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Upregulation of peroxisome proliferator activated receptor alpha by fenofibrate in induced testicular ischemia reperfusion
Biomedicine & Pharmacotherapy 98 (2018) 507–515
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Upregulation of peroxisome proliferator activated receptor alpha by fenofibrate in induced testicular ischemia reperfusion
T
Marwa M.M. Refaie Department of Pharmacology, Faculty of Medicine, Minia University, 61511, Minia, Egypt
A R T I C L E I N F O
A B S T R A C T
Keywords: Testicular ischemia reperfusion Fenofibrate Peroxisome proliferator activated receptor alpha Testosterone
Torsion of the spermatic cord is a common urological emergency among infants and adolescents that can lead to testicular necrosis and infertility. We investigated the effect of fenofibrate (FEN) on induced testicular ischemia reperfusion (testicular (I-R). FEN (100, 300 mg/kg/day) was administered orally in presence or absence of testicular (I-R). We measured testicular weight changes and serum testosterone level. In addition; Testicular tissue reduced glutathione (GSH), malondialdehyde (MDA), total nitrites (NOx) and superoxide dismutase (SOD) activity were measured. Moreover; tumor necrosis factor alpha (TNF-α), nuclear factor kappa B (NF-κB) immunoexpressions and histopathology were evaluated. Testicular (I-R) induced group showed significant decrease in serum testosterone level and testicular weight with increase in testicular tissue MDA and NOx levels. Testicular (I-R) induced group showed the histopathological changes of marked testicular damage according to Johnsen's score. In addition, there was significant reduction in GSH and SOD testicular tissue levels but significant increase in TNF-α and NF-κB immunoexpressions. FEN was able to markedly improve testicular (I-R) induced changes through its action on peroxisome proliferator activated receptor alpha (PPARα), anti-oxidant, anti-inflammatory and anti-apoptotic effects.
1. Introduction Testicular Torsion is considered as urological emergency which requires early diagnosis and surgical intervention to prevent testicular damage. Management of testicular torsion may be accompanied by further damage to the testes. Reperfusion of ischemic tissue leads to sequence of events that injure the tissue [1–3]. These injuries can be more severe than injuries induced by ischemia [4]. Ischemia-reperfusion of testis stimulates an intracellular signaling cascade in the endothelial cells that leads to neutrophil recruitment, reactive oxygen species (ROS) formation, inflammation, release of cytokines, such as interleukin-1b, tumor necrosis factor alpha (TNF-α) and nuclear factor kappa B (NF-κB). Recruitment of neutrophils and macrophages causes testicular atrophy, disruption of spermatogenesis and germ cell apoptosis [4]. ROS can cause tissue damage or death through cell membrane lipid peroxidation, protein denaturation and DNA impairment [5]. Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors. The group includes three isotypes encoded by different genes: PPAR α, β, γ, δ. These receptors are known for their involvement in fatty acid, lipoprotein metabolism,
glucose homeostasis, cellular proliferation and differentiation and the immune response [6]. Accordingly, PPARs are important targets in treatment of metabolic disorders such as type 2 diabetes mellitus and inflammatory diseases. Different studies identified other functions of PPARs in blood pressure regulation, neuroinflammation, neuroprotection and inflammatory pain reduction [7]. The fibrate family contains several compounds that are all peroxisome proliferator activated receptor alpha (PPARα) agonists. PPARα is one of the three subtypes of the nuclear receptor PPAR [8]. PPARα activation leads to the expression of target genes such as Cu/Zn SOD or glutathione peroxidases, glutathione reductase and glutathione Stransferase which increase the redox state. Moreover, PPARα activation represses NF-κB and activator protein-1 signaling pathways thereby down regulates the inflammatory response and oxidative stress [9]. 1.1. Purpose of the study In current study we tried to evaluate the role of upregulation of PPARα receptors in induced testicular (I-R) model by using a potent PPARα agonist, anti-inflammatory, antioxidant and anti-apoptotic drug
E-mail addresses: [email protected], [email protected]. https://doi.org/10.1016/j.biopha.2017.12.093 Received 9 September 2017; Received in revised form 15 December 2017; Accepted 19 December 2017 0753-3322/ © 2017 Elsevier Masson SAS. All rights reserved.
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NF-κB antibody and Immunostaining Detection Kit were from Lab Vision Laboratories, USA.
Table 1 Effect of FEN on testicular weights and serum testosterone in testicular (I-R) induced rats. Group
Testicular weight/g
Serum testosterone (nmol/ml)
Control FEN1 FEN2 testicular (I-R) testicular (I-R) + FEN1 testicular (I-R) + FEN2
1.80 ± 0.04 1.9 ± 0.02 1.9 ± 0.02 1.2 ± 0.04a 1.6 ± 0.06a,b 1.8 ± 0.02b
2.3 2.1 2.4 0.2 1.9 2.0
± ± ± ± ± ±
2.2. Animals and experimental design
0.08 0.06 0.14 0.02a 0.06a,b 0.05b
Adult male Wistar albino rats weighing 250–300 g were from the animal house, Giza, Egypt. Animals were left in standard housing conditions in cages, 3 rats/cage, and were left to acclimatize. Rats were given laboratory chow and tap water. This work was in the Pharmacology Departement, Faculty of Medicine, Minia University, Egypt and the animal experimental protocol was approved by the faculty board in accordance with European (EU) directive 2010/63/EU. Rats were randomly divided into 6 groups (n = 6 each group) Group I was sham group. It was subjected to all operative procedures, except vessels occlusion. This group was given vehicle (1% carboxymethylcellulose) once orally [10]. Group II was treated with FEN (100 mg/kg) once orally [11]. Group III was treated with FEN (300 mg/kg) once orally [12]. Group IV was testicular (I-R) induced group [4]. Group V was treated with FEN (100 mg/kg) once orally [11] +testicular (I-R) [4]. Group VI was treated with FEN (300 mg/kg) once orally [12] +testicular (I-R) [4]. Explanation of the operative procedures in each group: All rats were weighed then groups I, IV, V, VI were anesthetized using ip injection of 20% Urethane hydrochloride. After anesthesia, the rats were kept in a supine position and underwent antisepsis of the scrotal region with 2% iodine alcohol. Surgery was performed through a left scrotal incision. Unilateral testicular torsion was created in groups IV, V, VI by twisting the left testis 1080° in a clockwise direction and fixed within hemiscrotum with a 3/0 silk suture. The incision was then closed using 2/0 silk suture in groups I, IV, V, VI. Testicular (I-R) induced rats were subjected to 1hour of ischemia and 3h of reperfusion. Groups V, VI were received single dose of FEN (100, 300 mg/kg) respectively 30 min before reperfusion of the testes orally. Rats were sacrificed immediately after 3 h.
Values are representation of 6 observations in each group as means ± S.E.M. Results are considered significantly different when P < .05. a Significant difference compared to control sham operated group. b Significant difference compared to testicular ischemia reperfusion induced group. FEN1 is fenofibrate given group (100 mg/kg), FEN2 is fenofibrate given group (300 mg/ kg). Testicular (I-R) is testicular ischemia reperfusion induced group and testicular (I-R) +FEN1 is the testicular ischemia reperfusion and fenofibrate given group (100mg/kg). Testicular (I-R)+FEN2 is the testicular ischemia reperfusion and fenofibrate given group (300 mg/kg). Table 2 Effect of FEN on testicular tissue GSH and SOD levels in testicular (I-R) induced rats. Group
GSH (μmol/g tissue)
SOD (unit/mg tissue)
Control FEN1 FEN2 testicular (I-R) testicular (I-R) + FEN1 testicular (I-R) + FEN2
81.7 ± 3.1 82.97 ± 3.2 84.7 ± 4.3 18.2 ± 4.1a 55.2 ± 1.7a,b 80.6 ± 2.6b
3.3 3.2 3.4 2.4 2.9 3.2
± ± ± ± ± ±
0.15 0.11 0.16 0.13a 0.02a,b 0.04b
Values are representation of 6 observations in each group as means ± S.E.M. Results are considered significantly different when P < .05. a Significant difference compared to control sham operated group. b Significant difference compared to testicular ischemia reperfusion induced group. FEN1 is fenofibrate given group (100 mg/kg), FEN2 is fenofibrate given group (300 mg/ kg). Testicular (I-R) is testicular ischemia reperfusion induced group and testicular (I-R) +FEN1 is the testicular ischemia reperfusion and fenofibrate given group (100 mg/kg). Testicular (I-R)+FEN2 is the testicular ischemia reperfusion and fenofibrate given group (300 mg/kg).
