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Objective: The aim of this study was to investigate the pharmacological activity of an antioxidant, α-tocopherol (vitamin E, VE) in streptozotocin-induced diabetic rats and study its role in modulating the transforming growth factor β1 (TGF-β1). Methods: Male Sprague-Dawley rats were treated with streptozotocin to induce diabetes. VE and/or insulin (INS) were administered daily during treatment periods of 3, 5, 7 and 10 days. Plasma glucose and fructosamine were measured in diabetic rats at the end of each treatment period. Samples of plasma, urine and renal cortex were analyzed for changes in protein and lysozyme excretion, reduced glutathione and malondialdehyde formation. TGF-β1 was determined by ELISA and expression of TGF-β1 mRNA was investigated by RT-PCR and Northern blot analysis. Results: Diabetes-induced glycemic stress was suppressed by INS, VE or a combination of INS and VE. Diabetes-induced increases of glucose, protein and lysozyme excretion were markedly depressed after 10-day treatment with INS, VE and the combination of INS and VE. Decreased glutathione content in the renal cortex of diabetic rats recovered towards control values, especially after 10-day treatment. Malondialdehyde content increased in diabetic rats and was reduced towards control value following 7- and 10-day treatments. Treatment of diabetic rats with INS, VE or the combination of INS and VE decreased elevated TGF-β1 in plasma, decreased excretion of TGF-β1 in urine, and decreased renal cortex TGF-β1 mRNA levels. Conclusions: Diabetes-induced overexpression of TGF-β1 mRNA was suppressed by VE and INS after 5-, 7- and 10-day treatments. The results obtained with the antioxidant VE suggest that oxidative stress is involved in the development of diabetic nephropathy. Therefore, VE treatment may be effective in early stages of diabetic nephropathy to decrease or prevent pathological complications. Copyright © 2005 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Oxidative stress triggered by testicular torsion and detorsion in young males could negatively impact future fertility. Using a rat animal model for testicular IRI (tIRI), we aim to study the induction of autophagy (ATG) during testicular ischemia and tIRI and the role of oxidative-stress-induced c-Jun N-terminal Kinase (JNK) as a cytoprotective mechanism. Sixty male Sprague-Dawley rats were divided into five groups: sham, ischemia only, ischemia+SP600125 (a JNK inhibitor), tIRI only, and tIRI+SP600125. The tIRI rats underwent an ischemic injury for 1 h followed by 4 h of reperfusion, while ischemic rats were subjected to 1 h of ischemia only without reperfusion. Testicular-ischemia-induced Beclin 1 and LC3B expression was associated with decreased p62/SQSTM1 expression, increased ATP and alkaline phosphatase (AP) activity, and slightly impaired spermatogenesis. SP600125 treatment improved p62 expression and reduced the levels of Beclin 1 and LC3B but did not affect ATP or AP levels. The tIRI-induced apoptosis lowered the expression of the three ATG proteins and AP activity, activated caspase 3, and caused spermatogenic arrest. SP600125-inhibited JNK during tIRI restored sham levels to all investigated parameters. This study emphasizes the regulatory role of JNK in balancing autophagy and apoptosis during testicular oxidative injuries.

The male reproductive system requires the pleiotropic activity of JAK/STAT to maintain its function, especially spermatogenesis. The study aims to investigate the effect of JAK2 signaling on the expression of the Keap1/Nrf2 axis, spermatogenesis, and the Sertoli cells (Sc) junctions in an animal model of testicular ischemia reperfusion injury (tIRI). Testes subjected to tIRI exhibited increased JAK2/STAT3 activity associated with spermatogenic arrest and reduced expression of the Sc junctions. In addition, there was an increased protein expression of Keap1 and decreased Nrf2., which was coupled with the downregulation of gene expression of antioxidant enzymes. Reduced SOD and CAT activities were accompanied by increased lipid peroxidation and protein carbonylation during tIRI. Increased caspase 9 activity and Bax / Bcl2 ratio indicated initiation of apoptosis. Inhibition of JAK2 activity by AG490 maintained the integrity of spermatogenesis and SC junctions, normalized the expression of the Keap1/Nrf2 axis and its downstream antioxidant enzymes, and prevented germ cell apoptosis. The results further emphasized the regulatory role of JAK2/STAT3 on spermatogenesis, Keap1/Nrf2 signaling, and maintenance of the testicular redox balance to combat testicular dysfunction and male infertility.

Oxidative stress, inflammation and germ cell death are the main characteristics of testicular ischemia reperfusion injury (tIRI), which is considered as the underlying mechanism for testicular torsion and detorsion. The study aimed to examine the effect of tIRI-activated NADPH oxidase (NOX) on the expression of the NLRP3 inflammasome pathway components. Three groups of male Sprague-Dawley rats ( n = 12 each) were studied: sham, unilateral tIRI only and tIRI treated with apocynin, a NOX-specific inhibitor. The tIRI rat model was subjected to 1 h of ischemia followed by 4 h of reperfusion. H&E staining, real time PCR, biochemical assays, and Western blot were utilized to evaluate spermatogenic damage, gene expression, oxidative stress markers, and NLRP3 pathway components, respectively. As a result of tIRI, decreased total antioxidant capacity and suppressed activities of superoxide dismutase and catalase were associated with spermatogenic arrest. The components of the NLRP3 inflammasome pathway (TXNIP, NLRP3, ASC, caspase-1, GSDMD, MMP-9) were upregulated transcriptionally and post-transcriptionally during tIRI. In parallel, tissue inflammation was demonstrated by a marked increase in the concentrations of myeloperoxidase, IL-1β, and IL-18. Apocynin treatment prevented testicular oxidative stress and inflammation. Thus, NOX inhibition by apocynin prevented ROS accumulation, proinflammatory cytokine overexpression and NLRP3 inflammasome activation during tIRI.

