Exercise Training Ameliorates The Tramadol-Induced Toxicity on VEGF/VEGFRs/PI3K/AKT Signaling Pathway in Testicular Tissue after Tramadol Withdrawal

Abstract

Background:

The study investigated how exercise training protocols (ETPs) with different intensities can ameliorate the tramadol (TRA)-induced toxicity in the testicular tissue by upregulating VEGF/VEGFRs/PI3K/AKT signaling pathway in the post-withdrawal period.

Materials and Methods:

In this experimental study, thirty-six mature Wistar rats were included in two groups: one being a sedentary control group, and the other receiving TRA treatment (40 mg/kg, daily, ip). After 60 days, six rats from the TRA-received group were excluded (TRA-sole; euthanized after 60 days). The TRA administration was stopped and the rats were further divided into four subgroups: TRA/withdrawal (TRA/W), low (LICT), moderate (MICT), and high (HICT)-intensity continuous exercise training-received groups (n=6/each group). The expression levels of phosphoinositide 3-kinases (PI3K), protein Kinase B (AKT), vascular endothelial growth factor (VEGF), VEGF receptor 1 (VEGF R1) and 2 (VEGF R2) were assessed.

Results:

The expression levels of PI3K, AKT, VEGF, VEGF R1, and VEGF R2 were decreased in the TRA-sole group. No significant alterations were noted in the PI3K, AKT, VEGF, and VEGF R1 expressions after 60 days of withdrawal compared to the TRA-sole rats. However, this situation was reversed and the expression levels of PI3K, AKT, VEGF, VEGF R1, and VEGF R2 increased in the MICT and HICT-received groups.

Conclusion:

TRA disrupts the PI3K/AKT interaction, and this disruption is not easily reversed shortly after withdrawal. However, ETPs exert positive effects on PI3K/AKT signaling by promoting the expression of VEGF, VEGFR1, and R2. These results highlight the potential of ETPs to ameliorate the adverse effects of TRA on VEGF/VEGF R1, R2/PI3K/AKT signaling, even a short time after withdrawal.

Introduction

Spermatogenesis involves a multifaceted sequence of events that converts diploid spermatogonial stem cells (SSCs) into mature haploid spermatozoa through wellcoordinated processes of mitosis, meiosis, and differentiation. Taking place within the seminiferous tubules of the testes, this process is tightly regulated, scheduled, continuous, and necessary for male fertility (1). The fundamental aspect ensuring regular and consecutive spermatogenesis is the dual capability of SSCs to continually undergo self-renewal and differentiation (2).

The pivotal regulation of this step primarily involves various kinases, notably including phosphoinositide 3-kinases (PI3K). The PI3K is part of the PI3K/AKT (protein Kinase B) pathway, which plays a role in regulating various cellular processes including germ cell proliferation, survival, migration, and metabolism (3). Within the testis, various mediators such as glial cell line-derived neurotrophic factor (GDNF), stem cell factor (SCF), c-Kit, Foxo-1, and vascular endothelial growth factor (VEGF) trigger the PI3K-Akt signaling pathway at different stages of the spermatogenic process (4). Therefore, several

scholarly trials have focused on elucidating the significance of PI3K/Akt signaling in regulating distinct facets of male reproductive function, including gonadotropin release and gonadal development, spermatogenesis development, spermatogonia self-renewal, proliferation and anti-apoptotic role of AKT through multiple downstream signaling molecules (3).

VEGF, a potent angiogenic factor, is known to initiate PI3K/AKT signaling in various cell types, including cancer cells, neural cells, and endothelial cells (5). Upon binding to its receptors VEGFR1 and VEGFR2 on the cell membrane, VEGF activates PI3K by recruiting it to the plasma membrane, where it generates 3,4,5-trisphosphate (PIP3) from PIP2 (6). Following this, PIP3 recruits AKT to the plasma membrane, where it undergoes phosphorylation and activation by PDK1 and mTORC2 (7, 8). Activated AKT phosphorylates downstream targets, such as FOXO1, BAD, and GSK3β, leading to their inactivation and promoting cell survival and proliferation (9). AKT also activates mTORC1, promoting protein synthesis and cell growth (10).

VEGF plays a crucial role in the development and maintenance of reproductive cells, including Sertoli and Leydig cells. Research indicates that VEGF enhances the proliferation of spermatogenic cells and improves sperm motility. Additionally, the presence of VEGF in semen is believed to contribute to the fertilization process (11, 12).

