吸入一氧化氮對致敏大鼠氣道阻力以及氣道炎癥的影響

1、    吸入一氧化氮對致敏大鼠氣道阻力以及氣道炎癥的影響        摘要 目的:探討吸入一定劑量一氧化氮(NO)對哮喘氣道阻力以及氣道炎癥的影響。方法:采用免疫組織化學技術(shù)(ABC法),觀察卵蛋白致敏的氣道炎癥大鼠模型吸入40×10-6 NO對氣道阻力、氣道嗜酸性粒細胞(Eos)、淋巴細胞及mIL-2R陽性淋巴細胞的影響。結(jié)果:吸入40×10-6 NO 1 h或連續(xù)6 d每日吸入1 h均可明顯減低氣道阻力(P0.05)。短時吸入40×10-6

2、 NO(吸入1 h)對氣道炎性細胞浸潤無明顯影響(P0.05),連續(xù)6 d每日吸入NO 1 h,氣道淋巴細胞數(shù)無明顯變化,但mIL-2R陽性淋巴細胞明顯高于未吸入NO組(P0.05)。結(jié)論:吸入40×10-6 NO有明顯擴張支氣管的作用;短時吸入40×10-6 NO對氣道炎癥無影響,但反復間斷吸入40×10-6 NO后可引起氣道活化T淋巴細胞增加,可能會加重哮喘氣道炎癥,值得在臨床應(yīng)用中引起重視。主題詞一氧化氮；投藥，吸入；哮喘； T-淋巴細胞 Effects of inhaled nitric oxide on the airways resistance an

3、d inflammation in sensitized ratsXUE Jian-Min, XU Yong-Jian, ZHANG Zhen-XiangDepartment of Respiratory Medicine, Tongji Hospital,Tongji Medical University, Wuhan (430030) Abstract AIM: To investigate the effect of inhaled certain concentration of nitric oxide (NO) on the airway resistance and inflam

4、mation in bronchial asthma. METHODS: The influences of inhaled 40×10-6 NO on the airway resistance as well as on the cell numbers of eosinophils and lymphocytes, especially the numbers of membrane interleukin-2 receptor( mIL- 2R) positive lymphotytes,in ovalbumin- sensitized rats were observed

5、by physiological and immunohistochemical techniques. RESULTS: Inhalation of 40×10-6 NO both for 1h, and for 1h each day for 6 consecutive days attenuated the airways resistance significantly (both with P0.05). Short term (1h) inhalation of NO had no effects on the inflammatory cell numbers in t

6、he airways. On the contrary,prolonged use of inhaled NO (1h each day for 6 days) led to the significant increase in the numbers of mIL-2R positive lymphocytes in the airways (P0.05). CONCLUSION: The inhalation of 40×10-6 NO has bronchodilating effect. The airways inflammation is not affected by

7、 short term (1h) inhalation; but prolonged inhalation (1h each day for 6 days) may lead to exacerbation of airways inflammation, to which attention should be paid in the clinical practice when inhaled NO is used in the treatment of bronchial asthma.MeSH Nitric oxide；Administration inhalation;Asthma;

8、 T-lymphocyte支氣管哮喘(哮喘)是氣道慢性炎癥性疾病,支氣管平滑肌痙攣是其重要的臨床特征之一。一氧化氮(nitric oxide,NO)對氣道平滑肌有直接舒張作用,并調(diào)節(jié)支氣管的功能狀態(tài)1,因此有人設(shè)想把NO作為一種新型支氣管擴張劑在臨床使用,尤其是哮喘危重發(fā)作時使用。但吸入NO對哮喘氣道炎癥,尤其是淋巴細胞功能有無影響,報道尚少。我們擬通過觀察吸入40×10-6 NO對卵蛋白致敏大鼠的氣道阻力以及氣道炎癥和氣道淋巴細胞膜表面白介素2受體(mIL-2R)表達的影響,探討外源性NO(吸入NO)與哮喘氣道阻力及氣道炎癥的關(guān)系,為了解吸入NO治療哮喘的臨床應(yīng)用前景提供實驗資料。

