IL-21与STAT3在哮喘小鼠肺组织的表达及作用
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摘要
背景
支气管哮喘(简称哮喘)(bronchial asthma,asthma)是呼吸系统常见病、多发病之一,对人类健康构成了很大的威胁。世界卫生组织的统计结果显示,全球约有3亿哮喘患者,其中每年约有一千五百万人因为该病而丧失劳动能力。据不完全统计,我国至少也有1500万到2000万哮喘患者,世界各地支气管哮喘的发病率、死亡率都呈逐年增高趋势,每年造成了异常巨大的经济损失,哮喘已经成为严重的公共卫生问题。哮喘反复发作且病因复杂,在宿主易感因素与环境因素相互作用下,以慢性气道炎症和气道重塑为基本病理生理改变的综合征,是一种可以控制但很难治愈的疾病。研究哮喘的发病机理,预防和控制哮喘变态反应性炎症的发生和发展,受到越来越多研究者的关注。
在哮喘气道炎症发展过程中,CD4+T细胞群中的初始T细胞经变应原诱导激活后成为Th0细胞,并继续朝Th2细胞方向分化,生成大量的Th2细胞,Th2细胞占据优势地位,可大规模诱发下游的IgE和／或非IgE介导的Th2炎症。哮喘发病过程中,炎性细胞在气道组织中的浸润、聚集、激活和释放是其主要特征,在此过程中细胞因子发挥了重要作用。慢性炎症反复刺激和上皮结构的破坏可以引起哮喘的气道重塑。目前,气道炎症与气道重塑作为哮喘的两个病理特征逐渐被人们所接受。引起气道重塑的各种细胞因子、炎症介质等在气道重塑中发挥作用的机制尚未完全明确。
白细胞介素21(interleukin21,IL-21)是2000年发现的一种细胞因子,属于Ⅰ型细胞因子家族,主要是由CD4+Th2细胞合成和分泌,与IL-2、IL-4和IL-15具有同源性。自从发现了IL-21以后,关于它的生物学功能成为了研究的热点。IL-21的效应是多样的,主要是由于IL-21的受体分布广泛。IL-21开始时被认为是激活的外周血T细胞分泌的细胞因子,现在被认为可以由一系列分化型CD4+Th细胞合成,生物学功能主要是刺激T细胞增殖、NK细胞增殖和分化以及CD40特异性应答B细胞的增殖。IL-21受体(IL-21R)表达于淋巴造血细胞、成纤维细胞、角质细胞、肠上皮细胞。实验证明,IL-21在先天性免疫、适应性免疫及细胞内的免疫应答中发挥重要的作用,与多种自身免疫性疾病及炎性疾病有关,例如类风关、系统性红斑狼疮、系统性硬化病、炎症性肠病、过敏性疾病,对于这些疾病有重要的免疫调节作用。
IL—21在Th细胞的分化方面起了关键作用,支气管哮喘是一种免疫异常导致的变态反应性疾病,免疫功能紊乱在哮喘发病中起十分重要的作用,T细胞的不同方向的分化决定哮喘疾病是否发生,Th1/Th2失衡及Th17/Treg失衡理论一直是研究的热点。Th1/Th2细胞之间处于相互抑制状态,正常情况下,Th1/Th2细胞处于恒定状态,哮喘患者Th1细胞功能下降,Th2细胞功能异常增高。Treg细胞数量减少、其抑制炎症反应的功能降低,而Th17细胞过度表达介导气道炎症。IL—21是CD4+Th2型细胞分泌的细胞因子,可以促进Th17细胞的分泌,有人认为Th17细胞又是IL—21生成的一个重要来源,由此推断IL—21可能参与哮喘疾病的发病。2009年,Rajshekhar Chatterjee第一次报道了IL-21基因多态性与哮喘显著相关。
IL-21功能的发挥依靠其受体的共用γ链,主要通过JAK-STAT途径传递信号。IL-21R蛋白由538个氨基酸组成,与IL-2Rβ链、IL-4R α链和IL-9R有同源性,N端有4个高度保守的Cys残基,跨膜区有"WSXWS"序列,胞浆区存在经典的信号传递亚单位boxl和box2基序,表明该受体具有信号传导功能。IL-21与IL-21R结合时,主要通过与γ c的偶联,激活JAK1和JAK3(特别是JAK3),活化的JAK酪氨酸激酶使STAT1和STAT3发生磷酸化,磷酸化STAT移行到细胞核内,起到活化T细胞核因子(NFAT)的作用,诱导IL-21基因表达。JAK-STAT途径参与许多细胞因子信号转导而影响Th细胞分化和Th细胞免疫偏移,是哮喘发病重要的信号通路。哮喘小鼠肺组织IL-21及IL-21R表达未见报道。本研究建立慢性哮喘小鼠动物模型,用不同的实验方法观察IL-21、IL-21R、STAT3、P-STAT3在哮喘小鼠肺组织的表达,使用地塞米松及酪氨酸激酶(JAK,janus Kinnase)抑制剂AG490进行干预,观察IL-21对哮喘气道炎症及气道重塑的影响,探讨IL-21/STAT3信号通路在哮喘发病中所起的作用。
研究目的
1、用不同的实验方法观察IL-21、IL-21R在小鼠及哮喘小鼠肺组织中的表达,观察IL-21及其受体与气道炎症、气道重塑指标的关系。
2、观察STAT3、活化后的STAT3(p-STAT3)在哮喘小鼠的表达及其与气道炎症、气道重塑的关系。
3、使用JAK通路抑制剂AG490腹腔注射后观察哮喘小鼠气道炎症及气道重塑指标的变化,观察肺组织总STAT3、p-STAT3的表达变化,观察IL-21表达的变化。
4、使用地塞米松干预哮喘小鼠后,观察对哮喘小鼠气道炎症的改变及对IL-21/STAT3信号通路的影响。
研究方法
1、动物模型的建立和分组
SPF级BALB/C雌性小鼠,共44只,随机分为4组,分别为对照组、哮喘组、AG490组、地塞米松组,每组11只。哮喘组于第1天、第14天每只小鼠腹腔注射0.2mL致敏液,致敏液含卵蛋白(OVA, Grade V)10ug,氢氧化铝凝胶100ug,第21天开始,雾化吸入25g/L的OVA溶液30min,每周3次,共8周。对照组以生理盐水代替OVA。地塞米松组在每次雾化OVA溶液前0.5h腹腔注射10mg/kg地塞米松溶液。AG490组是于第21天开始腹腔注射AG490,按每只小鼠每次450ug,每周3次,连续8周,每5mgAG490用0.5mL二甲基亚砜(0.05%)、9.5mL双蒸水充分溶解。
2、IL-21及IL-21R在慢性哮喘小鼠肺组织的表达
在末次雾化吸入24h后,左肺行肺泡灌洗,收集灌洗液(bronchoalveolar lavage fluid, BALF),离断颈椎处死小鼠。左肺组织放入液氮罐用于提取RNA及总蛋白。右肺组织石蜡包埋切片行HE染色,并用免疫组化染色观察IL-21、 IL-21R在小鼠肺组织中的表达,采用荧光定量PCR法检测肺组织IL-21mRNA、 IL-21R mRNA的表达,western blot法检测IL-21蛋白、IL-21R蛋白的表达。
3、IL-21及IL-21R与气道炎症和气道重塑的关系
肺泡灌洗液离心后,取沉渣用血细胞计数板进行细胞分类及计数,重点观察细胞总数及嗜酸性粒细胞数、中性粒细胞计数变化：肺组织行HE染色观察气道及肺组织病理学改变；肺组织石蜡切片HE染色后,用IPP6.0图像分析软件分析单位长度基底膜气道壁面积(气道壁厚度,total bronchial wall area/basement membrane perimeter, WAt/Pbm)、单位长度基底膜的气道平滑肌面积(平滑肌厚度,the area of the wall occupied by smooth muscle/basement membrane perimeter, WAm/Pbm);分析IL-21、IL-21R与WAt/Pbm、WAm/Pbm指标的相关性。
4、STAT3、p-STAT3在慢性哮喘小鼠肺组织的表达及使用JAK通路抑制剂AG490干预后的表达变化
用western blot及免疫组化方法检测小鼠肺组织p-STAT3蛋白表达,用荧光定量PCR检测肺组织STAT3的表达；观察哮喘小鼠使用了AG490干预后p-STAT3的表达的变化,观察小鼠气道炎症的改变及气道重塑指标的变化。
