普通肝素雾化吸入对内毒素性肺损伤大鼠肺泡局部凝血及炎症反应的影响

Effect of inhalation of aerosolized unfractioned heparin on peri-alveolar coagulability and inflammatory response in endotoxin-induced acute lung injury rat model

目的 观察内毒素诱导急性肺损伤（ALI）大鼠雾化吸入普通肝素（UFH）后的肺部局部效应,探讨其对肺泡内凝血、纤溶和炎症反应的作用.方法 87只雄性Wistar大鼠按随机数字表法分为假损伤组、模型组、肝素治疗组（HT组）和肝素预防组（HP组）.静脉注射（静注）内毒素脂多糖（LPS）制备ALI模型.HP组和HT组分别于注射LPS前后给予UFH雾化吸入,模型组和假损伤组则雾化吸入生理盐水.各组分别于静注LPS后6、12和24 h处死大鼠进行肺泡灌洗,采用酶联免疫吸附法测定支气管肺泡灌洗液（BALF）中凝血酶-抗凝血酶复合物（TAT）、组织型纤溶酶原激活物（t-PA）、尿激酶型纤溶酶原激活物（u-PA）、纤溶酶原激活物抑制剂-1（PAI-1）、肿瘤坏死因子-α（TNF-α）、白细胞介素-6（IL-6）水平.结果 损伤后6 h,模型组BALF中TAT浓度（μg/L：3.346±0.585）最高,其次是HT组（2.764±0.100）,HP组（2.564±0.216）最低（均P＜0.05）;HP组t-PA（μg/L：3.037±0.524）最高,HT组（2.494±0.191）其次,模型组（1.716±0.125）最低（均P＜0.05）;HP组u-PA（μg/L）高于模型组（0.411±0.118比0.303±0.049,P＜0.05）;HP组PAI-1（μg/L）明显低于HT组和模型组（2.296±0.246比2.597±0.425、2.834±0.198,均P＜0.05）,直至12 h时HP组仍低于HT组（1.273±0.441比1.817±0.252,P＜0.05）;HT组和HP组TNF-α（ng/L）显著低于模型组（68.154±3.915、36.990±6.539比77.001±4.485）,且HP组低于HT组（均P＜0.05）,至12 h HP组（15.287±4.754）仍最低,与HT组和模型组（26.756±5.336、23.674±4.398）比较差异有统计学意义（均P＜0.05）.模型组、HT组和HP组各时间点IL-6水平均无明显差异.结论 内毒素性ALI大鼠雾化吸入UFH后起到了抑制局部凝血、减弱纤溶抑制、促进纤溶、降低炎症反应的作用,预防性吸人UFH比治疗性吸入效果更显著,最佳效应时间在注射后6 h.

Objective To observe the local changes in alveoli in intravenous endotoxin-induced acute lung injury （ALI） rat model after inhalation of aerosolized unfractioned heparin（UFH）, and to observe its effects on coagulability, fibrinolysis and inflammatory response. Methods Eighty-seven male Wistar rats were divided into groups according to table of random number： sham, model, heparin therapy （HT） and heparin prophylaxis（HP）. Endotoxin-induced ALI model was reproduced by intravenous administration of lipopolysaccharide （LPS）. Rats in Hp and HT groups received aerosolized UFH before and after injection with LPS respectively, while rats in both sham and model groups inhaled aerosolized normal saline. Rats in each group were respectively sacrificed at 6, 12 and 24 hours after intravenous administration of LPS, and bronchoalveolar lavage fluid （BALF） was collected. Enzyme-linked immunosorbent assay was used to measure the level of thrombin-antithrombin （TAT）, tissue-type plasminogen activator （t-PA）,urokinase-type plasminogen activator （u-PA）, plasminogen activator inhibitor-1 （PAI-1）, tumor necrosis factor- （TNF-α）, interleukin-6 （IL-6） in BALF. Results At.6 hours after injury, the level of TAT （μg/L） in model group （3.346±0. 585） was highest, that in HT group （22. 764±0. 100） was higher, and that in HP group （2. 564±0. 2216） was lowest in BALF （all P〈0. 05）. The t-PA（μg/L） concentration in HP group （3.037±0. 5224） was highest, that in HT group （22. 494±0. 191） was higher, and that in model group （1. 716±0. 1225） was lowest （all P〈0. 05）. Compared with model group, u-PA （μg/L） level in HP group dramatically enhanced （0. 411±0. 118 vs. 0. 303±0. 049, P〈0. 05）. The concentration of PAI-1 （μg/L） in HP group was significantly lower than that of HT and model groups （22. 2296 ± 0. 2246 vs. 22.597±0. 4225,2.834±0.198, both P〈0. 05）. In HP group, it was still lower than that in HT group at 12 hours （1.2273±0. 441 vs. 1. 817±0. 252, P〈0. 05）. TNF-α（ng/L） levels in HT and HP groups were markedly lower compared with model group （68. 154±3. 915, 36. 990±6. 539 vs. 77. 001±4. 485） at 6 hours, and the level in HP group was lower than that in HT group （all P〈0. 05）. TNF-α concentration in HP group was still the lowest at 122 hours （15.2287±4. 754）, and there was significant difference compared with HT and model groups （26. 756±5. 336, 23. 674±4. 398, both P〈0. 05）. The levels of IL-6 were not distinctively different among model, HT and HP groups at various time-points. Conclusion It was proved that inhalation of aerosolized UFH resulted in lowering local coagulability, alleviating fibrinolytic depression, improving fibrinolysis, and attenuating inflammation in endotoxin-induced ALI rat model. More prominent results will be obtained when it was use as a prophylactic measure. The optimal time of usage is 6 hours after endotoxin injection.