创伤弧菌经呼吸道感染小鼠早期靶器官损伤及炎性因子谱分析

The injury of major organs and expression of inflammatory cytokines in mice with early infection of Vibrio vulnificus through respiratory tract

目的观察创伤弧菌(V.vulnificus)经呼吸道感染小鼠模型早期靶器官损伤情况及炎性因子谱的变化,为相关防治策略的制订提供参考。方法24只8~10周龄健康雌性BALB/c小鼠,随机分为对照组、低浓度感染组、中浓度感染组和高浓度感染组,每组6只。采用滴鼻法建立低、中、高浓度V.vulnificus感染小鼠模型。观察感染早期(0~12h)小鼠的生存、排便情况,毛发色泽,咳喘和精神状态等指标;取小鼠不同系统的重要器官进行HE染色观察病理表现,采用免疫组化染色法检测靶器官中白细胞介素(IL)-6、IL-10、γ干扰素(IFN-γ)和肿瘤坏死因子-α(TNF-α)的表达变化,采用小鼠炎性多因子检测试剂盒结合流式细胞术检测血清中致炎-抑炎因子谱的变化情况。结果V.vulnificus经呼吸道感染12h内即可引起小鼠生存、排便、毛发、呼吸道和精神状态改变。HE染色结果显示,不同浓度感染组小鼠回肠、肺、肝和脾均出现炎性细胞浸润、细胞坏死等损伤表现,且高浓度感染组损伤表现更严重。免疫组化染色结果显示,中、高浓度感染组小鼠回肠、肺、肝和脾中IL-6、IL-10、IFN-γ和TNF-α的阳性表达率均明显高于对照组(P<0.05)。高通量多因子检测结合流式细胞术结果显示,与对照组比较,低、中、高浓度感染组TNF-α、单核细胞趋化蛋白(MCP)-1表达水平明显增高(P<0.05),IL-27表达水平明显降低(P<0.05),中、高浓度感染组IL-1β、IL-6、IL-17A、粒细胞-巨噬细胞集落刺激因子(GM-CSF)表达水平明显增高(P<0.05),高浓度感染组IL-1α、IL-23、IL-12p70、IFN-γ表达水平明显增高(P<0.05)。结论经呼吸道感染V.vulnificus早期小鼠病情进展迅速,肺、肠、肝和脾是主要靶器官,同时血清中多种炎性因子分泌增多,可引起机体严重的炎性反应。

Objective Establishing animal model of Vibrio vulnificus(V.vulnificus)early infection via respiratory tract,to observe the survival,pathological changes of target organs and the expression changes of inflammatory cytokines in model mice,and to provide references for the relevant prevention and treatment strategies.Methods A total of 24 healthy female BALB/c mice(8 to 10 weeks old)were randomly divided into 4 groups(6 mice per group),including control group and three infection groups(low,medium and high concentration of infection,respectively),based on which the V.vulnificus infection models were established by intranasal administration.The survival statistics,status of defecation,hair and respiration as well as mental state of mice were monitored during 0-12 h early infection progress.Hematoxylin-eosin(HE)staining was performed on the important organs of mice to analyze the pathological manifestations,and immunohistochemical staining was used to detect the expression of interleukin(IL)-6,IL-10,interferon-γ(IFN-γ)and tumor necrosis factor-α(TNF-α)in the target organs.Mouse inflammation panel combined with flow cytometry was used to further detect the changes of multiple inflammatory and anti-inflammatory cytokines in serum.Results V.vulnificus infection via respiratory tract caused mice deterioration in survival rate,defecation,hair status,respiratory tract and mental state within 12 h.HE pathological analysis showed inflammatory cell infiltration and cell necrosis in ileum,lung,liver and spleen of mice in different infection groups,and the injury was much severer in high concentration of infection group.Immunohistochemical results reflected the positive expression rates of IL-6,IL-10,IFN-γand TNF-αin ileum,lung,liver and spleen of mice in medium and high concentration of infection groups were significantly higher than those in control group(P<0.05).The results of mouse inflammation panel and flow cytometry showed that compared with the control group,the expression levels of TNF-αand monocyte chemotactic protein(MCP)-1 in all three infection groups were significantly increased(P<0.05),and the expression level of IL-27 significantly decreased(P<0.05);the expression levels of IL-1β,IL-6,IL-17A and granulocyte-macrophage colony-stimulating factor(GM-CSF)were significantly increased in medium and high concentration of infection groups(P<0.05);the expression levels of IL-1α,IL-23,IL-12p70 and IFN-γwere significantly increased in high concentration of infection group(P<0.05).Conclusions V.vulnificus infection progressed rapidly through respiratory tract in mice,and lung,intestine,liver and spleen were major target organs.Accompanied by increased secretion of multiple serum inflammatory cytokines,V.vulnificus infection through respiratory tract might further causes severe inflammatory reaction in hosts.