ANP信号调控CD4~+T细胞及其在过敏性哮喘中的作用研究
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摘要
支气管哮喘(简称哮喘),是一种复杂的慢性气道疾病,以持续的气道炎症和气道高反应性为特点。在哮喘气道炎症中,多种细胞及细胞因子参与其中,如嗜酸性粒细胞、肥大细胞、T淋巴细胞、中性粒细胞等。其中,CD4+T淋巴细胞家族被证实是哮喘免疫学发病机制的重要效应细胞,其各亚群均发育于同一前体细胞-Th0细胞(又称初始CD4+T细胞),最经典的Th1/Th2细胞亚群数量与功能失衡即Th2型应答优势化早已被公认为过敏性哮喘的重要发病机制。在过敏性哮喘中,被激活的Th2细胞产生大量的细胞因子如IL-4、IL-5等,而这些细胞因子在诱导气道嗜酸性粒细胞炎症中发挥主导作用。
Th17细胞,是近年新发现的、不同于Th1/Th2细胞亚群的CD4+T细胞家族成员,在机体炎症反应和免疫应答尤其是自身免疫方面发挥重要作用。在CD4+T细胞家族中,类似于Th1/IFN-γ,和Th2/IL-4,IL-17为Th17细胞的身份标识因子,也是代表性的细胞因子。实验发现Th17/IL-17也参与了过敏性哮喘的发病,但具体机制至今不完全清楚。
ANP (atrial natriuretic peptide)-心房利钠肽,又名心钠素,源于ANP激素原(pro-ANP),最早从心房组织分离鉴定的多效能血管活性肽,因具有扩血管、利钠、利尿等作用而闻名。其实,肺脏是除心血管系统外重要的ANP合成部位,同时也是ANP的靶器官之一。
研究表明ANP广泛存在于外周肺组织、气管上皮细胞、呼吸上皮、人胎肺组织等部位；通过放射核素定位和免疫细胞化学证实,肺部支气管上皮和肺泡细胞均存在ANP的效应受体NPRA (natriuretic peptide receptor A)及其清除受体NPRC (natriuretic peptide receptorC)；人、猪、鼠的肺部均表达NPRA及NPRC;而且与肾脏和肝脏组织匀浆相比较,肺组织匀浆能更大程度的通过中性肽链内切酶降解pro-ANP。上述ANP、高亲和性ANP作用受体及大量降解ANP的酶的存在,表明肺不仅仅是ANP的靶器官,它自身亦拥有着完善的ANP代谢系统,而不只是依赖于循环中的ANP。这暗示ANP信号可能在肺部生理、病理过程中有着不可忽视的作用。然而,迄今为止,关于ANP信号在肺部的生理病理学功能知之甚少。
其实,ANP信号除了其经典的调节机体容量-压力内稳态作用外,越来越多的证据表明ANP信号在炎症和免疫反应方面也可能发挥重要作用。如在猪、小鼠、大鼠的胸腺、脾脏、淋巴结里存在ANP激素原；在人、鸟、小鼠的胸腺和扁桃体均能测出ANP mRNA; ANP信号可抑制胸腺细胞的增殖和分化；体外实验发现ANP可以剂量依赖方式诱导腹腔肥大细胞释放组胺；ANP信号还可影响中性粒细胞、巨噬细胞及树突状细胞的功能活性等。此外,T淋巴细胞亦可表达ANP的作用受体NPRA。这些都说明了ANP信号确实与炎症反应与免疫应答密切相关。
有临床观察发现在哮喘患者急性发作期,外周血ANP水平较缓解期和正常人均有明显的升高,这提示ANP信号可能与哮喘发病相关,但其在哮喘发病中的具体效应及作用机制尚不明确。基于前述,我们提出假说：既然哮喘患者外周血中ANP水平有明显异常的变化,而且ANP信号也与免疫炎症反应密切相关,那么ANP信号是否有可能通过调节CD4+T细胞的功能活性而参与过敏性哮喘的发病呢?
本课题将以小鼠脾源性CD4+T细胞和过敏性哮喘小鼠模型作为研究对象进行观察以期达到以下研究目的：一、研究ANP信号是否调控CD4+T细胞的功能活性及其可能的作用通路；二、观察ANP信号是否通过调控CD4+T细胞功能活性而参与过敏性哮喘的发病。首先,利用免疫磁珠分选法获取纯化的小鼠脾脏CD4+T细胞行体外研究,观察ANP信号对CD4+T细胞分化及功能的调控作用；其次,利用小鼠成功建立过敏性哮喘模型,进行在体干预ANP信号后,观察对哮喘小鼠模型气道炎症及CD4+T细胞相应转录因子及细胞因子等的影响,初步阐明ANP信号通过调控CD4+T细胞功能活性而参与过敏性哮喘的发病,也为认识ANP信号在其他肺部疾病的作用提供参考。
第一章ANP信号对Th17细胞分化和功能的作用研究
目的
(1)体外观察ANP信号对Th17细胞分化和功能的影响；
(2)探讨ANP调控Th17细胞的信号通路。
方法
(1)无菌提取BALB/c小鼠脾脏,充分研磨制备单个核细胞悬液；
(2)利用MACS技术获取纯化的CD4+T细胞,计数并测定细胞活力；
(3)按原液组、定向分化未干预组(简称分化组)、定向分化+ANP组(简称ANP组)、定向分化+A71915(ANP/NPRA信号抑制剂)+ANP组(简称A71915组)及定向分化+KT5823(NPRA受体偶联PKG激酶的抑制剂)+ANP组(简称KT5823组)培养4天；
(4)干预ANP信号后,检测第二信使cGMP水平变化；定向分化培养4天后,流式检测IL-17+-CD4+T细胞(Thl7)数量、Western-Blot(WB)检测各组中Thl7细胞转录因子水平(RORyt及BATF)及ELISA检测培养上清液中细胞因子IL-17的变化。
结果
(1)相较于原液组,anti-IFN-y抗体、anti-IL-4抗体、TGF-β1、IL-6及IL-23细胞因子组成的定向分化诱导微环境能明显的诱导Th0细胞定向分化为Th17细胞,同时伴随着细胞因子IL-17量的明显增加(P<0.01)：
(2)相较于定向分化组,加入ANP后可抑制Th17细胞的分化数量且伴随着Th17细胞特异性转录因子RORγt及BATF表达下降与培养上清液中细胞因子IL-17含量的减少(P均<0.05),并呈现浓度依赖性(10-8-10-6M)(P<0.05)；
(3)提前加入ANP/NPRA抑制剂-A71915(10-6M)后,ANP对Th17细胞分化和功能的抑制效应被明显的阻断(P均<0.05)；
(4)同样,提前加入NPRA受体偶联的PKG激酶抑制剂-KT5823同样可抵消ANP对Th17细胞分化和功能的影响(P均<0.05)；
(5)ANP信号的干预变化均导致与NPRA耦联的第二信使cGMP水平的相应变化(P均<0.01)。
结论
(1)在体外,ANP信号可抑制Th17细胞的分化和功能,并呈浓度依赖性；
(2)NPRA-PKG信号通路在ANP调控Th17细胞分化和功能的过程中发挥重要作用。
第二章ANP信号对Th17/IL-17的影响及其在过敏性哮喘小鼠模型的作用探讨
目的
(1)建立并评估OVA诱导的过敏性哮喘小鼠模型；
(2)观察ANP及其受体NPRA在过敏性哮喘小鼠模型中的变化；
(3)观察ANP信号对过敏性哮喘小鼠模型气道炎症及肺组织中Th17细胞特异性转录因子蛋白表达和肺局部IL-17水平的影响。
方法
取40只SPF级BABL/c雌性小鼠,按在体实验目的随机分为五组：正常组、哮喘未干预组(简称哮喘组)、ANP雾化吸入组(简称ANP组)、ANP雾化吸入+A71915腹腔注射组(简称ANP+A71915组)及A71915腹腔注射组(简称A71915组),每组8只。利用10μgONA/只+0.2ml Al(OH)3/只腹腔注射致敏、5%OVA溶液雾化激发模拟建立过敏性哮喘模型,同时正常组则以等体积的PBS代替OVA行致敏、激发；ANP组在每次雾化OVA溶液前30min给予ANP干粉溶液(浓度为1μg/g小鼠)雾化吸入；ANP+A71915组则在每次雾化ANP干粉溶液前30min予以腹腔注射ANP信号的抑制剂-A71915(浓度0.5μg/g小鼠)；而A71915组即OVA雾化激发前30min腹腔注射A71915,其余操作同哮喘未干预组。致敏和雾化激发过程中注意观察记录小鼠的一般行为活动。完成最后一次激发24h后处理所有小鼠,实验内容包括：(1)麻醉小鼠,通过气管插管吸入不同浓度的乙酰甲胆碱溶液后,有创肺阻抗法测定各组小鼠的气道反应性(RL及Cdyn)变化；(2)利用支气管肺泡灌洗法收集支气管肺泡灌洗液(bronchoalveolar lavage fluid-BALF)行细胞总数计数及分类计数,并检测BALF上清液中ANP、IL-4及IL-17的含量；(3)肺组织病理学观察分析；(4)Western-blot (WB)检测正常与哮喘组小鼠肺组织和脾源性CD4+T细胞中ANP效应受体-NPRA的变化；(5)WB测定肺组织Th17细胞特异性转录因子RORyt/BATF表达水平的变化及肺组织匀浆上清液ANP. IL-4及IL-17的含量。
