吸烟对哮喘Th1/Th2平衡的影响及转录调节机制研究
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摘要
支气管哮喘(简称哮喘)是全球范围内严重影响公共健康的慢性疾病,其发病率和严重性逐年增高,但原因尚不清楚。流行病学研究认为与儿童期感染机率下降及环境中作为危险因素的污染物增多有关。其中吸烟(包括主动和被动吸烟)作为室内空气污染的主要来源,是哮喘发病中一个重要环境因素。国外流行病学资料显示至少三分之一的哮喘患者经常暴露在香烟烟雾环境中,有资料显示发达国家至少有50%的成人哮喘患者目前吸烟或曾经吸烟。
哮喘是多种细胞及细胞组份参与的气道慢性炎症,随病程延长可产生气道不可逆性缩窄和气道重塑。吸烟使哮喘本来就复杂的病理生理过程进一步复杂化,吸烟不仅可以引起哮喘频繁发作、导致哮喘患者的肺功能迅速下降,而且可以降低哮喘患者糖皮质激素的治疗效果,因此使哮喘患者病情不易控制。吸烟对哮喘发生发展及临床表现的影响是复杂的,相关的研究报道很少,具体机制尚不清楚。
研究表明吸烟和许多疾病如社区获得的肺炎,肺癌及呼吸道感染密切相关,因此认为可能与吸烟改变宿主的免疫反应有关。香烟烟雾中的主要成分之一尼古丁,具有免疫调节作用,能够影响免疫监视功能,并促进疾病的发生发展。
辅助性T细胞1(Th1)/辅助性T细胞2(Th2)失衡是哮喘发病的重要环节,无论气道炎症还是气道重塑都受到Thl/Th2平衡的调节。辅助性T细胞(CD4+T细胞),即Th细胞,可分为Thl和Th2两个亚群,Thl细胞主要分泌细胞因子γ干扰素(IFN-γ)、白细胞介素(IL)-2、IL-12等,可激活巨噬细胞,在抗病毒感染和胞内细菌感染的免疫应答中发挥作用。Th2细胞主要分泌细胞因子IL-4、IL-5、IL-6、IL-10、IL-13等,参与急性超敏反应,对B细胞合成所有抗体包括IgE有辅助作用,在对过敏原和寄生虫感染的免疫反应中起作用。
T-bet/GATA-3是近年来新发现的转录因子,分别是诱导Th1/Th2极性分化以及分泌相应效应性细胞因子的专有性转录因子。研究认为T-bet/GATA-3表达变化可间接反映体内Th1/Th2类细胞数量以及Th1/Th2类细胞因子表达水平的动态变化。CD4+CD25+调节性T细胞(regulatory T cell,Treg)是一类独特的CD4+T细胞,在抑制主动免疫及维持免疫耐受中起着关键性作用;叉状头/翅膀状螺旋转录因子(forkhead/winged helix transcription factor,Foxp3)在一定程度上可以反映CD4+CD25+Treg的水平和功能活性;新近研究认为,Treg可明显抑制Thl、Th2活化增殖,是在Th1/Th2网络平衡之上的更高层次调控者,并可能在控制支气管哮喘和过敏性疾病的发病中起重要作用。
吸烟对调控Th1/Th2平衡的关键转录因子T-bet、GATA-3的影响未见文献报道,吸烟对Treg的数量及Foxp3表达影响的研究报道鲜见。转录因子在转录水平调控细胞因子和炎症介质的产生,在哮喘等气道炎症性疾病的发病机制中成为研究热点,有望成为新的治疗靶点。鉴于上述研究背景和研究现状,本实验将通过体内、体外实验,从不同细胞水平研究吸烟及主要成份尼古丁对哮喘Th1/Th2免疫平衡的影响及其转录调节机制,一方面深入探讨吸烟加重哮喘的免疫学机制,另一方面也为哮喘治疗探寻新的靶点。
一、研究内容
1.香烟烟雾暴露对哮喘大鼠Th1/Th2细胞因子及转录因子T-bet/GATA-3表达的影响;
2.香烟烟雾提取物对卵蛋白致敏大鼠脾淋巴细胞Th1/Th2细胞因子及转录因子T-bet/GATA-3表达的影响;
3.尼古丁对卵蛋白致敏CD4+T淋巴细胞Th1/Th2细胞因子及转录因子T-bet/GATA-3表达的影响;
4.香烟烟雾暴露对哮喘大鼠CD4+CD25+调节性T细胞数量及转录因子Foxp3表达的影响。
二、研究方法
实验一:香烟烟雾暴露对哮喘大鼠Th1/Th2细胞因子及转录因子T-bet/GATA-3表达的影响
1.复制动物模型:雄性Wistar大鼠,随机分为4组:对照组、烟雾暴露组、哮喘组和哮喘组+烟雾暴露组,每组10只。建立慢性哮喘大鼠模型(1%OVA雾化激发8周,1次/天,5天/周)和慢性哮喘大鼠吸烟模型(吸烟和OVA雾化激发同时开始,1次/天,5天/周,共8周)。
2. HE染色观察各组大鼠气道炎性细胞浸润及支气管壁厚度变化。
3. Masson染色观察各组气道胶原含量。
4. ELISA法测定肺组织和血浆IFN-γ/IL-4含量。
5. Western blot法检测肺组织T-bet/GATA-3蛋白表达。
6. Quantitive real time RT-PCR检测肺组织T-bet/GATA-3mRNA表达。
实验二:香烟烟雾提取物对卵蛋白致敏大鼠脾淋巴细胞Th1/Th2细胞因子和转录因子T-bet/GATA-3表达的影响
1.大鼠OVA致敏和脾淋巴细胞的分离:6-8周龄雄性Wistar大鼠,第1天、8天分别腹腔内注射OVA和氢氧化铝混合液致敏,第15天麻醉动物无菌取脾,分离脾淋巴细胞。
2.不同浓度香烟烟雾提取物(cigarette smoke extract,CSE)的干预:将脾淋巴细胞随机分为4组:对照组、0.5%CSE组、1%CSE组、2%CSE组,同时各孔均加入OVA作为特异性刺激物,CSE作用24小时。
3. ELISA法检测细胞培养上清液IFN-γ/IL-4含量。
4. Quantitative real time RT-PCR检测培养细胞T-bet/GATA-3mRNA的表达。
实验三:尼古丁对OVA致敏大鼠CD4+T细胞Th1/Th2细胞因子和T-bet/GATA-3表达的影响
1.大鼠OVA致敏:方法同实验二
2. CD4+T细胞的分离及不同浓度尼古丁的干预:大鼠CD4+T纯化柱分离CD4+T细胞,将CD4+T细胞随机分为4组:对照组、1μg/ml尼古丁组、10μg/ml尼古丁组、100μg/ml尼古丁组,同时各孔均加入OVA作为特异性刺激物,尼古丁刺激24小时。
3. ELISA法检测细胞培养上清液IL-4和IFN-γ含量。
4. Quantitive real time RT-PCR检测培养细胞T-bet/GATA-3mRNA的表达。
实验四:香烟烟雾暴露对哮喘大鼠CD4+CD25+T细胞数量及转录因子Foxp3表达的影响
1.复制动物模型:同实验一。
2.流式细胞仪检测大鼠外周血CD4+CD25+Treg细胞占CD4+T细胞的比例。
3. Western blot法检测肺组织Foxp3蛋白的表达。
三、研究结果
实验一:香烟烟雾暴露对哮喘大鼠Th1/Th2细胞因子及转录因子T-bet/GATA-3表达的影响
1.各组大鼠肺组织和气道普通病理学变化
对照组细支气管纤毛排列整齐,管腔圆整,血管腔、肺泡腔、肺间质均未见到嗜酸性粒细胞浸润,基底膜较薄;哮喘组气道壁和肺组织中淋巴细胞为主并伴有EOS的炎症细胞浸润、粘膜皱褶增多、气道壁增厚;哮喘组+烟雾暴露组气道壁和肺组织中可见大量淋巴细胞及嗜酸性粒细胞和中性粒细胞浸润,气道壁明显增厚;吸烟组大鼠气道可见中性粒细胞和淋巴细胞浸润。
2.各组大鼠支气管管壁厚度的变化
哮喘组支气管管壁厚度(15.72±0.82μm2/μm)明显增加,与对照组(8.52±0.58μm2/μm)比较差异有统计学意义(P<0.01);哮喘+烟雾暴露组支气管管壁厚度(18.34±0.87μm2/μm)较哮喘组进一步增加,差异有统计学意义(P<0.01);烟雾暴露组支气管管壁厚度(9.89±1.07μm2/μm)较对照组增加,差异有统计学意义(P<0.05)。
3.各组大鼠气道胶原含量的变化
哮喘组气道胶原含量(16.41±1.08μm2/μm)较对照组(7.59±0.59μm2/μm)明显增加,差异有统计学意义(P<0.01);哮喘+烟雾暴露组胶原含量(19.15±0.54μm2/μm)较哮喘组明显增加,差异有统计学意义(P<0.01);烟雾暴露组(9.73±0.62μm2/μm)较对照组气道胶原含量增加,差异有统计学意义(P<0.01)。
4.各组大鼠血浆和肺组织IL-4/IFN-γ含量的比较
哮喘组血浆和肺组织IL-4含量(22.57±4.32 pg/ml,0.80±0.14 pg/ml)分别较对照组(11.38±2.90 pg/ml,0.27±0.08 pg/ml)明显增加,差异有统计学意义(P均<0.01),IFN-γ含量(58.83±19.57 pg/ml,0.58±0.23 pg/ml)分别较对照组(150.87±55.54 pg/ml,1.63±0.20 pg/ml)降低,差异有统计学意义(P均<0.01);哮喘+烟雾暴露组血浆和肺组织IL-4含量(34.07±6.11 pg/ml,1.41±0.31 pg/ml)分别较哮喘组升高,差异有统计学意义(P<0.01,P<0.05),血浆IFN-γ含量(9.53±3.28 pg/ml)较哮喘组进一步降低,差异有统计学意义(P<0.01),肺组织IFN-γ含量(0.48±0.10 pg/ml)较哮喘组有下降趋势,差异无统计学意义。烟雾暴露组血浆和肺组织匀浆IL-4、IFN-γ较对照组比较,差异无统计学意义。
