Thymol Preserves Spermatogenesis and Androgen Production in Cisplatin-Induced Testicular Toxicity by Modulating Ferritinophagy, Oxidative Stress, and the Keap1/Nrf2/HO-1 Pathway
Abstract
1. Introduction
2. Materials and Methods
2.1. Molecular Modeling Study
2.2. In Vivo Study
2.2.1. Animal Studies
2.2.2. Experimental Design
2.2.3. Testicular Tissue Collection, Preparation and Sperm Analysis
2.2.4. Histological and Immunohistochemical Analysis
2.2.5. Quantification of Serum Testosterone and Luteinizing Hormone (LH)
2.2.6. Assessment of Oxidative Stress Biomarkers
2.2.7. Assessment of Inflammatory Markers
2.2.8. Assessment of Ferrous Level in Testis
2.2.9. Quantification of mRNA Expression by Real-Time Polymerase Chain Reaction (RT-PCR) for Steroidogenesis and Ferritinophagy- Related Genes
2.2.10. Western Blot Analysis
2.3. In Vitro Studies
2.3.1. Cell Viability Assay
2.3.2. Selectivity Index (SI)
2.4. Statistical Analysis
3. Results
3.1. Molecular Docking Analysis Reveals Thymol’s Interaction with Key Protective Pathways
3.2. Thymol Attenuates CDDP-Induced Reductions in Body Weight Without Affecting Testis Weight
3.3. Thymol Restores Steroidogenic Gene Expression
3.4. Thymol Enhances Spermatogenic Recovery Despite Limited Structural Restoration in CDDP-Treated Animals
3.5. Thymol Protects Against CDDP-Induced Azoospermia and Morphological Defects
3.6. Thymol Modulates Oxidative Stress and Inflammatory Markers Expression Altered by CDDP
3.7. Thymol Ameliorates CDDP-Mediated Suppression of the Keap1/Nrf2/HO-1 Pathway
3.8. Thymol Inhibits Ferritinophagy-Related Genes mRNA Expression and Reduces Iron-Mediated Oxidative Damage
3.9. Thymol Upregulates GPx4, SOD2, and TfR1 Protein Expression in Testicular Tissues
3.10. Thymol Potentiates CDDP Anticancer Activity and Selectivity
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Gene | Primer Sequence (5′ → 3′) | Accession Number |
---|---|---|
ACSL4 | Forward: TCACCATTGTATTGCTGCCT Reverse: GAGCGATATGGACTTCCG | NM_053623.1 |
TFRC | Forward: ATCAAGCCAGATCAGCAT Reverse: GGGTTTTCTGACACTAGC | NM_022712 |
SLC7A11 | Forward: GAGCCACCTGGGCATGAGAA Reverse: CCACAGGCAGACCAGAACAC | NM_001107673 |
17β-HSD | Forward: CAACCTGCTCCCAAGTCA Reverse: AACCCCTACTCCCGAAGA | NM_054007.1 |
3β-HSD | Forward: GGTGCAGGAGAAAGAACT Reverse: GGCATCCAGAATATCTCC | NM_001007719.3 |
StAR | Forward: TTGGGCATACTCAACAACCA Reverse: CACCAGTTCTTCATAGAGTC | NM_031558 |
NCOA4 | Forward: ACGCGAGCTCCTCAAGTATT Reverse: AGTCCTGTGGGTTGGTACTG | NM_019744 |
Protein | Binding Energy (Kcal/mol) | Amino Acid Residues of Interaction | Types of Bonds (Distances Å) |
---|---|---|---|
HO-1 | −4.63 | Arg 136 | H-bond (2.18) |
Ala 28 | Pi-alkyl (4.73) | ||
Phe 207 | Pi-alkyl (4.42) | ||
Phe 214 | Pi-alkyl (4.89) | ||
ACSL4 | −4.76 | Glu 470 | H-bond (2.52) |