Testicular ischemia reperfusion injury (tIRI) causes oxidative stress-induced DNA damage leading to germ cell apoptosis (GCA). The aim of the study is to establish a direct link between JAK2 activation and the DNA damage response (DDR) signaling pathways and their role in tIRI-induced GCA using AG490, a JAK2 specific inhibitor. Male Sprague Dawley rats ( n = 36) were divided into three groups: sham, unilateral tIRI and tIRI + AG490 (40 mg/kg). During tIRI, augmentation in the phosphorylation levels of the JAK2/STAT1/STAT3 was measured by immunohistochemistry. Observed spermatogenic arrest was explained by the presence of considerable levels of DSB, AP sites and 8OHdG and activation of caspase 9, caspase 3 and PARP, which were measured by colorimetric assays and TUNEL. The ATM/Chk2/H2AX and ATR/Chk1 pathways were also activated as judged by their increased phosphorylation using Western blot. These observations were all prevented by AG490 inhibition of JAK2 activity. Our findings demonstrate that JAK2 regulates tIRI-induced GCA, oxidative DNA damage and activation of the ATM/Chk2/H2AX and ATR/Chk1 DDR pathways, but the cell made the apoptosis decision despite DDR efforts.

The aim of this study is to determine whether the c-Jun N-terminal kinase (JNK) signaling is a regulator of oxidative DNA damage, germ cell apoptosis (GCA), and mitochondrial dysfunction during testicular ischemia reperfusion injury (tIRI) using the JNK inhibitor SP600125. Male Sprague Dawley rats (n = 36) were equally divided into three groups: sham, tIRI only, and tIRI + SP600125 (15 mg/kg). Testicular ischemia was induced for 1 h followed by 4 h of reperfusion prior to animal sacrifice. Spermatogenesis was evaluated by light microscopy, while expression of oxidative stress and GCA-related mRNAs and proteins were evaluated by real-time polymerase chain reaction and colorimetric assays, respectively. Expressions of JNK, p53, and survivin were detected by immunofluorescence (IF) staining. Indicators of mitochondrial dysfunction were examined by western blot analysis and colorimetric assay. In comparison to sham, the tIRI testes showed a significant increase in lipid and protein oxidation products. Oxidative DNA damage was reflected by a significant increase in the number of DNA strand breaks, increased concentration of 8-OHdG, and elevated poly (ADP-ribose) polymerase activity. Spermatogenic damage was associated with the activation of caspase 3 and elevated Bax to Bcl2 ratio. This was also accompanied by a significantly heightened IF expression of the phosphorylated forms of JNK and p53 paralled with the suppression of survivin. Mitochondrial dysfunction was reflected by NAD+ depletion, overexpression of uncoupling protein 2, and increased level of cytochrome c. Such tIRI-induced modulations were all attenuated by SP600125 treatment prior to reperfusion. In conclusion, JNK signaling regulates oxidative DNA damage, GCA, and mitochondrial dysfunction through activation of p53 and suppression of survivin during tIRI., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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., (© 2020 The Author(s).)

To examine the role of the transcription factor nuclear factor-erythroid 2 (NF-E2)-related factor 2 (Nrf2) and the SAFE pathway (JAK/STAT) in the induction of germ cell apoptosis (GCA) and the protective role of epigallocatechin-3-gallate (EGCG) during testicular ischemia reperfusion injury (tIRI). Male Sprague-Dawley rats (n = 18) were divided into three groups: sham, unilateral tIRI, tIRI + epigallocatechin-3-gallate (EGCG, 50 mg/Kg). Unilateral tIRI was induced by 1-h ischemia followed by 4-h reperfusion, and EGCG was injected i.p. 30-min post ischemia. Immuno-histological analyses were used to detect spermatogenesis, oxidative DNA damage, and the immuno-expression of the JAK2, STAT3, and STAT1. Biochemical assays were used to investigate the oxidative, apoptosis proteins and enzymes, while Western blot was used to detect the expression of NOX and Nrf2. Expression of apoptosis-related genes was measured by real-time PCR. During tIRI, there was a clear damage to spermatogenesis associated with decreased activities of SOD, CAT, and GPx and increased levels of lipid peroxidation and oxidative DNA damage. In addition, GCA was indicated by the activation of caspase1, PARP, decreased gene expression of survivin and increased Bax to Bcl2 ratio. In contrast to lowered Nrf2 levels, NOX levels were augmented and phosphorylation of JAK2, STAT3, and STAT1 was increased. Pre-perfusion treatment with EGCG prevented the above modulations. The coordinated activation of the SAFE pathway and suppression of Nrf2 contribute to the tIRI-induced oxidative damages and GCA, which were modulated by EGCG.

