Thymoquinone protects the testes of hypothyroid rats by suppressing pro-inflammatory cytokines and oxidative stress and promoting SIRT1 testicular expression

Abstract

Background: Hypothyroidism has been linked to many testicular structural and dysfunctional changes in males. Thymoquinone (TQ) has shown a potent testicular protective effect through its antioxidant, anti-inflammatory, antiapoptotic, fertility-enhancing, and endocrine modulatory activities. Objectives: This study aimed to investigate the efficacy of TQ in preserving the testicular structure of a model of experimentally induced hypothyroidism in rats and identify the mechanism behind this effect. Materials and methods: Propylthiouracil (PTU) was used to induce hypothyroidism in adult male Wistar rats, who were then treated with TQ (50 mg/kg/body weight) for 4 weeks and compared to the untreated rats. Thyroid hormonal profile, oxidants/antioxidants profile, and serum testosterone levels were assessed. Gene expression and immune expression of SIRT1 and pro-inflammatory cytokines TNF-α and NF-κB were also assessed in the testicular tissue. Results: TQ administration successfully improved PTU-induced disturbance in the thyroid hormonal profile (T3, T4, and TSH), serum testosterone level, and pancreatic antioxidants compared to the untreated hypothyroid group. TQ significantly downregulated (p = 0.001, p ˂ 0.001) TNF-α and NF-κB transcription, while it significantly upregulated (p = 0.01) SIRT1 transcription in the testes of hypothyroid rats. TQ markedly relieved the histopathological testicular changes induced by PTU and significantly increased (p = 0.002, p = 0.01) the sectional area of seminiferous tubules and germinal epithelial height, respectively. TUNEL-positive apoptotic germinal cells were significantly decreased (p ˂ 0.001), while PCNA-positive proliferating germinal cells and androgen receptor expression were significantly increased (p ˂ 0.001) in the testes of TQ-treated hypothyroid rats. Conclusion: Thymoquinone could limit the hypothyroidism-induced structural changes in the testis, mostly through the upregulation of SIRT1 expression, which seems to mediate its promising antioxidant, anti-inflammatory and antiapoptotic effects that were evident in this study. Therefore, TQ is recommended as an adjuvant safe supplement in managing hypothyroidism, especially in males.

Keywords: NF-κB; PCNA; SIRT1; TNF-α; hypothyroidism; rat; testis; thymoquinone.

FIGURE 1

Effects of thymoquinone administration on the histological structure of the testes of hypothyroid rats (A). Control (a) and TQ-treated (b) groups show intact structure, while the hypothyroid group (c) shows smaller and irregular ST, widening of the interstitial space, and reduced number and vaculation of interstitial cells of Leydig (black arrow). The germinal epithelium showed multiple spaces and gaps between the cells, detachment, and reduced spermatocytes density (red arrow). The ST showed reduced number and scattered spermatids (star). These changes are less frequently observed in the hypothroid + TQ group (d). The graph shows the cross-sectional area of the seminiferous tubules (B) and the height of the germinal epithelium (B) of the studied groups. TQ, Thymoquinone. NC, Normal control. Data are presented as the mean ± SD. A comparison between groups was done using one-way ANOVA test followed by Bonforoni post hoc test (n = 6).

FIGURE 2

Effects of thymoquinone (TQ) administration on the immunoexpression of PCNA (A) and TUNEL (B). Graphs that show the quantitative assessment of the number of PCNA-positive (C) and TUNEL-positive cells (D) are presented. TQ, thymoquinone. NC, normal control. Data are presented as the mean ± SD. Comparison between groups was carried out using one-way ANOVA test followed by Bonferroni post hoc test (n = 6).

FIGURE 3

Effects of thymoquinone (TQ) administration on the expression of NF-κB (A) and TNF-α (B). Representative pictures of immunoexpression of each marker are presented (a–d), in addition to graphs that show the quantitative assessment of immunoexpression (e) using Image-Pro Plus software version 6.0 and gene expression (f) assessed using RT-PCR. TQ, thymoquinone. NC, normal control. Data are presented as the mean ± SD. A comparison between groups was made using one-way ANOVA test followed by Bonferroni post hoc test (n = 6).

FIGURE 4

Effects of thymoquinone (TQ) administration on the expression of SIRT (A) and AR (B). Representative pictures of immunoexpression of each marker are presented (a–d), in addition to graphs that show the quantitative assessment of immunoexpression. (e) using Image-Pro Plus software version 6.0 and gene expression (f) assessed using RT-PCR. TQ: thymoquinone. NC, normal control. AR, androgen receptors. Data are presented as the mean ± SD. A comparison between groups was made using one-way ANOVA test followed by Bonferroni post hoc test (n = 6).