Mechanism Of Selenium Attenuating Oxidative Stress In Bovine Mammary Epithelial Cells Through Modulation Of TrxR1/MAPK Signaling Pathway

Oxidative stress is inducing factors that cause dairy cow mastitis and reduction in milk quality and milk yield.For dairy cows in early lactation period and late perinatal period,selenium(Se)status and antioxidative status are not good,and it is very easy to induce oxidative stress,resulting in increased disease susceptibility.Bovine mammary epithelial cells(BMEC)are important sites for the synthesis and secretion of milk fat and milk protein,and the biosynthetic capacity of the cells determine the milk production capacity of the mammary glands.Therefore,investigation on the mechanism of Se attenuating oxidative stress in BMEC is significant for Se supplement in a scientifical and rational way in dairy industry,alleviation of oxidative stress in dairy cows,and optimizing antioxidant defense capability of dairy cows.In this thesis,we investigated possible mechanism on Se alleviating BMEC oxidative damage from TrxR1/mitogen-activated protein kinase(MAPK)/NO pathway by using exogenous nitric oxide(NO)to induce oxidative stress,using dinitrochlorobenzene(DNCB)to inhibit thioredoxin reductase(TrxR)activity and induce oxidative damage,using Interleukin-1 receptor antagonist(IL-1Ra)to inhibit interleukin-1(IL-1)biological activity,using inhibitors to block phosphorylation of MAPK pathway,as well as overexpression of TrxR1.This thesis is divided into 6 trials.Trial 1 was conducted using a single-factor completely randomized design to study the regulatory effects of different doses of Se(0,10,20,50,100,150,200 nmol/L)on the antioxidant function in BMEC,and explore the effect of Se on TrxR activity and NO synthesis under normal physiological condition,providing a basis for further investigation of its protective mechanism.The results showed that Se enhanced relative growth rate(RGR),activity and gene expression of TrxR and glutathione peroxidase(GPx),superoxide dismutase(SOD)activity,total antioxidant capacity(T-AOC)and inhibited content of malondialdehyde(MDA)and reactive oxygen species(ROS)in a dose-dependent manner,that is,Se promoted the antioxidant function of BMEC in a dose-dependent manner.Based on the results of multiple indexes,the protective effect of Se was optimal at a dose of 50 nmol/L and weakened at 100 to 200 nmol/L.In contrast,Se had no significant effect on IL-1,IL-6,tumor necrosis factor-α(TNF-α),NO content,and activity and its gene expression of inducible nitric oxide synthase(iNOS),as well as gene expression of nuclear transcription factor E2 related factor 2(Nrf2)under normal physiological conditions.Trial 2 was conducted using completely randomized design to establish NO-induced oxidative stress model in BMEC by judging RGR and antioxidant and inflammatory indexes.Different concentrations of NO(0,250,500,750,1000,1250,1500 μmol/L)and different action times(2,4,6,8,12 and 24 h)were involved to select the appropriate concentration and action time of NO for oxidative stress model.Overdose NO induced oxidative stress in BMEC using diethyl-enetriamine/nitrogen oxide polymer(DETA/NO)as exogenous NO,the optimal concentration action time of NO was 1000μmol/L and 6h,respectively.The RGR was reduced to 76.61%,and activity of SOD,CAT,GPx were decreased,and content of MDA,inflammatory factors,NO and iNOS activity were increased.It can be used as a suitable condition for establishing oxidative damage model in BMEC.Trial 3 was conducted using a single-factor completely randomized design to study protective effect and its possible mechanism of different doses of Se(0,10,20,50,100,150,and 200 nmol/L)on oxidative damage in BMEC caused by NO,and then screen out the optimal dose of Se.The results showed that NO-induced oxidative stress in BMEC,resulting in an significant decrease in RGR,activity of SOD,T-AOC,CAT,activity and its gene and protein expressions of GPx and TrxR.Gene expression of Nrf2 showed a similar change.While ROS activity,the content of MDA showed the opposite trend.In addition,the gene expression of p38 mitogen activated protein kinases(p38MAPK),c-Jun N-terminal kinase(JNK)and extracellular regulated protein kinases 1/2(ERK1/2)were also increased significantly,and so did the expressions of its downstream IL-1 and other inflammatory factors,iNOS,contributing to the excessive release of NO.The supplement of Se significantly reversed the changes of the above indicators,indicating that Se had a significant protective effect on NO-induced oxidative stress in BMEC through reducing the production of IL-1,the