Effects and mechanism of dibutyl phthalate on apoptosis of rat Leydig cells through AMPK/mTOR signaling pathway
10.3760/cma.j.cn101441-20200723-00412
- VernacularTitle:邻苯二甲酸二丁酯通过AMPK/mTOR信号通路对大鼠睾丸间质细胞凋亡的影响及其机制研究
- Author:
Xiaowei QU
1
;
Haibin GUO
;
Ke FENG
;
Yanqing XIA
;
Feng WAN
;
Juntao LI
Author Information
1. 河南省人民医院生殖医学中心，郑州 450003
- Publication Type:Journal Article
- Keywords:
Reproduction;
Testis;
Interstitial cells;
Apoptosis;
Dibutyl phthalate
- From:
Chinese Journal of Reproduction and Contraception
2022;42(3):268-276
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To explore the effect of dibutyl phthalate (DBP) on the rat testis Leydig cell apoptosis by AMP activated protein kinase/mammalian rapamycin target protein (AMPK/mTOR) signaling pathway.Methods:Rats with reproductive function impairment were divided into model (DBP) group of 17 rats, model+AMPK inhibitor [DBP+compound C (CC)] group of 17 rats, model+AMPK agonist [DBP+metformin (MF)] group of 17 rats, DBP+AMPK inhibitor+agonist (DBP+CC+MF) group of 17 rats by body mass ranking grouping method. Another 11 rats were taken as the blank group. The blank group and DBP group were intraperitoneally injected with the same amount of normal saline, while DBP+CC group and DBP+MF group were intraperitoneally injected with 20 mg/kg CC and 200 mg/kg MF respectively, and DBP+CC+MF group was intraperitoneally injected with CC and MF once a day for 4 weeks. Luteinizing hormone (LH), follicle-stimulating hormone (FSH) and testosterone (T) were measured by radioimmunoassay. Sperm quality was analyzed by automatic sperm quality analysis system. Leydig cell apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) and flow cytometry. The expressions of AMPK, mTOR, Caspase 3 mRNA and protein, p-AMPK and p-mTOR protein were detected by RT-PCR and Western blotting. Results:The serum level of FSH in DBP+MF group [(9.88±0.67) U/L] increased, while that in DBP+CC group [(6.82±0.60) U/L] decreased compared with DBP group [(9.07±0.52) U/L] (all P<0.001). The serum LH, T levels and sperm concentration, percentage of (a+b) grade sperm in DBP+MF group [(3.97±0.70) U/L, (2.96±0.11) mg/L, (13.15±2.63)×10 6/mL, (22.20±4.13)%], DBP+CC group [(6.52±0.71) U/L, (4.48±0.15) mg/L, (25.47±2.18)×10 6/mL, (45.60±4.78)%] increased compared with DBP group [(4.51±0.75) U/L, (3.25±0.11) mg/L, (16.46±3.40)×10 6/mL, (25.43±4.36)%] (DBP group vs. DBP+MF group PLH=0.038, the other all P<0.001). HE staining showed that the structure of testis in blank group was normal. In DBP group and DBP+CC+MF group, the epithelial cells of seminiferous tubules atrophied and twisted in irregular shape, and the disease became serious in DBP+MF group, and there were a lot of vacuoles around the nucleus. The number of apoptosis, p-AMPK/AMPK protein relative expression and Caspase 3 mRNA and protein relative expression of Leydig cells in DBP+MF group (286.60±30.17, 0.95±0.08, 2.17±0.18, 1.23±0.10) increased, and DBP+CC group (88.00±21.34, 0.42±0.04, 1.35±0.15, 0.54±0.06) decreased compared with those in DBP group (142.40±26.78, 0.70±0.07, 1.85±0.14, 0.80±0.09, all P<0.001). Compared with DBP group (0.45±0.06), the p-mTOR/mTOR of DBP+MF group (0.23±0.04) decreased, and the p-mTOR/mTOR of DBP+CC group (0.84±0.07) increased (all P<0.001). Conclusion:DBP can damage the reproductive system of rats and increase the apoptosis rate of Leydig cells, which may be related to AMPK activation and mTOR inhibition.
