【摘要】目的探讨一氧化氮(NO)及其合酶(NOS)在哮喘发病机制中的作用。方法采用哮喘豚鼠模型，将豚鼠分为4组：(1)哮喘组(13只)，用10%卵白蛋白腹腔注射1 ml致敏，2周后用1%卵白蛋白超声雾化吸入致其哮喘发作。(2)肾上腺皮质激素预防组（激素组13只）；诱喘同哮喘组，在每次诱喘前腹腔滴注地塞米松0.5 mg/kg。(3)硝基精氨酸甲酯预防组(L-NNA组13只)：诱喘同哮喘组，每次诱喘前腹腔注射LNNA 0.4 mg/kg。(4)正常对照组(13只)：用生理盐水代替诱喘剂。每组分别测定其血浆、支气管肺泡灌洗液(BALF)和肺组织/水平，肺组织诱导型(iNOS)和原生型(cNOS)一氧化氮合酶活性水平，并用组织化学染色法观察了NOS在豚鼠哮喘模型肺组织中的分布。结果4组豚鼠血浆/水平差异无显著性(P均＞0.05)。哮喘组BALF及肺组织中N/N和肺组织iNOS活性水平均明显高于其它3组(哮喘组BALF中/，t分别=7.37、4.95、4.48，P均＜0.01；哮喘组肺组织中/，t分别=23.34、18.10、16.12，P均＜0.01；哮喘组肺组织iNOS，t分别=7.42、7.51、6.93，P均＜0.01)；但这3组之间差异无显著意义(P均＞0.05)。LNNA组肺组织cNOS活性水平明显低于对照组(t=2.15，P＜0.05)，但与另两组间比较差异无显著性(P均＞0.05)，其余3组比较差异亦无显著性(P均＞0.05)。而且哮喘组肺组织iNOS活性水平与BALF和肺组织/水平呈高度正相关(r分别=0.714，0.842，P分别＜0.05，0.01)。对照组肺组织还原性辅酶Ⅱ硫辛酰胺脱氢酶(NADPHd)，组织化学方法显示的阳性产物主要分布于各级支气管上皮细胞，哮喘组及激素组阳性产物变化不明显，但是LNNA组可见阳性产物明显减少。结论哮喘豚鼠肺组织cNOS活性水平变化不明显，而iNOS活性水平升高，导致BALF及肺组织中/水平随之升高，从而使气道粘膜水肿，加重气道阻塞，并损伤气道组织，加重炎症反应，引起气道高反应性而导致和(或)加重哮喘发病。

【关键词】哮喘一氧化氮一氧化氮合酶

The role of nitric oxide and nitric oxide synthase in the pathogenesis of asthma

Jiang Dongbo, Li Huaqiang, Shi Yuan, et al. Institute of Surgery. Daping Hospital, Third Military Medical College, Chongqing 400042