The aim is to explore the mechanism of the apoptosis signal-regulating kinase-1 (ASK-1) signaling pathway and the involvement of the thioredoxin (Trx) system during testicular ischemia reperfusion injury (tIRI) by using ASK-1 specific inhibitor, NQDI-1. Male Sprague-Dawley rats (n = 36, 250-300 g) were equally divided into 3 groups: sham, tIRI, and tIRI + NQDI-1 (10 mg/kg, i.p, pre-reperfusion). For tIRI induction, the testicular cord and artery were occluded for 1 h followed by 4 h of reperfusion. Histological analyses, protein immunoexpression, biochemical assays, and real-time PCR were used to evaluate spermatogenesis, ASK-1/Trx axis expression, enzyme activities, and relative mRNA expression, respectively. During tIRI, ipsilateral testes underwent oxidative stress indicated by low levels of superoxide dismutase (SOD) and Glutathione (GSH), increased oxidative damage to lipids and DNA, and spermatogenic damage. This was associated with induced mRNA expression of pro-apoptosis genes, downregulation of antiapoptosis genes, increased caspase 3 activity and activation of the ASK-1/JNK/p38/survivin apoptosis pathway. In parallel, the expression of Trx, Trx reductase were significantly reduced, while the expression of Trx interacting protein (TXNIP) and the NADP + / nicotinamide Adenine Dinucleotide phosphate (NADPH) ratio were increased. These modulations were attenuated by NQDI-1 treatment. In conclusion, the Trx system is regulated by the ASK-1/Trx/TXNIP axis to maintain cellular redox homeostasis and is linked to tIRI-induced germ cell apoptosis via the ASK-1/JNK/p38/survivin apoptosis pathway., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Objective: To investigate whether exogenous angiotensin (Ang)-(1-7) administration can protect against the damaging consequences of testicular ischemia reperfusion (tIR) injury., Materials and Methods: Eighteen male Sprague-Dawley rats were divided equally among the following 3 groups: sham, unilateral tIR injury (1 hour of ischemic treatment and 4 hours of reperfusion), and tIR + Ang-(1-7) (0.3 mg/kg). Testicular tissues obtained from the rats were evaluated for the expression of testicular angiotensin-converting enzyme (tACE), Ang-(1-7), and the Ang-(1-7)-specific receptor Mas by immunohistochemistry and enzyme-linked immunosorbent assay. Reduced spermatogenesis, induction of the caspase-8 pathway, and nitric oxide (NO) generation were assessed. The effects of tIR and Ang-(1-7) treatment on the PI3K/Akt antiapoptosis pathway were also investigated., Results: Testicular morphological changes and reduced spermatogenesis associated with decreased expression of the tACE/Ang-(1-7)/Mas axis were observed during tIR. These effects were also accompanied by increased activity of caspase-3 and -8, downregulation of the survivin and BAD transcripts, and decreased NO formation. During tIR, PTEN expression was increased, leading to inactivation of the PI3K/Akt pathway. Acute treatment with Ang-(1-7) prior to reperfusion attenuated the tIR-induced damage described above., Conclusion: Expression of the tACE/Ang-(1-7)/Mas axis was downregulated during tIR. Administration of exogenous Ang-(1-7) prior to reperfusion rescued tACE and Mas expression and protected against germ cell apoptosis and oxidative stress. Increased NO generation and activation of the PI3K/Akt signaling pathway may have partially contributed to these effects. The tACE/Ang-(1-7)/Mas axis likely plays a role in the maintenance of normal testis physiology and spermatogenesis., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background. Testicular ischemia reperfusion injury (tIRI) is considered the mechanism underlying the pathology of testicular torsion and detorsion. Left untreated, tIRI can induce testis dysfunction, damage to spermatogenesis and possible infertility. In this study, we aimed to assess the activities and expression of glycolytic enzymes (GEs) in the testis and their possible modulation during tIRI. The effect of fructose 1,6-diphosphate (FDP), a glycolytic intermediate, on tIRI was also investigated. Methods. Male Sprague-Dawley rats were divided into three groups: sham, unilateral tIRI, and tIRI + FDP (2 mg/kg). tIRI was induced by occlusion of the testicular artery for 1 h followed by 4 h of reperfusion. FDP was injected peritoneally 30 min prior to reperfusion. Histological and biochemical analyses were used to assess damage to spermatogenesis, activities of major GEs, and energy and oxidative stress markers. The relative mRNA expression of GEs was evaluated by real-time PCR. ELISA and immunohistochemistry were used to evaluate the expression of p53 and TP53-induced glycolysis and apoptosis regulator (TIGAR). Results. Histological analysis revealed tIRI-induced spermatogenic damage as represented by a significant decrease in the Johnsen biopsy score. In addition, tIRI reduced the activities of hexokinase 1, phosphofructokinase-1, glyceraldehyde 3-phosphate dehydrogenase, and lactate dehydrogenase C. However, mRNA expression downregulation was detected only for hexokinase 1, phosphoglycerate kinase 2, and lactate dehydrogenase C. ATP and NADPH depletion was also induced by tIRI and was accompanied by an increased Malondialdehyde concentration, reduced glutathione level, and reduced superoxide dismutase and catalase enzyme activities. The immunoexpression of p53 and TIGAR was markedly increased after tIRI. The above tIRI-induced alterations were attenuated by FDP treatment. Discussion. Our findings indicate that tIRI-induced spermatogenic damage is associated with dysregulation of GE activity and gene expression, which were associated with activation of the TIGAR/p53 pathway. FDP treatment had a beneficial effect on alleviating the damaging effects of tIRI. This study further emphasizes the importance of metabolic regulation for proper spermatogenesis.

Testicular ischemia reperfusion injury (tIRI) is considered as the underlying mechanism of testicular torsion, which can cause male infertility. tIRI-induced damage was investigated by assessing the gene expression of spermatogenesis master transcription factors (TFs) and that of major adhesion molecules of the blood-testis barrier. The effect of lutein, a hydroxyl carotenoid, in alleviating tIRI-induced damage was also studied. Male Sprague-Dawley rats were divided into three groups: sham, unilateral tIRI, and tIRI + lutein (0.2 mg/kg). tIRI was induced by occlusion of the testicular artery for 1 h, followed by 4 h of reperfusion. Lutein was injected 15 min after the start of ischemia. Histological analysis and real-time polymerase chain reaction revealed significant decreases in tissue biopsy scores, reduced seminiferous tubule diameters, and downregulated the mRNA expression of the TFs cAMP-responsive element modulator (CREM), TATA box-binding protein-related factor 2 (TRF2), and regulatory factor X 2 (RFX2) compared with the sham group. Lutein treatment reversed these effects. The mRNA expression of the adhesion molecules N-cadherin, nectin-2, claudin-11, occludin, and connexin-43 was significantly downregulated during tIRI, but this change was prevented by lutein treatment. In addition, lutein normalized the tIRI-induced increase in total antioxidant capacity, increased malondialdehyde (MDA) levels, augmented number of TdT-mediated dUTP-X nick-end labeling (TUNEL)-positive nuclei, and activated caspase-8 pathway. The components of survivor activating factor enhancement (SAFE) were also activated during tIRI. Increased tissue expression of TNF-α and its receptor, TNFR1, was accompanied by increased phosphorylation of Janus kinase (JAK) and STAT3, which was prevented by lutein treatment. Our findings suggested that tIRI-induced spermatogenic damage may involve modulation of the SAFE pathway and could benefit from lutein treatment.

