The Effects And Mechanisms Of CSE/H₂S On Methionine Restriction Attenuates Lipopolysaccharide-induced Acute Lung Injury In Mice

Background and purposeAcute lung injury（ALI）/acute respiratory distress syndrome（ARDS）is a severe disorder in clinical featured with pulmonary epithelial and capillary endothelium damage,inflammatory response,vascular permeability increasing,pulmonary edema and refractory hypoxia.Sepsis/gram-negative associated pathogenesis is one of the most common cause of ALI/ARDS.Clinical treatment of this syndrome focuses on respiratory support,anti-infection,fluid management and general supportive measures.The absence of specific therapies often leads to a high mortality which is up to 40%.Methionine restriction（MR）is one of nutritional interventions which has been proved as a reliable method for offering various benefits in a set of organisms,which from yeast to mammals.In yeast,flies,worms and fish,the primary benefits of MR are extending the lifespan.In mammals,it enhances stress resistance,improves metabolic fitness,delays loss of function,reduces the incidence of some major diseases like diabetes,cardiovascular disease,cancer,or improves them.One of the underlying mechanisms is mediated through reduction in oxidative stress via multiple ways,such as induction of autophagy,decreases reactive oxygen species（ROS）production in mitochondria and increases the production of hydrogen sulfide（H₂S）which acts as gasotransmitter and plays an important protective role in various physiological and pathological processes including inflammatory,apoptotic and regulation of blood vessels.Previous researches of MR primarily focus on chronic or age-related diseases,however,recent studies showed that MR could also improve acute diseases such as ischemia/reperfusion-induced injury,kidney injury and colitis.To the best of our knowledge,there are no studies about the effects of MR on pulmonary diseases so far.Therefore,in the present study we investigated the effects of MR on lipopolysaccharide（LPS）-induced ALI in mice,and explored the relative mechanisms.Materials and methods1.Establishment of ALI model in miceThe methylene blue solution was given by inhalable intratracheal instillation or non-exposed light-mediated intratracheal instillation,and the lung tissue was removed after10 minutes to observe the methylene blue staining.The ideal administration method was selected according to the range and uniformity of staining,and LPS was given in this way to establish the ALI model of mice.The lungs were harvested at days 1,3,and 7 after LPS administration for HE staining to determine the time point with the most severe injury in this model.2.The effects of MR on LPS-induced ALI in miceThe mice were fed with MR diet for 7 days before LPS administration until the end of the research,and their daily body weights were recorded.HE staining was used to observe the changes of lung injury in different diet groups.Immunohistochemical staining was used to observe the damage of alveolar epithelial cells,inflammatory reaction and activation of TLR4/NF-κB/NLRP3 pathway.IL-1,IL-6 and TNF-αin alveolar lavage fluid were detected by enzyme-linked immunosorbent assay（ELISA）.The survival rates of mice after lethal dose of LPS administration were recorded.3.The effect of MR on H₂S levels and related enzymes that produce H₂SThe levels of H₂S in lung and circulation of mice were detected by colorimetry.Immunohistochemical staining was used to detect the expression of the enzymes that produce H₂S in the lungs,and q RT-PCR was used to detect the related gene expression.4.The role of CSE/H₂S in the protective effects of MR on ALISpecific inhibitor of CSE（PAG）and CSE gene knockout were used to observe the changes of the protective effects of MR on ALI mice.The changes of H₂S levels in lung and circulation of mice after CSE inhibition were determined by colorimetry.5.The effects of exogenous H₂S on ALI in CSE gene knockout miceHE staining was used to observe the changes of lung injury in different groups.The damage of alveolar epithelial cells,the infiltration of inflammatory cells and the activation of TLR4/NF-κB/NLRP3 pathway were observed by immunohistochemical staining.Results:1.After methylene blue administration by inhalable intratracheal instillation,the staining range of lung tissues was wider and more uniform.In the ALI model of mice established by this method,edema,hemorrhage,atelectasis and inflammatory cell infiltration in lung peaked at day 3 after 15mg/kg LPS administration.2.MR could reduce the edema,hemorrhage,atelectasis and inflammatory cell infiltration in the lung tissues of mice at day 3 after LPS administration,while the body weight of the mice did not decrease compared with the normal diet group.3.MR reduced the mortality of mice within 7 days after LPS administration.4.MR reduced LPS-induced alveolar epithelial cells injury,macrophages and neutrophils infiltration,inhibited the activation of TLR4/NF-κB/NLRP3 pathway,and decreased the levels of inflammatory cytokines including IL-1,IL-6 and TNF-αin alveolar lavage fluid.5.MR significantly increased the protein and gene expression of CSE in the lungs,and increased the H₂S levels in the lungs and circulation of mice,while there were no significant differences as to the cystathionineβ-synthase（CBS）and 3-mercaptopyruvate sulfurtransferase（MST）.6.The protective effects of MR on LPS-induced ALI were eliminated by the administration of the specific inhibitor of CSE（PAG）and CSE gene knockout,and the increase of H₂S levels in the lungs and circulation induced by MR was also reversed.7.Exogenous H₂S restored the protective effects of MR on CSE knockout mice,including reducing lung edema,hemorrhage,atelectasis,inflammatory cell infiltration and alveolar epithelial cell damage,and inhibiting activation of TLR4/NF-κB/NLRP3 pathway.Conclusions:1.Compared with non-exposed light-mediated intratracheal instillation,inhalable intratracheal instillation is a more ideal method because of its simpler operation,higher success rate,and more stable effects in ALI model of mice.The injury of the lungs peaked at day 3 after 15mg/kg LPS administration in this model.2.MR improves LPS-induced pulmonary edema,hemorrhage and atelectasis,reduces the levels of inflammatory factors,macrophages and neutrophil infiltration,attenuates alveolar epithelial cell injury,and decreases the mortality of mice without malnutrition.3.MR alleviates ALI by upregulation of the CSE/H₂S pathway,which is associated with inhibition of the TLR4/NF-κB/NLRP3 pathway.