【Abstract】ObjectiveTo investigate the role of nitric oxide (NO) and nitric oxide synthase (NOS) in the pathogenesis of asthma.Method52 guinea pigs were randomly divided into four groups of 13 each: (1) asthmatic group (Group A): Dunkin-Hartley guinea-pigs were injected celiacly with 1 ml of 10% ovalbumin (OA). After 14 days, the animals were inhaled with an aerosol of 1% OA for 40～60 seconds for 10 days every other day; (2) Corticosteroid prevention group (Group CT): As above, just before the animals were inhaled with an aerosol, 0.5 mg/kg dexamethasone were injected celiacly ; (3) N-nitro-L arginine prevention group (Group L): As Group A, just before the animals were inhaled with an aerosol, 0.4 mg/kg LNNA were injected celiacly; (4) Controls (Group C): and nitrate (/) levels in plasma, nitrite bronchoalveolar lavage fluid (BALF) and lung tissues were examined . At the same time, inducible nitric oxide synthase (iNOS) and constitute nitric oxide synthase (cNOS) activity levels in the lung tissues were examined, and the changes of cNOS in the guinea pig asthma model lung tissues were observed using histochemical detection. ResultAll groups had no significant alteration of / in the plasma (P＞0.05). Group A had increased amounts of / in the BALF and in the lung tissues compared with the other groups (BALF: Group A 10.2±1.3, Group CT 7.2±1.1, Group L 7.3±1.3, Group C 6.2±0.8 μmol/L respectively, all of P＜0.01; the lung tissues: Group A 0.89±0.07, Group CT 0.16±0.09, Group L 0.24±0.09, Group C 0.18±0.05 nmol/mg respectively, all of P＜0.01). Group A also showed increased amounts of iNOS levels in the lung tissues than the other groups (Group A 59±18, Group CT 10±5, Group L 12±7, Group C 10±5 pmol/mg respectively, all of P＜0.01). Group L showed decreased amounts of cNOS levels in the lung tissues than the Group C (0.8±0.4, 1.2±0.4 fmol/mg, P＜0.05). While there were no significant alterations in the other groups (P＞0.05). Elevation of iNOS in the lung tissues was correlated with / in the BALF and in the lung tissues (r=0.714, 0.842, respectively, P＜0.05, 0.01 respectively). NADPHd was found to be a histochemical marker reflecting cNOS activity. It was found that there was no marked alteration of cNOS activity in the Group A, Group CT and Group C, but lower in the Group L. ConclusionThere is increased production of iNOS in asthmatic guinea pigs, the iNOS produced could cause increased production of NO, and probably cause cytotoxicity and mediate airway hyperresponsiveness. NO and NOS may play an important role in the pathogenesis of asthma.

【Key words】AsthmaNitric oxideNitric oxide synthase

哮喘是一种气道慢性炎症反应，其特征是气道高反应性(AHR)，炎细胞浸润及细胞因子的增加。近年研究发现内源性一氧化氮(NO)具有广泛的生物学作用，既是细胞信使分子，又是细胞毒性分子^［1］。NO作用的多样性决定了其在哮喘发病机理中的复杂性。因此，NO及其合酶对哮喘发病机制的影响越来越多地受到重视。在体内，一氧化氮合酶(NOS)是催化生成NO的关键酶，NOS分为两种，即诱导型(iNOS)和原生型(cNOS)^［1］。不同的酶产生不同剂量的NO，其作用亦不一致，因此，NO及其合酶在哮喘的发病中可能有重要作用。为此，我们检测了过敏性哮喘豚鼠模型血浆及肺组织NO水平，肺组织iNOS和cNOS活性水平，并用还原性辅酶Ⅱ硫辛酰胺脱氢酶（NADPHd）组织化学染色法观察了NOS在豚鼠哮喘模型肺组织分布情况，以探讨NO及其合酶在哮喘发病机制中的作用。

材料与方法

1.实验动物: 健康豚鼠52只，体重250±20 g。雌雄各半，随机分为4组，每组13只。

(1)哮喘组：动物模型的制作按郭鹞^［2］法并略加改良。用10%卵白蛋白腹腔注射1 ml将动物致敏，2周后用1%卵白蛋白超声雾化吸入致其哮喘发作，所有动物均出现喘息，呼吸困难和青紫等。隔日诱喘1次，共10次，末次后于15分钟内采心脏血标本并处死动物，摘取肺脏，称重肺组织。8只行支气管肺泡灌洗，收集支气管肺泡灌洗液 (BALF) 6～7 ml，置4℃离心后，取上清液待测。然后剪碎肺组织，置10 ml离心管内，按1∶5加入PBS，组织匀浆器匀浆，10 000 r/min离心15分钟，收集上清液待测。5只行NADPHd组织化学染色。(2)肾上腺皮质激素预防组(激素组)：诱喘及操作方法同哮喘组，在每次诱喘前腹腔注射地塞米松0.5 mg/kg，其哮喘发作稍轻于(1)组。(3)硝基精氨酸甲酯组(LNNA组)：诱喘及操作方法同哮喘组，在每次诱喘前腹腔注射LNNA 0.4 mg