2.3. Evaluation of serum testosterone
Table 3 Effect of FEN on MDA and NOX levels in testicular (I-R) induced rats. Group
MDA (nmol/g tissue)
NOX (nmol/g tissue)
Control FEN1 FEN2 testicular (I-R) testicular (I-R) + FEN1 testicular (I-R) + FEN2
123.2 125.5 123.8 171.8 135.7 124.7
138.2 135.3 138.2 187.3 166.5 142.2
± ± ± ± ± ±
.1.5 .1.5 1.3 1.9a 1.4a,b 1.4b
± ± ± ± ± ±
Measurement of serum testosterone was measured according to testosterone ELISA kit (Cayman Chemicals., USA). This measurement is based on the competition between testosterone and a testosterone acetylcholine esterase conjugate (testosterone tracer).This antiserum testosterone complex binds to mouse monoclonal anti-rabbit IgG that has previously attached to the well. The plate is washed to remove any unbound reagents and then Ellman's Reagent is added. The reaction gives color measured at 412 nm.
1.9 1.9 1.8 4.9a 4.9a,b 2.9b
Values are representation of 6 observations in each group as means ± S.E.M. Results are considered significantly different when P < .05. a Significant difference compared to control sham operated group. b Significant difference compared to testicular ischemia reperfusion induced group. FEN1 is fenofibrate given group (100 mg/kg), FEN2 is fenofibrate given group (300 mg/ kg). Testicular (I-R) is testicular ischemia reperfusion induced group and testicular (I-R) +FEN1 is the testicular ischemia reperfusion and fenofibrate given group (100 mg/kg). Testicular (I-R)+FEN2 is the testicular ischemia reperfusion and fenofibrate given group (300 mg/kg).
2.4. Evaluation of testicular homogenate All of the testicular tissues were washed with saline solution then the testicles were weighed on Mettler Toledo scale, Swizer Land. Ipsilateral testes were kept at −80 °C. Testes were homogenized (GlasCol homogenizer, USA) and homogenate was prepared in ice cold Tris HCl buffer solution for biochemical analysis. The homogenate was centrifuged at 4000 rpm for 15 min at 4 °C in cooling centrifuge and the supernatant was kept at −80 °C till used.
fenofibrate (FEN) on induced testicular ischemia reperfusion (testicular (I-R) in rats.
2.5. Evaluation of testicular reduced glutathione (GSH) and superoxide dismutase (SOD) levels 2. Materials and methods Evaluation of testicular antioxidant defense mechanisms was done by assessment of testicular tissue GSH and SOD enzyme levels. GSH measurement depends on that the sulfhydryl groups of GSH react with5, 5-dithio-bis-2-nitrobenzoic acid (Ellman’s reagent). That gives a yellow colored 5-thio-2-nitrobenzoic acid. The color density was
2.1. Chemicals FEN powder was from Mina Pharm Company. Testosterone ELISA kit (Cayman Chemicals., USA). Polyclonal TNF-α antibody, polyclonal 508
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Fig. 1. Evaluation of testicular tissues histopathology. Hematoxylin and eosin histopathological evaluation of rat testicles: (A) is a Control group and (B), (C) are fenofibrate (100,300 mg/kg) given groups, respectively. Black arrow refers to normal architecture of the testis and Seminiferous tubules were round to oval with regular outlines. Red arrow refers to germinal epithelium at different stages of spermatogenesis and Sertoli cells. Germinal epithelium was formed of spermatogonia, spermatocytes, spermatozoa, rounded spermatids and elongated spermatids. Blue arrow refers to the interstitial spaces in-between the tubules contained Leydig cells and some blood vessels. (D) represents testicular ischemia reperfusion (testicular (I-R) induced group (group IV). Black arrow refers to disruption of testicular architecture in the form of marked reduction in number of germinal epithelium. Red arrow refers to the arrest of spermatogenesis. Blue arrow refers to the interstitial spaces that showed edema and congestion of blood vessels. (E) represents testicular ischemia reperfusion (testicular (I-R) + fenofibrate (100 mg/kg) (group V) showed mild changes of seminiferous tubules compared to the marked damage observed in Group IV. (F) represents testicular ischemia reperfusion (testicular (I-R) + fenofibrate (300 mg/kg) (group VI) with significant improvement of testicular ischemia reperfusion (testicular (I-R) induced damage. Black arrow refers to marked improvement of testicular architecture and normalization of the germinal epithelium. Red arrow refers to normal spermatogenesis. Blue arrow shows marked reduction of the interstitial spaces edema and congestion of blood vessels.
Nitrites and nitrates are an index of NOX production which depends on Griess reaction. It depends on the spectrophotometric measurement of NOX at 540 nm after conversion of nitrate to nitrite by copperized cadmium granules which expressed as nmol/g tissue [16].
detected at 405nm colorimetrically by using Beckman DU-64 UV/VIS spectrophotometer, USA. The results were expressed as μmol/g tissue [13]. The evaluation of testicular SOD levels depends on the ability of the enzyme to inhibit the phenazinemethosulphate-mediated reduction of nitrobluetetrazolium dye. Results were expressed as unit/mg tissue [14].
2.7. Histopathological evaluation 2.6. Assessment of testicular tissue malondialdehyde (MDA) and total nitrites (NOX) levels
Immediately after sacrifice, ipsilateral testicles were dissected and the adipose tissue was removed. They were fixed in Bouin’s solution for 24 h, processed and embedded in paraffin wax. Five μm serial sections were prepared and stained with hematoxylin and eosin. Assessment was performed with light microscopy using an Olympus microscope, Japan. The examiner was blinded to the treated groups.
Lipid peroxidation depends on thiobarbituric acid reacting substance and that is expressed as equivalents of MDA, by using 1, 1, 3, 3tetramethoxypropane as standard. The results were expressed as nmol/ g tissue [15].
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covered slipped. Each staining batch included both positive and negative control sections. In negative control; primary antibody was omitted and replaced by PBS.
Table 4 Effect of FEN on Johnsen's score in testicular (I-R) induced rats. Group
Johnsen's score
Control FEN1 FEN2 testicular (I-R) testicular (I-R) + FEN1 testicular (I-R) + FEN2
8.93 ± .83 8.33 ± .42 8.68 ± .48 2.2 ± .30a 6.2 ± .24a,b 7.97 ± .48b
2.10. Scoring of immunostaining Screening of sections was done under light microscope magnification X200. Each slide was individually evaluated and scored in a blinded fashion. To assess positive staining for TNF-α, tissue section was screened for positive cells, defined as cells with cytoplasmic staining and the cut-off for positivity was set on 5% [18]. Regarding NFκB, tissue section was screened for positive cells, defined as cells with nuclear staining considered positive when > 10% of the cells were positive [19].
Values are representation of 6 observations in each group as means ± S.E.M. Results are considered significantly different when P < .05. a Significant difference compared to control sham operated group. b significant difference compared to testicular ischemia reperfusion induced group. FEN1 is fenofibrate given group (100 mg/kg), FEN2 is fenofibrate given group (300 mg/kg). Testicular (I-R) is testicular ischemia reperfusion induced group and testicular (I-R) +FEN1 testicular ischemia reperfusion and fenofibrate given group (100 mg/kg). Testicular (I-R) +FEN2 testicular ischemia reperfusion and fenofibrate given group (300 mg/kg).
2.11. Statistical analysis Data was analyzed by one way ANOVA followed by Dunnett Multiple Comparison Test. The values are represented as means ± SEM. Statistical analysis was done using GraphPad Prism software (version 5). The differences were considered significant when the calculated P value is less than. 05.
2.8. Histological grading according to Johnsen’s scoring system [17] The testicular tissues were examined in random microscopic areas semi-quantitatively under 20 high power fields and the number of changes was assessed by counting of 3 non overlapped fields for the same slide of each animal. Hematoxylin and eosin stained sections of the testes were semiquantitavely graded according to Johnsen’s scoring system. Score–10 (Complete spermatogenesis with many spermatozoa present) Score–9 (Slightly impaired spermatogenesis with many late spermatids, disorganized epithelium) Score–8 (Less than five spermatozoa per tubule, few late spermatids) Score–7 (No spermatozoa, no late spermatids, many early spermatids) Score–6 (No spermatozoa, no late spermatids, few early spermatids) Score–5 (No spermatozoa or spermatids, many spermatocytes) Score–4 (No spermatozoa or spermatids, few spermatocytes) Score–3 (Spermatogonia only) Score–2 (No germinal cells, Sertoli cells only) Score–1 (No seminiferous epithelium)
3. Results 3.1. Effect of FEN on testicular weights and serum testosterone level in testicular (I-R) induced rats Testicular (I-R) significantly decreased testicular weights and serum testosterone levels compared to control group. Administration of FEN to testicular (I-R) induced rats significantly increased testicular weights and serum testosterone levels compared to testicular (I-R) induced group (Table 1). 3.2. Evaluation of testicular homogenate 3.2.1. Evaluation of testicular tissues GSH and SOD Testicular (I-R) caused significant decrease in testicular tissue GSH and SOD levels compared with control group (Table 2). Administration of FEN to testicular (I-R) induced rats significantly increased the levels of GSH and SOD compared to testicular (I-R) treated group. 3.2.2. Evaluation of testicular tissue MDA and NOX MDA was evaluated as an indicator of testicular lipid peroxidation. In addition; NOX levels were evaluated (Table 3). Testicular (I-R) significantly increased MDA and NOX levels compared to control group. Administration of FEN to testicular (I-R) induced rats significantly decreased MDA and NOX compared to testicular (I-R) group.
