HMG-CoA Reductase Inhibition Protects Testis and Sperm Quality from Testicular Ishemia and Reperfusion via Activation of Antioxidant Status and AKT Signaling

HMG-CoA Reductase Inhibition Protects Testis and Sperm Quality from Testicular Ishemia and Reperfusion via Activation of Antioxidant Status and AKT Signaling | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article HMG-CoA Reductase Inhibition Protects Testis and Sperm Quality from Testicular Ishemia and Reperfusion via Activation of Antioxidant Status and AKT Signaling Berna Yildirim, Oguzhan Baygul, Nursena Sengun, Unsal Veli Ustundag, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6611379/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 22 Nov, 2025 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract Testicular torsion (TT) is a urological emergency that results in ischemia/reperfusion (I/R) injury, leading to oxidative stress, cellular apoptosis, and impaired spermatogenesis. This study aimed to investigate the protective effects of the HMG-CoA reductase inhibitor rosuvastatin on TT-induced I/R injury and elucidate the underlying mechanisms. Male Balb/C mice (n = 28) were subjected to 720° testicular torsion for two hours, followed by 24 hours of detorsion. Rosuvastatin was administered either acutely post-torsion or prophylactically prior to injury. Histopathological analysis, oxidative stress parameters, sperm motility and morphology assessments, as well as western blot analysis of survival-related signaling proteins (pAKT, pJNK, pERK1/2, and Bcl-xL), were performed. Rosuvastatin treatment significantly reduced tissue damage decreased oxidative stress (as evidenced by increased TAS and reduced TOS/OSI), and improved sperm motility and morphology. Both treatment regimens enhanced cell survival by increasing pAKT and Bcl-xL levels and decreasing pERK1/2 activation, while also activating stress-responsive JNK1/2 signaling. These findings suggest that rosuvastatin mitigates I/R-induced testicular damage through modulation of key intracellular signaling pathways, notably PI3K/AKT, and supports its therapeutic potential in acute testicular injuries and related degenerative conditions. Biological sciences/Cell biology Health sciences/Biomarkers Health sciences/Medical research Health sciences/Molecular medicine Health sciences/Pathogenesis Health sciences/Risk factors Health sciences/Signs and symptoms Health sciences/Urology HMG Co-A reductase testicular torsion cell signaling sperm motility Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 INTRODUCTION Testicular torsion (TT) is an acute urological condition caused by rotation of the spermatic cord or improper fixation of the Tunica vaginalis , which reduces blood flow to the testicular vessels and carries a risk of tissue degeneration and infertility [ 1 ]. After testicular torsion, irreversible tissue degeneration is observed within a period of four to eight hours with detrimental effects on spermatogenesis [ 2 ]. Studies have shown that the spermatogonia and the primary spermatocytes are the first cells to be damaged by ischemia [ 3 ]. Increased reactive oxygen species (ROS) are generated particularly after reperfusion and may increase tissue damage further [ 4 ]. High levels of ROS may also affect sperm- motility, capacitation, the acrosome reaction, quality, and cause serious damage in sperm DNA. Oxidative stress or TT has been shown to cause poor sperm quality, reduce sperm concentration and mobility resulted also in the impaired sperm capacitation and viability in mice [ 5 – 8 ]. Oxidative stress in spermatozoa reduces also mitochondrial activity. It affects all cellular components [ 9 ]. It is thought that TT not only blocks blood flow to the testicles but also to the epididymis [ 3 ]. Oxidative stress and hypoxic conditions induced by ischemia- reperfusion (I/R) injury may affect sperm cells both during the production phase in the testis and during maturation in the epididymis through the contents of the epididymal lumen [ 3 ]. Therefore, the epididymis has an antioxidant enzyme capacity to prevent oxidative stress and damage to sperm structures [ 7 ]. On the other hand, oxidative stress increases ion permeability, inhibits enzymatic and receptor movement, disrupts the integrity of the sperm membrane, and leads to impaired motility [ 10 ]. This disrupts sperm- oocyte interaction [ 10 ]. Previous studies have shown that tubulin is highly oxidized when human spermatozoa are incubated under conditions of oxidative stress [ 9 ]. Many protective agents have been studied in testicular torsion over the years, but few of them had low side effects [ 11 , 12 ]. Studies suggest that treatment with immunosuppressive, anti-apoptotic, and anti-inflammatory agents may preserve tissue in TT to prevent testicular dysfunction [ 13 ]. In an experimental model of testicular TT injury, treatment with various antioxidants has been shown to reduce lipid peroxidation, oxidative stress, and germ cell apoptosis [ 13 ]. Both in vitro and in vivo evidences suggest that statins with the ability to inhibit HMG-CoA reductase have direct antioxidant properties [ 14 ]. Experimental studies investigating the effect of statins after cerebral ischemic and reperfusion have shown that statins have multiple effects such as anti-inflammatory, antioxidant, and angiogenesis [ 15 – 18 ]. There is evidence to suggest that statin therapy may activate the phosphatidylinositol 3-kinase (PI3K) protein kinase AKT pathway [ 19 , 20 ]. This activation is involved in the regulation of cell survival, growth, and proliferation [ 21 ]. The AKT signaling pathway regulates cell growth and survival by inhibiting pro-apoptotic proteins or signals [ 22 – 24 ]. When pAKT is active, it can have a therapeutic effect on ischemic injury by reducing oxidative stress, inflammation, and cell apoptosis [ 25 ]. Rosuvastatin is among the most potent statin and considered safe for a toxic mechanism such as HMG-CoA reductase inhibition [ 26 ]. Rosuvastatin can be metabolized in the body and this potent effect is due to the presence of active metabolites [ 26 ]. No toxicity has been observed in mice at various doses of rosuvastatin [ 27 ]. In this sense, here we studied the role of HMG-CoA reductase inhibitor rosuvastatin in tissue protection and functionality after TT. We hypothesized that increased AKT phosphorylation caused by rosuvastatin treatment is an important factor in the drug's multiple protective effects. Mice were treated to two hours of 720-degree torsion and then 24 hours of detorsion damage with the aim of testing this theory. Here, we demonstrated that rosuvastatin, both acutely and