陳艷妮，邱琴，閆珊，劉美娟，高福生(濰坊醫(yī)學(xué)院，山東濰坊261000)

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·基礎(chǔ)研究·

霧化吸入L-精氨酸對哮喘小鼠氣管急性炎癥的影響及機(jī)制

陳艷妮，邱琴，閆珊，劉美娟，高福生(濰坊醫(yī)學(xué)院，山東濰坊261000)

摘要：目的觀察霧化吸入L-精氨酸對支氣管哮喘(簡稱哮喘)小鼠氣管急性炎癥的影響，探討其作用機(jī)制。方法30只雄性BALB/c小鼠隨機(jī)分為三組，每組10只。L-精氨酸組、模型組分別于第1、8、15天腹腔注射含100 μg卵清蛋白干粉劑(OVA)和2 mg氫氧化鋁粉的混懸液0.3 mL致敏，對照組于相同時(shí)間腹腔注射PBS 0.3 mL。第21天起將三組分別放于自制玻璃霧化箱內(nèi)，L-精氨酸組、模型組以30 g/L OVA進(jìn)行霧化吸入，對照組霧化吸入PBS，均1次/d、30 min/次、連續(xù)12 d。每次霧化激發(fā)前30 min，L-精氨酸組霧化吸入L-精氨酸174 mg/mL。末次霧化吸入24 h放血處死所有小鼠，留取左肺組織及右肺支氣管肺泡灌洗液(BALF)。將左肺上葉組織切片進(jìn)行HE染色，觀察支氣管病理變化；取BALF沉渣，計(jì)數(shù)白細(xì)胞總數(shù)及嗜酸性粒細(xì)胞、淋巴細(xì)胞、巨噬細(xì)胞數(shù)量；采用ELISA法檢測BALF中IL-4、IFN-γ及NO。結(jié)果HE染色見模型組氣道上皮斷裂、脫落，黏膜水腫、皺褶增多；支氣管周圍有大量以嗜酸性粒細(xì)胞、淋巴細(xì)胞為主的炎性細(xì)胞浸潤。L-精氨酸組氣道周圍炎細(xì)胞浸潤及黏膜水腫均較模型組減輕。與對照組比較，模型組、L-精氨酸組BALF中白細(xì)胞總數(shù)及嗜酸性粒細(xì)胞、淋巴細(xì)胞、巨噬細(xì)胞數(shù)量均增多，L-精氨酸組上述細(xì)胞數(shù)量均較模型組減少，組間比較P均<0.05。與對照組比較，模型組及L-精氨酸組BALF中IL-4水平、IL-4/IFN-γ明顯升高，IFN-γ及NO降低；與模型組比較，L-精氨酸組BALF中IL-4、IL-4/IFN-γ降低，IFN-γ及NO升高；組間比較P均<0.05。結(jié)論L-精氨酸霧化吸入對哮喘小鼠氣管炎癥有抑制作用，其作用機(jī)制可能與抑制IL-4表達(dá)、促進(jìn)IFN-γ表達(dá)和NO產(chǎn)生有關(guān)。

關(guān)鍵詞：支氣管哮喘；L-精氨酸；霧化吸入；白細(xì)胞介素4；干擾素γ；小鼠

支氣管哮喘(簡稱哮喘)是臨床常見病，氣道慢性炎癥是哮喘的病理特征，多種炎癥細(xì)胞和炎癥因子參與哮喘的發(fā)生、發(fā)展[1～3]。糖皮質(zhì)激素是目前治療哮喘最有效的抗炎藥物之一，但大劑量或長期使用會導(dǎo)致基底膜增厚等不良反應(yīng)[4]。研究發(fā)現(xiàn)，精氨酸作為一種半必需氨基酸，其分解代謝失常參與哮喘的發(fā)病[5]。氣道中精氨酸缺乏可抑制NO產(chǎn)生，刺激炎癥細(xì)胞及炎癥因子產(chǎn)生，增強(qiáng)氣道炎癥反應(yīng)[6,7]。2015年4～6月，本研究觀察了霧化吸入L-精氨酸對哮喘小鼠氣道急性炎癥的影響，現(xiàn)分析結(jié)果并探討其作用機(jī)制。

