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

Abstract

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.

Fig. 1

Effect of apocynin on spermatogenesis. Rat ipsilateral testicular tissue sections stained with H&E demonstrating normal histological features of the STs and normal spermatogenesis in both sham (A) and apocynin-treated (C). The sections from the tIRI group (B) displayed disrupted germ layers indicating damage to spermatogenesis. Testicular sections were photographed at 10× (A, C and E) and 40× (B, D and F) magnifications.

Fig. 2

Apocynin activates NOX during tIRI. Bar Graphs representing the concentrations of (A) NADP⁺, (B) NADPH and (C) their ratio. While NADP displayed a significant increase in NADP⁺ concentration during tIRI, NADPH levels were reduced as compared to sham and apocynin-treated groups. This resulted in a significant increase in the NADP⁺/NADPH ratio during tIRI indirectly suggesting NOX activation during tIRI. No significant difference was obtained between contralateral testes (P < 0.05). Data are presented as mean ± SD (n = 6/group). *tIRI compared to sham and ^#apocynin compared to tIRI. I = Ipsilateral and C = Contralateral.

Fig. 3

Apocynin prevents testicular oxidative stress. Ipsilateral tIRI-subjected rats had significantly (A) low SOD activity (%), (B) increased MDA concentration (μM) and (C) heightened PCC levels (nmole) as compared to sham and apocynin-treated groups, which showed baseline levels of SOD, MDA and PCC. No significant difference was measured among contralateral testes (p > 0.05). Data are presented as mean ± SD (n = 6/group). *tIRI compared to sham and ^#apocynin-treated compared to tIRI. I = Ipsilateral and C = Contralateral.

Fig. 4

DNA damage and repair is attenuated by apocynin. (A-C) Rat ipsilateral testicular tissue sections labeled by TUNEL showing increased DNA damage in tIRI testes in comparison to sham and apocynin-treated groups at 40x magnifications. (D-F) Ipsilateral testicular tissue sections subjected to IF staining for γ-H2AX (yellow) showing increased immune-expression in the spermatogonia of tIRI compared to sham and apocynin-treated groups at 40x magnification. (G) Bar Graph representing 8-OHdG concentration (ng) showing that ipsilateral testes of tIRI had significantly higher concentration of 8-OHdG in comparison to sham and apocynin-treated groups. No significant difference was obtained among contralateral testes (p-value > 0.05). Data are presented as mean ± SD (n = 6/group). *tIRI compared to sham and ^#apocynin-treated compared to tIRI. I = Ipsilateral and C = Contralateral.

Fig. 5

Apocynin inhibits activation of the ASK1/JNK signaling pathway. Ipsilateral testicular tissue sections subjected to IF staining against the phosphorylated forms of (A-C) ASK1 (green) and (D-F) JNK (red) showing an increased immune-expression in germ cells and spermatocytes of tIRI, respectively, compared to sham and apocynin-treated groups. (G-I) Sequential double IF staining of ipsilateral testis combining p-ASK1, p-JNK and DAPI where the overlap is displayed as yellow color. Images were captured at 40x magnification.

Fig. 6

Apocynin modulates the expression of ER stress markers by IF staining. NOX-induced oxidative and ER stresses through activation of the UPR pathway components in tIRI-subjected testes. Significant upregulation of UPR proteins is demonstrated as high fluorescence intensity in tIRI (B, E, H, K) compared to baseline expression in sham (A, D, G, J) and apocynin-treated groups (C, F, I, L). GRP78 (A-C, blue), p-eIF2α (D-F, green), CHOP (G-I, magenta), and caspase 12 (J-L, green). Images were captured at 40× magnification.

Fig. 7

Detection of ER stress markers by Western Blot. During tIRI, the immune-expression of GRP78, p-eIF2α and caspase 12 was increased by 57%, 52% and 54% as compared to sham (p > 0.001) and was higher by 35, 21% and 46% in comparison to apocynin-treated group (p > 0.001). Data are presented as mean ± SD (n = 6/group). *tIRI compared to sham and ^#apocynin-treated compared to tIRI. I = Ipsilateral.