5、使用地塞米松药物干预后气道重塑的改变及IL-21、IL-21R、p-STAT3的表达变化
用IPP6.0图像分析软件分析地塞米松干预组小鼠气道壁厚度(WAt/Pbm)、平滑肌厚度(WAm/Pbm),免疫组化法及western blot法检测地塞米松组IL-21、 IL-21R、p-STAT3蛋白在肺组织中的表达,采用荧光定量PCR法检测IL-21mRNA、IL-21RmRNA的表达。
6、统计学处理
数据用x±s表示,经SPSS16.0统计软件进行统计分析。两样本均数比较采用两独立样本t检验,多组间比较采用单因素方差分析,多组样本均数两两比较,方差齐者用Bonferroni检验,方差不齐者用Dunnett's T3法检验；两变量的相关分析采用Bivariate过程的等级Pearson相关法。P<0.05为差异有统计学意义。
研究结果
1、44只小鼠全部存活,OVA致敏激发后,成功建立了慢性哮喘小鼠模型。对照组小鼠雾化后无明显异常反应,哮喘组小鼠激发后,出现烦躁不安、呼吸急促、腹肌抽搐、二便失禁等症状,用听诊器听诊可闻及哮鸣音。地塞米松组及AG490组症状相似,但程度明显减轻。肺组织HE染色镜下所见符合哮喘的病理学特征,哮喘组小鼠细支气管及伴行血管周围大量炎症细胞浸润,气道壁和肺组织中可见以嗜酸性粒细胞、淋巴细胞为主的炎症细胞浸润,支气管管壁增厚、管腔狭窄,支气管管腔内可见粘液栓。
2、免疫组化法结果表明哮喘小鼠同对照组小鼠肺组织均有IL-21、IL-21R的表达,哮喘组的表达较对照组表达增强(P<0.05),与western blot检测结果一致；荧光定量PCR法检测结果同样显示哮喘组小鼠IL-21mRNA、IL-21RmRNA表达较对照组增强(P<0.05)。反应哮喘组小鼠气道重塑指标的WAm/Pbm、 WAt/Pbm较对照组增加(P<0.05)。
3、哮喘组、AG490组、地塞米松组小鼠肺泡灌洗液中细胞总数、中性粒细胞、嗜酸粒细胞和淋巴细胞计数均较正常对照组明显增多(P<0.01);地塞米松治疗组与哮喘组相比,细胞总数、中性粒细胞、嗜酸粒细胞和淋巴细胞计数明显减少(P<0.01),AG490组上述指标较哮喘组也有所下降(P<0.05)；地塞米松组肺泡灌洗液中细胞总数及嗜酸性粒细胞、淋巴细胞、中性粒细胞计数低于AG490组(P<0.05)。
4、免疫组化及western blot分析显示对照组几乎无p-STAT3蛋白表达,哮喘组小鼠肺组织p-STAT3蛋白表达较对照组增加(P<0.05),AG490组小鼠肺组织p-STAT3蛋白表达较哮喘组减弱(P<0.05)；荧光定量PCR显示哮喘组、AG490组小鼠肺组织STAT3mRNA较对照组增加(P<0.05),两组相比,无明显差异(P>0.05)。
5、地塞米松组小鼠气道重构指标WAm/Pbm、WAt/Pbm高于对照组(P<0.05),低于哮喘组(P<0.05)。Western blot法检测地塞米松组IL-21、IL-21R、 p-STAT3在肺组织中的表达较哮喘组下降(P<0.05)；AG490组小鼠IL-21的表达较哮喘组下降(P<0.05),与地塞米松组相比无统计学意义(P>0.05),AG490组小鼠肺组织p-STAT3的表达与地塞米松组相比,无统计学意义(P>0.05);AG490组小鼠肺组织STAT3mRNA的含量较对照增加(P<0.05),但与哮喘组小鼠相比,无统计学意义(P>0.05)。
6、相关性分析结果：用western blot法测定IL-21蛋白、IL-21R蛋白、p-STAT3蛋白,结果表明上述指标与小鼠肺泡灌洗液中细胞总数、嗜酸性粒细胞计数均呈正相关(r>0.5, p<0.01);IL-21、IL-21R、p-STAT3的蛋白表达量与小鼠气道重构指标WAm/Pbm、WAt/Pbm均呈正相关(r>0.5,p<0.01)。
研究结论
IL-21主要通过JAK-STAT途径传递信号,本实验首先用不同实验方法观察了IL-21及其受体在正常对照组及哮喘模型组小鼠肺组织中的表达,观察了总STAT3及活化后的形式p-STAT3在小鼠肺组织中的表达,在建立慢性哮喘小鼠动物模型基础上,使用JAK通路抑制剂AG490及地塞米松干预后观察上述指标的变化。得出以下结论：
1、IL-21及其受体在小鼠肺组织中有表达,哮喘小鼠肺组织的表达较对照组增高,IL-21在哮喘发病中是重要的促炎因子,IL-21与受体结合参与慢性哮喘小鼠发病,并参与了气道重塑过程；
2、STAT3及活化后的STAT3参与了哮喘发病,可能影响了气道重塑：
3、哮喘模型经AG490作用后STAT3的活化程度降低,气道炎症及重塑程度减轻,其疗效与地塞米松大致相当。
4、IL—21通过激活STAT3信号通路参与了哮喘疾病的发生,地塞米松下调了IL-21及其受体的表达,抑制了肺组织STAT3的活化,减轻了气道炎症和气道重塑,IL-21/STAT3信号转导通路可能为地塞米松治疗哮喘的作用靶点之一。
Background
Bronchial asthma (asthma) is one of common and frequently-occurring diseases in the respiratory system, and it's a great threat to human health. According to the statistical results of the world health organization, there are about300million asthma patients all over the world. About fifteen million people lose labor ability because of this disease every year. According to incomplete statistics, there are at least15million to20million asthma patients in our country.The morbidity and mortality of bronchial asthma all over the world are increasing year by year, this kind of situation causes enormous economic losses every year, asthma has become a serious public health problem. Asthma episodes frequently and the cause is complex. Under the interaction of host susceptibility and environmental factors,bronchial asthma is a syndrome which basic pathophysiological change is chronic airway inflammation and airway remodeling.It's a kind of disease that can be controlled but difficult to cure. Now, more and more researchers study about asthma's pathogenesis,do their best to prevent and control the allergic inflammation and the development about asthma.
