Protective effect of Huangqi-Guizhi-Wuwutang against cyclophosphamide-induced spermatogenesis dysfunction in mice by promoting steroid hormone biosynthesis

The primary adverse effect of cyclophosphamide (CTX) chemotherapy to cancer in male patients of reproductive age is a significant impairment of reproductive function. Huangqi-Guizhi-Wuwutang (HGW), a classical traditional Chinese medicine formula, is designed to exert a salutary effect on qi and promote blood circulation, thereby eliminating blood stasis and promoting spermatogenesis, and it has been recorded as a treatment for oligospermia. However, its potential in mitigating the adverse impact of CTX on male spermatogenesis remains unexplored. The present investigation aims to elucidate the potential protective effects and underlying mechanisms of HGW against CTX - induced spermatogenic dysfunction in mice. C57BL/6J mice that received intraperitoneal injections of CTX were employed to induce dysfunction in spermatogenesis. Pharmacological experimentation was conducted to evaluate the potential effect of HGW in mitigating spermatogenic toxicity induced by CTX. Additionally, mRNA sequencing was utilized to identify genes exhibiting differential expression between the untreated and HGW treated groups, thereby elucidating the comprehensive underlying mechanisms involved. The most significantly enriched potential pathways were identified and subsequently validated in vivo. The administration of HGW significantly increased the testicular index, epididymal index, sperm concentration and sperm motility in mice with spermatogenesis dysfunction. Furthermore, HGW effectively ameliorated the observed tissue damage in pathological sections of the testes, elevated serum levels of inhibin B (INH-B) and testosterone (T), while reduced levels of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) compared to the Model group. An analysis on the genes associated with HGW treatment for CTX-induced oligospermia revealed a significantly enhanced functional enrichment in steroid hormone biosynthesis signaling pathways. Experimental results demonstrated that HGW significantly enhanced cellular activity and T levels in TM3 cell injury model using phosphoramide mustard (PM, the active ingredient of CTX in vitro) and upregulates protein expression of key enzymes involved in hormonal steroid synthesis within testicular tissues, including 3β-hydroxysteroid dehydrogenase (3β-HSD), steroidogenic acute regulatory protein (StAR), cytochrome P450 family 17 subfamily A member 1 (CYP17A1) and ytochrome P450 family 11 subfamily A member 1 (CYP11A1). Our data has demonstrated the effectiveness of HGW in improving CTX-induced spermatogenic dysfunction by enhancing hormonal steroid synthesis in a model system, indicating the potential chemoprotective effects of HGW against CTX-induced spermatotoxicity.