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

doi:10.11843/j.issn.0366-6964.2024.09.014

摘要/Abstract

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、10 μg·mL^-1)、不同浓度的槲皮素组(0、5、10、20 μmol·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、10 μg·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(5 μmol·L^-1)、高浓度LTA+中浓度槲皮素组(10 μmol·L^-1)、高浓度LTA+高浓度槲皮素组(20 μmol·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(50 μmol·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10 μg·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。

关键词: 槲皮素, 奶牛乳腺上皮细胞, 脂磷壁酸, 自噬, 紧密连接

Abstract:

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, 10 μg·mL^-1), different concentrations of quercetin group (0, 5, 10, 20 μmol·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 10 μg·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 (5 μmol·L^-1), a high concentration of LTA+medium concentration of quercetin group (10 μmol·L^-1), and a high concentration of LTA+high concentration of quercetin group (20 μmol·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 (50 μmol·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 10 μg·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 5-20 μmol·L^-1 quercetin had no significant effect on MAC-T viability (P>0.05), and the optimal action concentration was further screened by a follow-up experiment on the basis of CCK-8 results. The expression of tight junction proteins of MAC-T was reduced and the level of autophagy was elevated after LTA induction; the expression of tight junction proteins of the cells was elevated and the level of autophagy was reduced after the action of quercetin on MAC-T, the expression of tight junction proteins was elevated and the autophagy level was reduced after CQ treatment. LTA can lead to over-activation of MAC-T autophagy and disruption of tight junction function, whereas quercetin can restore tight junction function by inhibiting autophagy. The results of this study provide new research ideas and theoretical basis for the treatment of mastitis in dairy cows.

Key words: quercetin, mammary alveolar cells-large T antigen, lipoteichoic acid, autophagy, tight junction

中图分类号:

S823.91

李相辰, 王林楠, 于正青, 张莉, 杨晨晨, 宋亮丽. 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[J]. 畜牧兽医学报, 2024, 55(9): 3887-3896.

Xiangchen LI, Linnan WANG, Zhengqing YU, Li ZHANG, Chenchen YANG, Liangli SONG. Quercetin Inhibits Autophagy to Restore LTA-induced Tight Junction Function in Mammary Alveolar Cells-large T Antigen[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(9): 3887-3896.

图/表 4

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