In the development of airway inflammation in asthma, the naive T cells in CD4+T cells become ThO cells by the allergen-induced activation. ThO cells continue to differentiate in the direction of the Th2cells and generate a lot of Th2cells. Th2cells in the dominant status, they can induce Th2inflammation on a large scale.,the inflammation mediate by the downstream of the IgE and/or not the IgE.In the onset process of asthma, the infiltration, aggregation, activation and release of inflammatory cells in airway tissue are the main features, cytokines play an important role in the process. The repeated stimulation of chronic inflammation and the destruction of the epithelial structure can cause asthma airway remodeling. At present, the airway inflammation and airway remodeling in asthma as two pathological features are gradually accepted by people. Various cytokines and inflammatory mediators can lead to the airway remodeling.but the mechanism is not fully clear.
Interleukin21(IL-21) is a cytokine found in2000, belongs to a type I cytokine family, mainly secreted and synthesed by CD4+Th2cell, and have homology with IL-2, IL-4and IL-15. Since IL-21is found, about its biological functions has become a research hot spot. The effect of IL-21is various, mainly due to IL-21receptors are widely distributed. IL-21is considered to be secretion of cytokines activated by peripheral blood T cell, now be considered to be synthesized from a series of differentiated CD4+Th cells. Biological functions mainly to stimulate T cell proliferation, NK cell proliferation and differentiation and CD40specific response of B cell proliferation. IL-21receptors (IL-21R) express in lymphatic hematopoietic cells, fibroblasts, keratinocytes, intestinal epithelial cells. Experiments show that IL-21plays an important role in innate immunity, adaptive immunity and cellular immune response.IL-21is associated with a variety of autoimmune diseases and inflammatory diseases, such as rheumatoid disease, systemic lupus erythematosus, systemic sclerosis disease, inflammatory bowel disease, allergic disease, it plays an important role in immune regulationfor these diseases.
IL-21plays a key role in Th cell differentiation, bronchial asthma is a kind of allergic disease caused by immune abnormalities, immune dysfunction plays an important role in asthma incidence.T cell differentiate in different direction to determine whether asthma disease occurs or not, Th1/Th2imbalance and imbalance of Th17/Treg theory have always been research hot spot. Th1/Th2cells are in a state of reciprocal inhibition. Under normal circumstances, Th1/Th2cells in a constant state, but in patients with asthma,the Th1cells function decline and the function of Th2cells increase abnormally. When the mumber of treg cells decline, the inhibition of inflammatory response function is reduced.The overexpression of Th17cells mediate airway inflammation. IL-21is a cytokine secreted by Th2CD4+cells, can promote the secretion of Th17cells, some people think that Th17cells are an important generation source of IL-21, thus infer that IL-21may involved in the pathogenesis of asthma disease. In2009, Rajshekhar Chatterjee for the first time reported IL-21gene polymorphism was significantly associated with asthma.
The functions of IL-21depend on its receptor shared gamma chain, IL-21mainly transmit signal through JAK-STAT signal pathway. IL-21R protein is composed of538amino acids, and have homology with IL-2R beta chain, IL-4R alpha chain and IL-9R, N side has four highly conservative Cys residue, transmembrane region has "WSXWS sequence, cytoplasm area exists classic signaling subunits boxl and box2motif, show that the receptor has signaling function.IL-21combines with IL-21R,mainly through coupling with gamma c, after that,they activate the JAK1and JAK3(especially JAK3), activation of the JAK tyrosine kinase phosphorylate STAT1and STAT3, phosphorylation STAT transfers to the nucleus, have the effect of activating T cell nucleus factor (NFAT) and induce expression of IL-21gene. JAK-STAT signal participates in transduction pathways of many cytokines, influences Th cell differentiation and Th cell immune deviation,it's an important signaling pathways in asthma. But the expression of IL-21and IL-21R in lung tissue of asthmatic mice is not reported. We set up animal model of chronic asthma mice and observe the expression of IL-21,IL-21R, STAT3, p-STAT3in lung tissue of asthmatic mice, intervene the asthmatic mice by using dexamethasone and tyrosine kinases (JAK, janus Kinnase) inhibitors AG490.We study the influence of IL-21on asthmatic airway inflammation and airway remodeling, discusse the mechanism of IL-21/STAT3signal pathway in the pathogenesis of asthma.
Objectives
1To observe the expression of IL-21and IL-21R in lung tissue of mice and asthmatic mice with different experimental methods, observe the relationship between IL-21, its receptor and airway inflammation, airway remodeling.
2To observe the expression of STAT3and activation of STAT3(p-STAT3) in asthmatic mice,observe the relationship between these index and airway inflammation, airway remodeling.
3After intraperitoneal injection of JAK pathway inhibitor AG490,To observe the changes in asthma airway inflammation and airway remodeling index, and observe the expression changes of total STAT3and p-STAT3in lung tissue.
4To observe the changes of airway inflammation in asthma mice and the influence on IL-21/STAT3signal pathways by using of dexamethasone to intervent the asthmatic mice.
Methods
1The establishment and group of animal models
44SPF BALB/C female mice were randomly divided into4groups, control group, asthma group, AG490group and dexamethasone group,11mice each group. In asthma group, mice were sensitized by means of intraperitoneal injection of OVA (10μg) precipitated with aluminum hydroxide (100μg) on days1and14,from21st day,25g/L of OVA solution were atomized inhalation for30min, three times a week for8weeks. The control group with normal saline instead of OVA. In dexamethasone group,0.5h before aerosolized OVA, each mouse was given10mg/kg dexamethasone by means of intraperitoneal injection. In AG490group,each mouse was injected AG490, per mouse450ug/each time, three times a week, every5mg AG490with0.5mL dimethyl phosphite maple (0.05%),9.5mL dual steaming water dissolves.
2The expression of IL-21and IL-21R in lung tissue of chronic asthma mice
Tweenty-four hours after the last atomization inhalation, the left lungs were lavaged, the bronchoalveolar lavage fluid(BALF) were collectted. The mice were sacrificed,right lung tissue were putted in liquid nitrogen tank that can be used to extract total RNA and protein. Left lung tissue paraffin embedding sectioning lines for HE staining.The expression of IL-21and IL-21R in the lung tissue were analyzed by immunohistochemical method. The mRNA expression of IL-21and IL-21R were detected by fluorescent quantitative PCR method.Western blotting method were used to detect the protein expression of IL-21and IL-21R.
