Regulation of Keap-1/Nrf2/AKT and iNOS/NF-κB/TLR4 signals by apocynin abrogated methotrexate-induced testicular toxicity: Mechanistic insights and computational pharmacological analysis.

AIM

Male reproductive toxicity is becoming of growing significance due to clinical chemotherapy usage. Methotrexate (MTX) is an anti-folate used on a large scale for different tumors and autoimmune conditions. Despite its wide clinical use, MTX is associated with severe testicular intoxication. The exact underlying mechanism is unclear.

METHODS

Our study was conducted to explore the pathogenesis mechanism of MTX-induced testicular damage and the potential testicular protective effects of apocynin (APO) on testicular injury induced by single i.p. MTX (20 mg/kg). APO was administered orally (100 mg/kg) for ten days.

RESULTS

As compared to rats given MTX alone, co-administration of MTX with APO demonstrated multiple beneficial effects evidenced by a marked increase in testosterone, FSH, and LH and significantly restored testes histopathological alterations. Mechanistically, APO restored antioxidant status through up-regulation of Nrf2, cytoglobin, PPAR-γ, SIRT1, AKT, and p-AKT, while effectively lowering Keap-1. Moreover, APO significantly attenuated inflammation by down-regulating NF-κB-p65, iNOS, and TLR4 expressions confirmed by in-silico evidence. Additionally, network pharmacology analysis, a bioinformatics approach, was used to decipher various cellular processes' molecular mechanisms.

SIGNIFICANCE

The current investigation proves the beneficial effects of APO in MTX-associated testicular damage through activation of cytoglobin, Keap-1/Nrf2/AKT, PPAR-γ, SIRT1, and suppressing of TLR4/NF-κB-p65 signal. Our data collectively encourage extending the investigation to the clinical setting to explore APO effects in MTX-treated patients.