5.各组大鼠肺组织T-bet/GATA-3蛋白表达的比较
Western blot分析显示,在蛋白质相对分子量为62000、50000和43000处有表达条带,分别是T-bet、GATA-3和β-actin蛋白表达。哮喘组T-bet蛋白表达(0.47±0.06)较对照组(0.91±0.07)显著降低,两组比较差异有统计学意义(P<0.01);哮喘+烟雾暴露组T-bet蛋白表达(0.17±0.03)较哮喘组明显降低,两组比较差异有统计学意义(P<0.01)。烟雾暴露组T-bet蛋白表达(0.82±0.05)与对照组比较差异有无统计学意义。哮喘组GATA-3蛋白表达(0.87±0.04)较对照组(0.45±0.06)显著增加,两组比较差异有统计学意义(P<0.01);哮喘+烟雾暴露组GATA-3蛋白表达(1.29±0.08)较哮喘组增加,两组比较差异有统计学意义(P<0.01)。烟雾暴露组GATA-3蛋白表达(0.65±0.03)较对照组增加,差异有统计学意义(P<0.01)。
6.各组大鼠肺组织T-bet/GATA-3mRNA表达的比较
实时荧光定量RT-PCR结果分析显示,哮喘组T-betmRNA表达(0.34±0.07)较对照组(0.95±0.12)显著降低,两组比较差异有统计学意义(P<0.01);哮喘+烟雾暴露组T-betmRNA表达(0.13±0.05)较哮喘组显著降低,两组比较差异有统计学意义(P<0.05)。烟雾暴露组T-betmRNA表达(1.02±0.10)较对照组比较差异无统计学意义。哮喘组GATA-3mRNA表达(1.86±0.07)较对照组(0.99±0.13)显著增加,两组比较差异有统计学意义(P<0.01),哮喘+烟雾暴露组GATA-3mRNA表达(2.20±0.08)较哮喘组增加,两组比较差异有统计学意义(P<0.01)。烟雾暴露组GATA-3mRNA表达(1.32±0.07)较对照组增加,差异有统计学意义(P<0.01)。
实验二:香烟烟雾提取物对卵蛋白致敏大鼠脾淋巴细胞Th1/Th2细胞因子和T-bet/GATA-3表达的影响
1.不同浓度CSE对脾淋巴细胞IFN-γ/IL-4分泌的影响
对照组IFN-γ含量为(111.97±8.32 pg/ml),IL-4含量为(28.10±2.03 pg/ml),随CSE干预浓度的增加,IFN-γ的分泌呈CSE浓度依赖性减少(90.69±7.26 pg/ml,64.61±4.52 pg/ml,55.62±4.09 pg/ml),而IL-4的分泌呈CSE浓度依赖性增加(34.66±1.61 pg/ml,38.97±1.62 pg/ml,54.28±2.78pg/ml),其中2%CSE干预组IFN-γ分泌最少,IL-4水平最高,不同浓度CSE干预组IFN-γ和IL-4含量与对照组比较,差异有统计学意义(P均<0.05),不同浓度CSE干预组组间比较,差异均有统计学意义(P均<0.05)。
2.不同浓度CSE对脾淋巴细胞T-bet/GATA-3mRNA表达的影响
随着CSE干预浓度的增加,T-betmRNA表达逐渐降低,0.5%、1.0%、2.0%CSE干预组T-betmRNA表达(0.77±0.04,0.56±0.03,0.37±0.04)与对照组(1.01±0.14)比较,差异均有统计学意义(P均<0.05),不同浓度CSE干预组组间比较,差异均有统计学意义(P均<0.05)。随着CSE干预浓度的增加,GATA-3mRNA表达逐渐增加,0.5%、1.0%、2.0%CSE干预组GATA-3mRNA表达(1.92±0.18,3.16±0.32,4.56±0.44)与对照组(1.18±0.17)比较,差异均有统计学意义(P均<0.05),不同浓度CSE干预组组间比较,差异均有统计学意义(P均<0.05)。
实验三:尼古丁对卵蛋白致敏大鼠CD4+T细胞Th1/Th2细胞因子和T-bet/GATA-3表达的影响
1.不同浓度尼古丁对CD4+T细胞IFN-γ/IL-4分泌的影响
对照组IFN-γ的浓度为142.30±5.89 pg/ml,IL-4的浓度为36.91±3.24 pg/ml,随尼古丁干预浓度的增加,IFN-γ的分泌呈尼古丁浓度依赖性减少(113.78±6.06pg/ml,70.31±7.26pg/ml , 20.00±2.14pg/ml ),而IL-4的分泌呈尼古丁浓度依赖性增加(69.49±3.91pg/ml,93.63±4.56 pg/ml,50.97±3.07pg/ml),不同浓度尼古丁干预组IFN-γ和IL-4含量与对照组比较,差异有统计学意义(P均<0.05),100μg/ml尼古丁干预组IL-4水平较10μg/ml干预组有所下降,但仍高于对照组。
2.不同浓度尼古丁对CD4+T细胞T-bet/GATA-3mRNA表达的影响
对照组T-betmRNA表达为0.98±0.09,对照组GATA-3mRNA为1.00±0.07,随尼古丁干预浓度的增加,GATA-3mRNA表达呈浓度依赖性增加,1μg/ml、10μg/ml、100μg/ml尼古丁干预组GATA-3mRNA表达分别为4.31±0.26、5.16±0.23、1.56±0.14;不同浓度尼古丁干预组GATA-3mRNA表达与对照组比较,差异均有统计学意义(P均<0.05),100μg/ml尼古丁干预组GATA-3mRNA表达较10μg/ml尼古丁干预组有所下降,但仍高于对照组。T-betmRNA表达呈尼古丁浓度依赖性减少,1μg/ml、10μg/ml、100μg/ml尼古丁干预组T-betmRNA表达分别为0.73±0.03、0.57±0.04、0.31±0.04,不同浓度尼古丁干预组T-bet mRNA表达与对照组比较,差异有统计学意义(P均<0.05)。
实验四:香烟烟雾暴露对哮喘大鼠CD4+CD25+Treg细胞数量及转录因子Foxp3表达的影响
1.各组大鼠外周血CD4+CD25+Treg细胞占CD4+T细胞的比例
各组大鼠外周血CD4+CD25+Treg细胞经流式细胞仪检测结果显示,对照组大鼠CD4+CD25+Treg百分比为(9.93±1.006)%,哮喘组为(6.44±0.956)%,较对照组明显降低,差异有统计学意义(P<0.01);哮喘+烟雾暴露组CD4+CD25+Treg百分比为(3.31±0.846)%,与对照组和哮喘组比较均明显降低,差异有统计学意义(P均<0.01)。烟雾暴露组CD4+CD25+Treg百分比为(26.05±5.269)%,与对照组比较显著增高,差异有统计学意义(P<0.01)。
2.各组大鼠肺组织Foxp3蛋白表达的比较
Western blot分析显示,在蛋白质相对分子量为48000和43000处有表达条带,分别是Foxp3、β-actin蛋白表达。对照组Foxp3蛋白表达为10.27±0.33,哮喘组Foxp3蛋白表达为8.18±0.26,与对照组比较明显降低,差异有统计学意义(P<0.01);哮喘+烟雾暴露组Foxp3蛋白表达为6.36±0.38,较哮喘组表达减少,两组比较差异有统计学意义(P<0.05)。烟雾暴露组与对照组Foxp3蛋白比较,差异有无统计学意义。
四、研究结论
1.香烟烟雾暴露可能通过调控转录因子T-bet/GATA-3表达,进而加重哮喘大鼠Th1/Th2失衡,在哮喘的气道炎症及气道重塑中扮演重要角色。
2.尼古丁在吸烟加重哮喘的Th1/Th2失衡中起重要作用。
3. 2个月卵蛋白雾化激发哮喘模型中CD4+CD25+Treg细胞占CD4+T细胞的比例下调,Foxp3蛋白表达下降,二者可能在哮喘发病中发挥作用。
4.香烟烟雾暴露降低转录因子的Foxp3蛋白表达及下调CD4+CD25+Treg细胞比例,可能为吸烟加重哮喘Th1/Th2失衡的上游调控机制。
Asthma is a serious global health problem. Worldwide prevalence and severity of asthma is increasing in the general population. Although this increase is probably the result of several factors, epidemiological studies have implicated both a decrease in childhood infections and an increase in environmental pollution as risk factors. Cigarette smoking (including passive and active smoking) is the major risk factor for astham. Epidemiological studies have inplicated that almost one third of asthmatic patients are often exposured to cigarette smoking. It is estimated that around 50% of adult asthmatics in the developed world probably are or have been smokers.
