Inhibition of NADPH oxidase alleviates germ cell apoptosis and ER stress during testicular ischemia reperfusion injury

Testicular torsion and detorsion (TTD) is a serious urological condition affecting young males that is underlined by an ischemia reperfusion injury (tIRI) to the testis as the pathophysiological mechanism. During tIRI, uncontrolled production of oxygen reactive species (ROS) causes DNA damage leading to germ cell apoptosis (GCA). The aim of the study is to explore whether inhibition of NADPH oxidase (NOX), a major source of intracellular ROS, will prevent tIRI-induced GCA and its association with endoplasmic reticulum (ER) stress. Sprague-Dawley rats (n = 36) were divided into three groups: sham, tIRI only and tIRI treated with apocynin (a NOX inhibitor). Rats undergoing tIRI endured an ischemic injury for 1 h followed by 4 h of reperfusion. Spermatogenic damage was evaluated histologically, while cellular damages were assessed using real time PCR, immunofluorescence staining, Western blot and biochemical assays. Disrupted spermatogenesis was associated with increased lipid and protein peroxidation and decreased antioxidant activity of the enzyme superoxide dismutase (SOD) as a result of tIRI. In addition, increased DNA double strand breaks and formation of 8-OHdG adducts associated with increased phosphorylation of the DNA damage response (DDR) protein H2AX. The ASK1/JNK apoptosis signaling pathway was also activated in response to tIRI. Finally, increased immuno-expression of the unfolded protein response (UPR) downstream targets: GRP78, eIF2-α1, CHOP and caspase 12 supported the presence of ER stress. Inhibition of NOX by apocynin protected against tIRI-induced GCA and ER stress. In conclusion, NOX inhibition minimized tIRI-induced intracellular oxidative damages leading to GCA and ER stress.

Keywords: 8-OHdG, 8-hydroxy-2′-deoxyguanosine; ANOVA, analysis of variance; ASK1, apoptosis signaling kinase 1; ATF, activating transcription factor; ATM, ataxia telangiectasia mutated; BSA, bovine serum albumin; BTB, blood-testis barrier; CHOP, CCAAT-enhancer-binding protein homologous protein; Chk, checkpoint kinase; DAPI, diamidino phenylindole; DDR, DNA damage response; DMSO, dimethyl sulfoxide; DNA, deoxyribonucleic acid; ECL, electrochemiluminescence; ELISA, enzyme-linked immunosorbent assay; ER stress; ER, endoplasmic reticulum; GCA, germ cell apoptosis; GRP78, glucose-related protein 78; Germ cell apoptosis; H&E, hematoxylin and eosin; H2AX, histone variant; H2O2, hydrogen peroxide; IAP, inhibitors of apoptosis; IF, immunofluorescence; IRE1, inositol requiring kinase 1; JNK, c-Jun N-terminal Kinase; MDA, malondialdehyde; NADP, nicotinamide adenine dinucleotide phosphate; NADPH oxidase; NOX, NADPH oxidase; O2, molecular oxygen; O2−, superoxide anion; OS, oxidative stress; Oxidative stress; PARP, poly ADP-ribose polymerase; PCC, protein carbonyl content; PCR, polymerase chain reaction; PERK, pancreatic ER kinase; PVDF, polyvinylidene difluoride; RIPA, radioimmunoprecipitation assay; RNA, ribonucleic acid; ROS, reactive oxygen species; RT, reverse transcription; SD, standard deviation; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; SOD, superoxide dismutase; ST, seminiferous tubule; TOS, testicular oxidative stress; TRAF-2, tumor-necrosis-factor receptor-associated factor 2; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; Testicular ischemia Reperfusion Injury; UPR, unfolded protein response; cDNA, complementary DNA; eIF2α1, eukaryotic initiation factor 2α1; gDNA, genomic DNA; i.p., intraperitoneal; kDa, kilodalton; mRNA, messenger ribonucleic acid; p-, phosphorylated; phox, phagocyte oxidase; γ-H2AX, 139 serine-phosphorylated histone variant.