Examination of Apoptotic and Autophagic Effects of Chronic Roumilast Use on Rat Testicular Tissue by Immunohistochemical and Immunouorescence Methods

Roumilast (ROF) (3-cyclo-propylmethoxy-4-difuorome-thoxy-N-[3,5-di-chloropyrid-4-yl] benzamide) is a second generation and forcible phosphodiesterase-4 (PDE4) inhibitor. This study aims to investigate the effects of chronic Roumilast in different doses on testicular tissue and testosterone levels in healthy Sprague-Dawley rats. 36 male rats were into 4 (ROF) and Hematoxylin-Eosin (H&E) staining for histopathological examinations in testicular tissue, immunohistochemical and immunouorescence examinations for Caspase-3, Apoptosis Inducing Factor (AIF) and Light Chain 3β (LC3B) expression levels, and ELISA method used to determine serum testosterone levels. Data were analyzed using SPSS v.22 with Kruskal-Wallis, Mann Whitney-U, and Wilcoxon tests.

Phosphodiesterases (PDEs) are phosphohydrolase enzymes that cause hydrolysis of the 3'-phosphate bridges of intracellular secondary messenger molecules, cyclic guanosine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP), that is, they regulate the continuity and termination of cAMP and cGMP signals in the cell (Drobnis and Nangia 2017). As PDEs have a critical effect on signaling pathways in various pharmacological processes, including cell function, they have drawn the attention of researchers relevant to many disease pathologies (Ouyang et al. 2021). PDEs, which are a large family of enzymes, reportedly consist of 11 subtypes (PDE1-PDE11) and over 40 isoforms (Nabavi et al. 2019).
Phosphodiesterase inhibitors such as ro umilast act by inhibiting the breakdown of intracellular cAMP, reducing in ammation, suppressing cell proliferation, cytokine production, and chemotaxis. Ro umilast, which was used as an antidepressant in the 1980s, is the rst selector PDE4 inhibitor approved for human usage in 2010, with the primary indication being chronic obstructive pulmonary disease (COPD) (Giembycz and Field 2010;Wedzicha 2013). In the later years, it started being used in the elds of arthritis, neurodegenerative diseases, liver and dermatology (Fala 2015;Nabavi et al. 2019;Fleming et al. 2020).
Apoptosis and autophagy are phenomena that occur in many cases as cell death mechanisms (Zheng et al. 2016). Apoptosis is the mechanism in which the cell self-destructs, regulated by genes, needs protein synthesis and energy, and maintains the balance in the organism (Voss and Strasser 2020). This mechanism of death is divided into two as caspase-dependent and caspase-independent apoptosis. In the caspase-dependent apoptosis, caspase-3 is in caspase-independent apoptosis; To induce programmed cell death, the AIF protein plays a role in the initiation of the caspase-independent apoptotic pathway by triggering chromatin condensation and DNA fragmentation in the cell and regulating the permeability of the mitochondrial membrane (Yu et al. 2015). Autophagy, on the other hand, is a catabolic mechanism that sends not only cytosolic proteins, but also intracellular cytosolic components, organelles and aggregates to lysosomes for degradation, prevents the accumulation of misfolded proteins and destroys unwanted organelles (García-Prat et al. 2016). LC3B protein, which is a reagent of autophagy, is effective in initiating the autophagic pathway (Herb, Gluschko, and Schramm 2020). Studies have shown that apoptosis and autophagy interact with Ro umilast (Bajpai et al. 2020).
Usage doses of Ro umilast have been determined as 250, 500 and 1000 µg/kg, and it is reportedly more effective in medium and high doses (Murad et al. 2017). In the pharmacology review published by the FDA in 2011, it was reported that headache, gastrointestinal disorders, dizziness, palpitations, u, and arterial hypotension were observed after a single oral application of 2.5 and 5 mg in Phase 1 studies of Ro umilast (FDA 2011).
When the studies are examined in the literature, the protective properties of Ro umilast in different tissues have been reported against experimentally induced damages and some toxic agents. However, there is no in-vivo scienti c study to investigate healthy testicular tissue toxicity after chronic use. Chronic use of Ro umilast, which has a widespread clinical use, was found to be apoptotic, caspase-dependent (Caspase-3), caspase-independent (AIF), autophagic, expressions of (LC3B) proteins and their effects were investigated by analyzing serum testosterone levels on rat testicular tissue with this study.

