Abstract:

Objective To investigate the effect of bisphenol AF （BPAF） on the senescence in the human endometrial stromal cells （hESCs）， and to elucidate the inhibitory effect of rapamycin （Rapa） on the BPAF-induced senescent hESCs. Methods The hESCs were cultured in vitro and treated with 0， 7.5， 15.0， 30.0， 60.0， and 120.0 μmol·L^-1 BPAF. MTT assay was used to detect the cell survival rates of the hESCs after treated with different concentrations of BPAF. The cells were divided into control group （normal medium）， BPAF group （medium containing 30.0 μmol·L^-1 BPAF）， and Rapa group （medium containing both 30.0 μmol·L^-1 BPAF and 100.0 nmol·L^-1 Rapa）. Senescence-associated β-galactosidase （SA-β-gal） staining was used to assess the percentages of SA-β-gal positive cells in various groups； flow cytometry was used to analyze the percentages of cells at different cell cycles in various groups； real-time fluorescence quantitative PCR （RT-qPCR） method was used to assess the expression levels of p16， p21 and p53 mRNA in the cells in various groups； Western blotting method was used to detect the expression levels of senescence-related proteins （p16， p21 and p53）， autophagy-related proteins p62， microtubule-associated protein 1-light chain 3 （LC3）-Ⅱ and LC3-Ⅰ in the cells in various groups. Results The MTT assay results showed that the cell survival rate was 75% when the BPAF concentration was 30.0 μmol·L^-1； therefore， this concentration was selected for subsequent senescence induction experiments. Compared with control group， the percentage of SA-β-gal positive cells in BPAF group was significantly increased（P<0.01）， the percentage of the cells at G₁ phase was significantly increased（P<0.01）， and the expression levels of p16， p21， and p53 mRNA and proteins were significantly increased （P<0.01）. Compared with BPAF group， the percentage of SA-β-gal positive cells in Rapa group was significantly decreased （P<0.01）， the percentage of the cells at G₁ phase was significantly decreased （P<0.01）， the expression levels of p16， p21， and p53 mRNA and proteins in the cells were significantly decreased （P<0.01）， and the expression level of the autophagy-related protein p62 in the cells was significantly decreased （P<0.01） while the LC3-Ⅱ/LC3-Ⅰ ratio was significantly increased （P<0.01）. Conclusion High concentrations of BPAF can inhibit the proliferation of the hESCs and induce cellular senescence. Rapa can suppress BPAF-induced cellular senescence， and its mechanism may be related with the activation of autophagy.

Key words: Endometrial stromal cell, Bisphenol AF, Cellular senescence, Rapamycin, Autophagy, Senescence-associated β-galactosidase