Establishment Of Mouse Models And Inflammatory Mechanisms Of Decompression-Induced Lung Injury

Humans are exploring remote worlds through extensive underwater activities in unstable ambient pressure,and the reduction of ambient pressure may induce the formation of bubbles in the blood vessels and other tissues due to the inert gas supersaturation.It has been confirmed that the bubbles are a major cause of decompression sickness(DCS).However,generally,the amount of bubbles does not correlate directly with the clinical manifestations of DCS,and bubbles can cause damage to human body without causing severe DCS.Bubbles are more likely to form in the veins than in other sites where inert gas supersaturates.These bubbles forming in the vein are finally filtered by the lung through the pulmonary circulation.Thus,the lung is a target organ of bubbles formed in the blood,and susceptible to bubble induced injury.The decompression-induced lung injury(DILI)has been reported in divers after repetitive dive without DCS.The pathogenesis of DILI is still poorly understood,and inflammation and oxidative stress induced by circulating bubbles may be the potential mechanisms.Macrophages belonging to the mononuclear phagocyte system(MPS)are an important participant in the innate immunity which is an effective immune response to general signs of infection.As a key cellular component of the innate immunity,macrophages are a major player in the firstline defense against the pathogens and the modulation of homeostatic and inflammatory responses.Macrophages are not homogenous,and they can be categorized into two subsets as the classically activated(M1)and the alternatively activated(M2).Pro-inflammatory stimuli may result in the shift to M1-cells,which are responsible for the clearance of infected or transformed cells,but simultaneously contribute to tissue injury.Conversely,antiinflammatory signals may induce the formation of M2-cells,promoting tissue regeneration and wound healing.This phenomenon,termed polarization,results from different microenvironments,governing macrophage functionality within the hosting tissues.The polarization of macrophages has been found to be related to some diseases in humans.Studies have shown that bubbles formed in the blood can induce the inflammatory responses,but the activation of macrophages following decompression is poorly understood.Our previous study showed decompression could induce the activation of macrophages in the blood and lung as shown by the elevated production of factors related to macrophage activation and the increase in the proportion of M1 macrophages in the blood.Hydrogen is the simplest molecule in nature.In recent years,hydrogen has been found to protect against various diseases,mainly by suppressing oxidative stress,inhibiting inflammation,and compromising cell apoptosis.Our previous study has demonstrated that high concentrations of hydrogen(HCH)gas(67% H2,33% O2)can affect the polarization of macrophages in a stroke model.Whether hydrogen gas is also protective on the DILI via inhibiting macrophage polarization is unclear.This study aimed to establish a model of DILI secondary to repetitive diving in mice,investigate the lung macrophage polarization in the DILI and explore the protective effect of HCH on the DILI.This study aimed to establish an animal model of decompression-induced lung injury(DILI)secondary to repetitive diving in mice and explore the role of macrophages in DILI and the protective effects of high concentration hydrogen(HCH)on DILI.Part I The animal model of decompression-induced lung injuryMethods:We developed a decompression protocol(once a day for 4 consecutive days),detected bubbles formed in mice and evaluated the structure and function of liver,kidney,and brain.We detected changes in lung function at different time points after each decompression,and selected the most obvious time points for pathophysiological index detection.Results: According to the decompression protocol of this experiment,bubbles can be detected in mice.The structure and function of liver,kidney,and brain did not change significantly.Single decompression did not cause significant changes in the lung function.6 h after each time of repeated decompression,total lung capacity(TLC),chord compliance(Cchord),Functional residual capacity(FRC)reached the lowest value,as the number of decompression increases,the index decreases more obviously,but the index did not continue to decrease after 4 times of consecutive decompression.The lung water content(LWC)and the Evans Blue content in the alveolar lavage fluid of mice increased after repeated decompression,and did not continue to increase after 4 times of consecutive decompression.Conclusion: By choosing a decompression protocol,we have successfully constructed DILI mouse models.Bubbles were observed in mice,and caused lung injury without affecting other important organs.DILI aggravated as the number of decompression increases,but adaptation was found in mice.Part II: The role of inflammation and macrophage and the protective effect of HCH in DILIMethods: We detected the differential genes in the lungs of DILI mice,detected the expression of inflammatory factors,anti-inflammatory factors and adhesion chemokines in the lung and the expression of macrophage marker proteins.Then we detected the protective effects of inhaled HCH intervention on the structure and function of the lung,detected the influence of HCH on the expression of inflammatory factors,anti-inflammatory factors,adhesion chemokines,and macrophage marker proteins in the lung.Results: GO analysis results show that the lung immune response and immune regulation-related genes are obviously differentially expressed after DILI.Repeated decompression caused tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),IL-10 and transforming growth factor-β(TGF-β)increased in the lungs of mice,but stopped increasing after 4 consecutive days of decompression treatment.After HCH intervention,TNF-α and IL-1β were significantly reduced,while IL-10 and TGF-β were significantly increased.The expression of intracellular adhesion factor-1(ICAM-1),monocyte chemotactic protein-1(MCP-1)and endothelin-1(endothelin-1,ET-1)increased and reached the maximum after 3 times of decompression.After inhaling HCH,these indicators were significantly reduced.Macrophage markers F4/80,i NOS,CD11 c,CD206,and Arg-1 all increased after repeated decompression,but the timepoints of the increase markers of were different.After inhaled HCH treatment,the expression of F4/80,i NOS and CD11 c in the lung decreased significantly,while the expression of CD206 and Arg-1 increased significantly.In addition,the structure and function of the lung were significantly improved after inhaling HCH.Conclusion: The occurrence of DILI is closely related to the activation of inflammatory response.As an important component of the immune system,macrophages play an important role in the occurrence and repairment of DILI.Hydrogen can protect DILI through anti-inflammatory effects.