Abstract:

This study was to detect the effects of Daidzein (DZ) on the synthesis of lactose, milk protein, milk fat and cell proliferation of mouse mammary epithelial cells EpH4-Ev to investigate the associated regulatory roles. EpH4-Ev cells were treated with different concentrations of DZ (0 to 1 000 μmol·L^-1) for 6, 12, 24, 48 h and the concentration of DZ and time were determined by detecting cell viability; they were divided into a control group (0 μmol·L^-1 DZ treatment), a low-concentration group (10 μmol·L^-1 DZ treatment), a medium-concentration group (20 μmol·L^-1 DZ treatment), and a high-concentration group (40 μmol·L^-1 DZ treatment), and physiological dose of estradiol (E2) was used as a positive control, and the cells were incubated for 12 h at 37 ℃ and 5% CO₂ as follows experiments were performed: 1) Determination of triglyceride (TG) and glucose (GLU) content in cells and supernatants; 2) Cell proliferation and apoptosis-related proteins, milk component synthesis-related proteins and PI3K/AKT-mTOR signaling pathways were detected, and milk lipid synthesis was probed by combining with lipid droplet staining. The apoptosis rate and cell cycle distribution were analyzed by flow cytometry. The results showed that: 1) Compared with the control group, between 2.5-80.0 μmol·L^-1 DZ treatments could significantly improve the cell viability of EpH4-Ev cells (P < 0.01), and 20 μmol·L^-1 DZ treatment had the most significant improvement effect. Combined with the results of the pre-laboratory period, the low, medium and high concentrations of DZ action were selected to be 10, 20, and 40 μmol·L^-1. 2) Compared with the control group, medium and high concentration of DZ and E2 treatments significantly increased the content of TG and GLU in EpH4-Ev and supernatants and promoted the synthesis of lipid droplets (P < 0.01). 3) Compared with the control group, the expression of glucose transporter carrier 1 (GLUT1) and β-casein were both highly significantly elevated after treatment with different concentrations of DZ (P < 0.01); meanwhile, both DZ and E2 treatments increased the expression of fatty acid synthetase (FASN), cholesterol regulatory element-binding protein 1 (SREBP1), peroxisome proliferator-activated receptor γ(PPAR-γ), and acetyl coenzyme A carboxylase (ACC) expression. 4) Compared with the control group, the proportion of cells in G2/M phase and S phase, the expression of proliferating cell nuclear antigen (PCNA), cell cycle proteins D1 and D3 (CyclinD1 and D3), and anti-apoptotic protein Bcl-2 were increased after treatment with different concentrations of DZ and E2, and the medium concentration of DZ group highly significantly increased the Bcl-2/Bax ratio (P < 0.01) and decreased the apoptosis rate. 5) Compared with the control group, three concentrations of DZ and E2 treatment increased the phosphorylation levels of p-PI3K, p-mTOR, p-AKT and promoted the activation of the PI3K/AKT-mTOR signaling pathway. These results suggested that DZ can promote the proliferation of mammary epithelial cells and the synthesis of milk components. The mechanism is related to the up-regulation of the expression of cell proliferation proteins, the reduction of apoptosis rate, and the activation of PI3K/AKT-mTOR pathway.

Key words: phytoestrogen, daidzein, mouse mammary epithelial cells, milk component synthesis, mammary epithelial cell proliferation