Materials And Methods
In the study, a total of 36 male 40 days old, 180-200 g, Sprague-Dawley rats were used. The groups were randomly formed. They were fed as ad libitum with tap water in an environment with 12 hours of darkness and 12 hours of lighting and kept in standard cages. The ROF groups were given 0.5 and 1 mg/kg Ro umilast by oral gavage at the same time every day for 4 weeks (Table 1) (Botros et al. 2020). At the end of the experiment, rats were deeply sedated with Sevo urane (Sevorane®, Abbott Lab. Istanbul, Turkey) and cervical dislocation was performed, and intracardiac blood and testicular tissue samples were collected. Body weights and testes weights of rats in all groups were measured. Testicular tissue samples were placed in 10% buffered neutral formalin solution, and after routine histological procedures, they were blocked in para n.

Histological Studies
H&E staining technique was applied to the sections taken from the blocks to examine the general structure of testicular tissue. The preparations were examined under the light microscope (Nikon BX51), 6 selected elds chosen randomly, and the experimental groups were compared by scoring according to the Johnsen criteria in order to reveal the possible severity of Ro umilast damage to the testicular seminiferous tubule cells (Johnsen 1970;Erboga et al. 2016).

Immunohistochemical Studies
The 5 µm sections taken from the blocks were passed through xylol and alcohol series and washed with PBS for 10 minutes in 3% H2O2 and treated with antigen retrieval solution. Afterwards, a protein block was applied to completely cover the sections. Then the sections of Table 2   The procedures speci ed in immunohistochemical staining were applied to the prepared sections until incubation with primary antibody. The sections were then incubated at 37°C for 45 minutes with the primary antibodies (1/200 dilution ratio) indicated in Table 2. After washing with PBS, the sections were incubated again with the uorescence-linked secondary antibodies in Table 3 (1/50 dilution ratio) for 45 min at 37°C in the dark. DAPI was dripped onto the tissue samples and covered with a coverslip and examined under a uorescent microscope (Zeiss Axio scope). Immunohistochemical and immuno uorescence positivities in 10 randomly selected areas at 20x magni cation, by using the image analysis computer program named as Image J 1.43, according to the intensity of staining: none (0), mild (1), moderate (2), and severe (3).

Biochemical Analysis
Serum testosterone levels were analyzed with the trademark ELISA kit (Biovision, Catalog No: K7418) according to the kit procedure. Results are given in ng/ml for testosterone.
To measure the diameters of the seminiferous tubules, 2 measurements were made from 10 randomly selected round and near-circular seminiferous tubules from each animal at 20x magni cation and their averages were taken (Mouro et al. 2018). The measurement was made using the image analysis computer program called as Image J 1.43 (Rasband 2014).

Statistical analysis
Non-parametric statistics were used in the study. Kruskal Wallis H Test was used to compare body weight, seminiferous tubule diameter and Johnsen Testicular Biopsy Scores between groups, and Mann Whitney U test was performed between paired groups for differences between signi cant variables. However, Wilcoxon Test was used for the difference in body weights of the groups on the 1st and 30th days.
Analyses were carried out using SPSS v.22.

