Revista Internacional de Andrología. 2024; 22(1): 1-7. doi: 10.22514/j.androl.2024.001
Original Research

Gallic acid attenuates torsion/detorsion-induced testicular injury in rats through suppressing of HMGB1/NF-κB axis and endoplasmic reticulum stress
El ácido gálico atenúa la lesión testicular inducida por torsión/detorsión en ratas mediante la supresión del eje HMGB1/NF-κB y el estrés del retículo endoplásmico

Selim Demir1,*,, Ilke Onur Kazaz2, Gokcen Kerimoglu3, Nihal Turkmen Alemdar4,5, Fatih Colak2, Tugba Arici3, Ahmet Mentese6, Yuksel Aliyazicioglu6

1Department of Nutrition and Dietetics, Faculty of Health Sciences, Karadeniz Technical University, 61080 Trabzon, Turkey

2Department of Urology, Faculty of Medicine, Karadeniz Technical University, 61080 Trabzon, Turkey

3Department of Histology and Embryology, Faculty of Medicine, Karadeniz Technical University, 61080 Trabzon, Turkey

4Department of Medical Biochemistry, Graduate School of Health Sciences, Karadeniz Technical University, 61080 Trabzon, Turkey

5Department of Medical Services and Techniques, Vocational School of Health Services, Recep Tayyip Erdogan University, 53100 Rize, Turkey

6Department of Medical Biochemistry, Faculty of Medicine, Karadeniz Technical University, 61080 Trabzon, Turkey

*Corresponding Author(s):selim.demir@ktu.edu.tr (Selim Demir)

History Submitted: 02 July 2022 | Accepted: 30 August 2023 | Published: 30 March 2024
Copyright:  ©2024 The Author(s). Published by MRE Press.
This is an open access article under the CC BY 4.0 license (https://creativecommons.org/licenses/by/4.0/).

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Abstract

It was aimed to evaluate whether gallic acid (GA) have a beneficial effect in the testicular ischemia/reperfusion injury (IRI) model in rats for the first time. Testicular malondialdehyde, 8-hydroxy-2′-deoxyguanosine, superoxide dismutase, catalase, high mobility group box 1 protein, nuclear factor kappa B, tumor necrosis factor-alpha, interleukin-6, myeloperoxidase, 78-kDa glucose-regulated protein, activating transcription factor 6, CCAAT-enhancer-binding protein homologous protein and caspase-3 levels were determined using colorimetric methods. The oxidative stress, inflammation, endoplasmic reticulum stress and apoptosis levels increased statistically significantly in the IRI group compared with the sham operated group (p < 0.05). GA application improved these damage significantly (p < 0.05). Moreover, it was found that the results of histological examinations supported the biochemical results to a statistically significant extent. Our findings suggested that GA may be evaluated as a protective agent against testicular IRI.

Resumen
El objetivo era evaluar si el ácido gálico (GA) tenía un efecto beneficioso en el modelo de lesión por isquemia/reperfusión testicular (IRI) en ratas por primera vez. Malondialdehído testicular, 8-hidroxi-2′-desoxiguanosina, superóxido dismutasa, catalasa, proteína del grupo de alta movilidad caja 1, factor nuclear kappa B, factor de necrosis tumoral alfa, interleucina-6, mieloperoxidasa, proteína regulada por glucosa de 78 kDa, activadora el factor de transcripción 6, la proteína homóloga de la proteína de unión al potenciador de CCAAT y los niveles de caspasa-3 se determinaron mediante métodos colorimétricos. El estrés oxidativo, la inflamación, el estrés del retículo endoplásmico y los niveles de apoptosis aumentaron de manera estadísticamente significativa en el grupo IRI en comparación con el grupo operado de forma simulada (p < 0.05). La aplicación de GA mejoró significativamente estos daños (p < 0.05). Además, se encontró que los resultados de los exámenes histológicos respaldaron los resultados bioquímicos en un grado estadísticamente significativo. Nuestros hallazgos sugieren que GA puede evaluarse como un agente protector contra IRI testicular.

