陈艳妮，邱琴，闫珊，刘美娟，高福生(潍坊医学院，山东潍坊261000)



·基础研究·

雾化吸入L-精氨酸对哮喘小鼠气管急性炎症的影响及机制

陈艳妮，邱琴，闫珊，刘美娟，高福生(潍坊医学院，山东潍坊261000)

摘要：目的观察雾化吸入L-精氨酸对支气管哮喘(简称哮喘)小鼠气管急性炎症的影响，探讨其作用机制。方法30只雄性BALB/c小鼠随机分为三组，每组10只。L-精氨酸组、模型组分别于第1、8、15天腹腔注射含100 μg卵清蛋白干粉剂(OVA)和2 mg氢氧化铝粉的混悬液0.3 mL致敏，对照组于相同时间腹腔注射PBS 0.3 mL。第21天起将三组分别放于自制玻璃雾化箱内，L-精氨酸组、模型组以30 g/L OVA进行雾化吸入，对照组雾化吸入PBS，均1次/d、30 min/次、连续12 d。每次雾化激发前30 min，L-精氨酸组雾化吸入L-精氨酸174 mg/mL。末次雾化吸入24 h放血处死所有小鼠，留取左肺组织及右肺支气管肺泡灌洗液(BALF)。将左肺上叶组织切片进行HE染色，观察支气管病理变化；取BALF沉渣，计数白细胞总数及嗜酸性粒细胞、淋巴细胞、巨噬细胞数量；采用ELISA法检测BALF中IL-4、IFN-γ及NO。结果HE染色见模型组气道上皮断裂、脱落，黏膜水肿、皱褶增多；支气管周围有大量以嗜酸性粒细胞、淋巴细胞为主的炎性细胞浸润。L-精氨酸组气道周围炎细胞浸润及黏膜水肿均较模型组减轻。与对照组比较，模型组、L-精氨酸组BALF中白细胞总数及嗜酸性粒细胞、淋巴细胞、巨噬细胞数量均增多，L-精氨酸组上述细胞数量均较模型组减少，组间比较P均<0.05。与对照组比较，模型组及L-精氨酸组BALF中IL-4水平、IL-4/IFN-γ明显升高，IFN-γ及NO降低；与模型组比较，L-精氨酸组BALF中IL-4、IL-4/IFN-γ降低，IFN-γ及NO升高；组间比较P均<0.05。结论L-精氨酸雾化吸入对哮喘小鼠气管炎症有抑制作用，其作用机制可能与抑制IL-4表达、促进IFN-γ表达和NO产生有关。

关键词：支气管哮喘；L-精氨酸；雾化吸入；白细胞介素4；干扰素γ；小鼠

支气管哮喘(简称哮喘)是临床常见病，气道慢性炎症是哮喘的病理特征，多种炎症细胞和炎症因子参与哮喘的发生、发展[1～3]。糖皮质激素是目前治疗哮喘最有效的抗炎药物之一，但大剂量或长期使用会导致基底膜增厚等不良反应[4]。研究发现，精氨酸作为一种半必需氨基酸，其分解代谢失常参与哮喘的发病[5]。气道中精氨酸缺乏可抑制NO产生，刺激炎症细胞及炎症因子产生，增强气道炎症反应[6,7]。2015年4～6月，本研究观察了雾化吸入L-精氨酸对哮喘小鼠气道急性炎症的影响，现分析结果并探讨其作用机制。

1材料与方法

1.1材料近交系SPF级、健康雄性BALB/c小鼠30只，6～8周龄，体质量18～20 g，购自北京华阜康生物科技股份有限公司，实验动物许可证号：SCXK(京)2014-0004。卵清蛋白干粉剂(OVA)，氢氧化铝粉，IL-4及IFN-γ ELISA试剂盒，NO ELISA试剂盒。

1.2动物分组及处理将小鼠随机分为L-精氨酸组、模型组、对照组，每组10只。根据文献[7,8]方法，L-精氨酸组、模型组制备哮喘模型：分别于第1、8、15天腹腔注射含100 μg OVA和2 mg氢氧化铝粉的混悬液0.3 mL；对照组于相同时间腹腔注射PBS 0.3 mL。第21天起将三组分别放于体积为3 L的自制玻璃雾化箱内，L-精氨酸组、模型组以30 g/L OVA进行雾化吸入，对照组雾化吸入PBS，均1次/d、30 min/次、连续12 d。每次雾化吸入前30 min，L-精氨酸组雾化吸入174 mg/mL L-精氨酸[8]。三组末次雾化吸入24 h后腹腔注射100 g/L水合氯醛(0.5 mg/kg)麻醉，放血处死小鼠，留取左肺组织；结扎左主支气管，0.4 mL PBS冲洗右肺，取支气管肺泡灌洗液(BALF)，1 500 r/min离心10 min，取上清液，-80 ℃冰箱保存。