Asthma is a chronic inflammatory disorder of the airways in which many cells and cellular elements play a role, sometimes associated with airway structural changes, often described as airway remodeling. Smoking favors the development of a complex pathophysiology of inflammatory airway disease. Tobacco smoking makes asthma more difficult to control, results in more frequent exacerbations and produces a more rapid decline in lung function and poorly responsive to glucocorticosteroids. However, the mechanisms whereby cigarette smoking influence the development and expression of asthma are complex and interactive, so still poorly understood today.
Epidemiological studies have linked cigarette smoking to community-acquired pneumonia, higher incidences of lung cancer, respiratory infections. It is well accepted that many of these health consequences may be attributable to cigarette smoke-induced changes in host immunity. Cigarette smoke contains nicotine, an immunomodulatory component that is thought to affect immune surveillance and increase the progression of diseases.
Imbalance between Th1 and Th2, higher levels of Th2 cytokine IL-4, 1L-5 and IL-13 and lower levels of Thl cytokine IFN-γis prominent in the pathogenesis of asthma. More recent studies clearly demonstrated that Th2 cells and their typical cytokines play a critical role not only in airway inflammation but also in the development of airway remodeling. T helper cell (CD4+T cell, Th) differentiate into two subsets, T helper cell type 1(Thl) and T helper cell type 2 (Th2), each with distinct functions and cytokine profiles. Interferon (IFN)-γis the signature cytokine of Thl cells. Interleukin (IL)-4 is the corresponding signature cytokine of Th2 cells, which also secrete IL-5 and IL-13, these two subgroups of T helper arise in response to different immunogenic stimuli and cytokines. T box expressed in T cells (T-bet) and GATA binding protein-3(GATA-3), as the specific transcription factors of Thl and Th2 respectively and the upstream factors of Thl/Th2 balance are an active area of research. Recently significant progress has been made in identifying the transcription factors that control the transcription of a T helper cell to a Th2 or Thl cell, the results show that there is significantly a positive correlation between the level of T-bet/GATA-3 expression and IFN-γ/IL-4. A subset of CD4 cells, known collectively as CD4+CD25+regulatory T cells (Treg), are a central component of active immune suppression. Forkhead transcription facter3 (Foxp3) is a critical transcription factor in regulatory T cells development. The pathogenesis of asthma has been regarded as between Thl and Th2 cells, which is now considered as superficies. In fact, reason for the activation and proliferation of CD4+T cells and differentiation towards Th2 cells is the dysfunction of CD4+CD25+Treg in patients with asthma.
So the objective of this study is to investigate the impact of cigarette smoking on Th1/Th2 banlance and its mechanism of transcription regulatory in the pathogenesis of asthma.
Contents 1. Effect of cigarette smoke exposure on the expression of Th1/Th2 cytokines and transcription factor T-bet/GATA-3 in asthmatic rat.
2. Effect of cigarette smoke extract on the expression of Th1/Th2 cytokines and transcription factor T-bet/GATA-3 in cultured splenocytes of rat sensitized by ovalbumin.
3. Effect of nicotine on the expression of Th1/Th2 cytokines and transcription factor T-bet/GATA-3 in cultured CD4+ T of rat sensitized by ovalbumin.
4. Effect of cigarette smoke exposure on the percentage of CD4+CD25+T cell and the expression of transcription factor Foxp3 in asthmatic rat.
Methods
Part one: Effect of cigarette smoke exposure on the expression of Th1/Th2 cytokines and transcription factor T-bet/GATA-3 in asthmatic rat.