结果
(1)哮喘组及其他三个药物干预组小鼠在雾化激发时均有不同程度的哮喘样发作症状,如身体蜷缩、烦躁不安、挠耳抓鼻、呼吸急促、大小便失禁等表现,而正常组则表现基本正常,建模和在体干预过程中未出现小鼠死亡现象；
(2)与正常组比较,哮喘组的肺阻力(RL)对不同浓度的乙酰甲胆碱(Mch)反应曲线明显上移(P<0.01)；同时肺动态顺应性(Cdyn)则明显下移(P<0.01),提示成功模拟过敏性哮喘的气道高反应性；
(3)相较于正常组,(?)ANP+A71915及A71915组的肺功能检测发现气道高反应性仍存在(P均<0.05),但较哮喘组有所缓解(P均<0.05)；而ANP组小鼠的肺阻力和肺动态顺应性无显著变化,接近于正常组水平(P均>0.05)；
(4)哮喘组的BALF中细胞总数及嗜酸性粒细胞、淋巴细胞等分类计数均明显高于正常组(P均<0.01)；然而ANP组BALF中细胞总数计数及分类计数不仅明显高于正常组(P均<0.01),甚至还高于哮喘组(P均<0.05)；A71915组则较哮喘组明显减少(P<0.05)；而A71915+ANP组中细胞总数及嗜酸性粒细胞计数较ANP组减少(P均<0.05)；
(5)相较于正常组,哮喘组BALF上清液中IL-4水平明显升高(P<0.01),IL-17含量亦增加(P<0.01)；ANP组BALF中IL-4水平升高更显著(P<0.01),甚至高于哮喘组(P<0.05),但未见IL-17水平的明显下降(P>0.05)；在A71915组,IL-4、IL-17水平相比于哮喘组均下降(P均<0.05)；A71915+ANP组较ANP组IL-4有所下降(P<0.05)但IL-17变化亦不显著(P>0.05)；
(6)与正常组比较,哮喘组小鼠BALF及肺组织匀浆上清液中ANP的含量均升高(P均<0.01)；
(7)哮喘组小鼠肺组织中ANP作用受体NPRA表达水平较正常组明显升高(P<0.05)；而且,哮喘小鼠脾源性CD4+T细胞中NPRA的表达亦高于正常小鼠(P<0.05)；
(8)与正常组相比,哮喘组支气管-肺组织病理学检测发现支气管及血管周围分布着大量的炎症细胞(炎症评分,P<0.01),并且PAS染色可见气道上皮周围有许多的粘液分泌(粘液评分,P<0.01)。而吸入ANP后,支气管及血管周围的炎性细胞浸润及粘液分泌程度比哮喘组更严重(P<0.05)；ANP+A71915组较ANP组均有所缓解(P均<0.05)；A71915组则较哮喘组明显改善(P均<0.01)；
(9)与正常组比较,哮喘组小鼠肺组织中Th17细胞转录因子RORyt及BATF均表达升高(P均<0.05)；吸入ANP后肺组织中RORyt及BATF的表达则较哮喘组有所下降(P均<0.05)；而阻断ANP信号即使用A71915后,RORyt的表达情况有所逆转(P<0.05)但BATF的表达差异无统计学差异(P>0.05)；
(10)各组肺组织匀浆上清液中IL-4、IL-17水平的变化趋势则与BALF中的测定结果基本一致。
结论
(1)过敏性哮喘小鼠模型建立成功；
(2)ANP信号参与了过敏性哮喘的发病；
(3)ANP信号可缓解过敏性哮喘的气道高反应性；
(4)ANP信号虽可下调肺局部Th17细胞RORyt及BATF转录因子的表达,但IL-17水平变化不明显；虽与体外观察结果不完全一致,但仍可能是ANP信号加剧过敏性哮喘气道炎症的机制之一；
(5)ANP信号加重过敏性哮喘气道炎症反应。
第三章ANP信号对Th1/Th2细胞功能平衡的影响及其在过敏性哮喘气道炎症中的作用探讨
目的
(1)体外观察ANP信号对Th1/Th2细胞功能平衡的影响；
(2)结合在体实验,初步探讨ANP信号通过促进Th2细胞功能优势化而加重过敏性哮喘气道炎症。
方法
无菌提取哮喘小鼠脾脏并研磨分离制备单个核细胞悬液后,利用免疫磁珠阳性分选获取脾源性CD4+T细胞行体外干预实验。计数、测定活力后按一定比例接种培养。依不同的试剂干预分为原液组、ANP组、SNP+A71915组。加入ConA(终浓度为5μg/m1)刺激培养24小时后收集细胞。Western-Blot法检测T-bet/GATA3蛋白的表达变化,ELISA法测定培养上清液中IFN-y/IL-4因子含量以评估ANP信号对Th1/Th2细胞功能平衡的影响。
在体实验方面,各组小鼠接受最后一次激发结束24h后取肺组织,测定其中Th1/Th2细胞特异性转录因子T-bet/GATA3蛋白表达有无变化及BALF与肺组织匀浆上清液中IFN-y水平的含量变化测定,在体实验操作均与第二章实验同步开展。
结果
(1)相较于正常组,哮喘组小鼠肺组织中GATA3表达明显增高(P<0.05)；而且相较于哮喘组,ANP吸入组小鼠肺组织中GATA3蛋白表达更明显(P<0.05),相反T-bet表达则下降(P<0.05)；阻断ANP信号(A71915)后,转录因子GATA3表达有所下降(P<0.05),而T-bet的表达则有所恢复(P<0.05)；
(2)与哮喘组比较,ANP组肺组织匀浆上清液中与T-bet表达变化基本一致(P<0.05),但BALF中IFN-y水平变化不明显(P>0.05)；在A71915+ANP组中变化也不显著(P>0.05)；
(3)在体外干预哮喘小鼠脾源性CD4+T细胞实验中,加入ANP刺激后,相较于原液组,GATA3蛋白表达亦增加(P<0.05),同时伴随T-bet的表达下降(P<0.05),变化趋势基本同在体肺组织中的观察结果；而提前加入ANP信号抑制剂A71915后,GATA3/T-bet的变化趋势则被逆转(P<0.05)；
(4)相比较于原液组,ANP组细胞培养上清液中IL-4水平升高(P<0.05),而IFN-γ减少(P<0.05)；而提前加入拮抗剂A71915后,IL-4及IFN-γ含量的变化趋势与ANP组比较均有所恢复(P<0.05)。
结论
(1)在体外,ANP信号影响Th1/Th2功能平衡,即可促进Th2型反应而抑制Thl型反应；
(2)在体内,ANP信号可促进肺局部Th2型应答,结合体外观察结果,这很可能是ANP信号加重过敏性哮喘气道炎症反应的机制之一。
Background
Bronchial asthma (hereinafter referred to asthma), is a complex chronic airway disease which is characterized by persistent airway inflammation and hyperresponsiveness. A variety of cells and cytokines such as eosinophils, mast cells, T lymphocytes, neutrophils, and so on, play an important role in the process of airway inflammatory reaction in asthma, in which CD4+T cells have been considered as major effector cells in the immunological pathogenesis of asthma. As we know, CD4+T cells comprise several subsets such as the most classic Thl/Th2cells which are all differentiated from the same progenitor cells-ThO cells (also named naive CD4+T cells). Th2cell dominance has been considered to be the significant mechanism of allergic asthma for a long time, because activation and dominance of Th2cells can produce lots of cytokines such as IL-4, IL-5which play a dominant role in inducing an eosinophilic airway inflammation of allergic asthma.