2.9. Immunohistochemistry Five μm sections were placed on positively charged slides (CITOGLAS). Sections were de-paraffinized with xylene, hydrated through 99.9%, 95%, and 70% ethanol, and then treated with 3% hydrogen peroxide for 30 min to inactivate endogenous peroxides, then washed in phosphate-buffered saline (PBS) solution. For antigen retrieval, sections were treated for 20 min in citrate buffer (pH 6.0) by microwave, and then allow cooling. After rinse in PBS, primary antibodies were incubated overnight in a humidity chamber using NF-κB and TNF-α primary antibodies, then washed with PBS before applying the biotinylated secondary antibody for 30 min. Sections were washed in PBS and incubated with the streptavidin-biotin complex reagent for 30 min. A brown color was developed with 3, 3-diaminobenzidinetetra hydrochloride (DAB) for 5 min, then washed in distilled water, counterstained with haematoxylin, dehydrated, cleared in xylene, mounted and
3.3. Histopathological evaluation As shown in Fig. 1, groups I–III which represent control, FEN (100 mg/kg), FEN (300mg/kg) respectively showed normal architecture of the testis, Seminiferous tubules were round to oval with regular outlines and they were lined by layers of germinal epithelium at different stages of spermatogenesis and Sertoli cells. Germinal epithelium was formed of spermatogonia, spermatocytes, spermatozoa, rounded spermatids and elongated spermatids and the interstitial spaces in-between the tubules contained Leydig cells and some blood vessels (Fig. 1A–C) respectively. Testicular (I-R) induced group (group IV) showed disruption of
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Fig. 2. tumor necrosis factor alpha (TNF-α) immunoexpression in testicular tissues. (A) represents control group, (B) represents fenofibrate (100 mg/kg) (group II). (C) represents fenofibrate (300 mg/kg) (group III). There were negative. immunoexpression of tumor necrosis factor alpha (TNF-α) in these groups. (D) represents testicular ischemia reperfusion (testicular (I-R) induced group (group IV). Black arrow showed positive immunoexpression of tumor necrosis factor alpha (TNF-α). € represents testicular ischemia reperfusion (testicular (I-R) + fenofibrate (100 mg/kg) (group V). Black arrow showed mild immunoexpression of tumor necrosis factor alpha (TNF-α). (F) represents testicular ischemia reperfusion (testicular (I-R) + fenofibrate (300 mg/kg) (group VI) showed negative immunoexpression of tumor necrosis factor alpha (TNF-α).
number of germinal epithelium and spermatogenesis. There is marked reduction of the interstitial spaces edema and congestion of blood vessels (Fig. 1F).
testicular architecture in the form of marked reduction in number of germinal epithelium, arrest of spermatogenesis and the interstitial spaces showed edema and congestion of blood vessels (Fig. 1D). The animals given FEN (100 mg/kg) with induction of testicular (IR) (group V) showed mild changes of seminiferous tubules compared to the marked damage observed in the animals in Group IV (Fig. 1E). The animals given FEN (300 mg/kg) with induction of testicular (IR) (group VI) showed marvelous reduction of testicular (I-R) induced damage in form of marked improvement of testicular architecture and
3.4. Histopathological scoring of H&E according to Johnsen's score Evaluation of histopathological changes according to Johnsen's score showed significant decrease in Johnsen's score in testicular (I-R) induced group in comparison with normal control group. On
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3.8. Semiquantitive analysis of NF-κB immunoexpression (Fig. 5) There is significant increase in NF-κB immunoexpression in testicular (I-R) induced group in comparison with control group. Administration of FEN showed significant decrease in NF-κB immunoexpression compared to testicular (I-R) induced group. 4. Discussion Spermatic cord torsion-detorsion is an ischemia reperfusion process of the testis. In such cases ROS are overproduced which is considered the main cause of tissue injury [2]. Our study showed significant decrease of serum testosterone and testicular weights in testicular (I-R) induced group that is due to marked testicular damage, apoptosis and necrosis that occur not only in ipisilateral testicle but also in the contralateral one [5]. These findings are in line with [5] who found significant decrease in testicular weight in testicular (I-R) induced group and in agreement with [20] who found significant decrease in serum testosterone in testicular (I-R) induced group. Coadministration of FEN showed significant increase in serum testosterone and testicular weights compared to testicular (I-R) group. This indicates the protective effect of FEN due to its anti-inflammatory, antioxidant and anti-apoptotic properties which leads to marked preservation of the testicular tissue against testicular (I-R) induced damage [21]. Oxidative stress has a main role in testicular (I-R). Ischemia stimulates chemotactic factors and leads to the migration of polymorphonuclear leukocytes to the ischemic region, which also generates ROS after reperfusion [22]. Oxygen increases during the first 60–90 min of reperfusion, which causes the toxic burst of ROS. The enzymatic antioxidant defense system, which includes SOD, catalase and GSH react to scavenge the free radicals to protect tissues from injury [22]. SOD catalyses the superoxide anion (O2.–) to hydrogen peroxide then it is converted to water by catalase or GSH [2]. GSH is the predominant low molecular weight thiol in mammalian cells and it is present in high concentration in adult mouse testis. This tripeptide has different functions which contribute to maintain cell defense and protection. This includes free radical scavenger, coenzyme for several antioxidant enzymes, maintenance of the thiol-disulfide status, and detoxication of electrophilic xenobiotics via conjugation [23]. That is detected in our study which showed decrease in SOD and GSH in testicular (I-R) induced group due to overproduction of ROS and free radicals. On administration of FEN to testicular (I-R) induced group; SOD and GSH markedly increased. Elevation of these enzymes is due to antioxidant properties of FEN [21]. The production of ROS in excessive amounts reacts with membrane lipids and results in lipid peroxidation and eventually the loss of cellular components of the tissue [22]. These free radicals react with lipid in cellular and mitochondrial membranes forming lipid peroxides. Membrane lipid peroxidation disrupts or changes membrane permeability and cell integrity. Lipid peroxidation is probably the most extensively investigated process induced by free radicals. O2%− reacts with lipid to form lipid peroxides followed by β-oxidation to form MDA which is an important indicator for the state of lipid peroxidation [24]. The same is detected in our results which showed significant increase in testicular MDA levels compared to sham operated group. That is in agreement with Parlaktas et al. [3] and Yıldız et al. [2]. NOx is water and lipid soluble which plays an important role in regulating blood flow in normal and pathological states. Ischemia causes an increase in NO synthetase activity and changes in NOx levels [25]. Reperfusion leads to the generation of the O.2–, and the
Fig. 3. Semiquantitive analysis of TNF-α immunoexpression. There is significant increase in tumor necrosis factor alpha (TNF-α) immunoexpression in testicular ischemia reperfusion (testicular (I-R) induced group in comparison with control group. Administration of fenofibrate (FEN) showed significant decrease in tumor necrosis factor alpha (TNF-α) immunoexpression compared to testicular ischemia reperfusion (testicular (I-R) induced group. Values are representation of 6 observations in each group as means ± S.E.M. Results are considered significantly different when P < .05. a Significant difference compared to control sham operated group, b significant difference compared to testicular ischemia reperfusion induced group. FEN1 is fenofibrate given group (100 mg/kg), FEN2 is fenofibrate given group (300 mg/kg). Testicular (I-R) is testicular ischemia reperfusion induced group and testicular (I-R)+FEN1 is the testicular ischemia reperfusion and fenofibrate given group (100 mg/kg). Testicular (I-R)+FEN2 is the testicular ischemia reperfusion and fenofibrate given group (300 mg/kg).