1. Forty male Wistar rats were randomly divided into four groups: control group, aerosolized ovalbumin exposure group, cigarette smoke exposure group and OVA combined cigarette, smoke exposure group. Rats were exposed to either aerosolized OVA, tobacco smoke, or both tobacco smoke and OVA.
2. The change of airway histology was observed by HE and Masson.
3. The concentration of interleukin-4 (IL-4) and interferon-γ(INF-γ) in peripheral blood and lung hemogenates were measured by enzyme-linked immunosorbent assay (ELESA).
4. The nucleus protein expression of T-bet and GATA-3 in the lungs were detected by Western blot.
5. The mRNA expression of T-bet and GATA-3 in the lungs were detected by quantitative real time polymerase chain reaction (real time PCR) using ready-made fluorogenic probes and primers.
Part two: Effect of cigarette smoke extract on the expression of Th1/Th2 cytokines and transcription factor T-bet/GATA-3 in cultured splenocytes of rat sensitized by ovalbumin.
1. Two weeks after immunization by ovalbumin, spleens were excised aseptically from rats and after lyses of RBCs Splenocytes were obtained from singled-cell suspension of spleen.
2. Splenocytes cultured in 0.5, 1, 2.5 percent cigarette smoke extract (CSE) solution prepared with RPMI, with which splenocytes were treated simultaneously with ovalbumin, supernatants and cell pellets were harvested after cultured for 24 hours.
3. The concentration of INF-γ/IL-4 in Supernatants was measured by enzyme-linked immunosorbent assay.
4. The mRNA expression of T-bet/GATA-3 in Splenocytes were detected by quantitative real time polymerase chain reaction using ready-made fluorogenic probes and primers.
Part three: Effect of nicotine on the expression of Th1/Th2 cytokines and transcription factor T-bet/GATA-3 in cultured CD4+ T of rat sensitized by ovalbumin.
1. Two weeks after immunization by ovalbumin, spleens were excised aseptically from rats and after lyses of RBCs splenocytes were obtained from singled-cell suspension of spleen.
2. Splenic CD4+ T cells were purified using CD4+T cell enrichment kit.
3. Purified CD4+ T cells were stimulated in ovalbumin with or without nicotine. Supernatants and cell pellets were harvested after cultured for 24 hours.
4. The concentration of INF-γ/IL-4 in supernatants was measured by enzyme-linked immunosorbent assay.
5. The mRNA expression of T-bet/GATA-3 in CD4+T cells was detected by quantitative real time polymerase chain reaction using ready-made fluorogenic probes and primers.
Part four: Effect of cigarette smoke exposure on the percentage of CD4+CD25+T cell and the expression of transcription factor Foxp3 in asthmatic rat.
1. Forty male Wistar rats were randomly divided into four groups: a control group, aerosolized ovalbumin exposure group, cigarette smoke exposure group and ovalbumin combined cigarette, smoke exposure group. Rats were exposed to either aerosolized ovalbumin, tobacco smoke, or both tobacco smoke and ovalbumin.
2. Percentage of CD4+CD25+T cell was determined by flow cytometry analysis.
3. The protein expression of Foxp3 in the lungs was detected by Western blot.
Results
1. Pathological change of the rat groups
The thickness of airway wall and expression of collagen in OVA combined cigarette smoke exposure group was higher than that in aerosolized OVA exposure and the control group(P<0.01).
2. Protein levels of INF-γ/IL-4 in plasma and lung
The plasma and lung levels of IL-4 in aerosolized OVA exposure were significantly higher than those in control group (P<0.05), respectively. The plasma and lung level of IFN-γwas significantly lower than that in control group (P<0.05), The plasma and lung levels of IL-4 in Combining OVA with cigarette smoke exposure group were significantly higher than those in aerosolized OVA exposure group (P<0.05),respectively, The plasma and lung levels of IFN-γin asthma group was significantly lower than that in aerosolized OVA exposure group(P<0.05),and there was no statistical difference between the control group and the cigarette exposure group (P >0.05).
3. Protein expression of T-bet/GATA-3 in lung
Nucleus protein expression of T-bet in OVA combined cigarette smoke exposure group were lower than that in the control group and aerosolized OVA exposure group(all P<0.01), meanwhile nucleus protein expression of GATA-3 in OVA combined cigarette smoke exposure group were higher than that in the control group and aerosolized OVA exposure group(all P<0.01).
4. mRNA expression of T-bet/GATA-3 in lung
The T-bet mRNA expression in lung in OVA combined cigarette smoke exposure group was significantly attenuated than those in aerosolized OVA exposure group and control group(P<0.05),meanwhile the GATA-3 mRNA expression was significantly enhanced(P<0.05) respectively.
5. The effect of cigarette smoke extract (CSE) on the production of INF-γ/IL-4 in cultured splenocytes
The production of INF-γin CSE treatment groups were significantly lower than that in the control group, P<0.05, and there were significant difference in 0.5, 1, 2.5 percent CSE treatment group in term of INF-γproduction, P<0.01. CSE treatment cause dose-dependent repression of INF-γproduction. The production of IL-4 in CSE treatment groups were significantly higher than that in the control group, P<0.01, and there were significant difference in 0.5, 1, 2.5 percent CSE treatment groups in term of IL-4 production, P<0.05. CSE treatment cause dose-dependent stimulated of IL-4 production.
6. The effect of cigarette smoke extract (CSE) on mRNA expression of T-bet/GATA-3 in cultured splenocytes
The mRNA expression of T-bet in CSE treatment groups were significantly lower than that in the control group, P<0.05, and there were significant difference in 0.5, 1, 2.5 percent CSE treated group in term of T-bet The mRNA expression, all P<0.01. CSE treatment cause dose-dependent repression of T-bet the mRNA expression. The mRNA expression of GATA-3 in CSE treatment groups were significantly higher than that in the control group, P<0.01, and there were significant difference in 0.5, 1, 2.5 percent CSE treatment groups in term of GATA-3 mRNA expression, all P<0.05. CSE treatment cause dose-dependent stimulated of IL-4 production.
7. The effect of nicotine on the production of INF-γ/IL-4 in cultured CD4+T
When challenged simultaneously with OVA, exposure of nicotine not only induced IL-4 but also inhibits INF-γproduction in a dose dependent manner.
8. The effect of nicotine on mRNA expression of T-bet/GATA-3 in cultured CD4+T cells
When challenged simultaneously with OVA, exposure of nicotine not only induced mRNA expression of GATA-3 but also inhibits mRNA expression of T-bet in a dose dependent manner.
9. Percentage of CD4+CD25+T cell in peripheral blood
Percentage of CD4+CD25+T cell in aerosolized OVA group was significantly lower than that in the control group (P<0.01), meanwhile percentage of CD4+CD25+T cell in OVA combined cigarette smoke exposure group was lower than that in aerosolized OVA exposure group(P<0.01). Percentage of CD4+CD25+T cell in cigarette exposure group was significantly higher than that in the control group and the (P<0.01).
10. Protein expression of Foxp3 in lung
Protein expression of Foxp3 in aerosolized OVA group were lower than that in the control group(P<0.01), meanwhile protein expression of Foxp3 in OVA combined cigarette smoke exposure group were lower than that in the control group and aerosolized OVA exposure group(all P<0.01). There was no statistical difference between the control group and the cigarette exposure group.
Conclusions
1. In this study cigarette smoke exposure play a key role in the development of Th2-type allergic inflammation in asthma by promoting over-expression of transcription factor GATA-3 and repressing the expression of T-bet.
