclear and then adjust the activation of transcription factors and gene expression of inflammatory cytokines. Among them,p38 signaling pathway is considered "stress induced MAPK".It is closely related with the mechanism of regulation of inflammatory reaction and may play an important role in lung tissue in early stage of ALI.Ulinastatin is a urine trypsin inhibitor(UTI) developed in recent years and widely used in clinical.Data shows that UTI can inhibit the release of inflammatory medium, prevent cytokines cascade and block the excessive activation of white cell.Therefore UTI plays an important role in the early blocking development of SIRS to MODS by inhibitting vicious circle between cytokines and white cells,but the mechanism has not been clearly showed.The existing research is mainly on the effect of UTI to inflammatory cytokines,little is about the signal transduction mechanism. We can not find relative report about whether the protective of UTI has the relationship with p38MAPK, and its mechanism is expected to study.Endotoxin namely lipopolysaccharide is the main pathogenic componengt on the wall of G negative bacteria.When released into the circulation, LPS can activate toll-like receptor (TLR)-4 which has the signal transduction function, and then activate p38 MAPK signalling pathways in the cells through a series of cascade signals.At last, LPS acts on the gene expression of inflammatory cytokines such as TNF-a and IL-1, and all kinds of adhesion molecules.In this study,we have established the rat model of LPS-induced ALI and intervented it by intravenous injection of UTI.On the one hand,we tried to reveal the protective effect on the lung tissue through observing the ation of lung tissue pathology, wet/dry weight ratio and the changes in serum cytokines; on the other hand,we examined the level of TNF-a mRNA in lung tissue by Real time RT-PCR detection the expression of phosphorylated p38 MAPK in the lung tissue by Western blot and immunohistochemical staining method to reveal the molecular biologic mechanism of action of UTI,we hoped to provide certain theoretical and experimental basis for the clinical application of UTI.Methods1 Establishment and grouping of animal model:6-8-week-aged male Wistar rats were randomly divided intdo:control group, model group, and the intervention group. Control group was divided into three subgroups by 1,3,6h,the model and intervention group were divided into four subgroups by 0.5,1,3, and 6h.There was a total of 11 groups, and each group was 6 rats.Rats in model group were given 5mg/kg LPS through tail intravenous;rats in intervention group were given 50,000 units/kg UTI 30min before 5mg/kg LPS through the tail intravenous; rats in control group were given the equal amount of saline solution(NS) through tail intravenous.The experimental rats were all fed standard feed during experiment,they were no food and free water 12h before experiment.2. Collection of specimen:all the animals in the different groups were given 3% pentobarbital sodium by intraperitoneal injection to anesthesia.When chest cavity was exposed and opened,we observed the pathological changes of lung tissue and got the blood sample by puncturing from left ventricular.After taking blood samples,we got the left lung tissue to calculate the wet/dry weight ratio (W/D),got the right upper lobe fixed in 10% formalin for HE and immunohistochemical staining,at last,we got the right middle and lower lobe freeze-stored in liquid nitrogen and preservated at 80 degrees below zero for Real Time RT-PCR and Western blot inspection.3. Pathology examination by conventional HE staining and to calculate the score of lung tissue pathologic damage.4. The levels of TNF alpha and IL-10 were detected quantitatively by ELISA method.5. The relative expression of TNF alpha mRNA in lung tissue were examined by real time RT-PCR method.6 The expression of phosphorylated p38 MAPK in lung tissue were detected by immunohistochemical staining method.7. The expression of phosphorylated p38 MAPK in lung tissue were detected by western blot method.8. Statistical analysis:all data were expressed as means±S.D and analyzed by statistical package SPSS 13.0 for windows.One-way analysis of variance (ANOVA) was used to determine statistically significant differences among different groups and followed pairwise comparison by LSD inspection.A level of p< 0.05 was condidered statistically significant.Results1. General health state:rats in control group all survived and breathed smoothly, could escape quickly when grabbed.The lungs were pink without of abnormal changes after anatomy. Rats in model group were shortness of breath(>100/min),lip cyanosis,listlessness,less food and dynamic,could not to escape when grabbed.Two rats in 6h group and 3h group died,they appeared dying twitching, incontinence,deep breathing and stiff before death.The lungs increased in size, visceral pleura was with high tension,surface were dark red,include subcapsular pointlike, flake bleeding,and the dissection was loose with yellow or pink liquid spilled. Performance of rats in intervention group was relatively