Involvement of hypoxia-inducible factor1-alpha in the protective effect of rivaroxaban against testicular ischemia-reperfusion in rats

Abstract

This study investigated the protective effects of Rivaroxaban (RVX) against testicular ischemia-reperfusion (IR) injury in rats with a secondary aim of studying the involvement of hypoxia-inducible factor1-alpha testicular protection against ischemic insults. Twenty-four male rats were divided into four groups: sham control, testicular IR, and two RVX treatment groups (7 and 14 mg/kg) administered for one week prior to IR. Testicular IR led to significant impairment in testicular function, evidenced by an 86.5% reduction in testosterone levels and marked oxidative stress with an 189.3% increase in malondialdehyde (MDA). IR injury also triggered substantial elevations in apoptotic markers (271% increase in Bax (Bcl2-associated X protein) and 65.9% decrease in BCL2 (B-cell lymphoma 2), and inflammatory mediators (285.7% increase in NFκB (nuclear factor-kappa B). Additionally, angiogenic markers showed dramatic increases, with VEGF (vascular endothelial growth factor) and HIF-1 (Hypoxia inducible factor-1) rising by 431.8% and 519%, respectively. RVX treatment demonstrated dose-dependent protective effects, with the 14 mg/kg dose showing superior outcomes compared to 7 mg/kg. The higher dose significantly improved hormonal function (486.8% increase in testosterone), reduced oxidative stress (51.8% reduction in MDA), modulated apoptotic markers (68.3% decrease in BAX, 159.1% increase in BCL2), and normalized angiogenic factors (71.8% reduction in HIF-1). In conclusion, RVX demonstrated significant therapeutic potential in protecting against testicular IR injury, with the 14 mg/kg dose showing optimal protective effects. Reduction in HIF-1α and VEGF protein expression mediated RVX's anti-oxidant, anti-inflammatory, and anti-apoptotic effect in rats subjected to testicular IR.

Keywords: Apoptosis; HIF-1α, VEGF, oxidative stress; Rivaroxaban; Testicular ischemia-reperfusion.

Fig. 1

Effects of rivaroxaban on serum testosterone against testicular ischemia-reperfusion injury in rats. Each bar represents the mean ± SEM of 6 rats: * vs. corresponding pairwise at p< 0.05, ** vs. corresponding bar at p< 0.01, *** vs. corresponding bar at p< 0.001, **** vs. corresponding bar at p< 0.0001.

Fig. 2

Effects of rivaroxaban on testicular (a) MDA and (b) GPX content against testicular ischemia-reperfusion injury in rats. Each bar represents the mean ± SEM of 6 rats: * vs. corresponding pairwise at p< 0.05, ** vs. corresponding bar at p< 0.01, *** vs. corresponding bar at p< 0.001, **** vs. corresponding bar at p< 0.0001.

Fig. 3

Effects of rivaroxaban on testicular (a) BAX and (b) BCL2 (c) NF-ĸb p65 content against testicular ischemia-reperfusion injury in rats. E Each bar represents the mean ± SEM of 6 rats: * vs. corresponding pairwise at p< 0.05, ** vs. corresponding bar at p< 0.01, *** vs. corresponding bar at p< 0.001, **** vs. corresponding bar at p< 0.0001.

Fig. 4

Effects of rivaroxaban on testicular (a) VEGF and (b) HIF1 content against testicular ischemia-reperfusion injury in rats. Each bar represents the mean ± SEM of 6 rats: * vs. corresponding pairwise at p< 0.05, ** vs. corresponding bar at p< 0.01, *** vs. corresponding bar at p< 0.001, **** vs. corresponding bar at p< 0.0001.

Fig. 5

Effects of rivaroxaban on histopathology and immunohistochemistry of caspase-3 and TNF-alpha in testicular ischemia-reperfusion injury in rats. (a) Normal histological structure of seminiferous tubules with full spermatogenesis in normal group. (b) Impaired spermatogenesis, distorted germinal epithelium and few early spermatids in IR group. (c) The spermatogenesis was slightly improved and the germinal epithelium lining seminiferous tubules showed moderate alteration in RVX (7 mg/kg). (d) The spermatogenesis was slightly impaired, and seminiferous tubules had many late spermatids in RVX (14 mg/kg). hematoxylin and eosin stain (X200). (e) Caspase-3 immunostaining was weak in normal group, (f) severe in Leydig cells, many spermatocytes and few spermatogonia in seminiferous tubules of IR group, (g) moderate in spermatocytes and Leydig cells of RVX (7 mg/kg). (h) few positive spermatocytes and Leydig cells in RVX (14 mg/kg). (Immunoperoxidase X400). (i) TNF-α was expressed weakly in Leydig cells and interstitial tissue of Normal group, (j) severe in IR group, (k) moderate in RVX (7 mg/kg) group, (l) and mild in RVX (14 mg/kg) group. (Immunoperoxidase X200).

Fig. 6

Effects of rivaroxaban on (a) Cosentino’s score, (b) Johnsen’s score, (c) Testicular caspase-3 Expression (d) Testicular TNF-alpha against testicular ischemia-reperfusion injury in rats. Each bar represents the mean ± SEM of 6 rats: * vs. corresponding pairwise at p< 0.05, ** vs. corresponding bar at p< 0.01, *** vs. corresponding bar at p< 0.001, **** vs. corresponding bar at p< 0.0001.

Fig. 7

Summary on the involvement of hypoxia-inducible factor1-alpha in the protective effect of Rivaroxaban against testicular ischemia-reperfusion in rats.