Hydrogen Gas Alleviates The Lung Injury Caused By Severe Sepsis In Mice By Increasing The Expression Of Heme Oxygenase-1

Objective:Sepsis is the result of a systemic inflammatory response syndrome(SIRS) caused by infection. It is a common critical illness. Acute lung injury(ALI), which has a high mortality, is one of the most serious complications of sepsis. Hydrogen(H2) is the smallest molecule in nature. It is a kind of diatomic gas which is colorless, odorless and tasteless. In recent years, many researches have found that H2 has many effects such as: anti-oxidation, anti-inflammation, anti-apoptosis and the therapeutic function on many illnesses. Heme oxygenase-1(HO-1), which is also called heat shock protein 32(hsp32), is an antioxidant enzyme that distributes widely in body tissues. Our previous research has certified that HO-1 mediates the anti-inflammatory effect of H2 in RAW264.7 macrophages stimulated by lipopolysaccharide(LPS). However, the mechanism of the anti-inflammatory effect of H2 in vivo remains unknown. In this study, we aim to demonstrate the protective effect of a specific HO-1 induction with H2 on sepsis-induced lung injury. Methods:Adult male ICR mice weighing 20 to 25 g were randomly divided into six groups: sham, sham+H2, severe sepsis, severe sepsis+H2, severe sepsis+ZnPPIX and severe sepsis+H2+ZnPPIX groups. Cecal ligation and puncture(CLP) model was used to induce severe sepsis. The animals in the sham+H2, severe sepsis+H2 and severe sepsis+H2+ZnPPIX groups maintained 2% H2 inhalation for 1 h at 1 h and 6 h after CLP or sham operation, respectively. As a control, the mice in the sham, severe sepsis and severe sepsis+ZnPPIX groups received room air at similar time points. The severe sepsis+ZnPPIX and severe sepsis+H2+ZnPPIX groups received an intraperitoneal injection of ZnPPIX(40 mg/kg) 1 h before CLP.Part 1: A total of 120 mice were randomly divided into six groups(n = 20 per group) as described above. The survival rate was observed on days 1, 2, 3, 5, and 7 after CLP or sham operation. An additional 108 mice were randomly assigned to six groups(n =18 per group) as described above. The inflammatory cytokines tumor necrosis factor-α(TNF-α), high mobility group box1(HMGB1) and interleukin-10(IL-10) in the serum and lungs were detected at 6 h, 12 h and 24 h using ELISA. Lung histopathological scores, wet/dry(W/D) weight ratio and oxygen pressure/fraction of inspiration oxygen(PaO2/FIO2) ratio were observed at 24 h after CLP or sham operation.Part 2: An additional 108 mice were randomly assigned to six groups(n = 18 per group) as described above. The late inflammatory cytokine HMGB1 in the lungs, as well as antioxidant molecules, nuclear factor-erythroid 2-related factor 2(Nrf2) and HO-1 in the lungs, were detected at 6 h, 12 h and 24 h using western blot and quantitative real-time PCR(qPCR). The HO-1 activity was measured by bilirubin generation. Simultaneously, the HMGB1 changes in different groups were also observed using immunofluorescence staining at 24 h after CLP or sham operation. Results:Part 1: There was no death of mice in the sham or sham+H2 groups. While in the severe sepsis group, the survival rate of mice was 85% at 24 h and dropped to 40% at 48 h; Therapy with 2% H2 increased the 7-day survival rate(P<0.05); In addition, ZnPPIX decreased the survival rate of mice in the severe sepsis+H2 group(P<0.05). Compared with the sham group, septic mice showed an increase in the histopathological scores and W/D weight ratio of lung and a decrease in the PaO2/FIO2 ratio(P<0.05). These abnormal changes were significantly attenuated by H2 treatment(P<0.05). However, compared with the severe sepsis+H2 group, in the severe sepsis+H2+ZnPPIX group, the histopathological scores and W/D weight ratio increased and the PaO2/FIO2 ratio decreased(P<0.05). Moreover, after the H2 treatment, the expression of TNF-α and HMGB1 in the serum and lungs of the severe sepsis group decreased(P<0.05), the expression of IL-10 increased(P<0.05). However, this effect was removed by ZnPPIX(P<0.05).Part 2: Compared with the sham group, the protein and mRNA expression of HO-1 and Nrf2, and HO-1 activity increased in the severe sepsis group(P<0.05). H2 further increased the expression of HO-1, Nrf2 and HO-1 activity(P<0.05). The levels of HMGB1 protein and mRNA in the severe sepsis group increased compared with the sham group at 12 h and 24 h(P<0.05). After using H2, the elevation of HMGB1 was inhibited(P<0.05). Whereas Zn PPIX inhibited the HO-1 activity, increased the expression of HMGB1(P<0.05) and reversed the therapeutic effect of H2. Conclusion:H2 plays a significant role in regulating the release of the inflammatory cytokines in septic mice, which is partially mediated through the activation of HO-1 as a downstream molecule of Nrf2.