Isatin improves oligoasthenospermia caused by busulfan by regulating GSH/GPX4 axis to inhibit ferroptosis

Abstract

Introduction: Ferroptosis, induced by iron overload and an imbalance in redox homeostasis, promotes the generation of reactive oxygen species (ROS), leading to iron-dependent lipid peroxides (LPO) and oxidative stress. Lipid peroxidation induced by reactive oxygen species is essential for the progression of spermatogenesis. However, its imbalance can lead to reproductive system damage and oligoasthenospermia, a critical cause of oligoasthenospermia. Isatin (ISA) is a naturally occurring compound that is widely distributed in lobsters, crustaceans, shellfish and various plants. It exhibits significant antioxidant and anti-aging properties, suggesting its potential as a therapeutic agent for the treatment of oligoasthenospermia. This study aimed to investigate the effects and mechanisms of ISA on oligoasthenospermia and to elucidate the underlying molecular pathways.

Methods: All mice were divided into normal group, model group and treatment group. Both model group and treatment group received a single intraperitoneal injection of 30 mg/kg BUS to create the model of oligoasthenospermia. After 2 weeks, the treatment group received different doses of 25, 50 and 100 mg/kg ISA by gavage for 28 days, and then mice were sacrificed and tested.

Results: The results demonstrated that ISA effectively reversed busulfan-induced reproductive system damage in mice. This included the restoration of testicular histomorphology, improvement in sperm concentration and motility, regulation of serum sex hormone levels, and normalization of various oxidative indices in testicular tissue. Furthermore, ISA successfully reversed testicular ferroptosis by restraining the translocation of nuclear factor erythroid 2-related factor 2 (NRF2) into the nucleus and improved oligoasthenospermia through the glutathione (GSH)/glutathione peroxidase 4 (GPX4) axis.

Discussion: ISA was found to effectively ameliorate oligoasthenospermia in mice, presenting a potential therapeutic option for patients with this condition.

Keywords: GSH/GPX4 axis; ferroptosis; isatin; oligoasthenospermia; spermatogenesis.

FIGURE 1

Effects on testicular organ index and sperm parameters. (A): Morphology of the testes from 8-week-old mice after treatment with saline, BUS 30 mg/kg, BUS + ISA 50 mg/kg, (B): Testicular weight, (C): Ratio of testis weight to body weight, (D): Sperm motility, (E): Sperm concentration, (F): Rapid progressive motility, (G): Graph of sperm count, scale bar: 200 μm *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, n ≥ 8.

FIGURE 2

Histological examination of mice testes and epididymis. (A): Morphology of testis. Blue arrow: vacuolization. (B): Morphology of epididymis tail. Scale bars, 50 μm.

FIGURE 3

(A): Changes of the cytoskeleton in different groups by immunofluorescence, scale bar: 100 μm. (B): Changes of the cytoskeleton in sperm of different groups by immunofluorescence, scale bar: 50 μm.

FIGURE 4

Immunofluorescent staining of ZO-1 protein in the testes of different groups. Green: ZO-1, Blue: DAPI, scale bar: 100 μm.

FIGURE 5

Effect of ISA on hormone levels. (A): T concentration, (B): FSH concentration. (C): LH concentration. *P < 0.05, **P < 0.01, ***P < 0.001, n ≥ 3.

FIGURE 6

Effects of ISA on oxidative indices. (A): MDA concentration, (B): ROS concentration. (C): SOD concentration. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, n ≥ 3.

FIGURE 7

High-throughput sequencing. (A): The number of differentially expressed genes (B): Volcano map of differentially expressed genes between oligoasthenospermia and ISA treatments. (C): GO enrichment analysis. (D) Verification of sequencing results by RT-qPCR. **P < 0.01, ***P < 0.001, ****P < 0.0001, n = 3.

FIGURE 8

Effect of ISA on System Xc-. (A): GSH activity. (B): Immunofluorescence of GPX4. (C, D): Western blot of System Xc-relative proteins. **P < 0.01, ****P < 0.0001, n = 3, scale bar: 200 μm.

FIGURE 9

Effect of ISA on transport of Fe²⁺ and PUFA. (A): Total iron concent. (B): Immunofluorescence of TFR1. (C, D): Western blot of Fe²⁺ and PUFA transport relative proteins. Ns: no significance, ***P < 0.001, ****P < 0.0001, n = 3, scale bar: 100 μm.

FIGURE 10

Effect of ISA on Nrf2-HMOX1 pathway. (A, B): Western blot of Nrf2-HMOX1 pathway relative proteins. (C): Immunofluorescence of Nrf2. (D): Molecular docking of ISA with Nrf2. Grid score is −8.6349. (E): Interaction plot between ISA and Nrf2. (F): Interactive visualization of ISA binding to Nrf2 Val465, Tyr520, and Cys513 sites via hydrogen bonding. **P < 0.01, ****P < 0.0001, n = 3, scale bar: 100 μm.

FIGURE 11

Possible mechanism of ISA on oligoasthenospermia mice induced by BUS.