Riboflavin recovery of spermatogenic dysfunction via a dual inhibition of oxidative changes and regulation of the PINK1-mediated pathway in arsenic-injured rat model

Abstract

Arsenic trioxide (As2O3) poisoning and associated potential lesions are of a global concern. Inversely, riboflavin (vitamin B2, VB2) as a component of flavoproteins could play a vital role in the spermatogenic enzymatic reactions. Thus, this research aimed to explore potential beneficial roles of VB2 during As2O3-injured-toxicity. Rats were randomly allocated into 4 groups (n=8/group) and challenged as follows (for 30 days continuously): Group 1 received normal saline; Group 2 was treated with 3 mg As2O3/L; Group 3 received 40 mg VB2/L; Group 4 received 3 mg As2O3/L + 40 mg VB2/L. Both As2O3 and VB2 were dissolved in deionized water. Malondialdehyde (MDA), Glutathione Peroxidase (GSH-Px), Superoxide dismutase (SOD), and Catalase (CAT) were assessed for the oxidative profile, while TAS (Total Antioxidative Status) levels were evaluated for the antioxidant system, in both serum and testicular tissue. P<0.05 was considered statistically significant. The results show that As2O3 significantly decreased the body weight, testicular weight and testis volume, semen quality and testicular cell count (p<0.05). Furthermore, MDA content in the testicular tissue of the As2O3 group rats was significantly higher in comparison to the vehicle group (p<0.05). Likewise, TAS and the activities of GSH-Px, CAT and SOD were reduced (p<0.05) when compared to the control. As(2)O(3) induced testicular damage and seminiferous tubular atrophy. Monodansylcadaverine assays mirrored the histopathology observations. Meanwhile, As2O3 upregulated the expression of mitophagy-related genes including PINK1, Parkin, USP8, LC3-I, Fis1 and Mfn2. The p38 gene, responsible to stress stimuli, was also upregulated by As2O3 administration. Meanwhile, exposure to VB2 led to a significant decrease of the expression levels of mitophagy related genes. Our study revealed that VB2 supplementation protected testicular structures against As2O3-induced injury via a dual inhibition of oxidative changes and a regulation of the PINK1-mediated pathway.

Fig. 1

Experimental design. Healthy rats were treated with As₂O₃ (3 mg/kg) and 40 mg/l vitamin B2 daily for 30 days continuously. Following treatment, testicular structure and function were evaluated with a special focus on the dual mitophagy pathway and oxidative stress markers.

Fig. 2

Histopathological changes in the testicular tissue of rats (H&E staining). The magnification is ×40. Symbols: black arrows - extensive atrophy in the seminiferous tubules and destruction of the germinal epithelium, gray arrows - shrinkage of the basal lamina, asterisks - Increased interstitial space, hallow arrow head - congested blood vessels and vascular hyperemia

Fig. 3

Qualitive monodansylcadaverine (MDC)-labeled autophagic vacuoles in rat testis. As₂O₃-induced autophagy was detectable by autofluorescence emitted by MDC staining (400×magnification, Bar=10 μm).

Fig. 4

Changes in the expressions of mitophagy key markers in mRNA induced by As₂O₃, mRNA expressions levels of PINK1, Parkin, USP8, LC3-I, Rab7, Fis1, Mfn2 and p38 as detected by qPCR. All data were expressed as relative values against their respective control group. β-actin was used as an internal control. The values are presented as mean ± SEM (n=5). The same superscripts (a–c) are not significantly different from each other (p<0.05).