prophylactically, reduced the damaging effects of testicular torsion injury on sperm and testicular tissue, increased the levels of phosphorylated AKT, Bcl-xL, phosphorylated JNK1/2 and decreased the levels of phosphorylated ERK1/2 proteins. In conclusion, the data provided here points to the possibility that rosuvastatin's various preventive characteristics reduce the negative effects of testicular torsion damage via molecular processes. MATERIALS AND METHOD Ethical approval and animal care All procedures involving animals were conducted in accordance with the National Institutes of Health (NIH) guidelines for the care and use of laboratory animals. Ethical approval for this study was obtained from the Istanbul Medipol University Animal Experiments Local Ethics Committee (IMU HADYEK) (25/10/2021, E-38828770-772.02-5440), and permission was granted by the local government authorities. Animals were housed and maintained at the Istanbul Medipol University Medical Research Center (MEDITAM) under standard laboratory conditions with a 12-hour light/dark cycle (lights on daily at 7:00 a.m.). Male adult Balb/C mice, aged 8–12 weeks and weighing 20–25 g, were randomly assigned to four groups (n = 7 per group): sham (i), control (torsion/detorsion injury) (ii), acute rosuvastatin (Crestor, AstraZeneca; 20 mg/kg) administered immediately after detorsion (iii), and prophylactic rosuvastatin (Crestor, AstraZeneca; 20 mg/kg) (iv) [28]. The Surgical Procedures Surgical procedures for torsion induction were based on previous studies and included anesthesia, torsion for 2 hours, detorsion, and euthanasia after 24 hours [29,30]. Anesthesia was administered intraperitoneally using ketamine (80–100 mg/kg) and xylazine (8–10 mg/kg). Testicular ischemia was induced by rotating the right testis 720° clockwise and maintaining this position for 2 hours [31,32]. After ischemia, the testis was detorsioned by rotating it 720° counterclockwise, repositioned into the scrotum, and the incision was sutured [33,34]. Mice were then returned to their cages and allowed to recover for 24 hours to permit reperfusion. At the end of the experiment, 24 hours after detorsion, all animals were euthanized under deep anesthesia by administering an overdose of ketamine (50 mg/kg) and xylazine (10 mg/kg). All anesthesia and euthanasia procedures were conducted in accordance with the American Veterinary Medical Association (AVMA) Guidelines for the Euthanasia of Animals (2020). No outdated or non-standard agents such as chloral hydrate, ether, or chloroform were used. Experimental procedures were reported in accordance with the ARRIVE guidelines (PLoS Biol 8(6), e1000412, 2010). One testis from each animal was placed in 10% neutral buffered formalin (NBF) for histopathological analysis, and the other was stored at –80°C for Western blot analysis. All statin doses were based on previous studies [28], and rosuvastatin was dissolved in drinking water using an ultrasonic bath (Bandelin Sonorex RK 52 Ultrasonic Bath, Berlin, Germany). Sperm Analysis The epididymis of the torsion/detorsion testis was used to assess sperm function parameters. For this purpose, the epididymis was cut with small sharp scissors and placed in a Petri dish containing RPMI medium. The sample was placed in an incubator at 37°C for 10 minutes to facilitate semen drainage. Semen was collected from the epididymis and poured into a small Petri dish containing 5 ml RPMI 1640 (Sigma-Aldrich, Munich, Germany) [35]. Sperm concentration and motility analyses were evaluated using a Makler Counting Camera (Sefi Medical Instruments LTT, Haifa, Israel). For concentration, sperm were counted per hundred squares of each sample. Results are expressed in millions/ml. Spermatozoa were categorized into four groups for motility analysis: progressively motile (A motility), slow-moving (B motility), motile but unable to move forward (C motility), and immotile (D motility). The results are presented as percentages (%) [36]. To assess sperm morphology, Diff-3 staining was performed on sperm samples. For this purpose, 10 µl of sperm sample was dropped onto a positively charged slide. After drying, staining was performed, and images were captured under a 100X light microscope using immersion oil. A total of 100 spermatozoa were analyzed from each mouse. Spermatozoa were graded as normal and abnormal according to Kruger's strict criteria [37]. Abnormalities were classified as head, acrosome, neck, and tail abnormalities. The results were expressed as (%). Histopathological Analyses The follow-up procedure for testicular tissue was based on previously published studies [12,38]. Testicular tissues removed after surgery and sacrifice and fixed in 10% neutral buffered formalin (NBF) were used for histopathological evaluation. Specimens were stained with hematoxylin and eosin (Bio-Optica Mayer's Hematoxylin and Eosin Y Plus) according to the manufacturer's protocol to examine general histological structures. According to Johnsen's quantitative approach, all cells involved in spermatogenesis in all experimental groups of the study were scored on a 10-point scale to assess spermatogenesis [39]. According to this scoring, 50 seminiferous tubule sections from each sample were scored from 1 to 10. The Cosentino method was used to assess testicular damage and necrosis [40]. This method includes necrosis in the testicular tissue, organization of the germ cells, and 4 grades categorized according to hemorrhage. According to this scoring, 40 seminiferous tubules from each sample were graded from 1 to 4. Total Antioxidant Status / Total Oxidant Status Testicular tissues from the groups were homogenized in 0.9% NaCl. The supernatants were separated by centrifugation at 3000 rpm for 10 min. The parameters of oxidative stress, total antioxidant status / total oxidant status (TAS)/(TOS), were measured in the supernatants and the oxidative stress index (OSI) was determined. TAS measurement The antioxidant activity of the sample against the hydroxyl radical was determined by the method of Erel A. ABTS (2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid)) is converted to a radical by H 2 O 2 in an acidic medium and its antioxidants then neutralize the ABTS radicals. The absorbance of the products was determined using a BioTek Synergy HTX multimode reader (BioTek, Inc., USA) at 658 nm. Results are expressed as mmol Trolox eq. /L [41]. TOS measurement The method of Erel A. et al. was used to determine the total oxidant status. Briefly, this method oxidizes ferrous ions to ferric ions in the presence of various oxidative species under acidic conditions. The absorbance of the resulting-colored