2. Nicotine play a key role in the development of Th2-type allergic inflammation in asthma.
3. The ratio of CD4+CD25+Treg and the expressions of Foxp3 decreased in athmatic rats which may play an important role in the pathogenesis of asthma.
4. Cigarette smoke exposure can downregulate the ratio of CD4+CD25+Treg and the expression of Foxps which was the upstream factors in cigarette smoking-induced Th1/Th2 imbalance.
哮喘是多种细胞及细胞组份参与的气道慢性炎症,随病程延长可产生气道不可逆性缩窄和气道重塑。吸烟使哮喘本来就复杂的病理生理过程进一步复杂化,吸烟不仅可以引起哮喘频繁发作、导致哮喘患者的肺功能迅速下降,而且可以降低哮喘患者糖皮质激素的治疗效果,因此使哮喘患者病情不易控制。吸烟对哮喘发生发展及临床表现的影响是复杂的,相关的研究报道很少,具体机制尚不清楚。
研究表明吸烟和许多疾病如社区获得的肺炎,肺癌及呼吸道感染密切相关,因此认为可能与吸烟改变宿主的免疫反应有关。香烟烟雾中的主要成分之一尼古丁,具有免疫调节作用,能够影响免疫监视功能,并促进疾病的发生发展。
辅助性T细胞1(Th1)/辅助性T细胞2(Th2)失衡是哮喘发病的重要环节,无论气道炎症还是气道重塑都受到Thl/Th2平衡的调节。辅助性T细胞(CD4+T细胞),即Th细胞,可分为Thl和Th2两个亚群,Thl细胞主要分泌细胞因子γ干扰素(IFN-γ)、白细胞介素(IL)-2、IL-12等,可激活巨噬细胞,在抗病毒感染和胞内细菌感染的免疫应答中发挥作用。Th2细胞主要分泌细胞因子IL-4、IL-5、IL-6、IL-10、IL-13等,参与急性超敏反应,对B细胞合成所有抗体包括IgE有辅助作用,在对过敏原和寄生虫感染的免疫反应中起作用。
T-bet/GATA-3是近年来新发现的转录因子,分别是诱导Th1/Th2极性分化以及分泌相应效应性细胞因子的专有性转录因子。研究认为T-bet/GATA-3表达变化可间接反映体内Th1/Th2类细胞数量以及Th1/Th2类细胞因子表达水平的动态变化。CD4+CD25+调节性T细胞(regulatory T cell,Treg)是一类独特的CD4+T细胞,在抑制主动免疫及维持免疫耐受中起着关键性作用;叉状头/翅膀状螺旋转录因子(forkhead/winged helix transcription factor,Foxp3)在一定程度上可以反映CD4+CD25+Treg的水平和功能活性;新近研究认为,Treg可明显抑制Thl、Th2活化增殖,是在Th1/Th2网络平衡之上的更高层次调控者,并可能在控制支气管哮喘和过敏性疾病的发病中起重要作用。
吸烟对调控Th1/Th2平衡的关键转录因子T-bet、GATA-3的影响未见文献报道,吸烟对Treg的数量及Foxp3表达影响的研究报道鲜见。转录因子在转录水平调控细胞因子和炎症介质的产生,在哮喘等气道炎症性疾病的发病机制中成为研究热点,有望成为新的治疗靶点。鉴于上述研究背景和研究现状,本实验将通过体内、体外实验,从不同细胞水平研究吸烟及主要成份尼古丁对哮喘Th1/Th2免疫平衡的影响及其转录调节机制,一方面深入探讨吸烟加重哮喘的免疫学机制,另一方面也为哮喘治疗探寻新的靶点。
一、研究内容
1.香烟烟雾暴露对哮喘大鼠Th1/Th2细胞因子及转录因子T-bet/GATA-3表达的影响;
2.香烟烟雾提取物对卵蛋白致敏大鼠脾淋巴细胞Th1/Th2细胞因子及转录因子T-bet/GATA-3表达的影响;
3.尼古丁对卵蛋白致敏CD4+T淋巴细胞Th1/Th2细胞因子及转录因子T-bet/GATA-3表达的影响;
4.香烟烟雾暴露对哮喘大鼠CD4+CD25+调节性T细胞数量及转录因子Foxp3表达的影响。
二、研究方法
实验一:香烟烟雾暴露对哮喘大鼠Th1/Th2细胞因子及转录因子T-bet/GATA-3表达的影响
1.复制动物模型:雄性Wistar大鼠,随机分为4组:对照组、烟雾暴露组、哮喘组和哮喘组+烟雾暴露组,每组10只。建立慢性哮喘大鼠模型(1%OVA雾化激发8周,1次/天,5天/周)和慢性哮喘大鼠吸烟模型(吸烟和OVA雾化激发同时开始,1次/天,5天/周,共8周)。
2. HE染色观察各组大鼠气道炎性细胞浸润及支气管壁厚度变化。
3. Masson染色观察各组气道胶原含量。
4. ELISA法测定肺组织和血浆IFN-γ/IL-4含量。
5. Western blot法检测肺组织T-bet/GATA-3蛋白表达。
6. Quantitive real time RT-PCR检测肺组织T-bet/GATA-3mRNA表达。
实验二:香烟烟雾提取物对卵蛋白致敏大鼠脾淋巴细胞Th1/Th2细胞因子和转录因子T-bet/GATA-3表达的影响
1.大鼠OVA致敏和脾淋巴细胞的分离:6-8周龄雄性Wistar大鼠,第1天、8天分别腹腔内注射OVA和氢氧化铝混合液致敏,第15天麻醉动物无菌取脾,分离脾淋巴细胞。
2.不同浓度香烟烟雾提取物(cigarette smoke extract,CSE)的干预:将脾淋巴细胞随机分为4组:对照组、0.5%CSE组、1%CSE组、2%CSE组,同时各孔均加入OVA作为特异性刺激物,CSE作用24小时。
3. ELISA法检测细胞培养上清液IFN-γ/IL-4含量。
4. Quantitative real time RT-PCR检测培养细胞T-bet/GATA-3mRNA的表达。
实验三:尼古丁对OVA致敏大鼠CD4+T细胞Th1/Th2细胞因子和T-bet/GATA-3表达的影响
1.大鼠OVA致敏:方法同实验二
2. CD4+T细胞的分离及不同浓度尼古丁的干预:大鼠CD4+T纯化柱分离CD4+T细胞,将CD4+T细胞随机分为4组:对照组、1μg/ml尼古丁组、10μg/ml尼古丁组、100μg/ml尼古丁组,同时各孔均加入OVA作为特异性刺激物,尼古丁刺激24小时。
3. ELISA法检测细胞培养上清液IL-4和IFN-γ含量。
4. Quantitive real time RT-PCR检测培养细胞T-bet/GATA-3mRNA的表达。
实验四:香烟烟雾暴露对哮喘大鼠CD4+CD25+T细胞数量及转录因子Foxp3表达的影响
1.复制动物模型:同实验一。
2.流式细胞仪检测大鼠外周血CD4+CD25+Treg细胞占CD4+T细胞的比例。
3. Western blot法检测肺组织Foxp3蛋白的表达。
三、研究结果
实验一:香烟烟雾暴露对哮喘大鼠Th1/Th2细胞因子及转录因子T-bet/GATA-3表达的影响
1.各组大鼠肺组织和气道普通病理学变化
对照组细支气管纤毛排列整齐,管腔圆整,血管腔、肺泡腔、肺间质均未见到嗜酸性粒细胞浸润,基底膜较薄;哮喘组气道壁和肺组织中淋巴细胞为主并伴有EOS的炎症细胞浸润、粘膜皱褶增多、气道壁增厚;哮喘组+烟雾暴露组气道壁和肺组织中可见大量淋巴细胞及嗜酸性粒细胞和中性粒细胞浸润,气道壁明显增厚;吸烟组大鼠气道可见中性粒细胞和淋巴细胞浸润。
2.各组大鼠支气管管壁厚度的变化
哮喘组支气管管壁厚度(15.72±0.82μm2/μm)明显增加,与对照组(8.52±0.58μm2/μm)比较差异有统计学意义(P<0.01);哮喘+烟雾暴露组支气管管壁厚度(18.34±0.87μm2/μm)较哮喘组进一步增加,差异有统计学意义(P<0.01);烟雾暴露组支气管管壁厚度(9.89±1.07μm2/μm)较对照组增加,差异有统计学意义(P<0.05)。
3.各组大鼠气道胶原含量的变化
哮喘组气道胶原含量(16.41±1.08μm2/μm)较对照组(7.59±0.59μm2/μm)明显增加,差异有统计学意义(P<0.01);哮喘+烟雾暴露组胶原含量(19.15±0.54μm2/μm)较哮喘组明显增加,差异有统计学意义(P<0.01);烟雾暴露组(9.73±0.62μm2/μm)较对照组气道胶原含量增加,差异有统计学意义(P<0.01)。
4.各组大鼠血浆和肺组织IL-4/IFN-γ含量的比较
哮喘组血浆和肺组织IL-4含量(22.57±4.32 pg/ml,0.80±0.14 pg/ml)分别较对照组(11.38±2.90 pg/ml,0.27±0.08 pg/ml)明显增加,差异有统计学意义(P均<0.01),IFN-γ含量(58.83±19.57 pg/ml,0.58±0.23 pg/ml)分别较对照组(150.87±55.54 pg/ml,1.63±0.20 pg/ml)降低,差异有统计学意义(P均<0.01);哮喘+烟雾暴露组血浆和肺组织IL-4含量(34.07±6.11 pg/ml,1.41±0.31 pg/ml)分别较哮喘组升高,差异有统计学意义(P<0.01,P<0.05),血浆IFN-γ含量(9.53±3.28 pg/ml)较哮喘组进一步降低,差异有统计学意义(P<0.01),肺组织IFN-γ含量(0.48±0.10 pg/ml)较哮喘组有下降趋势,差异无统计学意义。烟雾暴露组血浆和肺组织匀浆IL-4、IFN-γ较对照组比较,差异无统计学意义。
5.各组大鼠肺组织T-bet/GATA-3蛋白表达的比较