The aim of the study is to examine the role of matrix metalloproteinases (MMPs) and their inhibitors (TIMP) during testicular ischemia/reperfusion (t I/R). The involvement of the Notch pathway, and their modulation by the antioxidant genistein is also studied. Three groups of male Sprague-Dawley rats were used: sham rats, t I/R rats, and genistein-treated rats (10 mg/kg). The t I/R rat model underwent testicular artery occlusion of the left testis and was subjected to 60 min ischemia followed by 4 h reperfusion. Protein expression of MMP-2, MMP-9, TIMP-1, and TIMP-2 were measured in testicular tissue. Histological examination was performed to assess spermatogenesis. Protein levels of Notch 2, Jagged 1, and hairy/enhancer of split 1 (hes-1) was quantified. The degree of testicular oxidative stress, DNA damage and germ cell apoptosis were also evaluated. T I/R induced severe tubular damage, a significant increase in MMP- 2 and MMP-9 expression and decreased expression TIMP-1 and TIMP-2. Genistein treatment normalized the MMP-TIMP imbalance. Rats subjected to t I/R had low total antioxidant capacity of the testis, decreased superoxide dismutase activity, and increased oxidative DNA damage. Enhanced activities of caspase 8, caspase 3 and PARP were also observed during t I/R. Genistein reversed the t I/R-induced suppression of the Notch 2/Jagged 1/hes-1 pathway. Genistein was also able to salvage the testicular structure and function through restoring the MMP-TIMP anti-proteolytic balance, suppressing spermatogenic damage, alleviating oxidative stress and apoptosis. The Notch pathway is partly involved in inhibiting the t I/R-induced testicular impairment.

Our previous studies have established that (-)-epigallocatechin-3-gallate (EGCG) has both neuroprotective and -regenerative capacity after sciatic nerve injury. Moreover, this improvement was evident on the behavioral level. The aim of this study was to investigate the central effects of ECGC on spinal cord motor neurons after sciatic nerve injury. Our study showed that administering 50 mg/kg intraperitoneally i.p. of EGCG to sciatic nerve-injured rats improved their performance on different motor functions and mechanical hyperesthesia neurobehavioral tests. Histological analysis of spinal cords of EGCG-treated sciatic nerve-injured (CRUSH+ECGC) animals showed an increase in the number of neurons in the anterior horn, when compared to the naïve, sham, and saline-treated sciatic nerve-injured (CRUSH) control groups. Additionally, immunohistochemical study of spinal cord sections revealed that EGCG reduced the expression of glial fibrillary acidic protein and increased the expression of growth-associated protein 43, a marker of regenerating axons. Finally, EGCG reduced the ratio of B-cell lymphoma 2 (Bcl-2)-associated X protein/Bcl-2 and increased the expression of survivin gene. This study may shed some light on the future clinical use of EGCG and its constituents in the treatment of peripheral nerve injury.

Introduction: The objective of this study is to investigate the molecular mechanisms underlying the effects of zinc deficiency on spermatogenesis in the Sprague-Dawley (SD) rat., Materials and Methods: Three groups of eight adult male SD rats were maintained for 4 weeks on a normal diet as control, zinc deficient diet and zinc deficient diet with zinc supplementation of 28 mg zinc/kg body weight respectively. Using standard techniques, the following parameters were compared between the three groups of experimental animals at the end of 4 weeks: (a) Serum zinc, magnesium (Mg), copper (Cu), selenium (Se) and cadmium (Cd), (b) serum sex hormones, malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GPX), (c) interleukin-4 (IL-4), tumor necrosis factor-alpha (TNF-α), Bcl-2, Bax and caspase-3 expression in the testes, (d) assessment of apoptosis of testicular cells using electron microscopy and (e) testicular volume and histology using the orchidometer and Johnsen score, respectively., Results: The zinc deficient group showed a reduction of testicular volume, serum concentrations of Zn, Cu, Se, Mg, SOD, GPX, IL-4, Bcl-2 and testosterone (P < 0.05), as well as increased levels of serum Cd, MDA and tissue TNF-α, Bax, caspase-3 and apoptosis of the germ cells (P < 0.05) compared with control and zinc supplementation groups., Conclusion: Zinc deficiency is associated with impaired spermatogenesis because of reduced testosterone production, increased oxidative stress and apoptosis. These findings suggest that zinc has a role in male reproduction.

The anti-apoptotic effect of (-)-epigallocatechin-3-gallate (EGCG) during unilateral testicular torsion and detorsion (TT/D) was established in our previous study. In mice, the smallest inhibitor of apoptosis, survivin, is alternatively spliced into three variants, each suggested to have a unique function. Here, we assessed how EGCG exerts its protective effect through the expression of the different survivin splice variants and determined its effect on the morphology of the seminiferous tubules during TT/D. Three mouse groups were used: sham, TT/D+vehicle and TT/D treated with EGCG. The expression of the survivin variants (140 and 40) and other apoptosis genes (p53, Bax and Bcl-2) was measured with semi-quantitative RT-PCR. Histological analysis was performed to assess DNA fragmentation, damage to spermatogenesis and morphometric changes in the seminiferous tubules. In the TT/D+vehicle group, survivin 140 expression was markedly decreased, whereas survivin 40 expression was not significantly different. In parallel, there was an increase in the mRNA level of p53 and the Bax to Bcl-2 ratio in support of apoptosis induction. Histological analyses revealed increased DNA fragmentation and increased damage to spermatogenesis associated with decreased seminiferous tubular diameter and decreased germinal epithelial cell thickness in the TT/D+vehicle group. These changes were reversed to almost sham levels upon EGCG treatment. Our data indicate that EGCG protects the testis from TT/D-induced damage by protecting the morphology of the seminiferous tubules and modulating survivin 140 expression.