product was evaluated at 658 nm using the BioTek Synergy HTX multimode reader (BioTek, Inc., USA). The results are expressed as μmol H 2 O 2 eq. /L [42]. Oxidative Stress Index Measurement OSI was defined as the ratio of TOS to TAS. Specifically, OSI = TOS (μmol H 2 O 2 eq./L) /TAS (mmol Trolox eq./L). Western blot Western blot analysis was performed to determine the levels of post-injury stress (pJNK1/2) and survival (pAKT, pERK 1/2) kinases and anti-apoptotic Bcl-xL protein in isolated testicular tissues. The western blotting was carried out as described previously [43]. Briefly, testis tissue samples were harvested from the mice after 24 hours detorsion. Tissue samples of the same group were pooled, homogenized, sonicated, and treated with protease inhibitor cocktail and phosphatase inhibitor cocktail. Total protein content was evaluated using Qubit 2.0 Fluorometer according to the manufacturer's protocol (Invitrogen, Life Technologies Corporation, Carlsbad, CA, USA). Equal amounts of protein (20 µg) were size fractionated using any-kD Mini-Protean TGX gel electrophoresis and then transferred to a nitrocellulose membrane using the Trans-Blot TurboTransfer System (Bio-Rad, Life Sciences Research). Next, membranes were blocked in 5% nonfat milk in 50 mMol Tris-buffered saline containing 0.1% Tween (TBS-T; blocking solution) for 1 h at room temperature, were washed in 50 mMol TBS-T, and were incubated overnight with monoclonal rabbit anti-phosphorylated AKT (Thr308; 13038S; Cell Signaling), monoclonal rabbit anti-Bcl-xL (2764S; Cell Signaling), polyclonal rabbit anti-phosphorylated pERK1/2 (Thr202/Tyr204; 9101L; Cell Signaling), monoclonal rabbit anti- phosphorylated pJNK1/2 (Thr183/Tyr185; 9255L; Cell Signaling) antibody (1:1000). The next day, membranes were washed with 50 mM TBS-T and were incubated with horseradish peroxidase-conjugated goat-anti 657 rabbit (31460; Thermo Scientific) antibody (1:2500) for 1 h at room temperature. Each blot was performed in 3 or more replicates. Protein loading was controlled with polyclonal rabbit anti-β-actin antibody (4967; Cell Signaling Technology). Blots were developed using Clarity Western ECL Substrate kit (Bio-Rad; Life Sciences Research) and visualized using the ChemiDoc MP System (Bio-Rad; Life Sciences Research). Blot intensities were analyzed densitometrically using ImageJ. Protein levels were normalized to the β-actin levels and were presented as percentage of the rosuvastatin treated mice. Statistical analysis SPSS software (version 26, SPSS Inc., Chicago, USA) was used for statistical analysis. The data obtained were analyzed by one-way analysis of variance (ANOVA). Differences between groups were analyzed using the least significant difference (LSD) test. All values are expressed as mean ± standard deviation and *p<0.05 was considered statistically significant (*p<0.05, **p<0.01, ***p<0.001, ****p<0.000). Analyses were visualized using GraphPad Prism (version 9.5.0). RESULTS It was observed that the Johnsen score of the TT group decreased statistically significantly compared to the sham group (p < 0.000), (Fig. 1 A, 1 B, 1 C, 1 D, 1 E). Acute rosuvastatin and prophylactic rosuvastatin groups increased statistically significantly compared to the TT group (p < 0.001), (Fig. 1 E). These results show that TT increases tissue damage and impairs spermatogenesis, as reported in the literature. According to Cosentino scoring, the seminiferous tubules in the TT group did not contain all spermatogenic cells, and tissue irregularity was observed. When the TT group was compared with the sham group, the Cosentino score was statistically significantly higher (p < 0.000), (Fig. 1 F). When the treatment groups were analyzed, there was a non-statistically significant decrease in the Cosentino score in both the acute and prophylactic rosuvastatin groups compared to the TT group (Fig. 1 F). In our study, it was observed that there was no statistically significant difference in sperm concentration between the TT and sham groups (data not shown). There was also no statistically significant difference between the treatment and TT groups. (data not shown). According to the sperm motility analyses of our study, when comparing the sham and TT groups, it was observed that A motility decreased statistically significantly in the injury group compared to the sham group (p < 0.000), (Fig. 2 B). There was also statistical significance between the acute rosuvastatin and TT groups (p < 0.05), (Fig. 2 B). A statistically significant difference was observed between TT and acute rosuvastatin groups for B motility (p < 0.05), (Fig. 2 C). No statistically significant difference was observed between groups for C motility (Fig. 2 D). When the percentage of A, B, and C motility was considered as total motility, a statistically significant difference was observed between the sham and TT groups (p < 0.000), (Fig. 2 A). When the TT group was compared with the acute and prophylactic rosuvastatin groups, both the acute and prophylactic groups were statistically significant compared with the TT group (p < 0.01), (Fig. 2 A). When comparing the groups for D motility, a statistically significant difference was observed between the sham and TT groups (p < 0.000), (Fig. 2 E). When comparing the acute and prophylactic rosuvastatin groups with TT, a statistically significant difference was observed between the acute and prophylactic rosuvastatin groups and the TT group (p < 0.01), (Fig. 2 E). According to the morphological analysis of our study, there was a statistically significant difference between the sham and TT groups in the percentage of sperm with head abnormalities (p < 0.01), (Fig. 3 A, 3 B). When comparing the acute rosuvastatin group with the TT group, there was no statistical difference in the percentage of head abnormalities. When the TT group was compared with the prophylactic rosuvastatin group, a statistically significant change in head abnormality was observed (p < 0.05), (Fig. 3 A). When the groups were compared for acrosome anomaly, a statistically significant difference was observed between the TT group and both the acute and prophylactic rosuvastatin groups (p < 0.05), (Fig. 3 C, 3 D). There was no statistically significant difference between groups about neck anomaly (Fig. 3 E, 3 F). When all groups were compared for the percentage of sperm with normal morphology, a statistically significant difference was observed between the sham group and the TT group (p < 0.000), (Fig. 3 G, 3 H). When the groups were compared in terms of total antioxidant status (TAS), a statistically significant difference was observed between the sham and TT groups (p < 0.01), (Fig. 4 A). Statistical significance was observed when comparing the acute and prophylactic rosuvastatin groups with the TT group (p < 0.001 and p < 0.000), (Fig. 4 A). When the groups were compared in terms of total oxidant status (TOS), a statistically significant difference was observed between the sham and TT groups (p < 0.000), (Fig. 4 B). A statistically significant difference was observed between the TT group and the acute- and prophylactic rosuvastatin groups (p < 0.000), (Fig. 4 B). When the experimental groups were compared in terms of oxidative stress index (OSI), a statistically significant difference was observed between the sham group and the TT group (p < 0.000), (Fig. 4 C). When the acute rosuvastatin and prophylactic rosuvastatin groups were compared with the TT group, a statistically significant difference was observed (p < 0.000), (Fig. 4 C). In this study, when the sham and TT groups were compared for pAKT expression, a statistically significant difference was observed between the two groups (p < 0.000), (Fig. 5 A). In the acute and prophylactic rosuvastatin groups, pAKT expression was significantly increased compared to the TT group (p < 0.001), (Fig. 5 A). When the expression of Bcl-xL, an anti-apoptotic gene, was compared between groups, a statistically significant difference was observed between the sham and TT groups (p < 0.000), (Fig. 5 B). Acute and prophylactic use of rosuvastatin caused an increase in Bcl-xL expression along with an increase in pAKT expression (p < 0.05), (Fig. 5 B). In this study, when groups were compared for pERK1 expression, a statistically significant difference was observed between sham and TT groups (p < 0.05), (Fig. 5 C). When the acute and prophylactic rosuvastatin groups were compared with the TT group, a statistically significant difference was observed between both the prophylactic- and acute rosuvastatin groups and the TT group (p < 0.000), (Fig. 5 C). pERK2 expression showed a statistically significant difference between the sham and TT groups (p < 0.05), (Fig. 5 D). A statistically significant decrease was observed when the acute and prophylactic rosuvastatin groups were compared with the TT group (p < 0.000), (Fig. 5 D). In this study, when the groups were compared in terms of pJNK1 expression, a statistically significant difference was observed between the sham and TT groups (p < 0.000), (Fig. 5 E). When the acute and prophylactic rosuvastatin groups were compared with the TT group, a statistically significant difference was observed (p < 0.001), (Fig. 5 E). When groups were compared for pJNK2 expression, a statistically significant difference was observed between sham and TT groups (p < 0.000), (Fig. 5 F). When comparing acute- and prophylactic rosuvastatin with the TT group, statistically significant differences were observed (p < 0.001 and p < 0.01), (Fig. 5 F). DISCUSSION Based on previous findings showing that HMG-CoA reductase inhibitor rosuvastatin reduces ishemic injury following cerebral ischemia by regulating signaling pathways such as survival kinases, we examined the role of rosuvastatin in tissue protection, functionality and associated signaling pathways after TT [ 15 ]. Torsion at 720° for 2 hours is sufficient to disrupt the seminiferous epithelium and germ cell-specific degeneration [ 31 , 44 ]. In our study, the Johnsen score was used to monitor the effect of I/R injury on spermatogenesis [ 37 ]. According to the Johnsen score result of our study, it was observed that prophylactic- and acute statin administration alleviated the damage caused by TT, which were associated with increased activation of AKT signaling and antiapoptotic Bcl-xL. To assess the effect of torsion-induced I/R injury on testicular tissue pathology through necrosis, the Cosentino scoring system was used to score the irregularity, vacuolation, and coagulation in the seminiferous tubules between 1 and 4 [ 40 , 45 , 46 ]. In our study, it was observed that statins suppressed hypoxia-induced necrosis and ROS increase caused by TT damage in testicular tissue by exerting antioxidant properties. Consistent with the literature, statin treatment significantly improved histological criteria in the I/R injured groups [ 13 ]. Both prophylactic and acute administration of rosuvastatin reduced testicular dysfunction and histopathological deterioration in injured testicular tissue. As in previous studies, this study used epididymal sperm samples [ 4 ]. Spermatogenesis in mice takes approximately 30–35 days [ 47 ]. Therefore, it is thought that the sperm count in the epididymis may not have been much affected by TT damage. Although there was no statistically significant difference between the TT group and the treatment groups in this study, it is thought that statins may protect epididymal sperm concentration through antioxidant effects in the long term after torsion. Oxidative stress impairs sperm motility and capacitation; plasma membrane lipids and flagellum proteins are oxidized and unable to perform their functions [ 48 ]. Increased levels of S-glutathionylation and protein oxidation and decreased percentage of motile spermatozoa with oxidative stress suggest that key proteins associated with the motility mechanism may be affected by oxidative stress-related protein modifications [ 9 ]. These results suggest that increased lipid peroxidation due to oxidative stress is suppressed by rosuvastatin treatment and that statins may have an antioxidant effect in the epididymis. This study observed a statistically significant decrease in the percentage of morphological abnormalities due to TT injury with statin treatment. The decrease in head and acrosome abnormalities in the TT group with statin treatment suggests that statins exert antioxidant effects via plasmalogens in the sperm membrane. In agreement with previous studies, our results suggest that the ischemia/reperfusion process may damage epididymal stored spermatozoa and that prophylactic and acute statin use may improve sperm motility and morphology. Total antioxidant status (TAS) and total oxidant status (TOS) provide a comprehensive measure of oxidative stress [ 41 , 42 , 49 ]. This study investigated the effects of statins on total oxidant and antioxidant levels in testicular torsion. These findings confirm that statins affect TAS (Total Antioxidant Status), TOS (Total Oxidant Status), and OSI (Oxidative Stress Index) levels in testicular torsion. These results highlight the potential effects of statins in reducing oxidative stress, in line with the literature. Studies have shown that the suppressing effect of statins on ischemia is likely to be related to PI3K/AKT