Protective Effect And Mechanism Of Astragaloside ? Against Oxidative Stress And Apoptosis Induced By Ammonia In Bovine Mammary Epithelial Cells

Ammonia,produced mainly from the deamination of amino acids and glutamine,is one of the major toxic components in blood and tissues that may affect bovine health.Some reports have provides insights and evidence that increased ammonia can disturb many organ and cell types and hence lead to dysfunction.However,the physiological and pathological roles of ammonia in the mammary glands are not understood clearly.The mammary epithelial cells are the major places where the milksynthesized.The function and the status of the bovine mammary epithelial cells are critical for the yield and quality of milk.In the present study,the bovine mammary epithelial cell line?MAC-T?was utilized as an in vitro model to determine the effects of ammonia on bovine mammary gland.These data suggest that ammonia exposure induces apoptosis and oxidative stress in bovine mammary epithelial cells.In addition,astragaloside IV,extracted from astragalus,is well known for its antioxidant activity.In the present study,the cytoprotective effects of astragaloside IV against ammonia in vitro were explored.Finally,we studied the effects of high blood ammonia on mammary tissue and the protective effects of astragaloside IV on ammonia induced oxidative damage of mammary gland in mice using mice as model in vivo.In the present study,we focused on these problems to study.Main results obtained were as followed:1.Effects of ammonia on apoptosis and oxidative stress in bovine mammary epithelial cellsIn the present study,we demonstrated the effects of ammonia on the bovine mammary epithelial cells firstly.To explore the effects of ammonia on the bovine mammary epithelial cells,we measured cell viability,cell apoptosis rate,reactive oxygen species,mitochondrial membrane potential???m?,and intracellular free-Ca²⁺of cells.The results shown that ammonia stimulated the production of intracellular reactive oxygen species,decreased mitochondrial membrane potential,interrupted intracellular calcium ion(Ca²⁺)homeostasis,and induced cell apoptosis.In addition,the mRNA expression of inflammatory factors?IL-6 and IL-8?and endoplasmic reticulum?ER?stress markers?CHOP and GPR78?were significantly increased with ammonia treatment.In conclusion,Ammonia induced bovine mammary epithelial cells apoptosisa and oxidative stress.Meanwhile,in order to determine the molecular pathway of ammonia-induced mammary epithelial cell apoptosis and oxidative stress,we detected the apoptosis-related factors using qPCR and Western blot assays.The results shown that the mRNA expression of expressions of BAX,caspase 8,caspase 9,and caspase 3 were significantly increased with ammonia treatment.In addition,the level of p53 phosphorylation and the protein expression of BAX,caspase 8,caspase9,and caspase 3 were significantly increased with ammonia treatment.Ammonia induced cell apoptosis via activation of the p53 pathway and the mitochondrial apoptotic pathway.Interestingly,bumetanide,a specific Na⁺K⁺2Cl^--cotransporter inhibitor,dramatically abolished the damaging effects of ammonia on the cells.These data suggest that ammonia exposure induces apoptosis in bovine mammary epithelial cells via p53 signaling pathway and the mitochondrial apoptotic pathway,and that these effects involved the Na⁺K⁺2Cl^--cotransporter.2.Protective effects of astragaloside IV against ammonia–induced bovine mammary epithelial cell apoptosis and oxidative stressIn order to confirm whether astragaloside IV have a protective effect on ammonia-induced bovine mammary epithelial cell damage,we carried out these following experiments.First,various concentrations of AS IV were applied the MAC-T cells for 24h.The results shown that cells treated with astragaloside IV at various concentrations?0,5,10,and 20?M?for different times showed no effect on bovine mammary epithelial cell growth.Pretreatment of cells with astragaloside IV at various concentrations of?0,5,10,and 20?M?for 4 h,and then added NH₄Cl for designed time periods.We detected the cell viability,the level of intracellular reactive oxygen species?ROS?and the rate of cell apoptosis.The results demonstrated that pretreatment of MAC-T cells with astragaloside IV could potently suppress the increase in the level of intracellular reactive oxygen species?ROS?and the rate of cell apoptosis,and rescue the decrease of cell viability.Furthermore,we detected the mRNA expression of inflammatory factors?IL-6and IL-8?and endoplasmic reticulum?ER?stress markers?CHOP and GPR78?using qPCR assay.The results shown that pretreatment of MAC-T cells with astragaloside IV could potently inhibit the ammonia-induced inflammatory responses and prevented ammonia-induced endoplasmic reticulum stress.In conclusion,astragaloside IV can protect breast epithelial cells from ammonia-induced cell damage.3.Mechanisms of astragaloside IV against ammonia-induced oxidative stress and apoptosis of bovine mammary epithelial cellsIn order to further study the mechanisms of astragaloside IV against ammonia-induced cell apoptosis of bovine mammary epithelial cells,we detect the level of apoptosis related factors using qPCR assay and Western blot assay.Astragaloside IV prevented ammonia-induced