Tramadol (TRA), a synthetic analogue of codeine, is a widely used analgesic medication for the management of osteoarthritis when non-steroidal anti-inflammatory drugs (NSAIDs) are not recommended due to contraindications (13). Unlike NSAIDs, which reduce pain by inhibiting cyclooxygenase enzymes, TRA's mechanism involves both opioid receptor activation and modulation of neurotransmitters, making it distinct from traditional opioids due to its lower risk of dependence and respiratory depression. With a dual mechanism of action comprising both opioid and non-opioid components, it stands as a primary treatment for alleviating pain. However, it is also prevalent among male subjects with a history of substance abuse as one of the most commonly misused opioids (14). Indeed, the overconsumption of opioids can have negative effects on male reproductive potential. Accordingly, due to its low plasma protein binding ratio (20%) and wide distribution in several tissues, TRA has been shown to have negative effects on spermatogenesis (15). This is consistent with earlier animal studies that demonstrated the prolonged TRA treatment adversely affects sperm normal morphology, count, and viability (16). Esua et al. (17) also found similar results when administering 50 and 100 mg/kg of TRA. Oxidative stress (OS) and the excessive expression of nitric oxide synthase (NOS) are commonly discussed as potential pathophysiological factors contributing to tramadol-induced suppression of spermatogenesis and sperm parameters (13). Since, Ibrahim and Salah-Eldin showed that TRA (40 mg/kg) induces testicular apoptosis by triggering the intrinsic pathway through the induction of OS (18). Recently, we demonstrated that chronic consumption of TRA has negative effects on sperm parameters by significantly disrupting testicular antioxidant capacity by affecting relative expression levels of miR-126-3p and miR-181a and reducing SIRT1 and Nrf2 expressions (15).

Recently, there has been an increasing focus on the positive impacts of exercise training on spermatogenesis. Experimental research has demonstrated that exercise training protocols (ETPs) characterized by low and moderate intensity levels can enhance the testicular endocrine system, improve antioxidant status, enhance the spermatogenesis process, and improve semen quality (19-21). Moreover, exercise training has been found to have a positive impact on VEGF expression, which in turn leads to increased angiogenesis and improved blood flow to tissues. For instance, Tryfonos et al. (22) demonstrated that high-intensity interval training (HIIT) or incorporated HIIT with strength training is capable of up-regulating angiogenic factors related to muscle capillarization. In another study, Tang et al. (23) found that a treadmill running session at 24 M/min and a 10-degree incline for 1 hour resulted in increased VEGF expression in muscles, lungs, and the brain. Additionally, another study has shown that exercise training-induced proangiogenic effects are associated with the PI3K/AKT and AKT/ERK1/2 pathways, which suggests that exercise training promotes the boosting of the VEGF/PI3K/AKT signaling pathway (24). Taking into account these and other research findings, it can be concluded that exercise training has a positive impact, with variations depending on the intensity and duration of the protocols utilized.

Taking into account the previously mentioned information, our study had several aims. Firstly, we aimed to investigate whether TRA has any effect on the VEGF/ PI3K/AKT signaling pathway in testicular tissue. Secondly, we aimed to determine whether the negative effects induced by TRA administration are reversible after discontinuation of the drug. Moreover, we aimed to explore whether low, moderate, and high-intensity continuous ETPs could expedite the recovery process after TRA withdrawal. Finally, considering that a complete spermatogenesis cycle in rat models takes at least 48 days (25). To achieve these aims, we studied TRA addiction and stopped the TRA administration to observe any changes in VEGF, VEGF R1, VEGF R2, PI3K, and AKT expression levels, both with and without exercise training. Finally, all the findings were compared among the corresponding groups with the same experimental criteria.

Materials and Methods

Experimental design and grouping

To achieve the aims of the current original experimental study, 36 mature male Wistar rats, 8 weeks old and weighing between 180-220 g, were sourced from the Animal Resource Center at Urmia University (ARCUU). Approval for the experimental procedures was granted by the Ethics Committee of the Urmia University of Medical Sciences (IR.UMSU.REC.1398.191). Following one week of acclimatization to standard conditions, including unrestricted access to food and water, and exposure to a 12-hour dark/light cycle at 25°C, the rats were segregated into two groups: control (n=6) and experimental (n=30). The experimental group was further divided into five subgroups: TRA-sole (n=6), TRA withdrawal (TRA/W, n=6), low-intensity continuous training (TRA+LICT, n=6), moderate-intensity continuous training (TRA+MICT, n=6), and high-intensity continuous training (TRA+HICT, n=6). All experimental groups received TRA (Millipore Sigma, USA, Cat N:36282-47-0; 40 mg/kg, IP) to induce TRA addiction (15). The TRA-sole group was included to assess the detrimental effects of TRA within 60 days, which is the time required for one complete cycle of spermatogenesis in rat models (25). Following the 60-day period, TRA administration was stopped, and the rats in the TRA-sole group were euthanized. The TRA/W group underwent TRA administration for 60 days. After this period, TRA administration was discontinued, and the rats were observed for an additional 60 days without TRA, receiving normal saline instead (in an equivalent volume to the TRA solvent). This group was included to explore the recovery process without any intervention following TRA withdrawal. All TRA+exercise (TRA+LICT, TRA+MICT, and TRA+HICT) groups underwent running ETPs without receiving TRA in the additional 60 days (15). The rats were euthanized 24 hours after the last TRA injection and ETP induction.

