五步蛇毒蛋白C激活剂在脓毒症大鼠早期适应性免疫应答中的作用

Effect of protein C activator from Agkistrodon acutus venom on early adaptive immune response of septic rats

目的探讨五步蛇毒蛋白C激活剂(PCA)在脓毒症大鼠早期适应性免疫应答中的作用。方法采用SPF级SD大鼠78只,用随机数字表法分组:对照组(n=6,腹腔注射生理盐水);通过腹腔注射脂多糖(LPS 10 mg/kg)复制脓毒症大鼠模型,模型组以LPS注射后4、6、8、12、16、24 h时的标本采集时间分为6组,6只/组;余36只大鼠于LPS注射30 min后使用药物干预,分为1-磷酸鞘氨醇受体1(S1PR1)激动剂SEW2871干预组(0.5 mg/kg,腹腔注射)和PCA干预组(0.1 mg/kg,腹腔注射),各干预组以LPS注射后6、12、24 h时的标本采集时间分为3组,每组6只。采用ELISA法检测血浆IL-4、S1P、IL-12和IFN-γ水平,应用免疫荧光法检测肠系膜淋巴结S1PR1和CD103表达的变化。结果与对照组比较,脓毒症大鼠在注射LPS后的24 h内,血浆S1P、IL-12、IL-4和IFN-γ水平明显增高(P<0.05),LPS注射后6 h内IFN-γ/IL-4比值逐渐增高,之后逐渐降低;肠系膜淋巴结中S1PR1和CD103的表达明显增高(P<0.05)。SEW2871明显增加血浆S1P、IL-12和IFN-γ的浓度,降低血浆IL-4水平(P<0.05),且明显减少淋巴结中S1PR1和CD103的表达(P<0.05)。蛇毒PCA干预后,血浆IL-4水平明显增高,淋巴结S1PR1表达显著增多(P<0.05)。结论五步蛇毒PCA对维持脓毒症早期机体炎症和免疫反应的平衡具有调节作用,其机制可能与S1P-S1PR1通路有关。

Objective To investigate the effect of protein C activator(PCA)from Agkistrodon acutus venom(AAV)in modulating early adaptive immune response of septic rats.Methods Rat models of sepsis were established by intraperitoneal injection of lipopolysaccharide(LPS;10 mg/kg)in 36 SD rats,which were divided into 6 groups(n=6)for sample collection at 4,6,8,12,16 and 24 h after LPS injection,with 6 rats injected with saline as the control group.Another 36 rats were divided into two groups,and 30 min after LPS injection,the rats were treated with SEW2871(a sphingosine-1-phosphate receptor 1 agonist;0.5 mg/kg)or PCA group(0.1 mg/kg),and each group was divided into 3 groups(n=6)for sample collection at 6,12 and 24 h after LPS injection.Plasma IL-4,S1P,IL-12 and IFN-γlevels of the rats were detected using ELISA,and the expressions of S1PR1 and CD103 in the mesenteric lymph nodes were detected with immunofluorescence assay.Results The plasma levels of S1P,IL-12,IL-4 and IFN-γ(P<0.05)and the expressions of S1PR1 and CD103 in the mesenteric lymph nodes(P<0.05)all increased significantly in the rats 24 h after LPS injection;IFN-γ/IL-4 ratio increased progressively within 6 h after LPS injection and then subsided gradually.Compared with those in the corresponding sepsis model subgroups,the levels of S1P,IL-12 and IFN-γincreased while IL-4 level decreased significantly(P<0.05),and the expression of S1PR1 and CD103 were reduced significantly(P<0.05)in SEW2871-treated rats;both the plasma level of IL-4 and the expression of S1PR1 in the mesenteric lymph nodes increased significantly in PCA-treated rats(P<0.05).Conclusion PCA can regulate the balance of inflammation and immune response in the early stage of sepsis in rats possibly through the S1P-S1PR1 pathway.