3The relationship between IL-21,IL-21R and airway inflammation,airway remodeling.
After centrifugation of alveolar lavage fluid, collected sediment to classifyt and count the cells by blood cells count plate.Observed mainly the total amount of cells and the count of eosinophils and neutrophils. Pathology change of airway and lung tissue were observed by HE staining. After HE staining,we used IPP image analysis software6.0to analyse unit length basement membrane airway wall area (airway wall thickness, total bronchial wall area/basement membrane perimeter, WAt/Pbm), area per unit length basement membrane of airway smooth muscle (airway smooth muscle thickness,the area of the wall occupied by smooth muscle/basement membrane perimeter, WAm/Pbm). The correlation between the index of IL-21,IL-21R and WAt/Pbm,WAm/Pbm were analysed.
4The expression of STAT3and p-STAT3in chronic asthmatic mice and the changes after the intervention with AG490(JAK pathway inhibitor)
The protein expression of p-STAT3in lung tissue were detected by western blotting and immunohistochemical methods.The expression of STAT3were observed by fluorescence quantitative PCR. After using AG490,we observed the expression changes of STAT3and p-STAT3, the changes in the airway inflammation and airway remodelling in mice.
5The airway remodeling changes and the index of IL-21,IL-21R and p-STAT3after the intervention of dexhmesone
To analyse the airway wall thickness (WAt/Pbm) and smooth muscle thickness (WAm/Pbm) in dexamethasone intervention group by using IPP6.0image analysis software. Immunohistochemical method and western blotting method were used to detect the protein expression of IL-21, IL-21R and p-STAT3in lung tissue of dexamethasone group. The expression of IL-21mRNA and IL-21RmRNA in the lung tissue were detected by fluorescent quantitative PCR method.
6Statistical processing
All data were expressed as means±SD. All statistical analysis was operated with SPSS16.0statistic software.Difference among two independent groups was analyzed by t-test. Differences among one-way designed groups were analyzed by one-way ANOVA followed by Bonferroni test. When homogeneity of variance can not achieve, multiple comparisons were analyzed by Dunnett's T3test. Correlations were analyzed by Pearson test. A value of P<0.05was considered statistically significant.
Results
1.44mice were all alive, after OVA sensitized/challenged,the mouse model of chronic asthma was successfully established.Control group mice had no obvious abnormal reaction after atomization, asthma group of mice appeared symptoms such as dysphoria, shortness of breath, abdominal muscle twitching, mainland incontinence, and could be heard the wheezing sound with a stethoscope auscultation. Dexamethasone group and AG490group had similar but significantly gentle symptoms as asthmatic group. The lung tissue by HE staining seen microscopically conformed to the pathological features of asthma.Around bronchioles and vessels,there were a large number of inflammatory cells infiltration in asthmatic mice.Airway wall and lung tissue visible was priority to eosinophilic granulocyte and lymphocyte of inflammatory cell infiltration, bronchial wall thickening and luminal stenosis, bronchial lumen mucous plug.
2.The results showed that IL-21and IL-21R were all expressed in lung tissue of asthma group and control group by immunohistochemical method.Compare with the control group, the expression of IL-21and IL-21R enhanced in asthma group (P<0.05), and the result was consistent with the means of western blot.It also showed that the mRNA expression of IL-21and IL-21R in asthma group were much more than control group (P<0.05). The index of WAm/Pbm and WAt/Pbm about airway remodeling in asthma mice were higher than the control group (P<0.05).
3.In group of asthma, AG490and dexamethasone, the total number of cells, neutrophils, acidophilic granulocyte and lymphocyte count in alveolar lavage fluid were significantly increased compared with normal control group (P<0.01). Compared with the asthma group, the total number of cells, neutrophils, acidophilic granulocyte and lymphocyte count in dexamethasone treatment group were significantly reduced (P<0.01), the index in AG490group also fell comaring with asthmatic group (P<0.05).Compared AG490group with dexamethasone group, total number of cells, eosinophils, lymphocytes and neutrophils in alveolar lavage fluid in dexamethasone group were lower than AG490group (P<0.05).
4.Immunohistochemistry and western blot analysis showed that the control group almost without the protein expression of P-STAT3.In contrast to control group, increased protein expression of P-STAT3wer detected in lung tissue (P<0.05).The expression of P-STAT3protein in lung tissue of AG490group were weaker than in the asthma group (P<0.05).Compared to the control group Fluorescence quantitative PCR showed that the expression of STAT3mRNA in asthma group and AG490group increased (P<0.05), between the two groups, there were no significant differences(P>0.05).
5.The index of WAm/Pbm and WAt/Pbm about airway remodelling in dexamethasone group was higher than the control group (P<0.05), lower than the asthma group (P<0.05). The protein expression of IL-21, IL-21R and p-STAT3in the lung tissue of dexamethasone group declined compared with the asthma group by using western blot method (P<0.05).The expression of IL-21in AG490group fell compared with the asthma group (P<0.05).No statistical significance were observed between the AG490group and dexamethasone group (P>0.05). For the protein expression of p-STAT3,there was no statistical significance between AG490group and dexamethasone group (P>0.05). Compared to control group,the level of STAT3mRNA in lung tissue of AG490group increased(P<0.05). However, there was no statistical significance between asthma group and AG490group (P>0.05).
6.Correlation analysis:The protein expression of IL-21, IL-21R, p-STAT3were detected by using western blot method, the results showed that these indicators and the total number of cells, eosinophil count in the mice alveolar lavage fluid were positively correlated (r>0.5, P<0.01), the protein expression of IL-21, IL-21R,p-STAT3and the index of WAm/Pbm, WAt/Pbm were positively correlated (r>0.5, P<0.01).
Conclusions
IL-21mainly transmit signal through the JAK-STAT pathway, the experiment firstly used different methods to observe the expression of IL-21and its receptor in the lung tissue in control group and asthma group. The expression changes of STAT3and activation of STAT3in lung tissue of mice were observed. After the intervention of JAK pathway inhibitors AG490and dexamethasone, the index changes were observed. Draw the following conclusions:
1.IL-21and its receptor express in the lung tissue of mice. Compare to the control group, the expression in asthmatic group is increased.IL-21is an important proinflammatory factor in the incidence of asthma. IL-21that combines with it's receptor involve in the onset of chronic asthma and participate in the process of airway remodeling.
2.STAT3and activation of STAT3involve in the pathogenesis of asthma, may affect the airway remodeling.
3.After the intervention of AG490in asthma model, the expression of p-STAT3is reduced, degree of airway inflammation and airway remodeling is reduced, its curative effect are roughly equal to dexamethasone.
4.IL-21involve in the onset of asthma disease by activating STAT3signaling pathway, dexamethasone downgrade the expression of IL-21and its receptors, inhibit the activation of STAT3in the lung tissue, relieve the airway inflammation and airway remodeling, IL-21/STAT3signal transduction pathway may be one of the targets of dexamethasone in treatment of asthma.
