Animal treatment

Female C57BL/6 mice of 27-week-old were purchased from Beijing HFK Bio-Technology Co., Ltd. (Beijing, PR China) and fed freely for one week to adapt to the environment. The vaginal smears of these mice were taken every morning for consecutive seven days. One week later, mice with regular estrous cycles and normal body weight (30.59±1.70g) were chosen and divided into two groups, the control group (CON, n=21) and the metformin-treated group (MET, n=22). Mice in the CON group were fed with food and water freely, and those in the MET group were fed with the chows supplemented with 100mg/kg metformin. Body weight was measured every week, and mice were sacrificed at the end of the experiment. Blood samples were centrifuged at 3000 rpm for 5 min, and serum was collected and stored at −80 °C until analysis. And heart, liver, spleen, kidney, uterus, ovary, and brain were separated and weighed. All procedures and protocols conducted on animals were approved by the ethics committee of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology in the People's Republic of China.