| The objective of this paper was to investigate the retard effect of selenium(Se) on mammary gland epithelial cells(BMECs) injured by diethylenetriamine/nitrogen monoxide polymerase(NO/DETA) via Nrf2/GPX1-PPARy-NF-KB signaling pathway in order to provide a theoretical basis for scientifically supplementing Se in the practice and improving the antioxidant function of mammary gland in dairy cows.The paper is divided into three experiments.The first experiment was designed as a single factor randomized trial,and the 3rd generation BMECs were divided into 8 treatments with 6 replicates.The control(CON) group without Selenium and NO/DETA were cultured for 30 hours.Groups 2 to 8 were treated with NO/DETA and different doses of Se(0,10,20,50,100,150,200 nmol/L) for 24h,and then treated with 1000 ?mol/L NO/DETA and different Se concentration for 6h.It was to investigate the pre-protection effect and mechanism of different doses of Se on BMECs injured by NO according to the expression of selenoproteinase,inflammatory factor genes and enzymes,activities and expression levels of genes related to peroxisome proliferator-activated receptor(PPAR?),transcription factor NF-E2 related factor 2(Nrf2) and nuclear factor-kappaB(NF-?B) signaling pathway to select the optimal selenium concentration.The test 2 concluded two parts,the single factor randomized design was conducted in this test.The first part was added different doses of PPARy inhibitor(GW9662) for 6 hours and selected the appropriate GW9662 concentration by determining cell proliferation rate.In the second part,PPAR? was blocked by GW9662(concentration determined according to the first part).The groups was CON group,NO/DETA injury group,GW9662 inhibitor group(The culture was carried out under the culture conditions of the CON group for 24 hours,then the GW9662 was added for another 6 hours,Se alone(Se concentration according to the test 1),Se+DETA group,Se+GW9662 and Se+DETA+GW9662.It was to investigate whether Se could alleviate cellular oxidative damage caused by NO through PPAR?-NF-?B signaling pathway.The silent GPX1 technique was used in the third experiment.The experiment included 7 treatment groups,CON group,NO/DETA injury group,Se treatment group,Se pre-protection(Se+DETA) group,and Se+GPX1 shRNA group,Se+DETA+GPX1shRNA group to investigate the mechanism of Se mitigating the oxidative stress of BMECs through Nrf2/GPX1-PPARy-NF-KB signaling pathway.The results of experiment 1 showed that different doses of Se had different alleviated effect on oxidative damaged BMECs injured by excessive NO,and increased GPX,thioredoxin reductase(TrxR) and total antioxidant enzyme(T-AOC),superoxide dismutase(SOD),catalase(CAT)activity and gene expression of GPX,TrxR,PPARy,the activation of Nrf2 signaling pathway and inhibited the activation of NF-?B in the inflammatory pathway,Malondialdehyde(MDA) content,reactive oxygen species(ROS) activity,activities and its genes expression of inflammatory factors like interleukin-1?(IL-1?),interleukin-6(IL-6),anti-tumor necrosis factor(TNF-?),and nitric oxide synthase(iNOS).Based on all the resuls,the addition of 50?100 nmol/L Se showed the better effect on the alleviated effect on oxidative stress,and the 50 nmol/L Se had the best effect.The results of the second experiment showed compared with the Se+DETA group,that added GW2.5 inhibitor in the Se+DETA group significantly inhibited the gene expression of PPARy and the antioxidant enzymes of GPX,T-AOC,T-SOD,CAT activity,TrxR,SelP mRNA levels and enzyme activities(P<0.01),and increased inflammatory factors IL-1?,IL-6,TNF-?and NF-?Bp50 gene expression levels,and MDA,NO contents,ROS and iNOS activities also showed similar changes.It is indicated that when PPARy was inhibited,antioxidative function of Se was inbibited,indicating that Se alleviated oxidative stress by activating PPARy,and inhibiting the activation of NF-?B signaling pathway and the production of inflammatory factors and NO production.The results of the 3rd experiment showed that the Se+DETA group silenced by GPX1 significantly inhibited the expression of PPARy and Nrf2 compared with the Se+DETA group and decreased the mRNA level and enzyme activity of antioxidant enzymes of GPX1,TrxR,T-AOC and CAT.The activity and gene expression of NF-?Bp65 gene and inflammatory factors IL-1?,TNF-a and iNOS activities were remarkablely increased,and ROS activity,MDA and NO content also showed similar results.These results indicated that GPX1 was a key gene for the anti-oxidation function of Se.When GPX1 gene is silenced,the expression of PPARy gene was reduced and NF-?B signaling pathway and the activity of downstream inflammatory factors and gene expression,was activated then the antioxidant function was reduced to decrease the antioxidant function of Se on cells.In summary,this paper explored the alleviative effect and mechanism of Se on excess NO-induced oxidative stress in BMECs by Nrf2/GPX1-PPAR?-NF-?B signaling pathway,that is Se promoted GPX1 by activating the signaling pathway Nrf2 and increased the gene expression and enzymatic activity of PPAR? to inhibit the activation of NF-?B signaling pathway and the release of inflammatory factor IL-1? then decreased the excessive production of NO and protected cells from oxidative stress. |
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