Exercise adaptation and protocols

After a week of treadmill adaptation (Danesh Yakhte, Iran) with a running intensity of 15 m/minutes for 5-20 minutes, the Maximum velocity test (Vmax) was conducted. The test involved a 5-minute warm-up period at a speed of 10-15 m/minutes, followed by a stepwise increase in speed (5 m/minutes, every 3 minutes) Until the rats reached a point where they were either incapable or unwilling to proceed further (26). The LICT protocol intensity was conducted at 50-60% of the Vmax for 45 minutes, while the MICT and HICT protocols intensities were performed at 60-75% and 80-85% of the Vmax for 45 min, respectively. The ETPs time intervals were designed based on the principles of overload, starting with 20 minutes in the first week and gradually increasing to 45 minutes by week 8. The warm-up and cool-down phases were set at 40-45% and 35-40% of the Vmax, respectively. Exercise training sessions were carried out five days a week for a total of 60 days (15).

Euthanasia and tissue sampling

A euthanasia solution of Ketamine and Xylazine (Alfason, Netherlands) was used to euthanize the rats, and the testicular tissue was collected after euthanasia. Histological analyses were conducted on the testicles from the left side, while the testicles from the right side were utilized for biochemical and molecular investigations.

mRNA extraction, cDNA synthesis, and quantitative reverse transcription polymerase chain reaction

For the extraction of total RNA from 0.1 g of tissue, the TRIZOL-chloroform-based method (Sina-Gen, Iran) was employed (27). The extracted RNA was assessed for quantity and quality by a nanodrop spectrophotometer (Thermo ScientificTM, USA) at 260 nm and an A260/280 ratio of 1.8-2.0. The synthesis of cDNA followed the instructions provided by the manufacturer (Fermentas, GmbH, Germany). Subsequently, a polymerase chain reaction (PCR) reaction master mixture was prepared by combining 0.5 μl (5-10 ng) of cDNA template, 10 μl of 1X SYBR GREEN master mix (High ROX, Noavaran TebBeinolmelal, Iran), and 0.5 μl (600 nM) of both reverse and forward primers for the target genes. PCR conditions were set as follows: initial denaturation at 95°C for 5 minutes, followed by 45 cycles of 95°C for 20 seconds, annealing temperature (53°C for AKT; 53°C for PI3K; 55°C for VEGF and VEGF R1; VEGF R2, 63°C for GAPDH) for 15 s, and elongation at 72 °C for 1 minute with a final extension at 72°C for 5 minutes. The PCR threshold cycle (CT) values were determined from triplicate targets and normalized by subtracting GAPDH CT values. the equation: 2^−(ΔΔCt) was employed to calculate the relative mRNA expression levels of the target genes. The primers used to assess the expression of PI3K, AKT, VEGF, VEGFR1, VEGFR2, and GAPDH (as an internal control) genes possessed the following sequences:

PI3K-F: 5'- CTT GAC ACG GAG GGA AGC AT-3' R: 5'- CGG CCC AGA ATA TAC CCG AA-3'

AKT-F: 5'-CTA CGG TGC GGA GAT TGT GT-3' R: 5'-CAC AGC CCG AAG TCC GTT AT-3'

VEGF-F: 5'- TGA ACT TTC TGC TCT CTT GG-3' R: 5'- AAC AAA TGC TTT CTC CGC TC-3'

VEGFR1-F: 5'-GGT GCA TGG CTA CTC GTT A-3' R: 5'-AGA GTG GCT GTG AGG TTT CT-3'

VEGFR2-F: 5'-TTG GAA ACT GAA TGG CAC CG-3' R: 5'-TGA CGA GCT GCT TGA CTA GG-3'

GAPDH-F: 5'-CAA TTC CAT CCC AGA CCC CA-3' R: 5'-TTA TTG ATG GTA TTC GAG AGA AGG G-3'

Immunohistochemical staining

In brief, 4-5 µm sections of tissue were deparaffinized, rehydrated, and heated in a hot air oven (Venticell, MMM, Einrichtungen, Germany) for 25 minutes at 60°C. Sodium citrate buffer (28.7 ml sodium citrate+6.3 ml acid citric+315 ml distilled water, pH=7.2) was utilized for antigen retrieval, while endogenous peroxidases were neutralized by treatment with 3% hydrogen peroxide for 5 minutes. Super blocker solution in buffer PBS (Thermo Scientific, Cat N: 37580, USA) was used to block nonspecific antigens for 10 minutes. The primary antibody (VEGF: 1:50, Cat N: E-AB-64131; Elabsciences, USA, PI3K, 1:300, Cat N: E-AB-67781; AKT: 1:100, Cat N: E-AB-63467) was applied on slides, and the slides were left to incubate overnight at 4°C in a humidified chamber. The peroxidase/HRP-conjugated Goat Anti-Rabbit IgG (Cat N: E-AB1003; Elabsciences, USA) served as the secondary antibody, and the slides underwent a 1-hour incubation at 37°C. Protein staining was achieved using diaminobenzidine (DAB, ScyTek Laboratories, Logan, USA), while hematoxylin (Richard-Allen Scientific, Kalamazoo, USA) was employed for counterstaining nuclei. For the negative control, normal IgG was employed in place of the primary antibody. In each cross-section, twenty seminiferous tubules (meeting the same criteria) were analyzed and the number of VEGF⁺, PI3K⁺, and AKT⁺ cells was recorded per tubule.