支气管哮喘(简称哮喘)(bronchial asthma,asthma)是呼吸系统常见病、多发病之一,对人类健康构成了很大的威胁。世界卫生组织的统计结果显示,全球约有3亿哮喘患者,其中每年约有一千五百万人因为该病而丧失劳动能力。据不完全统计,我国至少也有1500万到2000万哮喘患者,世界各地支气管哮喘的发病率、死亡率都呈逐年增高趋势,每年造成了异常巨大的经济损失,哮喘已经成为严重的公共卫生问题。哮喘反复发作且病因复杂,在宿主易感因素与环境因素相互作用下,以慢性气道炎症和气道重塑为基本病理生理改变的综合征,是一种可以控制但很难治愈的疾病。研究哮喘的发病机理,预防和控制哮喘变态反应性炎症的发生和发展,受到越来越多研究者的关注。
在哮喘气道炎症发展过程中,CD4+T细胞群中的初始T细胞经变应原诱导激活后成为Th0细胞,并继续朝Th2细胞方向分化,生成大量的Th2细胞,Th2细胞占据优势地位,可大规模诱发下游的IgE和／或非IgE介导的Th2炎症。哮喘发病过程中,炎性细胞在气道组织中的浸润、聚集、激活和释放是其主要特征,在此过程中细胞因子发挥了重要作用。慢性炎症反复刺激和上皮结构的破坏可以引起哮喘的气道重塑。目前,气道炎症与气道重塑作为哮喘的两个病理特征逐渐被人们所接受。引起气道重塑的各种细胞因子、炎症介质等在气道重塑中发挥作用的机制尚未完全明确。
白细胞介素21(interleukin21,IL-21)是2000年发现的一种细胞因子,属于Ⅰ型细胞因子家族,主要是由CD4+Th2细胞合成和分泌,与IL-2、IL-4和IL-15具有同源性。自从发现了IL-21以后,关于它的生物学功能成为了研究的热点。IL-21的效应是多样的,主要是由于IL-21的受体分布广泛。IL-21开始时被认为是激活的外周血T细胞分泌的细胞因子,现在被认为可以由一系列分化型CD4+Th细胞合成,生物学功能主要是刺激T细胞增殖、NK细胞增殖和分化以及CD40特异性应答B细胞的增殖。IL-21受体(IL-21R)表达于淋巴造血细胞、成纤维细胞、角质细胞、肠上皮细胞。实验证明,IL-21在先天性免疫、适应性免疫及细胞内的免疫应答中发挥重要的作用,与多种自身免疫性疾病及炎性疾病有关,例如类风关、系统性红斑狼疮、系统性硬化病、炎症性肠病、过敏性疾病,对于这些疾病有重要的免疫调节作用。
IL—21在Th细胞的分化方面起了关键作用,支气管哮喘是一种免疫异常导致的变态反应性疾病,免疫功能紊乱在哮喘发病中起十分重要的作用,T细胞的不同方向的分化决定哮喘疾病是否发生,Th1/Th2失衡及Th17/Treg失衡理论一直是研究的热点。Th1/Th2细胞之间处于相互抑制状态,正常情况下,Th1/Th2细胞处于恒定状态,哮喘患者Th1细胞功能下降,Th2细胞功能异常增高。Treg细胞数量减少、其抑制炎症反应的功能降低,而Th17细胞过度表达介导气道炎症。IL—21是CD4+Th2型细胞分泌的细胞因子,可以促进Th17细胞的分泌,有人认为Th17细胞又是IL—21生成的一个重要来源,由此推断IL—21可能参与哮喘疾病的发病。2009年,Rajshekhar Chatterjee第一次报道了IL-21基因多态性与哮喘显著相关。
IL-21功能的发挥依靠其受体的共用γ链,主要通过JAK-STAT途径传递信号。IL-21R蛋白由538个氨基酸组成,与IL-2Rβ链、IL-4R α链和IL-9R有同源性,N端有4个高度保守的Cys残基,跨膜区有"WSXWS"序列,胞浆区存在经典的信号传递亚单位boxl和box2基序,表明该受体具有信号传导功能。IL-21与IL-21R结合时,主要通过与γ c的偶联,激活JAK1和JAK3(特别是JAK3),活化的JAK酪氨酸激酶使STAT1和STAT3发生磷酸化,磷酸化STAT移行到细胞核内,起到活化T细胞核因子(NFAT)的作用,诱导IL-21基因表达。JAK-STAT途径参与许多细胞因子信号转导而影响Th细胞分化和Th细胞免疫偏移,是哮喘发病重要的信号通路。哮喘小鼠肺组织IL-21及IL-21R表达未见报道。本研究建立慢性哮喘小鼠动物模型,用不同的实验方法观察IL-21、IL-21R、STAT3、P-STAT3在哮喘小鼠肺组织的表达,使用地塞米松及酪氨酸激酶(JAK,janus Kinnase)抑制剂AG490进行干预,观察IL-21对哮喘气道炎症及气道重塑的影响,探讨IL-21/STAT3信号通路在哮喘发病中所起的作用。
研究目的
1、用不同的实验方法观察IL-21、IL-21R在小鼠及哮喘小鼠肺组织中的表达,观察IL-21及其受体与气道炎症、气道重塑指标的关系。
2、观察STAT3、活化后的STAT3(p-STAT3)在哮喘小鼠的表达及其与气道炎症、气道重塑的关系。
3、使用JAK通路抑制剂AG490腹腔注射后观察哮喘小鼠气道炎症及气道重塑指标的变化,观察肺组织总STAT3、p-STAT3的表达变化,观察IL-21表达的变化。
4、使用地塞米松干预哮喘小鼠后,观察对哮喘小鼠气道炎症的改变及对IL-21/STAT3信号通路的影响。
研究方法
1、动物模型的建立和分组
SPF级BALB/C雌性小鼠,共44只,随机分为4组,分别为对照组、哮喘组、AG490组、地塞米松组,每组11只。哮喘组于第1天、第14天每只小鼠腹腔注射0.2mL致敏液,致敏液含卵蛋白(OVA, Grade V)10ug,氢氧化铝凝胶100ug,第21天开始,雾化吸入25g/L的OVA溶液30min,每周3次,共8周。对照组以生理盐水代替OVA。地塞米松组在每次雾化OVA溶液前0.5h腹腔注射10mg/kg地塞米松溶液。AG490组是于第21天开始腹腔注射AG490,按每只小鼠每次450ug,每周3次,连续8周,每5mgAG490用0.5mL二甲基亚砜(0.05%)、9.5mL双蒸水充分溶解。
2、IL-21及IL-21R在慢性哮喘小鼠肺组织的表达
在末次雾化吸入24h后,左肺行肺泡灌洗,收集灌洗液(bronchoalveolar lavage fluid, BALF),离断颈椎处死小鼠。左肺组织放入液氮罐用于提取RNA及总蛋白。右肺组织石蜡包埋切片行HE染色,并用免疫组化染色观察IL-21、 IL-21R在小鼠肺组织中的表达,采用荧光定量PCR法检测肺组织IL-21mRNA、 IL-21R mRNA的表达,western blot法检测IL-21蛋白、IL-21R蛋白的表达。
3、IL-21及IL-21R与气道炎症和气道重塑的关系
肺泡灌洗液离心后,取沉渣用血细胞计数板进行细胞分类及计数,重点观察细胞总数及嗜酸性粒细胞数、中性粒细胞计数变化：肺组织行HE染色观察气道及肺组织病理学改变；肺组织石蜡切片HE染色后,用IPP6.0图像分析软件分析单位长度基底膜气道壁面积(气道壁厚度,total bronchial wall area/basement membrane perimeter, WAt/Pbm)、单位长度基底膜的气道平滑肌面积(平滑肌厚度,the area of the wall occupied by smooth muscle/basement membrane perimeter, WAm/Pbm);分析IL-21、IL-21R与WAt/Pbm、WAm/Pbm指标的相关性。
4、STAT3、p-STAT3在慢性哮喘小鼠肺组织的表达及使用JAK通路抑制剂AG490干预后的表达变化
用western blot及免疫组化方法检测小鼠肺组织p-STAT3蛋白表达,用荧光定量PCR检测肺组织STAT3的表达；观察哮喘小鼠使用了AG490干预后p-STAT3的表达的变化,观察小鼠气道炎症的改变及气道重塑指标的变化。