endoplasmic reticulum stress.Astragaloside IV also significantly suppressed the levels of BAX,caspase 3 and p53 phosphorylation in ammonia-induced MAC-T cells.In conclusion,astragaloside IV inhibits ammonia-induced cell apoptosis by inhibiting the p53 signaling pathways in MAC-T cells.At the same time,in order to further explore which key molecules mediate the protective effect of astragaloside IV on the damage of bovine mammary epithelial cells,the following studies were conducted.To attain further insight into the antioxidative effect of astragaloside IV,expressions of genes encoding antioxidant/detoxificant enzymes?Nrf2,HO-1 and xCT?involved in cytoprotection against oxidative stress were observed in the present study.In the present study,astragaloside IV increased the expressions of Nrf2,HO-1,and xCT and promoted the nuclear protein level of Nrf2 in MAC-T cells,indicating that Nrf2 mediated the induction of HO-1 and xCT by astragaloside IV.In addition,the nuclear protein levels of Nrf2 were significantly increased with the astragaloside IV treatment using Western blot assay.The results showed that astragaloside IV stimulated Nrf2 protein levels in the cytoplasm and nucleus of MAC-T cells.In addition,the role of Nrf2 in the cytoprotective effects of astragaloside IV on oxidative stress ammonia induced was detected by an Nrf2 siRNA transfection assay.The expression of GPR78 and CHOP mRNAs in the Nrf2 knockdown group increased significantly compared with that in the cells transfected with a control siRNA and challenged with ammonia.In addition,an inhibitory effect of astragaloside IV on the mRNA expression of CHOP was not shown in the Nrf2siRNA-transfected cells.Furthermore,knockdown of Nrf2 abolished the protective effect of astragaloside IV against an ammonia-induced decrease in cell viability.Thus,it can be concluded that nuclear factor erythroid 2-related factor 2?Nrf2?was essential to cytoprotective effects of astragaloside IV in MAC-T cells,as knockdown of Nrf2 dramatically abolished the prosurvival effects of astragaloside IV on treated cells.To attain further insight into the molecular pathway by which astragaloside IV upregulates Nrf2 to protect MAC-T cells from ammonia–induced cell damage,the following studies were conducted.In the present study,it was also found that astragaloside IV could activate the ERK and AKT signaling pathways involved in MAC-T cell survival upon oxidative stress induced by ammonia.Furthermore,the astragaloside IV-induced expressions of Nrf2,HO-1,and xCT were inhibited by specific inhibitors of ERK and AKT,indicating that the ERK and AKT pathways play an important role in mediating the cytoprotective effects of astragaloside IV.The protective effects of astragaloside IV against oxidative damage induced by ammonia in MAC-T cells might be mediated via activation of the ERK and AKT signaling pathways.4.Protective effects and mechanisms of astragaloside IV against ammonia-induced oxidative damage of mammary gland in miceWe studied the effects of high blood ammonia on mammary tissue and the protective effects of astragaloside IV on ammonia induced oxidative damage of mammary gland in mice using mice as model in vivo.High and low doses of astragaloside IV were administered to mice four days before each week,followed by intraperitoneal injection of ammonium chloride for 4 weeks.We measured the level of lipid peroxide level and the mRNA expression of apoptosis-related genes of the mammary gland.Results shown that,high blood ammonia would induce the oxidative stress of mammary gland of mice.Astragaloside IV had protective effects on ammonia-induced oxidative damage of mammary gland in mice.To attain further insight into the molecular pathway of the cytoprotective effects of astragaloside IV on oxidative stress in mammary gland in mice,we detected the protein expression on Nrf2 and HO-1 using Western blot.The results suggested that astragaloside IV may alleviate high blood ammonia induced oxidative damage through Nrf2signaling pathway.In summary,ammonia stimulated the production of intracellular reactive oxygen species,decreased mitochondrial membrane potential,interrupted intracellular calcium ion(Ca²⁺)homeostasis,and induced ER stress,inflammatory responses,and cell apoptosis.In addition,ammonia exposure induces apoptosis in bovine mammary epithelial cells via activation of the p53pathway and the mitochondrial apoptotic pathway,and that these effects involved the Na⁺K⁺2Cl^--cotransporter.In addition,astragaloside IV played a beneficial role against ammonia-induced damage of MAC-T cells.In addition,the vivo studies had also confirmed the protective effects of astragaloside IV on ammonia-induced oxidative damage and highlighted its effects of the Nrf2 signaling pathway.Astragaloside IV may regulate the activation of Nrf2 by activating the PI3K/AKT and ERK/MAPK pathways and protective cells against oxidative damage induced by ammonia.The purpose of this study was to provide a theoretical basis for the pathologic effects of high ammonia on cow mammary gland and its mechanism.In addition,the present work may provide a possibility for the therapeutic application of astragaloside IV in the protection of the mammary gland against ammonia exposure.This remains to be tested by an in vivo study of dairy cows.