黄花蒿醇提物对LPS诱导奶牛乳腺细胞损伤的干预机制

Mastitis is recognised as one of the most important diseases affecting dairy cattle worldwide, It has adverse effects on the economics of milk production by reducing the quantity and quality of milk. Mastitis can be caused by a large number of bacterial species, but the most prevalent pathogens isolated from clinical cases include Staphylococcus aureus, Escherichia coli and Streptococcus agalactiae. The previous studies showed that the isolation rate of Escherichia coli was the highest in raw milk, and the Artemisia annua ethanol extracts could reduce the number of somatic cells in raw milk. Therefore, Artemisia annua distributed in the desert grassland more in Inner Mongolia and the bovine mammary epithelial cells (BMECs) will be used as the research object in this study, and the LPS-injured BMECs modle will be preliminarily established. On the basis, The effects of Artemisia annua ethanol extracts will be studied on cell ultrastructure, cell apoptosis, tight junctional protein Occludin and ZO-1 in BMECs induced by LPS by scanning electron microscopy (SEM), transmission electron microscope (TEM), flow cytometry (FCM), RT-PCR and the Western Blot. Furthermore, scavenger receptor CD36 expression will be manipulated to explore the effects of CD36 on NF-κB IκBa, NF-κB p65, MAPKs p38, MAPKs JNK, MAPKs ERK1/2, and IL-8, IL-1β, IL-6, TNF-a, and in order to reveal the effects and mechanisms of Artemisia annua ethanol extracts against inflammation injury of BMECs induced by LPS, which will provide theoretical basis for the prevention and control of mastitis in production.

奶牛乳房炎是导致奶牛养殖业经济损失常见病之一，引起该病主要病原微生物包括金黄色葡萄球菌、大肠杆菌及无乳链球菌等。前期研究表明，生鲜乳中大肠杆菌分离率最高，且黄花蒿醇提物能降低乳中体细胞。鉴于此，本研究选取内蒙古荒漠化草原分布较多的黄花蒿，以奶牛乳腺上皮细胞为研究对象，通过大肠杆菌主要致病毒力因子LPS诱导建立细胞损伤模型；在此基础上，采用扫描电镜、透射电镜、流式细胞、RT-PCR及Western Blot等技术研究黄花蒿醇提物对LPS诱导损伤奶牛乳腺上皮细胞超微结构、细胞凋亡及紧密连接蛋白Occludin和ZO-1影响，进一步通过超表达或干扰CD36研究其对NF-κB信号通路IκBa、p65和MAPKs信号通路p38、JNK、ERK1/2及细胞因子IL-8、IL-1β、IL-6和TNF-a的影响，揭示黄花蒿醇提物对LPS诱导损伤奶牛乳腺细胞干预机制，为其在生产中防控奶牛乳房炎提供理论依据。

乳房炎是制约奶业发展最严重的疾病之一，不仅影响奶牛的生产性能，还导致抗生素残留和耐药性产生。由于常用的抗生素治疗存在诸多安全隐患，迫切需要寻找防治奶牛乳房炎的新方案。植物提取物具有丰富的生物多样性和巨大的药用潜力，能够作为抗生素替代物预防奶牛乳房炎。菊科植物黄花蒿广泛分布于内蒙古部分荒漠化草原地区，具有重要药用价值。课题组前期研究表明，生鲜牛乳中大肠杆菌分离率最高，且黄花蒿乙醇提取物（AAE）能降低乳中体细胞。因此本研究旨在探讨AAE对大肠杆菌性奶牛乳房炎的部分作用机理，明确AAE对LPS诱导损伤奶牛乳腺细胞（bMECs）的保护作用是否与抑制CD36介导的炎症信号通路有关。本试验以bMECs为研究对象，添加3，6，12μg/mL AAE处理bMECs 3h后，与10μg/mL脂多糖（LPS）共孵育12h后，采用扫描电镜、透射电镜、流式细胞术、转录组、RT-PCR、western blotting及基因干扰或过表达等技术，评估AAE对LPS诱导损伤bMECs的预保护作用及机制。结果表明，LPS能显著增加CD36 mRNA和蛋白质表达量，诱导NF-κB和MAPK信号通路关键基因表达和蛋白磷酸化，提高TNF-α、IL-1β和IL-6、IL-8 mRNA水平，进而导致bMECs结构及超微结构损伤，紧密连接蛋白（claudin-1、occludin 和ZO-1）表达减少，细胞活力降低。相反，AAE能以剂量依赖性方式显著抑制LPS刺激的bMECs中CD36的表达，减弱NF-κB和MAPK信号通路的活性，并下调炎症因子的水平，其中12μg/mL AAE 效果最为明显。综上可知，AAE能有效保护bMECs免受LPS诱导炎症损伤，其中CD36发挥了核心调控作用。本研究的开展对于寻找抗生素替代物、开发新型绿色饲料添加剂、降低奶牛乳房炎发病率、助力奶业振兴具有重要意义。