Purpose: A steady increase in the incidence of septicemia after prostate biopsy in our unit between 2001 and 2005 prompted us to review our prophylactic antibiotic regimen. We compared the incidence of septicemia in patients undergoing prostate biopsy between 2001 and 2005 when only oral ciprofloxacin was used prophylactically (group 1) to the incidence among patients undergoing biopsy between 2006 and 2010 when a single dose of intravenous amikacin was added to ciprofloxacin (group 2)., Materials and Methods: In group 1 the 300 patients were given 500 mg oral ciprofloxacin twice daily 1 day before and for 2 days after the biopsy while in group 2 the 897 patients, in addition to the ciprofloxacin previously mentioned, received 500 mg intravenous amikacin 30 minutes before the biopsy. Patients admitted to the hospital with septicemia after prostate biopsy had urine and blood culture and sensitivity tests. The number of patients in whom septicemia developed in each group after prostate biopsy and the microorganisms isolated from the urine and blood of such patients were compared using the chi-square test., Results: Septicemia was seen in 24 of 300 (8%) and 15 of 897 (1.7%) patients in groups 1 and 2, respectively (p <0.001). In group 1 the rate of septicemia after prostate biopsy was 2.1% and 13% in 2001 and 2005, respectively (p <0.001). In group 2 the rate of septicemia was 1.5% in 2006 and 1.6% in 2010 (p <0.25). Escherichia coli resistant to quinolones was responsible for 33 of 39 (84.6%) septicemic cases., Conclusions: The addition of amikacin to ciprofloxacin prophylaxis significantly reduces the incidence of septicemia after prostate biopsy., (Copyright © 2013 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

Recently, we have shown that green tea (GT) consumption improves both reflexes and sensation in unilateral chronic constriction injury to the sciatic nerve. Considering the substantial neuroprotective properties of GT polyphenols, we sought to investigate whether (-)-epigallocatechin-3-gallate (EGCG) could protect the sciatic nerve and improve functional impairments induced by a crushing injury. We also examined whether neuronal cell apoptosis induced by the crushing injury is affected by EGCG treatment. Histological examination of sciatic nerves from EGCG-treated (50mg/kg; i.p.) showed that axonotmized rats had a remarkable axonal and myelin regeneration with significant decrease in the number of myelinated axonal fibers compared to vehicle-treated crush group. Similarly, ultrastructural evaluation of EGCG-treated nerves displayed normal unmyelinated and myelinated axons with regular myelin sheath thickness and normalized appearance of Schmidt-Lantermann clefts. Extracellular matrix displayed normal collagen fibers appearance with distinctively organized distribution similar to sham animals. Analysis of foot position and extensor postural thrust test showed a progressive and faster recovery in the EGCG-treated group compared to vehicle-treated animals. EGCG-treated rats showed significant increase in paw withdrawal thresholds to mechanical stimulation compared to vehicle-treated crush group. EGCG treatment also restored the mRNA expression of Bax, Bcl-2 and survivin but not that of p53 to sham levels on days 3 and 7 post-injury. Our results demonstrate that EGCG treatment enhanced functional recovery, advanced morphological nerve rescue and accelerated nerve regeneration following crush injury partly due to the down regulation of apoptosis related genes., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Objective: To assess whether epidermal growth factor receptor (EGFR) and survivin immunostaining of tumour cells in urinary cytology and tissue of patients with bladder cancer has a prognostic significance., Design: Prospective study, Setting: Department of Surgery (Division of Urology), Mubarak Al-Kabeer Teaching Hospital and Faculty of Medicine, Kuwait University, Kuwait, Subjects: Urine cytology smears obtainedpriorto cystoscopy in patients with transitional cell carcinoma (TCC) of the bladder were immunostained for EGFR and survivin. Bladder cancer tissue resected at surgery was also immunostained for EGFR and survivin expression. Tissue expression of EGFR and survivin in TCC of the bladder was compared to their expression in urine cytology and relationship to tumour grade and stage., Results: 178 patients were studied (43 newly diagnosed bladder cancer, 58 with recurrent TCC and 77 in disease remission). Twenty five patients with normal urothelium served as controls. The mean sensitivity of urine cytology, tissue survivin immunohistochemistry (IHC) and tissue EGFR IHC was 30.5%, 62% and 59% respectively. The corresponding mean specificity was 95%, 79% and 38% respectively. For grades 1, 2 and 3 bladder tumors, tissue expression positivity for EGFR was 47.8%, 92.9%, 100% and for tissue survivin it was 27.8%, 18.2% and 33.3% respectively. For grades 1, 2 and 3 bladder tumors, urine expression positivity for EGFR was 35.7%, 40% and 67.7% and for urine survivin it was 8.3%, 42.9% and 33.3% respectively., Conclusion: Positive EGFR immunostaining of urine cytology specimen or tumour tissue increases with histological grade of TCC of the bladder. Survivin expression is less consistent in both urine cytology specimen and tissue samples. EGFR immunostaining may provide a useful tool in the grading of bladder TCC and aid in the selection of patients that may benefit from administration of EGFR inhibitors.

Aim: To determine whether the measurement of tissue mRNA levels of AMACR and survivin has a role in distinguishing prostate cancer (PCa) from benign lesions and high risk from low-risk PCa in men with suspected PCa., Methods: TRUS prostate biopsies from 170 patients with suspected PCa were used to measure the mRNA levels of AMACR and survivin using semi-quantitative RT-PCR technique. The diagnosis, staging and risk stratification of PCa was based on established clinical criteria. The ability of tissue mRNA levels to distinguish benign from malignant prostate and high- and low-risk PCa was assessed. The diagnostic value for the two genes was evaluated by calculating their individual and combined sensitivity and specificity, which were compared with that of PSA., Results: Histological examination showed 90/170 (53%) of patients had benign prostate pathology, while 80/170 (47%) had PCa. Tissue mRNA levels of both AMACR and survivin were able to distinguish benign from PCa biopsies (p<0.0001) and also high-risk from low-risk PCa cases (p<0.02, p<0.05, respectively). Compared with serum PSA levels, the combined use of tissue mRNA levels of AMACR and survivin yielded a higher detection specificity (84 vs. 22%)., Conclusion: Based on AMACR and survivin combined sensitivity and specificity, these mRNA markers can be used as an adjunct to distinguish patients with and without PCa and in men with PCa may help to identify those with low- or high-risk PCa.