activity of iNOS,and the over-release of NO.This may be related to increased TrxR activity and the its gene and protein expression,which in turn inhibited the activation of the MAPK signaling pathway.The protective effect of Se on oxidative stress induced by excessive NO was dose-dependent.Among them,the protective effect of 20～100 nmol/L Se was better,especially of 50 nmol/L Se.However,excessively high concentrations of Se failed to reverse NO-induced oxidative stress and even caused cell damage.Trial 4 was conducted with a completely randomized design to explore mechanism of Se relieving NO-induced oxidative stress in BMEC from the TrxR/IL-1/NO pathway.DNCB was used to inhibit TrxR and induce oxidative stress,and IL-Ra was used to inhibit IL-1 biological activity.BMEC was randomly divided into 8 groups,namely:control group(CON),Se group(Se),DNCB group(DNCB),IL-1Ra group(IL-1Ra),Se+DNCB group(S+D),Se+IL-1Ra group(S+I),DNCB+IL-1Ra group(D+I),Se+DNCB+IL-1Ra group(S+D+I).The results showed that DNCB inhibited the activity of TrxR and the expression of its gene and protein,and contributing to increase in the activity of ASK-1,which activated the p38MAPK and JNK signaling pathways and increased its downstream IL-1 and NO concentrations,further causing oxidative stress in BMEC.IL-1Ra inhibited the activity of IL-1,and reduced the excessive production of NO and relieved oxidative stress caused by DNCB,indicating that IL-1 was a key factor that induced production of large amounts of NO and caused oxidative stress in BMEC.Se also had a mitigating effect on DNCB-induced oxidative damage.The addition of Se promoted TrxR activity and the expression of its genes and proteins,and it can reverse the activation of p38MAPK and JNK signaling pathways caused by DNCB,thereby inhibiting IL-1 production and iNOS activity as well as excessive production of NO.It showed that Se exerts its mitigation effect on oxidative stress in BMEC through TrxR1 enhancement inhibiting IL-1 activity.Trial 5 was conducted with a completely randomized design to explore the mechanism of Se mitigating NO-induced oxidative damage in BMEC from the TrxR/MAPK/NO pathway,using DNCB as an inhibitor of TrxR activity and inducer of oxidative stress,using SB203580,SP600125,PD98059 as an inhibitor of p38MAPK,JNK,and ERK1/2 signaling pathways respectively.The third-generation BMEC was randomly divided into 7 treatments,namely:control group,DNCB group,Se group,DNCB+Se group,DNCB+SB203580 group,DNCB+SP600125 group,DNCB+PD98059 group.The results showed that DNCB inhibited TrxR,increased ASK-1 activity,and then downstream p38MAPK and JNK pathways were activated,contributing to over-release of IL-1 which induced oxidative stress.Se reversed the oxidative damage in BMEC,and enhancement of TrxR expression suppressed ASK-1 activity and p38MAPK/JNK pathway phosphorylation,mitigating the excessive release of IL-1 and suppressing its downstream iNOS-NO cascade.When the p38MAPK and JNK pathways were inhibited by SB203580 and SP600125,DNCB-induced oxidative damage was inhibited,and its downstream factors,such as content of IL-1,activity and expression of iNOS,were significantly inhibited,thereby protecting BMEC from the damage caused by NO accumulation.It suggested that Se alleviated the oxidative damage in BMEC by TrxR enhancement inhibiting the activation of p38MAPK and JNK signaling pathway.Trial 6 was conducted using a completely randomized design,inducing oxidative damage to BMEC with NO,and over-expressing TrxR1.BMEC was randomly divided into 4 groups,namely:control group,NO group,NO+Se group,NO+TrxR1 OE group.We further studied the effect of TrxR1 on the expressions of iNOS,IL-1β and other inflammatory factors,as well as p38MAPK and JNK signaling pathway in BMEC to verify whether TrxR1 gene is a key gene for Se relieving BMEC oxidant damage.The results showed that overexpression of TrxR1 gene significantly reduced the gene expression and phosphorylation level of p38MAPK and JNK signaling pathway and its downstream IL-1β and iNOS gene and protein expressions,alleviating NO-induced oxidative damage,further indicating that TrxR1 was a key gene for Se protecting BMEC from oxidative stress.In summary,this study explored the mechanism of Se promoting BMEC antioxidant function and relieving NO-induced oxidative stress in BMEC from the TrxR1/MAPK/NO pathway,that is,Se protected the BMEC from oxidative stress by promoting gene and protein expression of TrxR1,and then inhibiting activation of p38MAPK/JNK,reducing the gene and protein expressions of IL-1β and iNOS,as well as inhibiting the excessive release of NO.