Keywords:Apoptosis;Endoplasmic reticulum stress;Gallic acid;Inflammation;Oxidative stress;Testicular torsion
Palabras Clave:Apoptosis;Estrés del retículo endoplásmico;Ácido gálico;Inflamación;Estrés oxidativo;Torsión testicular
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Cite this article

Selim Demir, Ilke Onur Kazaz, Gokcen Kerimoglu, Nihal Turkmen Alemdar, Fatih Colak, Tugba Arici, Ahmet Mentese, Yuksel Aliyazicioglu. Gallic acid attenuates torsion/detorsion-induced testicular injury in rats through suppressing of HMGB1/NF-κB axis and endoplasmic reticulum stress. Revista Internacional de Andrología. 2024; 22(1): 1-7. doi: 10.22514/j.androl.2024.001

1. Introduction

Testicular torsion (TT) is a condition that results from the rotation of the spermatic cord around its own axis and requires emergency surgical intervention [1]. TT causes biochemical and histological changes in the tissue [2]. The severity of damage to the gonadal structures is directly related to the degree and duration of torsion, and delayed treatment leads to harmful consequences, such as subfertility and infertility [1, 3]. The only treatment for TT is surgical detorsion [4]. The testicular salvage rate of the surgical detorsion procedure performed within 6 h from the beginning of the torsion is 90%. Unfortunately, this rate decreases to 50% after 12 h and to 10% after 24 h [5]. However, as a result of increased perfusion after detorsion, a greater damage occurs in the tissue than ischemia-induced damage, and is known as ischemia/reperfusion injury (IRI) [2]. IRI can lead to testicular atrophy by increasing reactive oxygen species (ROS), lipid peroxidation, inflammation, endoplasmic reticulum (ER) stress and apoptosis [3]. Recent researches have therefore focused on antioxidant molecules that can prevent testicular IRI [2].

The ER is a central organelle involved in protein synthesis and folding [6]. Pathological conditions, such as hypoxia and nutritional deficiencies, trigger the accumulation of unfolded/misfolded proteins in the ER lumen, and this is termed “ER stress” [7]. Mild ER stress includes activation of the unfolded protein response (UPR) and works entirely to keep the cell alive. If UPR activation fails to overcome ER stress, the cell is driven to apoptosis [8]. Increasing evidence indicates that ER stress have a significant potential to induce IRI-induced tissue damage [9].

Investigation of various pharmacological action mechanisms of phenolic compounds obtained from natural products has attracted great interest in recent years [10]. Gallic acid (GA) is a phenolic acid and found in red wine, green tea, strawberry, pineapple, banana, lemon and saffron [11]. GA has been reported to exhibit many biological activities, including antimicrobial, anticancer, anti-inflammatory, antioxidant, antimutagenic and anti-apoptotic properties [12, 13]. Today, GA is therefore widely used drug, food and dyeing industries [12]. Although GA has been shown to protect kidney [14], brain [15], heart [16] and liver [17] tissues against IRI in experimental models, to our best knowledge, no study has been found examining the effect of GA against testicular IRI. Protecting male reproductive health against IRI is very important for the continuation of fertility. This study therefore aimed to evaluate whether GA had a beneficial effect in the testicular IRI model in rats for the first time.

2. Material and methods

2.1 Experimental design

The 18 rats were divided into three groups (n = 6): sham control, torsion/detorsion (T/D) and T/D + GA (50 mg/kg). The time-dependent procedures performed in the study were summarized in Table 1. In the sham control group, the left testicle was removed and placed back into the scrotum to create surgical stress. In both T/D and T/D + GA groups, the left testicle removed by incision was rotated 720° clockwise and fixed to the scrotum using the method described previously [18]. The suture was removed after 4 h and testicular reperfusion was achieved for 2 h based on previously described methods [19, 20, 21]. In the GA treatment group, 30 min before detorsion, rats were given GA (50 mg/kg) via intraperitoneal route. GA dose (50 mg/kg) was determined considering previous studies and prepared by dissolving in sterile saline [17, 22, 23]. After 2 h of detorsion, orchiectomy was performed. The removed testicles were homogeneously divided longitudinally in two pieces, and one portion was frozen at −80 °C for biochemical analysis and other parts were immersed in Bouin’s solution for histological evaluation.

Table 1.A summary of the procedures in the experimental groups.
Groups
Sham ControlT/DT/D + GA
Torsion 0 min−++
210 min after torsionsterile salinesterile salineGA (50 mg/kg)
Detorsion (240 min after torsion)−++
Orchiectomy (360 min after torsion)+++
GA: gallic acid; T/D: torsion/detorsion.

