| Mastitis is a common animal disease in dairy farming,also named as cow mastitis.Physical,chemical,pathogenic microorganisms and other factors can lead to the occurrence of mastitis,in which intra-mammary infection of pathogenic microorganisms is the most important cause.In the treatment of mastitis,people usually focus on the use of antibiotics to kill pathogenic microorganisms and often ignore the importance of inhibiting the inflammatory response.Excessive inflammatory response is an important cause of damage of mammary gland.Studies have shown that the combination of antibiotics and anti-inflammatory drugs effectively kills pathogenic microorganisms,controls the inflammatory response and greatly shortens the treatment cycle.Therefore,in the research work of mastitis therapeutic drugs,we should not only pay attention to the development of new antibiotics,but also emphasize the search for new and effective anti-inflammatory drugs.Myricetin,known as bayberry bark and bayberry flavonoids,is a flavonol compound extracted from the bark of the bayberry plant.It has been found that myricetin has anti-inflammatory effects,but whether myricetin plays an anti-inflammatory role in mastitis has not been reported.Therefore,this experiment established Lipopolysaccharide(LPS)-induced mice mastitis model and mammary epithelial cells inflammatory response model,systematically studied the effects of myricetin on LPS-induced mice mastitis model and its mechanism.Firstly,we studied the effect of myricetin on LPS-induced mastitis in mice.We successfully constructed mice mastitis model by intra-mammary perfusion of LPS,and intraperitoneal injection of myricetin at the corresponding time points.Mammary gland samples were collected 24 hours after LPS injection into the mammary duct.One part of the mammary gland was fixed,sliced and Hematoxylin-eosin(H&E)stained.The damage of the mammary gland and the infiltration of inflammatory cells in the mammary gland were observed by microscope and another part of the mammary gland was ground,the Myeloperoxidase(MPO)activity was detected by the MPO activity detection kit,the protein levels of the pro-inflammatory cytokines TNF-α,IL-1β and IL-6 were detected by ELISA kits.The results showed that myricetin can significantly attenuated the damage of mammary gland caused by LPS,decreased the activity of MPO and the concentration of pro-inflammatory cytokines.To further clarifying the anti-mammary effect of myricetin,we applied the LPS-induced mice mammary epithelial cell line(mMECs)inflammatory response model to study the effect of myricetin on the inflammatory response of mammary epithelial cells in vitro,We found that myricetin can significantly reduced LPS-induced the gene levels of TNF-α,IL-1β,IL-6 as well as increased levels of Inducible nitric oxide synthase(iNOS)and Cyclooxygenase-2(COX-2)proteins in mMECs.These results indicated that myricetin has an anti-inflammatory effect in the LPS-induced mice mastitis model.In order to clarify the potential anti-inflammatory mechanism of myricetin in LPS-induced mice mastitis model,we examined the effect of myricetin on the activation of key inflammatory signaling molecules in inflammation in LPS-induced mice mastitis model and mammary epithelial inflammatory response model.Western blot results of mammary gland and mammary epithelial cells showed that myricetin can inhibit AKT/IKK/NF-κB signaling pathways and inhibit the phosphorylation of P38 and ERK.These results indicated that myricetin inhibits the production of pro-inflammatory mediators by inhibiting the phosphorylation of AKT/IKK/NF-κB signaling pathways and P38 and ERK proteins,thereby inhibiting the inflammatory response of mammary gland.Blood-milk barrier integrity was destroyed in mastitis,and repairing blood-milk barrier can improve the inflammatory state of mammary gland.Further studies have found that myricetin can repair the integrity of blood-milk barrier by increasing the levels of tight junction-associated proteins ZO-1,Occludin and Claudin-3 in the LPS-induced mice mastitis model.These results indicated that myricetin can also relieve mammary inflammation by repairing the integrity of the blood-milk barrier.In summary,myricetin exerts protective effect in the LPS-induced mastitis model.Its protective mechanism was on the one hand by inhibiting the AKT/IKK/NF-κB signaling pathway and the phosphorylation levels of P38 and ERK proteins;on the other hand,by maintaining the integrity of blood-milk barrier structure and function.Therefore,myricetin has the potential to be an effective anti-inflammatory drug for the treatment of mastitis. |