Western blot分析显示,在蛋白质相对分子量为62000、50000和43000处有表达条带,分别是T-bet、GATA-3和β-actin蛋白表达。哮喘组T-bet蛋白表达(0.47±0.06)较对照组(0.91±0.07)显著降低,两组比较差异有统计学意义(P<0.01);哮喘+烟雾暴露组T-bet蛋白表达(0.17±0.03)较哮喘组明显降低,两组比较差异有统计学意义(P<0.01)。烟雾暴露组T-bet蛋白表达(0.82±0.05)与对照组比较差异有无统计学意义。哮喘组GATA-3蛋白表达(0.87±0.04)较对照组(0.45±0.06)显著增加,两组比较差异有统计学意义(P<0.01);哮喘+烟雾暴露组GATA-3蛋白表达(1.29±0.08)较哮喘组增加,两组比较差异有统计学意义(P<0.01)。烟雾暴露组GATA-3蛋白表达(0.65±0.03)较对照组增加,差异有统计学意义(P<0.01)。
6.各组大鼠肺组织T-bet/GATA-3mRNA表达的比较
实时荧光定量RT-PCR结果分析显示,哮喘组T-betmRNA表达(0.34±0.07)较对照组(0.95±0.12)显著降低,两组比较差异有统计学意义(P<0.01);哮喘+烟雾暴露组T-betmRNA表达(0.13±0.05)较哮喘组显著降低,两组比较差异有统计学意义(P<0.05)。烟雾暴露组T-betmRNA表达(1.02±0.10)较对照组比较差异无统计学意义。哮喘组GATA-3mRNA表达(1.86±0.07)较对照组(0.99±0.13)显著增加,两组比较差异有统计学意义(P<0.01),哮喘+烟雾暴露组GATA-3mRNA表达(2.20±0.08)较哮喘组增加,两组比较差异有统计学意义(P<0.01)。烟雾暴露组GATA-3mRNA表达(1.32±0.07)较对照组增加,差异有统计学意义(P<0.01)。
实验二:香烟烟雾提取物对卵蛋白致敏大鼠脾淋巴细胞Th1/Th2细胞因子和T-bet/GATA-3表达的影响
1.不同浓度CSE对脾淋巴细胞IFN-γ/IL-4分泌的影响
对照组IFN-γ含量为(111.97±8.32 pg/ml),IL-4含量为(28.10±2.03 pg/ml),随CSE干预浓度的增加,IFN-γ的分泌呈CSE浓度依赖性减少(90.69±7.26 pg/ml,64.61±4.52 pg/ml,55.62±4.09 pg/ml),而IL-4的分泌呈CSE浓度依赖性增加(34.66±1.61 pg/ml,38.97±1.62 pg/ml,54.28±2.78pg/ml),其中2%CSE干预组IFN-γ分泌最少,IL-4水平最高,不同浓度CSE干预组IFN-γ和IL-4含量与对照组比较,差异有统计学意义(P均<0.05),不同浓度CSE干预组组间比较,差异均有统计学意义(P均<0.05)。
2.不同浓度CSE对脾淋巴细胞T-bet/GATA-3mRNA表达的影响
随着CSE干预浓度的增加,T-betmRNA表达逐渐降低,0.5%、1.0%、2.0%CSE干预组T-betmRNA表达(0.77±0.04,0.56±0.03,0.37±0.04)与对照组(1.01±0.14)比较,差异均有统计学意义(P均<0.05),不同浓度CSE干预组组间比较,差异均有统计学意义(P均<0.05)。随着CSE干预浓度的增加,GATA-3mRNA表达逐渐增加,0.5%、1.0%、2.0%CSE干预组GATA-3mRNA表达(1.92±0.18,3.16±0.32,4.56±0.44)与对照组(1.18±0.17)比较,差异均有统计学意义(P均<0.05),不同浓度CSE干预组组间比较,差异均有统计学意义(P均<0.05)。
实验三:尼古丁对卵蛋白致敏大鼠CD4+T细胞Th1/Th2细胞因子和T-bet/GATA-3表达的影响
1.不同浓度尼古丁对CD4+T细胞IFN-γ/IL-4分泌的影响
对照组IFN-γ的浓度为142.30±5.89 pg/ml,IL-4的浓度为36.91±3.24 pg/ml,随尼古丁干预浓度的增加,IFN-γ的分泌呈尼古丁浓度依赖性减少(113.78±6.06pg/ml,70.31±7.26pg/ml , 20.00±2.14pg/ml ),而IL-4的分泌呈尼古丁浓度依赖性增加(69.49±3.91pg/ml,93.63±4.56 pg/ml,50.97±3.07pg/ml),不同浓度尼古丁干预组IFN-γ和IL-4含量与对照组比较,差异有统计学意义(P均<0.05),100μg/ml尼古丁干预组IL-4水平较10μg/ml干预组有所下降,但仍高于对照组。
2.不同浓度尼古丁对CD4+T细胞T-bet/GATA-3mRNA表达的影响
对照组T-betmRNA表达为0.98±0.09,对照组GATA-3mRNA为1.00±0.07,随尼古丁干预浓度的增加,GATA-3mRNA表达呈浓度依赖性增加,1μg/ml、10μg/ml、100μg/ml尼古丁干预组GATA-3mRNA表达分别为4.31±0.26、5.16±0.23、1.56±0.14;不同浓度尼古丁干预组GATA-3mRNA表达与对照组比较,差异均有统计学意义(P均<0.05),100μg/ml尼古丁干预组GATA-3mRNA表达较10μg/ml尼古丁干预组有所下降,但仍高于对照组。T-betmRNA表达呈尼古丁浓度依赖性减少,1μg/ml、10μg/ml、100μg/ml尼古丁干预组T-betmRNA表达分别为0.73±0.03、0.57±0.04、0.31±0.04,不同浓度尼古丁干预组T-bet mRNA表达与对照组比较,差异有统计学意义(P均<0.05)。
实验四:香烟烟雾暴露对哮喘大鼠CD4+CD25+Treg细胞数量及转录因子Foxp3表达的影响
1.各组大鼠外周血CD4+CD25+Treg细胞占CD4+T细胞的比例
各组大鼠外周血CD4+CD25+Treg细胞经流式细胞仪检测结果显示,对照组大鼠CD4+CD25+Treg百分比为(9.93±1.006)%,哮喘组为(6.44±0.956)%,较对照组明显降低,差异有统计学意义(P<0.01);哮喘+烟雾暴露组CD4+CD25+Treg百分比为(3.31±0.846)%,与对照组和哮喘组比较均明显降低,差异有统计学意义(P均<0.01)。烟雾暴露组CD4+CD25+Treg百分比为(26.05±5.269)%,与对照组比较显著增高,差异有统计学意义(P<0.01)。
2.各组大鼠肺组织Foxp3蛋白表达的比较
Western blot分析显示,在蛋白质相对分子量为48000和43000处有表达条带,分别是Foxp3、β-actin蛋白表达。对照组Foxp3蛋白表达为10.27±0.33,哮喘组Foxp3蛋白表达为8.18±0.26,与对照组比较明显降低,差异有统计学意义(P<0.01);哮喘+烟雾暴露组Foxp3蛋白表达为6.36±0.38,较哮喘组表达减少,两组比较差异有统计学意义(P<0.05)。烟雾暴露组与对照组Foxp3蛋白比较,差异有无统计学意义。
四、研究结论
1.香烟烟雾暴露可能通过调控转录因子T-bet/GATA-3表达,进而加重哮喘大鼠Th1/Th2失衡,在哮喘的气道炎症及气道重塑中扮演重要角色。
2.尼古丁在吸烟加重哮喘的Th1/Th2失衡中起重要作用。
3. 2个月卵蛋白雾化激发哮喘模型中CD4+CD25+Treg细胞占CD4+T细胞的比例下调,Foxp3蛋白表达下降,二者可能在哮喘发病中发挥作用。
4.香烟烟雾暴露降低转录因子的Foxp3蛋白表达及下调CD4+CD25+Treg细胞比例,可能为吸烟加重哮喘Th1/Th2失衡的上游调控机制。
Asthma is a serious global health problem. Worldwide prevalence and severity of asthma is increasing in the general population. Although this increase is probably the result of several factors, epidemiological studies have implicated both a decrease in childhood infections and an increase in environmental pollution as risk factors. Cigarette smoking (including passive and active smoking) is the major risk factor for astham. Epidemiological studies have inplicated that almost one third of asthmatic patients are often exposured to cigarette smoking. It is estimated that around 50% of adult asthmatics in the developed world probably are or have been smokers.