2.2 Histological analysis

Routine histological tissue follow-ups were performed for testicular specimens fixed from Bouin’s solution. Paraffin blocks were cut in 5 μm sections, stained with hematoxylin and eosin (H&E) and evaluated under a light microscope (Olympus BX51, Olympus Co., Tokyo, Japan) [24]. Later, seminiferous tubule architecture and the levels of spermatogenesis were graded with the scoring system defined by Johnsen [25]. After the first seminiferous tubule was chosen randomly, the remaining part was observed by moving it clockwise. Analyzes were performed by a histologist unfamiliar with the groups using the coding system.

2.3 Biochemical analysis

Tissue samples were homogenized in 2 mL of phosphate buffered saline at 9500 rpm using a homogenizer (IKA, T25 Ultra-Turrax, Staufen im Breisgau, Germany) and the homogenates were centrifuged at 1800×g for 10 min at 4 °C to obtain supernatants. Protein levels of the supernatants were determined using a commercial kit (Thermo Scientific, Pierce BCA Protein Assay Kit, Cat No: 23225, Rockford, IL, USA) according to the manufacturer’s instructions. Bovine serum albumin was used as a standard, and the protein levels of the supernatants were calculated in mg/mL using the albumin standard graph. The biochemical parameters measured in the supernatants were proportioned to the amount of protein and expressed per mg of protein.

Testicular malondialdehyde (MDA) levels of all groups were determined according to the method described previously [26], while total oxidant status (TOS) (Cat No: RL0024) and total antioxidant status (TAS) (Cat No: RL0017) levels were determined using commercial colorimetric kits (Rel Assay Diagnostics, Gaziantep, Turkey). The oxidative stress index (OSI) was determined using the following formula [27]:

OSI(arbitraryunit)=(TOS/TAS)×100

The tissue amount of 8-hydroxy-2′-deoxyguanosine (8-OHdG) (Cat No: ER1487-HS), superoxide dismutase (SOD) (Cat No: ER0332), catalase (CAT) (Cat No: ER0264), high mobility group box 1 (HMGB1) (Cat No: ER0291), nuclear factor kappa B protein 65 (NF-κB p65) (Cat No: ER1187), tumor necrosis factor-alpha (TNF-α) (Cat No: ER1393), interleukin-6 (IL-6) (Cat No: ER0042), myeloperoxidase (MPO) (Cat No: ER0142), 78-kDa glucose-regulated protein (GRP78) (Cat No: ER0562), activating transcription factor 6 (ATF6) (Cat No: ER1645), CCAAT-enhancer-binding protein homologous protein (CHOP) (Cat No: ER0694) and caspase-3 (Cat No: ER0143) were determined using commercial sandwich enzyme-linked immunosorbent assay (ELISA) kits (Finetest, Wuhan, China).

2.4 Statistical analysis

All data are expressed as mean ± standard deviation (SD). Statistical differences between groups were assessed by analysis of variance (ANOVA) followed by Tukey’s post-hoc test. p < 0.05 is considered statistically significant.

3. Results

The MDA, TOS, OSI and 8-OHdG levels were significantly higher in the T/D group compared with sham control group (p = 0.0001, p = 0.003, p = 0.001 and p = 0.0001, respectively). GA application reduced the MDA, TOS, OSI and 8-OHdG levels significantly compared with T/D group (p = 0.0001, p = 0.002, p = 0.001 and p = 0.0001, respectively) (Table 2).