Muscle degeneration and impairment following nerve injury could lead to apoptosis as a result of increased levels of reactive oxygen species. This activates the apoptotic cascade through mitochondrial dysfunction and damage to lipids, proteins, and DNA. In considering of the multifactorial protective properties of green tea polyphenols (-)-epigallocatechin-3-gallate (EGCG), this study investigates whether EGCG treatment does improve skeletal muscle function impairments, induced by crushing of the sciatic nerve. Compared to the saline-treated injured group of animals, EGCG treatment of axonotomized animals showed significant motor enhancement in the toe spread and foot positioning analysis and gain in the percentage motor deficit. The proprioceptive function expressed by the hopping response showed significant progression in the EGCG-treated group. Recovery of sensory innervation was followed by a slowly retreating neuropathic pain-like syndrome in the EGCG-treated animals. Muscle tissues from injured limb showed severe histopathological alterations that were significantly attenuated by EGCG treatment at the end of week 3 post-surgery. Semi-quantitative desmin immunohistochemistry revealed intense staining in the saline-treated injured animals, whereas EGCG treatment decreased the desmin immunoreactivity back to sham control levels. Using RT-PCR, EGCG treatment induced a significant anti-apoptotic effect in injured muscle tissues by normalizing the Bax/Bcl-2 ratio back to baseline levels and inhibiting overexpression of the p53 apoptotic gene at days 3 and 7 post-surgery. In conclusion, our results demonstrate that EGCG enhances functional recovery, protects muscle fibers from cellular death by activating anti-apoptotic signaling pathway, and improves morphological recovery in skeletal muscle after nerve injuries.

Background and Objectives: Enhancer of zeste homolog 2 (EZH2) has been recently found to regulate several genes involved in immunoresponse and autocrine inflammation network. The aim of the study was to quantitate EZH2 messenger ribonucleic acid (mRNA) expression, evaluate its relation to conditions of prostatitis associated with benign prostatic hyperplasia (BPH), and correlate it with the levels of the inflammatory marker interlukin 6 (IL-6)., Design and Setting: Cross-sectional study in Middle Eastern men with BPH and prostatitis or BPH only., Patients and Methods: Transrectal ultrasound-guided prostate biopsies were collected from 106 patients suspected of having prostate cancer; however, the histology revealed BPH. Upon further pathological examination, 56 of these cases were identified as BPH with prostatitis and classified as: acute prostatitis (n=13); active chronic prostatitis (n=32); and, chronic inactive prostatitis (n=12). Serum IL-6 levels and EZH2 mRNA expression were measured and compared between patient groups., Results: EZH2 mRNA was overexpressed in BPH with prostatitis patients compared to BPH only patients (P<.0001). BPH with active chronic prostatitis had higher EZH2 expression than BPH with acute or chronic inactive prostatitis compared to BPH only (P=.05 and .73, respectively). EZH2 mRNA expression showed a negative correlation with IL-6 concentrations in BPH with prostatitis patients (rs=-0.31, P=.02). EZH2 overexpression was associated with an increased risk of having BPH with prostatitis (crude odds ratio 0.20, 95% CI 0.06-0.65, P=.0076)., Conclusions: EZH2 mRNA expression correlates positively with prostatitis conditions associated with BPH and negatively with serum IL-6 levels. This supports the possible involvement of EZH2 mRNA overexpression in the development of prostate inflammation, and its new regulatory role in suppressing the expression of some inflammatory network genes.

Testicular torsion (TT) is a urologic emergency that may result in future infertility problems. The pathologic process of TT is similar to an ischemia reperfusion injury (IRI). The purpose of this study was to evaluate the effect of epigallocatechin-3-gallate (EGCG) on reversing the damaging consequences of TT-induced IRI by examining its inhibitory effects on the expression of inflammatory and apoptosis mediators in a unilateral TT rat model. Eighteen male Sprague-Dawley rats were divided into 3 groups. Group 1 underwent a sham operation of the left testis under general anesthesia. Group 2 underwent ischemia for 1h followed by 4h reperfusion in the presence of saline. The third group was similar to group 2, however, EGCG (50 mg/kg) was injected i.p. 30 min after ischemia induction. The in vivo protective effect of EGCG was tested by measuring testicular levels of TNF-α, IL-6 and IL-1β by ELISA and mRNA expression of iNOS, MCP-1, p53, Bax, Bcl-2 and survivin by real-time PCR. Also, testicular morphological changes and damage to spermatogenesis were evaluated using H&E staining and Johnsen's scoring system, respectively. EGCG treatment improved testicular structures in the ipsilateral testis, markedly inhibited germ cell apoptosis (GCA) and significantly decreased testicular cytokine levels. In addition, EGCG was able to down regulate the mRNA expression of iNOS, MCP-1 and pro-apoptosis genes in favor of cell survival. For the first time we show that in vivo EGCG treatment rescued the torsed testes from IRI-induced inflammation, GCA and damage to spermatogenesis thus suggesting a new preventive approach to inhibiting the inflammatory and apoptotic consequences of TT-induced IRI., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Objective: To compare the diagnostic performance of urine cytology (UC), survivin mRNA expression, and the NMP22 BladderChek® (NMP22BC) test for the detection, grading and staging of transitional cell carcinoma (TCC) of the bladder., Materials and Methods: Voided urine samples collected from 25 healthy controls and 80 patients diagnosed with TCC of the bladder were subjected to UC, the NMP22BC test and reverse-transcription real-time PCR for survivin mRNA expression., Results: Survivin mRNA expression showed the highest sensitivity (87.5%) followed by the NMP22BC test (61.3%) while UC exhibited the lowest sensitivity (40%). All three urine markers had a similar specificity of 96% (95% CI 80.5-99.3%). Survivin mRNA expression was the only urine marker that showed a significant difference in relation to tumour histological grade (χ(2) 8.5, p = 0.015). None of the three urine markers was significantly related to tumour pathological stages., Conclusion: The diagnostic sensitivity of urinary survivin mRNA expression was superior to that of UC and the NMP22BC test and correlates with tumour pathological grade but not stage., (Copyright © 2012 S. Karger AG, Basel.)