Western blot

The testicular samples were homogenized using RIPA lysis buffer (Santa Cruz, sc-24948) and followed by centrifugation at 12,000 rpm for 15 minutes at 4°C. The protein concentration of the extracted samples was determined using Lowry’s method (28), and Equal quantities of protein were combined with loading buffer and then heated at 95°C for 5 minutes. The samples were subsequently applied onto a 10% sodium dodecyl sulfate-polyacrylamide gel (for B-Actin) and a 4% sodium dodecyl sulfate-polyacrylamide gel (for target proteins) and electrophoresed at 120 V to separate the proteins based on their molecular weights. After the electrophoresis process, the proteins were transferred onto a nitrocellulose membrane, which was then washed and placed in a blocker solution (5% skim milk) for incubation (Sigma, Cat N:70166) at 4°C, overnight. Following that, the membrane underwent incubation with the primary antibody (B-Actin: 1:300, SC-47778, SantaCruz, PI3K: 1:300, AKT: 1:200, VEGF: 1:200, VEGF R1: 1:200, Cat N: E-AB-93035, Elabsciences, USA; VEGF R2: 1:300, Cat N: 26415-1-AP, Proteintech, USA) for 1 hour at 4°C, this was followed by incubation with the suitable secondary antibody (anti-mouse, CAT N: E-AB-1001 or anti-rabbit, CAT N: E-AB-1003) conjugated with HRP for 90 minutes at room temperature. After each step, the membrane was rinsed in Tween 20-Tris buffer saline (TTBS). Finally, the immunoblots were observed utilizing enhanced chemiluminescence (ECL) and assessed using the ChemDoc system (1708265, Bio-Rad, USA).

Software analyses and image presentation

To assess the intensity of brown-stained reactions (representing AKT, PI3K, and VEGF-positive reactions) at the pixel level, photomicrographs with a resolution of 20 megapixels were captured (n=5 per cross-section, 15 per group), and the brown-stained pixels ratio relative to the total pixels was then evaluated within area of 3000 µm×3000 µm from the tissue. The photomicrographs were captured using a Canon onboard camera (Japan) and were standardized using Adobe Photoshop CC 2018 (version: 19).

Statistical analyses

Statistical analysis was conducted using One-way ANOVA followed by Tukey’s multiple comparison post hoc test in SPSS software version 11.00 (California, USA). The normality of data distribution was assessed using the Shapiro–Wilk test. Results are expressed as mean ± SD, and statistical significance was considered at P<0.05. A priori power analysis was performed using G*Power software to determine the minimum sample size required for detecting statistically significant differences with a power of 80% and an alpha level of 0.05, which justified the use of six rats per group.

Results

Exercise training protocols could amplify TRA reduced AKT and PI3K expression at the mRNA level after the withdrawal

The qRT-PCR analyses demonstrated a significant reduction in mRNA levels of AKT and PI3K in the experimental group compared to the control group (P=0.001). However, in the experimental group compared to the TRA-sole group, the mRNA level of AKT was found to be increased (P=0.001) after TRA withdrawal. There was no significant alteration observed in the AKT mRNA level following withdrawal compared to the TRA-sole group. Conversely, both MICT and HICT ETPs remarkably elevated the mRNA level of PI3K compared to the TRA/W group (P=0.001). Although all types of ETPs could increase the PI3K mRNA level compared to the TRA-sole group (P=0.001), no significant variances were noted among the LICT, MICT, and HICT groups (Fig .1A, B).

Fig.1.

Quantitative real-time polymerase chain reaction (qRT-PCR) analysis of PI3K and AKT mRNA expression levels. Mean changes in the mRNA levels of A. Phosphoinositide 3-kinases (PI3K) and B. Protein kinase B (AKT) in different groups. All data are presented in the mean ± SD. Different letters represent significant differences between groups (P<0.05, n=6/each group). TRA/Sole; Rats received tramadol for 60 days without further treatment, TRA/W; Rats that received tramadol for 60 days continued without tramadol treatment for an additional 60 days, LICT; Rats subjected to low-intensity continuous training, MICT; Rats subjected to moderate-intensity continuous training, and HICT; Rats were subjected to high-intensity continuous training, which discontinued tramadol treatment and continued training for 60 days after withdrawal.