Th17cells, recently discovered, a new important member different from Th1/Th2cells in CD4+T cells family, have been recognised to play a significant role in inflammatory and immune response especially in autoimmune reaction. In CD4+T cells family, similar to Thl/IFN-γ and Th2/IL-4, IL-17is an identifying and the representative cytokine of Thl7cells. Previous studies have confirmed that Th17/IL-17is also involved in the course of allergic asthma, but the mechanism is still unclear.
Atrial natriuretic peptide (ANP), a multifunctional peptide cleaved from pro-ANP hormone, was firstly isolated from tissue of heart atrium and famous for its biological actions on cardiovascular system including a stimulation of natriuresis, diuresis, vasorelaxation, and so forth. Actually, lung is also an important organ which can produce ANP besides the cardiovascular system, and meanwhile lung is one of the target organs of ANP signaling.
Many investigations show ANP is localized in peripheral lungtissue, tracheal epithelial cells, respiratory epithelium, human fetal lung tissue, etc; effecting receptor-natriuretic peptide receptor A (NPRA) and its clearance receptor-natriuretic peptide receptor C (NPRC) of ANP are all expressed in bronchial and alveolar cells via radionuclide location and immunocytochemical analyses; NPRA and NPRC are also discovered in the lungtissue of humans, pigs and mice; in addition, the lung homogenates could degrade pro-ANP with neutral endopeptidases to a greater degree compared with the kidney and liver homogenates, and so on. ANP, receoptors system with high affinity and masses of peptidases for ANP metabolism exist in the lung suggest that lung is not only the target organ of ANP, but it also owns an integrated metabolic system for ANP, in which effects of ANP are not only dependent on ANP production from circulation. These data indicate that ANP signaling may play a nonnegligible role in the physiological and pathological responses of the lung. However, so far, little has been known about it.
In fact, accumulating evidence suggest ANP signaling is closely related with the inflammatory and immune reactions besides its regulation in the homeostasis of volume-pressure. For example, pro-ANP is all found in the immune organs such as thymus, spleen, lymph nodes from pigs, mice and rats; ANP mRNA also can be detected in the thymus and tonsil of human and mouse; ANP signaling can attenuate the proliferation and differentiation of thymocytes; a study discovered that ANP could dose-dependently induce the release of histamine from mast cells of peritoneum; ANP signaling also affects the activities of neutrophilis, macrophages, dendritic cells. Moreover, NPRA also can be expressed on T lymphocyes. These data demonstrate that ANP signaling is indeed implicated in the inflammatory and immune responses.
Some observations from clinic found levels of ANP in peripheral blood of asthmatics in exacerbation were significantly higher than those of asthmatics in remission and the healthy, which suggests ANP signaling is correlated with asthma, but the effect and mechanism are still unknow. Based on the above, we have a hypothesis:now that abnormal change of ANP levels in peripheral blood of asthmatics were happened and ANP signaling is closed related with immune and inflammatory response, whether ANP signaling may play a role in allergic asthma via regulating the activities of CD4+T cells?
Mouse splenic CD4+T cells and mouse models with allergic asthma are performed in this study. One purpose of this study is to define whether ANP signaling could regulate the activities of CD4+T cells and to explore the mechanism; the other purpose is to observe whether ANP signaling is implicated in allergic asthma by influencing the activities of CD4+T cells. Firstly, we are going to investigate what ANP signaling's effect on the differentiation and function of CD4+T cells in vitro which isolated from spleens by immune magnetic beads sorting. Then, we will observe the effects of ANP signaling on airway inflammation of mouse models with allergic asthma and changes of transcription factors and related cytokines of CD4+T cells after activating or preventing ANP signaling. By studying in vitro and in vivo, we want to initially elucidate ANP signaling indeed plays an important role in allergic asthma via modulating CD4+T cells and meanwhile to provide an evidence for further recognising the role of ANP signaling in other pulmonary diseases.
Part I Effects of ANP signaling on the differentiation and capacity of Th17cells
Objectives
(1) To observe effects of ANP signaling on the differentiation and capacity of Th17cells in vitro;
(2) To probe the signaling pathway of ANP to regulate Th17cells.
Methods
(1) To aseptically excise and grind the spleens of BALB/c mice and to prepare the splenic mononuclear cells suspension;
(2) CD4+T cells were isolated from splenic mononuclear cells by positive selection with immuno-magnetic beads (MACS technology) and then counting and detecting the vitality of CD4+T cells;
(3) To define the expression of NPRA in CD4+T cells by WB;
(4) CD4+T cells were cultured for4days as control group (C), directional differentiation group (D), ANP addition group (ANP), A71915plus ANP group (A7), KT5823plus ANP group (KT);
(5) To detect the changes of the second messenger cGMP after intervening ANP signaling; cultured for4days, to measure the rate of IL-17-producing CD4+T cells by FACS; to detect the expression of RORγt and BATF (specific transcription factors for Th17cells) by WB; to confirm the changes of IL-17in supernatant.
Results
(1) Compared with the control group, the rate of IL-17-producing CD4+T cells could be significantly augmented in the cytokines microenvironment for directional differentiation comprising anti-IFN-y, anti-IL-4, TGF-β1, IL-6and IL-23, meanwhile along with increased production of IL-17in supernatant (P<0.01);
(2) Compared with differentiation group, addition of ANP could decrease the proportion of IL-17-producing CD4+T cells (P<0.01). Accordingly, the expression of RORyt and BATF (P<0.05) and the levels of IL-17in supernatant were also reduced (P<0.05); and the inhibitory effects showed dose-dependent (10-8-10-6M ANP);
(3) When using the ANP/NPRA signaling antagonist-A71915, the inhibitory effects of ANP on the differentiation and capacity of Th17cells were mostly reversed (respectively P<0.05);
(4) Similarly, KT5823were performed in advance, the inhibitory effects of ANP on the differentiation and capacity of Th17cells were also mostly recovered (respectively P<0.05);
(5) Changes of cGMP coupled NPRA were consistent following the intervention of ANP signaling(respectively P<0.01).
