Rho／ROCK信号通路在氢气改善脓毒症小鼠急性肺损伤中的作用

Role of Rho/ROCK signaling pathway in the protective effects of hydrogen against acute lung injury in septic mice

目的探讨Ras同源基因（Rho）／Rho激酶（ROCK）信号通路在氢气（H2）改善脓毒症小鼠急性肺损伤（ALI）中的作用。方法按照随机数字表法将雄性C57BL／6小鼠分为假手术（Sham）组、H2对照组（sham＋H2吸入）、脓毒症模型组和H2治疗组（脓毒症＋H2吸入），每组20只。采用盲肠结扎穿孔术（CLP）制备脓毒症模型；Sham组不进行盲肠结扎和穿孔。H2吸入两组分别于术后1h、6h吸入2％H2 1h。术后24h每组取10只小鼠，静脉注射伊文思蓝（EB）评价内皮通透性；处死另外10只小鼠，取支气管肺泡灌洗液（BALF）测定蛋白、肿瘤坏死因子-α（TNF-α）和白细胞介素-1β（IL-1β）含量并计数多形核中性粒细胞（PMN）；取肺组织测定丙二醛（MDA）含量和超氧化物歧化酶（SOD）活性以及肺湿／干质量比值（W／D）；苏木素-伊红（HE）染色后光镜下观察肺组织病理学改变；蛋白质免疫印迹试验（Western Blot）检测Rho／ROCK通路活性和紧密连接蛋白ZO-1表达；免疫荧光法测定ZO-1的表达和分布。结果Sham组和H2对照组各指标比较差异均无统计学意义。与Sham组比较，模型组BALF中蛋白、TNF-α．和IL-1β含量及PMN计数明显增高，肺组织EB、MDA含量增高，SOD活性下降，肺W／D比值升高，肺组织Rho—GTP／总Rho比值、ROCKl和ROCK2表达水平、磷酸化肌球蛋白磷酸酯酶目标亚基1占肌球蛋白磷酸酯酶目标亚基1的比值（p-MYPT1／MYPT1）明显升高，ZO-1表达水平明显降低。与模型组比较，H2治疗组BALF中蛋白、TNF—α和IL-1β含量及PrvIN计数明显降低[蛋白（以）：3．12±0．33比6．37±0．56，TNF—α（ng／L）：128．45±17．33比563．83±61．72，IL-1β（ng／L）：75．76±14．35比245．52±30．56，PMN计数（×105／L）：7．46±1．34比18．55±5．73]；肺通透性明显降低[EB含量（μg／g）：73．33±6．98比144．83±12．38]，肺组织MDA含量降低（mmol／g：3．66±0．53比6．04±1．13），SOD活性增高（kU／g：18．58±1．68比13．31±2．20），肺W／D比值降低（5．02±0．34比7．26±0．56），肺组织Rho—GTP／总Rho比值、ROCKl和ROCK2表达水平及P—MYPT1／MYPT1比值明显降低[Rho—GTP／总Rho：（43．12±4．69）％比（68．82±5．44）％，ROCK1（灰度值）：2．42±0．42比6．03±0．64，ROCK2（灰度值）：2．56±0．52比4．85±0．53，P—MYPT1／MYPT1比值：（57．83±8．67）％比（112．50±13．43）％j，ZO-1表达水平上调（灰度值：0．61±0．07比0．32±0．06，荧光强度：0．77±0．06比0．54±0．05），各指标比较差异均有统计学意义（均P〈0．05）。HE染色显示，模型组出现明显的肺损伤，也治疗组肺损伤较模型组有所减轻。结论H2可能通过抑制Rho／ROCK通路活性，改善内皮通透性并降低肺组织炎症反应和氧化应激反应，从而减轻脓毒症导致的小鼠ALI。

