氯胺酮对致敏原诱导哮喘模型大鼠的肺保护

目的观察氯胺酮对哮喘模型大鼠气道高反应性及炎症的影响。方法56只Brown Norway大鼠随机分成阴性对照组（A组）、哮喘模型组（B组）和不同浓度氯胺酮雾化吸入预处理组（C组、D组和E组）及不同剂量氯胺酮腹腔用药预处理组（F和G组）。采用卵蛋白（OVA）辅以百日咳杆菌菌苗和氢氧化铝为佐剂注射致敏大鼠。雾化吸入10mg／ml OVA激发。氯胺酮预处理组大鼠在激发前分别给予12．5mg／ml、25mg／ml或50mg／ml浓度的氯胺酮雾化吸入或50μg/kg,100μg／kg剂量的氯胺酮腹腔注射。A组采用磷酸盐缓冲液替代OVA进行雾化吸入。最后1次激发后24h,测定气道反应性。并取肺组织作诱导性一氧化氮合酶（iNOS）的基因和蛋白表达,一氧化氮（NO）生成量及病理组织学检测。结果（1）B组的呼气阻力（Re）增长百分率的剂量反应曲线向左上移位,而且PC100（Re增长达100％幅度时所需乙酰胆碱的激发剂量）,PC200及PC400值显著低于A组（分别为14．65±1．19vs32．28±1．43,15．17±1．19vs38．91±1．39及16．28±1．18vs56．53±1．38）差异有统计学意义（P〈0．01）。氯胺酮预处理组的Re剂量反应曲线向右下移位,而PC100,PC200及PC400值均明显高于B组（P〈0．05）。（2）B组可见明显的气道炎症性病理改变。氯胺酮治疗组炎症细胞浸润及上皮细胞损伤程度明显减轻,肺间质水肿改善。（3）B组iNOS基因表达与A组相比明显增强（1．00±0．07vs0．48±0．07,P〈0．01）。与B组相比,iNOS基因的表达在C组（0．65±0．07）,D组（0．58±0．09）,E组（0．56±1．00）及F组（0．66±0．06）均减低,差异有统计学意义（P〈0．05）。（4）与A组相比,B组iNOS蛋白表达（0．54±0．08）明显增强（P〈0．05）,而与B组相比,iNOS蛋白表达量在C组（0．20±0．03）,D组（0．18±0．03）及F组（0．21±0．04）均减低,差异有统计学意义（P〈0．05）。（5）B组肺组织NO产量[（0．39±0．04）μmol／g蛋白]显著高于A组（P〈0．01）。肺组织NO产量在C组[（0．19±0．03）μmol／g蛋白],D组[（0．17±0．03）μmol／g蛋白]及F组[0．16±0．04）μmol／g蛋白]明显低于B组,差异有统计学意义（P〈0．05）。结论氯胺酮明显抑制致敏原诱发的肺iNOS的高表达,降低NO的过度产生,从而减轻炎性损伤,抑制气道高反应性,对哮喘模型大鼠具有肺保护作用。

Protective effects of ketamine on allergen-induced airway inflammatory injure and high airway reactivity in asthma: experiment with rats

OBJECTIVE: To observe the effects of ketamine on bronchial hyperresponsiveness and airway inflammation in equal asthma. METHODS: 56 Brown-Norway rats were randomly assigned to seven groups: negative control group (Group A), asthma model group (Group B) and inhalation groups with nebulized ketamine at different concentrations (Group C, D, E) and intraperitoneal injection groups with ketamine at different doses (Group F, G). The rats were sensitized by injection of ovalbumin (OVA) together with aluminum hydroxide and Bordetella pertussis as adjuvants, then challenged by repeated intermittent (thrice weekly) exposure to aerosolized OVA for two weeks. Before challenge, the sensitized rats were exposed to an aerosol of phosphate buffered saline (PBS) or ketamine at the concentrations of 12.5 mg/ml, 25 mg/ml and 50 mg/ml respectively in Groups B, C, D and E. The sensitized rats were intraperitoneally injected with ketamine at the doses of 50 microg/kg or 100 microg/kg respectively in Group F and G. The sensitized rats in Group A received phosphate buffered solution (PBS) by inhalation. The airway reactivity to acetylcholine (ACH) was assessed in vivo 24 hr after the last OVA challenge, then the lungs were removed for measurement of the mRNA and protein expression of iNOS and production of NO and lung sections for histopathologic examination. RESULTS: (1) In the OVA-sensitized and challenged rats, the dose-response curve of the expiratory resistance (Re) shifted to the upper-left +/- ward compared with that of PBS control rats. In addition, the provocation doses required to increase the Re by 100%, 200% and 400% for OVA-sensitized and challenged rats in Group B were significantly lower than those of the PBS control rats (14.65 +/- 1.19 vs 32.28 +/- 1.43, 15.17 +/- 1.19 vs 38.91 +/- 1.39, and 16.28 +/- 1.18 vs 56.53 +/- 1.38, all P < 0.01). The OVA-sensitized rats treated with ketamine before OVA challenge demonstrated a significant decrease in AHR by a rightward shift of the dose-response curves to ACH and significant higher provocation doses compared with that of the OVA control rats (P < 0.05). (2) Marked inflammatory changes in the airways of Group B were present, while obviously lessen inflammatory cell infiltration in peribronchial and perialveolar tissues and improved lung edema were observed in the groups treated with ketamine. (3) Quantitation by densitometry showed that the relative density of iNOS mRNA bands normalized to beta-actin was significantly higher in the OVA control than the PBS control (1.0 +/- 0.07 vs 0.48 +/- 0.07, P < 0.01). Treatment with ketamine significantly decreased the expression of iNOS mRNA in Group C (0.65 +/- 0.07), Group D (0.58 +/- 0.09), Group E (0.56 +/- 1.00), and Group F (0.66 +/- 0.06) when compared with Group B (all P < 0.05). (4) The relative iNOS protein levels (ratios of iNOS/beta-actin) determined by densitometry analysis showed a 4-fold increase in Group A compared with those in the negative group (0.54 +/- 0.08 vs 0.13 +/- 0.08, P < 0.05). When compared with those of the OVA control, the levels of relative iNOS protein expression showed a significant decrease in the lungs from the rats treated with ketamine inhalation at the doses of 12.5 mg/ml (0.20 +/- 0.03) and 25 mg/ml (0.18 +/- 0.03) and with ketamine and intraperitoneally the at dose of 50 microg/kg (0.21 +/- 0.04) (P < 0.05). (5) NO production in pulmonary tissues was significantly higher in the OVA-treated rats compared to the PBS controls (0.39 +/- 0.04 micromol/g protein vs 0.13 +/- 0.01 micromol/g protein, P < 0.01), but this OVA-triggered NO production was significantly decreased by treatment with 12.5 and 25 mg/ml inhaled ketamine (0.19 +/- 0.03 micromol/g and 0.17 +/- 0.03 micromol/g, both P < 0.05) and 50 microg/kg i.p.-injected ketamine (0.16 +/- 0.04 micromol/g, P < 0.05) when compared with the OVA-treated rats. CONCLUSION: Both inhalation and systemic administration of ketamine attenuate inflammatory the lung injury and airway hyperreactivity of the OVA-induced asthma model. The protective effects of ketamine is achieved by inhibiting OVA-provoked over-expression of mRNA and protein of iNOS and reducing the production of NO in pulmonary tissues.