Abstract

Background

   Valproic Acid (VPA), a widely used anticonvulsant, is known to induce
   oxidative stress, contributing to male infertility. This study explores
   the potential of micronutrient antioxidants to improve fertility in
   VPA-treated individuals.

Methods

   Six-week-old male mice were treated with VPA and supplemented with
   antioxidants, including l-Arginine (120 mg/kg), N-Acetylcysteine (NAC)
   (2 mg/kg), Taurine (200 mg/kg), L-Tryptophan (0.5 mg/kg), Zinc chloride
   (ZnCl2) (1.5 mg/kg), and Selenium (0.5 mg/kg). The dosing regimen
   lasted for 34 days. Sperm quality, oxidative stress, and inflammatory
   biomarkers were assessed through gene expression analysis, western
   blotting, histological assessments, TUNEL assays, and
   immunohistochemistry. Additionally, GC-2spd(ts) and HepG2 cell lines
   were used to examine the testicular and systemic effects of VPA and
   antioxidants. Network pharmacology was applied to identify key
   molecular targets and pathways.

Results

   Antioxidant supplementation significantly improved sperm count, with
   l-Arginine showing an approximately 296.1 % increase, NAC a 270.7 %
   increase, and Taurine a 255.9 % increase compared to the VPA-only
   group. Furthermore, antioxidants enhanced semen volume, testosterone
   levels, sperm motility, morphology, and viability. Gene expression
   analysis revealed significant upregulation of key oxidative
   stress-related proteins such as SOD1, HO-1, NRF2, and NQO1. Western
   blot and histological analyses showed a reversal of oxidative stress
   and preservation of seminiferous tubule integrity. TUNEL assays
   demonstrated a reduction in apoptotic damage, and IHC confirmed an
   increase in HO-1 and SOD1. In vitro studies with GC-2spd(ts) and HepG2
   cells confirmed that antioxidants alleviated VPA-induced oxidative
   stress. Network pharmacology identified key molecular targets, such as
   GPX4, SOD1, HO-1, and NRF2, which are involved in oxidative stress,
   apoptosis, and inflammation pathways, that were modulated by
   antioxidants.

Conclusion

   Micronutrient antioxidants effectively reduce VPA-induced oxidative
   stress and improve male fertility. These results suggest that
   antioxidant supplementation could be a promising strategy to mitigate
   oxidative damage and enhance fertility in individuals undergoing VPA
   therapy.

   Keywords: Valproic acid-induced infertility, NRF2/HO-1 pathway,
   Micronutrient antioxidants, Oxidative stress, GC-2spd(ts) and HepG2
   cells, Network pharmacology

Graphical abstract

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Highlights

     * •
       Antioxidants improve sperm count in VPA-treated mice.
     * •
       l-Arginine, NAC, Taurine, and L-Tryptophan enhanced sperm motility
       and morphology.
     * •
       Antioxidant supplementation reduced oxidative stress and testicular
       damage in both animal and cell models.
     * •
       Gene expression showed increased levels of SOD1, HO-1, and NRF2.
     * •
       Network pharmacology identified key targets like SOD1, HO-1, and
       GPX4.

1. Introduction

   Valproic Acid (VPA) is a widely prescribed antiepileptic drug,
   recognized for its broad-spectrum anticonvulsant efficacy and
   affordability. It serves as a first-line treatment for epilepsy,
   particularly in young and reproductive-age patients, where it is
   frequently used as initial monotherapy in approximately 20 % of cases
   [[45][1], [46][2], [47][3]]. In addition to epilepsy, VPA is also
   prescribed for the management of migraine, bipolar disorder, neurogenic
   conditions, and certain cancers, with emerging research and ongoing
   clinical trials exploring its potential for other diseases [[48]4].
   Despite its broad therapeutic applications, VPA is linked to multiple
   adverse effects, particularly reproductive toxicity and male
   infertility, raising growing concerns among clinicians and researchers
   [[49]5,[50]6]. Male infertility, affecting approximately 15 % of
   couples worldwide, is aggravated by medications such as VPA, which has
   been shown to impair various aspects of male reproductive health
   [[51]7,[52]8].

   Preclinical and clinical studies indicate that VPA contributes to a
   range of systemic disorders, particularly male infertility, with the
   underlying mechanisms primarily involving oxidative stress,
   mitochondrial dysfunction, and endocrine disruption [[53]9,[54]10]. In
   addition, our previous systematic review, meta-analysis, and network
   pharmacology study comprehensively analysed VPA-induced oxidative
   stress in rodent models, demonstrating a significant increase in
   malondialdehyde (MDA) levels and a concurrent decrease in major
   antioxidant markers [[55]10]. These findings strengthen oxidative
   stress as a central mechanism of VPA-induced reproductive toxicity,
   highlighting the urgent need to explore antioxidant-based therapeutic
   strategies.

   Micronutrient antioxidants have emerged as potential therapeutic agents
   for counteracting oxidative stress and improving sperm function
   [[56]11]. Studies indicate that antioxidants may mitigate oxidative
   damage and enhance sperm count, motility, and morphology
   [[57]11,[58]12]. Previous studies have demonstrated the potential
   reproductive health benefits of several amino acids and compounds,
   including Taurine, NAC, L-Tryptophan, and l-Arginine. Taurine has been
   linked to improved sperm motility and overall reproductive function
   [[59]13]. NAC, known for its antioxidant properties, has shown promise
   in protecting against oxidative stress-related reproductive issues
   [[60]14]. L-Tryptophan, a precursor to serotonin, may play a role in
   regulating reproductive hormones, while l-Arginine is recognized for
   its effects on nitric oxide production, which is essential for optimal
   blood flow in the reproductive system [[61]15,[62]16]. However, no
   research has specifically addressed the efficacy of these compounds in
   protecting against VPA-induced oxidative stress and reproductive
   toxicity.