administration of FEN; results showed significant increase in the score in comparison with testicular (I-R) induced group (Table 4). 3.5. Evaluation of TNF-α immunoexpression As shown in Fig. 2, group I,II,III which represent control, FEN (100 mg/kg), FEN (300mg/kg) respectively showed negative immunoexpression of TNF-α (Fig. 2 A,B,C). Testicular (I-R) induced group (group IV) showed positive immunoexpression of TNF-α (Fig. 2D). FEN (100 mg/kg) administered group with induction of testicular (IR) showed mild immunoexpression of TNF-α (Fig. 2E). FEN (300 mg/kg) administered group with induction of testicular (IR) showed negative immunoexpression of TNF-α (Fig. 2F). 3.6. Semiquantitive analysis of TNF-α immunoexpression (Fig. 3) There is significant increase in TNF-α immunoexpression in testicular (I-R) induced group in comparison with control group. Administration of FEN showed significant decrease in TNF-α immunoexpression compared to testicular (I-R) induced group. 3.7. Evaluation of NF-κB immunoexpression As shown in Fig. 4, group I,II,III which represent control, FEN (100 mg/kg), FEN (300 mg/kg) respectively showed negative expressions of NF-κB (Fig. 4A–C). Testicular (I-R) induced group (group IV) showed positive expression of NF-κB immunoexpression (Fig. 4D). FEN (100 mg/kg) given group with induction of testicular (I-R) showed mild immunoexpression of NF-κB (Fig. 4E). FEN (300 mg/kg) given group with induction of testicular (I-R) showed negative immunoexpression of NF-κB (Fig. 4F).
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Fig. 4. Evaluation of nuclear factor kappa B (NF-κB) immunoexpression. (A) represents control group (group I), (B) represents fenofibrate (100 mg/kg) (group II), (C) represents fenofibrate (300 mg/kg) (group III). There were negative immunoexpression of nuclear factor kappa B (NF-κB) in these groups. (D) represents testicular ischemia reperfusion (testicular (I-R) induced group (group IV). Black arrow showed positive immunoexpression of nuclear factor kappa B (NF-κB). (E) represents testicular ischemia reperfusion (testicular (I-R) + fenofibrate (100 mg/kg) (group V). Black arrow showed mild immunoexpression of nuclear factor kappa B (NF-κB). (F) expresses testicular ischemia reperfusion (testicular (I-R) + fenofibrate (300 mg/kg) (group VI) and showed negative immunoexpression of nuclear factor kappa B (NF-κB).
interaction between NO and O.2– produces a specific type of ROS; peroxynitrite, which further promotes cellular damage [25].That was found in our study which showed significant increase in NOX levels in testicular (I-R) induced group. Coadministration of FEN to testicular (I-R) induced group results in significant decrease in MDA and NOX levels compared to testicular (I-R) induced group indicating the protective effect of FEN against membrane lipid peroxidation and nitric oxide formation This effect is due to
its PPARα agonist effect, antioxidant and anti-inflammatory properties of FEN This is in line with Ibrahim et al. [26]. Our study showed the typical histopathological features of testicular (I-R) and scored according to Johnsen's score as in Al-Maghrebi and Renno. [27]; Asghari et al. [22]. These changes were restored to normal with administration of FEN. Testicular (I-R) induced ROS formation is accompanied by different cytokines release as TNF-α and NF-κB. Theses cytokines are responsible
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Bordet, Recovery of brain biomarkers following peroxisome proliferator-activated receptor agonist neuroprotective treatment before ischemic stroke, Proteome Sci. 12 (2014) 24. [9] P. Losey, E. Ladds, M. Laprais, B. Geuvel, L. Burns, R. Bordet, D.C. Anthony, The role of PPAR activation during the systemic response to brain injury, J. Neuroinflamm. 12 (2015) 99. [10] A.M. Yousaf, D.W. Kim, Y.K. Oh, C.S. Yong, J.O. Kim, H.G. Choi, Enhanced oral bioavailability of fenofibrate using polymeric nanoparticulated systems: physicochemical characterization and in vivo investigation, Int. J. Nanomed. 10 (2015) 1819–1830. [11] V.H. Oidor-Chan, E. Hong, F. Pérez-Severiano, S. Montes, J.C. Torres-Narváez, L.D. Valle-Mondragón, G. Pastelín-Hernández, A. Sánchez-Mendoza, Fenofibrate Plus Metformin Produces Cardioprotection in a Type 2 Diabetes and Acute Myocardial Infarction Model vol. 2016, Hindawi Publishing Corporation PPAR Research, 2016 Article ID 8237264, 14 pages. [12] Y. Han, M. Do, M.S. Kim, E. Seo, M.K. Park, D.K. Kim, H.J. Lee, S.Y. Seo, Fenofibrate reduces age-related hypercholesterolemia in normal rats on a standard diet, Korean J. Physiol. Pharmacol. 14 (2010) 77–81. [13] E. Beutler, O. Duron, B.M. Kelly, Improved method for the determination of blood glutathione, J. Lab Clin. Med. 61 (1963) 882–888. [14] M. Nishikimi, N. Appaji, K. Yagi, The occurrence of superoxide anion in the reaction of reduced phenazine methosulfate and molecular oxygen, Biochem. Bioph. Res. Common. 46 (2) (1972) 849–854. [15] M. Mihara, M. Uchiyama, Properties of thiobarbituric acid-reactive materials obtained from lipid peroxide and tissue homogenate, Chem. Pharm. Bull. 31 (2) (1983) 605–611. [16] S. Sogut, S.S. Zoroglu, H. Ozyurt, H.R. Yilmaz, F. Ozugurlu, E. Sivasli, O. Yetkin, M. Yanik, H. Tutkun, H.A. Savas, M. Tarakcioglu, O. Akyol, Changes in nitric oxide levels and antioxidant enzyme activities may have a role in the pathophysiological mechanisms involved in autism, Clin. Chim. Acta 331 (1–2) (2003) 111–117. [17] S.G. Johnsen, Testicular biopsy score count - a method for registration of spermatogenesis in human testis: normal values and results in 325 hypogonadal males, Hormones 1 (1970) 1–24. [18] G. Soria, M. Ofri-Shahak, I. Haas, N. Yaal-Hahoshen, L. Leider-Trejo, T. LeibovichRivkin, P. Weitzenfeld, T. Meshel, E. Shabtai, M. Gutman, A. Ben-Baruch, Inflammatory mediators in breast cancer: coordinated expression of TNFα & IL-1β with CCL2 & CCL5 and effects on epithelial-to-mesenchymal transition, BMC Cancer 12 (11) (2011) 130. [19] S.O. Yoon, B. Kim, H.S. Lee, G.H. Kang, W.H. Kim, Y.A. Kim, J.E. Kim, M.S. Chang, Differential protein immunoexpression profiles in appendiceal mucinous neoplasms: a special reference to classification and predictive factors, Mod. Pathol. 22 (2009) 1102–1112. [20] M. Güzel, M.F. Sönmez, O. Baştuğ, N.F. Aras, A.B. Öztürk, M. Küçükaydın, C. Turan, Effectiveness of lycopene on experimental testicular torsion, J. Pediatr. Surg. 51 (7) (2016) 1187–1191. [21] M.M. Helmy, M.W. Helmy, M.M. El-Mas, Additive renoprotection by pioglitazone and fenofibrate against inflammatory, oxidative and apoptotic manifestations of cisplatin nephrotoxicity: modulation by PPARs, PLoS One 10 (11) (2015) e0142303. [22] A. Asghari, G. Akbari, A. Meghdadi, P. Mortazavi, Protective effect of metformin on testicular ischemia/reperfusion injury in rats, Acta Cirúrgica Brasileira 31 (6) (2016) 2411. [23] S.B. Guimarães, O.S. Kimura, P.R. Leitão de Vasconcelos, Dimethylsulfoxide attenuates ischemia-reperfusion injury in rat testis, Acta Cirúrgica Brasileira. 25 (2010) 4. [24] M.M. Refaie, E.F. Amin, N.F. El-Tahawy, A.M. Abdelrahman, Possible protective effect of diacerein on doxorubicin-induced nephrotoxicity in rats, J. Toxicol. (2016)
Fig. 5. Semiquantitive analysis of nuclear factor kappa B (NF-κB) immunoexpression. There is significant increase in nuclear factor kappa B (NF-κB) immunoexpression in testicular ischemia reperfusion (testicular (I-R) induced group in comparison with control group. Administration of fenofibrate (FEN) showed significant decrease in nuclear factor kappa B (NF-κB) immunoexpression compared to testicular ischemia reperfusion (testicular (I-R) induced group. Values are representation of 6 observations in each group as means ± S.E.M. Results are considered significantly different when P < .05. a Significant difference compared to control sham operated group, b significant difference compared to testicular ischemia reperfusion induced group. FEN1 is fenofibrate given group (100 mg/kg), FEN2 is fenofibrate given group (300mg/kg). Testicular (I-R) is testicular ischemia reperfusion induced group and testicular (I-R)+FEN1 is the testicular ischemia reperfusion and fenofibrate given group (100 mg/kg). Testicular (I-R)+FEN2 is the testicular ischemia reperfusion and fenofibrate given group (300mg/kg).
for mediation of apoptosis, inflammation and subsequent tissue damage [28]. That is found in our study which showed significant increase in TNF-α and NF-κB immunoexpressions in testicular (I-R) group in comparison with normal control group. That is in line with Zhang et al. [28] and Ozcan et al. [5]. On addition of FEN to testicular (I-R) group showed significant decrease in their immunoexpressions which indicates the anti-inflammatory and anti-apoptotic effects of FEN.
