The Molecular Mechanisms Of Nrf2-ARE Signaling Pathway Against Oxidative Stress And Its Regulation By Quercetin In Mammary Gland Of Mice

The function of lactation is essential for the survival and reproduction of mammals.Lactation not only ensures the survival of newborn mammals,but also provides nutrients for human growth and development.During lactation,a variety of diseases and sub-health conditions affect mammary health.Oxidative stress is a common factor that impairs mammary health and inhibits mammary function,resulting in decreased milk production and milk quality.Bioactive substances existing in nature have been widely attentioned due to their antioxidant,anti-inflammatory and anti-cytotoxic activities.Nrf2-ARE is one of the most important antioxidant signaling pathway.Whether bioactive substances alleviate oxidative stress via Nrf2-ARE pathway in mammary glands deserves further study.Therefore,this research used Nrf2 knockout mice to analyse the mechanisms of Nrf2-ARE pathway against oxidative stress in the mammary glands,and used mammary epithelial cells as model to explore the active substance relieving oxidative injury through Nrf2-ARE pathway,and investigated its molecular mechanisms.The study aimed to provide a theoretical basis of molecular mechanisms of Nrf2-ARE pathway defensing mammary oxidative injury,and to lay the scientific foundation of developing antioxidant additives which directionally control,prevention and treatment oxidative stress in mammary glands.1 Effects of Nrf2 on growth,reproduction and mammary health in miceIn this study,the genotype of mice was identified to confirm that Nrf2 genotype did not affect the growth and reproductive performance of female mice aged 6-8 weeks and the average milk yield of female mice aged 10-12 weeks during lactation 6-11 days.Nrf2 knockout mice can be used in the study of mammary health.Next,WT,Nrf2（+/-）and Nrf2（-/-）mice aged 10-12 weeks in early lactation（day 3）were uesd（n=6）.Each mouse was injected with 50μL LPS（0.2 mg/ml）to one side of the fourth mammary gland as the LPS group,and 50μL PBS to the other side of the fourth mammary gland as the control group.The fourth pair of mammary gland tissues were collected 24 h after injection.In addition,WT,Nrf2（+/-）and Nrf2（-/-）mice aged 10-12 weeks in mid-lactation（day 12）were used（n=5）and milk samples were collected 3 h after LPS and PBS injection.The mammary tissue appearance,milk protein content,expression of inflammatory factors and redox balance related genes in mammary samples were analyzed to evaluate the mammary health comprehensively.The results showed that the expression ofβ-casein in mammary tissues of Nrf2（-/-）mice was significantly increased and the expression ofκ-casein was significantly decreased under stress,while these genes of WT mice was not significantly affected.The results of transcriptome showed that m RNA abundance of heme oxygenase 1（HO-1）,glutamate/cysteine reverse transporter（x CT）and glutamate cysteine ligase regulatory subunit（GCLM）in mammary glands of Nrf2（-/-）mice were significantly lower than those of WT mice after LPS treatment,and total antioxidant capacity was significantly decreased in Nrf2（-/-）mice.In addition,Nrf2（-/-）mice showed more up-regulated expression of inflammatory factors in mammary glands.These results suggested that Nrf2（-/-）mice had disturbed redox balance in the mammary glands,and the mammary health of Nrf2（-/-）mice was more adversely affected by external stimuli than WT mice.2 The mechanisms of Nrf2-ARE signaling pathway against oxidative damage in mouse mammary glandsThis experiment used the fourth of mammary tissue samples of the early lactation（3 days）mice injected with LPS or PBS for 24 h.The study detected the expression of antioxidant enzyme,glutathione（GSH）content,GSH/reducing glutathione（GSSG）ratio,the expression of GSH synthesis and metabolism genes,cell apoptosis related genes and endoplasmic reticulum stress markers of mammary glands.The results showed that LPS stimulation decreased the expressions of catalase and peroxide-reductase 1 in Nrf2（-/-）mice,but had no significant effect in WT mice.LPS significantly increased the expression of Nrf2 and its downstream gene HO-1 in WT mice,but there were no such change in Nrf2（-/-）mice.Nrf2（+/-）and Nrf2（-/-）mice did not significantly upregulate GSH content and GSH/GSSG ratio after LPS induction,as WT mice did.The m RNA expression abundance of glutamate cysteine ligase catalytic subunit（GCLC）,GCLM and x CT in Nrf2（-/-）mice was also lower than those in WT mice under LPS stress.In addition,LPS treatment increased the expression of pro-apoptotic factor（BAX）,the BAX/Bcl-x L ratio and the expression of endoplasmic reticulum stress marker（GPR78）in Nrf2（-/-）mice,but not in WT mice.These results suggested that Nrf2-ARE signaling pathway regulated both enzymatic and non-enzymatic antioxidants,and regulated the synthesis and consumption of GSH via