Table 2.The levels of biochemical parameters and Johnsen scores in testicular tissues of control and experimental rats (n = 6 rats/per group).
ControlT/DT/D + GA
MDA (nmol/mg protein)5.67 ± 0.5817.94 ± 2.49a5.81 ± 0.34b
TOS (µM H2O2 equivalent/L)21.62 ± 3.7451.71 ± 11.61a20.40 ± 10.08b
TAS (mM trolox equivalent/L)1.24 ± 0.030.68 ± 0.21a1.22 ± 0.04b
OSI (arbitrary unit)1.75 ± 0.328.34 ± 3.56a1.68 ± 0.86b
8-OHdG (ng/mg protein)4.40 ± 1.1812.28 ± 2.56a4.70 ± 0.85b
SOD (ng/mg protein)2.02 ± 0.570.48 ± 0.10a1.83 ± 0.48b
CAT (mIU/mg protein)15.80 ± 3.995.88 ± 0.74a14.30 ± 7.62b
HMGB1 (pg/mg protein)55.57 ± 8.16172.18 ± 6.90a60.40 ± 2.80b
NF-κB p65 (pg/mg protein)90.03 ± 17.11234.22 ± 18.10a84.42 ± 24.81b
TNF-α (pg/mg protein)0.26 ± 0.151.05 ± 0.16a0.23 ± 0.22b
IL-6 (pg/mg protein)40.47 ± 6.10262.01 ± 19.74a45.25 ± 9.36b
MPO (ng/mg protein)0.57 ± 0.091.54 ± 0.29a0.61 ± 0.13b
GRP78 (pg/mg protein)21.65 ± 3.8484.90 ± 18.28a19.82 ± 3.99b
ATF6 (pg/mg protein)36.38 ± 9.2499.28 ± 15.54a34.10 ± 4.36b
CHOP (pg/mg protein)159.80 ± 29.90353.90 ± 76.14a160.78 ± 16.86b
Caspase 3 (ng/mg protein)0.40 ± 0.131.70 ± 0.25a0.48 ± 0.26b
Johnsen scores9.48 ± 0.326.68 ± 0.83a8.68 ± 0.54b
T/D: torsion/detorsion; GA: gallic acid; MDA: malondialdehyde; TOS: total oxidant status; TAS: total antioxidant status; OSI: oxidative stress index; 8-OHdG: 8-hydroxy-2′-deoxyguanosine; SOD: superoxide dismutase; CAT: catalase; HMGB1: high mobility group box 1; NF-κB: nuclear factor kappa B; TNF-α: tumour necrosis factor alpha; IL-6: interleukin-6; MPO: myeloperoxidase; GRP78: 78-kDa glucose-regulated protein; ATF6: activating transcription factor 6; CHOP: CCAAT-enhancer-binding protein homologous protein. p-values according to one-way ANOVA test, post-hoc Tukey test. Data were expressed as mean ± SD. ap < 0.05 compared with control group, bp < 0.05 compared with T/D group.

The TAS, SOD and CAT levels were found to be significantly decreased in the testicular tissues of the T/D group compared to the sham control group (p = 0.0001, p = 0.0001 and p = 0.007, respectively). GA application increased TAS, SOD and CAT levels significantly compared with T/D group (p = 0.0001, p = 0.0001 and p = 0.025, respectively) (Table 2).

The HMGB1, NF-κB p65, TNF-α, IL-6 and MPO levels were significantly increased in the T/D group compared to the sham control group (all p = 0.0001). GA application decreased HMGB1, NF-κB p65, TNF-α, IL-6 and MPO levels significantly compared with T/D group (all p = 0.0001) (Table 2).

The GRP78, ATF6, CHOP and caspase-3 levels of the T/D group was significantly increased compared to the sham control group (all p = 0.0001). GA application decreased GRP78, ATF6, CHOP and caspase-3 levels significantly compared with T/D group (all p = 0.0001) (Table 2).

Johnsen scores were significantly lower in the T/D group compared to sham control group (p = 0.0001), and GA treatment increased these scores significantly compared to the T/D group (all p = 0.0001) (Table 2). Microscopic images were presented in Fig. 1. (A) Sham control group; testicular tissue showed normal seminiferous tubule germinal epithelial structure and normal intertubular space morphology (B) T/D group; irregularities, degeneration and decreased germinal epithelial cells were observed in the seminiferous tubule epithelium. The tubule structure with spermatozoa in the lumen was considerably reduced. Widespread severe edema were observed in the intertubular area (C) T/D + GA group; compared to the T/D group, testicular tissue was observed close to the control group, where the damage was largely eliminated. Although the degree of edema decreased compared to the T/D group, it still continued.

Histopathological images of testicular tissues of groups (×200, H&amp;E staining). Control Group (A) P: normal peritubular tissue, S: spermatozoon in the lumen of the seminiferous tubule. T/D Group (B) DT: degenerative seminiferous tubule structure, black arrow: spilled immature germinal cells into the lumen of the seminiferous tubule, arrowhead: vacuolization in tubule epithelium, black star: edema in peritubular tissue. T/D + GA Group (C) Black arrow: spilled immature germinal cells into the lumen of the seminiferous tubule, black star: edema in peritubular tissue.

