Zhang Kai, Zhang Ruizhen, Zhang Yuanyuan, Zhang Min, Su Hong, Zhao Feifei, Wang Daqing, Cao Guifang, Zhang Yong
College of Veterinary Medicine, Inner Mongolia Agricultural University, Hohhot 010010, China.
Animal Embryo and Developmental Engineering Key Laboratory of Higher Education, Institutions of Inner Mongolia Autonomous Region, Hohhot 010011, China.
Life (Basel). 2025 Jan 9;15(1):69. doi: 10.3390/life15010069.
Lactoferrin (LF), a member of the transferrin family, is widely present in mammalian milk and other secretions, exhibiting anti-inflammatory, antibacterial, and anti-infective properties. Although the biological functions of LF have been extensively studied, there are few reports on its effects and molecular mechanisms concerning bovine mastitis caused by bacterial infection. This study used bovine mammary epithelial cells (BMECs) cultured in vitro as the research model. An inflammatory injury model was established by stimulating BMECs with LPS to investigate whether LF at different concentrations (10, 50, 100, and 200 μg·mL) could inhibit the inflammatory response before and after the onset of inflammation. The expression of inflammatory cytokines IL-1β, IL-6, IL-8, and TNF-α at both the gene and protein levels was detected using RT-qPCR and ELISA. Western blotting was employed to evaluate the phosphorylation levels in the inflammatory signaling pathways MAPK/P38/ERK and NF-κB/P65, while RT-qPCR was used to examine the impact on TLR4 receptor gene expression. The results display that pretreatment with LF prior to LPS-induced inflammation in BMECs reduced the expression of inflammatory cytokines IL-1β, IL-6, IL-8, and TNF-α at both the gene and protein levels ( < 0.05). LF also inhibited the phosphorylation of NF-κB/P65 and MAPK/P38/ERK signaling pathways and downregulated TLR4 receptor gene expression ( < 0.05). However, when LF was added after the onset of LPS-induced inflammation, inflammatory cytokine expression and phosphorylation levels in the NF-κB/P65 and MAPK/P38/ERK pathways remained elevated, along with high expression of the TLR4 receptor gene ( < 0.05). These findings show that LF can antagonize LPS-induced inflammatory responses in BMECs and reduce cytokine expression, exhibiting anti-inflammatory effects when administered before inflammation. Conversely, when LF is added post-inflammation, it appears to enhance cytokine expression, potentially promoting the recruitment of more cells or factors to resolve inflammation rapidly. Both effects are mediated through the TLR4 receptor and the NF-κB/P65 and MAPK/P38/ERK signaling pathways.
乳铁蛋白（LF）是转铁蛋白家族的成员，广泛存在于哺乳动物乳汁及其他分泌物中，具有抗炎、抗菌和抗感染特性。尽管对LF的生物学功能已进行了广泛研究，但关于其对细菌感染引起的牛乳腺炎的影响及分子机制的报道较少。本研究以体外培养的牛乳腺上皮细胞（BMECs）作为研究模型。通过用脂多糖（LPS）刺激BMECs建立炎症损伤模型，以研究不同浓度（10、50、100和200μg·mL）的LF在炎症发生前后是否能抑制炎症反应。使用RT-qPCR和ELISA检测炎症细胞因子IL-1β、IL-6、IL-8和TNF-α在基因和蛋白水平的表达。采用蛋白质印迹法评估炎症信号通路MAPK/P38/ERK和NF-κB/P65中的磷酸化水平，同时用RT-qPCR检测对TLR4受体基因表达的影响。结果显示，在LPS诱导BMECs炎症之前用LF预处理可降低炎症细胞因子IL-1β、IL-6、IL-8和TNF-α在基因和蛋白水平的表达（<0.05）。LF还抑制了NF-κB/P65和MAPK/P38/ERK信号通路的磷酸化，并下调了TLR4受体基因的表达（<0.05）。然而，当在LPS诱导的炎症发生后添加LF时，NF-κB/P65和MAPK/P38/ERK通路中的炎症细胞因子表达和磷酸化水平仍然升高，同时TLR4受体基因高表达（<0.05）。这些发现表明，LF可拮抗LPS诱导的BMECs炎症反应并降低细胞因子表达，在炎症发生前给药具有抗炎作用。相反，当在炎症后添加LF时，它似乎会增强细胞因子表达，可能促进更多细胞或因子的募集以快速解决炎症。这两种作用均通过TLR4受体以及NF-κB/P65和MAPK/P38/ERK信号通路介导