5、使用地塞米松药物干预后气道重塑的改变及IL-21、IL-21R、p-STAT3的表达变化
用IPP6.0图像分析软件分析地塞米松干预组小鼠气道壁厚度(WAt/Pbm)、平滑肌厚度(WAm/Pbm),免疫组化法及western blot法检测地塞米松组IL-21、 IL-21R、p-STAT3蛋白在肺组织中的表达,采用荧光定量PCR法检测IL-21mRNA、IL-21RmRNA的表达。
6、统计学处理
数据用x±s表示,经SPSS16.0统计软件进行统计分析。两样本均数比较采用两独立样本t检验,多组间比较采用单因素方差分析,多组样本均数两两比较,方差齐者用Bonferroni检验,方差不齐者用Dunnett's T3法检验；两变量的相关分析采用Bivariate过程的等级Pearson相关法。P<0.05为差异有统计学意义。
研究结果
1、44只小鼠全部存活,OVA致敏激发后,成功建立了慢性哮喘小鼠模型。对照组小鼠雾化后无明显异常反应,哮喘组小鼠激发后,出现烦躁不安、呼吸急促、腹肌抽搐、二便失禁等症状,用听诊器听诊可闻及哮鸣音。地塞米松组及AG490组症状相似,但程度明显减轻。肺组织HE染色镜下所见符合哮喘的病理学特征,哮喘组小鼠细支气管及伴行血管周围大量炎症细胞浸润,气道壁和肺组织中可见以嗜酸性粒细胞、淋巴细胞为主的炎症细胞浸润,支气管管壁增厚、管腔狭窄,支气管管腔内可见粘液栓。
2、免疫组化法结果表明哮喘小鼠同对照组小鼠肺组织均有IL-21、IL-21R的表达,哮喘组的表达较对照组表达增强(P<0.05),与western blot检测结果一致；荧光定量PCR法检测结果同样显示哮喘组小鼠IL-21mRNA、IL-21RmRNA表达较对照组增强(P<0.05)。反应哮喘组小鼠气道重塑指标的WAm/Pbm、 WAt/Pbm较对照组增加(P<0.05)。
3、哮喘组、AG490组、地塞米松组小鼠肺泡灌洗液中细胞总数、中性粒细胞、嗜酸粒细胞和淋巴细胞计数均较正常对照组明显增多(P<0.01);地塞米松治疗组与哮喘组相比,细胞总数、中性粒细胞、嗜酸粒细胞和淋巴细胞计数明显减少(P<0.01),AG490组上述指标较哮喘组也有所下降(P<0.05)；地塞米松组肺泡灌洗液中细胞总数及嗜酸性粒细胞、淋巴细胞、中性粒细胞计数低于AG490组(P<0.05)。
4、免疫组化及western blot分析显示对照组几乎无p-STAT3蛋白表达,哮喘组小鼠肺组织p-STAT3蛋白表达较对照组增加(P<0.05),AG490组小鼠肺组织p-STAT3蛋白表达较哮喘组减弱(P<0.05)；荧光定量PCR显示哮喘组、AG490组小鼠肺组织STAT3mRNA较对照组增加(P<0.05),两组相比,无明显差异(P>0.05)。
5、地塞米松组小鼠气道重构指标WAm/Pbm、WAt/Pbm高于对照组(P<0.05),低于哮喘组(P<0.05)。Western blot法检测地塞米松组IL-21、IL-21R、 p-STAT3在肺组织中的表达较哮喘组下降(P<0.05)；AG490组小鼠IL-21的表达较哮喘组下降(P<0.05),与地塞米松组相比无统计学意义(P>0.05),AG490组小鼠肺组织p-STAT3的表达与地塞米松组相比,无统计学意义(P>0.05);AG490组小鼠肺组织STAT3mRNA的含量较对照增加(P<0.05),但与哮喘组小鼠相比,无统计学意义(P>0.05)。
6、相关性分析结果：用western blot法测定IL-21蛋白、IL-21R蛋白、p-STAT3蛋白,结果表明上述指标与小鼠肺泡灌洗液中细胞总数、嗜酸性粒细胞计数均呈正相关(r>0.5, p<0.01);IL-21、IL-21R、p-STAT3的蛋白表达量与小鼠气道重构指标WAm/Pbm、WAt/Pbm均呈正相关(r>0.5,p<0.01)。
研究结论
IL-21主要通过JAK-STAT途径传递信号,本实验首先用不同实验方法观察了IL-21及其受体在正常对照组及哮喘模型组小鼠肺组织中的表达,观察了总STAT3及活化后的形式p-STAT3在小鼠肺组织中的表达,在建立慢性哮喘小鼠动物模型基础上,使用JAK通路抑制剂AG490及地塞米松干预后观察上述指标的变化。得出以下结论：
1、IL-21及其受体在小鼠肺组织中有表达,哮喘小鼠肺组织的表达较对照组增高,IL-21在哮喘发病中是重要的促炎因子,IL-21与受体结合参与慢性哮喘小鼠发病,并参与了气道重塑过程；
2、STAT3及活化后的STAT3参与了哮喘发病,可能影响了气道重塑：
3、哮喘模型经AG490作用后STAT3的活化程度降低,气道炎症及重塑程度减轻,其疗效与地塞米松大致相当。
4、IL—21通过激活STAT3信号通路参与了哮喘疾病的发生,地塞米松下调了IL-21及其受体的表达,抑制了肺组织STAT3的活化,减轻了气道炎症和气道重塑,IL-21/STAT3信号转导通路可能为地塞米松治疗哮喘的作用靶点之一。
Background
Bronchial asthma (asthma) is one of common and frequently-occurring diseases in the respiratory system, and it's a great threat to human health. According to the statistical results of the world health organization, there are about300million asthma patients all over the world. About fifteen million people lose labor ability because of this disease every year. According to incomplete statistics, there are at least15million to20million asthma patients in our country.The morbidity and mortality of bronchial asthma all over the world are increasing year by year, this kind of situation causes enormous economic losses every year, asthma has become a serious public health problem. Asthma episodes frequently and the cause is complex. Under the interaction of host susceptibility and environmental factors,bronchial asthma is a syndrome which basic pathophysiological change is chronic airway inflammation and airway remodeling.It's a kind of disease that can be controlled but difficult to cure. Now, more and more researchers study about asthma's pathogenesis,do their best to prevent and control the allergic inflammation and the development about asthma.
