慢性血吸虫感染对脓毒症小鼠保护作用的研究

The protective effect of chronic schistosoma japonica infestation against sepsis in mice

目的初步探讨慢性血吸虫（SJ）感染对脓毒症小鼠的保护作用及其机制。方法选择BALB／c雄性小鼠，按随机数字表法分组进行三部分实验。实验1：经腹部皮肤接种SJ尾蚴感染8周建立慢性SJ感染模型，分为正常组和SJ组，每组10只；用酶联免疫吸附法（ELIsA）检测血清白细胞介素（IL-4和IL-10）、肿瘤坏死因子-α（TNF-α）、γ-干扰素（IFN-γ）水平，实时荧光定量聚合酶链反应（PcR）检测腹腔巨噬细胞IL-10和TNF-α的mRNA表达，了解慢性SJ感染小鼠免疫状态。实验2：以脂多糖（LPS）腹腔注射诱导小鼠脓毒症模型，分为LPS组和SJ—LPS组，每组15只；用ELISA法动态观察注射LPS后0、24、48和72h细胞因子的变化，0h的水平相当于正常小鼠和SJ感染8周水平，观察慢性SJ感染对脓毒症过程的影响。实验3：分别以盲肠结扎穿孔术（CLP）和LPS诱导两种不同的脓毒症模型，评价慢性SJ感染对脓毒症小鼠72h存活率的影响。结果实验1：SJ组血清抗炎因子IL-4（（151．35±12．24）ng／L3和IL-10（（133．22±11．09）ng／L3水平较正常组（IL-4（56．32±8．66）ng／L，IL-10（48．17±7．23）ng／L3显著升高（均P〈0．05），并可使巨噬细胞向替代活化性巨噬细胞分化，慢性SJ感染使腹腔巨噬细胞高表达IL-10mRNA（SJ组4．46±1．82，正常组1．52±0．60），抑制TNF-αmRNA表达（SJ组1．61±0．93，正常组2。32±1．03，均P〈0．05）。实验2、3：慢性SJ感染小鼠血清IL-4、IL-10于注射LPS后0h即显著升高，随后下降，至72h仍明显高于LPS组（IL-4（ng／L）：92．2±7．6比41．5±4．5；IL-10（ng／L）：92．1±7．8比35．6±4．0，均P〈0．053；TNF-α、IFN-γ均于24h达峰值后逐渐下降，至72hSJ—LPS组仍显著低于LPS组（TNF-α（ng／L）：82．9±5．6比91．5±5．2；IFN-γ（ng／L）：44．1±4．8比52．6±4．0，均P〈0．053。慢性SJ感染可明显改善CLP或LPS所致脓毒症小鼠的存活率（CLP：80％比20％，LPS：70％比30％，均P（0．05）。结论慢性SJ感染可使脓毒症小鼠血清抗炎因子升高，存活率上升，从而起到保护作用。

Objective To preliminarily study the protective effect of chronic schistosoma japonica （SJ） infestation against sepsis in mice and its mechanism. Methods BALB/c male mice were used, and the experiment was divided into three parts. Experiment 1： chronic SJ infestation model was reproduced by SJ cercaria inoculation through abdominal skin for 8 weeks. Twenty mice were randomly grouped into normal group （n=10） and SJ group （n= 10）. The levels of interleukins （IL-4, IL-10）, tumor necrosis factor-α （TNF-α） and interferon-γ （IFN-γ） in serum were detected by enzyme linked immunosorbent assay （ELISA）. Real-time polymerase chain reaction （PCR） was employed to detect the levels of It-10 mRNA and TNF-α mRNA in abdominal macrophages. This experiment was meant to evaluate immune state in mice with chronic SJ infestation. Experiment 2.. lipopolysaccharide （LPS） was intraperitoneally injected to reproduce sepsis model. Thirty mice were randomly grouped into LPS group （n= 15） and SJ-LPS group （n= 15）. The levels of cytokines were determined by ELISA at 0, 24, 48 and 72 hours after LPS injection. This experiment was meant to detect the effect of chronic SJ infestation in mice during the septic process. Experiment 3.. two types of sepsis model were reproduced by cecal ligation and puncture （CLP） and LPS injection, respectively. The survival rate of mice with chronic SJ infestation in 72 hours in either type of sepsis was evaluated. Results Experiment 1： compared with normal group （IL-4 （56.32 ± 8. 66） ng/L, IL-10 （48. 17 ± 7.23） ng/L3, chronic SJ infestation showed an increase in serum It-4 [（151.35±12.24） ng/L3 and IL-10 C（133.22± 11.09） ng/L, both P〈0. 053. Chronic SJ infestation also resulted in an increase in IL-10 mRNA expression （SJ group 4.46±1.82, normal group 1.52±0. 60） and inhibited TNF-α mRNA expression （SJ group 1.61± 0. 93, normal group 2.32±1.03） in abdominal macrophages （both P〈0. 05）, indicating that macrophages could be differentiated into alternative activated macrophages. Experiments 2 and 3 showed that the levels of serum It-4 and It-10 were increased at 0 hour after LPS injection, and then gradually decreased in SJ-LPS group, but the levels were still higher than those in LPS group at 72 hours （IL-4 （ng/L）： 92.2±7. 6 vs. 41.5±4.5; IL-10 （ng/L） ; 92.1±7.8 vs. 35.6±4.0, both P〈0. 053; the levels of TNF-α and IFN-γ were increased at 24 hours, and then decreased in SJ-LPS group, and the levels were lower than those in LPS group at 72 hours （TNF-α （ng/L） ： 82.9±5.6 vs. 91.5±5.2; IFN-γ （ng/L）.. 44. 1±4.8 vs. 52.6±4.0, both P〈0. 05]. Therefore, chronic SJ infestation could improve the survival rate of mice with sepsis induced by CLP or LPS （CLP： 80% vs. 20%, LPS： 70% vs. 30%, both P〈0.05）. Conclusion Chronic SJ infestation could elevate anti-inflammatory factors in septic mice, thus ameliorating the survival rate, so it has protective effect on mice with sepsis.