   GC-2spd(ts) cells, a spermatocyte-like cell line, provide a relevant
   model for exploring local oxidative stress and spermatogenesis
   [[63]17]. Similarly, HepG2 cells have been widely used to study
   oxidative stress mechanisms and cellular toxicity, making them an ideal
   model for investigating the systemic effects of VPA [[64]18]. Given
   that VPA undergoes first-pass metabolism in the liver, HepG2 cells are
   particularly relevant for studying the systemic oxidative stress
   induced by this drug and its effects on cellular function across the
   body, including the reproductive system [[65]19]. Together, these cell
   lines enable a comprehensive approach to understanding the systemic and
   reproductive impacts of VPA and the potential therapeutic role of
   antioxidants in mitigating oxidative stress in male infertility.

   Network pharmacology combines systems biology, bioinformatics, and
   pharmacology to provide a comprehensive understanding of drug actions
   by identifying molecular targets and key signalling pathways [[66]20].
   It constructs interaction networks and performs pathway enrichment
   analyses to reveal how antioxidants may mitigate VPA-induced oxidative
   damage and male infertility.

   Despite growing evidence of the role of oxidative stress in male
   infertility, there is a notable gap in research regarding the
   comprehensive mechanisms of VPA toxicity and the efficacy of
   antioxidants in alleviating VPA-induced reproductive toxicity. This
   study addresses the concern over oxidative stress and male infertility
   induced by VPA, a widely used antiepileptic drug. We provide novel
   insights into how micronutrient antioxidants can mitigate VPA-induced
   infertility by reducing oxidative damage. Our approach is unique, as
   these antioxidants typically studied individually, are proposed here
   for the first time to be used with VPA to counteract its adverse
   effects. Importantly, these nutrients have shown minimal long-term
   toxicity, making them suitable for clinical use without significant
   risks. Given VPA's widespread prescription, especially in Asian
   countries to young children and individuals of childbearing age, our
   study offers a safe and effective strategy to preserve fertility in
   patients. This research fills a critical gap in understanding
   VPA-induced oxidative stress and provides a strong foundation for
   developing therapeutic interventions in male reproductive health.
   Additionally, this is the first study to present a comprehensive
   mechanistic analysis, integrating animal models, HepG2 and GC-2spd(ts)
   cell models, and network pharmacology to explore the molecular pathways
   involved in VPA-induced oxidative stress and male infertility, making
   this study a significant contribution to the field. Given VPA's
   clinical relevance, it could also serves as an ideal model for studying
   the long-term effects of medications on male fertility and developing
   safer therapeutic strategies.

2. Materials and methods

2.1. Materials

   VPA was purchased from Bide Pharmaceutical (Shanghai, China; Cat. No.
   DNU064). In addition, the following key chemicals and reagents were
   used in the study: l-Arginine (Hefei Hongrui Biotechnology Co., Ltd.,
   Hefei, Anhui, China), Taurine (Hebei Qiansheng Biotechnology Co., Ltd.,
   Shijiazhuang, Hebei, China), L-Tryptophan (Shanghai Maclean Biochemical
   Technology Co., Ltd., Shanghai, China), NAC (ACERBLEND Ingredients Co.,
   Ltd., Guangzhou, Guangdong, China), Zinc chloride (ZnCl[2]) (Shanghai
   McLean Biochemical Technology Ltd., Shanghai, China), Selenium
   (Shanghai Yuanye Biotechnology Co., Ltd., Shanghai, China), MDA, SOD
   (Superoxide dismutase), and GPx (Glutathione peroxidase) assay kits
   (Nanjing Jiancheng Technology Co., Ltd., Nanjing, Jiangsu, China).

2.2. Animal experiments

Ethical approval

   All animal experiments were performed in compliance with institutional
   guidelines and were approved by the Institutional Animal Care and Use
   Committee (IACUC) of Chongqing Medical University (Approval No.
   IACUC-CQMU-2025-0079).

2.2.1. Experiment groups and drug administration

   Six-week-old male Kunming (KM) mice (25–30 g), purchased from the
   Laboratory Animal Center of Chongqing Medical University (Chongqing,
   China), were housed in a standard pathogen-free (SPF) facility. Mice
   were randomly assigned to the following experimental groups: (1)
   control group (corn oil), (2) model group (VPA 400 mg/kg), (3)
   VPA + l-Arginine group (l-Arginine 120 mg/kg), (4) VPA + Taurine group
   (Taurine 200 mg/kg), (5) VPA + L-Tryptophan group (L-Tryptophan
   0.5 mg/kg), (6) VPA + NAC group (NAC 2 mg/kg), (7) VPA + ZnCl[2] group
   (ZnCl[2] 1.5 mg/kg), and (8) and VPA + Selenium group (Selenium
   0.5 mg/kg). The doses of antioxidants used in this study were based on
   preliminary experiments conducted on mice, where multiple doses were
   tested to determine the most effective and clinically relevant doses
   for mitigating VPA-induced oxidative stress. We focused on using low
   doses to maximize therapeutic benefits while ensuring safety for
   clinical use. Although higher doses have been used in previous studies
   in different contexts, we selected doses that provide efficacy without
   significant toxicity, with references included to support the dose