【作者】 刘梅；

【导师】 姚尚龙；

【作者基本信息】 华中科技大学 ， 麻醉学， 2013， 博士

【摘要】 第一部分脂多糖诱导的小鼠急性肺损伤模型的构建目的：探索一种准确性高、创伤小、高效的经口气管内插管方法并滴定脂多糖建立小鼠急性肺损伤模型。方法：40只雄性BALB/C小鼠随机分为两组(n=20)：脂多糖组(LPS组)和对照组(NS组)。气管内插管成功后分别滴定脂多糖(3mg/kg)和生理盐水(1.5ml/kg),记录气管内插管一次成功率、最终成功率、存活率；24h后,动脉血气分析测定血氧分压(Pa02)并计算氧合指数；肺组织烘干后计算湿/干重比值；在光学显微镜下观察肺组织病理学形态改变及计数肺泡灌洗液(BALF)中总细胞数,瑞氏染色后进行细胞分类计数；BCA法测定BALF中总蛋白浓度；ELISA法测定BALF中TNF-α和IL-6浓度。结果：插管一次成功率92.5%,最终成功率和存活率均为100%；与NS组比较,LPS组动脉血Pa02和氧合指数均明显降低(P<0.01)；LPS组肺组织湿／干比值明显升高(P<0.001)；在光学显微镜下看到LPS组有大量肺泡萎陷、肺泡壁变厚、肺间质和肺泡中有大量中性粒细胞和蛋白液浸润、肺水肿、出血和肺充血,但NS组小鼠肺组织未看到以上病理改变；LPS组BALF中总细胞数和多形核粒细胞数均明显增加(P<0.01),但是单个核细胞数无明显变化；BALF中总蛋白浓度、TNF-α和IL-6浓度均显著增加(P<0.01),差异有统计学意义。结论：小鼠颈透视下经口气管内插管成功率高、损伤小,且滴定LPS3mg/kg成功建立了急性肺损伤模型。第二部分阿司匹林诱生型脂氧素A4对脂多糖诱导的小鼠急性肺损伤炎症反应的影响目的：本实验旨在研究阿司匹林诱生型脂氧素A4(ATL)对脂多糖诱导的小鼠急性肺损伤炎症反应的影响,以便进一步探讨其作用机制。方法：80只雄性BALB/C小鼠随机平分为四组(n=20)：对照组(control),急性肺损伤组(ALI),1微克ATL预处理组(ATL1μg-LPS)和5微克ATL预处理组(ATL5ng-LPS)。小鼠经气管内滴定脂多糖3mg/kg诱导急性肺损伤,滴定无菌生理盐水1.5ml/kg作为对照组,在滴定脂多糖前30min经小鼠尾静脉注射阿司匹林诱生型脂氧素A4。24h后,动脉血气分析计算氧合指数；肺组织烘干后计算湿／干重比值；在光学显微镜下观察各组肺组织病理学形态改变并进行肺组织损伤评分；在光学显微镜下计数支气管肺泡灌洗液(BALF)中总细胞数、多形核粒细胞数和单个核细胞数；BCA蛋白测定试剂盒测定BALF中总蛋白浓度；MPO试剂盒检测肺组织髓过氧化物酶(MPO)活性；ELISA法测定BALF中TNF-α、IL-6、 MCP-1和IL-10的浓度。结果：与对照组相比,ALI组小鼠动脉血氧合指数明显降低(P<0.01)；肺组织湿／干比值增高(P<0.01)；肺组织损伤评分明显升高(P<0.01)；BALF中总细胞数、多形核粒细胞数、单个核细胞数和总蛋白浓度显著增加(P<0.01)；肺组织MPO活性增强(P<0.01); BALF中促炎细胞因子(TNF-a、IL-6和MCP-1)表达显著上调(P<0.01),抗炎细胞因子IL-10水平明显下调(P<0.01)。与ALI组相比,小鼠静脉注射ATL后,动脉血氧合指数明显升高(P<0.01)；肺组织湿／干比值下降(P<0.01); ATL极大地改善肺组织病理形态,引起肺组织损伤评分明显下降(P<0.01); ATL剂量依赖性地减少BALF中总细胞数、多形核粒细胞数和总蛋白浓度(P<0.05),对单个核细胞数没有影响；只有5微克ATL明显降低肺组织中MPO活性(P<0.05); ATL抑制BALF中促炎细胞因子TNF-a, IL-6和MCP-1表达(P<0.05),但是1微克ATL明显增加抗炎细胞因子IL-10水平(P<0.01)。结论：本研究提示外源性ATL改善小鼠肺氧合功能和肺组织形态,抑制肺组织MPO活性,减少白细胞浸润,抑制促炎细胞因子,促进抗炎细胞因子,从而减轻脂多糖诱导的小鼠急性肺损伤时肺部炎症反应,对其发挥保护作用,且大剂量ATL效果更明显。第三部分阿司匹林诱生型脂氧素A4对急性肺损伤MAPK/AP-1和NF-κB信号通路的影响目的：研究ATL对LPS诱导的小鼠ALI的保护作用是否通过抑制LPS激活的丝裂原激活蛋白激酶(mitogen-activated protein kinases, MAPKs)/激活蛋白-1(activator protein-1, AP-1)和核因子-κB (nuclear factor-kappaB, NF-κB)信号通路实现,以探讨ATL减轻LPS诱导的小鼠急性肺损伤炎症反应的机制。方法：30只雄性BALB/C小鼠随机平分为三组(n=10)：对照组(control),急性肺损伤组(ALI)和5微克ATL预处理组(ATL5μg-LPS)。Western blotting测定肺组织总蛋白中p38、c-Jun氨基末端激酶(c-Jun N-terminal kinase, JNK)和细胞外调节蛋白激酶(extracellular regulated protein kinasesl/2, ERK1/2)表达及其磷酸化水平,肺组织胞浆蛋白NF-κB p65和核因子κB抑制因子(inhibitor of kappaB, IicB-a)表达水平；凝胶电泳迁移实验(electrophoretic mobility shift assays, EMS A)检测肺组织中转录因子AP-1和NF-κB p65结合DNA的活性。结果：Western blotting结果显示：与对照组相比,ALI组小鼠肺组织中p38.JNK和ERKl/2蛋白表达没有差异,磷酸化的p38、JNK和ERKl/2蛋白表达水平明显增高(P<0.01),给予5微克ATL明显抑制其磷酸化水平(P<0.05)；ALI组肺组织胞浆蛋白中NF-κB p65和IκB-α蛋白表达水平明显比对照组中低(P<0.05),5微克ATL预处理明显增强NF-κB p65和IκB-α在胞浆蛋白中的表达(P<0.05)。EMSA结果显示：与对照组相比,ALI组肺组织中AP-1和NF-κB p65DNA结合活性增强,5微克ATL处理减弱肺组织中AP-1和NF-κB p65的DNA结合活性。结论：ATL抑制LPS诱导的p38、JNK和ERK1/2磷酸化,增强胞浆蛋白中IκB表达,减少胞浆蛋白中NF-κB p65核转位,降低转录因子AP-1和NF-κB p65的DNA结合活性。因此,研究显示ATL对LPS诱导的小鼠急性肺损伤的保护作用至少部分是通过抑制MAPK/AP-1和NF-κB信号转导通路实现的。更多还原

【Abstract】 Part oneThe model of LPS-induced acute lung injury in miceObjective: To explore a reliable, minimally invasive and efficient method for endotracheal intubation, and instill lipopolysaccharide (LPS) to induce acute lung injury (ALI) in mice.Methods: Forty male BALB/C mice were randomly distributed into two groups (n=20/group):lipopolysaccharide group (LPS) and normal saline group (NS). After successfully endotracheal intubation, mice were instilled of LPS (3mg/kg) or NS (1.5ml/kg). Record the once success rate, final success rate of the endotracheal