ETPs increased the VEGF, VEGF R1, and VEGF R2 mRNA levels after the withdrawal

There was a significant decrease in the mRNA level of VEGF observed in the TRA-sole group compared to the control rats (P=0.001). This condition remained unchanged even after withdrawal in the TRA/W group. Furthermore, the mRNA levels of VEGF R1 and VEGF R2 were also diminished in the TRA-sole group compared to the control rats (P=0.001). While the mRNA level of VEGF R2 was increased after withdrawal (P=0.001), there was no remarkable increase observed in the mRNA level of VEGF R1 in the TRA/W group compared to the TRA-sole rats. However, the groups influenced by ETPs demonstrated a notable increase in VEGF mRNA levels in comparison to both the TRA/W and TRA-only groups (P=0.001). After the induction of LICT (P=0.001) and HICT (P=0.001), there was an increase in the mRNA levels of VEGF R1 and R2 compared to the TRA-sole group. This situation remained unchanged regarding the mRNA level of VEGF R2 between TRA/W and the TRA+MICT groups (Fig .2A-C).

Fig.2.

Quantitative real-time polymerase chain reaction (qRT-PCR) analysis of VEGF, VEGF R1, and VEGF R2 mRNA expression levels. Mean changes in the mRNA levels of A. Vascular endothelial growth factor (VEGF), B. VEGF receptor R1 (VEGF R1), and C. VEGF receptor R2 (VEGF R2) in different groups. All data are presented in the mean ± SD. Different letters represent significant differences between groups (P<0.05, n=6/each group). TRA/Sole; Rats received tramadol for 60 days without further treatment, TRA/W; Rats that received tramadol for 60 days continued without tramadol treatment for an additional 60 days, LICT; Rats subjected to low-intensity continuous training, MICT; Rats subjected to moderate-intensity continuous training, and HICT; Rats were subjected to high-intensity continuous training, which discontinued tramadol treatment and continued training for 60 days after withdrawal.

ETPs increased the global AKT protein expression after the withdrawal

To evaluate the impact of TRA and ETPs before and after withdrawal on AKT protein expression in Leydig, Sertoli, and germ cells, both western blotting and IHC staining were utilized. Western blotting was utilized to analyze the global protein levels of AKT in different groups, while the IHC staining was used to examine the mean distributions of AKT⁺ Leydig, Sertoli, and germ cells. The results showed a notable decrease in the global AKT protein level in the TRA-sole group compared to the control rats (P=0.001), which remained unchanged after withdrawal. However, ETPs (with no remarkable differences between ETP-induced groups) up-regulated the global AKT protein levels after withdrawal (P=0.002), with no notable differences between ETP-induced and control groups. The IHC analysis showed a notable decrease in the mean distribution of AKT⁺ Leydig, Sertoli, and germ cells in comparison to the control group (P=0.001) in the TRA-sole group. This reduction persisted after withdrawal. In contrast, the lower-intensity LICT group (with lower effectiveness) and higher-intensity MICT and HICT groups (with higher impact) showed increased AKT⁺ Leydig, Sertoli, and germ cell distribution in the seminiferous tubules. Finally, the mean pixels of brown reactions (indicating a positive reaction for corresponding proteins) were investigated to minimize the examination errors. Observations indicated similar results, reflecting a significant decrease in the total intensities of positive reactions within the TRA-sole and TRA/W groups (versus the control rats, P=0.001) and an increment in the ETPs-induced groups (P=0.001) compared to the TRA-sole and TRA/W groups (Fig .3A-D).

Fig.3.

Immunohistochemistry staining and Western Blot analyses for Assessment of AKT protein expression. The immunoreactivity of protein Kinase B (AKT) in different groups: A. and B. Cross-sections of seminiferous tubules. Significant reduction of AKT⁺ reactions (represented by brown-stained cells) in the TRA sole group, which is impressively increased after low, moderate, and high-intensity continuous training consideration in the post-withdrawal period. The AKT⁺ Leydig (L), Sertoli (S), and germ (G) cells are marked in the photomicrographs with higher magnifications (scale bar A: 100 µm, B: 50 µm). C. Mean numbers of AKT⁺ Leydig cells per total cells in one mm², and AKT⁺ Sertoli and germ cells per total cells of one seminiferous tubule across experimental groups. Decreased SUM pixel-based brown-stained reactions (representing AKT) in the TRA-sole and TRA/W groups versus control rats, which are remarkably increased in the exercise training-received groups. D. Protein bands for AKT (protein kinase B), and mean changes in the protein contents of AKT in different groups. All data are presented in the mean ± SD. Different letters represent significant differences between groups (P<0.05, n=6/each group). TRA/Sole; Rats received tramadol for 60 days without further treatment, TRA/W; Rats that received tramadol for 60 days continued without tramadol treatment for an additional 60 days, LICT; Rats subjected to low-intensity continuous training, MICT; Rats subjected to moderate-intensity continuous training, HICT; Rats were subjected to high-intensity continuous training, which discontinued tramadol treatment and continued training for 60 days after withdrawal.