5. Conclusion Upregulation of PPAR-α by FEN showed marked protective effect in induced testicular (I-R). FEN (300mg/kg) showed more improvement than low dose (100 mg/kg). Further studies are recommended to evaluate the suspected protective effect of fenofibrate in urgent cases of testicular torsion in males.
Conflicts of Interest None.
Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Acknowledgement Many thanks to Dr. Maram El-hussieny, Lecturer of Pathology, Faculty of Medicine, Minia University, for her generous help in this study. 514
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[27] M. Al-Maghrebi, W.M. Renno, Altered expression profile of glycolytic enzymes during testicular ischemia reperfusion injury is associated with the p53/TIGAR pathway: effect of fructose 1,6-diphosphate, Peer J. 4 (2016) e2195. [28] Y. Zhang, Y. Lv, Y.J. Liu, C. Yang, H.J. Hu, X.E. Meng, M.X. Li, S.Y. Pan, Hyperbaric oxygen therapy in rats attenuates ischemia-reperfusion testicular injury through blockade of oxidative stress, suppression of inflammation and reduction of nitric oxide formation, Urology 82 (2) (2013) 489 e9-489.e15.
9507563. [25] G. Ozmerdiven, B. Coskun, O. Kaygisiz, B.A. Vuruskan, B. Asiltas, Kilicarslan, The protective effect of L-arginine, tadalafil, and their combination in rat testes after ischemia and reperfusion injury, Can. Urol. Assoc. J. 11 (1–2) (2017) E19–E25. [26] M.A. Ibrahim, A.A. El-Sheikh, H.M. Khalaf, A.M. Abdelrahman, Protective effect of peroxisome proliferator activator receptor (PPAR)-α and -γ ligands against methotrexate-induced nephrotoxicity, Immunopharmacol. Immunotoxicol. 36 (2) (2014) 130–137.
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Upregulation of peroxisome proliferator activated receptor alpha by fenofibrate in induced testicular ischemia reperfusion
T
Marwa M.M. Refaie Department of Pharmacology, Faculty of Medicine, Minia University, 61511, Minia, Egypt
A R T I C L E I N F O
A B S T R A C T
Keywords: Testicular ischemia reperfusion Fenofibrate Peroxisome proliferator activated receptor alpha Testosterone
Torsion of the spermatic cord is a common urological emergency among infants and adolescents that can lead to testicular necrosis and infertility. We investigated the effect of fenofibrate (FEN) on induced testicular ischemia reperfusion (testicular (I-R). FEN (100, 300 mg/kg/day) was administered orally in presence or absence of testicular (I-R). We measured testicular weight changes and serum testosterone level. In addition; Testicular tissue reduced glutathione (GSH), malondialdehyde (MDA), total nitrites (NOx) and superoxide dismutase (SOD) activity were measured. Moreover; tumor necrosis factor alpha (TNF-α), nuclear factor kappa B (NF-κB) immunoexpressions and histopathology were evaluated. Testicular (I-R) induced group showed significant decrease in serum testosterone level and testicular weight with increase in testicular tissue MDA and NOx levels. Testicular (I-R) induced group showed the histopathological changes of marked testicular damage according to Johnsen's score. In addition, there was significant reduction in GSH and SOD testicular tissue levels but significant increase in TNF-α and NF-κB immunoexpressions. FEN was able to markedly improve testicular (I-R) induced changes through its action on peroxisome proliferator activated receptor alpha (PPARα), anti-oxidant, anti-inflammatory and anti-apoptotic effects.
1. Introduction Testicular Torsion is considered as urological emergency which requires early diagnosis and surgical intervention to prevent testicular damage. Management of testicular torsion may be accompanied by further damage to the testes. Reperfusion of ischemic tissue leads to sequence of events that injure the tissue [1–3]. These injuries can be more severe than injuries induced by ischemia [4]. Ischemia-reperfusion of testis stimulates an intracellular signaling cascade in the endothelial cells that leads to neutrophil recruitment, reactive oxygen species (ROS) formation, inflammation, release of cytokines, such as interleukin-1b, tumor necrosis factor alpha (TNF-α) and nuclear factor kappa B (NF-κB). Recruitment of neutrophils and macrophages causes testicular atrophy, disruption of spermatogenesis and germ cell apoptosis [4]. ROS can cause tissue damage or death through cell membrane lipid peroxidation, protein denaturation and DNA impairment [5]. Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors. The group includes three isotypes encoded by different genes: PPAR α, β, γ, δ. These receptors are known for their involvement in fatty acid, lipoprotein metabolism,
glucose homeostasis, cellular proliferation and differentiation and the immune response [6]. Accordingly, PPARs are important targets in treatment of metabolic disorders such as type 2 diabetes mellitus and inflammatory diseases. Different studies identified other functions of PPARs in blood pressure regulation, neuroinflammation, neuroprotection and inflammatory pain reduction [7]. The fibrate family contains several compounds that are all peroxisome proliferator activated receptor alpha (PPARα) agonists. PPARα is one of the three subtypes of the nuclear receptor PPAR [8]. PPARα activation leads to the expression of target genes such as Cu/Zn SOD or glutathione peroxidases, glutathione reductase and glutathione Stransferase which increase the redox state. Moreover, PPARα activation represses NF-κB and activator protein-1 signaling pathways thereby down regulates the inflammatory response and oxidative stress [9]. 1.1. Purpose of the study In current study we tried to evaluate the role of upregulation of PPARα receptors in induced testicular (I-R) model by using a potent PPARα agonist, anti-inflammatory, antioxidant and anti-apoptotic drug
E-mail addresses: [email protected], [email protected]. https://doi.org/10.1016/j.biopha.2017.12.093 Received 9 September 2017; Received in revised form 15 December 2017; Accepted 19 December 2017 0753-3322/ © 2017 Elsevier Masson SAS. All rights reserved.
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NF-κB antibody and Immunostaining Detection Kit were from Lab Vision Laboratories, USA.
Table 1 Effect of FEN on testicular weights and serum testosterone in testicular (I-R) induced rats. Group
Testicular weight/g
Serum testosterone (nmol/ml)
Control FEN1 FEN2 testicular (I-R) testicular (I-R) + FEN1 testicular (I-R) + FEN2
1.80 ± 0.04 1.9 ± 0.02 1.9 ± 0.02 1.2 ± 0.04a 1.6 ± 0.06a,b 1.8 ± 0.02b
2.3 2.1 2.4 0.2 1.9 2.0
± ± ± ± ± ±
2.2. Animals and experimental design
0.08 0.06 0.14 0.02a 0.06a,b 0.05b
Adult male Wistar albino rats weighing 250–300 g were from the animal house, Giza, Egypt. Animals were left in standard housing conditions in cages, 3 rats/cage, and were left to acclimatize. Rats were given laboratory chow and tap water. This work was in the Pharmacology Departement, Faculty of Medicine, Minia University, Egypt and the animal experimental protocol was approved by the faculty board in accordance with European (EU) directive 2010/63/EU. Rats were randomly divided into 6 groups (n = 6 each group) Group I was sham group. It was subjected to all operative procedures, except vessels occlusion. This group was given vehicle (1% carboxymethylcellulose) once orally [10]. Group II was treated with FEN (100 mg/kg) once orally [11]. Group III was treated with FEN (300 mg/kg) once orally [12]. Group IV was testicular (I-R) induced group [4]. Group V was treated with FEN (100 mg/kg) once orally [11] +testicular (I-R) [4]. Group VI was treated with FEN (300 mg/kg) once orally [12] +testicular (I-R) [4]. Explanation of the operative procedures in each group: All rats were weighed then groups I, IV, V, VI were anesthetized using ip injection of 20% Urethane hydrochloride. After anesthesia, the rats were kept in a supine position and underwent antisepsis of the scrotal region with 2% iodine alcohol. Surgery was performed through a left scrotal incision. Unilateral testicular torsion was created in groups IV, V, VI by twisting the left testis 1080° in a clockwise direction and fixed within hemiscrotum with a 3/0 silk suture. The incision was then closed using 2/0 silk suture in groups I, IV, V, VI. Testicular (I-R) induced rats were subjected to 1hour of ischemia and 3h of reperfusion. Groups V, VI were received single dose of FEN (100, 300 mg/kg) respectively 30 min before reperfusion of the testes orally. Rats were sacrificed immediately after 3 h.
