| Dairy cows are stimulated by a variety of stressors during the peripartum period,which can cause elevated cortisol and metabolic disorders.The decreased disease resistance increases the incidence of postpartum uterine diseases(such as metritis and endometritis)in dairy cows.Oxidative stress is an important part of the occurrence and development of diseases,which can cause changes in endometrial morphology and function.Escherichia coli(E.coli)is a common pathogen causing uterine infection.Lipopolysaccharide(LPS),its pathogenic component,can induce inflammatory response and oxidative stress in endometrial cells,eventually leading to endometrial injury.Selenium(Se)has an antioxidant effect,and appropriate supplementation of Se during the perinatal period can enhance the resistance to disease in dairy cows.The aim of this study is to investigate the effects of cortisol(COR)on oxidative stress in bovine endometrial epithelial cells(BEEC)of dairy cows and whether selenium protects cells from oxidative damage under the background of high cortisol,so as to provide a theoretical basis for the prevention and control of stress intrauterine bacterial infection in dairy cows.(1)Effect of COR on LPS-induced oxidative stress response in BEECIn this study,first,the effects of three concentrations of COR(5,15,30 ng/mL)alone on the oxidative stress response ability of BEEC were observed.Then,1 μg/mL LPS was used to establish an in vitro model of BEEC injury,and three concentrations of cortisol were added together for 90 min.The oxidative stress injury of BEEC induced by LPS under high cortisol was observed.The signaling pathway protein expression of Nuclear factor erythroid-2 related factor 2(Nrf2),Kelch-like ECH-associated protein 1(Keapl),Heme oxygenase-1(HO-1),NADPH quinone oxidoreductase 1(NQO1)and other oxidative damage related indicators were detected.The results showed that the levels of reactive oxygen species(ROS)and malondialdehyde(MDA)decreased(P<0.05),and the level of lactate dehydrogenase(LDH)did not change(P>0.05).COR of 15 or 30 ng/mL upregulated(P<0.01)the activities of catalase(CAT),glutathione peroxidase(GSH-Px)and thioredoxin reductase(TrxR).COR of 15 ng/mL promoted(P<0.01)the activities of superoxide dismutase(SOD)and total antioxidant capacity(T-AOC),the protein levels of Nrf2,HO-1 and NQO1,and decreased(P<0.05)the protein level of Keapl.The effect of 5 ng/mL COR was relatively weak.The production of ROS,MDA and LDH increased(P<0.01)in endometrial epithelial cells stimulated by 1 μg/mL LPS.The activities of CAT(P<0.01),GSH-Px(P<0.05),TrxR(P<0.05)and T-AOC(P<0.01)were decreased,but SOD had no significant change(P>0.05).Compared with LPS group,addition of 15 or 30 ng/mL COR reduced(P<0.01)the levels of ROS(P<0.01),MDA(P<0.01)and LDH(P<0.05)in BEEC.The activities of CAT,SOD,GSH-Px and TrxR and the level of T-AOC were significantly increased(P<0.01).LPS down-regulated the protein expression of Nrf2,NQO-1 and HO-1(P<0.05),and upregulated the protein expression of Keapl(P<0.05).In cells co-treated with LPS+COR(15 or 30 ng/mL),the protein levels of Nrf2,HO-1 and NQO1 were increased(P<0.05),and the protein levels of Keapl were decreased(P<0.05),and the changes in Nrf2,HO-1 and NQO1 were consistent with the changes in antioxidant enzymes.Addition of 5 ng/mL COR had no significant effect on LPS-induced injury of BEEC.(2)Protective effect of selenium on oxidative stress injury of BEECIn this study,firstly,the effects of 1,2,and 4 μM selenium on the oxidative stress response of BEEC were detected,and then the effect of high concentration of selenium on the oxidative stress response of BEEC under high cortisol level was observed.LPS was used to construct a BEEC injury model,and three concentrations of selenium were added to observe the protective effect of selenium on LPS-induced oxidative damage of BEEC.The protein expressions of Nrf2,Keap1,HO-1 and NQO1,and Nrf2 protein translocation into nucleus were detected after 90 min of stimulation,and the changes of other indicators related to oxidative damage were detected at 12 h.The results showed that the activities of CAT and SOD increased in BEEC treated with 1,2 or 4 μM Na2SeO3 alone(P<0.01).The level of T-AOC and the activities of GSH-Px and TrxR increased(P<0.01),whereas the levels of MDA and LDH were not affected(P>0.05).Na2SeO3 of 2 or 4 μM caused the decreased ROS level(P<0.01)and the increased Nrf2,HO-1 and NQO1 protein levels(P<0.01).Na2SeO3 of 1 μM increased the protein expression of Nrf2 and NQO1,but had no significant effect on Keapl and HO-1(P>0.05).After treatment with high concentrations of Na2SeO3 and high levels of COR,the activities of CAT,SOD,GSH-Px and TrxR,T-AOC levels and Nrf2,HO-1 and NQO1 protein levels in BEEC were significantly increased(P<0.01).The levels of Keapl protein and ROS decreased(P<0.01),but the levels of MDA and LDH were not significantly changed(P>0.05).On the basis of LPS and COR stimulation,the levels of T-AOC,CAT and SOD in BEEC did not change significantly after 1 μM Na2SeO3 treatment(P>0.05),but increased(P>0.05)after 2 or 4 μM Na2SeO3 treatment.The levels of T-AOC,CAT and SOD were significantly increased(P<0.01).Compared with LPS and COR groups,the activities of GSH-Px and TRXR in selenium group increased(P<0.01),and showed an upward trend with the increase of Na2SeO3 concentration.The protein expression of Nrf2,HO-1 and NQO1 increased(P<0.01),and the protein expression of Keapl decreased(P<0.05).In conclusion,cortisol increased the antioxidant capacity of BEEC and reduced the LPSinduced oxidative stress response of BEEC.Na2SeO3 of 1,2 or 4 μM increased the antioxidant capacity of BEEC treated with high cortisol and LPS,activated Nrf2 signaling pathway,increased the level of cellular antioxidant indicators,and reduced the related indicators of BEEC oxidative response.This antioxidant capacity of selenium is not affected by LPS and 30 ng/mL COR. |
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