Live Weight Findings
There was no statistically signi cant difference between the groups in terms of the weight of the rats on day 1 (χ2 = 0.284, SD = 3, p > 0.05). When the body weights of the rats were compared between the groups, a signi cant difference was observed (χ2 = 29.856, SD = 3, p < 0.001) at day 30. It was determined that there was no statistically signi cant difference between the control group and the sham group (p = 0.171), while the differences between all other paired groups were statistically signi cant (p < 0.01).
Control group (z = 2.521, p < 0.05), sham group (z = 2.524, p < 0.05), 0.5 mg/kg ROF (z = 2.623, p < 0.01) and 1 mg/kg ROF A statistically signi cant difference was found between the 1st and 30th day weights of the groups (z = 2.814, p < 0.01). When the change in body weight within 30 days was examined, a statistically signi cant increase at the end of 30 days in the control group and sham group; A statistically signi cant decrease was observed at the end of 30 days in the 0.5 mg/kg and 1 mg/kg ROF groups (Table 4). There was a statistically signi cant difference between the groups in terms of seminiferous tubule diameter of rats (χ2 = 23.959, SD = 3, p < 0.001). There was no statistically signi cant difference between the control and sham groups in terms of seminiferous tubule diameter of rats (p > 0.05), but it was observed that there was statistically signi cant difference between the control group and 0.5 mg/kg ROF (p < 0.05) and 1 mg/kg ROF (p < 0.001) groups. However, there was no statistically signi cant difference between the sham group and 0.5 mg/kg ROF group (p > 0.05), but there was a statistically signi cant difference between the mg/kg ROF group and the sham group and 1 mg/kg ROF group (p < 0.001) and 0.5 mg/kg and 1 (p < 0.001) ( Table 5). According to the Johnsen Testicular Biopsy Score, there was no signi cant difference between the control and sham groups (p > 0.05), but there was a signi cant difference between the 0.5 mg/kg and 1 mg/kg ROF groups and the control and sham groups (p > 0.05). <0.05). Statistical analysis results according to Johnsen Testicular Biopsy Score are given in Table 6.

Histopathological Findings
It was observed that the seminiferous tubules and spermatogenic and Sertoli cells in these tubules were normal in the testicular tissues of the rats in the control and sham groups. Interstitial connective tissue and interstitial cells were found between the seminiferous tubules. It was observed that the full structures of the seminiferous tubules began to disappear and the normal ordered structures of the spermatogenic cells began to deteriorate in the testicular tissues of the rats in the 0.5 and 1 mg/kg ROF groups. In the 0.5 mg/kg ROF group, shedding of the seminiferous epithelium and local degenerations in the interstitial area were observed, while in the 1 mg/kg ROF group, intercellular separation, desquamation, interstitial edema and degenerative changes were observed (Fig. 1).

Immunohistochemical Findings
Caspase-3, AIF and LC3B immunoreactivity (Fig. 2) was not statistically signi cant in testicular tissues of control and sham group rats (p > 0.05).
Mild Caspase-3, AIF and LC3B immunoreactivity was observed in the interstitial area in testicular tissues of rats in the 0.5 mg/kg ROF group, while mild LC3B immunoreactivity was also observed in the seminiferous epithelium (Fig. 3). Caspase-3 and AIF immunoreactivity was mild in the interstitial area, and LC3B immunoreactivity was severe in the interstitial area and seminiferous epithelium in testicular tissues of rats in the 1 mg/kg ROF group (Fig. 3). There was a statistically signi cant difference between the control and sham groups and the 0.5 and 1 mg/kg ROF groups (Table 7, p < 0.05).

Immuno uorescence Findings
It was not observed a signi cant immunopositivity in terms of Caspase-3, AIF and LC3B in testicular tissues of rats in the control and sham groups (Fig. 4) and no statistical difference (p > 0.05). It was determined that Caspase-3, AIF and LC3B expression levels in testicular tissues of rats in the 0.5 mg/kg ROF group were increased compared to the control and sham groups and were mildly severe in the interstitial area. Caspase-3, AIF and LC3B immunopositivity in testicular tissues of rats in the 1mg/kg ROF group was moderate in the interstitial area (Fig. 5). There was a signi cant difference between the control and sham groups and the 0.5 and 1 mg/kg ROF groups (Table 8, p < 0.05). In terms of serum testosterone levels, there was no statistically signi cant difference between the control and sham groups (p = 0.144), the 0.5 mg/kg ROF group was lower than the control and sham groups (p = 0.001 and p = 0.014, respectively), and the 1 mg/kg ROF group was found to be lower compared with the control, sham and 0.5 mg/kg ROF groups (p = 0.000, p = 0.000, p = 0.027, respectively) ( Table 9).