Raf kinase inhibitory protein (RKIP) is a physiologic inhibitor of c-RAF kinase and nuclear factor κB signaling that represses tumor invasion and metastasis. Glycogen synthase kinase-3β (GSK3β) suppresses tumor progression by downregulating multiple oncogenic pathways including Wnt signaling and cyclin D1 activation. Here, we show that RKIP binds GSK3 proteins and maintains GSK3β protein levels and its active form. Depletion of RKIP augments oxidative stress-mediated activation of the p38 mitogen activated protein kinase, which, in turn, inactivates GSK3β by phosphorylating it at the inhibitory T390 residue. This pathway de-represses GSK3β inhibition of oncogenic substrates causing stabilization of cyclin D, which induces cell-cycle progression and β-catenin, SNAIL, and SLUG, which promote epithelial to mesenchymal transition. RKIP levels in human colorectal cancer positively correlate with GSK3β expression. These findings reveal the RKIP/GSK3 axis as both a potential therapeutic target and a prognosis-based predictor of cancer progression., (©2011 AACR.)

Objective: To evaluate the expression of the apoptotic genes survivin, Bax and Bcl-2 in vasectomized rabbits and to determine their relation with vasectomy-induced spermatogenic impairment and germ cell apoptosis., Materials and Methods: Twelve adult rabbits (6-12 months old) were divided into three groups: sham control, unilateral vasectomy or bilateral vasectomy. Six months after vasectomy, testicular tissue was analyzed for germ cell apoptosis and DNA fragmentation by the TUNEL assay and gel electrophoresis, respectively. Spermatogenesis was assessed using the Johnsen score. The relative gene expression of survivin, Bax and Bcl-2 was measured using reverse transcription followed by real-time PCR., Results: Compared to sham animals, a significant decrease in testicular survivin mRNA levels was measured in the two vasectomy animal groups (p < 0.05). This was accompanied by a significant increase in the Bax:Bcl-2 ratio in the vasectomized animals (p < 0.05). In addition, these data showed positive correlation with enhanced apoptotic index, damage to spermatogenesis and DNA fragmentation after vasectomy., Conclusion: These findings demonstrate that vasectomy-induced damage to spermatogenesis due to testicular apoptosis may be associated with survivin downregulation and Bax overexpression., (Copyright © 2011 S. Karger AG, Basel.)

Testicular torsion is associated with damage to the testicular tissue as a result of ischemia-reperfusion injury (IRI) and induction of apoptosis leading to progressive damage to spermatogenesis. Survivin is suggested to be an important regulator in the control of the mitochondrial apoptotic pathway, although its role in torsion-induced IRI is unknown. Therefore, we sought to evaluate testicular survivin expression after long term IRI induced by testicular torsion. Survivin expression was measured by real-time PCR in 6-12 month old New Zealand white rabbits divided into three groups (4 animals/group): group (A) sham control, group (B) ischemia alone for 60 min and group (C) ischemia for 60 min followed by reperfusion for 6 months. Germ cell apoptosis was evaluated by TUNEL assay, Bax/Bcl-2 ratio and DNA fragmentation. The Johnsen score was used to assess testicular morphological damage, while lipid peroxidation was used as an indicator for oxidative stress. Survivin expression was detected in all testicular tissue samples. The rate of survivin expression after IRI was significantly higher (p<0.05) compared with ischemic only and sham control testes. Its expression in IRI samples was inversely correlated with the significant increase (p<0.05) in apoptosis, oxidative levels and spermatogenic damage. In conclusion, down-regulation of testicular survivin expression after long term IRI to the testis and its association with apoptosis induction suggests its involvement in the regulation of this apoptotic pathway. These findings also identify survivin as a potential new target for the prevention of germ cell death during testicular torsion., (Copyright (c) 2010. Published by Elsevier Inc.)

Angiotensin-(1-7) [Ang-(1-7)] is a vasodilator peptide with cardiac and vascular protective properties. We examined the influence of Ang-(1-7), both endogenous and after chronic treatment with the peptide (576microg/(kgday)), on ischemia/reperfusion (I/R)-induced cardiac dysfunction in streptozotocin-treated spontaneously hypertensive rats (diabetic SHR). In isolated perfused hearts, recovery of left ventricular function from 40min of global ischemia was improved significantly in Ang-(1-7)- or captopril-treated diabetic SHR and worsened in animals treated with A779, an Ang-(1-7) receptor (AT((1-7))) antagonist. The beneficial effect of captopril on cardiac recovery was reduced when co-administered with A779. Cardiac NF-kappaB activity appears to be higher in diabetic SHR and treatment with Ang-(1-7) or captopril decreased NF-kappaB activity in diabetic SHR, an effect partially reversed by co-administration of A779. Real-time PCR-based gene array analysis of cardiac tissue revealed that Ang-(1-7) or captopril treatment may reduce expression of several genes of inflammation involved in the NF-kappaB signalling pathway. The data provide for the first time a role for endogenous Ang-(1-7) as well as confirmation that exogenous treatment with the peptide produces cardioprotection. Whether potential anti-inflammatory and transcriptional factor changes are directly linked to the cardioprotection produced by Ang-(1-7) in diabetic SHR remains to be determined.