light, all animals were alive, could escape when grabbed.Changes of lungs were similar with those of lungs of model rats after anatomy, but relatively light.2.Changes of lung tissue wet/dry weight ratio (W/D):the lung tissue W/D weight ratio of rats in 3h and 6h were significantly higher in model group than control group(p< 0.01), the lung tissue W/D weight ratio of rats in intervention group at the above timepoint were lower than the model group(p< 0.05).The lung tissue W/D weight ratio of rats in control group、1h model group and 1h intervention group has no change.3. Pulmonary pathologic examination3.1 HE staining:lung tissue structure of rats in control group was complete, the alveolar cavity was clear without exudate, the alveolar walls were not thickening and no inflammatory cells infiltrated in the alveolar interstitial.The lung tissue structure of rats in model group were damaged, the alveolar cavity were filled with bleeding, inflammatory cells and liquid.Large gap surrounding vessels widened to pale pink mesh structure. There were many inflammatory cells infiltratde in the alveolar space, mainly neutrophils, lymphocytes, monocyte macrophages, and so on.Part of lung interval breakaged to be emphysema.Compared with the model group, damage of lung tissue of rats in the intervention group were relative light.3.2 Results of pathologic damage score:the pathologic damage score of rats in model group (5.90±0.48)was obviously higher than that in the control group(1.60±0.28),and reduced after intervention UTI(3.62±0.46),the difference was statistically significant (p< 0.01).4. Serum concentrations of cytokines4.1 Changes of TNF alpha concentration in serum:the serum concentrations of TNF alpha of rats in model group were significantly higher in each timepoint than control group, peaked at 3h and began to decline at 6h.After UTI injection, the serum concentrations of TNF alpha of rats in intervention group were below in each timepoint than model group (p< 0.01).4.2 Changes of IL-10 concentration in serum:the serum concentrations of IL-10 of rats in control group were below the level, after LPS injection, concentrations of IL-10 increased, arrived the highest level at 6h.The serum concentrations of IL-10 of rats in interention group were lower than those in model group (p< 0.01).5.Expression of lung tissue TNF alpha mRNA:the relative expression of lung tissue TNF alpha mRNA of rats in model group were higher than those in the control group, while the most obvious, was 128 folds with control group at lh.After the injection of UTI,the relative expression of TNF alpha mRNA decreased,the highest level was 58 folds with control group.Compared with the model group, the relative expression level of TNF alpha mRNA of rats in intervention group reduced at each timepoint,the difference was statistically significant (0.5 h, 1h p< 0.01,3h p< 0.05).6. Expression of lung tissue phosphorylated p38 MAPK protein6.1 Results of immunohistochemical staining:p-p38 MAPK in lung tissue of rats in control group were negatively expression.While in model group,p-p38 MAPK in lung tissue expressed as nuclei (or merge the cytoplasm) yellowish-brown particles,positive cells mainly conclude neutrophils and macrophages in the pulmonary intersitital and alveolar epithelial cells and vascular endothelial cells.Expression of p-p38 in UTI intervention group was significantly reduced.6.2 Results of Western blot detection:the expression of p-p38 MAPK in control group was very weak, after injection of LPS, the expression of p-p38 MAPK increased obviously, peaked at 1h, then began to decline, there were significantly difference (p< 0.01) at each timepoint compared with controls.After UTI intervention, the expression of p-p38 MAPK were lower than model group at each timepoint(p< 0.01).Conclusions1. The concentrations of both proinflammatory cytokines and anti-inflammatory cytokines elevated in the early stage of lipopolysaccharide-induced ALI.Compared to the concentrations of proinflammatory cytokines, the concentrations of anti-inflammatory cytokines were lower and later, there was imbalance between inflammatory reaction and anti-inflammatory reaction.2. It could reduce the pathological damage and pulmonary edema of LPS-induced ALI by injection UTI intravenously.3. UTI inhibited proinflammatory cytokines and apronounced anti-inflammatory cytokines, it played its protective role through regulate the imbalance between inflammatory reaction and anti-inflammatory reaction.4. P38 MAPK signaling pathways involved in the process of LPS-induced ALI. 5. UTI suppressed the proinflammatory cytokine TNF alpha at transcription level.6. UTI may be reduce the TNF alpha mRNA through inhibitting p38 MAPK signaling pathway.Innovation and significance1. We described the protective role of UTI in LPS-inducde ALI in this study, which provided a new idea for clinical therapy method.2. We clarified the feasible molecular biological machaniam of UTI inhibition TNF alpha, it may be through inhibitting the p38 MAPK signaling pathway, which provided a theoretical and experimental basis for the clinical application of UTI.更多还原