全身炎症反应对慢性阻塞性肺疾病气道、肺血管重构的影响

  本文关键词：全身炎症反应对慢性阻塞性肺疾病气道、肺血管重构的影响，，由笔耕文化传播整理发布。

        目的：研究慢性阻塞性肺疾病(chronic obstructive pulmonary disease,COPD)大鼠模型发病过程中,炎症因子表达水平的动态变化以评估COPD大鼠全身炎症反应的程度及其与气道及肺血管重塑情况的关系,同时探讨NF-κ B抑制剂吡咯烷二硫代氨基甲酸盐(pyrrolidine dithiocarbamate, PDTC))对慢性阻塞性肺疾病大鼠模型肺组织中炎症因子表达的影响及其干预后COPD大鼠模型气道及肺血管重塑情况的变化,从而了解全身炎症反应在慢性阻塞性肺疾病发病过程中的作用及PDTC对慢性阻塞性肺疾病全身炎症反应的影响,为慢性阻塞性肺疾病的治疗提供新的思路。方法：实验分为两部分。第一部分检测慢性阻塞性肺疾病(COPD)大鼠模型发病过程中炎症因子：高迁移率族蛋白B1(high mobility group protein B1, HMGB1),核因子-κB (the nuclear factor-κ B, NF-κB), TNF-α、IL-8、血管内皮生长因子(Vascular endothelial growth factor, VEGF)表达水平的动态变化及其与气道重构和肺血管重塑情况的关系。实验采用香烟雾暴露叠加低氧复制COPD大鼠模型(共分三组,每组随机分配SD大鼠8只：正常对照组(A)：第1d和第14d分别经气道内注入生理盐水,6周后检测；COPD组(B)：第1d和第14d分别经气道注入LPS,200μg/次,香烟烟雾暴露1h/d,共6周；COPD并低氧组(C)：第1d和第14d分别经气道注入LPS,200μg/次,香烟烟雾暴露1h/d,,共6周,实验的最后两周同时给予叠加18%低氧(8h/d)。第二部分检测PDTC对慢性阻塞性肺疾病大鼠模型炎症因子表达的影响：即干预前后肺组织中NF-κB蛋白水平,HMGB1mRNA和蛋白表达,血清、BALF中TNF-α、IL-8、VEGF的含量变化；干预后COPD大鼠模型气道重构和肺血管重塑情况。与第一部分方法相同设立A、B、C共三个实验模型组,每组随机分配SD大鼠8只：PDTC干预组为：A1(空白对照组)、B1(COPD干预组)、C1(COPD并低氧干预组)。动物模型制作同各对应组,从实验第3周起给各药物干预组腹腔注射干预药物PDTC,剂量为100mg· kg-1· d-1。Al组自实验第三周起腹腔注射与PDTC相同剂量的生理盐水。各组动物模型完成造模后均检测以下指标：①HE染色观察肺组织病理改变；②VG+维多利亚蓝特殊染色检测气道重构及肺血管重塑指标：气道重塑指标(细支气管管壁厚度与气管外径比值(MT%),管壁面积和气管总面积比值(MA%))；肺血管重塑指标(肺小动脉血管壁厚度与血管外径比(WT%),血管壁面积与血管总面积比(WA%))。