pathways [ 50 ]. In testicular tissue, increased AKT phosphorylation has been shown to prevent oxidative damage and apoptosis caused by testicular I/R injury and to support the survival of testicular spermatogonium [ 51 ]. AKT supports cell survival by protecting mitochondrial integrity, inhibiting Cytochrome C release and changes in mitochondrial membrane potential in a caspase-independent way [ 52 ]. It is thought that rosuvastatin, a potent statin, promotes glycolysis by increasing pAKT expression and preserves mitochondrial membrane potential [ 53 ]. Bcl-xL also supports the survival of growth factor-deprived cells by maintaining mitochondrial integrity and function [ 53 ]. pAKT-expressing cells are more metabolically active and depend on external energy sources to maintain high levels of metabolism. On the other hand, cells expressing Bcl-xL can survive despite limited external sources of nutrients [ 53 – 55 ]. Rosuvastatin can affect the energy metabolism of cells, reducing metabolic activity and helping them to develop metabolic adaptations to survive. These findings suggest that rosuvastatin may enable cells to survive in a low-energy state. It is known that there are important cross-talks between the Raf-MEK-ERK signaling pathway and the Phosphoinositol 3-Kinase (PI3K) and AKT signaling pathways at the level of Raf1 [ 56 , 57 ]. In our study, decreased ERK1/2 expression is thought to be caused by inhibition of Ras by pAKT, whose expression is increased to high levels by statins. Activated AKT inhibits apoptosis by maintaining mitochondrial integrity. In addition, it is thought that statins increase pAKT expression at high levels and may cause inhibition of pERK1/2 together with inhibition of Ras in the ERK1/2 pathway. These findings are in line with previous studies [ 58 ]. Extracellular stressors or pro-inflammatory cytokines stimulate the protein kinase/c-Jun N-terminal kinase (SAP/JNK) signaling pathway [ 59 – 61 ]. The JNK pathway mediates an immediate early response to a wide range of cellular abnormalities or stressors [ 62 ]. These findings suggest that increased JNK activation with statin treatment may play an important role in the regulation of intracellular ROS levels [ 63 ]. The JNK signaling pathway also plays an important role in controlling apoptosis, and statin treatment has been observed to increase pJNK1/2 expression and activation of this pathway. These findings suggest that statin treatment induces protective mechanisms and supports cell survival by exerting different effects on intracellular signaling pathways. In this study, rosuvastatin was used either preventively (prophylactically, for 15 days before the torsion) or therapeutically (acutely, immediately after the torsion). The results of this study suggest that the protective effects of both methods are still important. However, more studies are needed to understand the whole situation. CONCLUSION In conclusion, this study shows that prophylactic- and acute statin administration has preventive and curative effects against I/R damage caused by testicular torsion. In our study, when the damage caused by testicular torsion was examined histopathologically, acute and prophylactic statin use was observed to have protective effects on testicular tissue and spermatogenesis. In testicular torsion, statin treatment activates the PI3K/AKT pathway and is thought to increase pAKT phosphorylation, which plays a role in regulating cell survival, growth, and division. Rosuvastatin may also focus on a different mechanism that promotes survival in an energy-limited environment rather than mitochondrial membrane integrity via Bcl-xL. In addition, increased pAKT expression by rosuvastatin is thought to reduce the expression of pERK1/2. The stress-activated JNK pathway also plays an important role in the control of apoptosis, and statin treatment is thought to increase pJNK1/2 expression and activate this pathway. These findings suggest that statin treatment induces protective mechanisms and promotes cell survival by different effects on intracellular signaling pathways. Further studies are needed to better understand the potential protective effects of statins and to use them more effectively. Declarations Funding Statement: The Istanbul Medipol University BAP Commission funded this thesis (2022/32) and supported by the Turkish Academy of Sciences (TUBA). Author Contributions: B.Y. and E.K. conceived and designed the study. B.Y., O.B., N.S., U.V.U. and M.C.B. performed the experiments and collected data. Z.B., N.A., and I.K. contributed to data analysis and interpretation. B.Y. drafted the manuscript. E.K. supervised the study and critically revised the manuscript. All authors read and approved the final version of the manuscript. Conflict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Data Availability Statements: The data underlying this article are available in the article and in its online supplementary material. References Yecies, T., Bandari, J., Schneck, F. & Cannon, G. Direction of Rotation in Testicular Torsion and Identification of Predictors of Testicular Salvage. Urology 114 , 163–166. https://doi.org/10.1016/j.urology.2017.11.034 (2018). Korkes, F. et al. Testicular torsion and weather conditions: analysis of 21,289 cases in Brazil, Int. Braz J Urol Off. J. Braz. Soc. Urol. 38 222–228; discussion 228–229. (2012). https://doi.org/10.1590/s1677-55382012000200010 Gevrek, F., Biçer, Ç., Kara, M. & Erdemir, F. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6611379","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":465478999,"identity":"de8d7c19-a9f1-4be1-81e9-85a7fb42baca","order_by":0,"name":"Berna Yildirim","email":"","orcid":"","institution":"Istanbul Atlas University","correspondingAuthor":false,"prefix":"","firstName":"Berna","middleName":"","lastName":"Yildirim","suffix":""},{"id":465479000,"identity":"da121dbd-f5e5-48ec-9c6f-e107b54738e7","order_by":1,"name":"Oguzhan Baygul","email":"","orcid":"","institution":"Istanbul Medeniyet University","correspondingAuthor":false,"prefix":"","firstName":"Oguzhan","middleName":"","lastName":"Baygul","suffix":""},{"id":465479002,"identity":"51027921-f644-44ef-9c11-7fbe2cc44cdf","order_by":2,"name":"Nursena Sengun","email":"","orcid":"","institution":"Istanbul Medeniyet University","correspondingAuthor":false,"prefix":"","firstName":"Nursena","middleName":"","lastName":"Sengun","suffix":""},{"id":465479004,"identity":"78021199-4509-4ee4-ac02-ea63745ef588","order_by":3,"name":"Unsal Veli Ustundag","email":"","orcid":"","institution":"Istanbul Medipol