In the development of airway inflammation in asthma, the naive T cells in CD4+T cells become ThO cells by the allergen-induced activation. ThO cells continue to differentiate in the direction of the Th2cells and generate a lot of Th2cells. Th2cells in the dominant status, they can induce Th2inflammation on a large scale.,the inflammation mediate by the downstream of the IgE and/or not the IgE.In the onset process of asthma, the infiltration, aggregation, activation and release of inflammatory cells in airway tissue are the main features, cytokines play an important role in the process. The repeated stimulation of chronic inflammation and the destruction of the epithelial structure can cause asthma airway remodeling. At present, the airway inflammation and airway remodeling in asthma as two pathological features are gradually accepted by people. Various cytokines and inflammatory mediators can lead to the airway remodeling.but the mechanism is not fully clear.
Interleukin21(IL-21) is a cytokine found in2000, belongs to a type I cytokine family, mainly secreted and synthesed by CD4+Th2cell, and have homology with IL-2, IL-4and IL-15. Since IL-21is found, about its biological functions has become a research hot spot. The effect of IL-21is various, mainly due to IL-21receptors are widely distributed. IL-21is considered to be secretion of cytokines activated by peripheral blood T cell, now be considered to be synthesized from a series of differentiated CD4+Th cells. Biological functions mainly to stimulate T cell proliferation, NK cell proliferation and differentiation and CD40specific response of B cell proliferation. IL-21receptors (IL-21R) express in lymphatic hematopoietic cells, fibroblasts, keratinocytes, intestinal epithelial cells. Experiments show that IL-21plays an important role in innate immunity, adaptive immunity and cellular immune response.IL-21is associated with a variety of autoimmune diseases and inflammatory diseases, such as rheumatoid disease, systemic lupus erythematosus, systemic sclerosis disease, inflammatory bowel disease, allergic disease, it plays an important role in immune regulationfor these diseases.
IL-21plays a key role in Th cell differentiation, bronchial asthma is a kind of allergic disease caused by immune abnormalities, immune dysfunction plays an important role in asthma incidence.T cell differentiate in different direction to determine whether asthma disease occurs or not, Th1/Th2imbalance and imbalance of Th17/Treg theory have always been research hot spot. Th1/Th2cells are in a state of reciprocal inhibition. Under normal circumstances, Th1/Th2cells in a constant state, but in patients with asthma,the Th1cells function decline and the function of Th2cells increase abnormally. When the mumber of treg cells decline, the inhibition of inflammatory response function is reduced.The overexpression of Th17cells mediate airway inflammation. IL-21is a cytokine secreted by Th2CD4+cells, can promote the secretion of Th17cells, some people think that Th17cells are an important generation source of IL-21, thus infer that IL-21may involved in the pathogenesis of asthma disease. In2009, Rajshekhar Chatterjee for the first time reported IL-21gene polymorphism was significantly associated with asthma.
The functions of IL-21depend on its receptor shared gamma chain, IL-21mainly transmit signal through JAK-STAT signal pathway. IL-21R protein is composed of538amino acids, and have homology with IL-2R beta chain, IL-4R alpha chain and IL-9R, N side has four highly conservative Cys residue, transmembrane region has "WSXWS sequence, cytoplasm area exists classic signaling subunits boxl and box2motif, show that the receptor has signaling function.IL-21combines with IL-21R,mainly through coupling with gamma c, after that,they activate the JAK1and JAK3(especially JAK3), activation of the JAK tyrosine kinase phosphorylate STAT1and STAT3, phosphorylation STAT transfers to the nucleus, have the effect of activating T cell nucleus factor (NFAT) and induce expression of IL-21gene. JAK-STAT signal participates in transduction pathways of many cytokines, influences Th cell differentiation and Th cell immune deviation,it's an important signaling pathways in asthma. But the expression of IL-21and IL-21R in lung tissue of asthmatic mice is not reported. We set up animal model of chronic asthma mice and observe the expression of IL-21,IL-21R, STAT3, p-STAT3in lung tissue of asthmatic mice, intervene the asthmatic mice by using dexamethasone and tyrosine kinases (JAK, janus Kinnase) inhibitors AG490.We study the influence of IL-21on asthmatic airway inflammation and airway remodeling, discusse the mechanism of IL-21/STAT3signal pathway in the pathogenesis of asthma.
Objectives
1To observe the expression of IL-21and IL-21R in lung tissue of mice and asthmatic mice with different experimental methods, observe the relationship between IL-21, its receptor and airway inflammation, airway remodeling.
2To observe the expression of STAT3and activation of STAT3(p-STAT3) in asthmatic mice,observe the relationship between these index and airway inflammation, airway remodeling.
3After intraperitoneal injection of JAK pathway inhibitor AG490,To observe the changes in asthma airway inflammation and airway remodeling index, and observe the expression changes of total STAT3and p-STAT3in lung tissue.
4To observe the changes of airway inflammation in asthma mice and the influence on IL-21/STAT3signal pathways by using of dexamethasone to intervent the asthmatic mice.
Methods
1The establishment and group of animal models
44SPF BALB/C female mice were randomly divided into4groups, control group, asthma group, AG490group and dexamethasone group,11mice each group. In asthma group, mice were sensitized by means of intraperitoneal injection of OVA (10μg) precipitated with aluminum hydroxide (100μg) on days1and14,from21st day,25g/L of OVA solution were atomized inhalation for30min, three times a week for8weeks. The control group with normal saline instead of OVA. In dexamethasone group,0.5h before aerosolized OVA, each mouse was given10mg/kg dexamethasone by means of intraperitoneal injection. In AG490group,each mouse was injected AG490, per mouse450ug/each time, three times a week, every5mg AG490with0.5mL dimethyl phosphite maple (0.05%),9.5mL dual steaming water dissolves.
2The expression of IL-21and IL-21R in lung tissue of chronic asthma mice
Tweenty-four hours after the last atomization inhalation, the left lungs were lavaged, the bronchoalveolar lavage fluid(BALF) were collectted. The mice were sacrificed,right lung tissue were putted in liquid nitrogen tank that can be used to extract total RNA and protein. Left lung tissue paraffin embedding sectioning lines for HE staining.The expression of IL-21and IL-21R in the lung tissue were analyzed by immunohistochemical method. The mRNA expression of IL-21and IL-21R were detected by fluorescent quantitative PCR method.Western blotting method were used to detect the protein expression of IL-21and IL-21R.
