| Sepsis is the leading cause of death in intensive care unit(ICU),but the effective treatment is very limited[1].sepsis is an immune response of the host caused by infectious factors(including fungi,bacteria,viruses,etc.),and it is a syndrome mainly caused by systemic inflammation.Severe sepsis is closely related to the increase of short-term mortality.Current studies[2]have confirmed that severe sepsis can rapidly deteriorate the general condition of patients and make them into immunosuppressive state,resulting in various immune-related complications.In recent years,the incidence of severe sepsis has increased.Although advances in intensive care technology have reduced short-term mortality,they also increase the risk of adverse prognosis.The mechanism of systemic inflammatory response in sepsis is complex.Inflammation is beneficial to the body in a certain range and intensity,but it may cause serious damage to the body beyond a certain limit.More than 100 randomized clinical trials have shown that the survival rate of patients can be improved by regulating the immune response to sepsis,but these schemes have not become available choice of clinical treatment[3].At present,sepsis research is facing a dilemma,On the one hand,the update of sepsis concept is lagging behind,On the other hand,some ineffective research models are still adhered to.With regard to the pathogenesis of sepsis,studies have shown that oxidative stress,inflammation and mitochondrial dysfunction play an important role in the pathogenesis of sepsis[4-7].In 2007,Japanese scholar Oshawa[8]confirmed that low concentration of hydrogen can treat focal cerebral ischemia-reperfusion injury through selective antioxidant mechanism.In recent years,more and more research has been done on the application of hydrogen in medical field,and hydrogen has been widely recognized as a new type of medical gas molecule[1-3].At present,a large number of studies have confirmed that hydrogen inhalation or saturated hydrogen-rich water can improve the mortality of sepsis animal models,and significantly reduce the damage of important organs,and improve organ function.In recent years,with the application of new high-concentration hydrogen devices,more and more studies have been carried out on the treatment of various diseases with high-concentration hydrogen(67%),and good clinical results have been achieved[12,13].Whether the effect of high-concentration hydrogen in the treatment of sepsis is better than that of low-concentration hydrogen,and the specific mechanism of the therapeutic effect of high-concentration hydrogen need to be further confirmed.In this study,different concentrations of hydrogen(67%,2%)were used to intervene sepsis animal models.The protective effects of inhalation of high and low concentrations of hydrogen on sepsis mice and the therapeutic effects on sepsis lung injury in mice were observed,and the protective mechanism of high concentrations of hydrogen on sepsis lung injury was discussed.PART 1:Protective effect of high concentration hydrogen inhalation on lung injury in sepsis miceObjecitve:To compare the therapeutic effects of inhalation of high concentration hydrogen(67%)and low concentration hydrogen(2%)on sepsis mice and the protective effects on lung injury in sepsis mice.Methods:Moderate sepsis and severe sepsis mice models were prepared by cecum ligation puncture method.Imediately after the CLP models were successfully made 67%and 2%hydrogen were inhaled at time-point 1 h and 6 h for 1h.The arterial blood gas of mice during hydrogen inhalation was monitored in real time.The survival of moderate and severe sepsis mice models was recorded 7 days after different concentrations of hydrogen inhalation treatment.The pathological changes of the viscera(lung,liver,kidney)and the ratio of dry to wet weight of the lungs;the concentration changes of oxidized products,antioxidant enzymes and pro-inflammatory cytokines in the lungs and blood at different time points;the specific changes of Nrf2 expression in the lungs were observed and the relationship between hydrogen inhalation therapy and the survival of sepsis animals were observed by Nrf2 knocked-out mice.Results:1.High concentration(67%)of hydrogen and low concentration(2%)of hydrogen inhalation can improve the survival rate of sepsis mice,and high concentration of hydrogen can significantly improve the 7-day survival rate from 80%to 90%compared with low concentration of hydrogen(P<0.05);67%H2 can improve the severe CLP sepsis 7d survival rate significantly(P<0.05).2.High concentration(67%)of hydrogen and low concentration(2%)of hydrogen inhalation can alleviate the lung injury of sepsis,including alleviating pathological changes of lung tissue,reducing pulmonary myeloperoxidase activity.Wet/dry weight ratio,protein content in bronchoalveolar lavage fluid(P<0.05),the protective effect of high concentration hydrogen on lung is more significant than that of low concentration hydrogen.3.The therapeutic effect of hydrogen inhalation on sepsis and the increase of antioxidant enzyme activity are related to the concentration of 8-iso-PGF2 ? in blood and lung tissues and the expression of proinflammatory cytokine HMGB1(P<0.05).The expression of Nrf2 in lung tissue of sepsis mice was significantly improved,but there was no significant effect of hydrogen inhalation on sepsis mice with Nrf2 gene knocked out.Conclusion:High concentration(67%)of hydrogen and low concentration of hydrogen(2%)inhalation can improve the survival rate of moderate CLP sepsis mice,and high concentration of hydrogen inhalation can improve the survival rate of moderate CLP sepsis mice better;67%hydrogen can improve the survival rate of severe CLP sepsis