【作者】 马希刚；

【导师】 张珍祥；

【作者基本信息】 华中科技大学 ， 内科学， 2007， 博士

【摘要】 研究背景急性肺损伤(acute lung injury,ALI)是机体由于严重感染、创伤、休克等打击后,出现以弥漫性肺泡毛细血管内皮细胞及肺泡上皮细胞广泛损伤导致的肺水肿和肺不张为病理特征,其严重阶段是急性呼吸窘迫综合征(ARDS)。临床表现为进行性呼吸窘迫和顽固性低氧血症。ALI/ARDS的发病机制错综复杂,至今尚未完全阐明。近年来强调炎症反应起关键作用,是多种原因引起的肺部失控性炎症反应的结果,是全身炎症反应在肺部的表现。有多种效应细胞、炎症介质、细胞因子和粘附分子参与疾病的发生发展。目前的研究表明阻断或减轻炎症级联反应能够阻止急性肺损伤的发展。祖国传统中药火把花根片,是采用中药原料火把花根,经现代制药工艺加工而成,实验研究及临床应用均证明该药具有良好的抗炎调节免疫、改善微循环、降低毛细血管通透性等作用,提示火把花根可能对ALI具有治疗作用。临床已应用火把花根片成功治疗肾病综合征、类风湿关节炎等多种疾病。但对急性肺损伤有无保护作用,国内尚未开展研究。本研究在油酸诱导大鼠急性肺损伤形成过程中给予火把花根干预,旨在观察火把花根对急性肺损伤有无保护作用,并初步探讨其作用机制,为火把花根在临床治疗急性肺损伤提供理论依据。方法1、健康Wistar大鼠36只,雌雄不拘,体重200-250g。简单随机分成对照组、ALI组,和火把花根组,每组12只。ALI组:尾静脉注射油酸(0.04ml/kg),复制大鼠ALI模型后,4h采集样品;火把花根预防组:火把花根片研磨后溶于2ml生理盐水中,连续灌胃10天(600mg/kg),每天二次,第10天灌胃后1h和模型组一样注射油酸,4h后采集样品;对照组:尾静脉注射生理盐水0.04ml/kg,4h后采集样品。2、采用亚硝酸盐法、硫代巴比妥酸比色法和二硫代二硝基苯甲酸法,分别测定血浆和支气管肺泡灌洗液(BALF)中谷胱甘肽过氧化酶(GSH-PX)、超氧化物歧化酶(SOD)和脂质过氧化产物丙二醛(MDA)的水平。3、测定肺组织湿/干质量比(W/D)、肺通透指数(LPI),BALF中白细胞(WBC)计数及中性粒细胞(PMN)百分比。光镜和电镜下分别观察各组动物肺组织形态学改变,并进行肺损伤评分(Lung Injury Score,LIS)。4、采用链霉素亲和素-生物素-过氧化物酶复合物法(SABC),分别测定肺组织NF-κBp65和TNF-α的表达水平。5、采用链霉素抗生物素蛋白-过氧化物酶(SP)和流式细胞法,分别测定肺组织中ICAM-1和PMN表面CD11a、CD11b和CD18的表达水平。结果1、火把花根对肺损伤大鼠肺组织病理形态学的影响光镜观察:对照组肺泡结构完整清晰,肺泡腔干净,肺泡隔无增宽及充血,无炎性细胞浸润及出血现象;ALI组可见肺灶性出血,肺泡壁明显增厚,肺泡和肺间质水肿,大量炎性细胞浸润,肺泡腔内有渗出。火把花根组改变较ALI组程度明显减轻。ALI组LIS较对照组明显升高(P<0.01),火把花根组LIS低于ALI组(P<0.01),但仍高于对照组(P<0.01)。电镜观察:对照组肺微血管内皮细胞及基底膜形态和结构正常;ALI组可见肺微血管内皮细胞严重肿胀,基底膜缺损、疏松、不清晰;火把花根组微血管内皮细胞形态略有肿胀,形态基本正常,基底膜较完整。2、火把花根对肺损伤大鼠血浆及BALF中GSH-Px、SOD和MDA水平的影响ALI组血浆及BALF中GSH-Px和SOD活力较对照组明显降低(P<0.01),火把花根组血浆及BALF中GSH-Px和SOD活力高于ALI组(P<0.01),但仍低于对照组(P<0.05)。ALI组血浆及BALF中MDA含量较对照组明显升高(P<0.01),火把花根组血浆及BALF中MDA含量低于ALI组(P<0.01),但仍高于对照组(P<0.05)。3、火把花根对肺损伤大鼠肺W/D、BALF中WBC计数、PMN百分比及LPI的影响ALI组肺W/D、BALF中WBC计数、PMN百分比及LPI较对照组明显升高(P<0.01),火把花根组肺W/D、BALF中WBC计数、PMN百分比及LPI明显低于ALI组(P <0.01),除LPI外,余指标仍高于对照组(P<0.05)。4、火把花根对肺损伤大鼠肺组织NF-κBp65和TNF-α的表达的影响ALI组肺组织NF-κBp65和TNF-α达强度明显高于对照组(P<0.01),火把花根组肺组织NF-κBp65和TNF-α表达明显受到抑制,低于ALI组(P<0.01),但NF-κBp65的表达仍高于对照组(P<0.05),TNF-α的表达与对照组无差异。5、火把花根对肺组织中ICAM-1和PMN表面CD11a、CD11b和CD18的表达的影响ALI组肺组织中ICAM-1的表达高于对照组(P<0.01),火把花根组肺组织中ICAM-1的表达明显低于ALI组(P<0.01) ,但仍高于对照组(P<0.05)。ALI组PMN表面的CD11a、CD11b和CD18表达高于对照组(P<0.01),火把花根组PMN表面的CD11a、CD11b和CD18明显低于ALI组(P<0.01) ,但仍高于对照组(P<0.05)。结论1、油酸型大鼠肺损伤体内存在明显氧化/抗氧化平衡失调,不仅有氧化物生成增多的因素,还有抗氧化能力下降的因素。2、火把花根可以减轻肺损伤大鼠肺组织病理学改变,其机制可能是由于减少了机体氧化物的产生,同时也改善了机体的抗氧化能力,使氧化/抗氧化平衡失衡程度减轻。3、火把花根可以抑制肺损伤大鼠肺组织NF-κB和TNF-α的过渡表达,减轻肺部炎症程度,对肺损伤具有保护作用。4、火把花根通过对下调肺损伤大鼠肺组织中ICAM-1和PMN表面CD11a、CD11b和CD18的表达,减少中性粒细胞的活化和聚集,对肺损伤具有保护作用。更多还原

【Abstract】 BACKGROUNDThe pathological characteristic of acute lung injury（ALI） is diffused alveolar-capilary membrane injury ,which were caused by severe infection,trauma,shock,et al.The severe stage is acute respiratory distress syndrome（ARDS）,the clinical situation is respiratory distress and intractable hypoxemia. The pathogenesis is very complicated ,it is still not clear although many studies have been done.Recently the attention was paid to inflammatory response,and it is regarded