Conclusions
(1) ANP signaling could suppress the differentiation and capacity of Th17cells in vitro and displayed a dose-dependent effect;
(2) NPRA/PKG pathway played an important role in the process of ANP effecting on Thl7cells.
Part Ⅱ Effects of ANP signaling on Th17/IL-17and its role in mouse models with allergic asthma
Objectives
(1) To establish and evaluate mice models with allergic asthma;
(2) To observe changes of ANP and NPRA in mice with allergic asthma;
(3) To observe effects of ANP signaling on airway inflammation of mice models with allergic asthma, local expression of transcription factor for Th17cells and changes of IL-17in lung.
Methods
40SPF female BALB/c mice were randomly divided into five groups according to the study design:normal group, asthma group, ANP inhalation group (ANP), A71915intraperitoneal injection(i.p) plus ANP inhalation group (ANP+A71915), A71915(i.p) alone group. Every group contains8mice. As the scheme, mice models with allergic asthma were established by sensitized with10μg OVA/mouse+0.2ml A1(OH)3/mouse by i.p and then aerosol challenge with5%OVA via repeated inhalation in asthma group while the same volume of PBS were performed as the substitution of OVA in the normal group. In addition, aerosol nebulization of solution with ANP dry powder (dose1μg/g mouse) at30min before every OVA-challenge in ANP group and similarly, A71915 (dose0.5μg/g mouse) was used by i.p in A71915group at30min before every ANP inhalation. Other operations were same as the asthma group and behaviours of mice in every group should be observed during establishing the models. After24hours of the last challenge, operations were carried out as following:(1) after anesthesia, mice were implemented with tracheal intubation and changes of airway response were determined by recording lung resistance(RL) and lung dynamic compliance (Cdyn) stimulated with Methacholine of different doses (Mch);(2) total cells count, differential counts and production of ANP, IL-4, IL-17in bronchoalveolar lavage fluid (BALF) were detected;(3) observation and analysis of bronchial tube and lungtissue were completed by pathological staining;(4) expressions of NPRA for ANP signaling in lungtissue and splenic CD4+T cells from normal and asthma group were quantified by Western-blot;(5) RORyt and BATF proteins were also detected by WB in lungtissue and changes of ANP, IL-4, IL-17levels in lung homogenates.
Results
(1) Asthmatic symptoms of varying degrees were emerged in mice of asthma group, ANP group and A71915group such as tremble, unease, breathlessness, incontinence and so on while the normal group showed basically normal. In addition, no death of mice appeared during establishing model of asthma and drug treatment in vivo;
(2) Compared with the normal group, the dose-reponse curve of RL was significantly shifted up (P<0.01) while the curve of Cdyn was apparently shifted down (P<0.01) in asthma group, which suggests airway hyperresponsiveness (AHR) was successfully established.
(3) Compared with the normal group, airway hyperresponsiveness of A71915+ANP and A71915group was also obvious (respectively P<0.05) but reduced in contrast with asthma group (respectively P<0.05); while AHR of ANP group was not evident which was similar to normal group (respectively P>0.05);
(4) Total cells counts, differential cells counts of eosinophils, etc in BALF of asthma group were increased than those in BALF of normal group (respectively P<0.01); however, those in ANP group were not only significantly more than normal group (P<0.01) but even more seriuos than asthma group (P<0.05); while those in A71915group were obviously reduced compared with asthma (P<0.05); those in A71915+ANP group were also decreased compared with ANP group(P<0.05);
(5) Compared with normal group, levels of IL-4, IL-17in BALF of asthma group were apparently augmented (respectively P<0.01); while increased production of IL-4in BALF of ANP group (P<0.01) was more outstanding and the mean value of ANP group was even more higher than that of asthma group (P<0.05), but the levels of IL-17showed no obvious decline (P>0.05); in A71915group, the levels of IL-4, IL-17in BALF were all decreased compared with asthma group (P<0.05); in A71915+ANP group, levels of IL-4decreased (P<0.05) but change of IL-17was not significant compared with normal group (P>0.05);
(6) Production of ANP in BALF and lungtissue homogenates of asthma group were significantly higher than those in normal group (respectively P<0.01);
(7) Levels of NPRA protein in lungtissue of mice with asthma were much more than the normal group (P<0.05) and those in splenic CD4T cells were similar (P<0.05);
(8) Numerous inflammatory cells infiltrated and diffused around the bronchus and vessel, and lots of mucus secreted around airway compared with the normal group(respectively P<0.01); when inhalation of ANP, the above became more serious compared with asthma group (respectively P<0.05); and A71915+ANP group showed relaxative than ANP group (respectivelyP<0.05); while this situation in A71915group was attenuated compared with asthma group (respectively P<0.05);
(9) Expression of RORyt and BATF of Th17cells in mice lungtissue of asthma group were increased than the normal group (respectively P<0.05); while the RORyt and BATF were decreased after inhalation of ANP (respectively P<0.05) and expression of RORyt and BATF became much less in A71915+ANP group (P<0.05);
(10) The changes of IL-4, IL-17in mice lung homogenates of every group were similar to those of in BALF.
Conclusions
(1) Mice models with allergic asthma were successfully established based on the data;
(2) ANP signaling plays a role in the course of allergic asthma;
(3) ANP signaling could remit AHR of mice with allergic asthma;
(4) Not completely consistent with experimental data in vitro, ANP signaling could reduce expression of transcription factors of Thl7cells in lungtissue not along with significantly decreased IL-17in vivo, which also may be one of mechanisms;
(5) ANP signaling aggravate airway inflammation of allergic asthma.
Part III Effects of ANP signaling on functional balance of Th1/Th2cells and its role in airway inflammation of allergic asthma
Objectives
(1) To investigate the effects of ANP signaling on functional balance of Thl/Th2cells in vitro;
(2) To explore ANP signaling aggravating airway inflammation of allergic asthma by promoting dominance of Th2cells based on study in vivo.
Methods
To aseptically excise and grind the spleens of BALB/c mice with allergic asthma and to prepare the splenic mononuclear cells suspension. Then, CD4+T cells were isolated through positive selection with immuno-magnetic microbeads. CD4+T cells were cultured as the design after counting and detecting the vitality. All experiments in vitro were divided into control group, ANP group, A71915+ANP group according to the different intervention. Cells were harvested after24h by stimulated ConA (end concentration-5μg/ml). Then, for the sake of defining the effects of ANP signaling on the functional balance of Thl/Th2cells, to detect changes of T-bet/GATA3expression by WB and to measure levels of IFN-y/IL-4in supernatant were performed.
In vivo, detection of T-bet/GATA3for Thl/Th2cells in lungtissue of mice via WB and measurement of IFN-γ levels in BALF and lung homogenates were finished, and other experiments were processed in Part Ⅱ.
Results
(1) Compared with noraml group, expressions of GATA3in asthma group were obviously more higher; meanwhilie, expression of GATA3showed much more in ANP inhalation group than asthma group while T-bet was decreased (respectively P<0.05); when blockade of ANP signaling, expression of GATA3was decreased while that of T-bet was increased (respectively P<0.05);
(2) IFN-y levels in lung homogenates decreased compared with asthma group which was consistent with changes of T-bet (P<0.05) but not in BALF (P>0.05); and no obvious change of IFN-y levels happened in ANP+A71915group (P>0.05);
(3) In vitro, expression of GATA3in splenic CD4+T cells from mice with asthma was higher than control group after stimulated by ANP for24h along with reduced expression of T-bet (respectively P<0.05), in which the trend of changes were basically consistent with the results from in vivo; whlie A71915performed in advance, effects of ANP signaling on expressions of T-bet/GATA3were neutralized (respectively P<0.05);
(4) Level of IFN-y in supernatant was decreased while IL-4was increased in ANP group compared with the control (respectively P<0.05); When ANP signaling inhibitor was used, the changes of IFN-y/IL-4were also reversed (respectively P<0.05).