Leu 468 | Pi-alkyl (5.28) | ||
Lys 690 | Pi-alkyl (5.26) | ||
Leu 690 | Pi-cation (4.91) |
Body Weight Changes (g) | Testis Weight (g) | Testosterone (ng/mL) | LH (mIU/mL) | |
---|---|---|---|---|
Control | 17.89 ± 4.56 | 1.6 ± 0.17 | 7.86 ± 0.55 | 79.69 ± 7.09 |
CDDP (8 mg/kg) | −41.00 ± 17.59 a | 1.62 ± 0.16 | 3.81 ± 0.30 a | 23.49 ± 3.31 a |
CDDP (8 mg/kg) + Thymol (60 mg/kg) | −7.50 ± 8.01 a,b | 1.43 ± 0.32 | 6.07 ± 0.70 a,b | 64.9 ± 4.56 a,b |
Thymol (60 mg/kg) | 15.25 ± 3.01 b | 1.73 ± 0.20 | 7.57 ± 0.63 b | 75.06 ± 7.31 b |
MDA (nmol/g Tissue) | SOD (U/g Tissue) | GPX (U/g Tissue) | TNF-α (pg/mL) | IL-6 (pg/mL) | |
---|---|---|---|---|---|
Control | 111 ± 9.86 | 104.0 ±17.82 | 1.80 ± 0.09 | 33.19 ± 3.51 | 59.69 ± 5.84 |
CDDP (8 mg/kg) | 210 ± 7.50 a | 57.00 ± 4.47 a | 1.10 ± 0.07 a | 62.75 ± 7.71 a | 34.91 ± 2.89 a |
CDDP (8 mg/kg) + Thymol (60 mg/kg) | 187 ± 12.40 a,b | 77.00 ± 2.09 a,b | 1.71 ± 0.15 a,b | 42.0 ± 1.69 a,b | 44.52 ± 4.21 a,b |
Thymol (60 mg/kg) | 101 ± 12.1 b | 107.4 ± 8.23 b | 1.56 ± 0.25 b | 35.29 ± 6.37 b | 55.40 ± 4.56 b |
Treatment | IC50 MCF-7 | IC50 MCF10A | Selectivity Index (SI) |
---|---|---|---|
CDDP | 5.35 ± 1.55 | 6.23 ± 04 | 1.16 |
Thymol | 108.35 ± 15.48 | >1000 | ND |
CDDP + Thymol | 4.29 ± 1.58 | 19.43 ± 1.65 | 4.51 |
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Badr, A.M.; Aloyouni, S.; Mahran, Y.; Henidi, H.; Elmongy, E.I.; Alsharif, H.M.; Almomen, A.; Soliman, S. Thymol Preserves Spermatogenesis and Androgen Production in Cisplatin-Induced Testicular Toxicity by Modulating Ferritinophagy, Oxidative Stress, and the Keap1/Nrf2/HO-1 Pathway. Biomolecules 2025, 15, 1277. https://doi.org/10.3390/biom15091277
Badr AM, Aloyouni S, Mahran Y, Henidi H, Elmongy EI, Alsharif HM, Almomen A, Soliman S. Thymol Preserves Spermatogenesis and Androgen Production in Cisplatin-Induced Testicular Toxicity by Modulating Ferritinophagy, Oxidative Stress, and the Keap1/Nrf2/HO-1 Pathway. Biomolecules. 2025; 15(9):1277. https://doi.org/10.3390/biom15091277
Chicago/Turabian StyleBadr, Amira M., Sheka Aloyouni, Yasmin Mahran, Hanan Henidi, Elshaymaa I. Elmongy, Haya M. Alsharif, Aliyah Almomen, and Sahar Soliman. 2025. "Thymol Preserves Spermatogenesis and Androgen Production in Cisplatin-Induced Testicular Toxicity by Modulating Ferritinophagy, Oxidative Stress, and the Keap1/Nrf2/HO-1 Pathway" Biomolecules 15, no. 9: 1277. https://doi.org/10.3390/biom15091277
APA StyleBadr, A. M., Aloyouni, S., Mahran, Y., Henidi, H., Elmongy, E. I., Alsharif, H. M., Almomen, A., & Soliman, S. (2025). Thymol Preserves Spermatogenesis and Androgen Production in Cisplatin-Induced Testicular Toxicity by Modulating Ferritinophagy, Oxidative Stress, and the Keap1/Nrf2/HO-1 Pathway. Biomolecules, 15(9), 1277. https://doi.org/10.3390/biom15091277