The Role And Mechanism Of Neutrophil Extracellular Traps(NETs) In The Regulation Of Innate Immunity Related To Acute Exhaustive Exercise And Chronic Aerobic Exercise

Background:Neutrophil extracellular traps(NETs)are made of processed chromatin bound to granular and selected cytoplasmic proteins.NETs are released by white blood cells called neutrophils,maybe as a last resort,to control microbial infections However,excessive NETs release has been shown to participate in the pathological process of many diseases;therefore,too weak or too strong NETs release capacity in the body will cause the body health to be out of balance.Similarly,the research consensus reached in the field of exercise immunology is:excessive exercise causes the body to be temporarily immunosuppressed and the risk of infection is increased;regular aerobic exercise improves the body's immune function and reduces the risk of disease.However,there are only a handful of researches related to NETs and the consensus on these two points of research.Therefore,this topic will start from the two major consensus of sports immunology and explore the functions of NETs in two parts:First,NETs release during acute exhaustion exercise in peripheral circulation and its possible mechanism;Second,the role and mechanism of NETs in regular aerobic exercise to alleviate the inflammatory response of the disease.In the second part of the study,we selected the clinically common respiratory diseases,acute lung injury(ALI)as the research model,which is typically characterized by an uncontrolled inflammatory response of the airways and the whole body.Previous studies have reported that NETs participate in the process of ALI airway inflammation,and regular aerobic exercise can reduce the ALI airway inflammation response;this has laid a good foundation for this part of the study.Therefore,this subject will study the two parts to reveal the role and mechanism of NETs in health and the ALI body under different exercise modes,so as to enrich the cognitive perspective of exercise-induced immunosuppression and provide new insights into the treatment of ALI.TargetMethods:In the first part of the study,we selected 7-week-old male C57 BL/6 mice and randomly divided them into a control group(Group C),running with increasing load to 60 min(E60 group),and running with increasing load to exhaustion group EE group.,Recovery 1.5 hours group(1.5E group),recovery 3 hours group(3E group)after exhaustion;at different time points during the acute exhaustion treadmill exercise,detect lactic acid accumulation,NETs release,and explore through in vitro experiments Situation and mechanism of lactic acid inhibition of NETs In the second part of the study,7-week-old male C57BL/6 mice were randomly divided into a control group(CON group),an LPS-induced acute lung injury group(LPS group),an aerobic exercise+acute lung injury group(EXE+LPS group),Acute lung injury combined with DNase injection degraded NETs group(DNase+LPS group).Twenty-four hours after exercise intervention and drug injection,materials were taken to detect the infiltration of neutrophils and M1 type alveolar macrophages in lung tissue,and the release of NETs.Key proteins of MAPK signaling pathway and NF-?B protein activation;In addition,purified NETs induced MH-S polarization of mouse alveolar macrophage cell line,and detected the expression of proinflammatory factors and activation of key proteins after stimulationResults:The results of Study 1 are:1.Changes in blood lactic acid concentration during acute exhaustion:The blood lactic acid concentration was at a low level during the first 60 minutes of the incremental treadmill exercise;when it continued for 90 minutes,a significant increase occurred and remained at a high platform level2.Release of plasma NETs during acute exhaustion:The MPO-DNA complex in plasma showed a gradual inhibitory trend during exhaustive exercise,and there was no recovery 3 hours after exercise3.The ability of circulating neutrophils to release NETs in vitro after acute exhaustion exercise:Whether without stimulation or with 100nM PMA,the ability of neutrophils to release NETs in peripheral blood of mice after acute exhaustion exercise was significantly lower than the control group4.Correlation between blood lactic acid concentration and plasma NETs release during and immediately after exhaustive exercise:Plasma MPO-DNA complex and blood lactic acid showed a significant negative correlation5.Different concentrations of lactic acid in vitro stimulated neutrophils to release NETs:The ability of neutrophils to release NETs under the stimulation of 10,15 and 20mM lactic acid combined with 100nM PMA was significantly lower than that of the control group6.Possible mechanism of lactic acid inhibition of NETs release:Under the stimulation of 100nM PMA,with the increase of lactic acid stimulation concentration,the expression of ROS in neutrophils showed a significant declineThe results of Study 2 are:1.Lung tissue inflammatory response:LPS group has more neutrophils and M1 type alveolar macrophages infiltration than CON group,and proinflammatory cytokines in alveolar lavage fluid increase significantly;The inflammation response of EXE+LPS group was more relieved than LPS group2.Release of NETs in lung tissue:LPS group released more NETs in the lung than CON group.The expression of NETs in the lung of EXE+LPS group and DNase+LPS group was significantly reduced compared with LPS group3.Possible mechanism of aerobic exercise for alleviating ALI airway inflammation:the levels of phosphorylation of ERK,JNK,p38 and NF-?B proteins in alveolar macrophages in the LPS group were significantly higher than those in the CON group;the above-mentioned protein phosphorylation levels in the exercise group and DNase group All were significantly lower than the LPS group4.The effect and mechanism of NETs on MH-S cell polarization:In the early stage of stimulating MH-S cells to undergo M1-type polarization,ERK protein was activated first,and in the later stage of stimulation,the activation effect of NF-?B protein appeared.Conclusion:1.Conclusion of Study 1:The accumulation of blood lactic acid during acute exhaustion exercise inhibits the ability of NETs to release in peripheral blood of mice;the extracellular strong lactic acid environment can inhibit the release of NETs by neutrophils depending on the ROS pathway2.Conclusion of Study 2:Regular aerobic exercise can reduce the airway inflammatory response by inhibiting the excessive release of airway NETs and reduce the M1-type polarization of alveolar macrophages;aerobic exercise can reduce the ALI airway inflammatory response by inhibiting ERK and NF-?B related signaling pathways.