Values are representation of 6 observations in each group as means ± S.E.M. Results are considered significantly different when P < .05. a Significant difference compared to control sham operated group. b Significant difference compared to testicular ischemia reperfusion induced group. FEN1 is fenofibrate given group (100 mg/kg), FEN2 is fenofibrate given group (300 mg/ kg). Testicular (I-R) is testicular ischemia reperfusion induced group and testicular (I-R) +FEN1 is the testicular ischemia reperfusion and fenofibrate given group (100mg/kg). Testicular (I-R)+FEN2 is the testicular ischemia reperfusion and fenofibrate given group (300 mg/kg). Table 2 Effect of FEN on testicular tissue GSH and SOD levels in testicular (I-R) induced rats. Group
GSH (μmol/g tissue)
SOD (unit/mg tissue)
Control FEN1 FEN2 testicular (I-R) testicular (I-R) + FEN1 testicular (I-R) + FEN2
81.7 ± 3.1 82.97 ± 3.2 84.7 ± 4.3 18.2 ± 4.1a 55.2 ± 1.7a,b 80.6 ± 2.6b
3.3 3.2 3.4 2.4 2.9 3.2
± ± ± ± ± ±
0.15 0.11 0.16 0.13a 0.02a,b 0.04b
Values are representation of 6 observations in each group as means ± S.E.M. Results are considered significantly different when P < .05. a Significant difference compared to control sham operated group. b Significant difference compared to testicular ischemia reperfusion induced group. FEN1 is fenofibrate given group (100 mg/kg), FEN2 is fenofibrate given group (300 mg/ kg). Testicular (I-R) is testicular ischemia reperfusion induced group and testicular (I-R) +FEN1 is the testicular ischemia reperfusion and fenofibrate given group (100 mg/kg). Testicular (I-R)+FEN2 is the testicular ischemia reperfusion and fenofibrate given group (300 mg/kg).
2.3. Evaluation of serum testosterone
Table 3 Effect of FEN on MDA and NOX levels in testicular (I-R) induced rats. Group
MDA (nmol/g tissue)
NOX (nmol/g tissue)
Control FEN1 FEN2 testicular (I-R) testicular (I-R) + FEN1 testicular (I-R) + FEN2
123.2 125.5 123.8 171.8 135.7 124.7
138.2 135.3 138.2 187.3 166.5 142.2
± ± ± ± ± ±
.1.5 .1.5 1.3 1.9a 1.4a,b 1.4b
± ± ± ± ± ±
Measurement of serum testosterone was measured according to testosterone ELISA kit (Cayman Chemicals., USA). This measurement is based on the competition between testosterone and a testosterone acetylcholine esterase conjugate (testosterone tracer).This antiserum testosterone complex binds to mouse monoclonal anti-rabbit IgG that has previously attached to the well. The plate is washed to remove any unbound reagents and then Ellman's Reagent is added. The reaction gives color measured at 412 nm.
1.9 1.9 1.8 4.9a 4.9a,b 2.9b
Values are representation of 6 observations in each group as means ± S.E.M. Results are considered significantly different when P < .05. a Significant difference compared to control sham operated group. b Significant difference compared to testicular ischemia reperfusion induced group. FEN1 is fenofibrate given group (100 mg/kg), FEN2 is fenofibrate given group (300 mg/ kg). Testicular (I-R) is testicular ischemia reperfusion induced group and testicular (I-R) +FEN1 is the testicular ischemia reperfusion and fenofibrate given group (100 mg/kg). Testicular (I-R)+FEN2 is the testicular ischemia reperfusion and fenofibrate given group (300 mg/kg).
2.4. Evaluation of testicular homogenate All of the testicular tissues were washed with saline solution then the testicles were weighed on Mettler Toledo scale, Swizer Land. Ipsilateral testes were kept at −80 °C. Testes were homogenized (GlasCol homogenizer, USA) and homogenate was prepared in ice cold Tris HCl buffer solution for biochemical analysis. The homogenate was centrifuged at 4000 rpm for 15 min at 4 °C in cooling centrifuge and the supernatant was kept at −80 °C till used.
fenofibrate (FEN) on induced testicular ischemia reperfusion (testicular (I-R) in rats.
2.5. Evaluation of testicular reduced glutathione (GSH) and superoxide dismutase (SOD) levels 2. Materials and methods Evaluation of testicular antioxidant defense mechanisms was done by assessment of testicular tissue GSH and SOD enzyme levels. GSH measurement depends on that the sulfhydryl groups of GSH react with5, 5-dithio-bis-2-nitrobenzoic acid (Ellman’s reagent). That gives a yellow colored 5-thio-2-nitrobenzoic acid. The color density was
2.1. Chemicals FEN powder was from Mina Pharm Company. Testosterone ELISA kit (Cayman Chemicals., USA). Polyclonal TNF-α antibody, polyclonal 508
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Fig. 1. Evaluation of testicular tissues histopathology. Hematoxylin and eosin histopathological evaluation of rat testicles: (A) is a Control group and (B), (C) are fenofibrate (100,300 mg/kg) given groups, respectively. Black arrow refers to normal architecture of the testis and Seminiferous tubules were round to oval with regular outlines. Red arrow refers to germinal epithelium at different stages of spermatogenesis and Sertoli cells. Germinal epithelium was formed of spermatogonia, spermatocytes, spermatozoa, rounded spermatids and elongated spermatids. Blue arrow refers to the interstitial spaces in-between the tubules contained Leydig cells and some blood vessels. (D) represents testicular ischemia reperfusion (testicular (I-R) induced group (group IV). Black arrow refers to disruption of testicular architecture in the form of marked reduction in number of germinal epithelium. Red arrow refers to the arrest of spermatogenesis. Blue arrow refers to the interstitial spaces that showed edema and congestion of blood vessels. (E) represents testicular ischemia reperfusion (testicular (I-R) + fenofibrate (100 mg/kg) (group V) showed mild changes of seminiferous tubules compared to the marked damage observed in Group IV. (F) represents testicular ischemia reperfusion (testicular (I-R) + fenofibrate (300 mg/kg) (group VI) with significant improvement of testicular ischemia reperfusion (testicular (I-R) induced damage. Black arrow refers to marked improvement of testicular architecture and normalization of the germinal epithelium. Red arrow refers to normal spermatogenesis. Blue arrow shows marked reduction of the interstitial spaces edema and congestion of blood vessels.
Nitrites and nitrates are an index of NOX production which depends on Griess reaction. It depends on the spectrophotometric measurement of NOX at 540 nm after conversion of nitrate to nitrite by copperized cadmium granules which expressed as nmol/g tissue [16].
detected at 405nm colorimetrically by using Beckman DU-64 UV/VIS spectrophotometer, USA. The results were expressed as μmol/g tissue [13]. The evaluation of testicular SOD levels depends on the ability of the enzyme to inhibit the phenazinemethosulphate-mediated reduction of nitrobluetetrazolium dye. Results were expressed as unit/mg tissue [14].
2.7. Histopathological evaluation 2.6. Assessment of testicular tissue malondialdehyde (MDA) and total nitrites (NOX) levels
Immediately after sacrifice, ipsilateral testicles were dissected and the adipose tissue was removed. They were fixed in Bouin’s solution for 24 h, processed and embedded in paraffin wax. Five μm serial sections were prepared and stained with hematoxylin and eosin. Assessment was performed with light microscopy using an Olympus microscope, Japan. The examiner was blinded to the treated groups.
Lipid peroxidation depends on thiobarbituric acid reacting substance and that is expressed as equivalents of MDA, by using 1, 1, 3, 3tetramethoxypropane as standard. The results were expressed as nmol/ g tissue [15].
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covered slipped. Each staining batch included both positive and negative control sections. In negative control; primary antibody was omitted and replaced by PBS.