University","correspondingAuthor":false,"prefix":"","firstName":"Unsal","middleName":"Veli","lastName":"Ustundag","suffix":""},{"id":465479006,"identity":"2a7a95a9-ed55-4113-b915-326f27c32a61","order_by":4,"name":"Nilay Ateş","email":"","orcid":"","institution":"Istanbul Medeniyet University","correspondingAuthor":false,"prefix":"","firstName":"Nilay","middleName":"","lastName":"Ateş","suffix":""},{"id":465479007,"identity":"34b62d97-5acf-45b0-9d21-781a48b7bc91","order_by":5,"name":"Zeynep Balcikanli","email":"","orcid":"","institution":"Demiroglu Bilim University","correspondingAuthor":false,"prefix":"","firstName":"Zeynep","middleName":"","lastName":"Balcikanli","suffix":""},{"id":465479008,"identity":"74343391-0cf2-4c9c-8871-19edd03c02a3","order_by":6,"name":"Mustafa Caglar Beker","email":"","orcid":"","institution":"Istanbul Medeniyet University","correspondingAuthor":false,"prefix":"","firstName":"Mustafa","middleName":"Caglar","lastName":"Beker","suffix":""},{"id":465479009,"identity":"7d83f7ac-08c5-4c10-9ebe-ad567d9e1e2c","order_by":7,"name":"Ilknur Keskin","email":"","orcid":"","institution":"Istanbul Medipol University","correspondingAuthor":false,"prefix":"","firstName":"Ilknur","middleName":"","lastName":"Keskin","suffix":""},{"id":465479010,"identity":"8bbf3611-fa61-45e2-a3c0-b1bc204f03f9","order_by":8,"name":"Ertugrul Kilic","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxUlEQVRIiWNgGAWjYBACCWbmhgMMPGxyDAw8RGthBGqR4TMmQQsDYwMDg41cYgPRWiTbGRsP/MgxS99w/OzBBx8Y7OR0GwhokQY67GDPmbTcDWfykg1nMCQbmx0goEUOqOUwY8+x3A0HcsykeRgOJG4jTsu//+kG598QqQXksMPAQE4wuEGsLZLNIL/wsBnOvPHG2HCGARF+kTh/+PCHHzxs8nzncwwffKiwkyOoBQ4UwCoNiFUOAvINpKgeBaNgFIyCEQUAVwRBE/LC5sQAAAAASUVORK5CYII=","orcid":"","institution":"Istanbul Medeniyet University","correspondingAuthor":true,"prefix":"","firstName":"Ertugrul","middleName":"","lastName":"Kilic","suffix":""}],"badges":[],"createdAt":"2025-05-07 11:08:22","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6611379/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6611379/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-29283-w","type":"published","date":"2025-11-22T15:57:47+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":83821570,"identity":"462276a3-e1f3-4a44-837d-0aac8246cc15","added_by":"auto","created_at":"2025-06-03 09:07:39","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":425679,"visible":true,"origin":"","legend":"\u003cp\u003eHistopathological effects of rosuvastatin. \u003cstrong\u003eA-D: \u003c/strong\u003eCross-sections of the seminiferous tubules of the groups \u003cstrong\u003eA: \u003c/strong\u003eSham experimental group, \u003cstrong\u003eB:\u003c/strong\u003e Control (TT injury) group. \u003cstrong\u003eC:\u003c/strong\u003e Acute rosuvastatin experimental group, \u003cstrong\u003eD:\u003c/strong\u003e Prophylaxis rosuvastatin experimental group. Light microscopy (Nikon), 40X, Bar: 50 μm. H-E staining. \u003cstrong\u003eE:\u003c/strong\u003e Johnsen scoring between groups. \u003cstrong\u003eF:\u003c/strong\u003e Cosentino scoring between groups.\u003c/p\u003e","description":"","filename":"image1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6611379/v1/b540e3cfaa3cd2e434fa97e6.jpeg"},{"id":83821573,"identity":"d8791cd0-1822-41ca-a3ad-ceaa286e4e7d","added_by":"auto","created_at":"2025-06-03 09:07:39","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":189447,"visible":true,"origin":"","legend":"\u003cp\u003eDetermination of sperm motility \u003cstrong\u003eA:\u003c/strong\u003e Total Motility, the sum of A, B, and C motility, \u003cstrong\u003eB:\u003c/strong\u003e A Motility for progressively motile sperm, \u003cstrong\u003eC:\u003c/strong\u003e B Motility for slow-moving sperm, \u003cstrong\u003eD:\u003c/strong\u003eC Motility for motile spermatozoa that cannot move forward, \u003cstrong\u003eE:\u003c/strong\u003e D Motility for immotile spermatozoa. \u003cstrong\u003eA-E:\u003c/strong\u003e Results are expressed as percentage (%). Data are presented as mean ± standard deviation. p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001, ****p\u0026lt;0.000 indicates statistical significance compared to the TT injury group.\u003c/p\u003e","description":"","filename":"image2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6611379/v1/7c0c0ca717364fc962f570aa.jpeg"},{"id":83822369,"identity":"45fcead5-0fe5-47c3-b023-de05e64fd89b","added_by":"auto","created_at":"2025-06-03 09:15:39","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":426517,"visible":true,"origin":"","legend":"\u003cp\u003eDetermination of sperm morphology. \u003cstrong\u003eA:\u003c/strong\u003e Head abnormality analyses between groups. \u003cstrong\u003eB:\u003c/strong\u003e Head abnormalities observed in sperm smear preparations\u003cstrong\u003e. C:\u003c/strong\u003e Acrosome abnormality analyses between groups. \u003cstrong\u003eD:\u003c/strong\u003e Acrosome abnormalities observed in sperm smear preparations. \u003cstrong\u003eE:\u003c/strong\u003eNeck abnormality analyses between groups. \u003cstrong\u003eF:\u003c/strong\u003e Neck abnormalities observed in sperm smear preparations. \u003cstrong\u003eG:\u003c/strong\u003e Sperm with normal morphology analyses between groups. \u003cstrong\u003eH:\u003c/strong\u003e Sperm with normal morphology observed in sperm smear preparations. Light microscope (Nikon). Magnification: 100X, Bar: 10 μm. Diff-3 Staining. Data are presented as mean ± standard deviation. Results are expressed as percentage (%). p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001, ****p\u0026lt;0.000 indicates statistical significance compared to the TT injury group.\u003c/p\u003e","description":"","filename":"image3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6611379/v1/f8999ba43a41cbdeb21828f9.jpg"},{"id":83821568,"identity":"d51e90b5-623e-4a36-9158-36cdcfd9e17b","added_by":"auto","created_at":"2025-06-03 09:07:39","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":58463,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of rosuvastatin on TAS-TOS and various protein amounts. \u003cstrong\u003eA:\u003c/strong\u003e Total Antioxidant Status (TAS), the results are expressed as mmol Trolox eq. /L. \u003cstrong\u003eB:\u003c/strong\u003e Total Oxidant Status (TOS), the results are expressed as μmol H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e eq. /L. \u003cstrong\u003eC:\u003c/strong\u003e Oxidative Stress Index (OSI) = TOS (μmol H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e eq./L) /TAS (mmol Trolox eq./L). Data are presented as mean ± standard deviation. p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001, ****p\u0026lt;0.000 indicates statistical significance compared with the control group.