9、材料和方法1.主要藥品與試劑: 40×10-6 NO根據(jù)400×10-6 NO標準氣體(以高純N2為母氣貯存于鋁合金罐內(nèi),由北京氦普氣體工業(yè)有限公司提供)調(diào)配;卵蛋白(OVA)為Sigma產(chǎn)品;氫氧化鋁A1(OH)3為上海金山化工廠產(chǎn)品;百日咳桿菌疫苗為上海生物制品研究所提供;小鼠抗大鼠mIL-2R單克隆抗體購于北京邦定醫(yī)學生物公司。2.動物模型及分組: 雄性SD大鼠30只,體重(180±45) g,分為:正常對照組(A組,6只),一次激發(fā)組(B組,6只);反復激發(fā)組(C組,6只);吸入NO 1h組(D組,6只);吸入NO 1 h/d×6 d組(E組,6

10、只)。B、C、D、E各組大鼠的致敏參照文獻2方法進行:腹腔注射OVA 1mg,百日咳桿菌疫苗5×109個及A1(OH)3100 mg,2周后用1%OVA生理鹽水超聲霧化吸入激發(fā),每次20 min。A組以生理鹽水代替抗原進行注射和霧化吸入;B組為一次激發(fā)組,于第一次霧化吸入OVA后即終止吸入。C組為反復激發(fā)組,反復霧化吸入OVA每天1次,連續(xù)6 d。D組為OVA霧化吸入后于自制密閉容器內(nèi)吸入40×10-6 NO 1 h;E組為每天于1%OVA吸入后即吸入40×10-6 NO 1 h/d,連續(xù)6 d。以上各組大鼠均于生理鹽水,或OVA超聲霧化吸入,或吸入NO等前及吸入

11、后10 min,用3%戊巴比妥鈉進行麻醉后,用SB-40生理四導儀測定氣道阻力(E組為吸入NO第6 d進行測定),然后處死大鼠,留取右肺中葉組織用于HE染色及mIL-2R的免疫組化染色。3.免疫組化技術(shù)(ABC法)檢測大鼠肺組織mIL-2R陽性細胞:取右肺中葉組織,用恒冷切片機制成20 m切片,冷丙酮固定10 min,用ABC法3進行肺組織mIL-2R陽性細胞染色。用PBS取代羊抗小鼠血清作為空白對照。結(jié)果用醫(yī)用TYTJ-300 像分析系統(tǒng)處理,每張切片隨機取10個視野,在400×光鏡下測定mIL-2R陽性細胞(呈棕色)表達的積分光密度值(A值)。取10個視野的均值作為該片的代表值。

12、4.致敏大鼠氣道炎性細胞浸潤的觀察: 常規(guī)HE染色,400×光鏡下隨機觀察5個視野,計數(shù)支氣管粘膜下Eos、淋巴細胞數(shù),計算每個視野平均值。5.統(tǒng)計學方法: 數(shù)據(jù)用均數(shù)±標準差(±s)表示,組間差異的顯著性用t檢驗法檢驗。結(jié)果一、各組大鼠氣道阻力變化：由表1可見一次激發(fā)組和反復激發(fā)組激發(fā)后氣道阻力均明顯高于激發(fā)前(P0.05), 激發(fā)組激發(fā)后氣道阻力均顯著高于正常組(P0.05);吸入NO 1 h組激發(fā)后較一次激發(fā)組激發(fā)后,吸入NO 1 h/d×6 d組激發(fā)后第6 d較反復激發(fā)組激發(fā)后第6 d氣道阻力均減低(P0.05),吸入NO組激發(fā)前后氣道阻力變化無

13、顯著差異(P0.05)。表1各組大鼠氣道阻力變化Tab 1 Changes of airway resistance in rats(±s,n=6)GroupR(kPa.s/mL)Before stimulationAfter stimulationA0.062±0.0090.065±0.004B0.068±0.0120.151±0.018C0.075±0.0110.194±0.022D0.077±0.0040.082±0.005E0.084±0.0030.090±0.003*P0.0

14、5,vs B; *P0.05,vs CA:control;B:stimulated once; C:repeatedly stimulated;D:inhalation of NO for 1 h; E:inhalation of NO, 1 h/d,6 d 表2各組大鼠氣道炎性細胞數(shù)的變化Tab 2 Changes of airway inflammation cell numbers in rats airway (±s,n=6)GroupEos(cells/HP)Lymphocyte(cells/HP)mIL-2R+lymphocyte(A)A05.32±2.5611