1.3相关指标观察①气道病理学观察：取左肺上叶组织，4%多聚甲醛固定24 h，脱水，二甲苯透明，石蜡包埋，制备厚3 μm的标准切片，HE染色，光学显微镜下观察。②BALF中细胞计数：PBS重悬BALF沉渣，取200 μL重悬液涂片，Wright-Giemsa染色，光镜下计数白细胞总数及嗜酸性粒细胞、淋巴细胞、巨噬细胞数量。③ BALF中IL-4、IFN-γ、NO检测：采用ELISA法检测BALF中IL-4、IFN-γ、NO水平，步骤均参照试剂盒说明书。

2结果

2.1三组气管病理学变化HE染色见对照组气管上皮平整，黏膜无明显水肿，气管及周围血管组织无明显炎性细胞浸润，支气管管腔光滑。模型组气管上皮断裂、脱落，黏膜水肿，皱褶增多；支气管周围有大量以嗜酸性粒细胞、淋巴细胞为主的炎性细胞浸润。L-精氨酸组气管周围炎细胞浸润较模型组减少，黏膜水肿较模型组减轻。见插页Ⅱ图3。

2.2三组BALF中细胞计数比较与对照组比较，模型组、L-精氨酸组BALF中白细胞总数及嗜酸性粒细胞、淋巴细胞、巨噬细胞数量均增多，L-精氨酸组上述细胞数量均较模型组减少，组间比较P均<0.05。见表1。

表1　三组BALF中细胞计数比较

注：与对照组比较，*P<0.05；与模型组比较，#P<0.05。

2.3三组BALF中IL-4、IFN-γ、NO水平比较与对照组比较，模型组及L-精氨酸组BALF中IL-4、IL-4/IFN-γ明显升高，IFN-γ及NO升高；与模型组比较，L-精氨酸组BALF中IL-4、IL-4/IFN-γ降低，IFN-γ及NO升高，P均>0.05。见表2。

3结论

哮喘是由多种细胞及细胞组分参与的气道慢性炎症性疾病，以嗜酸性粒细胞、T淋巴细胞为代表的多种炎症细胞在气道局部聚集并释放炎性介质是哮喘发病的关键因素。研究表明，辅助性T淋巴细胞亚群Th1/Tp比例和功能失衡是哮喘的主要免疫学发病机制，其中IL-4 和IFN-γ在哮喘的发病过程中发挥重要作用[9]，并与哮喘的严重程度相关[10]。Tp细胞分泌的IL-4可以上调嗜酸性粒细胞趋化蛋白1和嗜酸性粒细胞趋化蛋白3 mRNA的表达，增强呼吸道内嗜酸性粒细胞等炎症细胞的聚集、活化；增加精氨酸酶活性，竞争性抑制一氧化氮合酶(NOS)活性，导致精氨酸代谢失衡，参与哮喘的发病[11]。Th1细胞分泌的IFN-γ抑制Tp类细胞因子产生，抑制IL-4 诱导的B细胞增殖及嗜酸粒细胞活化、聚集，减轻呼吸道炎症反应[12]。IL-4、IFN-γ作为哮喘发生及发展过程中的重要细胞因子并非独立存在，而是相互联系，互相影响。因此，纠正Th1/Tp 比例和功能失衡对哮喘治疗具有较大价值。