③采用不同方法检测各炎症因子的表达情况：Western blot法检测各组肺组织NF-κB蛋白表达情况、RT-PCR及Western blot分别检测各组肺组织匀浆中HMGB1mRNA及蛋白表达情况,ELISA法分别检测各组血及清肺泡灌洗液(BALF)中TNF-α、IL-8、VEGF的表达情况。④分析上述各炎症因子的表达情况与气道重构、肺血管重塑指标的相关性,以及PDTC对各炎症因子表达的影响。结果：1.气道重塑指标(MT%、MA%):与A组比较,B、C组气道重构指标细支气管管壁厚度与气管外径比值(MT%)、管壁面积和气管总面积比值(MA%)均明显增高(P<0.05)。B、C组间MT%、MA%无差异。B1组与B组,C1组与C组比较,细支气管MT%、MA%均下降(P<0.05)。2.肺血管重塑指标(WT%、WA%):与A组相比,B组肺血管重构仅表现为肌化型动脉百分比增高,肌化型动脉管壁厚度尚未增加,未见肌化型动脉WT%、WA%明显改变(P>0.05),而C组不仅出现肌化动脉百分比增高,并且出现肌化型动脉WT%、WA%值增高(P<0.05)。使用NF-κB抑制剂吡咯烷二硫代氨基甲酸盐(PDTC))干预后,B1组较B组,C1组较C组的WT%、 WA%降低(P<0.05)。而A1组WA%与对应的试验组A组比较无明显差异(P>0.05)。3.各炎症因子的表达情况3.1NF-κ B的表达情况：Western blot电泳结果显示,B、C组NF-κB蛋白表达与A组相比明显增强(P<0.05),药物PDTC干预组B1、C1组NF-κB蛋白表达比较B、C组降低；3.2HMGB1的表达情况：RT-PCR及Western blot结果说明, B、C组HMGB1mRNA、蛋白表达与A组相比均明显增强(P<0.05),且B组、C组表达有逐渐增加的趋势,药物PDTC干预组B1、CI组与对应的实验组B、C组比较HMGB1mRNA、蛋白表达均降低(P<0.05)；3.3TNF-α、IL-8、VEGF的表达情况：ELISA结果表明：B组和C组BALF及血清中TNF-α、IL-8、VEGF表达水平高于A组,药物PDTC干预组B1、C1组TNF-α、IL-8、VEGF表达较对应B、C组下降,(P<0.05)。4.各炎症因子的表达情况与气道重构、肺血管重塑指标的相关性分析：炎症因子NF-κB、HMGB1的表达量与气道重构指标(MT%、MA%)及肺血管重塑指标(WT%、WA%)成正相关；大鼠肺组织内NF-κB蛋白的表达量与大鼠肺组织内HMGB1的表达量及肺泡灌洗液(BALF)中IL-8、TNF-α、VEGF的表达成正相关；大鼠肺组织内HMGB1的表达量与肺泡灌洗液(BALF)中IL-8、TNF-α、VEGF的表达成正相关。使用NF-κB抑制剂吡咯烷二硫代氨基甲酸盐(PDTC))干预后,各炎症因子的表达量下降,气道重构、肺血管重塑部分缓解。结论：①COPD的炎症不仅局限于肺部,同时存在全身炎症反应；全身炎症反应参与了COPD病情发生发展的全过程,并随着COPD病情的加重,炎症因子的表达逐渐增高。全身炎症反应可能通过影响肺血管、气道重构参与COPD病情的进展。②NF-κB可通过调节HMGB1、TNF-α、IL-8、VEGF的表达影响气道、肺血管的重构,NF-κB在细胞因子网络的调节中的起核心作用。

