The Role Of NK Cells In The Protection Of Hyperbaric Oxygen Treatment Against Zymosan-induced Sepsis In Mice

Objective: Sepsis is one of the main causes of death in patients with severe disease,which is caused by infection factors,characterized by fulminant,persistent and systemic inflammatory reaction.At present,there is no effective treatment for it.In the development of sepsis,various immune cells and cytokines are involved,of which the most important one is NK cell.As a multifunctional sentinel,NK cell is the main source of inflammation cytokines,recruits and activates other immune cells by secreting various cytokines,amplifies inflammatory reaction,and aggravates the systemic organ injury.Previous studies have found that HBO can significantly reduce the inflammatory response,protect organ function,and improve the survival in animal models of sepsis.It has also been found that HBO has immunosuppressive effect by inhibiting the release of pro-inflammatory cytokines at the beginning of inflammatory state.In addition,HBO has been applied for infection,trauma,even surgery in clinical,but the exact mechanism is not clear.We intend to clarify the mechanism of HBO in sepsis by treating the mice of sepsis by HBO,explore the changes of NK cells and cytokines related,provide clues for the further application of HBO in the treatment of sepsis.Methods: In this study,SPF adult male C57BL/6 mice were used,randomly divided into 4 groups: group I–“saline + air”,group II-“sepsis + air”,group III-“saline + HBO”,group IV-“sepsis + HBO”.Group II and group IV were injected intraperitoneally with500mg/kg ZY to induce sepsis model,while group I and group III were given the same amount of normal saline.4h and 11 h after the injection,group III and group IV were treated with HBO respectively,while group I and group II were given no intervention.The clinical symptoms of poisoning,animal survival rate were observed,the biochemical parameters(ALT,AST,Cr and BUN),arterial gas and serum lactic acid,inflammatory factors(TNF-α,IL-6,IFN-γ,IL-1,IL-10)and other indicators were tested.Mononuclear cells in peripheral blood of mice were separated the molecular phenotypes and molecular functions of NK cells were detected by flow cytometry.Results: 1.compared with the sepsis + air group,the mice in sepsis + HBO group were significantly improved in poisoning symptoms(P<0.05),blood biochemical parameters was significantly decreased(P<0.05),tissue oxygenation capacity was significantly improved(P<0.05),blood lactate levels were significantly decreased(P<0.05).The survival rate of 72 h was significantly increased(P<0.05).In addition the HBO can significantly reduce the levels of pro-inflammatory cytokines such as TNF-α,IL-6,IFN-γ,IL-1 in peripheral blood(P<0.05),and up-regulate the level of anti-inflammatory cytokines IL-10 obviously.2.Sepsis can significantly increase the percentage of NK cells in peripheral blood of mice(P<0.05).However,the ratio of NK cells can be significantly reduced after HBO therapy(P<0.05).3.The level of CD94 and NKG2 A on the surface of NK cells was significantly decreased(P<0.05)in sepsis,but HBO could significantly increase the expression level(P<0.05).4.The level of NKp46 and NKG2 D on the surface of NK cells was significantly increased in sepsis(P<0.05),but and the HBO could decrease level significantly(P<0.05).5.The level of costimulatory molecules PD-1 and PD-L1 on the surface of NK cells was significantly up-regulated(P<0.05)in sepsis,was not significantly affected by HBO(P>0.05).6.The level of CD107 a,TRAIL and Fas L on the surface of NK cells was significantly increased in sepsis(P<0.05),and HBO could significantly decrease the level(P<0.05).7.The level of TLR2 and TLR4 on the surface of NK cells was significantly increased under the condition of sepsis,HBO could significantly decrease the level(P<0.05).8.HBO has no effect on NK cells,the inhibitory receptor CD94 and NKG2 A,activating receptors molecules NKp46 and NKG2 D,costimulatory molecules PD-1 and PD-L1,surface functional molecules such as CD107 a,TRAIL,Fas L,TLRs surface molecules TLR2 and TLR4 in mice under natural conditions(P>0.05).Conclusion: We intervented the zymosan induced mouse model of sepsis by HBO early.We found that HBO can significantly improve the symptoms of sepsis and organ function in mice,reduce inflammation,improve the survival rate;the number of NK cells of mice in early sepsis stage and activity of pro-inflammatory are increased significantly;while HBO can reduce the number of NK cells in the early stage of sepsis in mice,inhibit its proinflammatory activity and immune function;therefore,HBO could improve the symptoms and prognosis of sepsis by its effects on NK cell.