1材料與方法

1.1材料近交系SPF級、健康雄性BALB/c小鼠30只，6～8周齡，體質(zhì)量18～20 g，購自北京華阜康生物科技股份有限公司，實(shí)驗(yàn)動物許可證號：SCXK(京)2014-0004。卵清蛋白干粉劑(OVA)，氫氧化鋁粉，IL-4及IFN-γ ELISA試劑盒，NO ELISA試劑盒。

1.2動物分組及處理將小鼠隨機(jī)分為L-精氨酸組、模型組、對照組，每組10只。根據(jù)文獻(xiàn)[7,8]方法，L-精氨酸組、模型組制備哮喘模型：分別于第1、8、15天腹腔注射含100 μg OVA和2 mg氫氧化鋁粉的混懸液0.3 mL；對照組于相同時(shí)間腹腔注射PBS 0.3 mL。第21天起將三組分別放于體積為3 L的自制玻璃霧化箱內(nèi)，L-精氨酸組、模型組以30 g/L OVA進(jìn)行霧化吸入，對照組霧化吸入PBS，均1次/d、30 min/次、連續(xù)12 d。每次霧化吸入前30 min，L-精氨酸組霧化吸入174 mg/mL L-精氨酸[8]。三組末次霧化吸入24 h后腹腔注射100 g/L水合氯醛(0.5 mg/kg)麻醉，放血處死小鼠，留取左肺組織；結(jié)扎左主支氣管，0.4 mL PBS沖洗右肺，取支氣管肺泡灌洗液(BALF)，1 500 r/min離心10 min，取上清液，-80 ℃冰箱保存。

1.3相關(guān)指標(biāo)觀察①氣道病理學(xué)觀察：取左肺上葉組織，4%多聚甲醛固定24 h，脫水，二甲苯透明，石蠟包埋，制備厚3 μm的標(biāo)準(zhǔn)切片，HE染色，光學(xué)顯微鏡下觀察。②BALF中細(xì)胞計(jì)數(shù)：PBS重懸BALF沉渣，取200 μL重懸液涂片，Wright-Giemsa染色，光鏡下計(jì)數(shù)白細(xì)胞總數(shù)及嗜酸性粒細(xì)胞、淋巴細(xì)胞、巨噬細(xì)胞數(shù)量。③ BALF中IL-4、IFN-γ、NO檢測：采用ELISA法檢測BALF中IL-4、IFN-γ、NO水平，步驟均參照試劑盒說明書。

2結(jié)果

2.1三組氣管病理學(xué)變化HE染色見對照組氣管上皮平整，黏膜無明顯水腫，氣管及周圍血管組織無明顯炎性細(xì)胞浸潤，支氣管管腔光滑。模型組氣管上皮斷裂、脫落，黏膜水腫，皺褶增多；支氣管周圍有大量以嗜酸性粒細(xì)胞、淋巴細(xì)胞為主的炎性細(xì)胞浸潤。L-精氨酸組氣管周圍炎細(xì)胞浸潤較模型組減少，黏膜水腫較模型組減輕。見插頁Ⅱ圖3。

2.2三組BALF中細(xì)胞計(jì)數(shù)比較與對照組比較，模型組、L-精氨酸組BALF中白細(xì)胞總數(shù)及嗜酸性粒細(xì)胞、淋巴細(xì)胞、巨噬細(xì)胞數(shù)量均增多，L-精氨酸組上述細(xì)胞數(shù)量均較模型組減少，組間比較P均<0.05。見表1。

表1　三組BALF中細(xì)胞計(jì)數(shù)比較

注：與對照組比較，*P<0.05；與模型組比較，#P<0.05。

2.3三組BALF中IL-4、IFN-γ、NO水平比較與對照組比較，模型組及L-精氨酸組BALF中IL-4、IL-4/IFN-γ明顯升高，IFN-γ及NO升高；與模型組比較，L-精氨酸組BALF中IL-4、IL-4/IFN-γ降低，IFN-γ及NO升高，P均>0.05。見表2。