Conclusions
(1) In vitro, ANP signaling could affect functional balance of Thl/Th2cells, that is promoting Th2-type response while inhibiting Th1-type response;
(2) In vivo, ANP signaling could aggravate Th2cells response in lung, which is probably one of the mechanisms for ANP signaling to exacerbate airway inflammation of allergic asthma.
Th17细胞,是近年新发现的、不同于Th1/Th2细胞亚群的CD4+T细胞家族成员,在机体炎症反应和免疫应答尤其是自身免疫方面发挥重要作用。在CD4+T细胞家族中,类似于Th1/IFN-γ,和Th2/IL-4,IL-17为Th17细胞的身份标识因子,也是代表性的细胞因子。实验发现Th17/IL-17也参与了过敏性哮喘的发病,但具体机制至今不完全清楚。
ANP (atrial natriuretic peptide)-心房利钠肽,又名心钠素,源于ANP激素原(pro-ANP),最早从心房组织分离鉴定的多效能血管活性肽,因具有扩血管、利钠、利尿等作用而闻名。其实,肺脏是除心血管系统外重要的ANP合成部位,同时也是ANP的靶器官之一。
研究表明ANP广泛存在于外周肺组织、气管上皮细胞、呼吸上皮、人胎肺组织等部位；通过放射核素定位和免疫细胞化学证实,肺部支气管上皮和肺泡细胞均存在ANP的效应受体NPRA (natriuretic peptide receptor A)及其清除受体NPRC (natriuretic peptide receptorC)；人、猪、鼠的肺部均表达NPRA及NPRC;而且与肾脏和肝脏组织匀浆相比较,肺组织匀浆能更大程度的通过中性肽链内切酶降解pro-ANP。上述ANP、高亲和性ANP作用受体及大量降解ANP的酶的存在,表明肺不仅仅是ANP的靶器官,它自身亦拥有着完善的ANP代谢系统,而不只是依赖于循环中的ANP。这暗示ANP信号可能在肺部生理、病理过程中有着不可忽视的作用。然而,迄今为止,关于ANP信号在肺部的生理病理学功能知之甚少。
其实,ANP信号除了其经典的调节机体容量-压力内稳态作用外,越来越多的证据表明ANP信号在炎症和免疫反应方面也可能发挥重要作用。如在猪、小鼠、大鼠的胸腺、脾脏、淋巴结里存在ANP激素原；在人、鸟、小鼠的胸腺和扁桃体均能测出ANP mRNA; ANP信号可抑制胸腺细胞的增殖和分化；体外实验发现ANP可以剂量依赖方式诱导腹腔肥大细胞释放组胺；ANP信号还可影响中性粒细胞、巨噬细胞及树突状细胞的功能活性等。此外,T淋巴细胞亦可表达ANP的作用受体NPRA。这些都说明了ANP信号确实与炎症反应与免疫应答密切相关。
有临床观察发现在哮喘患者急性发作期,外周血ANP水平较缓解期和正常人均有明显的升高,这提示ANP信号可能与哮喘发病相关,但其在哮喘发病中的具体效应及作用机制尚不明确。基于前述,我们提出假说：既然哮喘患者外周血中ANP水平有明显异常的变化,而且ANP信号也与免疫炎症反应密切相关,那么ANP信号是否有可能通过调节CD4+T细胞的功能活性而参与过敏性哮喘的发病呢?
本课题将以小鼠脾源性CD4+T细胞和过敏性哮喘小鼠模型作为研究对象进行观察以期达到以下研究目的：一、研究ANP信号是否调控CD4+T细胞的功能活性及其可能的作用通路；二、观察ANP信号是否通过调控CD4+T细胞功能活性而参与过敏性哮喘的发病。首先,利用免疫磁珠分选法获取纯化的小鼠脾脏CD4+T细胞行体外研究,观察ANP信号对CD4+T细胞分化及功能的调控作用；其次,利用小鼠成功建立过敏性哮喘模型,进行在体干预ANP信号后,观察对哮喘小鼠模型气道炎症及CD4+T细胞相应转录因子及细胞因子等的影响,初步阐明ANP信号通过调控CD4+T细胞功能活性而参与过敏性哮喘的发病,也为认识ANP信号在其他肺部疾病的作用提供参考。
第一章ANP信号对Th17细胞分化和功能的作用研究
目的
(1)体外观察ANP信号对Th17细胞分化和功能的影响；
(2)探讨ANP调控Th17细胞的信号通路。
方法
(1)无菌提取BALB/c小鼠脾脏,充分研磨制备单个核细胞悬液；
(2)利用MACS技术获取纯化的CD4+T细胞,计数并测定细胞活力；
(3)按原液组、定向分化未干预组(简称分化组)、定向分化+ANP组(简称ANP组)、定向分化+A71915(ANP/NPRA信号抑制剂)+ANP组(简称A71915组)及定向分化+KT5823(NPRA受体偶联PKG激酶的抑制剂)+ANP组(简称KT5823组)培养4天；
(4)干预ANP信号后,检测第二信使cGMP水平变化；定向分化培养4天后,流式检测IL-17+-CD4+T细胞(Thl7)数量、Western-Blot(WB)检测各组中Thl7细胞转录因子水平(RORyt及BATF)及ELISA检测培养上清液中细胞因子IL-17的变化。
结果
(1)相较于原液组,anti-IFN-y抗体、anti-IL-4抗体、TGF-β1、IL-6及IL-23细胞因子组成的定向分化诱导微环境能明显的诱导Th0细胞定向分化为Th17细胞,同时伴随着细胞因子IL-17量的明显增加(P<0.01)：
(2)相较于定向分化组,加入ANP后可抑制Th17细胞的分化数量且伴随着Th17细胞特异性转录因子RORγt及BATF表达下降与培养上清液中细胞因子IL-17含量的减少(P均<0.05),并呈现浓度依赖性(10-8-10-6M)(P<0.05)；
(3)提前加入ANP/NPRA抑制剂-A71915(10-6M)后,ANP对Th17细胞分化和功能的抑制效应被明显的阻断(P均<0.05)；
(4)同样,提前加入NPRA受体偶联的PKG激酶抑制剂-KT5823同样可抵消ANP对Th17细胞分化和功能的影响(P均<0.05)；
(5)ANP信号的干预变化均导致与NPRA耦联的第二信使cGMP水平的相应变化(P均<0.01)。
结论
(1)在体外,ANP信号可抑制Th17细胞的分化和功能,并呈浓度依赖性；
(2)NPRA-PKG信号通路在ANP调控Th17细胞分化和功能的过程中发挥重要作用。
第二章ANP信号对Th17/IL-17的影响及其在过敏性哮喘小鼠模型的作用探讨
目的
(1)建立并评估OVA诱导的过敏性哮喘小鼠模型；
(2)观察ANP及其受体NPRA在过敏性哮喘小鼠模型中的变化；
(3)观察ANP信号对过敏性哮喘小鼠模型气道炎症及肺组织中Th17细胞特异性转录因子蛋白表达和肺局部IL-17水平的影响。
方法
取40只SPF级BABL/c雌性小鼠,按在体实验目的随机分为五组：正常组、哮喘未干预组(简称哮喘组)、ANP雾化吸入组(简称ANP组)、ANP雾化吸入+A71915腹腔注射组(简称ANP+A71915组)及A71915腹腔注射组(简称A71915组),每组8只。利用10μgONA/只+0.2ml Al(OH)3/只腹腔注射致敏、5%OVA溶液雾化激发模拟建立过敏性哮喘模型,同时正常组则以等体积的PBS代替OVA行致敏、激发；ANP组在每次雾化OVA溶液前30min给予ANP干粉溶液(浓度为1μg/g小鼠)雾化吸入；ANP+A71915组则在每次雾化ANP干粉溶液前30min予以腹腔注射ANP信号的抑制剂-A71915(浓度0.5μg/g小鼠)；而A71915组即OVA雾化激发前30min腹腔注射A71915,其余操作同哮喘未干预组。致敏和雾化激发过程中注意观察记录小鼠的一般行为活动。完成最后一次激发24h后处理所有小鼠,实验内容包括：(1)麻醉小鼠,通过气管插管吸入不同浓度的乙酰甲胆碱溶液后,有创肺阻抗法测定各组小鼠的气道反应性(RL及Cdyn)变化；(2)利用支气管肺泡灌洗法收集支气管肺泡灌洗液(bronchoalveolar lavage fluid-BALF)行细胞总数计数及分类计数,并检测BALF上清液中ANP、IL-4及IL-17的含量；(3)肺组织病理学观察分析；(4)Western-blot (WB)检测正常与哮喘组小鼠肺组织和脾源性CD4+T细胞中ANP效应受体-NPRA的变化；(5)WB测定肺组织Th17细胞特异性转录因子RORyt/BATF表达水平的变化及肺组织匀浆上清液ANP. IL-4及IL-17的含量。