Table 4 Effect of FEN on Johnsen's score in testicular (I-R) induced rats. Group
Johnsen's score
Control FEN1 FEN2 testicular (I-R) testicular (I-R) + FEN1 testicular (I-R) + FEN2
8.93 ± .83 8.33 ± .42 8.68 ± .48 2.2 ± .30a 6.2 ± .24a,b 7.97 ± .48b
2.10. Scoring of immunostaining Screening of sections was done under light microscope magnification X200. Each slide was individually evaluated and scored in a blinded fashion. To assess positive staining for TNF-α, tissue section was screened for positive cells, defined as cells with cytoplasmic staining and the cut-off for positivity was set on 5% [18]. Regarding NFκB, tissue section was screened for positive cells, defined as cells with nuclear staining considered positive when > 10% of the cells were positive [19].
Values are representation of 6 observations in each group as means ± S.E.M. Results are considered significantly different when P < .05. a Significant difference compared to control sham operated group. b significant difference compared to testicular ischemia reperfusion induced group. FEN1 is fenofibrate given group (100 mg/kg), FEN2 is fenofibrate given group (300 mg/kg). Testicular (I-R) is testicular ischemia reperfusion induced group and testicular (I-R) +FEN1 testicular ischemia reperfusion and fenofibrate given group (100 mg/kg). Testicular (I-R) +FEN2 testicular ischemia reperfusion and fenofibrate given group (300 mg/kg).
2.11. Statistical analysis Data was analyzed by one way ANOVA followed by Dunnett Multiple Comparison Test. The values are represented as means ± SEM. Statistical analysis was done using GraphPad Prism software (version 5). The differences were considered significant when the calculated P value is less than. 05.
2.8. Histological grading according to Johnsen’s scoring system [17] The testicular tissues were examined in random microscopic areas semi-quantitatively under 20 high power fields and the number of changes was assessed by counting of 3 non overlapped fields for the same slide of each animal. Hematoxylin and eosin stained sections of the testes were semiquantitavely graded according to Johnsen’s scoring system. Score–10 (Complete spermatogenesis with many spermatozoa present) Score–9 (Slightly impaired spermatogenesis with many late spermatids, disorganized epithelium) Score–8 (Less than five spermatozoa per tubule, few late spermatids) Score–7 (No spermatozoa, no late spermatids, many early spermatids) Score–6 (No spermatozoa, no late spermatids, few early spermatids) Score–5 (No spermatozoa or spermatids, many spermatocytes) Score–4 (No spermatozoa or spermatids, few spermatocytes) Score–3 (Spermatogonia only) Score–2 (No germinal cells, Sertoli cells only) Score–1 (No seminiferous epithelium)
3. Results 3.1. Effect of FEN on testicular weights and serum testosterone level in testicular (I-R) induced rats Testicular (I-R) significantly decreased testicular weights and serum testosterone levels compared to control group. Administration of FEN to testicular (I-R) induced rats significantly increased testicular weights and serum testosterone levels compared to testicular (I-R) induced group (Table 1). 3.2. Evaluation of testicular homogenate 3.2.1. Evaluation of testicular tissues GSH and SOD Testicular (I-R) caused significant decrease in testicular tissue GSH and SOD levels compared with control group (Table 2). Administration of FEN to testicular (I-R) induced rats significantly increased the levels of GSH and SOD compared to testicular (I-R) treated group. 3.2.2. Evaluation of testicular tissue MDA and NOX MDA was evaluated as an indicator of testicular lipid peroxidation. In addition; NOX levels were evaluated (Table 3). Testicular (I-R) significantly increased MDA and NOX levels compared to control group. Administration of FEN to testicular (I-R) induced rats significantly decreased MDA and NOX compared to testicular (I-R) group.
2.9. Immunohistochemistry Five μm sections were placed on positively charged slides (CITOGLAS). Sections were de-paraffinized with xylene, hydrated through 99.9%, 95%, and 70% ethanol, and then treated with 3% hydrogen peroxide for 30 min to inactivate endogenous peroxides, then washed in phosphate-buffered saline (PBS) solution. For antigen retrieval, sections were treated for 20 min in citrate buffer (pH 6.0) by microwave, and then allow cooling. After rinse in PBS, primary antibodies were incubated overnight in a humidity chamber using NF-κB and TNF-α primary antibodies, then washed with PBS before applying the biotinylated secondary antibody for 30 min. Sections were washed in PBS and incubated with the streptavidin-biotin complex reagent for 30 min. A brown color was developed with 3, 3-diaminobenzidinetetra hydrochloride (DAB) for 5 min, then washed in distilled water, counterstained with haematoxylin, dehydrated, cleared in xylene, mounted and
3.3. Histopathological evaluation As shown in Fig. 1, groups I–III which represent control, FEN (100 mg/kg), FEN (300mg/kg) respectively showed normal architecture of the testis, Seminiferous tubules were round to oval with regular outlines and they were lined by layers of germinal epithelium at different stages of spermatogenesis and Sertoli cells. Germinal epithelium was formed of spermatogonia, spermatocytes, spermatozoa, rounded spermatids and elongated spermatids and the interstitial spaces in-between the tubules contained Leydig cells and some blood vessels (Fig. 1A–C) respectively. Testicular (I-R) induced group (group IV) showed disruption of
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Fig. 2. tumor necrosis factor alpha (TNF-α) immunoexpression in testicular tissues. (A) represents control group, (B) represents fenofibrate (100 mg/kg) (group II). (C) represents fenofibrate (300 mg/kg) (group III). There were negative. immunoexpression of tumor necrosis factor alpha (TNF-α) in these groups. (D) represents testicular ischemia reperfusion (testicular (I-R) induced group (group IV). Black arrow showed positive immunoexpression of tumor necrosis factor alpha (TNF-α). € represents testicular ischemia reperfusion (testicular (I-R) + fenofibrate (100 mg/kg) (group V). Black arrow showed mild immunoexpression of tumor necrosis factor alpha (TNF-α). (F) represents testicular ischemia reperfusion (testicular (I-R) + fenofibrate (300 mg/kg) (group VI) showed negative immunoexpression of tumor necrosis factor alpha (TNF-α).
number of germinal epithelium and spermatogenesis. There is marked reduction of the interstitial spaces edema and congestion of blood vessels (Fig. 1F).
