主 办:北 京 中 医 药 大 学
ISSN 1006-2157 CN 11-3574/R

JOURNAL OF BEIJING UNIVERSITY OF TRADITIONAL CHINESE MEDICINE ›› 2021, Vol. 44 ›› Issue (6): 510-518.doi: 10.3969/j.issn.1006-2157.2021.06.005

• Chinese Medicinal Pharmacology • Previous Articles     Next Articles

Promotion of kidney-tonifying therapy on maturation of mouse oocyte in vitro based on PI3K/AKT/mTOR signaling pathway*

Tong Xue1, Liang Xiao1, Su Zi2, Yan Mengxuan1, Duan Yancang1,3,4#   

  1. 1 School of Integrated Traditional Chinese and Western Medicine, Hebei University of Chinese Medicine, Hebei 050091, China;
    2 School of Basic Medicine, Hebei University of Chinese Medicine, Hebei 050091, China;
    3 Hebei Collaborative Innovation Center of Integrated Chinese and Western Medicine on Reproductive Disease, Hebei 050091, China;
    4 Hebei Key Laboratory of Integrative Medicine on Liver-kidney Patterns, Hebei 050091, China
  • Received:2020-12-07 Online:2021-06-30 Published:2021-06-25
  • Contact: Prof. Duan Yancang, Ph.D., Doctoral Supervisor. Hebei University of Chinese Medicine. No. 326 Xinshi South Road, Qiaoxi District, Shijiazhuang 050091. E-mail: duanyancang@163.com
  • Supported by:
    National Natural Science Foundation of China (No. 81973893)

Abstract: Objective To explore the promotion of kidney-tonifying therapy on maturation of oocyte invitro and its relationship with PI3K/AKT/mTOR signaling pathways. Methods Female rats(6-8 weeks old) were given Bushen Tiaojing Fang Ⅲ (Kidney-tonifying Menstruation-regulating Formulas Ⅲ, BSTJF Ⅲ) to prepare serum containing BSTJF Ⅲ. Female mice (3-4 weeks old) were randomly divided into two groups: Group A and Group B. The two groups were given distilled water and a series of BSTJFs(BSTJF Ⅱ and BSTJF Ⅲ) by gavage respectively for 12 days. On the 11th day, all the mice were intraperitoneally injected with 5 IU of gonadotropin each and 48 hours later, the ovaries were removed and cumulus-oocyte complexes(COCs) were taken out for in vitro culture. COCs of Group A were divided into the blank control group and the inhibitor group (25 μmol/L of LY294002), and those of Group B were divided into the kidney-tonifying group (serum containing 10% of BSTJF Ⅲ) and kidney-tonifying + inhibitor group (serum containing 10% of BSTJF Ⅲ+25 μmol/L of LY294002). After 18 hours of culture in vitro, the number of discharged first polar bodies of the two groups were counted under microscope and the discharge rates were calculated. Real-time fluorescence quantitative PCR (RT-PCR) was used to detect the expressions of mRNA of Bcl-2, Bax, PI3K, AKT and mTOR in COCs of all groups. Immunofluorescence staining was used to detect the expressions of Bcl-2, Bax, PI3K, AKT, mTOR, p-PI3K, p-AKT and p-mTOR proteins in COCs of all groups. Results The first polar body discharge rate in the kidney-tonifying group was higher than that in the blank control group (P<0.05), that of the inhibitor group was lower than that of the blank control group (P<0.05), and that of the kidney-tonifying+inhibitor group was lower than that of the kidney-tonifying group (P<0.05). RT-PCR results showed that compared with the blank control group, the expressions of mRNA of Bcl-2, PI3K, AKT, and mTOR and Bcl-2/Bax of COCs increased (P<0.05) and the expressions of Bax mRNA decreased (P<0.05) in the kidney-tonifying group and the expressions of mRNA of Bcl-2, PI3K, AKT, and mTOR and Bcl-2/Bax of COCs decreased (P<0.05) and the expressions of Bax mRNA increased (P<0.05) in the inhibitor group. Compared with the kidney-tonifying group, the expressions of mRNA of Bcl-2, PI3K, AKT, and mTOR and Bcl-2/Bax of COCs decreased (P<0.05), and the expressions of Bax mRNA increased (P<0.05) in the kidney-tonifying+inhibitor group. Immunofluorescence staining results showed that there was no significant difference in the expressions of total protein of PI3K, AKT, and mTOR between the groups (P>0.05). Compared with the blank control group, the expression of Bcl-2 protein, p-PI3K/PI3K, and p-AKT/AKT of COCs increased (P<0.05) while the expression of Bax protein was decreased (P<0.05) in the kidney-tonifying group, and the expression of Bcl-2 protein, p-PI3K/PI3K, p-AKT/AKT, and p-mTOR/mTOR of COCs decreased (P<0.05 ) while the expression of Bax protein was increased (P<0.05) in the inhibitor group. Compared with the kidney-tonifying group, the expression of Bcl-2 protein, p-PI3K/PI3K, p-AKT/AKT, and p-mTOR/mTOR of COCs decreased (P<0.05) and Bax protein expression increased (P<0.05) in the kidney-tonifying+inhibitor group. Conclusion BSTJFs can promote mouse oocyte maturation by regulating the PI3K/AKT/mTOR signaling pathways, up-regulating the mRNA and ratio of protein expressions of Bcl-2 and Bax, and inhibiting granulosa cell apoptosis.

Key words: a series of Bushen Tiaojing Fang, cumulus oocyte complex, PI3K/AKT/mTOR signaling pathway, mice, rats

CLC Number: 

  • R285.5