College of Veterinary Medicine, Jilin University, Changchun 130062, China.
Int Immunopharmacol. 2022 Sep;110:109009. doi: 10.1016/j.intimp.2022.109009. Epub 2022 Jul 8.
Neutrophil extracellular traps (NETs) are an important means by which the body fights against exogenous bacteria. However, studies have shown that excessive NETs release can damage other cells. Accumulating evidence has shown that butyric acid can alleviate the inflammatory response of cells. However, the effect of butyric acid on Staphylococcus aureus-induced NETs formation and its underlying mechanism are still unclear. In this study, western blotting, immunofluorescence and CCK-8 assays were used to examine the effect of NETs formation by sodium butyrate (NaB). The results showed that NaB suppressed the release of S. aureus-induced NETs formation, as indicated by decreases in the levels of DNA, histones, myeloperoxidase, and neutrophil elastase. S. aureus can induce autophagy, and autophagy plays a key role in the formation of NETs. Our data showed that NaB activated mammalian target of rapamycin (mTOR) and the kinases protein kinase B (AKT) and unc-51 like kinase 1 (ULK1) at Ser757 and inhibited AMP-activated protein kinase (AMPK). To explore whether NaB inhibited the formation of NETs by inhibiting autophagy, we added 3-methyladenine (autophagy inhibitor) (3-MA, 5 mM) to bovine neutrophils, and the results showed that 3-MA significantly inhibited NETs release. Furthermore, we found that NETs and their component histones exhibited significantly increased the cytotoxic effects on bovine mammary epithelial cells (BMECs), indicating that NETs and their component histones play a key role in BMEC damage. In conclusion, NaB can reduce the excessive formation of NETs by inhibiting autophagy, thus reducing the damaging effect of NETs on BMECs.
中性粒细胞胞外诱捕网（NETs）是机体抵御外源性细菌的重要手段。然而，研究表明，NETs 的过度释放会损害其他细胞。越来越多的证据表明，丁酸可以减轻细胞的炎症反应。然而，丁酸对金黄色葡萄球菌诱导的 NETs 形成的影响及其潜在机制尚不清楚。在本研究中，采用 Western blot、免疫荧光和 CCK-8 检测丁酸钠（NaB）对 NETs 形成的影响。结果表明，NaB 抑制了金黄色葡萄球菌诱导的 NETs 形成，表现为 DNA、组蛋白、髓过氧化物酶和中性粒细胞弹性蛋白酶水平降低。金黄色葡萄球菌可以诱导自噬，自噬在 NETs 的形成中起着关键作用。我们的数据表明，NaB 通过激活哺乳动物雷帕霉素靶蛋白（mTOR）和蛋白激酶 B（AKT）和 UNC-51 样激酶 1（ULK1）在丝氨酸 757 位的激酶以及抑制 AMP 激活的蛋白激酶（AMPK）来诱导自噬。为了探讨 NaB 是否通过抑制自噬来抑制 NETs 的形成，我们向牛中性粒细胞中添加了 3-甲基腺嘌呤（自噬抑制剂）（3-MA，5 mM），结果表明 3-MA 显著抑制了 NETs 的释放。此外，我们发现 NETs 及其成分组蛋白对牛乳腺上皮细胞（BMECs）的细胞毒性作用显著增加，表明 NETs 及其成分组蛋白在 BMEC 损伤中起着关键作用。总之，NaB 可以通过抑制自噬来减少 NETs 的过度形成，从而减少 NETs 对 BMECs 的损伤作用。
