槲皮素抑制自噬恢复LTA诱导的奶牛乳腺上皮细胞紧密连接功能  

Quercetin Inhibits Autophagy to Restore LTA-induced Tight Junction Function in Mammary Alveolar Cells-large T Antigen

作　　者：李相辰 王林楠 于正青 张莉[1] 杨晨晨 宋亮丽 LI Xiangchen;WANG Linnan;YU Zhengqing;ZHANG Li;YANG Chenchen;SONG Liangli(College of Animal Science and Technology,Ningxia University,Yinchuan 750021,China)

机构地区：[1]宁夏大学动物科技学院,银川750021

出　　处：《畜牧兽医学报》2024年第9期3887-3896,共10页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：宁夏自然科学基金资助项目(2021AAC03040);宁夏重点研发(引才专项)资助(2020BEB04035)。

摘　　要：旨在探究槲皮素(quercetin)修复脂磷壁酸(lipoteichoic acid,LTA)诱导的奶牛乳腺上皮细胞(mammary alveolar cells-large T antigen,MAC-T)紧密连接损伤的作用机制。本试验以MAC-T为研究对象,设置不同浓度的LTA组(0、0.1、1、10μg·mL^(-1))、不同浓度的槲皮素组(0、5、10、20μmol·L^(-1))、空白组,检测不同浓度LTA和槲皮素对MAC-T细胞活力的影响。设置对照组(不做任何处理)、低、中、高浓度LTA组(0.1、1、10μg·mL^(-1)),筛选LTA最佳作用浓度;设置对照组(不做任何处理)、高浓度LTA+低浓度槲皮素(5μmol·L^(-1))、高浓度LTA+中浓度槲皮素组(10μmol·L^(-1))、高浓度LTA+高浓度槲皮素组(20μmol·L^(-1)),筛选槲皮素最佳作用浓度。在自噬与紧密连接关系的研究中,将细胞分为对照组(不做任何处理)、高浓度LTA组、高浓度LTA+中浓度槲皮素组、高浓度LTA+自噬抑制剂氯喹(chloroquine,CQ)组(50μmol·L^(-1))。每组均进行3次重复,槲皮素与氯喹均在LTA作用前加入,即使用槲皮素和氯喹分别孵育细胞3 h后,再加入10μg·mL^(-1)LTA继续培养12 h,通过CCK-8法检测不同浓度LTA和槲皮素对MAC-T活力的影响,利用Western blot和免疫荧光检测细胞紧密连接蛋白(Occludin、ZO-1、Claudin-1)和自噬相关蛋白(Beclin-1、LC3、P62)的表达水平。结果显示,中、高浓度LTA对MAC-T活力有显著影响,但细胞活性维持在90%以上,不同浓度槲皮素对MAC-T活力均无显著影响。此外,LTA诱导后的MAC-T中紧密连接蛋白表达降低,自噬水平升高;槲皮素作用MAC-T后,细胞紧密连接蛋白表达升高,自噬水平降低;自噬抑制剂CQ作用后,自噬水平被抑制,紧密连接蛋白表达升高。LTA可导致MAC-T自噬过度激活,破坏紧密连接功能,而槲皮素可通过抑制自噬恢复紧密连接功能。该研究结果为奶牛乳腺炎的治疗提供了新的研究思路和理论依据。The aim of this study was to investigate the mechanism of action of quercetin in repairing lipoteichoic acid(LTA)-induced tight junction damage in mammary alveolar cells-large T antigen(MAC-T)of dairy cows.MAC-T was used as the research object,and different concentrations of LTA group(0,0.1,1,10μg·mL^(-1)),different concentrations of quercetin group(0,5,10,20μmol·L^(-1)),and blank wells were set up to detect the effects of different concentrations of LTA and quercetin on the viability of MAC-T cells;A control group(without any treatment),low,medium and high LTA concentration groups(0.1,1 and 10μg·mL^(-1))were set up to screen for the optimal concentration of LTA action;A control group(without any treatment),a high concentration of LTA+low concentration of quercetin(5μmol·L^(-1)),a high concentration of LTA+medium concentration of quercetin group(10μmol·L^(-1)),and a high concentration of LTA+high concentration of quercetin group(20μmol·L^(-1))were set up to screen for the optimal action concentration of quercetin.In the study of the relationship between autophagy and tight junctions,the cells were divided into a control group(without any treatment),a high-concentration LTA group,a high-concentration LTA+medium-concentration quercetin group,and a high-concentration LTA+autophagy inhibitor chloroquine(CQ)group(50μmol·L^(-1)).Each group was subjected to 3 replications,and both quercetin and chloroquine were added before the action of LTA,the cells were incubated with quercetin and chloroquine for 3 h respectively,and then 10μg·mL^(-1)LTA was added to continue the incubation for 12 h.The effects of different concentrations of LTA and quercetin on the viability of MAC-T were detected by CCK-8 assay;the expression levels of cellular tight junction proteins(Occludin,ZO-1,Claudin-1)and autophagy-associated proteins(Beclin-1,LC3,P62)were detected by Western blot and immunofluorescence.The results showed that the MAC-T cell activity was maintained at 90%with the increase of LTA concentration,and 5-20μmol·

关 键 词：槲皮素 奶牛乳腺上皮细胞 脂磷壁酸 自噬 紧密连接 

分 类 号：S823.91[农业科学—畜牧学]