环磷酰胺处理雄性小鼠后不同时间生殖损伤及致病机制OA

Objective To establish an oligoasthenospermia(OAS)mouse model induced by cyclophosphamide(CTX),determine the optimal modeling time point,and provide a suitable experimental model for further OAS research.In addition,to predict potential pathogenic targets and mechanisms using network pharmacology and to validate them ex-perimentally in vivo.Methods Forty-eight specific pathogen-free(SPF)6-week-old male ICR mice were ran-domly divided into a control group and a model group.The model group received intraperitoneal injections of cyclophos-phamide(40 mg/kg),while the control group received normal saline,once daily for 5 consecutive days.After completion of CTX administration,mice were maintained for 14,28,35,and 42 days.Body weight was recorded,and mice were sacrificed after orbital venous blood collection.Testes were harvested and weighed to calculate the reproductive organ in-dex,and sperm counts were determined.Histopathological changes in testicular tissue were examined using hematoxylin-eosin(HE)staining,and sperm quality parameters were assessed.Serum reproductive hormone levels were measured by ELISA to evaluate the CTX-induced OAS model.Network pharmacology and molecular docking approaches were applied to explore the underlying pathogenic mechanisms,and key targets and signaling pathways were further validated in vivo.Results Compared with the control group,no significant differences were observed in body weight or testicular index at 14,28,35,or 42 days after completion of CTX treatment.However,sperm counts at 14 and 28 days were significantly reduced in the model group compared with controls.At 28 days,serum testosterone,luteinizing hormone(LH),and in-hibin B levels were significantly lower in the model group(P＜0.05).At 35 days,testosterone and inhibin B levels were significantly decreased(P＜0.05),LH levels were increased,and no significant difference was observed in follicle-stimulating hormone(FSH)levels.At 42 days,testosterone,LH(P＜0.05),and inhibin B levels were lower,whereas FSH levels were higher than those in the control group.Gene Ontology(GO)enrichment analysis based on network phar-macology revealed that the core targets epidermal growth factor receptor(EGFR)and cytochrome P450 family 19 subfamily A member 1(CYP19A1)were mainly involved in biological processes related to aldosterone,cortisol,and testosterone bi-osynthesis.Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis indicated that these targets were closely associated with metabolic pathways,steroid hormone biosynthesis,and the hypoxia-inducible factor-1(HIF-1)signaling pathway.Conclusion Intraperitoneal injection of cyclophosphamide at 40 mg/(kg·d)for 5 consecutive days,with evaluation at 28 days after treatment,represents the optimal time point for establishing a CTX-induced oli-goasthenospermia mouse model.Network pharmacology,molecular docking,and in vivo experiments suggest that CTX-induced oligoasthenospermia may be associated with targets such as EGFR and CYP19A1 and involvement of the HIF-1 signaling pathway.