mice.Compared with low concentration of hydrogen,high concentration of hydrogen has better protective effect on lung injury in sepsis mice,and its mechanism may be related to activating Nrf2 signaling pathway,reducing early release of proinflammatory factors and alleviating lung oxidative damage in sepsis mice.Part2:The role of Nrf2 pathway in the treatment of LPS-induced acute lung injury in mice with high concentration hydrogen inhalationObjective:To observe the protective effect of high concentration(67%)hydrogen on acute lung injury induced by lipopolysaccharide(LPS),and further explore the role of Nrf2 signaling pathway in the process of hydrogen treatment of acute lung injury.Methods:Acute lung injury model was induced by LPS inhalation.Group LPS+67%H2 were treated by 67%hydrogen at time-point 1 and 6 hours after operation for 1 hour.The pathological damage and apoptosis of lung tissue were assessed after high concentration hydrogen treatment.The concentration of various pro-inflammatory cytokines in bronchoalveolar lavage fluid was monitored at each time point.The expression of Nrf2 were detected by Western-Blot,and chemokines were monitored.The activity of NF-?B in lung organs was also testedResults:1.Inhalation of 67%hydrogen can significantly alleviate LPS-induced lung injury in mice,alleviate the pathological changes and scores of lung tissue,and improve the oxygenation function of lung(P<0.05);reduce the protein concentration in bronchoalveolar lavage fluid,reduce the wet/dry weight ratio of lung tissue,and reduce the myeloperoxidase reaction activity of lung tissue(P<0.05);2.Inhalation of high concentration hydrogen can significantly improve LPS-induced lung injury.Apoptosis of lung cells resulted in a significant decrease in Caspase-3 activity and the number of TUNEL-positive cells(P<0.05).3.High concentration of hydrogen inhalation improved the inflammation of lung induced by lipopolysaccharide,decreased the total cell content and polymorphonuclear granulocyte content in bronchoalveolar lavage fluid,and decreased the contents of pro-inflammatory cytokines,Nrf2 and chemokines.High concentration of hydrogen inhalation decreased the activity of NF-kappa B in LPS-induced lung injury,which was related to the regulation of inflammatory response.Conclusion:Hydrogen can effectively improve LPS-induced acute lung injury,which may be related to activation of Nrf2 signaling pathway and regulation of inflammatory response and cell apoptosis mediated by NF-?B.Part 3:Mitochondrial dynamic mechanism of protective effect of high concentration hydrogen inhalation on lung injury in sepsis miceObjective:To evaluate the effect of inhalation of high concentration hydrogen on mitochondrial function in lung tissue of sepsis mice and to explore its protective mechanism.Methods:The sepsis mice model was induced by cecal ligation and puncture(CLP).Twenty four mice were divided into four groups(n=6):sham group,sham group+high concentration hydrogen treatment group(Sham+67%H2 group),cecal ligation puncture group(CLP group),cecal ligation puncture+high concentration hydrogen treatment group(CLP+67%H2 group)inhaled hydrogen administration scheme:67%hydrogen inhalation for 1 h and 6 h after operation respectively.Sham group and CLP group did not receive hydrogen inhalation treatment.Twenty-four hours after the successful preparation of the model,a small amount of lung tissue was taken from anesthetized mice and mitochondria were isolated and extracted.Respiratory control rate(RCR),mitochondrial membrane potential(MMP)and the activity of mitochondrial respiratory chain complex(MRC)were measured and evaluated.The expression levels of mitochondrial fission protein drpl and mitochondrial fusion protein Mfn2 in lung tissues of four groups of mice were measured at 24 hours after operation.Western blot was used to determine the expression levels of these proteins.Results:1.Compared with Sham group,the activity of mitochondrial respiratory chain complex I,respiratory control rate(RCR)and membrane potential(MMP)of lung mitochondria in CLP group were significantly decreased(P<0.05).Compared with CLP+H2 group and CLP group,there was no significant difference in the activity of mitochondrial respiratory chain complex ? between the two groups(P>0.05).Mitochondrial RCR and MMP increased significantly(P<0.05).2.Compared with Sham group,the expression of drpl protein increased and Mfn2 protein decreased in CLP group(P<0.05).Compared with CLP group,the expression of drpl protein decreased and Mfn2 protein increased in CLP+67%H2 group(P<0.05).Conclusion:1.The function and structure of mitochondria in lung tissue of sepsis mice are decreased and abnormal.Hydrogen inhalation therapy can significantly improve the function and structure of mitochondria in lung tissue2.Hydrogen inhalation can remove irreversibly damaged mitochondria from lung tissue of sepsis mice through modulating mitochondrial dynamics and alleviate lung tissue damage in sepsis mice.Summary1.High concentration hydrogen inhalation can effectively improve the survival rate and organ function of moderate and severe sepsis mice.Its mechanism is related to activating Nrf2 signaling pathway,regulating inflammation and oxidative stress2.High concentration hydrogen inhalation can effectively improve LPS-induced acute lung injury,which is related to Nrf2 related mechanism and regulation of inflammatory response and cell apoptosis mediated3.Mitochondrial dynamics plays an important role in the protection of lung injury in sepsis mice.Hydrogen therapy may be achieved by regulating mitochondrial fission and fusion to maintain the balance of mitochondrial dynamics. |
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