3結(jié)論

哮喘是由多種細(xì)胞及細(xì)胞組分參與的氣道慢性炎癥性疾病，以嗜酸性粒細(xì)胞、T淋巴細(xì)胞為代表的多種炎癥細(xì)胞在氣道局部聚集并釋放炎性介質(zhì)是哮喘發(fā)病的關(guān)鍵因素。研究表明，輔助性T淋巴細(xì)胞亞群Th1/Tp比例和功能失衡是哮喘的主要免疫學(xué)發(fā)病機(jī)制，其中IL-4 和IFN-γ在哮喘的發(fā)病過程中發(fā)揮重要作用[9]，并與哮喘的嚴(yán)重程度相關(guān)[10]。Tp細(xì)胞分泌的IL-4可以上調(diào)嗜酸性粒細(xì)胞趨化蛋白1和嗜酸性粒細(xì)胞趨化蛋白3 mRNA的表達(dá)，增強(qiáng)呼吸道內(nèi)嗜酸性粒細(xì)胞等炎癥細(xì)胞的聚集、活化；增加精氨酸酶活性，競爭性抑制一氧化氮合酶(NOS)活性，導(dǎo)致精氨酸代謝失衡，參與哮喘的發(fā)病[11]。Th1細(xì)胞分泌的IFN-γ抑制Tp類細(xì)胞因子產(chǎn)生，抑制IL-4 誘導(dǎo)的B細(xì)胞增殖及嗜酸粒細(xì)胞活化、聚集，減輕呼吸道炎癥反應(yīng)[12]。IL-4、IFN-γ作為哮喘發(fā)生及發(fā)展過程中的重要細(xì)胞因子并非獨(dú)立存在，而是相互聯(lián)系，互相影響。因此，糾正Th1/Tp 比例和功能失衡對哮喘治療具有較大價(jià)值。

表2　　　　三組BALF中IL-4、IFN-γ、NO水平

注：與對照組比較，*P<0.05；與模型組比較，#P<0.05。

精氨酸作為一種半必需氨基酸，其在氣管中的代謝失衡參與哮喘的發(fā)病。哮喘患者氣管中精氨酸經(jīng)精氨酸酶(Arg)途徑和NOS途徑進(jìn)行分解代謝[13,14]，經(jīng)Arg途徑產(chǎn)生多胺類物質(zhì)，經(jīng)NOS途徑產(chǎn)生NO。內(nèi)生性NO參與哮喘氣管的擴(kuò)張及抗炎癥反應(yīng)[15]。研究發(fā)現(xiàn)，小鼠哮喘模型氣管中IL-4、IL-10等細(xì)胞因子通過轉(zhuǎn)錄因子STAT6、環(huán)腺苷酸-蛋白激酶 A(cAMP-PKA)信號通路等途徑增強(qiáng)精氨酸酶的活性，與NOS競爭性作用于共同底物精氨酸，導(dǎo)致氣管內(nèi)精氨酸的生物利用度降低，發(fā)生精氨酸代謝失衡。NO缺乏可刺激炎癥細(xì)胞及炎癥因子的產(chǎn)生，增強(qiáng)氣道炎癥反應(yīng)。研究發(fā)現(xiàn)，吸入L-精氨酸在減輕哮喘癥狀的同時(shí)，會使氣道局部NO濃度過高，引起氣道黏膜水腫、Tp細(xì)胞聚集、血管滲出等病理反應(yīng)，引起或加重氣管炎癥。霧化吸入L-精氨酸可在有效抑制氣管炎癥反應(yīng)的同時(shí)，降低由于NO增多產(chǎn)生的氣管局部不良反應(yīng)。