intubation and survival rate. At24hours after instillation, artery blood gas was tested. Wet/dry weight ratio (W/D ratio) was determined after the lung became dry. The histopathology of lung tissue was observed under a light microscope. Total cells in bronchoalveolar lavage fluid (BALF) were counted under a light microscope. The differential cells were counted after Wright-Geimsa stain. Total protein concentration in BALF was assayed with a BCA kit. Levels of tumor necrosis factor-alpha (TNF-a) and interleukin-6(IL-6) in BALF were quantified by ELISA.Results:The once success rate and final success rate of endotracheal intubation in mice were92.5%and100%, respectively. Survival rate of mice in the whole study was100%. PaO2of artery blood and Oxygenation index both decreased in LPS group in comparison to NS group (P<001). W/D ratio of lung tissue in LPS group was significantly elevated compared with NS group (P<0.001). Under a light microscope, it was observed that serious alveolar atelectasis, thickening of alveolar wells, significant neutrophil infiltration in the lung intersririal and alveoli, servere pulmonary interstitial edema, hemorrhage and alveolar congestion appeared in the lung tissues of LPS group mice. No pathological changes were presented in the lung tissue of NS group. Compared with NS group, the total cells and polymorphonulear leukocytes markedly increased (P<0.001), whereas there was no statistially significant difference of mononuclear leukocytes between the two groups. Total protein concentration, TNF-a level and IL-6level in BALF of LPS group were higher than these in BALF of NS group (P<0.05).Conclusion:Improved method for endotracheal intubation has high success rate and minimal injury, as well as instillation of LPS (3mg/kg) can induce ALI in mice successfully. Part twoEffects of ATL on the inflammation responses in LPS-induced ALI in miceObjective: The study aimed to investigate whether ATL alleviates excessive inflammation responses in LPS-induced ALI in mice.Methods: Eighty male BALB/C mice were randomly divided into four groups (n=20/group):control group, ALI group, ATL1μg-LPS group and ATL5μg-LPS group. Mice were instilled intratracheally with LPS (3mg/kg body weight) to induce ALI and endotoxin-free saline (1.5ml/kg body weight) as a control. ATL (1μg or5μg) or vehicle was injected via tail-vein injection30min prior to LPS instillation. After24hours of instillation, artery blood gas was tested for oxygenation index. W/D ratio was determined after all lung lobes became dry. Under a microscope, the histology changes of lung tissue were observed and then lung injure scores were quantified. Total cells, polymorphonuclear and mononuclear leukocytes in BALF were counted under a light microscope. Total protein concentration in BALF was assayed by using a BCA kit. Pulmonary myeloperoxidase (MPO) activity was measured by a MPO kit. Levels of TNF-a, IL-6, monocyte chemoattractant protein-1(MCP-1) and interleukin-10(IL-10) in BALF were analysed by ELISA.Results:Compared with control group, mice in ALI group had lower oxygenation index (P<0.01), higher W/D ratio and lung injury score (P<0.01). Total cells counts, polymorphonuclear and mononuclear leukocytes all markedly increased in ALI group (P<0.01). Pulmonary MPO activity was enhanced in ALI group (P<0.01). Total protein concertration and levels of pro-inflammatory