testicular architecture in the form of marked reduction in number of germinal epithelium, arrest of spermatogenesis and the interstitial spaces showed edema and congestion of blood vessels (Fig. 1D). The animals given FEN (100 mg/kg) with induction of testicular (IR) (group V) showed mild changes of seminiferous tubules compared to the marked damage observed in the animals in Group IV (Fig. 1E). The animals given FEN (300 mg/kg) with induction of testicular (IR) (group VI) showed marvelous reduction of testicular (I-R) induced damage in form of marked improvement of testicular architecture and
3.4. Histopathological scoring of H&E according to Johnsen's score Evaluation of histopathological changes according to Johnsen's score showed significant decrease in Johnsen's score in testicular (I-R) induced group in comparison with normal control group. On
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3.8. Semiquantitive analysis of NF-κB immunoexpression (Fig. 5) There is significant increase in NF-κB immunoexpression in testicular (I-R) induced group in comparison with control group. Administration of FEN showed significant decrease in NF-κB immunoexpression compared to testicular (I-R) induced group. 4. Discussion Spermatic cord torsion-detorsion is an ischemia reperfusion process of the testis. In such cases ROS are overproduced which is considered the main cause of tissue injury [2]. Our study showed significant decrease of serum testosterone and testicular weights in testicular (I-R) induced group that is due to marked testicular damage, apoptosis and necrosis that occur not only in ipisilateral testicle but also in the contralateral one [5]. These findings are in line with [5] who found significant decrease in testicular weight in testicular (I-R) induced group and in agreement with [20] who found significant decrease in serum testosterone in testicular (I-R) induced group. Coadministration of FEN showed significant increase in serum testosterone and testicular weights compared to testicular (I-R) group. This indicates the protective effect of FEN due to its anti-inflammatory, antioxidant and anti-apoptotic properties which leads to marked preservation of the testicular tissue against testicular (I-R) induced damage [21]. Oxidative stress has a main role in testicular (I-R). Ischemia stimulates chemotactic factors and leads to the migration of polymorphonuclear leukocytes to the ischemic region, which also generates ROS after reperfusion [22]. Oxygen increases during the first 60–90 min of reperfusion, which causes the toxic burst of ROS. The enzymatic antioxidant defense system, which includes SOD, catalase and GSH react to scavenge the free radicals to protect tissues from injury [22]. SOD catalyses the superoxide anion (O2.–) to hydrogen peroxide then it is converted to water by catalase or GSH [2]. GSH is the predominant low molecular weight thiol in mammalian cells and it is present in high concentration in adult mouse testis. This tripeptide has different functions which contribute to maintain cell defense and protection. This includes free radical scavenger, coenzyme for several antioxidant enzymes, maintenance of the thiol-disulfide status, and detoxication of electrophilic xenobiotics via conjugation [23]. That is detected in our study which showed decrease in SOD and GSH in testicular (I-R) induced group due to overproduction of ROS and free radicals. On administration of FEN to testicular (I-R) induced group; SOD and GSH markedly increased. Elevation of these enzymes is due to antioxidant properties of FEN [21]. The production of ROS in excessive amounts reacts with membrane lipids and results in lipid peroxidation and eventually the loss of cellular components of the tissue [22]. These free radicals react with lipid in cellular and mitochondrial membranes forming lipid peroxides. Membrane lipid peroxidation disrupts or changes membrane permeability and cell integrity. Lipid peroxidation is probably the most extensively investigated process induced by free radicals. O2%− reacts with lipid to form lipid peroxides followed by β-oxidation to form MDA which is an important indicator for the state of lipid peroxidation [24]. The same is detected in our results which showed significant increase in testicular MDA levels compared to sham operated group. That is in agreement with Parlaktas et al. [3] and Yıldız et al. [2]. NOx is water and lipid soluble which plays an important role in regulating blood flow in normal and pathological states. Ischemia causes an increase in NO synthetase activity and changes in NOx levels [25]. Reperfusion leads to the generation of the O.2–, and the
Fig. 3. Semiquantitive analysis of TNF-α immunoexpression. There is significant increase in tumor necrosis factor alpha (TNF-α) immunoexpression in testicular ischemia reperfusion (testicular (I-R) induced group in comparison with control group. Administration of fenofibrate (FEN) showed significant decrease in tumor necrosis factor alpha (TNF-α) immunoexpression compared to testicular ischemia reperfusion (testicular (I-R) induced group. Values are representation of 6 observations in each group as means ± S.E.M. Results are considered significantly different when P < .05. a Significant difference compared to control sham operated group, b significant difference compared to testicular ischemia reperfusion induced group. FEN1 is fenofibrate given group (100 mg/kg), FEN2 is fenofibrate given group (300 mg/kg). Testicular (I-R) is testicular ischemia reperfusion induced group and testicular (I-R)+FEN1 is the testicular ischemia reperfusion and fenofibrate given group (100 mg/kg). Testicular (I-R)+FEN2 is the testicular ischemia reperfusion and fenofibrate given group (300 mg/kg).
administration of FEN; results showed significant increase in the score in comparison with testicular (I-R) induced group (Table 4). 3.5. Evaluation of TNF-α immunoexpression As shown in Fig. 2, group I,II,III which represent control, FEN (100 mg/kg), FEN (300mg/kg) respectively showed negative immunoexpression of TNF-α (Fig. 2 A,B,C). Testicular (I-R) induced group (group IV) showed positive immunoexpression of TNF-α (Fig. 2D). FEN (100 mg/kg) administered group with induction of testicular (IR) showed mild immunoexpression of TNF-α (Fig. 2E). FEN (300 mg/kg) administered group with induction of testicular (IR) showed negative immunoexpression of TNF-α (Fig. 2F). 3.6. Semiquantitive analysis of TNF-α immunoexpression (Fig. 3) There is significant increase in TNF-α immunoexpression in testicular (I-R) induced group in comparison with control group. Administration of FEN showed significant decrease in TNF-α immunoexpression compared to testicular (I-R) induced group. 3.7. Evaluation of NF-κB immunoexpression As shown in Fig. 4, group I,II,III which represent control, FEN (100 mg/kg), FEN (300 mg/kg) respectively showed negative expressions of NF-κB (Fig. 4A–C). Testicular (I-R) induced group (group IV) showed positive expression of NF-κB immunoexpression (Fig. 4D). FEN (100 mg/kg) given group with induction of testicular (I-R) showed mild immunoexpression of NF-κB (Fig. 4E). FEN (300 mg/kg) given group with induction of testicular (I-R) showed negative immunoexpression of NF-κB (Fig. 4F).
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Fig. 4. Evaluation of nuclear factor kappa B (NF-κB) immunoexpression. (A) represents control group (group I), (B) represents fenofibrate (100 mg/kg) (group II), (C) represents fenofibrate (300 mg/kg) (group III). There were negative immunoexpression of nuclear factor kappa B (NF-κB) in these groups. (D) represents testicular ischemia reperfusion (testicular (I-R) induced group (group IV). Black arrow showed positive immunoexpression of nuclear factor kappa B (NF-κB). (E) represents testicular ischemia reperfusion (testicular (I-R) + fenofibrate (100 mg/kg) (group V). Black arrow showed mild immunoexpression of nuclear factor kappa B (NF-κB). (F) expresses testicular ischemia reperfusion (testicular (I-R) + fenofibrate (300 mg/kg) (group VI) and showed negative immunoexpression of nuclear factor kappa B (NF-κB).
interaction between NO and O.2– produces a specific type of ROS; peroxynitrite, which further promotes cellular damage [25].That was found in our study which showed significant increase in NOX levels in testicular (I-R) induced group. Coadministration of FEN to testicular (I-R) induced group results in significant decrease in MDA and NOX levels compared to testicular (I-R) induced group indicating the protective effect of FEN against membrane lipid peroxidation and nitric oxide formation This effect is due to
its PPARα agonist effect, antioxidant and anti-inflammatory properties of FEN This is in line with Ibrahim et al. [26]. Our study showed the typical histopathological features of testicular (I-R) and scored according to Johnsen's score as in Al-Maghrebi and Renno. [27]; Asghari et al. [22]. These changes were restored to normal with administration of FEN. Testicular (I-R) induced ROS formation is accompanied by different cytokines release as TNF-α and NF-κB. Theses cytokines are responsible
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Fig. 5. Semiquantitive analysis of nuclear factor kappa B (NF-κB) immunoexpression. There is significant increase in nuclear factor kappa B (NF-κB) immunoexpression in testicular ischemia reperfusion (testicular (I-R) induced group in comparison with control group. Administration of fenofibrate (FEN) showed significant decrease in nuclear factor kappa B (NF-κB) immunoexpression compared to testicular ischemia reperfusion (testicular (I-R) induced group. Values are representation of 6 observations in each group as means ± S.E.M. Results are considered significantly different when P < .05. a Significant difference compared to control sham operated group, b significant difference compared to testicular ischemia reperfusion induced group. FEN1 is fenofibrate given group (100 mg/kg), FEN2 is fenofibrate given group (300mg/kg). Testicular (I-R) is testicular ischemia reperfusion induced group and testicular (I-R)+FEN1 is the testicular ischemia reperfusion and fenofibrate given group (100 mg/kg). Testicular (I-R)+FEN2 is the testicular ischemia reperfusion and fenofibrate given group (300mg/kg).
for mediation of apoptosis, inflammation and subsequent tissue damage [28]. That is found in our study which showed significant increase in TNF-α and NF-κB immunoexpressions in testicular (I-R) group in comparison with normal control group. That is in line with Zhang et al. [28] and Ozcan et al. [5]. On addition of FEN to testicular (I-R) group showed significant decrease in their immunoexpressions which indicates the anti-inflammatory and anti-apoptotic effects of FEN.
5. Conclusion Upregulation of PPAR-α by FEN showed marked protective effect in induced testicular (I-R). FEN (300mg/kg) showed more improvement than low dose (100 mg/kg). Further studies are recommended to evaluate the suspected protective effect of fenofibrate in urgent cases of testicular torsion in males.
Conflicts of Interest None.
Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Acknowledgement Many thanks to Dr. Maram El-hussieny, Lecturer of Pathology, Faculty of Medicine, Minia University, for her generous help in this study. 514
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[27] M. Al-Maghrebi, W.M. Renno, Altered expression profile of glycolytic enzymes during testicular ischemia reperfusion injury is associated with the p53/TIGAR pathway: effect of fructose 1,6-diphosphate, Peer J. 4 (2016) e2195. [28] Y. Zhang, Y. Lv, Y.J. Liu, C. Yang, H.J. Hu, X.E. Meng, M.X. Li, S.Y. Pan, Hyperbaric oxygen therapy in rats attenuates ischemia-reperfusion testicular injury through blockade of oxidative stress, suppression of inflammation and reduction of nitric oxide formation, Urology 82 (2) (2013) 489 e9-489.e15.
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