苦参碱干预金黄色葡萄球菌主要毒力因子诱导奶牛乳腺上皮细胞凋亡机制研究

Abstract: Staphylococcus aureus is an important pathogenic bacterium in dairy cow mastitis. Many virulent factors secreted from Staphylococcus aureus are account for the apoptosis of mammary epitheial cells, which could directly result in the reduction of the production of milk, increase of the somatic cell number in milk,and pathological changes such as flammation in the mammary glands. In this proposal,we will investigate the impacts of matrine on bovine mammary epitheial cells and the underlying mechanism of bovine mammary epitheial cells apoptosis induced by main virulent factors of Staphylococcus aureus. The following aims will be pursued: based on the isolation and identification of Staphylococcus aureus isolates caused cow mastitis, we will first identify the distribution and characterics of main virulent factors; interrogate the signaling pathways involved in the virulent factor induced apoptosis of mammary epitheial cells; explore the functional mechanism of matrine on the main virulent factors of Staphylococcus aureus; With the amplification of the main virulent factors, the influence of matrine on virulent genes transcriptional level would be analyzed by real-time PCR. and we will establish and utilize a mammary epithelial cell culture model to reveal the underpinning mechanism of matrine in apoptosis of bovine mammary epithelial cells induced by major virulent factors of Staphylococcus aureus, by investigating the interactions of protein, matrine and cells using a strategy of overexpressing the bacterial virulent factors. An accomplishment of this study will provide novel targets for the prevention and treatment of dairy cow mastitis, as well as valuable information for the employment of herbal medicines in treatment of this disease in veterinary clinic.

金黄色葡萄球菌是引发奶牛乳房炎的一种重要致病菌，其毒力因子可致奶牛乳腺上皮细胞凋亡，使得奶牛产奶量下降、体细胞数增加、乳腺内环境发生炎症等病理变化。本研究以苦参碱为受试药，研究苦参碱对奶牛乳腺上皮细胞的影响及其干预金黄色葡萄球菌毒力因子诱导乳腺上皮细胞的凋亡机制。在分离鉴定乳房炎金黄色葡萄球菌基础上，检测金黄色葡萄球菌毒力基因和分布特点；建立并利用乳腺上皮细胞培养体系，研究毒力因子对乳腺上皮细胞的凋亡途径；检测苦参碱对金黄色葡萄球菌及主要毒力因子作用特点和机理；扩增细菌主要毒力基因，应用实时定量PCR分析药物对毒力基因转录调控水平的影响；检测苦参碱和毒力因子对乳腺上皮细胞通路调控影响；通过过表达细菌主要毒力蛋白，研究蛋白、药物和乳腺上皮细胞之间的相互作用规律，从而揭示苦参碱干预细菌主要毒力因子对乳腺上皮细胞凋亡的作用和机理。为奶牛乳房炎的防治研究提供新靶点并为中草药防治疾病提供依据。

金黄色葡萄球菌是引发奶牛乳房炎的一种重要致病菌，其毒力因子可致奶牛乳腺上皮细胞凋亡，使得奶牛产奶量下降、体细胞数增加、乳腺内环境发生炎症等病理变化。本研究内容分为奶牛乳腺上皮细胞细胞株的建立、苦参碱等药物干预产α-溶血素金黄色葡萄球菌对奶牛乳腺上皮细胞的损伤作用和苦参碱等药物干预产PV-杀白细胞素金黄色葡萄球菌及重组PVL对奶牛乳腺上皮细胞的损伤作用。通过乳汁分离法获得了BMECs，以传代12代的BMECs作为试验细胞株。金黄色葡萄球菌对奶牛乳腺上皮细胞的粘附作用的最佳时间为2.5 h，最适pH值7.0~7.5之间，最适温度为37℃，这些生理条件有利于金黄色葡萄球菌感染乳腺组织。产α-溶血素、杀白细胞素金黄色葡萄球菌株与α-溶血素、杀白细胞素基因缺失株对奶牛乳腺上皮细胞均有损伤作用，但产毒素的金黄色葡萄球菌株损伤奶牛乳腺上皮细胞的程度大于毒素基因缺失株。苦参碱显著干预了金黄色葡萄球菌诱导奶牛乳腺上皮细胞的损伤作用。在使用浓度内的苦参碱能够显著下调α-溶血素相关基因表达从而抑制金黄色葡萄球菌分泌α-溶血素，但苦参碱不能使α-溶血素失去活性。苦参碱通过调控NF-κB信号通路干预金黄色葡萄球菌入侵奶牛乳腺上皮细胞。苦参碱能抑制金黄色葡萄球菌LukS-PV蛋白表达，并呈浓度依赖性，这种抑制机制发生在蛋白表达方面，对基因转录方面没有影响。金黄色葡萄球菌rPVL通过caspase依赖途径诱导奶牛乳腺上皮细胞凋亡，黄芩苷通过下调细胞凋亡内源性途径中active caspase-9蛋白的表达，而苦参碱通过下调内源性和外源性途径active caspase-3、active caspase-8、active caspase-9蛋白的表达来干预rPVL诱导奶牛乳腺上皮细胞凋亡，这种效果是非常明显的。研究结果揭示了苦参碱干预细菌主要毒力因子对乳腺上皮细胞凋亡的作用和机理，为中药防治奶牛乳房炎的应用提供了科学依据。