The Mechanism Of Autophagy In Regulating Oxidative Stress In Mammary Epithelial Cells In Dairy Cows With Ketosis

Ketosis is a common metabolic disease in high-yielding dairy cows.During the perinatal period,the situation which refer to the insufficient energy intake in the diet and the increased energy demand caused by fetal development,parturition and lactation results in negative energy balance(NEB).NEB leads to massive fat mobilization and increased concentration of free fatty acids(FFA)in blood and the latter are incompletely oxidized in the liver to produce ketone bodies(mainly β-hydroxybutyric acid,BHB),which utimately induced ketosis..There exists systemic oxidative stress in ketotic cows,but prolonged oxidative stress could cause mammary injury,reduced milk production and milk quality.However,it is unclear whether high concentrations of FFA in blood of ketotic cows can induce oxidative stress in bovine mammary epithelial cells(BMEC).Autophagy is the main intracellular degradation system that eliminates excess ROS,oxidized biomolecules such as DNA,proteins,and lipids,and impaired cellular organelles in the presence of oxidative stress to maintain intracellular metabolic homeostasis.Therefore,the aim of this study was to investigate the effects of high concentrations of FFA in the blood of ketosis cows on the autophagy and oxidative stress in BMEC,and the mechanisms of autophagy modulation of oxidative stress in BMEC.In this study,we selected healthy cows(n = 15,BHB < 0.6 m M)and clinically ketotic cows(n = 15,BHB > 3.0 m M)and collected blood,milk and mammary tissue samples from day 3 to 15(mean = 7 days)of lactation.The blood composition,oxidative indicators and antioxidant indicators were measured in healthy and clinically ketotic cows;the milk composition was analyzed;and the expression levels of autophagy-related proteins in mammary tissue were measured.Compared with healthy cows,serum concentrations of FFA,BHB and malondialdehyde(MDA)were greater in clinically ketotic cows,but milk production,milk protein,activities of superoxide dismutase(SOD),catalase(CAT),and glutathione peroxidase(GSH-Px)were lower.The results indicate that clinical ketosis cows exhibited reduced lactation capacity and systemic oxidative stress.Compared to healthy cows,mammary autophagy levels were impaired in clinical ketosis cows,as manifested by lower m RNA and protein levels of the autophagy-related genes ATG5,ATG7 and LC3-II(MAP1LC3)and higher m RNA and protein levels of p62(SQSTM1)than those in healthy cows.For in vitro study bovine mammary epithelial cells(BMEC)isolated from healthy cows were treated with 0 m M,0.3 m M,0.6 m M or 1.2 m M FFA for 24 h.Furthermore,BMEC were pre-treated with 100 n M rapamycin,an autophagy activator,for 4 h or 50 m M 3-methyladenine(3-MA),an autophagy inhibitor,for 1 h,followed by treatment with or without FFA(1.2 m M)for another 24 h to measure oxidative indicators and antioxidant indicators and the abundance of autophagy-related protein.FFA treatment increased the content of MDA and ROS,but decreased the activities of SOD,CAT and GSH-Px.Compared with 0 m M-FFA group,abundance of ATG5,ATG7,LC3-II was greater,but p62 lower in the 0.6 m M FFA-treated cells.However,the group treated with high concentration of FFA(1.2 m M)showed decreased protein levels of ATG5,ATG7 and LC3-II and increased protein levels of p62.Therefore,the results showed that high concentrations of FFA(1.2 m M)inhibited the level of autophagy in BMEC,while low concentrations of FFA(0.6 m M)induced autophagy.And treatment with Rapa attenuated the oxidative stress induced by 1.2 m M FFA,while 3-MA exacerbated it.The results demonstrated that autophagy could regulate the level of oxidative stress in BMEC.Autophagy is a mechanism by which BMEC counteracts FFA-induced stress.This study reveals that high levels of FFA in cows with clinical ketosis is an important factor contributing to oxidative stress in mammary glands and that autophagy can be an effective way to regulate oxidative stress in mammary glands of cows.Thus,this study provides a theoretical basis and a novel drug target for the prevention and treatment of oxidative damage in mammary glands of cows with ketosis and mastitis.