氢对烧伤小鼠肺损伤炎症反应时自噬的影响

Effect of hydrogen on autophagy during inflammatory responses following lung injury in burned mice

目的评价氢对烧伤小鼠肺损伤炎症反应时自噬的影响。方法清洁级健康雄性ICR小鼠96只，6周龄，体重20～25g，采用随机数字表法分为4组（n=24）：假伤组（SH组）、氢气组（H2组）、烧伤组（B组）和烧伤＋氢气组（B＋H2组）。B组和B＋H，组小鼠背部按照40％全身体表面积Ⅲ度烫伤标准制备烧伤模型，SH组和H，组小鼠仅背部脱毛，用烫头以皮温接触。于烫伤后1和6h时，H2组和B＋H2组分别吸入2％氢气1h。于烫伤后24h时处死小鼠后取肺组织。称肺湿重，烘干后称重，计算肺湿干重比；采用Westernblot法检测自噬蛋白微管相关蛋白1轻链3（LC3）的表达．计算LC3-Ⅱ与LC3-I表达比值（LC3-II／LC3-I），采用髓过氧化物酶（MPO）试剂盒检测MPO活性，ELISA法检测IL-6和高迁移率族蛋白BI（HMGBl）含量；收集肺泡灌洗液，采用ELISA法检测IL-6和HMGBl浓度，计数中性粒细胞。结果与sH组比较，烫伤后24h时B组和B＋H，组肺湿干重比、肺组织LC3-Ⅱ／LC3-I、MPO、IL-6和HMGBl水平、肺泡灌洗液IL-6和HMGBl浓度、中性粒细胞计数均升高（P〈0．05）；与B组比较，烫伤后24h时B＋H，组肺湿干重比、MPO、IL-6和HMGBl水平、肺泡灌洗液IL-6和HMGBl浓度、中性粒细胞计数均降低，肺组织LC3-Ⅱ／LC3。I降低（P〈0．05）。结论氢可通过抑制炎症反应减轻烧伤小鼠肺损伤，其机制与增强自噬有关。

Objective To evaluate the effect of hydrogen on autophagy during inflammatory responses following lung injury in burned mice. Methods Ninety-six clean-grade healthy male ICR mice, aged 6 weeks, weighing 20-25 g, were divided into 4 groups （n=24 each） using a random number table： sham operation group （SH group） , H2 group （H2 group） , burn group （B group） and burn plus H2 group （B＋ H2 group）. Forty percent of the total body surface was shaved with 80 g/L sodium sulfide and then exposed to a 92℃scald device for 18 s in B and B＋H2 groups. Forty percent of the total body surface was shaved with 80 g/L sodium sulfide and then exposed to a scald device of skin temperature for 18 s in SH and H2 groups. Mice inhaled 2% H2 for 1 h starting from 1 and 6 h after burn in H2 and B＋H2 groups. The animals were sacrificed at 24 h after burn and lungs were removed for determination of wet/dry weight ratio （ W/D ra-tio）, expression of autophagy-related mi protein 1 light chain 3 （LC3） （ by Western blot）, activity of myeloperoxidase （MPO）, and contents of interleukin-6 （IL-6） and high mobility group box 1 （HMGB1） （by enzyme-linked immunosorbent assay）. The ratio of LC3-11 to LC3-I expression （LC3- H/LC3- I ） was calculated. The bronchoalveolar lavage fluid （BALF） was collected at 24 h after burn to detect the concentrations of IL-6 and HMGB1 and to count neutrophil. Results Compared with group SH, the W/D ratio, levels of LC3- II/LC3- I , MPO, IL-6 and HMGB1, concentrations of IL-6 and HMGB1 in BALF and neutrophil count were significantly increased at 24 h after scald in B and B＋H2 groups （P〈0.05）. Compared with group B, the W/D ratio, levels of LC3-11/LC3-I , MPO, IL-6 and HMGB1, concentrations of IL-6 and HMGB1 in BALF and neutrophil count were significantly decreased at 24 h after scald in group B＋H2 （P〈0. 05）. Conclusion Hydrogen can alleviate the lung injury in burned mice, and the mechanism is related to enhancing autophagy.