Discussion
Chronic bronchitis symptoms of Ro umilast and are the only PDE4 inhibitor for COPD patients with a history of exacerbation that has reached the market (Baye 2012). Several new PDE4 inhibitor compounds are in early clinical development, and there is not yet clear information about their e cacy and safety (Sugin et al. 2020). In preclinical and early-stage studies, it has been reported that PDE4 inhibitors improve memory and have curative effects on depressive-like behaviors caused by chronic mild stress, lipopolysaccharide or ethanol abstinence (Prickaerts, Heckman, and Blokland 2017;Yu et al. 2018). Ro umilast has been studied by many researchers to determine the mechanism and new indications.
Wang et al. were applied orally 5-10 mg/kg Ro umilast once a day for 30 days, and it was determined that it had anti-apoptotic and anti-in ammatory effects on nerve cells. It has also been reported that it can be an effective agent in memory impairment and in the treatment of depression ).
In cadmium (Cd)-derived nephrotoxic rats, they reported a signi cant increase in anti-oxidative enzymes such as Superoxide Dismutase, Catalase and Glutathione-S-transferase, and a decrease in oxidative parameters such as Malondialdehyde and ischemic modi ed albumin. However, it has been reported that the damage caused by Cd in kidney tissues is signi cantly recurred of Ro umilast treatment (0.5 and 1.5 mg/kg) (Ansari et al. 2019).
It has been asserted that Ro umilast, which is used orally once a day, does not cause arrhythmia and the most common side effects are diarrhea and weight loss (AYDEMİR 2018). It has been reported that weight loss, one of the most important side effects of Ro umilast, was observed in 83 adult COPD cases because of treatment with Ro umilast (500 µg tablet, 18 months, once a day) (Cilli, Bal, and Gunen 2019). We found that while an increase in rat body weight was observed in the control and sham groups in accordance with the months, the body weights of the rats exposed to Ro umilast decreased as the dose increased in accordance with the literature.
It has been reported that the dose and duration of application of phosphodiesterase inhibitors are determinative for e cacy, but long-term use at high doses causes adverse effects (Kızılay and Altay). In our study, in the 0.5 mg/kg Ro umilast group, the testicular tissue of the rats was narrowed in seminiferous tubule diameter and degeneration was observed in the interstitial area, while the damage become clearer in the 1 mg/kg Ro umilast group. It was observed that the narrowing of the tubule diameter was statistically signi cant, separation between cells, desquamation, degenerative changes and edematous areas in the interstitial connective tissue.
It has been reported that PDE4 increases cell viability and mitochondrial activity, and decreases cell death (Wedzicha, Calverley, and Rabe 2016;Kyung et al. 2018). It has been asserted that PDE4 may be a new therapeutic agent in liver brosis, increasing the level of cAMP, which suppresses collagen synthesis and broblast activation (Essam et al. 2019). The effects of Ro umilast on cerebral in ammation developing in the subarachnoid hemorrhage model in rats were investigated and it was shown that it signi cantly reduced neurological damage and in ammatory cytokines IL1β, IL-6 and TNFα levels and the number of apoptotic neurons (Wu et al. 2017). As a result of examining the cerebral cortices of rats applied Ro umilast by TUNEL method, it was determined that apoptosis was signi cantly reduced . In a study in which a sepsis model was created by cecal ligation and puncture surgery, Ro umilast (1 mg/kg and 3 mg/kg) was applied to mice once a day for 7 days, and it was concluded that 3 mg/kg Ro umilast could protect mice against kidney damage by partially reducing cell apoptosis . In the study, in which the PDE4 enzyme inhibitor rolipram was applied to rats (single dose 10 mg/kg) 15 minutes before detorsion and the effects on histopathological damage after testicular torsion/detorsion and the apoptotic pathways thought to cause damage; It was found that rolipram could not reverse histological damage, suppress the activated intrinsic apoptotic pathway, and did not have a protective effect against histopathological damage (Akdeniz E 2018.). We determined that caspase-3 expression increased in healthy testicular tissue, especially at high doses, in chronic use of Ro umilast in line with the results of this study.
