The synergistic activity of fisetin on quercetin improves testicular recover in ischemia-reperfusion injury in rats

Abstract

This study examines a potential treatment for testicular ischemia-reperfusion (I/R) injury using fisetin (FIS) and quercetin (QUE) in a rat model. Male rats were divided into five groups: a control group, a torsion/detorsion (T/D) group, and three experimental groups treated with FIS, QUE, or a combination of FIS + QUE. Sperm parameters, oxidative stress markers, histopathological features, RT-PCR analysis of apoptotic and antiapoptotic gene expression, and fertility index were evaluated. The results demonstrated that FIS + QUE, FIS, and QUE significantly improved sperm motility and concentration, leading to a higher fertility index, than the reduced metrics in the T/D group. Additionally, levels of MDA and NO were significantly lowered, while CAT, SOD, GPx, and TAC levels increased in the FIS + QUE, FIS, and QUE groups. Histopathological, RT-PCR and fertility analyses also revealed evidence of apoptosis and testicular damage in the T/D group, shown by significant increases in P53, Bax, and caspase-3, along with marked decreases in AKT, PI3K, and Bcl-2. Treatment with FIS and QUE, particularly in combination, significantly improved outcomes, indicating a strong synergistic effect that helps repair damage and enhance reproductive function after T/D injury.

Keywords: Fisetin; Ischemia-reperfusion; Oxidative stress; Quercetin; Rat; Testicular torsion.

Fig. 1

DNA damage assessment in different experimental groups with a scale bar of 65 μm. White arrow: Normal sperm (green), Black arrow: DNA-damaged sperm (yellow). (A) Control, (B) Torsion/Detorsion (T/D), (C) Fisetin + T/D, (D) Quercetin + T/D, (E) Fisetin + Quercetin + T/D. (Acridine Orange staining, 100x).

Fig. 2

Biochemical findings in different experimental groups. T/D: Torsion and detorsion; F: Fisetin; QE: Quercetin. (A) Total antioxidant capacity (TAC); (B) glutathione peroxidase (GPx); (C) superoxide dismutase (SOD); (D) Malondialdehyde (MDA); (E) nitric oxide (NO). Superscripts demonstrate significant differences (p ≤ 0.05; Mean ± SD).

Fig. 2

Biochemical findings in different experimental groups. T/D: Torsion and detorsion; F: Fisetin; QE: Quercetin. (A) Total antioxidant capacity (TAC); (B) glutathione peroxidase (GPx); (C) superoxide dismutase (SOD); (D) Malondialdehyde (MDA); (E) nitric oxide (NO). Superscripts demonstrate significant differences (p ≤ 0.05; Mean ± SD).

Fig. 2

Biochemical findings in different experimental groups. T/D: Torsion and detorsion; F: Fisetin; QE: Quercetin. (A) Total antioxidant capacity (TAC); (B) glutathione peroxidase (GPx); (C) superoxide dismutase (SOD); (D) Malondialdehyde (MDA); (E) nitric oxide (NO). Superscripts demonstrate significant differences (p ≤ 0.05; Mean ± SD).

Fig. 3

Fisetin and quercetin mitigate testicular damage induced by torsion/detorsion (T/D). Images (A–E) are magnified at 100x with a scale bar of 65 μm, while images (F–J) are magnified at 400x with a scale bar of 20 μm. The structure of testicular tissue was compared among the following experimental groups: (A, F) Control, (B, G) T/D, (C, H) Fisetin + T/D, (D, I) Quercetin + T/D, and (E, J) Fisetin + Quercetin + T/D. In the control group (A, F), germ cells were well-organized in columnar arrangements, with thick walls in the convoluted seminiferous tubules and a high density of sperm within the tubular lumens. White arrows indicate reduced sperm density in the lumen of the seminiferous tubules, particularly in the T/D group (B, G) and the treatment groups: Fisetin + T/D (C, H), Quercetin + T/D (D, I), and Fisetin + Quercetin + T/D (E, J). Asterisks highlight the disorganization and disruption of the germinal epithelium across different groups, with the most severe alterations observed in the T/D group (B, G), followed by the treatment groups: Fisetin + T/D (C, H), Quercetin + T/D (D, I), and Fisetin + Quercetin + T/D (E, J).

Fig. 4

Reverse transcription-polymerase chain reaction findings in different experimental groups. The mRNA expression levels using semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). T/D: Torsion and detorsion; F: Fisetin; QE: Quercetin. (A) P53; (B) Bax; (C) Caspase-3; (D) AKT; (E) PI3K; (F) Bcl-2. Superscripts demonstrate significant differences (p ≤ 0.05; Mean ± SD).

Fig. 4

Reverse transcription-polymerase chain reaction findings in different experimental groups. The mRNA expression levels using semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). T/D: Torsion and detorsion; F: Fisetin; QE: Quercetin. (A) P53; (B) Bax; (C) Caspase-3; (D) AKT; (E) PI3K; (F) Bcl-2. Superscripts demonstrate significant differences (p ≤ 0.05; Mean ± SD).

Fig. 4

Reverse transcription-polymerase chain reaction findings in different experimental groups. The mRNA expression levels using semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). T/D: Torsion and detorsion; F: Fisetin; QE: Quercetin. (A) P53; (B) Bax; (C) Caspase-3; (D) AKT; (E) PI3K; (F) Bcl-2. Superscripts demonstrate significant differences (p ≤ 0.05; Mean ± SD).

Fig. 5

The effectiveness of Fisetin and Quercetin on ischemia-reperfusion. The image was created using Microsoft Word 2019 (Version 16.0, Microsoft Corporation, https://www.microsoft.com).