Li Dengfu, Li Haixia, Wang Zhu, Huang Yuchen, Zhao Chenchong, Zhang Hongxia, Zhao Yanan, Zhu Naihui, Tang Xirong, Jin Yaping, Zhou Dong
College of Veterinary Medicine, Northwest A&F University, Xianyang, China.
Key Laboratory of Animal Biotechnology, Ministry of Agriculture and Rural Affairs, Northwest A&F University, Xianyang, China.
Vet Res. 2025 Jul 21;56(1):155. doi: 10.1186/s13567-025-01587-9.
Trueperella pyogenes is an opportunistic pathogen frequently associated with bovine endometritis, yet the mechanisms by which it induces uterine inflammation remain incompletely understood. In this study, we investigated the effects of T. pyogenes and its membrane vesicles (MVs) on bovine endometrial epithelial cells (BEECs) and explored the underlying inflammatory pathways involved. Bovine endometrial epithelial cells (BEECs) were treated with T. pyogenes (MOI = 100) or MVs at various concentrations (1 × 10, 1 × 10, or 1 × 10 particles/mL) for 6-24 h. Inflammatory cytokines (IL-1β, IL-6, IL-18, or TNF-α) and the activation of the NLRP3 and MyD88/NF-κB signalling pathways were analysed by ELISA, qRT‒PCR, and western blotting. Cell death mechanisms were assessed by flow cytometry and scanning electron microscopy. T. pyogenes significantly upregulated inflammatory cytokine mRNA expression at 6 and 12 h and protein expression at 12 and 24 h. Compared with bacterial stimulation at 12 h, MVs induced earlier activation of the NLRP3 inflammasome at 6 h. High-concentration MVs induced necrosis-like membrane disruption, whereas moderate concentrations promoted apoptosis and pyroptosis. Both T. pyogenes and its MVs activated the MyD88/NF-κB signalling pathway, with significantly increased phosphorylation of P65 at 12 h. Cytokine secretion exhibited time- and dose-dependent trends, aligning with transcriptional changes. Collectively, these findings demonstrate that T. pyogenes MVs contribute to endometrial inflammation through the NLRP3 and MyD88/NF-κB signalling pathways, with distinct forms of cell death determined by MV concentration. These findings highlight MVs as key virulence factors and potential therapeutic targets for bovine endometritis.
化脓放线杆菌是一种常与牛子宫内膜炎相关的机会致病菌，但其诱导子宫炎症的机制仍未完全阐明。在本研究中，我们研究了化脓放线杆菌及其膜泡（MVs）对牛子宫内膜上皮细胞（BEECs）的影响，并探讨了其中涉及的潜在炎症途径。用化脓放线杆菌（感染复数=100）或不同浓度（1×10、1×10或1×10颗粒/毫升）的MVs处理牛子宫内膜上皮细胞（BEECs）6至24小时。通过酶联免疫吸附测定（ELISA）、定量逆转录聚合酶链反应（qRT-PCR）和蛋白质免疫印迹法分析炎性细胞因子（白细胞介素-1β、白细胞介素-6、白细胞介素-18或肿瘤坏死因子-α）以及NLRP3和髓样分化因子88/核因子-κB（MyD88/NF-κB）信号通路的激活情况。通过流式细胞术和扫描电子显微镜评估细胞死亡机制。化脓放线杆菌在6小时和12小时显著上调炎性细胞因子mRNA表达，在12小时和24小时显著上调蛋白质表达。与12小时的细菌刺激相比，MVs在6小时更早激活NLRP3炎性小体。高浓度MVs诱导坏死样膜破坏，而中等浓度促进细胞凋亡和细胞焦亡。化脓放线杆菌及其MVs均激活MyD88/NF-κB信号通路，在12小时时P65磷酸化显著增加。细胞因子分泌呈现时间和剂量依赖性趋势，与转录变化一致。总体而言，这些发现表明，化脓放线杆菌MVs通过NLRP3和MyD88/NF-κB信号通路促进子宫内膜炎症，且MV浓度决定不同形式的细胞死亡。这些发现突出了MVs作为牛子宫内膜炎关键毒力因子和潜在治疗靶点的作用。
