依达拉奉在脂多糖所致小鼠急性呼吸窘迫综合征肺纤维化中的保护作用

Protective effect of edaravone on lung fibrosis in mice with lipopolysaccharides-induced acute respiratory distress syndrome

目的 探讨依达拉奉（edaravone,EDA）对脂多糖（lipopolysaccharides,LPS）所致小鼠急性呼吸窘迫综合征（acute respiratory distress syndrome,ARDS）肺纤维化的保护作用及其机制. 方法 72只雄性ICR小鼠按照完全随机分组法分为假致伤组（Sham组）、LPS组和EDA组,各组再分为1、3、7d和14d组,每组6只.LPS组和EDA组由气管内滴入LPS（5 mg/kg）;Sham组气管内滴入等量生理盐水;EDA组气管内滴入LPS前1h和后12h,分别予以腹腔注射EDA（5 mg/kg）.于各观察点（1、3、7d和14d）处死小鼠.苏木素-伊红（hematoxylin and eosin,HE）染色观察肺组织病理学改变,检测肺组织湿/干重比（wet/dry,W/D）、肿瘤坏死因子-α（tumor necrosis factor-α,TNF-α）、白介素（interleukin,IL）-1β、丙二醛（malondialdehyde,MDA）、髓过氧化酶（myeloperoxidase,MPO）、Ⅰ型胶原（collagen Ⅰ,Col Ⅰ）和羟脯氨酸（hydroxyproline,HYP）水平,免疫组化法检测肺组织内高迁移率族蛋白B1 （high mobility group box 1,HMGB1）,Western Blot检测HMGB1和α-平滑肌收缩蛋白（α-smooth muscle contractile protein,α-SMA）的表达. 结果 LPS组与同时间点Sham组比较,肺组织肺泡间隔明显增宽,可见大量炎性细胞浸润,部分肺泡腔萎陷不张;EDA组较LPS组小鼠肺组织炎性细胞浸润、肺间质肿胀程度明显减轻,正常肺组织结构破坏少.LPS组及EDA组肺组织W/D比值、TNF-α、IL-1β、MDA含量和MPO活性在1d时明显升高,随后逐渐降低;EDA组TNF-α和IL-1β含量明显少于同时间点LPS组（P＜0.05）;EDA-3、7、14 d组MDA含量分别为（6.94± 1.07）、（5.31±0.95） nmol/mg prot 和（3.16±0.37） nmol/mg prot,明显低于同时间点LPS组[（9.15±0.30）、（7.05±0.71） nmol/mg prot和（4.77±0.64） nmol/mg prot]（P＜0.05）;EDA-1、3、7d组MPO活性分别为（0.051 ±0.007）、（0.040±0.008） U/g和（0.032±0.005） U/g,显著低于同时间点LPS组[（0.066±0.011）、（0.050±0.014） U/g和（0.043±0.008） U/g] （P＜0.05）.LPS诱导后,HMGB1的表达增多,主要定位在胞质;EDA组HMGB1表达明显低于与同时间点LPS组（P＜0.05）.与同时间点Sham组比较,LPS组α-SMA和Col Ⅰ 、HYP显著增加（P＜0.05）;EDA-1、3、7d组α-SMA显著低于同时间点LPS组（P＜0.05）;EDA组Col Ⅰ、HYP显著低于同时间点LPS组（P＜0.05）. 结论 EDA可能通过抑制氧化应激反应,减少体内炎症因子的产生和释放,减缓或阻断炎症反应和氧化应激损伤的恶性循环,减轻肺泡上皮的损伤和异常修复,阻止肺纤维化的进展,进而对LP所致ARDS肺纤维化起到防治作用.

Objective The aim of this study is to explore the protective effect of edaravone （EDA） on lipopolysaccharides （LPS）-induced acute respiratory distress syndrome （ARDS） with pulmonary fibrosis in mice and their potential mechanism.Methods Seventy-two male ICR mice were divided into Sham group,LPS group and EDA group by completely randomized method.Each group was further divided into 1,3,7 d and 14 d group （n=6）.LPS（5 mg/kg） and equivalent saline was intratracheal instilled in LPS group and Sham group respectively.Intraperitoneal edaravone （5 mg/kg） was injected 1 h before and 12 h after intratracheal instillation of LPS in EDA group.At 1,3,7 d and 14 d,lungs were seized after animals were killed.Hematoxylin and eosin（HE） staining was used to observe of lung histopathology.Detection of lung wet/dry （W/D）,tumor necrosis factor-α （TNF-α）,interleukin （IL）-1 β,malondialdehyde （MDA）,myeloperoxidase （MPO）,collagen Ⅰ （Col Ⅰ） and hydroxyproline （HYP） levels was performed.Immunohistochemistry was used to detect high mobility group box 1 （HMGB1） in the lung tissue.The expression of HMGB1 and α-smooth muscle contractile protein （α-SMA） was detected by Western Blot.Results Compared with the same time point in Sham group,the lung tissue of LPS group showed significantly widened alveolar septa,a large number of inflammatory cell infiltration and partial alveolar collapse.Compared with the same time point in LPS group,the EDA group showed less inflammatory cell infiltration,interstitial lung exudates with significantly reduced damage of normal alveolar structure.Lung tissue W/D ratio,TNF-α,IL-1β,MDA content and MPO activity in LPS and EDA group was significantly elevated in 1 d,and then gradually decreased.EDA group TNF-α and IL-1β was significantly less than that in LPS group at the same time （P〈0.05）.TNF-α and IL-1β levels in mice lung tissue of EDA group were significantly less than that in LPS group at the same time （P〈0.05）.EDA-3,7,14 d group MDA levels of mice lung tissue was （6.94±1.07）,（5.31±0.95） nmol/mg prot and （3.16±0.37） nmol/mg prot,significantly lower than that in the LPS group [（9.15±0.30）,（7.05±0.71） nmol/mg prot and （4.77±0.64） nmol/mg prot] at the same time points （P〈0.05）.EDA-1,3,7 d group MPO lung tissue in mice were （0.051±0.007）,（0.040±0.008） U/g and （0.032±0.005） U/g,significantly lower than that in the LPS group [（0.066±0.011）,（0.050±0.014） U/g and （0.043±0.008） U/g] at the same time point （P〈0.05）.The expression of HMGB1 increased in LPS-induced mice,mainly located in the cytoplasm.HMGB1 expression was significantly lower in the the EDA group than that in LPS group at the same time point （P〈0.05）.Col Ⅰ,HYP levels of LPS group increased significantly in EDA group（P〈0.05）.α-SMA expression in EDA-1,3,7 d group was significantly lower than that in LPS group at the same time（P〈0.05）.Col Ⅰ,HYP content in mice lung tissue of EDA group was significantly lower （P〈0.05）.Conclusions EDA plays the vital role of prevention of LPS-induced ARDS with pulmonary fibrosis by reducing the abnormal alveolar epithelial injury and repair to prevent progression of lung fibrosis due to inhibition of oxidative stress,abatement of inflammatory cytokines and slowing down or breaking the vicious cycle of inflammatory response.