A Research On The Role Of Cell Pyroptosis In The Protection Against Sepsis By Hyperoxia And Its Mechanism

Sepsis is a systemic inflammatory response syndrome caused by infection.It is known as severe sepsis with organ dysfunction.In China,higher mortality and higher treatment costs often occur to patients with sepsis,which has become one of the major diseases that endanger human health.Therefore,it is urgent to find effective treatment strategies for sepsis! Our earlier studies confirmed in both in vitro and in vivo models that,100%oxygen could inhibit the abnormal inflammatory reaction after sepsis through inhibition of the nuclear translocation of NF-kappa B(Nuclear factor Kappa B,nuclear factor kappa B)after lipopolysaccharide stimulation,and reverse the up-regulation of part micro RNAs in sepsis,and thus have a protective effect on sepsis.Sepsis is accompanied by a large number of cell death,which is one of the main causes of excessive inflammatory response disorder.In recent years,the study on the relationship between host cell death and sepsis has attracted much attention.Cell pyroptosis is a new type of programmed cell death in recent years characterized by the caspase-1 dependent releases of pro-inflammatory cytokines IL-1 beta and IL-18 and the activation of immune cells.Based on the above,this project was scheduled to provide more experimental basis for the treatment of sepsis with hyperoxia by studying the roles and mechanisms of cell pyroptosis in the development of sepsis and in the protective action against sepsis by hyperoxia therapy in animals challenged by the cecal ligation and puncture procedure,and further studying the roles of NLRP3 inflammatory pathways in the protective action against sepsis by hyperoxia therapy through in-vitro experiments.These findings would provide new targets for the treatment of sepsis.Objective To explore the roles and mechanisms of cell pyroptosis in the development of sepsis and in the protective action against sepsis by hyperoxia therapy,and to study the efficacy of 40%,60% and 100% oxygen in the treatment of sepsis in lipopolysaccharide-stimulated bone marrow-derived macrophages.Materials and methods Part 1Experiment 1.The mice were randomly divided into two groups: Sham and CLP.CLP group used cecal ligation and puncture to establish sepsis model and Sham group only implemented laparotomy,no cecum ligation and puncture.Collect the lower right lung after 24 h after surgery for western blot detection.Observation target: The ratio of the activated fragment P10 of caspase-1 to pro-caspase-1.Experiment 2.The mice were randomly divided into four groups: Sham,CLP,CLP+DMSO,CLP+VX-765.Sham group only implemented laparotomy,no cecum ligation and puncture and the latter three groups used cecal ligation and puncture to establish sepsis model.Animals respectively were given caspase-1 inhibitor VX-765 or DMSO at 1.5 h after surgery.Collect lower right lung and liver,right kidney for HE pathological dyeing slice at 24 h after surgery.Experiment 3.The mice were randomly divided into six groups: Sham,Sham+100%,CLP,CLP+40%Oxy,CLP+60%Oxy,CLP+100%Oxy.The former two groups only implemented laparotomy,no cecum ligation and puncture and the latter four groups used cecal ligation and puncture to establish sepsis model.Treatment groups were respectively given 1 h hyperoxia inhalation 1 h and 6 h after surgery with 40%,60% and 100% oxygen and no treatment group inhaled air directly.Collect mice serum for detection of the inflammatory factor interleukin 1 beta level at 24 h after surgery.Part 2Experiment 1.Culture mice bone marrow derived macrophages(BMDMs)and randomly divided them into four groups: Control,LPS,ATP,LPS/ATP.LPS group provided LPS stimulation 24 h alone and ATP group provided ATP stimulation 4 h alone.LPS/ATP group used LPS to stimulate cells for 24 h and then ATP for 4 h and the control group gave the