Nrf2/GCL/GSH/GPx1 pathway,and also played an important role in cell apoptosis and endoplasmic reticulum stress.3 The mechanisms of quercetin against oxidative injury via Nrf2-ARE signaling pathway in mammary epithelial cells3.1 The effects of quercetin on resisting oxidative injury of mammary epithelial cellsThis study aimed to explore bioactive substances that may alleviate mammary oxidative stress via Nrf2-ARE pathway.Firstly,the model of oxidative damage was established by stimulating the mammary epithelial cells（HC11）with different concentrations of H₂O₂（50,100,250,500,750,1000μΜ）for 24 h.Then the HC11 cell tolerance concentration was determined by using different concentrations of quercetin（0,5,10,15,20,25μΜ）to stimulate HC11 cells for 24 h.Next,the HC11 cells were pretreated with quercetin for 2 h to detect the improvement effects of quercetin on cell proliferation and the release of lactate dehydrogenase（LDH）.Then,HC11 cells were divided into four groups.Quercetin group and quercetin pretreatment group were added with 20μΜquercetin to culture cells.2 h later,H₂O₂ and quercetin pretreatment group were added with 100μΜH₂O₂ to stimulate the cells for 24 hours.The control group was cultured in serum-free medium.Protein was extracted from the cells of the four groups,and the activity of antioxidant enzyme（CAT,SOD）,the expression of antioxidant genes（x CT,GCLM,TXNRD1）and apoptosis-related protein（BAX,BCL-2）were detected.The results showed that 100μΜH₂O₂ reduced the activity of HC11cells by about 60%and release LDH by about 24%in the control group,which was suitable as a research model.20μΜquercetin had the best protective effect on HC11cells,which significantly restored the decrease of T-AOC,improved the activity of antioxidant enzyme and expression of antioxidant protein,but had no significant effect on cell apoptosis.These results suggested that quercetin alleviated the cell proliferation,cytotoxicity and oxidative stress in HC11 cells.3.2 Molecular mechanism of quercetin’s resistance to oxidative damage via Nrf2-ARE signaling pathway in mammary epithelial cellsThe aim of this study was to elucidate the molecular mechanisms of quercetin improving oxidative damage via Nrf2-ARE signaling pathway in HC11 cells.Firstly,HC11 cells were divided into four groups and treated as described above to explore the effects of quercetin on the activation of Nrf2-ARE and MAPKs signaling pathways under stress state.Subsequently,signaling pathway inhibitors were used to explore the regulatory mechanism of Nrf2-ARE signaling pathway on MAPKs pathway regulating quercetin’effects,to clarify the relationship between Nrf2-ARE and MAPKs signaling pathway in quercetin’s resistance to oxidative stress.In the end,the cells were divided into 7 groups,including the control group,the H₂O₂ group and the quercetin pretreatment group as described above.HC11 cells in the four inhibitor groups were pretreated with Nrf2 inhibitor ML385（5μM）,p38 MAPK inhibitor SB203580（25μM）,ERK inhibitor PD98059（100μM）and JNK inhibitor SP600125（10μM）for 1 h,followed by quercetin for 2 h and H₂O₂ stimulation for 24 h.The changes of cell proliferation,cytotoxicity and antioxidant defense system in 7 groups were detected,to clarify the role of Nrf2-ARE and MAPKs signaling pathways in quercetin’s resistance to oxidative stress.The results showed that Nrf2-ARE,p38 MAPK,ERK and JNK signaling pathways were activated by H₂O₂.Quercetin pretreatment significantly restored the activation of Nrf2-ARE and MAPKs signaling pathway.ERK inhibitor significantly increased p-Nrf2/Nrf2 ratio,while p38 MAPK inhibitor significantly decreased the expression of p-Nrf2,suggesting that p38 MAPK and ERK signaling pathways may affected the antioxidant capacity of quercetin by regulating the activation state of Nrf2-ARE signaling pathway.All the four inhibitors significantly reduced the ameliorative effects of quercetin on cell proliferation and did not affect the anti-cytotoxicity of quercetin.As for the anti-oxidation defense system,the improvement effect of quercetin on T-AOC was mainly dependent on the Nrf2-ARE,p38 MAPK and ERK pathways,and the expression of x CT was mainly dependent on the Nrf2-ARE signaling pathway.The recovery effects of quercetin on CAT enzyme activity and expression of GCLM were regulated by Nrf2-ARE and MAPKs signaling pathways simultaneously.In conclusion,Nrf2-ARE signaling pathway regulated the expression of downstream antioxidant enzymes,mediated the Nrf2/GCL/GSH/GPx1 pathway to regulate the synthesis and consumption of GSH,and played a regulatory role in cell apoptosis and endoplasmic reticulum stress,thus maintained the redox balance of mammary glands.Nrf2-ARE signaling pathway mediated quercetin improving the proliferation and antioxidant defense ability of HC11 cells and restoring the redox balance.In addition,MAPKs signaling pathway was also involved in regulating the cell protective effects of quercetin.