Effect Of Ozone Pretreatment On Spleen NK Cells In Septic Mice

Objective:By constructing a lipopolysaccharide（LPS）-induced sepsis model in mice after ozone pretreatment,and observed the effect of ozone pretreatment on natural killer cells（NK）in spleen to investigate the protective mechanism of ozone pretreatment on sepsis model.Methods:SPF-grade male C57BL/6 mice,6～8 weeks old,weighing 18～22 g.The mice were randomly divided into four groups（n=6）according to the randomized scale method:blank control group（Control）,lipopolysaccharide group（LPS）,ozone+lipopolysaccharide group（O₃+LPS）,and air+lipopolysaccharide group（Air+LPS）.The O₃+LPS group was continuously injected intraperitoneally with ozone（concentration 30μg/ml）33.3 ml·kg^-1·d^-1for 5 d,and the Air+LPS group was injected with the same volume of air daily.10 mg/kg of LPS was injected intraperitoneally 24 h after the last pretreatment in the LPS,Air+LPS and O₃+LPS groups,and Control group was injected with the same volume of saline.Six mice in each group were observed for survival rate,body temperature and clinical symptom score within 72 h.In addition,six mice in each group were tested for the levels of the inflammatory factors interferon-γ（IFN-γ）and interleukin-10（IL-10）in the serum,using enzyme linked immunosorbent assay（ELISA）6 h and 24 h after LPS injection,respectively.Spleen specimens were taken from mice and NK cell levels in the spleen were detected using flow cytometry.Results:Compared with the Control group,the survival rate,ear temperature and symptom scores of the LPS,Air+LPS and O₃+LPS groups were reduced（P<0.05）.Compared with the LPS group,the survival rate,ear temperature and symptom scores of mice in the Air+LPS group were not statistically significant（P>0.05）,and the survival rate,ear temperature and symptom scores of mice in the O₃+LPS group were increased（P<0.05）;Compared with the Control group,the levels of inflammatory cytokines IFN-γand IL-10 in the serum of mice in the LPS,Air+LPS and O₃+LPS groups were elevated after 6 h and 24 h of LPS treatment（P<0.05）.The differences in the levels of inflammatory cytokines IFN-γand IL-10 in the serum of mice in the Air+LPS group after 6 h and 24 h of LPS treatment were not statistically significant compared with the LPS group（P>0.05）,and The levels of inflammatory cytokine IFN-γwere significantly lower and IL-10 levels were significantly higher in the O₃+LPS group mice after 6 h and 24 h of LPS treatment（P<0.05）.Compared with the Control group,the NK cell levels in the spleen of mice in the LPS,Air+LPS and O₃+LPS groups were elevated after 6 h of LPS treatment（P<0.05）,and the differences in the NK cell levels in the spleen of mice in the Air+LPS group after 6 h of LPS treatment were not statistically significant compared with the LPS group（P>0.05）,and the mice in the O₃+LPS group NK cell levels in the spleen after 6 h of LPS treatment were significantly reduced（P<0.05）;compared with the Control group,mice in the LPS group,Air+LPS group and O₃+LPS group all had reduced NK cell levels in the spleen after 24 h of LPS treatment（P<0.05）,and compared with the LPS group,the difference in NK cell levels in the spleen of mice in the Air+LPS group after 24 h of LPS treatment was not statistically significant（P>0.05）,and the levels of NK cells in the spleen of mice in the O₃+LPS group after 24 h of LPS treatment were significantly increased（P<0.05）.Conclusion:Ozone pretreatment may exert its protective mechanism against sepsis by improving the inflammatory response in septic mice and regulating the level of splenic NK cells to improve the survival rate of septic mice.