The present study was designed to see if acute local inhibition of Ras-GTPase before or after ischemia (during perfusion) would produce protection against ischemia and reperfusion (I/R)-induced cardiac dysfunction. The effect of glibenclamide, an inhibitor of cardiac mitochondrial ATP-sensitive potassium (mitoK(ATP)) channels, on Ras-GTPase-mediated cardioprotection was also studied. A 40 min episode of global ischemia followed by a 30 min reperfusion in perfused rat hearts produced significantly impaired cardiac function, measured as left ventricular developed pressure (P(max)) and left ventricular end-diastolic pressure (LVEDP). Perfusion with Ras-GTPase inhibitor FPT III before I/R [FPT(pre)], significantly enhanced cardiac recovery in terms of left ventricular contractility. P(max) was significantly higher at the end of 30 min reperfusion in FPT(pre)-treated hearts compared to pre-conditioned hearts. However, the degree of improvement in left ventricular contractility was significantly less when FPT III was given only after ischemia during reperfusion [FPT(post)]. Combination treatment with FPT III and glibenclamide before I/R resulted in significant reduction of FPT III-mediated cardioprotection. These data suggest that activation of Ras-GTPase signaling pathways during ischemia are critical in the development of left ventricular dysfunction and that opening of mitoK(ATP) channels, at least in part, contributes to cardioprotection produced by Ras-GTPase inhibition., ((c) 2005 John Wiley & Sons, Ltd.)

The aim of this study was to test the hypothesis that treatment with angiotensin-(1-7) [ANG-(1-7)] or ANG-(1-7) nonpeptide analog AVE-0991 can produce protection against diabetes-induced cardiovascular dysfunction. We examined the influence of chronic treatment (4 wk) with ANG-(1-7) (576 microg.kg(-1).day(-1) ip) or AVE-0991 (576 microg.kg(-1).day(-1) ip) on proteinuria, vascular responsiveness of isolated carotid and renal artery ring segments and mesenteric bed to vasoactive agonists, and cardiac recovery from ischemia-reperfusion in streptozotocin-treated rats (diabetes). Animals were killed 4 wk after induction of diabetes and/or treatment with ANG-(1-7) or AVE-0991. There was a significant increase in urine protein (231 +/- 2 mg/24 h) in diabetic animals compared with controls (88 +/- 6 mg/24 h). Treatment of diabetic animals with ANG-(1-7) or AVE-0991 resulted in a significant reduction in urine protein compared with vehicle-treated diabetic animals (183 +/- 16 and 149 +/- 15 mg/24 h, respectively). Treatment with ANG-(1-7) or AVE-0991 also prevented the diabetes-induced abnormal vascular responsiveness to norepinephrine, endothelin-1, angiotensin II, carbachol, and histamine in the perfused mesenteric bed and isolated carotid and renal arteries. In isolated perfused hearts, recovery of left ventricular function from 40 min of global ischemia was significantly better in ANG-(1-7)- or AVE-0991-treated animals. These results suggest that activation of ANG-(1-7)-mediated signal transduction could be an important therapeutic strategy to reduce cardiovascular events in diabetic patients.

Background: Wide evidence suggests the involvement of translation elongation factors (EFs) at the onset of oncogenesis. To investigate the potential role of the EF-1 subunits (A, Balpha and Bgamma) in the formation and progression of breast cancer, we quantified their expression in human breast tissues and cell lines., Materials and Methods: The mRNA levels of EF-1A, -1Balpha and -1Bgamma in human breast tissues and cell lines were measured by semi-quantitative RT-PCR and Northern blotting, respectively., Results: The mRNA expression of the three EF-1 subunits was significantly higher in cancerous over normal tissues. However, there was no significant difference in their expression between tumor grades. Overexpression of EF-1 mRNA in breast cancer cell lines were not caused by increased mRNA stability., Conclusion: Although not indicative of tumor grading, the elevated levels of EF-1 subunits are suggestive of their early role in the pathogenesis of breast cancer, possibly through their increased rate of transcription.

Translation elongation factor-1 (EF-1) forms a primary site of regulation of protein synthesis and has been implicated amongst others in tumorigenesis, diabetes and cell death. To investigate whether diabetes-induced oxidative stress affects EF-1 gene expression, we used a free radical scavenger, vitamin E. The following groups of rats (5/group) were studied: control, vitamin E control, diabetic and diabetic treated with vitamin E. Markers of hyperglycemia, kidney function, oxidative stress, and kidney hypertrophy were elevated in diabetic rats. Increased urinary protein excretion indicated early signs of glomerular and tubular dysfunction. The mRNA and protein levels of the three EF-1 subunits (A, Balpha, and Bgamma) were determined in renal cortex extracts using semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR), northern blot analysis and western blotting. EF-1A mRNA expression in renal cortex extracts was significantly increased by at least 2-fold (p < 0.002) in diabetic rats; however, there was no change in the mRNA levels of EF-1Balpha and EF-1Bgamma subunits. Similar results were observed at the protein level. Treatment of diabetic rats with vitamin E for 10 days suppressed both glycemic and oxidative stresses in renal cortex and kidney hypertrophy. EF-1A mRNA and protein levels were also reduced to control levels. In conclusion, EF-1A but not EF-1Balpha and EF-1Bgamma gene expression is significantly enhanced in the renal cortex of diabetic rats. Normalization of enhanced EF-1A expression by vitamin E treatment suggests a role for EF-1A during diabetes-induced oxidative stress.

Escherichia coli, lacking cytoplasmic superoxide dismutases, exhibits a variety of oxygen-dependent phenotypic deficits. Enrichment of the growth medium with Mn(II) relieved those deficits. Extracts of cells grown on Mn(II)-rich medium exhibited superoxide dismutase-like activity that was due partially to low-molecular-weight and partially to high-molecular-weight complexes. The high-molecular-weight activity was sensitive to proteolysis. Hence this activity is likely associated with low-affinity binding of Mn to proteins.