结果
(1)哮喘组及其他三个药物干预组小鼠在雾化激发时均有不同程度的哮喘样发作症状,如身体蜷缩、烦躁不安、挠耳抓鼻、呼吸急促、大小便失禁等表现,而正常组则表现基本正常,建模和在体干预过程中未出现小鼠死亡现象；
(2)与正常组比较,哮喘组的肺阻力(RL)对不同浓度的乙酰甲胆碱(Mch)反应曲线明显上移(P<0.01)；同时肺动态顺应性(Cdyn)则明显下移(P<0.01),提示成功模拟过敏性哮喘的气道高反应性；
(3)相较于正常组,(?)ANP+A71915及A71915组的肺功能检测发现气道高反应性仍存在(P均<0.05),但较哮喘组有所缓解(P均<0.05)；而ANP组小鼠的肺阻力和肺动态顺应性无显著变化,接近于正常组水平(P均>0.05)；
(4)哮喘组的BALF中细胞总数及嗜酸性粒细胞、淋巴细胞等分类计数均明显高于正常组(P均<0.01)；然而ANP组BALF中细胞总数计数及分类计数不仅明显高于正常组(P均<0.01),甚至还高于哮喘组(P均<0.05)；A71915组则较哮喘组明显减少(P<0.05)；而A71915+ANP组中细胞总数及嗜酸性粒细胞计数较ANP组减少(P均<0.05)；
(5)相较于正常组,哮喘组BALF上清液中IL-4水平明显升高(P<0.01),IL-17含量亦增加(P<0.01)；ANP组BALF中IL-4水平升高更显著(P<0.01),甚至高于哮喘组(P<0.05),但未见IL-17水平的明显下降(P>0.05)；在A71915组,IL-4、IL-17水平相比于哮喘组均下降(P均<0.05)；A71915+ANP组较ANP组IL-4有所下降(P<0.05)但IL-17变化亦不显著(P>0.05)；
(6)与正常组比较,哮喘组小鼠BALF及肺组织匀浆上清液中ANP的含量均升高(P均<0.01)；
(7)哮喘组小鼠肺组织中ANP作用受体NPRA表达水平较正常组明显升高(P<0.05)；而且,哮喘小鼠脾源性CD4+T细胞中NPRA的表达亦高于正常小鼠(P<0.05)；
(8)与正常组相比,哮喘组支气管-肺组织病理学检测发现支气管及血管周围分布着大量的炎症细胞(炎症评分,P<0.01),并且PAS染色可见气道上皮周围有许多的粘液分泌(粘液评分,P<0.01)。而吸入ANP后,支气管及血管周围的炎性细胞浸润及粘液分泌程度比哮喘组更严重(P<0.05)；ANP+A71915组较ANP组均有所缓解(P均<0.05)；A71915组则较哮喘组明显改善(P均<0.01)；
(9)与正常组比较,哮喘组小鼠肺组织中Th17细胞转录因子RORyt及BATF均表达升高(P均<0.05)；吸入ANP后肺组织中RORyt及BATF的表达则较哮喘组有所下降(P均<0.05)；而阻断ANP信号即使用A71915后,RORyt的表达情况有所逆转(P<0.05)但BATF的表达差异无统计学差异(P>0.05)；
(10)各组肺组织匀浆上清液中IL-4、IL-17水平的变化趋势则与BALF中的测定结果基本一致。
结论
(1)过敏性哮喘小鼠模型建立成功；
(2)ANP信号参与了过敏性哮喘的发病；
(3)ANP信号可缓解过敏性哮喘的气道高反应性；
(4)ANP信号虽可下调肺局部Th17细胞RORyt及BATF转录因子的表达,但IL-17水平变化不明显；虽与体外观察结果不完全一致,但仍可能是ANP信号加剧过敏性哮喘气道炎症的机制之一；
(5)ANP信号加重过敏性哮喘气道炎症反应。
第三章ANP信号对Th1/Th2细胞功能平衡的影响及其在过敏性哮喘气道炎症中的作用探讨
目的
(1)体外观察ANP信号对Th1/Th2细胞功能平衡的影响；
(2)结合在体实验,初步探讨ANP信号通过促进Th2细胞功能优势化而加重过敏性哮喘气道炎症。
方法
无菌提取哮喘小鼠脾脏并研磨分离制备单个核细胞悬液后,利用免疫磁珠阳性分选获取脾源性CD4+T细胞行体外干预实验。计数、测定活力后按一定比例接种培养。依不同的试剂干预分为原液组、ANP组、SNP+A71915组。加入ConA(终浓度为5μg/m1)刺激培养24小时后收集细胞。Western-Blot法检测T-bet/GATA3蛋白的表达变化,ELISA法测定培养上清液中IFN-y/IL-4因子含量以评估ANP信号对Th1/Th2细胞功能平衡的影响。
在体实验方面,各组小鼠接受最后一次激发结束24h后取肺组织,测定其中Th1/Th2细胞特异性转录因子T-bet/GATA3蛋白表达有无变化及BALF与肺组织匀浆上清液中IFN-y水平的含量变化测定,在体实验操作均与第二章实验同步开展。
结果
(1)相较于正常组,哮喘组小鼠肺组织中GATA3表达明显增高(P<0.05)；而且相较于哮喘组,ANP吸入组小鼠肺组织中GATA3蛋白表达更明显(P<0.05),相反T-bet表达则下降(P<0.05)；阻断ANP信号(A71915)后,转录因子GATA3表达有所下降(P<0.05),而T-bet的表达则有所恢复(P<0.05)；
(2)与哮喘组比较,ANP组肺组织匀浆上清液中与T-bet表达变化基本一致(P<0.05),但BALF中IFN-y水平变化不明显(P>0.05)；在A71915+ANP组中变化也不显著(P>0.05)；
(3)在体外干预哮喘小鼠脾源性CD4+T细胞实验中,加入ANP刺激后,相较于原液组,GATA3蛋白表达亦增加(P<0.05),同时伴随T-bet的表达下降(P<0.05),变化趋势基本同在体肺组织中的观察结果；而提前加入ANP信号抑制剂A71915后,GATA3/T-bet的变化趋势则被逆转(P<0.05)；
(4)相比较于原液组,ANP组细胞培养上清液中IL-4水平升高(P<0.05),而IFN-γ减少(P<0.05)；而提前加入拮抗剂A71915后,IL-4及IFN-γ含量的变化趋势与ANP组比较均有所恢复(P<0.05)。
结论
(1)在体外,ANP信号影响Th1/Th2功能平衡,即可促进Th2型反应而抑制Thl型反应；
(2)在体内,ANP信号可促进肺局部Th2型应答,结合体外观察结果,这很可能是ANP信号加重过敏性哮喘气道炎症反应的机制之一。
Background
Bronchial asthma (hereinafter referred to asthma), is a complex chronic airway disease which is characterized by persistent airway inflammation and hyperresponsiveness. A variety of cells and cytokines such as eosinophils, mast cells, T lymphocytes, neutrophils, and so on, play an important role in the process of airway inflammatory reaction in asthma, in which CD4+T cells have been considered as major effector cells in the immunological pathogenesis of asthma. As we know, CD4+T cells comprise several subsets such as the most classic Thl/Th2cells which are all differentiated from the same progenitor cells-ThO cells (also named naive CD4+T cells). Th2cell dominance has been considered to be the significant mechanism of allergic asthma for a long time, because activation and dominance of Th2cells can produce lots of cytokines such as IL-4, IL-5which play a dominant role in inducing an eosinophilic airway inflammation of allergic asthma.
