Mechanisms Underlying The Cytoprotective Effects Of Resveratrol Against Oxidative Stress In Bovine Mammary Epithelial Cells

The mammary epithelial cells (MECs) are the major places where the milksynthesized. The function and the status of the MECs are critical for the yield and the quality of the milk. In different physiological and pathological stages, MECs are susceptible to oxidative stress (OS) due to the intensive cell metabolism. In order to maintain a healthy mammary gland and to keep the productivity of the mammary gland, it is necessary for defending against OS as well as boosting intracellular antioxidant potential. Resveratrol is a well known natural antioxidant with versatile pharmacological activities despite of the fact that none of these studies focused on the mammary gland. In the present study, we used hydrogen peroxide-(H2O2-) to induce OS in cultured bovine MECs (MAC-T) and to evaluate the protective effects of resveratrol against OS in MECs. The molecular mechanisms underlying these protective effects were explored. Finally, lactating ICR mice were used to to test the safety of resveratrol to the lactating animals and to study the effects of resveratrol for the mammary gland redox homeostasis. The roles of resveratrol for activating the Nrf2-ARE signaling in the mammary gland were also investigated.Main Results obtained were as followed:1 Protection of Bovine Mammary Epithelial Cells from Hydrogen Peroxide-Induced Oxidative Cell Damage by Resveratrol1.1 To establish an oxidative stress model induced by H2O2 in cultured bovine MECs(MAC-T). Various concentrations of H2O2 for different time periods were used to study effects of H2O2 stimulation on the cell viability, cell death rates, Reactive oxygen species(ROS) accumulation levels, cellular redox status, gene expressions relating to mitochondrial damage (BAX and BCL2) and endoplasmic reticulum (ER) stress (GPR78 and CHOP). We found that treatment with H2O2 (500μM) of MAC-T cells for 24h lead to significant cell viability losses and increase cell apoptosis rates.Treatmentwith H2O2 for 12h raised ROS levels accompanied with decreases of the total antioxidant capacity and the superoxide dismutase (SOD) activity. Gene expressions of GPR78 and CHOP peaked at 4h after H2O2 treatment andgene expressions of BAX and BCL2 peaked at 24h after H2O2 treatment. MAC-T cells with H2O2 (500μM) treatment lead to cell viability losses, cell apotosis and damaged cellular redox homeostatis, accompanies with mitochondrial damage and ER stress. Based on these findings, we chose H2O2 (500μM) treatment in MAC-T cells to establish an OS induced cell damage model.1.2 To investigate the protective effects of resveratrol againstH2O2induced OS in cultured bovine MECs(MAC-T).Various concentrations of resveratrol were applied to the MAC-T cells for 24h. It is found that the maximum resveratrol concentration was 50μM. Based on our previous established OS model, MAC-T cells were pretreated with various concentrations of resveratrol (0-50μM) for 2h, then challenged with H2O2 (500μM) for designed time periods. Oxidative stress relating cell damage indicatorswere measured, including cell viability decreases, cell death rates, ROS accumulation levels, cellular redox status, gene expressions changes relating to mitochondrial damage (BAX and BCL2) and ERstress (GPR78 and CHOP). Compared with the H2O2 treated cells, resveratrol pretreatment showed potent cytoprotective effects by rescuing cell viability losses, inhibiting cellular apoptosis rates and ROS accumulation in a dose dependent manner. Resveratrol also alleviated mitochondrial damage and ER stress challenged by H2O2. It isconcluded that resveratrol protect the MECs against OS induced by H2O2 treatment.2 Molecular Mechanisms Underlying the Cytoprotective Effects by Resveratrol against Hydrogen Peroxide Induced Oxidative Cell Damage in Bovine Mammary Epithelial Cells.2.1 To investigate the roles of Nrf2-ARE signaling duing resveratrol and H2O2treatment in cultured bovine MECs(MAC-T).We determinated several key cellular antioxidant defense genes, TXNRD1, HO-1, XCT and NQO-1 after resveratrol treatment/resveratrol pretreatment MAC-T cells with/without H2O2 insults. It is found that resveratrol induced these key cellular antioxidant defense gene expressions significantly regardless of the H2O2 insults. Compared to H2O2 challenged cells, resveratrol pretreatment significant boosted key cellular antioxidant defense genes, peaked at 8h after H2O2 insults. Different concentrations of resveratrol treatment of MAC-T cells showed clear dose-dependent inductive effects on these antioxidant genes. It is also found that resveratrol treatment promoted the translocation of Nrf2 from the cytoplasm to the nuclei and activated ARE element.2.2 To clarify whether the protective effect of resveratrol for the MAC-T cells is Nrf2 dependent or not. We silenced Nrf2 gene by transfected the cells with siRNA and re-evaluated the cytoprotective effects by resveratrol. The inductive effects by resveratrol on several key cellular antioxidant defense genes were dismissed after Nrf2 gene was knockdown. The rescuing on the cell viability loss by resveratrol also disappeared in Nrf2 siRNA transfected MAC-T cells. It is indicated that the protective effects by resveratrol were mediated by Nrf2-ARE signaling.2.3 To investigate whether resveratrol regulates MAPK, PI3K/AKT signaling which affects the activation of Nrf2-ARE or not.The time-and dose effects of resveratrol and H2O2 treatment on the phosphorylation levels of AKT, ERK1/2, JNK1/2 and p38 were determined. It is found that H2O2 treatment activated all of these proteins and resveratrol pretreatment prolonged the phosphorylation of AKT and ERK1/2. Using selective inhibitors we found that the protective effects of resveratrol might dependent on the activation of ERK1/2, AKT and negatively regulated by p38 signaling.3 Effects of dietary supplemention ofresveratrol on the oxidative damage and growth performance in lactating periodIn this part, we added resveratrol into the diet (0,0.02%) and 0.2%) to the lactating ICR mice. Resveratrol had no effects on the daily food intake, body weight, organ indexes as well as mammary gland weights of the dams. Nevertheless, resveratrol had no effects onmilk productionas well as development of the pups, suggest that resveratrol inclusion is safe for the lactating animal.In addition, resveratrol significantly decreased the lipid peroxide concentrations and alleviated the oxidative damage in the mammary gland, but such effects were not observed in the liver and kidney. Gene expressions relating to the antioxidant defense (SOD2, GPX1, PRX1, TXNRD1) increased significantly in 0.2% resveratrol group. Expressions of TXNRD1、HO1、 Nrf2 increased in 0.02% resveratrol group. These data suggested that dietary supplemention of resveratrol may alleviate oxidative stress in the mammary gland of the mice by activating Nrf2-ARE singling.In summary, the present study established an oxidative stress model in mammary epithelial cells. Using this model we investigated the effects of resveratrol against oxidative stress in mammary epithelial cells and explored the underlying mechanisms. In vivo studies also demonstrated the beneficial effects of resveratrol for the mammary gland and highlighted its effects on the Nrf2-ARE signaling. The activations of PI3K/AKT and ERK signaling by resveratrol are involved with the Nrf2-ARE signaling which further regulate the mammary epithelial cells redox homeostasis. These data provide essential evidence for using resveratrol as a novel feed additive in dairy industry.