that ALI is the result of uncontrolled inflammatory response in the lung due to various causes.It is the pulmonary menifestation of systemic inflammatory response. . There are many effector cells , cytokines and adhension molecules to participate in this disease.It is belief to block or lessen inflammatory casecade can prevent the development of ALI successfully.Chinese traditional medicine colquhounia root tablets were purified from colquhounia root by moden pharmaceutical product technology.It was confimed that favourable anti- inflammatory effects by experimental studies and clinical application.But wheather it has the same effect on acute lung injury is not very clear.In this experiment ,rats were treated with colquhounia root tablets during the establishment of the models of ALI,we were to investigate its effects on the ALI and the mechanisms for applying theoretical evidence to treat ALI with colquhounia root tablets in clinic practice.METHODS1.36 healty Wistar rats weighing 200-250g,were randomly divided into three groups:the control group,the ALI model group and the colquhounia root treated group. In ALI group oleic acid 0.04ml.kg^-1 was injected into trail vein to establish ALI models,in colquhounia root tablet treated group colquhounia root tablet with 600mg.kg^-1 day^-1 were instilled into the stomach of each Wistar rat for ten consecutive days before intravenous injection of same dose oleic acid,in control group same dose saline was injected. After 4h of establishing model,all animals were sacrificed.2.Total superoxide dismutase （SOD） level of the plasma and broncho-alveolar lavage fluid （BALF） were measured by the nitrite method, glutathione peroxidase （GSH-Px） and malondialdehyde （MDA） activities were detected by 5,5’-dithionbis （2-nitrobenzoic acid） and whist thiobarbituric acid colorimrtric method respectively.3.Lung wet/dry ratio, lung permeability index （LPI） ,WBC number and percentage of active neutrophils in BALF were counted .Morphologic lung changes in the three groups were observed at both light and electron microscopic levels.4.The positive expression of Nuclear factor-κB and tumor necrosis-αwereperformed in the lung tissue by SABC in three groups.5.The positive expression of intercellular adhension molecule-1 in lung tissues by SP and CD11a,CD11band CD18 in neutrophil were quantitatively analyzed by flow cytometry using monoclonal antibodies in three groupsbbb.RESULT1. Effects of colquhounia root on pathologic changes of ALI in rats1.1 Light microscope（HE）: Alveoli appeared normal in the size and shape in the control group.,In the ALI group there were multiple lesions,including local hemorrhage ,diffuse sever congestion and edema in the interstitiun and alveoli with infiltration. of red cells, many inflammatory cell into the alveolar lumen and thickening of alveolar walls.The microscopic lesions observed in the colquhounia root treated group were lighter than the ones in the ALI group .The