臭氧预处理对脓毒症小鼠脾脏NK细胞水平的影响

Effect of ozone preconditioning on splenic natural killer cells in septic mice

目的评价臭氧预处理对脓毒症小鼠脾脏自然杀伤细胞(NK细胞)水平的影响。方法 SPF级雄性C57BL/6小鼠24只, 6~8周龄, 体质量18~22 g, 采用随机数字表法分为4组(n=6):对照组(C组)、脂多糖组(LPS组)、臭氧+脂多糖组(O_(3)+LPS组)、空气+脂多糖组(Air+LPS组)。采用腹腔注射LPS 10 mg/kg的方法制备小鼠脓毒症模型。在造模前5 d开始进行臭氧预处理:腹腔注射臭氧1 mg/kg, 1次/d, 连续5 d, Air+LPS组注射等量空气。LPS注射后72 h内, 观察小鼠生存情况, 记录脓毒症评分及耳温(每2 h记录1次, 取平均值)。LPS注射后6和24 h时, 取眼眶后静脉血样, 采用ELISA法检测血清IFN-γ和IL-10浓度。随后取脾脏, 制备脾脏单细胞悬液, 采用流式细胞术检测脾脏NK细胞百分比。结果与C组相比, LPS组、Air+LPS组和O_(3)+LPS组耳温、脓毒症评分和72 h生存率降低, LPS注射后各时点血清IFN-γ和IL-10浓度升高, 脾脏NK细胞百分比于LPS注射后6 h时升高, 24 h时降低(P<0.05);与LPS组相比, O_(3)+LPS组耳温、脓毒症评分和72 h生存率升高, LPS注射后各时点血清IFN-γ浓度降低, 血清IL-10浓度升高, 脾脏NK细胞百分比于LPS注射后6 h时降低, 24 h时升高(P<0.05), Air+LPS组上述指标差异无统计学意义(P>0.05)。结论臭氧预处理减轻小鼠脓毒症的机制可能与减轻机体炎症反应, 调节脾脏NK细胞水平有关。

Objective To evaluate the effect of ozone preconditioning on splenic natural killer(NK)cells in septic mice.Methods Twenty-four SPF-grade male C57BL/6 mice,aged 6-8 weeks,weighing 18-22 g,were divided into 4 groups(n=6 each)according to the random number table method:control group(group C),lipopolysaccharide(LPS)group,ozone+LPS group(O_(3)+LPS)and air+LPS group(Air+LPS).The sepsis was induced by intraperitoneal injection of LPS 10 mg/kg.Ozone preconditioning was started at 5 days before developing the model:ozone 1 mg/kg was intraperitoneally injected once a day for 5 consecutive days,the equal volume of air was injected in Air+LPS group.The survival was observed within 72 h after LPS injection,and sepsis score and ear temperature(once every 2 h,an average was calculated)were recorded.The posterior orbital venous blood samples were taken at 6 and 24 h after LPS injection for determination of serum interferon-γ(IFN-γ)and interleukin^(-1)0(IL-10)concentrations using enzyme-linked immunosorbent assay.The spleen was then taken,and a single cell suspension of the spleen was prepared for measurement of the percentage of NK cells in the spleen by flow cytometry.Results Compared with C group,the ear temperature,sepsis score and 72-h survival rate were significantly decreased,serum IFN-γand IL-10 concentrations were increased at each time point after LPS injection,and the percentage of splenic NK cells was increased at 6 h after LPS injection and decreased at 24 h after LPS injection in LPS,Air+LPS and O_(3)+LPS groups(P<0.05).Compared with LPS group,the ear temperature,sepsis score and 72-h survival rate were significantly increased,serum IFN-γconcentrations were decreased at each time point after LPS injection,serum IL-10 concentrations were increased at each time point after LPS injection,and the percentage of splenic NK cells was decreased at 6 h after LPS injection and increased at 24 h after LPS injection in O_(3)+LPS group(P<0.05),and no significant change was found in the parameters mentioned above in Air+LPS group(P>0.05).Conclusions The mechanism by which ozone preconditioning reduces sepsis may be related to reduction of inflammatory responses and regulation of splenic NK cell levels in septic mice.