Fig. 1.Histopathological images of testicular tissues of groups (×200, H&E staining). Control Group (A) P: normal peritubular tissue, S: spermatozoon in the lumen of the seminiferous tubule. T/D Group (B) DT: degenerative seminiferous tubule structure, black arrow: spilled immature germinal cells into the lumen of the seminiferous tubule, arrowhead: vacuolization in tubule epithelium, black star: edema in peritubular tissue. T/D + GA Group (C) Black arrow: spilled immature germinal cells into the lumen of the seminiferous tubule, black star: edema in peritubular tissue.

4. Discussion

The pathogenesis of IRI is associated with complex molecular processes, such as oxidative/nitrosative stress, inflammation and ER stress [2, 28]. Increased ROS cause damage to lipids, carbohydrates, proteins and DNA [2]. Peroxidation of lipids leads to the formation of unstable aldehyde compounds, including MDA, which are highly reactive for all biomolecules [22]. TAS and TOS are inexpensive and reliable cumulative markers to indirectly assess the degree of oxidative stress in a biological sample. TOS is an indicator of the total oxidant molecules in a sample, while TAS is a precise indicator of the total antioxidant capacity in a sample [17]. 8-OHdG is a biomarker of the oxidative DNA damage [29]. In this study, increased MDA, TOS, OSI and 8-OHdG levels and decreased TAS levels in the T/D group showed that testicular IRI is mediated by oxidative stress. Treatment with GA restored the levels of these parameters. Consistent with these results, GA has been reported to reduce IRI in various tissues, such as kidney, heart and brain, by reducing oxidative stress in various experimental models [14, 16, 22].

Under normal conditions, antioxidant enzymes play a very important role in maintaining fertility by protecting testicular tissue from free radical damage [30]. Various antioxidant enzymes work to eliminate the harmful effects of ROS in organisms. Among these enzymes, SOD forms the first line of defense and converts superoxide radicals to hydrogen peroxide (H2O2). H2O2 is then reduced to water by CAT [10]. Decreased levels of these enzymes cause an increase in ROS, and as a result, cell membranes lose their function and integrity over time [14]. The results showed that the protein levels of SOD and CAT were lower in the T/D group compared to the control group. It is known that increased ROS levels in the case of IRI decrease the expression of antioxidant enzymes [31, 32, 33, 34]. This may be the main reason for the decrease in SOD and CAT protein levels in the T/D group. In order to reach a definite conclusion, SOD and CAT mRNA levels should also be determined in future studies. The levels of antioxidant enzymes in the T/D + GA group increased significantly and this may be due to the fact that GA protects antioxidant enzymes from attack by ROS. Similar with these results, GA has been reported to reduce IRI in heart, liver and kidney tissues via elevating the levels of antioxidant enzymes [10, 14, 16].

Inflammation is an important biological process in response to external stimuli and it is the another major pathway that play critical role in the pathophysiology of testicular IRI [4, 5]. HMGB1 is a non-chromosomal nuclear protein involved in the initiation of the inflammatory cascade in the tissue damage response, including IRI [35]. After IRI, extracellular HMGB1 operates as a damage-associated molecular pattern (DAMP) through two major signaling pathways. It interacts with toll-like receptor 4 (TLR4) and receptor for advanced glycation end product (RAGE) receptors, inducing the activation of NF-κB. NF-κB activation increases the expression TNF-α and IL-6 [36]. Interestingly, it is reported that blocking HMGB1 can alleviate tissue damage caused by IRI [37, 38]. Neutrophil infiltration is another characteristic of IRI-induced inflammatory tissue damage [13]. In this study, increased levels of HMGB1, NF-κB p65, TNF-α, IL-6 and MPO in the T/D group showed that the testicular IRI is mediated by inflammation. Elevated levels of inflammatory markers as a result of T/D may be due to impaired antioxidant capacity, increased ROS amount and lipid peroxidation [36]. Treatment with GA significantly restored these inflammation parameters significantly. This may be due to the anti-inflammatory activity of GA resulting from its strong antioxidant property. Consistent with these results, GA has been reported to reduce IRI in various tissues, such as kidney and liver, by reducing inflammation [17, 39].