ETPs could increase global PI3K protein expression after the withdrawal

Similar to AKT, to explore the impact of TRA and ETPs on PI3K protein expression in Leydig, Sertoli, and germ cells, both western blotting and IHC staining techniques were considered. Observations indicated a notable (P=0.001) decrease in the mean global PI3K protein level of TRA-sole group when compared to the control rats. This condition persisted even after withdrawal. Conversely, MICT, and HICT could up-regulate the PI3K protein expression compared to the TRA-sole and TRA/W groups (P=0.001). There were no significant variances observed between the LICT and TRA/W groups. The IHC staining represented the same findings. Accordingly, the TRA-sole and TRA/W groups showed a remarkable (P=0.001) reduction in the mean distributions of PI3K⁺ Leydig, Sertoli, and germ cells compared to the control rats. However, the ETPs could significantly ameliorate the TRA-induced effect by increasing the PI3K expression in the Leydig, Sertoli, and germ cells when compared to those in the TRA-sole and TRA/W groups (P=0.001). No significant variances were observed within the ETPs group. The software analysis showed the same outcome. The SUM intensity of brown reactions (presenting PI3K protein) was decreased in the TRA-sole and TRA/W groups (P=0.001) compared to the control group. In contrast, the ETPs-induced groups showed a notable (P=0.001) increase in the SUM intensities of brown reactions versus the TRA-sole and TRA/W groups (Fig .4A-D).

Fig.4.

Immunohistochemistry staining and Western Blot analyses for assessment of PI3K protein expression. The immunoreactivity of phosphoinositide 3-kinases (PI3K) in different groups: A. and B. Cross-sections of seminiferous tubules. Significant reduction of PI3K⁺ reactions (represented by brownstained cells) in the TRA-sole group, which is impressively increased after low, moderate, and high-intensity continuous training consideration in the postwithdrawal period. The PI3K⁺ Leydig (L), Sertoli (S), and germ (G) cells are marked in the photomicrographs with higher magnifications (scale bar A: 100 µm, B: 50 µm). C. Mean numbers of PI3K⁺ Leydig cells per total cells in one mm², and PI3K⁺ Sertoli and germ cells per total cells of one seminiferous tubule across experimental groups. Decreased SUM pixel-based brown-stained reactions (representing PI3K) in the TRA-sole and TRA/W groups versus control rats which is remarkably increased in the exercise training-received groups. D. Protein bands for Phosphoinositide 3-kinase (PI3K), and Mean changes in the protein contents of PI3K in different groups. All data are presented in the mean ± SD. Different letters represent significant differences between groups (P<0.05, n=6/each group). TRA/Sole; Rats received tramadol for 60 days without further treatment, TRA/W; Rats that received tramadol for 60 days continued without tramadol treatment for an additional 60 days, LICT; Rats subjected to low-intensity continuous training, MICT; Rats subjected to moderate-intensity continuous training, and HICT; Rats were subjected to high-intensity continuous training, which discontinued tramadol treatment and continued training for 60 days after withdrawal.

ETPs could up-regulate the VEGF global expression after the withdrawal

The western blot analysis revealed a notable reduction in the global VEGF protein content in the TRA-sole and TRA/W groups compared to the control rats (P=0.001), which remained unchanged after withdrawal. In contrast, ETPs could increase the global VEGF protein content of testicles after withdrawal. The IHC staining demonstrated a significant decrease in the mean distributions of VEGF⁺ Leydig, Sertoli, and germ cells per one seminiferous tubule compared to the control rats. This condition was reversed within all groups induced by ETPs. Accordingly, the ETPs up-regulated the VEGF⁺ Leydig, Sertoli, and germ cells per one seminiferous tubule compared to the TRAS-sole and TRA/W groups (P=0.001). Similar findings were revealed after software analysis for brown reactions representing VEGF protein in one cross-section (Fig .5A-D).

Fig.5.

Immunohistochemistry staining and Western Blot analyses for Assessment of VEGF protein expression. The immunoreactivity of vascular endothelial growth factor (VEGF) in different groups: A. and B. Cross-sections of seminiferous tubules. Significant reduction of VEGF⁺ reactions (represented by brown-stained cells) in the TRA-sole group, which is impressively increased after low, moderate, and high-intensity continuous training consideration in the post-withdrawal period. The VEGF⁺ Leydig (L), Sertoli (S), and germ (G) cells are marked in the photomicrographs with higher magnifications (scale bar A: 100 µm, B: 50 µm). C. Mean numbers of VEGF⁺ Leydig cells per total cells in one mm², and VEGF⁺ Sertoli and germ cells per total cells of one seminiferous tubule across experimental groups. Decreased SUM pixel-based brown-stained reactions (representing VEGF) in the TRA-sole and TRA/W groups versus control rats which is remarkably increased in the exercise training-received groups. D. Protein bands for vascular endothelial growth factor (VEGF), and Mean changes in the protein contents of VEGF in different groups. All data are presented in the mean ± SD. Different letters represent significant differences between groups (P<0.05, n=6/each group). TRA/Sole; Rats received tramadol for 60 days without further treatment, TRA/W; Rats that received tramadol for 60 days continued without tramadol treatment for an additional 60 days, LICT; Rats subjected to low-intensity continuous training, MICT; Rats subjected to moderate-intensity continuous training, and HICT; Rats were subjected to high-intensity continuous training, which discontinued tramadol treatment and continued training for 60 days after withdrawal.