    Objective:To detect the changes of inflammatory cytokines expression in the development of COPD and To assess the degree of systemic inflammatory response in COPD,Find out the relationship between systemic inflammatory response and airway remodeling, also the relationship between systemic inflammatory response and the pulmonary vascular remodeling. To understand the effect of PDTC to inflammatory cytokines expression in animal mode lung tissue of COPD. To research the changes of airway remodeling and pulmonary vascular remodeling after use PDTC.Find out the effect of systemic inflammatory response in the development of COPD and the influence of the PDTC to airway remodeling and pulmonary vascular remodeling in COPD. Giving a new way to the treatment of COPD.Methods:There are two part of this research. Part1:To detect the changes of inflammatory cytokines(high mobility group protein B1,the nuclear factor-Kb, Tumor necrosis facto-a,Interleukin-8, Vascular endothelial growth factor)expression in the development of COPD. Find out the relationship between systemic inflammatory response and the airway and pulmonary vascular remodeling. The experiment uses the smog exposition with the low oxygen to make the animal mode of COPD.(There are three in this part of the research,8SD rat in each group randomly):Healthy control group (group A):0.2ml saline water was installed intratracheally once at first and forteenth day for all rats, testing after6weeks. COPD group (group B):200μg LPS was installed intratracheally once at first and forteenth day and the rats were exposed to cigarette smoke lh/d for6weeks. low oxygen with COPD group (group C):200μg LPS was installed intratracheally once at first and forteenth day and the rats were exposed to cigarette smoke1h/d for6weeks. In last2weeks chronic hypoxia (FiO2=0.18)8h/d were performed simultaneously.Part2:To understand the effect of NF-κB inhibitor pyrrolidine two dithiocarbamate (PDTC)), to inflammatory cytokines (HMGB1, IL-8, TNF-a, VEGF, NF-κB) expression in animal mode of COPD. To research the changes of airway and pulmonary vascular remodeling after use PDTC.(There are three group in this part of the research,8SD rat in each group randomly):PDTC intervention group include:A1(Healthy control group),B1(COPD intervented group), C1(low oxygen with COPD intervented group). Animal model were made the same as the corresponding group, PDTC100mg-kg-1·d-1were injected into the rats in each drug group from the third week. The saline water which was the same dose of PDTC was injected into the rats in A1group from the third week.We examine the following target of rat in each group after animal model made completely:HE staining of lung tissue and VG+Victoria blue triple staining was used to observe the airway and pulmonary vascular remodeling in pathological changes, includeing MT%and MA%,WT%and WA%. Using different methods to detect the expression of various inflammatory factors: Western blot detect the NF-κB protein expression. RT-PCR and Western blot were used to detect HMGB1mRNA and protein expression in each group in lung tissue, ELISA was used to detect TNF-a, IL-8, VEGF expression in lavage fluid (BALF) and serum in each group. Analysis of the expression of various inflammatory factors and airway remodeling, pulmonary vascular remodeling related indicators, as well as expression of PDTC on various inflammatory factors.Results:1.The target of airway remodeling(MT%and MA%):Compare to group A, the target of airway remodeling of group B and C were higher than that of group A(P<0.05). After using PDTC, the target of airway remodeling of group B1and C1were significantly decreased than that of the corresponding group B and C(P<0.05). The above target of group Al which were used saline were no different to that of the corresponding group A(P>0.05).2. The target of pulmonary vascular remodeling(WT%and WA%): Compare to group A, Pulmonary vascular remodeling expressed as the percentage of muscular arteries were increase in group B, But muscle type of artery wall thickness has not been increased.The target of pulmonary vascular remodeling of group C were higher than that of group A(P<0.05). After using PDTC, the target of pulmonary vascular remodeling of group B1and C1were significantly decreased than that of the corresponding group B and C(P<0.05). The above target of group A1which were used saline were no different to that of the corresponding group A(P>0.05).3. The expression of inflammatory factors:3.1The expression of NF-κB Western blot showed that NF-κB protein expression of group B and C were higher than that of group A(P<0.05); After using PDTC NF-κB protein expression of group B1and C1were significantly decreased than that of the corresponding group B and C(P<0.05).3.2The expression of HMGB1RT-PCR and Western blot shows that in group B, C, of HMGB1mRNA, protein expression compared with group A were significantly increased(P<0.05), The expression of NF-κB protein in group C is higher than group B(P<0.05). After using PDTC HMGB1mRNA and protein expression of group B1and C1were significantly decreased than that of the corresponding group B and C (P<0.05).3.3The expression of TNF-a, IL-8, VEGF ELISA results showed that These above target include concentration of TNF-a,IL-8, VEGF in BALF and in serum of group B and C were all higher than that of group A ((P<0.05). After using PDTC, Compared to the group B and C,the expression of TNF-a, IL-8, VEGF in BALF and in serum of group B1and C1, were decreased(P<0.05).4. The releationship between the expression of inflammatory cytokines and the airway and pulmonary vascular remodeling:There was positive correlation between the expressions of inflammatory cytokines (NF-κB,HMGB1) and airway remodeling index (MT%,MA%) and pulmonary vascular remodeling index (WT%,WA%) and inflammatory cytokines(IL-8,TNF-a,VEGF). There was positive correlation between the expressions of NF-κB and HMGB1. After using PDTC, the expression of inflammatory cytokines (NF-κB,HMGB1,IL-8, TNF-a,VEGF) were decreased and the airway remodeling and pulmonary vascular remodeling were improve partly.Conclusions:①With the development of the desease, the expression of TNF-α、 IL-8、VEGF in Serum and bronchoalveolar lavage fluid were gradually increased, there was positive correlation between the expressions of TNF-a, IL-8, VEGF and NF-κB,HMGB1. Prompted the inflammation of COPD is not only confined to the lungs, while there is a systemic inflammatory response. Systemic inflammatory response participated in all process of the development of COPD, Systemic inflammatory response expression advances gradually with the aggravation of COPD. It was possibly promotes the COPD course progress through the airway and pulmonary vascular remodeling.②NF-κB influence pulmonary vascular and airway reconstruction by regulating the expression of HMGB1, TNF-alpha, IL-8, VEGF. NF-κB plays an important role in COPD inflammation factor network.

        全身炎症反应对慢性阻塞性肺疾病气道、肺血管重构的影响

中文摘要4-8Abstract8-12英汉缩略词对照表13-14前言14-171 材料与方法17-242 结果24-473 讨论47-514 结论51-52参考文献52-56综述56-71    参考文献68-71攻读学位期间发表的学术论文目录71-72致谢72-73

  本文关键词：全身炎症反应对慢性阻塞性肺疾病气道、肺血管重构的影响，由笔耕文化传播整理发布。