Recent studies highlight that ER stress comes to the forefront as an important molecular mechanism in IRI-induced tissue damage [9]. Mild ER stress induces the UPR to activate cytoprotective mechanisms. In contrast, severe ER stress induces apoptosis to protect the organism [40]. There are three sensor proteins that detect ER stress and initiate the UPR pathway: ATF6, inositol requiring enzyme 1 (IRE-1) and protein kinase RNA-activated-like ER kinase (PERK) [6]. These sensors interact with GRP78 under non-stressed conditions and are kept inactive state. In cases of increased ER stress, sensor proteins are separated from GRP78 and thus UPR is activated [9]. ATF6 is activated by dissociation from GRP78 when ER stress is triggered. The released ATF6 is then transferred to the Golgi, where it is activated. Activated ATF6 migrates into nucleus where it elevates the expression level of proteins involved in optimizing protein folding, maturation and elimination of missense proteins. Therefore, there is no doubt that ATF6 is an important mediator during IRI [8]. In the case of excessive ER stress, ATF6 induces CHOP expression, resulting in apoptosis and elimination of the cell whose ER stress cannot be reduced. Under increased ER stress conditions, increased CHOP suppresses B-cell lymphoma 2 (Bcl-2) and glutathione levels, increases Bcl-2-associated X protein (Bax) and ROS levels, and causes caspase-3 activation [41]. Therefore, GRP78, ATF6 and CHOP levels were evaluated in testicular tissues to reveal ER stress, and caspase-3 levels were determined to show the level of apoptosis in this study. The results showed that increased ER stress and apoptosis contribute to testicular IRI, and GA attenuated this damage by ER stress inhibitor and anti-apoptotic activities. Similarly, Obafemi et al. [23] demonstrated that GA protects liver and pancreas tissues against diabetes-induced damage via alleviating the levels of ER stress, while Blas-Valdivia et al. [41] reported that GA exhibits neuroprotective effect against hypothyroid-induced oxidative and ER stress via decresing PERK, ATF6 and CHOP levels.

Histological evaluation is a very important method in evaluating tissue damage caused by IRI and revealing the degree of therapeutic effect of the investigated molecule [20, 42]. Histological data revealed the presence of pathological findings in T/D group and showed that GA application could eliminate these pathological findings in parallel with the biochemical results. Consistent with these results, previous studies have shown that GA can exhibit a testicular protective effect against damage caused by various chemicals by modulating oxidative stress and inflammation [13, 43, 44, 45, 46, 47].

GA is a natural phenolic acid and has powerful antioxidant activity due to chain breaking, hydrogen-donating, metal chelating and radical scavenging potential [12, 13]. It is also stated that GA functions by strengthening antioxidant enzymes [11, 29]. In addition, it has been demonstrated that it can exhibit anti-inflammatory activity by reducing the expression of inflammatory molecules [17, 39]. Therefore, we speculated that the protective effect of GA against testicular IRI is mainly due its antioxidant and anti-inflammatory potential.

There were also some limitations of our study. First, only the one dose of GA was evaluated based on previous literature data within the scope of this study. Second, the efficacy of GA at different times or in chronic T/D conditions was not evaluated in this study. Third, the effect of GA on sexual behavior and fertility level of rats were not evaluated in the study. We believe that demonstrating the protective efficacy of GA against testicular IRI in long-term studies together with physiological fertility behavior experiments will shed light on clinical stages.

5. Conclusions

The results showed that GA reduced testicular IRI in an experimental model for the first time. This protective effect of GA is thought to be due its antioxidant, anti-inflammatory, anti-apoptotic and ER stress inhibitor properties. The suitability of GA for clinical use needs to be supported by more detailed studies.

Availability of data and materials

The data presented in this study are available on reasonable request from the corresponding author.

Author contributions

SD, IOK, GK and AM—contributed to study design, data collection and research evaluation; IOK and FC—contributed to perform torsion/detorsion induction; SD, NTA and AM—contributed to perform the biochemical analysis; GK and TA—contributed to perform the histological analysis; SD, IOK, AM and YA—performed data analysis and drafted the manuscript. All authors accomplished the edition and approved the ultimate version of this manuscript for submission, contributed to the completion of the manuscript and affirmed the ultimate draft.

Ethics approval and consent to participate

Ethics committee approval was received for this study from the Animal Experiments Local Ethics Commitee of Karadeniz Technical University (Protocol number: 2019/53).

Acknowledgment

The authors wish to thank Sait Al and Ibrahim Aydin from Surgical Practice and Research Center of Karadeniz Technical University for professional assistance with the experimental studies. We also would like to acknowledge animal, biochemistry and histology laboratory staffs for their effort during the study.

Funding

This research received no external funding.

Conflict of interest

The authors declare no conflict of interest.

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