Morin hydrate protects against cisplatin-induced testicular toxicity by modulating ferroptosis and steroidogenesis genes' expression and upregulating Nrf2/Heme oxygenase-1

Abstract

Cisplatin is a widely used, effective chemotherapy drug. However, its application is often limited by severe side effects, including testicular toxicity. Cisplatin-induced testicular damage is primarily driven by oxidative stress and inflammation. Ferroptosis has recently been identified to contribute to cisplatin-testicular toxicity. Morin hydrate (MH) is a naturally occurring flavonoid known for its powerful antioxidant, anti-inflammatory, and anti-apoptotic properties. The study was designed to evaluate the protective effects of MH against cisplatin-induced testicular toxicity in Wistar albino rats. Rats were given MH 50 mg/kg, p.o. daily for fourteen days, seven days before the injection of cisplatin 8 mg/kg. Assessment of sperm quality, testosterone, luteinizing hormone levels, and oxidative stress markers were carried out. Also, steroidogenesis and ferroptosis-related gene expressions were assessed. Results: Our findings demonstrated that MH significantly corrected the antioxidant/oxidant balance, evidenced by increased superoxide dismutase, glutathione peroxidase, and Nrf2/heme oxygenase-1 (HO-1) expression and reduced malondialdehyde in testicular tissue. Also, MH ameliorated the negative changes in sperm quality, hormone levels, and testicular histology induced by cisplatin, and this was accompanied by upregulation of steroidogenesis gene expressions (17β-HSD, 3β-HSD, and star). Moreover, MH inhibited cisplatin-induced ferroptosis via the modulation of ferroptosis genes' expression (ACSL4, SLC7A11, and TFRC) and the reduction of iron accumulation in testicular tissue. Conclusion: MH effectively protected against cisplatin-induced testicular toxicity by reducing oxidative stress and inhibiting ferroptosis signalling. This study points out that MH might mitigate iron-mediated apoptosis through the downregulation of Nrf2/HO-1 signaling, providing a potential therapeutic strategy for preventing infertility in male patients undergoing cisplatin chemotherapy.

Keywords: Cisplatin; Ferroptosis; Morin hydrate; Oxidative stress; Steroidogenesis; Testicular toxicity.

Fig. 1

Effect of morin hydrate and cisplatin on changes in body weights and testicular weight. data are expressed as mean ± SEM, n = 6. a and b indicate statistical differences from the control and CIS group, respectively, (p < 0.05) using ANOVA followed by Tukey Kramer as a post hoc test. CIS cisplatin, CM cisplatin and morin hydrate, M morin hydrate.

Fig. 2

Effects of Morin Hydrate and Cisplatin on Sperm Morpholology and Testosterone and Luteinizing Hormone Levels in Rats. Photomicrograph of Sperm in Different Groups. (A) control epidydimal sperm showing normal rat sperm, head (black arrow), hook (green arrow), straight tail (red arrow). (B) epidydimal sperm treated with cisplatin displaying deformed rat sperm, distorted head (black arrow), and bent tail (red arrow). (C) photomicrograph of epidydimal sperm in cisplatin and morin hydrate treated group showing some deformed rat sperm, bent tail (red arrow), flattened hook (green arrow). (D) photomicrograph of epidydimal sperm treated with morin hydrate revealing healthy rat sperm, head (black arrow), hook (green arrow), straight tail (red arrow) (H&E-1000X). (E) Testosterone hormone level in different groups. (F) LH hormone level in different groups. Data were expressed as mean ± SEM (n = 6). a, b and c indicate statistical differences from the control, CIS and CM group, respectively (p < 0.05) using ANOVA followed by Tukey Kramer as a post hoc test. CIS cisplatin, CM cisplatin and morin hydrate, M morin hydrate, LH Luteinizing hormone.