3The relationship between IL-21,IL-21R and airway inflammation,airway remodeling.
After centrifugation of alveolar lavage fluid, collected sediment to classifyt and count the cells by blood cells count plate.Observed mainly the total amount of cells and the count of eosinophils and neutrophils. Pathology change of airway and lung tissue were observed by HE staining. After HE staining,we used IPP image analysis software6.0to analyse unit length basement membrane airway wall area (airway wall thickness, total bronchial wall area/basement membrane perimeter, WAt/Pbm), area per unit length basement membrane of airway smooth muscle (airway smooth muscle thickness,the area of the wall occupied by smooth muscle/basement membrane perimeter, WAm/Pbm). The correlation between the index of IL-21,IL-21R and WAt/Pbm,WAm/Pbm were analysed.
4The expression of STAT3and p-STAT3in chronic asthmatic mice and the changes after the intervention with AG490(JAK pathway inhibitor)
The protein expression of p-STAT3in lung tissue were detected by western blotting and immunohistochemical methods.The expression of STAT3were observed by fluorescence quantitative PCR. After using AG490,we observed the expression changes of STAT3and p-STAT3, the changes in the airway inflammation and airway remodelling in mice.
5The airway remodeling changes and the index of IL-21,IL-21R and p-STAT3after the intervention of dexhmesone
To analyse the airway wall thickness (WAt/Pbm) and smooth muscle thickness (WAm/Pbm) in dexamethasone intervention group by using IPP6.0image analysis software. Immunohistochemical method and western blotting method were used to detect the protein expression of IL-21, IL-21R and p-STAT3in lung tissue of dexamethasone group. The expression of IL-21mRNA and IL-21RmRNA in the lung tissue were detected by fluorescent quantitative PCR method.
6Statistical processing
All data were expressed as means±SD. All statistical analysis was operated with SPSS16.0statistic software.Difference among two independent groups was analyzed by t-test. Differences among one-way designed groups were analyzed by one-way ANOVA followed by Bonferroni test. When homogeneity of variance can not achieve, multiple comparisons were analyzed by Dunnett's T3test. Correlations were analyzed by Pearson test. A value of P<0.05was considered statistically significant.
Results
1.44mice were all alive, after OVA sensitized/challenged,the mouse model of chronic asthma was successfully established.Control group mice had no obvious abnormal reaction after atomization, asthma group of mice appeared symptoms such as dysphoria, shortness of breath, abdominal muscle twitching, mainland incontinence, and could be heard the wheezing sound with a stethoscope auscultation. Dexamethasone group and AG490group had similar but significantly gentle symptoms as asthmatic group. The lung tissue by HE staining seen microscopically conformed to the pathological features of asthma.Around bronchioles and vessels,there were a large number of inflammatory cells infiltration in asthmatic mice.Airway wall and lung tissue visible was priority to eosinophilic granulocyte and lymphocyte of inflammatory cell infiltration, bronchial wall thickening and luminal stenosis, bronchial lumen mucous plug.
2.The results showed that IL-21and IL-21R were all expressed in lung tissue of asthma group and control group by immunohistochemical method.Compare with the control group, the expression of IL-21and IL-21R enhanced in asthma group (P<0.05), and the result was consistent with the means of western blot.It also showed that the mRNA expression of IL-21and IL-21R in asthma group were much more than control group (P<0.05). The index of WAm/Pbm and WAt/Pbm about airway remodeling in asthma mice were higher than the control group (P<0.05).
3.In group of asthma, AG490and dexamethasone, the total number of cells, neutrophils, acidophilic granulocyte and lymphocyte count in alveolar lavage fluid were significantly increased compared with normal control group (P<0.01). Compared with the asthma group, the total number of cells, neutrophils, acidophilic granulocyte and lymphocyte count in dexamethasone treatment group were significantly reduced (P<0.01), the index in AG490group also fell comaring with asthmatic group (P<0.05).Compared AG490group with dexamethasone group, total number of cells, eosinophils, lymphocytes and neutrophils in alveolar lavage fluid in dexamethasone group were lower than AG490group (P<0.05).
4.Immunohistochemistry and western blot analysis showed that the control group almost without the protein expression of P-STAT3.In contrast to control group, increased protein expression of P-STAT3wer detected in lung tissue (P<0.05).The expression of P-STAT3protein in lung tissue of AG490group were weaker than in the asthma group (P<0.05).Compared to the control group Fluorescence quantitative PCR showed that the expression of STAT3mRNA in asthma group and AG490group increased (P<0.05), between the two groups, there were no significant differences(P>0.05).
5.The index of WAm/Pbm and WAt/Pbm about airway remodelling in dexamethasone group was higher than the control group (P<0.05), lower than the asthma group (P<0.05). The protein expression of IL-21, IL-21R and p-STAT3in the lung tissue of dexamethasone group declined compared with the asthma group by using western blot method (P<0.05).The expression of IL-21in AG490group fell compared with the asthma group (P<0.05).No statistical significance were observed between the AG490group and dexamethasone group (P>0.05). For the protein expression of p-STAT3,there was no statistical significance between AG490group and dexamethasone group (P>0.05). Compared to control group,the level of STAT3mRNA in lung tissue of AG490group increased(P<0.05). However, there was no statistical significance between asthma group and AG490group (P>0.05).
6.Correlation analysis:The protein expression of IL-21, IL-21R, p-STAT3were detected by using western blot method, the results showed that these indicators and the total number of cells, eosinophil count in the mice alveolar lavage fluid were positively correlated (r>0.5, P<0.01), the protein expression of IL-21, IL-21R,p-STAT3and the index of WAm/Pbm, WAt/Pbm were positively correlated (r>0.5, P<0.01).
Conclusions
IL-21mainly transmit signal through the JAK-STAT pathway, the experiment firstly used different methods to observe the expression of IL-21and its receptor in the lung tissue in control group and asthma group. The expression changes of STAT3and activation of STAT3in lung tissue of mice were observed. After the intervention of JAK pathway inhibitors AG490and dexamethasone, the index changes were observed. Draw the following conclusions:
1.IL-21and its receptor express in the lung tissue of mice. Compare to the control group, the expression in asthmatic group is increased.IL-21is an important proinflammatory factor in the incidence of asthma. IL-21that combines with it's receptor involve in the onset of chronic asthma and participate in the process of airway remodeling.
2.STAT3and activation of STAT3involve in the pathogenesis of asthma, may affect the airway remodeling.
3.After the intervention of AG490in asthma model, the expression of p-STAT3is reduced, degree of airway inflammation and airway remodeling is reduced, its curative effect are roughly equal to dexamethasone.
4.IL-21involve in the onset of asthma disease by activating STAT3signaling pathway, dexamethasone downgrade the expression of IL-21and its receptors, inhibit the activation of STAT3in the lung tissue, relieve the airway inflammation and airway remodeling, IL-21/STAT3signal transduction pathway may be one of the targets of dexamethasone in treatment of asthma.
引文
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