表2　　　　三组BALF中IL-4、IFN-γ、NO水平

注：与对照组比较，*P<0.05；与模型组比较，#P<0.05。

精氨酸作为一种半必需氨基酸，其在气管中的代谢失衡参与哮喘的发病。哮喘患者气管中精氨酸经精氨酸酶(Arg)途径和NOS途径进行分解代谢[13,14]，经Arg途径产生多胺类物质，经NOS途径产生NO。内生性NO参与哮喘气管的扩张及抗炎症反应[15]。研究发现，小鼠哮喘模型气管中IL-4、IL-10等细胞因子通过转录因子STAT6、环腺苷酸-蛋白激酶 A(cAMP-PKA)信号通路等途径增强精氨酸酶的活性，与NOS竞争性作用于共同底物精氨酸，导致气管内精氨酸的生物利用度降低，发生精氨酸代谢失衡。NO缺乏可刺激炎症细胞及炎症因子的产生，增强气道炎症反应。研究发现，吸入L-精氨酸在减轻哮喘症状的同时，会使气道局部NO浓度过高，引起气道黏膜水肿、Tp细胞聚集、血管渗出等病理反应，引起或加重气管炎症。雾化吸入L-精氨酸可在有效抑制气管炎症反应的同时，降低由于NO增多产生的气管局部不良反应。

本研究采用OVA致敏的哮喘小鼠作为研究对象，模拟哮喘发作的急性期，并对急性哮喘小鼠给予雾化吸入L-精氨酸治疗，结果显示L-精氨酸组较模型组BALF中IL-4、IL-4/IFN-γ降低，IFN-γ、NO升高，且支气管黏膜上皮损伤脱落及炎性细胞浸润程度均较模型组减轻。提示L-精氨酸雾化吸入对哮喘小鼠气道炎症有抑制作用，可减轻哮喘气管损伤，其作用机制可能与抑制IL-4表达、促进IFN-γ表达和NO产生有关。

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Effect of aerosol inhalation of L-arginine on acute airway inflammation in mice model with asthma and its mechanism

CHENYanni,QIUQin,YANShan,LIUMeijuan,GAOFusheng

(WeifangMedicalCollege,Weifang261000,China)

Abstract:ObjectiveTo observe the effect of aerosol inhalation of L-arginine on acute airway inflammation in mice model with asthma and to investigate the mechanism. MethodsThirty male BALB/c mice were randomly divided into three groups and 10 mice in each group: L-arginine group, model group and the control group. The L-arginine group and model group were sensitized by intraperitoneal (i.p.) injections of 0.3 mL suspension of 100 μg OVA and 2 mg AL(OH)3 , and the control group was injected with 0.3 mL PBS at the same time. Since the 21st day, these three groups were put into homemade glass atomization box. L-arginine group and model group were given 30 g/L OVA for aerosol inhalation and the control group with PBS for aerosol inhalation, once a day, thirty minutes one time and for 12 days. L-arginine group was given 174 mg/mL L-arginine for aerosol inhalation for 30 minutes before every sensitization. Twenty-four hours after the last sensitization, mice of the three groups were bled to death, and then we collected the left lung tissues and the bronchoalveolar lavage fluid. HE staining was used on the upper left lobe of lung tissue, and the pathological changes were observed. We counted the total number of leukocytes, esosinophils, lymphocytes and macrophages. The levels of interlerkin-4(IL-4), NO and interferon-γ (IFN-γ) in BALF were measured by ELISA. ResultsIn the model group, the airway epithelium fractured, fell off and had mucous edema with more folds under HE staining, and there was inflammatory cell infiltration with a large number of esosinophils and lymphocytes around the bronchus. The inflammatory cell infiltration and mucous edema in the L-arginine group was less than that of the model group. Compared with the control group, the leukocytes, esosinophils, lymphocytes and macrophages in BALF were increased in the L-arginine group and the model group, and the above indexes of the L-arginine group were less than those of the model group (all P<0.05). Compared with the control group, the levels of IL-4 and IL-4/IFN-γ were increased, but the levels of IFN-γ and NO were decreased in the L-arginine group and model group; compared with the model group, the levels of IL-4 and IL-4/IFN-γ declined, but IFN-γ and NO elevated in the L-arginine group, and significant difference was found among these groups (all P<0.05). ConclusionThe aerosol inhalation of L-arginine can inhibit the airway inflammation of asthmatic mice, and its mechanism may be related to inhibiting IL-4 expression, promoting IFN-γ expression and the production of NO.

Key words:bronchial asthma; L-arginine; aerosol inhalation; interleukin-4; interferon-γ; mice

(收稿日期：2015-09-07)

中图分类号：R562.2

文献标志码：A

文章编号：1002-266X(2016)12-0028-03

doi:10.3969/j.issn.1002-266X.2016.12.008

通信作者：高福生(E-mail: gaofs888@163.com)

基金项目：山东省自然科学基金资助项目(ZR2012HM094)。