Protective Effects of Topical Application of Nitrite on Testicular Ischemia-Reperfusion Injury in Rats

Abstract

Testicular torsion is a urologic emergency induced by torsion of the spermatic cord, interrupting blood circulation to the testis. Therapeutic options for testicular torsion, except surgical restoration of testis, are rarely applied in clinical practice. This study, therefore, investigated whether topical application of nitrite (NO₂ ^-) is beneficial in tissue damage due to testicular ischemia-reperfusion (I/R) injury in rats. Pubertal Sprague-Dawley rats were assigned to seven groups: group A, sham-operated control group; group B, I/R with no treatment; groups C, D, and E, I/R followed by treatment with three different doses of nitrite; group F, I/R followed by administration of nitrite and a NO scavenger, C-PTIO (2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt); and group G, I/R followed by administration of nitrate (NO₃ ^-). Unilateral testicular ischemia was maintained for 5 h, followed by reperfusion for 24 h. Nitrite and nitrate were topically administered before reperfusion. Compared to group A, germ cell apoptosis, oxidative stress, antioxidant enzymatic function, and lipid peroxidation were significantly increased, along with abnormal morphology and impaired spermatogenesis in group B (P < 0.05). In contrast, testicular damage was generally attenuated in the nitrite treatment groups due to a reduction in superoxide and peroxynitrite levels and the inhibition of caspase-3-dependent apoptosis (P < 0.05 vs. group B). These therapeutic effects of nitrite-derived NO were suppressed after injection of C-PTIO, which showed in group F. Taken together, our results demonstrate that topical application of nitrite may be one of the therapeutic strategies for testicular ischemia-reperfusion injury.

Figure 1

Effects of topically injected nitrite and nitrate on 3-nitrotyrosine (3-NT) and dihydroethidium (DHE) staining in the ipsilateral testis (×200). (a–d) Immunohistochemical staining of 3-NT in the left ischemic testes of groups A, B, C, and G. (e) The number of 3-NT-positive cells per seminiferous tubule in all groups. (f–i) DHE staining of in the left ischemic testes of groups A, B, C, and G. (j) The number of DHE-stained positive cells per seminiferous tubule in all groups. ^∗P < 0.05 vs. group A, ^#P < 0.05 vs. group B. Groups: A: control, B: ischemia/reperfusion (IR) injury, C: IR+0.12 nmol/g nitrite, D: IR+1.2 nmol/g nitrite, E: IR+12 nmol/g nitrite, F: IR+0.12 nmol/g nitrite+0.01 μmol/g C-PTIO, and G: IR+0.12 nmol/g nitrate.

Figure 2

Effects of topical application of nitrite and nitrate on malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), and cyclic guanosine monophosphate (cGMP) levels in the ipsilateral and contralateral testes. Color black, left testis; color gray, right testis. (a) MDA values are expressed as micromoles of MDA per milligram of protein (μmol/mg protein). Data are mean ± SD. ^∗P < 0.05 vs. group A left testis, ^#P < 0.05 vs. group B left testis. (b, c) SOD values are expressed as unit of SOD per milligram of protein (U/mg protein), and CAT values are expressed as nanomolar/minute/milligram protein. Data are mean ± SD. ^∗P < 0.05 vs. group B left testis. (d) cGMP values are expressed as femtomoles of cGMP per milligram of protein (fmol/mg protein). Data are mean ± SD. ^∗P < 0.05 vs. group A left testis, ^#P < 0.05 vs. group F left testis, and ^&P < 0.05 vs. group C right testis. Groups: A: control, B: ischemia/reperfusion (IR) injury, C: IR+0.12 nmol/g nitrite, D: IR+1.2 nmol/g nitrite, E: IR+12 nmol/g nitrite, F: IR+0.12 nmol/g nitrite+0.01 μmol/g C-PTIO, and G: IR+0.12 nmol/g nitrate.

Figure 3

Western blot analysis of apoptosis in the ipsilateral testis: (a) the relative caspase-3/β-actin expression in the left testis of groups A, B, C, and G; (b) the relative cleaved PARP/β-actin expression in the left testis of groups A, B, C, and G. ^∗P < 0.05 vs. group B. Group: A: control, B: ischemia/reperfusion (IR) injury, C: IR+0.12 nmol/g nitrite, and G: IR+0.12 nmol/g nitrate.