Department of Clinical Veterinary Medicine, College of Veterinary Medicine, China Agricultural University, Beijing, China.
Department of Production Animal Health, Faculty of Veterinary Medicine, University of Calgary, Calgary, AB, Canada.
Front Immunol. 2022 Sep 2;13:934819. doi: 10.3389/fimmu.2022.934819. eCollection 2022.
, a highly contagious pathogen, causes bovine mastitis, resulting in premature culling of affected cows and severe economic losses. Infection with caused oxidative stress and apoptosis in bovine mammary epithelial cells (bMECs); however, mechanisms underlying -induced autophagy remain unclear. Therefore, the autophagy flux induced by in bMECs was analyzed by Western blot and laser scanning confocal microscopy. Expression levels of proteins in the HIF-1α and AMPKα/ULK1 pathway, including HIF-1α, AMPKα, p-AMPKα, ULK1, p-ULK1, mTOR, and p-mTOR, plus expression of autophagy-related genes including SQSTM1/p62, Atg5, Beclin1, and LC3II/LC3I, were quantified with Western blot. Infection with induced autophagosomes and LC3 puncta in bMECs that were detected using transmission electron microscopy and laser scanning confocal microscopy, respectively. In addition, lysosome-associated proteins Rab7 and LAMP2a, and lysosomal activity were measured with Western blot and laser scanning confocal microscopy. Infection with induced an unobstructed autophagic flux, increased protein expression of LC3II/LC3I, and decreased SQSTM1/p62 protein expression at 6 hpi. Furthermore, upregulated protein expression in the HIF-1α and AMPKα/ULK1 pathway and increased the ratio of LC3II/LC3I, implying autophagy was activated in bMECs. However, deletion of AMPKα or ULK1 decreased LC3II/LC3I expression levels and LC3 puncta numbers, suggesting that autophagy was inhibited in bMECs. Additionally, deficiency of HIF-1α decreased protein expression of AMPKα and ULK1 as well as LC3 puncta numbers, and autophagy induced by was also inhibited in bMECs. At 6 hpi, lysosome-associated protein Rab7 was decreased and LAMP2a was increased, indicating normal autophagy. In contrast, at 12 hpi, expression of Rab7 and LAMP2a proteins indicated that autophagy was inhibited in bMECs at that time. Therefore, we confirmed that infection induced autophagy in bMECs the HIF-1α and AMPKα/ULK1 pathway, with involvement of lysosome-associated protein Rab7 and LAMP2a.
牛疱疹病毒 2 型是一种高传染性病原体，可引起牛乳腺炎，导致受感染奶牛提前淘汰和严重的经济损失。牛疱疹病毒 2 型感染会导致奶牛乳腺上皮细胞（bMEC）发生氧化应激和细胞凋亡；然而，其诱导自噬的机制尚不清楚。因此，本研究通过 Western blot 和激光共聚焦扫描显微镜分析了 bMEC 中牛疱疹病毒 2 型诱导的自噬流。Western blot 定量检测了 HIF-1α 和 AMPKα/ULK1 通路中的蛋白质表达水平，包括 HIF-1α、AMPKα、p-AMPKα、ULK1、p-ULK1、mTOR 和 p-mTOR，以及自噬相关基因 SQSTM1/p62、Atg5、Beclin1 和 LC3II/LC3I 的表达。透射电子显微镜和激光共聚焦扫描显微镜分别检测到 bMEC 中自噬体和 LC3 斑点。此外，Western blot 和激光共聚焦扫描显微镜测量了溶酶体相关蛋白 Rab7 和 LAMP2a 以及溶酶体活性。牛疱疹病毒 2 型感染诱导 bMEC 中自噬体和 LC3 斑点形成，电镜和激光共聚焦显微镜观察到 6 hpi 时 LC3II/LC3I 蛋白表达增加，SQSTM1/p62 蛋白表达减少。此外，牛疱疹病毒 2 型感染上调 HIF-1α 和 AMPKα/ULK1 通路蛋白表达，增加 LC3II/LC3I 比值，表明 bMEC 中自噬被激活。然而，AMPKα 或 ULK1 缺失降低了 LC3II/LC3I 的表达水平和 LC3 斑点数量，表明 bMEC 中的自噬受到抑制。此外，HIF-1α 缺失降低了 AMPKα 和 ULK1 的蛋白表达以及 LC3 斑点数量，bMEC 中的牛疱疹病毒 2 型诱导的自噬也受到抑制。在 6 hpi 时，溶酶体相关蛋白 Rab7 减少而 LAMP2a 增加，表明自噬正常。相比之下，在 12 hpi 时，Rab7 和 LAMP2a 蛋白的表达表明此时 bMEC 中的自噬受到抑制。因此，我们证实了牛疱疹病毒 2 型感染通过 HIF-1α 和 AMPKα/ULK1 通路诱导 bMEC 中的自噬，涉及溶酶体相关蛋白 Rab7 和 LAMP2a。
