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

中文关键词:  肠炎沙门菌  SpvD  NF-κ通路  免疫应答

英文关键词:Salmonella enteritidis  SpvD  NF-κB pathway  inflammatory responses

中文摘要:

      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。

英文摘要:

      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 complemented strains (P>0.05). In animal experiments, mice exhibited splenomegaly and hepatomegaly with increased tissue fragility at 5 days post-infection. Histopathological analysis revealed more severe inflammation and tissue damage in the liver and spleen of mice infected with the complemented strain compared to the wild-type strain. These findings indicate that the spvD gene exacerbates inflammation in vivo. Conclusion The spvD gene mediates the inflammatory response during Salmonella infection of macrophages through the NF-κB pathway and enhances inflammation in vivo.