\u003c/p\u003e","description":"","filename":"image4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6611379/v1/92455ba9a76bc9fa26430254.jpeg"},{"id":83821571,"identity":"7d1a6541-96d9-4add-911f-5b14a5171952","added_by":"auto","created_at":"2025-06-03 09:07:39","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":111693,"visible":true,"origin":"","legend":"\u003cp\u003eProtein levels between groups. \u003cstrong\u003eA:\u003c/strong\u003e p-AKT, \u003cstrong\u003eB:\u003c/strong\u003eBcl-xL, \u003cstrong\u003eC:\u003c/strong\u003e p-ERK1, \u003cstrong\u003eD:\u003c/strong\u003e p-ERK2, \u003cstrong\u003eE:\u003c/strong\u003e p-JNK, \u003cstrong\u003eF:\u003c/strong\u003e p-JNK2. Data are expressed as mean ± standard deviation. p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001, ****p\u0026lt;0.000 indicates statistical significance compared to the TT injury group.\u003c/p\u003e","description":"","filename":"image5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6611379/v1/b8e598ed8ac6b39b9387bb32.jpg"},{"id":96650282,"identity":"c3738fe1-a4db-4d29-a08d-77419083c953","added_by":"auto","created_at":"2025-11-24 16:10:46","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2002290,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6611379/v1/56b6053f-e782-4420-9ef9-24a1e5fde6a8.pdf"},{"id":83821574,"identity":"e7231f75-be10-4adb-abb2-01b8a48e08f8","added_by":"auto","created_at":"2025-06-03 09:07:41","extension":"pptx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":20682951,"visible":true,"origin":"","legend":"","description":"","filename":"bernasupplementarydata.pptx","url":"https://assets-eu.researchsquare.com/files/rs-6611379/v1/3f9eb4fa06d595eac939bd93.pptx"}],"financialInterests":"No competing interests reported.","formattedTitle":"HMG-CoA Reductase Inhibition Protects Testis and Sperm Quality from Testicular Ishemia and Reperfusion via Activation of Antioxidant Status and AKT Signaling","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eTesticular torsion (TT) is an acute urological condition caused by rotation of the spermatic cord or improper fixation of the \u003cem\u003eTunica vaginalis\u003c/em\u003e, which reduces blood flow to the testicular vessels and carries a risk of tissue degeneration and infertility [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. After testicular torsion, irreversible tissue degeneration is observed within a period of four to eight hours with detrimental effects on spermatogenesis [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Studies have shown that the spermatogonia and the primary spermatocytes are the first cells to be damaged by ischemia [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Increased reactive oxygen species (ROS) are generated particularly after reperfusion and may increase tissue damage further [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. High levels of ROS may also affect sperm- motility, capacitation, the acrosome reaction, quality, and cause serious damage in sperm DNA. Oxidative stress or TT has been shown to cause poor sperm quality, reduce sperm concentration and mobility resulted also in the impaired sperm capacitation and viability in mice [\u003cspan additionalcitationids=\"CR6 CR7\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOxidative stress in spermatozoa reduces also mitochondrial activity. It affects all cellular components [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. It is thought that TT not only blocks blood flow to the testicles but also to the epididymis [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Oxidative stress and hypoxic conditions induced by ischemia- reperfusion (I/R) injury may affect sperm cells both during the production phase in the testis and during maturation in the epididymis through the contents of the epididymal lumen [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Therefore, the epididymis has an antioxidant enzyme capacity to prevent oxidative stress and damage to sperm structures [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. On the other hand, oxidative stress increases ion permeability, inhibits enzymatic and receptor movement, disrupts the integrity of the sperm membrane, and leads to impaired motility [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. This disrupts sperm- oocyte interaction [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Previous studies have shown that tubulin is highly oxidized when human spermatozoa are incubated under conditions of oxidative stress [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMany protective agents have been studied in testicular torsion over the years, but few of them had low side effects [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Studies suggest that treatment with immunosuppressive, anti-apoptotic, and anti-inflammatory agents may preserve tissue in TT to prevent testicular dysfunction [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In an experimental model of testicular TT injury, treatment with various antioxidants has been shown to reduce lipid peroxidation, oxidative stress, and germ cell apoptosis [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e evidences suggest that statins with the ability to inhibit HMG-CoA reductase have direct antioxidant properties [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Experimental studies investigating the effect of statins after cerebral ischemic and reperfusion have shown that statins have multiple effects such as anti-inflammatory, antioxidant, and angiogenesis [\u003cspan additionalcitationids=\"CR16 CR17\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThere is evidence to suggest that statin therapy may activate the phosphatidylinositol 3-kinase (PI3K) protein kinase AKT pathway [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. This activation is involved in the regulation of cell survival, growth, and proliferation [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The AKT signaling pathway regulates cell growth and survival by inhibiting pro-apoptotic proteins or signals [\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. When pAKT is active, it can have a therapeutic effect on ischemic injury by reducing oxidative stress, inflammation, and cell apoptosis [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Rosuvastatin is among the most potent statin and considered safe for a toxic mechanism such as HMG-CoA reductase inhibition [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Rosuvastatin can be metabolized in the body and this potent effect is due to the presence of active metabolites [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. No