ETPs could up-regulate the VEGF R1 and VEGF R2 global expression after the withdrawal

The western blot analysis revealed a remarkable (P=0.001) reduction in VEGF R1 and VEGF R2 protein levels in the TRA-sole group compared to the control rats. After withdrawal, the protein levels of VEGF R1 remained unchanged, while an increase was observed for VEGF R2 compared to the TRA-sole rats (P=0.039). However, the ETPs led to an increase in global VEGF R1 and VEGF R2 protein levels compared to the TRA-sole (P=0.001) group. The VEGF R2 was increased in all groups after withdrawal, and no significant change was shown between TRA/W, LICT, MICT, and HICT groups (Fig .6A, B).

Fig.6.

Western Blot analyses for Assessment of VEGF R1 and VEGF R2 protein expression. ^A. Protein bands for vascular endothelial growth factor receptor R1 and R2 (VEGF R1 and VEGF R2), ^B. Mean changes in the protein contents of VEGF R1 and VEGF R2 in different groups. All data are presented in the mean ± SD. Different letters represent significant differences between groups (P<0.05, n=6/each group). TRA/Sole; Rats received tramadol for 60 days without further treatment. TRA/W: Rats that re ceived tramadol for 60 days continued without tramadol treatment for an additional 60 days, LICT; Rats subjected to low-intensity continuous training, MICT; Rats subjected to moderate-intensity continuous training, and HICT; Rats were subjected to high-intensity continuous training, which discontinued tramadol treatment and continued training for 60 days after withdrawal.

Discussion

TRA acts as a central opiate agonist, providing pain relief. However, prolonged use of TRA can result in addiction and adversely affect the testicular spermatogenesis potential, sperm parameters, and antioxidant status (13, 15). In this study, a rat model of TRA addiction was established , and after 60 days of cessation of TRA consumption, ETPs of different intensities were administered to determine which type of ETP could expedite the recovery process. The goal was to investigate the potential mechanism by which ETPs can alleviate the pathogenic effects induced by TRA on testicular tissue. Our results indicated that TRA notably diminishes the expression of AKT, PI3K, VEGF, VEGF R1, and VEGF R2 at both mRNA and protein levels. This situation is not reversible after a short time from withdrawal. In contrast, considering the ETPs with different intensities can remarkably ameliorate the TRA-induced detrimental effects.

Multiple kinases, including the PI3K, plays a vital role in sustaining spermatogenesis. The PI3K/AKT (protein Kinase B) pathway regulates various cellular processes, such as germ cell proliferation, survival, migration, and metabolism. Accordingly, a decline in PI3K expression and/or synthesis can significantly impact spermatogenesis by suppressing germ cell proliferation, survival, and metabolism (3). Previous research has demonstrated that long-term TRA consumption can negatively affect the PI3K expression in the lungs (29) and breast cancer cells (30), leading to the inactivation of the PI3K/AKT signaling pathway. A recent study by Kamranian et al (31) revealed that TRA (50 mg/kg, daily) can induce hippocampal neurodegenerative disorders by inactivating the PI3K/ AKT/mTOR network. Indeed, PI3K brings AKT to the cell membrane, where it generates phosphatidylinositol 3, 4, 5 trisphosphate PIP3 from PIP2 (6). AKT is then phosphorylated and activated by PDK1 and mTORC2 and phosphorylates downstream targets that promote cell survival and proliferation (9). Moreover, AKT activates mTORC1, which promotes protein synthesis and cell growth (10). Therefore, understanding the importance of PI3K/AKT interaction is crucial in exploring the molecular mechanism underlying TRA-induced pathogenesis in the testicular tissue. To explore the impact of TRA on testicular tissue, we analyzed the mRNA and protein levels of PI3K and AKT before and after TRA withdrawal and examined whether any changes occurred shortly after withdrawal. We also assessed the potential of LICT, MICT, and HICT ETPs in ameliorating the adverse ef fects of TRA. We found that TRA suppresses PI3K and AKT expression, which remained unchanged even af ter TRA withdrawal. However, different intensities of ETPs could accelerate recovery by up-regulating PI3K and AKT expression. Thus, considering these findings, it could be concluded that TRA besides other possible mechanism(s), disrupts spermatogenesis by negatively affecting PI3K/AKT interaction, which is not reversible shortly after withdrawal. In contrast, ETPs, mainly MICT and HICT, enhance PI3K and AKT expression, promoting spermatogenesis recovery. The impacts of various ETPs on PI3K and AKT expression/interactions have been shown previously. Accordingly, swimming exercise training (20 minutes/d, 6 days/week for two weeks) has been shown to rebalance the PI3K/AKT activity by increasing insulin sensitivity in polycystic ovarian conditions (32). Peng and co-workers demonstrated that aerobic exercise (12 m/minutes to 15 m/min, and the duration increased from 15 minutes to 45 minutes, maintaining a 0° incline°) reduces the apoptosis in the rat’s central nervous system by re-balancing PI3K/AKT/ GSK-3β signaling pathway (33). Nadi et al. (34) showed that aerobic exercise could be considered as a strong stimulator for PI3K and AKT expression in skeletal muscle, adipose tissue, liver, brain, and pancreas.