本研究采用OVA致敏的哮喘小鼠作為研究對象，模擬哮喘發(fā)作的急性期，并對急性哮喘小鼠給予霧化吸入L-精氨酸治療，結(jié)果顯示L-精氨酸組較模型組BALF中IL-4、IL-4/IFN-γ降低，IFN-γ、NO升高，且支氣管黏膜上皮損傷脫落及炎性細(xì)胞浸潤程度均較模型組減輕。提示L-精氨酸霧化吸入對哮喘小鼠氣道炎癥有抑制作用，可減輕哮喘氣管損傷，其作用機(jī)制可能與抑制IL-4表達(dá)、促進(jìn)IFN-γ表達(dá)和NO產(chǎn)生有關(guān)。

參考文獻(xiàn)：

[1] 廖述文,楊平,張通,等.藤黃酸對結(jié)腸癌 LoVo細(xì)胞增殖和侵襲能力的影響[J].國際中醫(yī)中藥雜志,2014,36(3):220-222.

[2] Hato T, Tabata M, Oike Y. The role of angiopoietin-like-proteins in angiogenesis and metabolism[J]. Trends Car-diovasc Med, 2008,18(1):6-14.

[3] 蔡暢,周美茜,陳成水,等.孟魯司特與甲潑尼龍緩解支氣管哮喘炎癥機(jī)制的對比實(shí)驗(yàn)研究[J].醫(yī)學(xué)研究雜志,2013,42(2):93-96.

[4] Shimoda T, Obase Y, Kishikawa R, et al. Impact of inhaled corticosteroid treatment on 15-year longitudinal respiratory functionchanges in adult patients with bronchial asthma [J]. Int Arch AllergyImmunol, 2013,162(4): 323-329.

[5] Scott JA, Grasemann H. Arginine metabolism in asthma[J]. Immunol Allergy Clin North Am, 2014,34(4):767-775.

[6] Benson RC, Hardy KA, Morris CR. Arginase and arginine dysregulation in asthma[J]. J Allergy (Cairo), 2011,2001:736319.

[7] Fan XY, Snoek M, Smids B, et al. Arginine deficiency augments inflammatory mediator production by airway epithelial cells in vitro[J]. Respir Res, 2009(10):62.

[8] Maarsingh H, Zuidhof AB, Bos IS, et al. Arginase inhibition protects against allergen-induced airway obstruction,hyperresponsiveness and inflammation[J]. Am J Respir Crit Care Med, 2008,178(6):565-573.

[9] Matsukura S, Odaka M, Kurokawa M, et al. Transforming growth factor-beta stimulates the expression of eotaxin /CC chemokine ligand11 and its promoter activity through binding site for nuclear factor-kappaB in airway smooth muscle cells[J]. Clin Exp Allergy, 2010,40(5):763-771.

[10] Tsuchiya K, Isogai S, Tamaoka M, et al. Depletion of CD8+T cells enhances airway remodeling in a rodent model of asthma[J]. Immunology, 2009,126(1):45-54.

[11] Ji NF, Xie YC, Zhang MS, et al. Ligustrazine corrects Th1/Tp and Treg/Th17 imbalance in a mouse asthma model [J]. Int Immunopharmacol, 2014,21(1):76-81.

[12] Kawaguchi M, Kokubu F, Huang SK, et al. The IL-17F signaling pathway is involved in the induction of IFN-gamma-inducible protein 10 in bronchial epithelial cells[J]. J Allergy Clin Immunol, 2007,119(6):1408-1414.

[13] Lara A, Khatri SB, Wang Z, et al. Alterations of the arginine metabolome in asthma[J]. Am J Respir Crit Care Med, 2008,178(7):673-681.

[14] Scott JA, Grasemann H. Arginine metabolism in asthma[J]. Immunol Allergy Clin North Am, 2014,34(4):767-775.

[15] Morris CR. Arginine and asthma[J]. Nestle Nutr Inst Workshop Ser, 2013(77):1-15.

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)

中圖分類號：R562.2

文獻(xiàn)標(biāo)志碼：A

文章編號：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)

基金項(xiàng)目：山東省自然科學(xué)基金資助項(xiàng)目(ZR2012HM094)。