毒力因子SpvD在调控NF-κB通路致肠炎沙门菌感染中的作用机制研究  

Mechanistic study of the effector protein SpvD in Salmonella enteritidis:Mediating inflammatory responses through NF-κB pathway regulation

作　　者：许增权 陈家良 侯悦 庄吉 褚洪迁 刘洁 XU Zeng-Quan;CHEN Jia-Liang;HOU Yue;ZHUANG Ji;CHU Hong-Qian;LIU Jie(School of Public Health,Hebei Medical University,Shijiazhuang 050017,China;Department of Microbiology Laboratory,Chaoyang District Center for Disease Control and Prevention,Beijing 100021,China;Department of Clinical Laboratory,Aerospace Center Hospital,Beijing 100049,China;Translational Medicine Center,Beijing Chest Hospital,Capital Medical University,Beijing 101149,China;Beijing Key Laboratory in Drug Resistant Tuberculosis Research,Beijing Tuberculosis and Thoracic Tumor Research Institute,Beijing 101149,China;School of Public Health,Baotou Medical College,Inner Mongolia University of Science and Technology,Baotou 014040,China)

机构地区：[1]河北医科大学公共卫生学院,石家庄050017 [2]北京市朝阳区疾病预防控制中心,北京100021 [3]航天中心医院检验科,北京100049 [4]首都医科大学附属北京胸科医院转化医学中心,北京101149 [5]北京市结核病与胸部肿瘤研究所北京市耐药结核病研究重点实验室,北京101149 [6]内蒙古科技大学包头医学院公共卫生学院,包头014040

出　　处：《食品安全质量检测学报》2024年第23期263-273,共11页Journal of Food Safety and Quality

基　　金：国家自然科学基金项目(82002206);北京市高层次公共卫生技术人才建设项目(学科骨干-02-02)。

摘　　要：目的探讨毒力因子SpvD在调控NF-κB通路致肠炎沙门菌感染中的作用机制。方法将肠炎沙门菌PLAGH-086野生株、spvD基因缺失株和回补株作为研究对象,进行Raw 264.7细胞和C57BL/6小鼠感染实验,比较3组间炎性因子RNA、分泌蛋白及病理结果差异。结果在Raw 264.7细胞感染实验中,缺失株侵袭率低于野生株和回补株(P<0.05)。计算不同时间点胞内菌量,缺失株在各时间点菌量均低于野生株和回补株,3组菌在12 h菌量较多,之后菌量减少。通过提取RNA探索核因子(nuclear factor erythroid 2-related factor 2,Nrf2)、醌氧化还原酶[NAD(P)H:quinone oxidoreductase 1,NQO1]、血红素加氧酶1(heme oxygenase,HO-1)、磷酸化核因子kappa B(phosphorylated nuclear factor kappa B,p-NF-κB)和髓样分化因子(myeloid differentiation factor 88,MyD88)的表达趋势,结果表明不同菌株的Nrf2、NQO1和HO-1的表达量趋势大致相同。不同时间点p-NF-κB和MyD88的表达量均呈现先下降后上升的趋势,与野生株相比,回补株的表达量在0、8、20、24 h均高于野生株(P<0.05)。关键炎性因子白细胞介素-6(interleukin-6,IL-6)、诱导型一氧化氮合酶(inducible nitric oxide synthase,iNOS)、白细胞介素-1(interleukin-1,IL-1)和环氧合酶-2(cyclooxygenase-2,COX-2)的表达量在感染12 h后逐渐上升,相较于野生株和缺失株,回补株上升趋势更明显,除iNOS之外,回补株IL-6、IL-1和COX-2的表达量在16~24 h内均高于野生株,差异具有统计学意义(P<0.05)。除8 h和16 h外,回补株的白介素-1β(interleukin-1β,IL-1β)均高于野生株(P<0.05),但其他分泌型炎性因子野生株和回补株浓度区别较小(P>0.05)。动物实验研究结果显示,感染5 d后小鼠的脾脏和肝脏增大且变脆。肝脏和脾脏的病理检查显示,与野生株相比,回补株导致更严重的炎症反应和组织损伤。以上结果表明,spvD基因在体内加剧了炎症反应。结论spvD基因在沙门菌感染巨噬细胞过程中Objective To elucidate the role of the virulence factor SpvD in regulating the NF-κB pathway during Salmonella enteritis infection.Methods The study utilized the wild-type strain PLAGH-086,spvD-deletion,and spvD-complemented strains of Salmonella enteritidis in infection experiments involving Raw 264.7 cells and C57BL/6 mice.Differences in inflammatory factor RNA levels,secreted proteins,and pathology outcomes were compared across 3 groups.Results In Raw 264.7 cells infection experiments,the invasion rate of the spvD-deletion strain was significantly lower than that of the wild-type and complemented strains(P<0.05).Quantification of intracellular bacterial loads at different time points revealed that the deletion strain consistently had lower bacterial counts,with all strains reached a high value 12 hours after infection and then decreased.RNA expression analysis showed that nuclear factor erythroid 2-related factor 2(Nrf2),NAD(P)H:quinone oxidoreductase 1(NQO1),and heme oxygenase(HO-1)levels were similar across strains.Phosphorylated nuclear factor kappa B(p-NF-κB)and myeloid differentiation factor 88(MyD88)exhibited time-dependent variation,showing an initial decrease followed by an increase.Compared to the wild-type strain,the complemented strain displayed significantly higher p-NF-κB and MyD88 levels at 0,8,20,and 24 hours(P<0.05).Key inflammatory factors,including interleukin-6(IL-6),inducible nitric oxide synthase(iNOS),interleukin-1(IL-1),and cyclooxygenase-2(COX-2),gradually increased after 12 hours of infection.The complemented strain exhibited a more pronounced rise than the wild-type and deletion strains.Except for iNOS,IL-6,IL-1,and COX-2 levels in the complemented strain were significantly higher than in the wild-type strain between 16 and 24 hours(P<0.05).Except the 8 and 16 hours,interleukin-1β(IL-1β)levels in the complementation strain were consistently higher than in the wild-type strain(P<0.05).Other secreted inflammatory factors showed minimal differences between the wild-type and compleme

关 键 词：肠炎沙门菌 SpvD NF-ΚB通路 免疫应答 

分 类 号：R574[医药卫生—消化系统] TS201.3[医药卫生—内科学]