cytokines (TNF-a, IL-6and MCP-1) in BALF of ALI group were significantly upregulated (P<0.01), whereas anti-inflammatory cytokine IL-10was downregulated (P<0.01). In comparision with ALI group, mice with ATL adminiatration had higher oxygenation index and lower W/D ratio (P<0.05). ATL markedly improved the lung histopathology with a lower lung injury score in contrast to ALI group (P<0.01). ATL led to a dose-dependent decrement in LPS-induce total cells counts, polymorphonuclear leukocytes and total protein concentration (P<0.05), but had no effect on mononuclear leukocytes. Only5μg ATL significantly decreased MPO activity of lung tissue compared with ALI group (P><0.05). ATL significantly reduced LPS-induced TNF-a, IL-6, and MCP-1levels in BALF (P<0.05), whereas1μg ATL dramatically promoted IL-10expression (P<0.01).Conclusion: This study suggests that exogenous ATL attenuates LPS-induced ALI and inflammation reponses in mice by improving lung oxygenation and histology, enhancing IL-10production, inhibiting LPS-induced MPO activity, leukocytic infiltration, TNF-a, IL-6and MCP-1. Part threeEffects of ATL on MAPK/AP-1and NF-κB signalling pathways following acute lung injury in miceObjective:In order to investigate whether ATL exerts protective effects in LPS-induce ALI in mice by inhibiting LPS-activated mitogen-activated protein kinases (MAPKs)/activator protein-1(AP-1) and nuclear factor-kappa B (NF-κB) signalling pathways.Methods: Thirty male BALB/C mice were randomly divided into three groups (n=10/group):control group, ALI group and ATL5μg-LPS group. Mice were intratracheally instilled with LPS (3mg/kg body weight) to induce ALI and endotoxin-free saline (1.5ml/kg body weight) as a control.5μg ATL or vehicle was injected via tail-vein injection30min before LPS instillation. After24hours of instillation, total and phosphorylations of p38, ERK1/2and JNK in the lung tissue were determined by Western blotting. NF-κB p65and inhibitor of κB-α (IκB-α) in the cytoplasmic protein fraction of lung tissue were also measured by Western blotting. DNA-binding activity of NF-κB p65and activator protein-1(AP-1) were assayed by electrophoretic mobility shift assay (EMSA).Results:Compared with control group, phosphorylations of p38MAPK, ERK1/2and JNK in the lung tissue were significanly enhanced in ALI group (P<0.01), whereas5μg ATL markedly inhibited LPS-induced phosphorylations of p38, ERK1/2and JNK (P<0.05). NF-κB p65and IκB-α in the cytoplasmic protein fraction of lung tissue were significant less than those in control group (P<0.05). Pretreatment with5μg ATL kept higher NF-κB p65and IκB-α expression in the cytoplasmic protein fraction of lung tissue in contrast to ALI group(P<0.05). Moreover, EMSA showed that DNA-binding activity of AP-1and NF-κB p65were increased in ALI group compared with control, whereas5μg ATL administration dramatically decreased LPS-induced DNA-binding activity of AP-1and NF-κB p65.Conclusion:ATL markedly inhibited LPS-induced phosphorylations of p38MAPK, ERK1/2and JNK, enhanced IκB-a expression in the cytoplasm, decreased the translocation of the NF-κB p65from the cytoplasm into the nucleus, blocked LPS-induced DNA-binding activity of AP-1and NF-κB p65. The current study illustrates that ATL exerts protective effects at least partly through blocking NF-κB and MAPKs/AP-1signaling transduction pathway in LPS-induced ALI in mice.更多还原