normal culture medium.Collect the cell culture supernatants for detection of the interleukin 1 beta level.Experiment 2.Culture mice BMDMs and randomly divided them into six groups:Control,LPS,ATP,LPS/ATP,LPS/ATP+YVAD,LPS/ATP+DMSO.The former four groups’ intervention was the same as Experiment 1.LPS/ATP+YVAD group was stimulated with caspase-1 inhibitor AC-YVAD-CMK 0.5 h before the ATP stimulation.LPS/ATP+DMSO group was stimulated with DMSO 0.5 h before the ATP stimulation.Collect the cell culture supernatants for detection of the inflammatory factor interleukin 1beta level.Experiment 3.Culture mice BMDMs and randomly divided them into seven groups:Control,LPS,ATP,LPS/ATP,LPS/ATP+40%Oxy,LPS/ATP+60%Oxy,LPS/ATP+100%Oxy.The groups’ intervention was the same as Experiment 1.Treatment groups gave 40%,60% or 100% oxygen inhalation 1.5 h at the beginning of the ATP stimulation 0.5 h.Collect the cell culture supernatants for detection of the interleukin 1beta level.Experiment 4.The experimental group was the same as Part 3.The groups’ intervention was the same as Experiment 3 except that ATP stimulation time for 1.5 h.Treatment groups gave 40%,60% or 100% oxygen inhalation 1.5 h at the beginning of the ATP stimulation.Use Et Br and Hoechst33342 to have a fluorescence staining after the ATP stimulation 1.5 h.Observe the number of pyroptosis pore in the fluorescence microscope.Experiment 5.Culture mice BMDMs and randomly divided them into five groups:Control,LPS/ATP,LPS/ATP+40%Oxy,LPS/ATP+60%Oxy,LPS/ATP+100%Oxy.The groups’ intervention was the same as Experiment 1 except that ATP stimulation time for 2h.Treatment group gave 40%,60% or 100% oxygen inhalation 1.5 h at the beginning of the ATP stimulation.Use assay kit to detect the concentrations of the intracellular potassium ions after the ATP stimulation 2 h.Results Part 1Result 1.The pyroptosis of mice lower right lung tissue cells at 24 h after CLP were significantly higher than Sham group(P<0.05).Result 2.Compared with the Sham group,the lung,liver and kidney of CLP mice had significant pathological changes(P<0.001).Compared with DMSO group,administration of caspase-1 inhibitor VX-765 at 1.5 h after CLP relieved the pathological changes in the lung(P<0.01),liver and kidney(P<0.001).Result 3.The level of interleukin 1 beta in mice serum at 24 hours after CLP were significantly higher than Sham group(P<0.05)and the level was significantly reduced after the intervention of 40%(P<0.001),60% and 100%(P<0.01)oxygen inhalation.Part 2Result 1.Compared with the control group,the levels of interleukin 1 beta in cell culture supernatants of LPS and ATP group were slightly higher but without statistical significance,but the LPS/ATP group was significantly higher than control group(P<0.01).Result 2.The former four groups’ results was the same as Result 1.Compared with LPS/ATP group,LPS/ATP+YVAD group was significantly reduced(P<0.001)and LPS/ATP+DMSO group was significantly higher(P<0.001).Result 3.The former four groups’ results was the same as Result 1.Compared with LPS/ATP group,60% and 100% oxygen inhalation group were significantly reduced(P<0.001).Result 4.Compared with the control group,the percent number of cells with pyroptosis pore of LPS and ATP group had no statistical significance,but the LPS/ATP group was significantly higher than control(P<0.001).Compared with LPS/ATP group,40%,60% and 100% oxygen inhalation group were significantly reduced(P<0.001).Result 5.Compared with the control group,the concentrations of intracellular potassium ions of LPS,ATP and LPS/ATP were not significantly different.Compared with LPS/ATP group,40%,60% and 100% oxygen inhalation group had no statistical significance.Conclusion Our results demonstrated that cell pyroptosis is involved in the mechanisms underlying tissue injury in sepsis,and hyperoxia improves the symptoms of sepsis by inhibition of cell pyroptosis after sepsis.