蜂胶对细菌脂多糖刺激下奶牛乳腺上皮细胞炎症相关基因mRNA转录水平和紧密连接蛋白的影响  

Effects of Propolis on Transcript Levels of Inflammation-related Genes and Tight Junction Proteins of Bovine Mammary Epithelial Cells Stimulated by Bacterial Lipopolysaccharide

作　　者：欧爱群 王凯[2] 吴黎明[2] 李江红 彭文君[2] OU Aiqun;WANG Kai;WU Liming;LI Jianghong;PENG Wenjun(College of Animal Science(College of Bee Science),Fujian Agriculture and Forestry University,Fuzhou 350002,China;Institute of Apicultural Research,Chinese Academy of Agricultural Sciences,Beijing 100093,China)

机构地区：[1]福建农林大学动物科学学院(蜂学学院),福州350002 [2]中国农业科学院蜜蜂研究所,北京100093

出　　处：《畜牧兽医学报》2020年第5期1149-1157,共9页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家自然科学基金青年科学基金(31702287)。

摘　　要：本试验旨在研究中国蜂胶乙醇提取物(ethanol extract of Chinese propolis,EECP)对细菌脂多糖(lipopolysaccharide, LPS)刺激下体外培养奶牛乳腺上皮细胞炎症相关基因mRNA转录水平和紧密连接渗透性的影响。EECP中总酚酸和总黄酮含量测定采用福林酚法和硝酸铝法,并建立LPS诱导奶牛乳腺上皮细胞(bovine mammary epithelial cells,MAC-T)炎症模型,采用CCK-8法测定EECP对MAC-T相对增殖率的影响,利用实时荧光定量PCR(RT-qPCR)评估EECP对LPS诱导的MAC-T细胞炎症相关因子(IL-6、IL-8、TNF-α和IL-1β)相对mRNA转录水平;以及对紧密连接蛋白(occludin、ZO-1)相对mRNA转录水平进行检测,并进一步利用免疫荧光技术对紧密连接膜蛋白进行定位,确定EECP对LPS诱导MAC-T细胞炎症紧密连接渗透性的影响。结果显示:EECP中总酚酸含量为106.35 mg没食子酸当量(GAE)·g^-1、总黄酮含量为320.85 mg芦丁当量(RE)·g^-1;CCK-8结果显示EECP的安全浓度为0~15μg·mL^-1,并可有效提高LPS刺激下MAC-T的活力;LPS刺激显著增加了细胞炎症相关因子IL-6、IL-8、TNF-α和IL-1βmRNA的转录量(P<0.001);但2.5~15.0μg·mL^-1 EECP预处理显著降低了IL-6、IL-8、TNF-α和IL-1βmRNA的转录量;与此类似,LPS刺激显著抑制了紧密连接蛋白基因(occludin、ZO-1)mRNA的转录量(P<0.01),而EECP预处理后紧密连接蛋白基因(occludin和ZO-1) mRNA的转录量显著增加(P<0.05);免疫荧光染色试验也证实EECP能通过上调紧密连接蛋白(occludin、ZO-1)的表达,缓解LPS诱导的乳腺上皮细胞屏障功能紊乱。该结果证实,EECP对细菌脂多糖诱导奶牛乳腺上皮细胞炎症具有良好的保护作用,这为利用中国蜂胶预防奶牛乳腺炎提供了试验基础。This study aimed to investigate the effects of ethanol extract of Chinese propolis(EECP)on transcript levels of inflammation-related genes and tight junctional permeability in bovine mammary epithelial cells stimulated by bacterial endotoxin(lipopolysaccharide,LPS).The total content of phenolic acids and flavonoids in the EECP was determined by Folin-phenol method and ALNO 3 colorimetry,respectively.Moreover,the inflammatory model of bovine mammary epithelial cells(MAC-T)was induced by bacterial lipopolysaccharide and the effect of EECP on the relative cell proliferation rate was detected via CCK-8 method.Real-time quantitative PCR(RT-qPCR)was used to evaluate LPS-induced bovine mammary epithelial cells inflammatory factors(IL-6,IL-8,TNF-αand IL-1β)relative mRNA transcription levels.To determine the effect of EECP on LPS-induced MAC-T cell,inflammation tight junction permeability.We used RT-qPCR to detect the relative mRNA transcription levels of tight junction proteins(occludin,ZO-1),and further used immunofluorescence to localize tight junction membrane proteins.The results showed that the content of total phenolic acids and total flavonoids in EECP were 106.35 mg GAE·g^-1 and 320.85 mg RE·g^-1,respectively.CCK-8 results showed that the safe concentration of EECP was 0-15μg·mL^-1,and can effectively increase the viability of MAC-T cells under LPS stimulation.LPS stimulation significantly increased the mRNA transcription levels of cellular inflammation-related factors IL-6,IL-8,TNF-αand IL-1β(P<0.001).However,the mRNA transcription of IL-6,IL-8,TNF-αand IL-1βwere significantly reduced under pretreatment with 2.5-15.0μg·mL^-1 EECP.Similarly,the LPS model group significantly inhibited(P<0.01)the mRNA transcription of tight junction proteins(ZO-1 and occludin),while the mRNA transcription of tight junction proteins was increased by EECP pretreatment(P<0.05).Immunofluorescence staining experiments also confirmed that EECP could alleviate LPS-induced mammary epithelial barrier dysfunction by up-regulating

关 键 词：中国蜂胶 抗炎 紧密连接 奶牛乳腺上皮细胞 

分 类 号：S857.26[农业科学—临床兽医学] S896.6[农业科学—兽医学]