Fig. 3

Photomicrograph of control testes showing normal seminiferous tubules, interstitial tissue (IT), Leydig cells (black arrow), spermatogonia (green arrow), spermatocyte A (blue arrow), spermatocyte B (yellow arrow), spermatozoa (brown arrow), and Sertoli cell (red arrow). Photomicrograph of testes tissues from rat in cisplatin group displaying minimized seminiferous tubules and necrotic foci (N), while the testes in cisplatin and morin hydrate treated group showing improved seminiferous tubules (ST), necrotic foci (N), testes from morin hydrate treated group revealing healthy seminiferous tubules, IT, spermatogonia (green arrow), spermatocyte A (blue arrow), spermatocyte B (yellow arrow), and spermatozoa (brown arrow) (H&E-400X).

Fig. 4

Morin hydrate attenuates oxidative stress and inflammatory response in cisplatin-induced testicular toxicity in rats. Treatment of rats with 8 mg/kg CIS on day 7 decreased (a) SOD & (b) GPX in the CIS group, increased testes, (c) MDA and (d) TNF-α, and decreased (e) IL-6 levels. Data are mean ± SEM (n = 6). a, b, and c were found to be statistically significant when compared to the control, CIS, and CM groups, respectively (P < 0.05), using ANOVA followed by Tukey-Kramer as a post hoc test. CIS cisplatin, CM cisplatin and morin hydrate, M morin hydrate, MDA malondialdehyde, SOD superoxide dismutase, GPX glutathione peroxidase, TNF-α tumor necrosis factor-α, IL-6 interleukin-6.

Fig. 5

Photomicrographs of Nuclear factor erythroid 2-related factor-2 (Nrf2) and Hemeoxygenase-1 (HO-1), immunostained testis sections, Scale bar 25 µ and 50 µ, respectively. (A) The control rat showed a normal immune reaction, and (B) the section from a rat exposed to CIS showed a marked decrease in the immune reactivity in testis cells. (C) Testis section from rats exposed to CIS and morin, showing recovery of the immune reactivity in the testis cells. (D) Quantitative immunoexpression of Nrf2. Data are mean ± SEM (n = 6). a and b were found to be statistically significant compared to the control and CIS groups, respectively, P < 0.05 using ANOVA followed by Tukey-Kramer as a post hoc test. CIS cisplatin, CM cisplatin and morin hydrate. Black Circle: high expression of Nrf-2, Red Star: High expression of HO-1.

Fig. 6

Morin Hydrate Increased (a) STAR, (b) 3β-HSD, (c) 17β-HSD, (d) TFRC, and (f) SLC7A11 mRNA, and Suppressed that of (g) ACSL4 in CIS-induced Testicular Toxicity in Rats. Data are mean ± SEM, (n = 3). a and b were found to be statistically significant when compared to the control and CIS groups, respectively, P < 0.05 using ANOVA followed by Tukey Kramer as a post hoc test. (h) MH decreased the ferrous overload induced by CIS in rats. Data were expressed as mean ± SEM (n = 6). a, b, and c were found to be statistically significant when compared to the control, CIS, and CM group, respectively, P < 0.05 using ANOVA followed by Tukey Kramer as a post hoc test. CIS cisplatin, CM cisplatin and morin hydrate, M morin hydrate.

Fig. 7

The effect of morin hydrate on the cytotoxicity of cisplatin against noncancerous breast cancer cell line (MCF10a) and human breast cancer cell line (MCF-7) using MTT Assay. CIS cisplatin, CM cisplatin and morin hydrate, M morin hydrate.

Fig. 8

A schematic diagram of the effect of morin on cisplatin-induced testicular toxicity. HO-1 Hemeoxygenase-1, MDA malondialdehyde, SOD superoxide dismutase, GPX glutathione peroxidase, TNF-α tumor necrosis factor-α, IL-6, interleukin-6, LH Luteinizing hormone, ACSL4 acyl-CoA synthetase long-chain family 4, TFRC transferrin receptor 1 (Tfr1) gene, SLC7A11 cystine/glutamate antiporter, 17β-HSD 17β-Hydroxysteroid dehydrogenases, 3β-HSD 3β-Hydroxysteroid dehydrogenases, STAR steroidogenic acute regulatory protein.