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