Th17cells, recently discovered, a new important member different from Th1/Th2cells in CD4+T cells family, have been recognised to play a significant role in inflammatory and immune response especially in autoimmune reaction. In CD4+T cells family, similar to Thl/IFN-γ and Th2/IL-4, IL-17is an identifying and the representative cytokine of Thl7cells. Previous studies have confirmed that Th17/IL-17is also involved in the course of allergic asthma, but the mechanism is still unclear.
Atrial natriuretic peptide (ANP), a multifunctional peptide cleaved from pro-ANP hormone, was firstly isolated from tissue of heart atrium and famous for its biological actions on cardiovascular system including a stimulation of natriuresis, diuresis, vasorelaxation, and so forth. Actually, lung is also an important organ which can produce ANP besides the cardiovascular system, and meanwhile lung is one of the target organs of ANP signaling.
Many investigations show ANP is localized in peripheral lungtissue, tracheal epithelial cells, respiratory epithelium, human fetal lung tissue, etc; effecting receptor-natriuretic peptide receptor A (NPRA) and its clearance receptor-natriuretic peptide receptor C (NPRC) of ANP are all expressed in bronchial and alveolar cells via radionuclide location and immunocytochemical analyses; NPRA and NPRC are also discovered in the lungtissue of humans, pigs and mice; in addition, the lung homogenates could degrade pro-ANP with neutral endopeptidases to a greater degree compared with the kidney and liver homogenates, and so on. ANP, receoptors system with high affinity and masses of peptidases for ANP metabolism exist in the lung suggest that lung is not only the target organ of ANP, but it also owns an integrated metabolic system for ANP, in which effects of ANP are not only dependent on ANP production from circulation. These data indicate that ANP signaling may play a nonnegligible role in the physiological and pathological responses of the lung. However, so far, little has been known about it.
In fact, accumulating evidence suggest ANP signaling is closely related with the inflammatory and immune reactions besides its regulation in the homeostasis of volume-pressure. For example, pro-ANP is all found in the immune organs such as thymus, spleen, lymph nodes from pigs, mice and rats; ANP mRNA also can be detected in the thymus and tonsil of human and mouse; ANP signaling can attenuate the proliferation and differentiation of thymocytes; a study discovered that ANP could dose-dependently induce the release of histamine from mast cells of peritoneum; ANP signaling also affects the activities of neutrophilis, macrophages, dendritic cells. Moreover, NPRA also can be expressed on T lymphocyes. These data demonstrate that ANP signaling is indeed implicated in the inflammatory and immune responses.
Some observations from clinic found levels of ANP in peripheral blood of asthmatics in exacerbation were significantly higher than those of asthmatics in remission and the healthy, which suggests ANP signaling is correlated with asthma, but the effect and mechanism are still unknow. Based on the above, we have a hypothesis:now that abnormal change of ANP levels in peripheral blood of asthmatics were happened and ANP signaling is closed related with immune and inflammatory response, whether ANP signaling may play a role in allergic asthma via regulating the activities of CD4+T cells?
Mouse splenic CD4+T cells and mouse models with allergic asthma are performed in this study. One purpose of this study is to define whether ANP signaling could regulate the activities of CD4+T cells and to explore the mechanism; the other purpose is to observe whether ANP signaling is implicated in allergic asthma by influencing the activities of CD4+T cells. Firstly, we are going to investigate what ANP signaling's effect on the differentiation and function of CD4+T cells in vitro which isolated from spleens by immune magnetic beads sorting. Then, we will observe the effects of ANP signaling on airway inflammation of mouse models with allergic asthma and changes of transcription factors and related cytokines of CD4+T cells after activating or preventing ANP signaling. By studying in vitro and in vivo, we want to initially elucidate ANP signaling indeed plays an important role in allergic asthma via modulating CD4+T cells and meanwhile to provide an evidence for further recognising the role of ANP signaling in other pulmonary diseases.
Part I Effects of ANP signaling on the differentiation and capacity of Th17cells
Objectives
(1) To observe effects of ANP signaling on the differentiation and capacity of Th17cells in vitro;
(2) To probe the signaling pathway of ANP to regulate Th17cells.
Methods
(1) To aseptically excise and grind the spleens of BALB/c mice and to prepare the splenic mononuclear cells suspension;
(2) CD4+T cells were isolated from splenic mononuclear cells by positive selection with immuno-magnetic beads (MACS technology) and then counting and detecting the vitality of CD4+T cells;
(3) To define the expression of NPRA in CD4+T cells by WB;
(4) CD4+T cells were cultured for4days as control group (C), directional differentiation group (D), ANP addition group (ANP), A71915plus ANP group (A7), KT5823plus ANP group (KT);
(5) To detect the changes of the second messenger cGMP after intervening ANP signaling; cultured for4days, to measure the rate of IL-17-producing CD4+T cells by FACS; to detect the expression of RORγt and BATF (specific transcription factors for Th17cells) by WB; to confirm the changes of IL-17in supernatant.
Results
(1) Compared with the control group, the rate of IL-17-producing CD4+T cells could be significantly augmented in the cytokines microenvironment for directional differentiation comprising anti-IFN-y, anti-IL-4, TGF-β1, IL-6and IL-23, meanwhile along with increased production of IL-17in supernatant (P<0.01);
(2) Compared with differentiation group, addition of ANP could decrease the proportion of IL-17-producing CD4+T cells (P<0.01). Accordingly, the expression of RORyt and BATF (P<0.05) and the levels of IL-17in supernatant were also reduced (P<0.05); and the inhibitory effects showed dose-dependent (10-8-10-6M ANP);
(3) When using the ANP/NPRA signaling antagonist-A71915, the inhibitory effects of ANP on the differentiation and capacity of Th17cells were mostly reversed (respectively P<0.05);
(4) Similarly, KT5823were performed in advance, the inhibitory effects of ANP on the differentiation and capacity of Th17cells were also mostly recovered (respectively P<0.05);
(5) Changes of cGMP coupled NPRA were consistent following the intervention of ANP signaling(respectively P<0.01).
Conclusions
(1) ANP signaling could suppress the differentiation and capacity of Th17cells in vitro and displayed a dose-dependent effect;
(2) NPRA/PKG pathway played an important role in the process of ANP effecting on Thl7cells.
Part Ⅱ Effects of ANP signaling on Th17/IL-17and its role in mouse models with allergic asthma
Objectives
(1) To establish and evaluate mice models with allergic asthma;
(2) To observe changes of ANP and NPRA in mice with allergic asthma;
(3) To observe effects of ANP signaling on airway inflammation of mice models with allergic asthma, local expression of transcription factor for Th17cells and changes of IL-17in lung.