changes were localized.1.2 Electron microscope: The pulmonary microvascular basement membrane and endothelial cells in the control group were normal. While in the ALI group, the pulmonary microvascular basement membrane defect and puff , endothelial cells edema seriously and not integral. The pulmonary microvascular basement membrane was integral relatively,endothelial cells mild edema in the colquhounia root treated group .2. Effects of colquhounia root on level of GSH-Px、MDA and SOD in plasma and BALF of ALI in ratsThe activies of SOD and GSH-Px in the ALI group in plasma and BALF deceased significantly compared with the control group（P<0.01）. in the treated group, the activies of SOD and GSH-Px in plasma and BALF increased significantly compared with the ALI group（P<0.01）, but higher than that in the control group （P<0.05）.The content of MDA in the ALI group in plasma and BALF increased significantly compared with the control group（P<0.01）, in the treated group, the content of MDA in the ALI group in plasma and BALF deceased significantly compared with the ALI group, but lowerr than that in the control group （P<0.05）.3. Effects of colquhounia root on W/D,LPI ,WBC number and percentage of neutrophils in BALF of ALI in ratsW/D,LPI ,WBC number and percentage of neutrophils in the ALI group increased significantly compared with the control group（P<0.01）.In the treated group , W/D,LPI ,WBC number and percentage of neutrophils deceased significantly compared with the ALI group（P<0.01）,but except the LPI,the others higher than that in the control group （P<0.05）.4. Effects of colquhounia root on expression of NF-κB and TNF-αin lung tissues of ALI in ratsThe expression of NF-κB and TNF-αin the ALI group increased significantly compared with the control group（P<0.01）.In the treated group , The expression of NF-κB and TNF-αdeceased significantly compared with the ALI group（P<0.01）,but higher than that in the control group （P<0.05）.5. Effects of colquhounia root on expression of ICAM-1 in lung tissues and CD11a,CD11b and CD18 in neutrophil of ALI in ratsThe expression of ICAM-1 and CD11a,CD11b and CD18 in the ALI group increased significantly compared with the control group（P<0.01）.In the treated group , The expression of The expression of ICAM-1 and CD11a,CD11b and CD18 deceased significantly compared with the ALI group（P<0.01）,but higher than that in the control group （P<0.05）.CONCLUSION1. There is significant diorder of oxidant and antioxidant in the lung tissue of oleic acid induced ALI in rats.2. Colquhounia root can alleviate the pathological injury of the lung of oleic acid induced ALI in rats ,It can balance the disorder of oxidant and antioxidant.3. Colquhounia root can lessen the degree of lung inflammatory by inhibiting the over activition of NF-κB and TNF-αin lung tissues4. Colquhounia root can reduce activition and aggregation of PMNs by downregulation the expression of ICAM-1 and CD11a,CD11b and CD18 .更多还原