15、.17±2.37B18.96±3.50*30.53±4.32*136.90±15.43*C22.67±4.86*33.23±4.33*279.40±69.42*D17.32±2.33*28.47±2.78*155.80±24.43*E23.11±3.45*34.87±3.47*424.4±54.18*P0.05, vs A; P0.05, vs B; P0.05,vs C Fig 1 Bronchial and lung tissue of inhalation N

16、O 1 h/d×6 d group rat (immunohistochemical method, ×200), the numbers of mIL-2R positive lymphocytes in the airway were significantly increased1吸入NO 1h/d×6 d組大鼠支氣管肺組織(免疫組化染色，×200)，支氣管周圍大量mIL-2R陽性淋巴細胞二、各組大鼠氣道細胞學變化(見表2)：兩激發(fā)組氣道Eos,淋巴細胞及mIL-2R陽性淋巴細胞數(shù)明顯多于正常對照組(P0.05);反復激發(fā)組較一次激發(fā)組氣道Eos、

17、淋巴細胞數(shù)無明顯差別(P0.05);而mIL-2R陽性淋巴細胞數(shù)則較多(P0.05);吸入NO 1h組較一次激發(fā)組3種炎細胞變化差異無顯著(均為P0.05),吸入NO 1 h/d×6 d組(1)氣道m(xù)IL-2R陽性淋巴細胞數(shù)明顯多于反復激發(fā)組(2)差異(P0.05),Eos、淋巴細胞數(shù)目無明顯變化(P0.05)。正常對照及空白對照均未見mIL-2R陽性細胞。Fig 2 Bronchial and lung tissue of repeatedly stimulation group rat (immunohistochemical method, ×200), there

18、were some mIL-2R positive lymphocytee2反復激發(fā)組大鼠支氣管肺組織(免疫組化染色，×200)，有較多mIL-2R陽性淋巴細胞討論研究表明氣道中唯一具有舒張支氣管平滑肌作用的非膽堿能非腎上腺能神經(jīng)(NANC)的遞質(zhì)即為NO4,NO的作用與其激活平滑肌細胞鳥苷酸環(huán)化酶,生成cGMP增多有關(guān)5。近年已認識到,哮喘是氣道慢性炎癥性疾病,氣道炎癥細胞浸潤及激活導致氣道高反應(yīng)性,在致敏原刺激下出現(xiàn)支氣管痙攣,氣道阻力增加。吸入NO能逆轉(zhuǎn)氣道收縮,我們以往人體實驗中觀察到,吸入40×10-6 NO可以明顯改善急性發(fā)作期哮喘患者的肺功能6,因此吸入NO可

19、能不失為治療哮喘,解除支氣管痙攣的有效手段之一。本實驗結(jié)果顯示,反復間斷吸入NO 6 d、每天1 h,有明顯的擴支氣管作用,同時氣道中活化T淋巴細胞明顯多于未吸入NO組,NO激活哮喘淋巴細胞的機制尚不清楚,可能包括cGMP依賴機制cGMP非依賴性機制:Taylor-Robinson等7認為NO可選擇性抑制Th1細胞分泌IFN-,因而可間接促進IL-4、IL-5增高,從而促進Th2細胞激活;Benbernou等8新近觀察到NO供體可明顯減少T細胞IFN- mRNA表達,而一氧化氮合酶抑制劑L-NMMA可促進IL-4 mRNA表達,從轉(zhuǎn)錄水平證實NO可影響T細胞因子之間的平衡。NO對細胞因子影響的

20、更深機制可能與NO直接影響核因子-KB等及各種細胞對NO調(diào)節(jié)作用的反應(yīng)性不同有關(guān)9,有必要進一步探討。參考文獻1Barnes PJ. Neural mechanisms in asthma. Br Med Bull, 1992, 48:149.2Waserman S, Xu LJ, Olivenstein R, et al. Association between late allergic bronchocostriction in the rat and allergen stimulated lymphocyte proliferation in vitro.Am J Respir Crit Care Med, 1995,151:470.3蔡文琴,王伯云. 實用免疫細胞化學.第1版.四川：科學技術(shù)出版社, 1988.116.4Barnes PJ, Belvisi MG. Nitric oxide and lung disease. Thorax, 1993,48:1034.5Schmidt HHW, Lohmann SM, Walter U, et al. The nitric oxide and cGMP signal tran