Methods
40SPF female BALB/c mice were randomly divided into five groups according to the study design:normal group, asthma group, ANP inhalation group (ANP), A71915intraperitoneal injection(i.p) plus ANP inhalation group (ANP+A71915), A71915(i.p) alone group. Every group contains8mice. As the scheme, mice models with allergic asthma were established by sensitized with10μg OVA/mouse+0.2ml A1(OH)3/mouse by i.p and then aerosol challenge with5%OVA via repeated inhalation in asthma group while the same volume of PBS were performed as the substitution of OVA in the normal group. In addition, aerosol nebulization of solution with ANP dry powder (dose1μg/g mouse) at30min before every OVA-challenge in ANP group and similarly, A71915 (dose0.5μg/g mouse) was used by i.p in A71915group at30min before every ANP inhalation. Other operations were same as the asthma group and behaviours of mice in every group should be observed during establishing the models. After24hours of the last challenge, operations were carried out as following:(1) after anesthesia, mice were implemented with tracheal intubation and changes of airway response were determined by recording lung resistance(RL) and lung dynamic compliance (Cdyn) stimulated with Methacholine of different doses (Mch);(2) total cells count, differential counts and production of ANP, IL-4, IL-17in bronchoalveolar lavage fluid (BALF) were detected;(3) observation and analysis of bronchial tube and lungtissue were completed by pathological staining;(4) expressions of NPRA for ANP signaling in lungtissue and splenic CD4+T cells from normal and asthma group were quantified by Western-blot;(5) RORyt and BATF proteins were also detected by WB in lungtissue and changes of ANP, IL-4, IL-17levels in lung homogenates.
Results
(1) Asthmatic symptoms of varying degrees were emerged in mice of asthma group, ANP group and A71915group such as tremble, unease, breathlessness, incontinence and so on while the normal group showed basically normal. In addition, no death of mice appeared during establishing model of asthma and drug treatment in vivo;
(2) Compared with the normal group, the dose-reponse curve of RL was significantly shifted up (P<0.01) while the curve of Cdyn was apparently shifted down (P<0.01) in asthma group, which suggests airway hyperresponsiveness (AHR) was successfully established.
(3) Compared with the normal group, airway hyperresponsiveness of A71915+ANP and A71915group was also obvious (respectively P<0.05) but reduced in contrast with asthma group (respectively P<0.05); while AHR of ANP group was not evident which was similar to normal group (respectively P>0.05);
(4) Total cells counts, differential cells counts of eosinophils, etc in BALF of asthma group were increased than those in BALF of normal group (respectively P<0.01); however, those in ANP group were not only significantly more than normal group (P<0.01) but even more seriuos than asthma group (P<0.05); while those in A71915group were obviously reduced compared with asthma (P<0.05); those in A71915+ANP group were also decreased compared with ANP group(P<0.05);
(5) Compared with normal group, levels of IL-4, IL-17in BALF of asthma group were apparently augmented (respectively P<0.01); while increased production of IL-4in BALF of ANP group (P<0.01) was more outstanding and the mean value of ANP group was even more higher than that of asthma group (P<0.05), but the levels of IL-17showed no obvious decline (P>0.05); in A71915group, the levels of IL-4, IL-17in BALF were all decreased compared with asthma group (P<0.05); in A71915+ANP group, levels of IL-4decreased (P<0.05) but change of IL-17was not significant compared with normal group (P>0.05);
(6) Production of ANP in BALF and lungtissue homogenates of asthma group were significantly higher than those in normal group (respectively P<0.01);
(7) Levels of NPRA protein in lungtissue of mice with asthma were much more than the normal group (P<0.05) and those in splenic CD4T cells were similar (P<0.05);
(8) Numerous inflammatory cells infiltrated and diffused around the bronchus and vessel, and lots of mucus secreted around airway compared with the normal group(respectively P<0.01); when inhalation of ANP, the above became more serious compared with asthma group (respectively P<0.05); and A71915+ANP group showed relaxative than ANP group (respectivelyP<0.05); while this situation in A71915group was attenuated compared with asthma group (respectively P<0.05);
(9) Expression of RORyt and BATF of Th17cells in mice lungtissue of asthma group were increased than the normal group (respectively P<0.05); while the RORyt and BATF were decreased after inhalation of ANP (respectively P<0.05) and expression of RORyt and BATF became much less in A71915+ANP group (P<0.05);
(10) The changes of IL-4, IL-17in mice lung homogenates of every group were similar to those of in BALF.
Conclusions
(1) Mice models with allergic asthma were successfully established based on the data;
(2) ANP signaling plays a role in the course of allergic asthma;
(3) ANP signaling could remit AHR of mice with allergic asthma;
(4) Not completely consistent with experimental data in vitro, ANP signaling could reduce expression of transcription factors of Thl7cells in lungtissue not along with significantly decreased IL-17in vivo, which also may be one of mechanisms;
(5) ANP signaling aggravate airway inflammation of allergic asthma.
Part III Effects of ANP signaling on functional balance of Th1/Th2cells and its role in airway inflammation of allergic asthma
Objectives
(1) To investigate the effects of ANP signaling on functional balance of Thl/Th2cells in vitro;
(2) To explore ANP signaling aggravating airway inflammation of allergic asthma by promoting dominance of Th2cells based on study in vivo.
Methods
To aseptically excise and grind the spleens of BALB/c mice with allergic asthma and to prepare the splenic mononuclear cells suspension. Then, CD4+T cells were isolated through positive selection with immuno-magnetic microbeads. CD4+T cells were cultured as the design after counting and detecting the vitality. All experiments in vitro were divided into control group, ANP group, A71915+ANP group according to the different intervention. Cells were harvested after24h by stimulated ConA (end concentration-5μg/ml). Then, for the sake of defining the effects of ANP signaling on the functional balance of Thl/Th2cells, to detect changes of T-bet/GATA3expression by WB and to measure levels of IFN-y/IL-4in supernatant were performed.
In vivo, detection of T-bet/GATA3for Thl/Th2cells in lungtissue of mice via WB and measurement of IFN-γ levels in BALF and lung homogenates were finished, and other experiments were processed in Part Ⅱ.
Results
(1) Compared with noraml group, expressions of GATA3in asthma group were obviously more higher; meanwhilie, expression of GATA3showed much more in ANP inhalation group than asthma group while T-bet was decreased (respectively P<0.05); when blockade of ANP signaling, expression of GATA3was decreased while that of T-bet was increased (respectively P<0.05);
(2) IFN-y levels in lung homogenates decreased compared with asthma group which was consistent with changes of T-bet (P<0.05) but not in BALF (P>0.05); and no obvious change of IFN-y levels happened in ANP+A71915group (P>0.05);
(3) In vitro, expression of GATA3in splenic CD4+T cells from mice with asthma was higher than control group after stimulated by ANP for24h along with reduced expression of T-bet (respectively P<0.05), in which the trend of changes were basically consistent with the results from in vivo; whlie A71915performed in advance, effects of ANP signaling on expressions of T-bet/GATA3were neutralized (respectively P<0.05);
(4) Level of IFN-y in supernatant was decreased while IL-4was increased in ANP group compared with the control (respectively P<0.05); When ANP signaling inhibitor was used, the changes of IFN-y/IL-4were also reversed (respectively P<0.05).
Conclusions
(1) In vitro, ANP signaling could affect functional balance of Thl/Th2cells, that is promoting Th2-type response while inhibiting Th1-type response;
(2) In vivo, ANP signaling could aggravate Th2cells response in lung, which is probably one of the mechanisms for ANP signaling to exacerbate airway inflammation of allergic asthma.
引文
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