Asthma is a chronic inflammatory disorder of the airways in which many cells and cellular elements play a role, sometimes associated with airway structural changes, often described as airway remodeling. Smoking favors the development of a complex pathophysiology of inflammatory airway disease. Tobacco smoking makes asthma more difficult to control, results in more frequent exacerbations and produces a more rapid decline in lung function and poorly responsive to glucocorticosteroids. However, the mechanisms whereby cigarette smoking influence the development and expression of asthma are complex and interactive, so still poorly understood today.
Epidemiological studies have linked cigarette smoking to community-acquired pneumonia, higher incidences of lung cancer, respiratory infections. It is well accepted that many of these health consequences may be attributable to cigarette smoke-induced changes in host immunity. Cigarette smoke contains nicotine, an immunomodulatory component that is thought to affect immune surveillance and increase the progression of diseases.
Imbalance between Th1 and Th2, higher levels of Th2 cytokine IL-4, 1L-5 and IL-13 and lower levels of Thl cytokine IFN-γis prominent in the pathogenesis of asthma. More recent studies clearly demonstrated that Th2 cells and their typical cytokines play a critical role not only in airway inflammation but also in the development of airway remodeling. T helper cell (CD4+T cell, Th) differentiate into two subsets, T helper cell type 1(Thl) and T helper cell type 2 (Th2), each with distinct functions and cytokine profiles. Interferon (IFN)-γis the signature cytokine of Thl cells. Interleukin (IL)-4 is the corresponding signature cytokine of Th2 cells, which also secrete IL-5 and IL-13, these two subgroups of T helper arise in response to different immunogenic stimuli and cytokines. T box expressed in T cells (T-bet) and GATA binding protein-3(GATA-3), as the specific transcription factors of Thl and Th2 respectively and the upstream factors of Thl/Th2 balance are an active area of research. Recently significant progress has been made in identifying the transcription factors that control the transcription of a T helper cell to a Th2 or Thl cell, the results show that there is significantly a positive correlation between the level of T-bet/GATA-3 expression and IFN-γ/IL-4. A subset of CD4 cells, known collectively as CD4+CD25+regulatory T cells (Treg), are a central component of active immune suppression. Forkhead transcription facter3 (Foxp3) is a critical transcription factor in regulatory T cells development. The pathogenesis of asthma has been regarded as between Thl and Th2 cells, which is now considered as superficies. In fact, reason for the activation and proliferation of CD4+T cells and differentiation towards Th2 cells is the dysfunction of CD4+CD25+Treg in patients with asthma.
So the objective of this study is to investigate the impact of cigarette smoking on Th1/Th2 banlance and its mechanism of transcription regulatory in the pathogenesis of asthma.
Contents 1. Effect of cigarette smoke exposure on the expression of Th1/Th2 cytokines and transcription factor T-bet/GATA-3 in asthmatic rat.
2. Effect of cigarette smoke extract on the expression of Th1/Th2 cytokines and transcription factor T-bet/GATA-3 in cultured splenocytes of rat sensitized by ovalbumin.
3. Effect of nicotine on the expression of Th1/Th2 cytokines and transcription factor T-bet/GATA-3 in cultured CD4+ T of rat sensitized by ovalbumin.
4. Effect of cigarette smoke exposure on the percentage of CD4+CD25+T cell and the expression of transcription factor Foxp3 in asthmatic rat.
Methods
Part one: Effect of cigarette smoke exposure on the expression of Th1/Th2 cytokines and transcription factor T-bet/GATA-3 in asthmatic rat.
1. Forty male Wistar rats were randomly divided into four groups: control group, aerosolized ovalbumin exposure group, cigarette smoke exposure group and OVA combined cigarette, smoke exposure group. Rats were exposed to either aerosolized OVA, tobacco smoke, or both tobacco smoke and OVA.
2. The change of airway histology was observed by HE and Masson.
3. The concentration of interleukin-4 (IL-4) and interferon-γ(INF-γ) in peripheral blood and lung hemogenates were measured by enzyme-linked immunosorbent assay (ELESA).
