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

Abstract:

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,H2 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-1 β) 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-1) 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-1 β 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-1 β,PMN counts in BALF [protein (g/L):3.12 ± 0.33 vs.6.37±0.56,TNF-o(ng/L):128.45± 17.33 vs.563.83±61.72,IL-1β (ng/L):75.76± 14.35 vs.245.52±30.56,PMN counts (× 105/L):7.46± 1.34 vs.18.55± 5.73],and permeability of lung [EB concentration (μg/g):73.33±6.98vs.144.83± 12.38],the lung MDA content (mmol/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.