Liu Chang, Zhang Daoliang, Ding Hongyan, Wang Xichun, Feng Shibin, Li Yu, Zhao Chang
College of Veterinary Medicine, Anhui Agricultural University, Hefei, 230036, China; Anhui Provincial Animal Epidemic Disease Prevention and Control Center Laboratory, Hefei, 230091, China.
College of Veterinary Medicine, Anhui Agricultural University, Hefei, 230036, China; Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei, 230031, China.
J Dairy Sci. 2025 Sep;108(9):9967-9983. doi: 10.3168/jds.2024-26085. Epub 2025 Jul 18.
During clinical and subclinical Escherichia coli mastitis, bovine mammary epithelial cells (BMEC) are stimulated by LPS, leading to cellular inflammatory response, oxidative stress, and autophagy. This study hypothesized that rutin might mitigate these damages in BMEC via modulation of specific signaling pathways. To simulate mammary gland inflammation, different concentrations of LPS were used to stimulate BMEC. Different concentrations of rutin were used to pretreat the BMEC. The results demonstrated that LPS stimulated the NLR family pyrin domain containing 3 (NLRP3) inflammasome and its downstream effector molecules to produce an inflammatory response. Lipopolysaccharide caused a decrease in antioxidant enzymes and the total antioxidant capacity, an increase in malondialdehyde (MDA) levels, and a decrease in silent mating type information regulation 2 homolog-1 (SIRT1) levels. Treatment with LPS altered the morphological structure of the cells, converted the autophagy marker microtubule-associated protein light chain 3 (LC3)-I into LC3-II, and the increased the level of Beclin-1. Rutin increased silent mating type information regulation 2 homolog-1 (SIRT1) activity, reduced reactive oxygen species (ROS) generation, and decreased specific oxidative stress markers MDA. Additionally, rutin enhanced antioxidant enzyme activities including total antioxidant capacity, superoxide dismutase, catalase, and glutathione peroxidase. Rutin also inhibited NLRP3 inflammasome activation, which is associated with ROS production and oxidative stress. The mechanism of the protective effect of rutin on BMEC was investigated using the SIRT1 specific inhibitor EX-527. EX-527 considerably weakened the regulating function of rutin and aggravated the LPS-induced damage to BMEC. Rutin inhibited NLRP3 inflammasome activation and the downstream effector molecule gasdermin D (GSDMD) through SIRT1, thus alleviating excessive autophagy and reducing the inflammatory damage of cells. This study confirms that rutin reduces LPS-induced inflammation, autophagy, and oxidative stress in BMEC through the SIRT1/NLRP3 pathway. These findings provide new insights into addressing mastitis in dairy cows and offer promising clinical applications for future therapeutic interventions.
在临床和亚临床大肠杆菌性乳腺炎期间，牛乳腺上皮细胞（BMEC）受到脂多糖（LPS）刺激，导致细胞发生炎症反应、氧化应激和自噬。本研究假设芦丁可能通过调节特定信号通路减轻BMEC中的这些损伤。为模拟乳腺炎症，使用不同浓度的LPS刺激BMEC。使用不同浓度的芦丁对BMEC进行预处理。结果表明，LPS刺激含NLR家族pyrin结构域蛋白3（NLRP3）炎性小体及其下游效应分子产生炎症反应。脂多糖导致抗氧化酶和总抗氧化能力下降，丙二醛（MDA）水平升高，沉默信息调节因子2同源物1（SIRT1）水平降低。LPS处理改变了细胞的形态结构，将自噬标志物微管相关蛋白轻链3（LC3）-I转化为LC3-II，并提高了Beclin-1的水平。芦丁增加了沉默信息调节因子2同源物1（SIRT1）活性，减少了活性氧（ROS）生成，并降低了特定氧化应激标志物MDA。此外，芦丁增强了包括总抗氧化能力、超氧化物歧化酶、过氧化氢酶和谷胱甘肽过氧化物酶在内的抗氧化酶活性。芦丁还抑制了与ROS产生和氧化应激相关的NLRP3炎性小体激活。使用SIRT1特异性抑制剂EX-527研究了芦丁对BMEC保护作用的机制。EX-527大大削弱了芦丁的调节功能，并加重了LPS诱导的对BMEC的损伤。芦丁通过SIRT1抑制NLRP3炎性小体激活及其下游效应分子gasdermin D（GSDMD），从而减轻过度自噬并减少细胞的炎症损伤。本研究证实芦丁通过SIRT1/NLRP3途径减轻LPS诱导的BMEC炎症、自噬和氧化应激。这些发现为解决奶牛乳腺炎提供了新的见解，并为未来的治疗干预提供了有前景的临床应用。