4. The nucleus protein expression of T-bet and GATA-3 in the lungs were detected by Western blot.
5. The mRNA expression of T-bet and GATA-3 in the lungs were detected by quantitative real time polymerase chain reaction (real time PCR) using ready-made fluorogenic probes and primers.
Part two: Effect of cigarette smoke extract on the expression of Th1/Th2 cytokines and transcription factor T-bet/GATA-3 in cultured splenocytes of rat sensitized by ovalbumin.
1. Two weeks after immunization by ovalbumin, spleens were excised aseptically from rats and after lyses of RBCs Splenocytes were obtained from singled-cell suspension of spleen.
2. Splenocytes cultured in 0.5, 1, 2.5 percent cigarette smoke extract (CSE) solution prepared with RPMI, with which splenocytes were treated simultaneously with ovalbumin, supernatants and cell pellets were harvested after cultured for 24 hours.
3. The concentration of INF-γ/IL-4 in Supernatants was measured by enzyme-linked immunosorbent assay.
4. The mRNA expression of T-bet/GATA-3 in Splenocytes were detected by quantitative real time polymerase chain reaction using ready-made fluorogenic probes and primers.
Part three: Effect of nicotine on the expression of Th1/Th2 cytokines and transcription factor T-bet/GATA-3 in cultured CD4+ T of rat sensitized by ovalbumin.
1. Two weeks after immunization by ovalbumin, spleens were excised aseptically from rats and after lyses of RBCs splenocytes were obtained from singled-cell suspension of spleen.
2. Splenic CD4+ T cells were purified using CD4+T cell enrichment kit.
3. Purified CD4+ T cells were stimulated in ovalbumin with or without nicotine. Supernatants and cell pellets were harvested after cultured for 24 hours.
4. The concentration of INF-γ/IL-4 in supernatants was measured by enzyme-linked immunosorbent assay.
5. The mRNA expression of T-bet/GATA-3 in CD4+T cells was detected by quantitative real time polymerase chain reaction using ready-made fluorogenic probes and primers.
Part four: Effect of cigarette smoke exposure on the percentage of CD4+CD25+T cell and the expression of transcription factor Foxp3 in asthmatic rat.
1. Forty male Wistar rats were randomly divided into four groups: a control group, aerosolized ovalbumin exposure group, cigarette smoke exposure group and ovalbumin combined cigarette, smoke exposure group. Rats were exposed to either aerosolized ovalbumin, tobacco smoke, or both tobacco smoke and ovalbumin.
2. Percentage of CD4+CD25+T cell was determined by flow cytometry analysis.
3. The protein expression of Foxp3 in the lungs was detected by Western blot.
Results
1. Pathological change of the rat groups
The thickness of airway wall and expression of collagen in OVA combined cigarette smoke exposure group was higher than that in aerosolized OVA exposure and the control group(P<0.01).
2. Protein levels of INF-γ/IL-4 in plasma and lung
The plasma and lung levels of IL-4 in aerosolized OVA exposure were significantly higher than those in control group (P<0.05), respectively. The plasma and lung level of IFN-γwas significantly lower than that in control group (P<0.05), The plasma and lung levels of IL-4 in Combining OVA with cigarette smoke exposure group were significantly higher than those in aerosolized OVA exposure group (P<0.05),respectively, The plasma and lung levels of IFN-γin asthma group was significantly lower than that in aerosolized OVA exposure group(P<0.05),and there was no statistical difference between the control group and the cigarette exposure group (P >0.05).
3. Protein expression of T-bet/GATA-3 in lung
Nucleus protein expression of T-bet in OVA combined cigarette smoke exposure group were lower than that in the control group and aerosolized OVA exposure group(all P<0.01), meanwhile nucleus protein expression of GATA-3 in OVA combined cigarette smoke exposure group were higher than that in the control group and aerosolized OVA exposure group(all P<0.01).
4. mRNA expression of T-bet/GATA-3 in lung
The T-bet mRNA expression in lung in OVA combined cigarette smoke exposure group was significantly attenuated than those in aerosolized OVA exposure group and control group(P<0.05),meanwhile the GATA-3 mRNA expression was significantly enhanced(P<0.05) respectively.
5. The effect of cigarette smoke extract (CSE) on the production of INF-γ/IL-4 in cultured splenocytes
The production of INF-γin CSE treatment groups were significantly lower than that in the control group, P<0.05, and there were significant difference in 0.5, 1, 2.5 percent CSE treatment group in term of INF-γproduction, P<0.01. CSE treatment cause dose-dependent repression of INF-γproduction. The production of IL-4 in CSE treatment groups were significantly higher than that in the control group, P<0.01, and there were significant difference in 0.5, 1, 2.5 percent CSE treatment groups in term of IL-4 production, P<0.05. CSE treatment cause dose-dependent stimulated of IL-4 production.
6. The effect of cigarette smoke extract (CSE) on mRNA expression of T-bet/GATA-3 in cultured splenocytes
The mRNA expression of T-bet in CSE treatment groups were significantly lower than that in the control group, P<0.05, and there were significant difference in 0.5, 1, 2.5 percent CSE treated group in term of T-bet The mRNA expression, all P<0.01. CSE treatment cause dose-dependent repression of T-bet the mRNA expression. The mRNA expression of GATA-3 in CSE treatment groups were significantly higher than that in the control group, P<0.01, and there were significant difference in 0.5, 1, 2.5 percent CSE treatment groups in term of GATA-3 mRNA expression, all P<0.05. CSE treatment cause dose-dependent stimulated of IL-4 production.
7. The effect of nicotine on the production of INF-γ/IL-4 in cultured CD4+T
When challenged simultaneously with OVA, exposure of nicotine not only induced IL-4 but also inhibits INF-γproduction in a dose dependent manner.
8. The effect of nicotine on mRNA expression of T-bet/GATA-3 in cultured CD4+T cells
When challenged simultaneously with OVA, exposure of nicotine not only induced mRNA expression of GATA-3 but also inhibits mRNA expression of T-bet in a dose dependent manner.
9. Percentage of CD4+CD25+T cell in peripheral blood
Percentage of CD4+CD25+T cell in aerosolized OVA group was significantly lower than that in the control group (P<0.01), meanwhile percentage of CD4+CD25+T cell in OVA combined cigarette smoke exposure group was lower than that in aerosolized OVA exposure group(P<0.01). Percentage of CD4+CD25+T cell in cigarette exposure group was significantly higher than that in the control group and the (P<0.01).
10. Protein expression of Foxp3 in lung
Protein expression of Foxp3 in aerosolized OVA group were lower than that in the control group(P<0.01), meanwhile protein expression of Foxp3 in OVA combined cigarette smoke exposure group were lower than that in the control group and aerosolized OVA exposure group(all P<0.01). There was no statistical difference between the control group and the cigarette exposure group.
Conclusions
1. In this study cigarette smoke exposure play a key role in the development of Th2-type allergic inflammation in asthma by promoting over-expression of transcription factor GATA-3 and repressing the expression of T-bet.
2. Nicotine play a key role in the development of Th2-type allergic inflammation in asthma.
3. The ratio of CD4+CD25+Treg and the expressions of Foxp3 decreased in athmatic rats which may play an important role in the pathogenesis of asthma.
4. Cigarette smoke exposure can downregulate the ratio of CD4+CD25+Treg and the expression of Foxps which was the upstream factors in cigarette smoking-induced Th1/Th2 imbalance.
引文
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