Timed Administration of Febuxostat Improved Testicular Function Following Testicular Ischemia-reperfusion Injury Via Inhibition of MDA/NO Pathway, Down-regulation of Toll-like Receptor 4 Expression and Restoration of Reproductive Hormones

Abstract

Introduction: Testicular ischemia-reperfusion injury (TIRI) generates reactive oxygen species (ROS) through xanthine oxidase (XO) activity in the ischemic phase and leukocyte infiltration to the site of injury during reperfusion leading to oxidative stress, inflammation, and disruption of male reproductive hormones. Febuxostat (FEB), a xanthine oxidase (XO) inhibitor has been proven to exhibit superior antioxidant, anti-inflammatory, cytoprotective and anti-apoptotic effect than other XO inhibitors. Methodology: Forty male Wistar rats (120-150 g) were divided into 5 groups (n=8 rats each): Group 1:  Sham operated (SO) rats underwent surgery without TIRI induction, Group 2: Torsion + Detorsion (TD) rats underwent left unilateral TT for one hour and detorsed immediately to induce reperfusion which lasted for 3 days, Group 3: Torsion + 5 mg/kg Febuxostat + Detorsion (TF30D), Group 4: Torsion + Detorsion + 5 mg/kg Febuxostatimm (TDFimm) and Group 5:   Torsion + Detorsion + 5 mg/kg Febuxostat30 (TDF30). TF30D, TDFimm and TDF30 received 5 mg/kg of FEB intraperitoneally 30 minutes after TT onset, immediately on detorsion and 30 minutes after detorsion respectively. Rats were euthanized with 40 mg/kg ketamine 3 days after reperfusion. Blood samples were used for the measurement of nitrite, myeloperoxidase enzyme, Tumor-necrosis factor (TNF-α), interleukin-1-beta (IL-1β),   and reproductive hormones (LH, FSH, testosterone and inhibin). Left testes were homogenized and used for the assessment of Toll-like receptor-4-expression (TLR-4), superoxide dismutase (SOD), catalase (CAT), malondialdehyde (MDA), glutathione (GSH), total protein (TP), non-protein thiol (NPSH) and protein thiol (PSH). Results: This study showed that TIRI significantly increased oxidative stress markers (MDA, serum nitrite) and inflammatory markers (TLR-4, TNF-α, IL-1β) decreased antioxidant enzymes (SOD, CAT, GSH, non-protein thiols) and altering reproductive hormones (increased LH, FSH, and decreased testosterone and inhibin level) when compared to SO group (p<0.01; 0.001). Febuxostat administered in the ischemic phase (TF30D)  significantly suppressed oxidative stress markers (MDA and serum nitrite), and improved antioxidant markers (SOD, CAT, TP, GSH, NPSH and PSH) when compared to  TDFimm and TDF30  groups (p<0.001). Additionally, all the febuxostat treated groups significantly reduced the level of TLR-4, TNF-α, and IL-1β, with MPO level only significantly reduced in TF30D and TDF30  groups  (p<0.001;0.05) while the level of reproductive hormones (LH, FSH, inhibin, testosterone) were restored in all febuxostat-treated groups  (p<0.01;0.05; 0.001). Conclusion: Febuxostat administered in the ischemic phase (TF30D) is best to prevent TIRI when compared to its administration immediately on detorsion and 30 minutes after detorsion. This treatment strategy may guide clinician to prevent TIRI in humans after surgical detorsion.