It is emphasized that in male reproduction of cAMP, while it is reported that the improvements of sperm motility with hyperactivity of the ability to enter the changes and the acrosome reaction and developments during the capacitation process is required, in case of increasing of cAMP levels in an uncontrollable way also may cause hyper-proliferation in the cellular processes including cancer development can cause problems in cellular process with many studies (Aitken and De Iuliis 2009;Yan et al. 2016). It has been stated that while apoptosis that occurs under physiological conditions in the immature testis is necessary for the development of germ cells, misactivation of apoptosis may impair spermatogenesis and cause reproductive defects (Kumar, Abbas, and Aster 2013;Qian et al. 2020). In the results of this study, it was observed that the intensity of Caspase-3 and AIF immunoreactivity, which are important apoptosis reagents in spermatogenesis, increased more in rat testis tissue in 1 mg/kg Ro umilast group compared to 0.5 mg/kg Ro umilast group. Researchings related on the subject show that chronic and high doses use of Ro umilast may affect the spermatogenesis process negatively.
Autophagy activation has been shown to play a regulatory role in the maintenance of spermatogenesis and the maintenance of spermatogenic stem cells. When the activation of LC3-2, LC3-2/LC3-1, Atg5 and Beclin-1 autophagic reagents were examined by spermatogenic stem cell culture study of organophosphate, which is toxic for the reproductive system; it has been reported that all these reagents are increased in rat spermatogenic stem cells ). In the study investigating the role of autophagy in testicular tissue, germ cell speci c Atg7 deletion was performed and the fertility of Atg7 mice was evaluated. It has been observed that there are very few sperms in the epididymis, most of the sperms exhibit round head anomaly, and the acrosome structure is damaged . In this study, we found that LC3B expression increased in rats exposed to 0.5 and 1 mg/kg Ro umilast for 4 weeks, and there was positivity in some spermatogenic cells, unlike caspase-3 and AIF. Based on these ndings, it was thought that structural and functional changes in testicular tissue might be associated with increased autophagy.
Testosterone is an important androgen in the development of the male reproductive system and sexual characteristics. It has been reported that testosterone regulates the cGMP pathway and thus affects endothelial function and endothelial progenitor cells, which are key to the endothelial repair system (Zgair et al. 2021). It has been asserted that decreased testosterone level causes structural and functional changes in Sertoli cells, which are important for germ cells (Dym and Madhwa Raj 1977). After long-term treatment with sildena l, a PDE5 inhibitor; it has been reported that the circulating testosterone level is normalized and contributes to the accumulation of cGMP in the testicular interstitial uid, resulting as an improvement in Leydig cell steroidogenic capacity and an increase in the number of Leydig cells, in addition to preventing the atrophy of the seminiferous tubules (Sokanovic et al. 2018). The inhibition of PDE4 enzymes is in testosterone production by the Leydig cells have been reported to cause a signi cant increase. In rats, it has been asserted that autophagy participates in the regulation of steroid synthesis in Leydig cells (Weckmann et al. 2018). It has been reported that PDE4 and PDE8 play important roles in the physiology of steroidogenic tissues and there is synergism between signaling compartments regulated by PDE4 and PDE8 to facilitate maximum steroid output ). It has been asserted that the repository of cAMP, which regulates androgen production, is controlled by PDE8s working together with PDE4, and that PDE inhibitor therapy should simultaneously target both PDE8 isozymes and PDE4 to be an effective stimulator of steroidogenesis ). In our ndings, it was revealed by ELISA method that the serum testosterone level was within the normal reference range in the control and sham groups (p = 0.001 and p = 0.014), but with the increase in the dose used in the Ro umilast groups, the testosterone level decreased statistically signi cantly (p = 0.000, p = 0.000, p = 0.027).
As a result, it is considered that degenerated and narrowed seminiferous tubule structures, increased caspase-3, AIF and LC3B immunoreactivity density, decrease in serum testosterone level with increasing dose; Ro umilast may have some anabolic effects and have negative effects on spermatogenesis and therefore male fertility due to changes in the mechanism of apoptosis and autophagy with this study.
However, more detailed experimental studies and clinical ndings are needed to determine the toxicities that may occur in chronic use of high doses of Ro umilast.

Declarations
Author Contributions: AG and EKS: Conceptualization, data curation, investigation, formal analysis, original draft preparation, pharmacokinetics and tissue distribution of Ro umilast in rats, methodology and writing, editing and review. All authors have published version of the manuscript and agreed to the read.