VEGF activates various intracellular pathways, including PI3K/AKT signaling (35). VEGF by binding to its receptors, VEGF R1 and VEGF R2, triggers downstream signaling through the PI3K/AKT pathway (6). Tian et al. (36) demonstrated that VEGF, specifically through VEGF R2, directly regulates germ cell proliferation and the testicular vascular system. Previous studies have also shown that VEGF via VEGF R2 controls initial spermatogenesis and via VEGF R1 maintains the germ cell differentiation during spermatogenesis (37). Therefore, we explored the expression levels of VEGF and its receptors, VEGFR1 and VEGFR2, in the testicles before and after withdrawal from TRA. Our results showed that TRA treatment sig nificantly suppressed VEGF, VEGF R1, and VEGF R2 expression in the testicles. Moreover, both mRNA and protein levels of VEGF and VEGF R1 remained un changed after TRA withdrawal, indicating that TRA had a sustained down regulatory effect on VEGF and VEGF R1 and R2. On the other hand, we found that different ETPs could significantly upregulate the VEGF, VEGF R1, and VEGF R2 mRNA levels as well as protein content in the testicles. Based on the stimulating effect of VEGF/VEG FR1 and VEGFR2 on PI3K activation by recruiting it to the plasma membrane to generate PIP3 from PIP2 (6), it is logical to suggest that TRA suppresses PI3K/AKT sign aling by downregulating the expression of VEGF, VEG FR1, and VEGFR2. Since, PIP3, in turn, recruits AKT to the cell membrane, where it undergoes phosphorylation and activation by PDK1 and mTORC2 (7, 8), leading to the phosphorylation and subsequent inactivation of down stream targets involved in cell survival and proliferation (9). The improved expression of VEGF, VEGFR1, and VEGFR2 following ETPs, particularly MICT and HICT, suggests that ETPs can restore the expression of VEGF and its receptors, leading to an enhancement of PI3K/ AKT signaling in germ cells. These characteristics have been demonstrated in previous studies in various tissues. Accordingly, acute endurance exercise has been shown to significantly stimulate the expression of VEGF in adipose tissue of rats during the initial phases of weight gain (38). The LICT, MICT, and HICT ETPs have been shown to remarkably increase the VEGF, VEGF R1, and VEGF R2 expression in the cardiac tissue (39). Moreover, aerobic training (25 m/minutes, 5° gradients, and 30 minutes each session for 8 weeks) has been shown to increase VEGF expression in cardiac tissue of streptozotocin-induced diabetic rats (26).

The study evaluates the effects only 60 days post-with- drawal, which may not fully capture the long-term recovery process or potential relapse effects. Further research is needed to understand the long-term implications of ETPs on AKT, PI3K, and VEGF expression. Moreover, although the study explored the expression levels of AKT, PI3K, and VEGF, the underlying molecular mechanisms involved in the observed effects were not fully elucidated. Further research is needed to investigate the specific signaling pathways and interactions involved.

Conclusion

Our findings indicate that TRA can disrupt spermatogenesis by negatively affecting the PI3K/AKT interaction, which is not reversible shortly after withdrawal. In contrast, ETPs, particularly MICT and HICT, improve PI3K and AKT expression, facilitating the recovery process. The beneficial effects of ETPs on PI3K/AKT signaling partially depend on their promoting effect on VEGF, VEGF R1, and VEGF R2 expression, supporting the promising potential of ETPs in ameliorating the adverse effects of TRA on VEGF/VEGF R1, R2/PI3K/AKT signaling in the germ cells even a short time after withdrawal. Although all types of ETPs produced roughly similar outcomes during the withdrawal period, the results following MICT and HICT induction were more promising compared to the LICT ETP.

Author’s Contributions.

V.S.; Methodology and Resources. A.T.; Investigation and Supervision; J.T.A.; Conceptualization, Formal anal ysis, Supervision, Project Administration, Data curation, Writing-original draft, Review, and Editing. M.R.; Con ceptualization, Data curation, Methodology, Validation, Writing-original draft, Review, and editing. All authors read and approved the final manuscript.