Objective To investigate the role of Rho/ROCK signaling pathway in the protective effects of hydrogen gas （H2） on acute lung injury （ALI） in a mouse model of sepsis. Methods Eighty male C57BL/6 mice were randomly divided into four groups （n = 20 per group）： sham surgery group, n2 control group （sham ± H2 inhalation）, sepsis model group and H2 treatment group （sepsis ± H2 inhalation）. The mouse model of sepsis was created by cecal ligation puncture （CLP）, and the mice in sham surgery group didn＇t undergo cecal ligation and puncture. The mice in the H2 inhalation groups received inhalation of 2% H2 for 1 hour at 1 hour and 6 hours after CLP or sham surgery, respectively. Ten mice in each group were selected and subjected to Evans blue （EB） test to evaluate the pulmonary endothelial permeability at 24 hours after CLP operation. The rest of 10 mice in each group were sacrificed at 24 hours after CLP operation, the bronchoalveolar lavage fluid （BALF） was collected for the measurement of protein concentration, tumor necrosis factor- α （TNF- α ） and interleukin- 1β （IL- 1β ） content, and polymorphonuclear neutrophils （PMN） counts. The lung tissues were obtained to determine the content of malonaldehyde （MDA） and the activity of superoxide dismutase （SOD）, the wet/dry lung weight ratio （W/D） was calculated, the lung pathological changes in hematoxylin and eosin （HE） stained sections were evaluated under a light microscope, the activity of Rho/ROCK signaling pathway and expression of zonula occluden 1 （ZO-I） were detected by Western Blot, and the distribution and expression of ZO-1 were also examined by immunofluorescence staining. Results There was no statistical difference in the above indexes between the sham surgery group and the H2 control group. Compared with the sham surgery group, the sepsis group demonstrated significant increases in the concentrations of protein, TNF-α, IL-1β and PMN counts in BALF, the lung EB and MDA content, W/D ratio, the ratio of Rho-GTP/total Rho, the expressions of ROCK1 and ROCK2, the ratio of phosphorylated-myosine phosphatae targeting subunit 1 （p-MYPT1）/MYPT1, and significant decreases in the lung SOD activity and ZO-1 expression. Compared with the sepsis group, the H2 treatment group showed statistically significant decreases in the concentrations of protein, TNF-α, IL-1β, PMN counts in BALF [protein （g/L）： 3.12 ± 0.33 vs. 6.37 ± 0.56, TNF- α （ng/L）： 128.45 ± 17.33 vs. 563.83 ± 61.72, IL-1 β （ng/L）： 75.76 ± 14.35 vs. 245.52± 30.56, PMN counts （×10^8/L）： 7.46± 1.34 vs. 18.55 ±5.73], and permeability of lung [EB concentration （μg/g）： 73.33±6.98 vs. 144.83 ± 12.38], the lung MDA content （retool/g： 3.66±0.53 vs. 6.04±1.13）, the lung W/D ratio （5.02±0.34 vs. 7.26± 0.56）, the ratio of Rho-GTP/total Rho, the expressions of ROCK1 and ROCK2, the ratio of p-MYPT1/MYPT1 [Rho-GTP/total Rho： （43.12±4.69）% vs. （68.82± 5.44）%, ROCK1 （gray value）： 2.42 ±0.42 vs. 6.03 ±0.64, ROCK2 （gray value）： 2.56 ± 0.52 vs. 4.85 ± 0.53, p-MYPT1/MYPT1： （57.83 ± 8.67）% vs. （112.50 ± 13.43）%], and statistically significant increases in the lung SOD activity （kU/g： 18.58± 1.68 vs. 13.31 ±2.20） as well as the expression of ZO-1 （gray value： 0.61 ±0.07 vs. 0.32±0.06, fluorescence intensity： 0.77 ±0.06 vs. 0.54±0.05; all P 〈 0.05）. Moreover, lung HE staining showed that there were obvious lung injuries in the sepsis group which were alleviated in the H2 treatment group. Conclusion H2 could improve endothelial permeability and suppress inflammation and oxidative stress to alleviate ALI in septic mice through inhibition of Rho/ROCK signaling pathway.