toxicity has been observed in mice at various doses of rosuvastatin [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this sense, here we studied the role of HMG-CoA reductase inhibitor rosuvastatin in tissue protection and functionality after TT. We hypothesized that increased AKT phosphorylation caused by rosuvastatin treatment is an important factor in the drug's multiple protective effects. Mice were treated to two hours of 720-degree torsion and then 24 hours of detorsion damage with the aim of testing this theory. Here, we demonstrated that rosuvastatin, both acutely and prophylactically, reduced the damaging effects of testicular torsion injury on sperm and testicular tissue, increased the levels of phosphorylated AKT, Bcl-xL, phosphorylated JNK1/2 and decreased the levels of phosphorylated ERK1/2 proteins. In conclusion, the data provided here points to the possibility that rosuvastatin's various preventive characteristics reduce the negative effects of testicular torsion damage via molecular processes.\u003c/p\u003e"},{"header":"MATERIALS AND METHOD","content":"\u003cp\u003e\u003cstrong\u003eEthical approval and animal care\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll procedures involving animals were conducted in accordance with the National Institutes of Health (NIH) guidelines for the care and use of laboratory animals. Ethical approval for this study was obtained from the Istanbul Medipol University Animal Experiments Local Ethics Committee (IMU HADYEK) (25/10/2021, E-38828770-772.02-5440), and permission was granted by the local government authorities. Animals were housed and maintained at the Istanbul Medipol University Medical Research Center (MEDITAM) under standard laboratory conditions with a 12-hour light/dark cycle (lights on daily at 7:00 a.m.). Male adult Balb/C mice, aged 8\u0026ndash;12 weeks and weighing 20\u0026ndash;25 g, were randomly assigned to four groups (n = 7 per group): sham (i), control (torsion/detorsion injury) (ii), acute rosuvastatin (Crestor, AstraZeneca; 20 mg/kg) administered immediately after detorsion (iii), and prophylactic rosuvastatin (Crestor, AstraZeneca; 20 mg/kg) (iv) [28].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe Surgical Procedures\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSurgical procedures for torsion induction were based on previous studies and included anesthesia, torsion for 2 hours, detorsion, and euthanasia after 24 hours [29,30]. Anesthesia was administered intraperitoneally using ketamine (80\u0026ndash;100 mg/kg) and xylazine (8\u0026ndash;10 mg/kg). Testicular ischemia was induced by rotating the right testis 720\u0026deg; clockwise and maintaining this position for 2 hours [31,32]. After ischemia, the testis was detorsioned by rotating it 720\u0026deg; counterclockwise, repositioned into the scrotum, and the incision was sutured [33,34]. Mice were then returned to their cages and allowed to recover for 24 hours to permit reperfusion.\u003c/p\u003e\n\u003cp\u003eAt the end of the experiment, 24 hours after detorsion, all animals were euthanized under deep anesthesia by administering an overdose of ketamine (50 mg/kg) and xylazine (10 mg/kg). All anesthesia and euthanasia procedures were conducted in accordance with the American Veterinary Medical Association (AVMA) Guidelines for the Euthanasia of Animals (2020). No outdated or non-standard agents such as chloral hydrate, ether, or chloroform were used. Experimental procedures were reported in accordance with the ARRIVE guidelines (PLoS Biol 8(6), e1000412, 2010).\u003c/p\u003e\n\u003cp\u003eOne testis from each animal was placed in 10% neutral buffered formalin (NBF) for histopathological analysis, and the other was stored at \u0026ndash;80\u0026deg;C for Western blot analysis. All statin doses were based on previous studies [28], and rosuvastatin was dissolved in drinking water using an ultrasonic bath (Bandelin Sonorex RK 52 Ultrasonic Bath, Berlin, Germany).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSperm Analysis\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe epididymis of the torsion/detorsion testis was used to assess sperm function parameters. For this purpose, the epididymis was cut with small sharp scissors and placed in a Petri dish containing RPMI medium. The sample was placed in an incubator at 37\u0026deg;C for 10 minutes to facilitate semen drainage. Semen was collected from the epididymis and poured into a small Petri dish containing 5 ml RPMI 1640 (Sigma-Aldrich, Munich, Germany) [35]. Sperm concentration and motility analyses were evaluated using a Makler Counting Camera (Sefi Medical Instruments LTT, Haifa, Israel). For concentration, sperm were counted per hundred squares of each sample. Results are expressed in millions/ml. Spermatozoa were categorized into four groups for motility analysis: progressively motile (A motility), slow-moving (B motility), motile but unable to move forward (C motility), and immotile (D motility). The results are presented as percentages (%) [36].\u003c/p\u003e\n\u003cp\u003eTo assess sperm morphology, Diff-3 staining was performed on sperm samples. For this purpose, 10 \u0026micro;l of sperm sample was dropped onto a positively charged slide. After drying, staining was performed, and images were captured under a 100X light microscope using immersion oil. A total of 100 spermatozoa were analyzed from each mouse. Spermatozoa were graded as normal and abnormal according to Kruger\u0026apos;s strict criteria [37]. Abnormalities were classified as head, acrosome, neck, and tail abnormalities. The results were expressed as (%).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHistopathological Analyses\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe follow-up procedure for testicular tissue was based on previously published studies [12,38]. Testicular tissues removed after surgery and sacrifice and fixed in 10% neutral buffered formalin (NBF) were used for histopathological evaluation. Specimens were stained with hematoxylin and eosin (Bio-Optica Mayer\u0026apos;s Hematoxylin and Eosin Y Plus) according to the manufacturer\u0026apos;s protocol to examine general histological structures.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAccording to Johnsen\u0026apos;s quantitative approach, all cells involved in spermatogenesis in all experimental groups of the study were scored on a 10-point scale to assess spermatogenesis [39]. According to this scoring, 50 seminiferous tubule sections from each sample were scored from 1 to 10.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe Cosentino method was used to assess testicular damage and necrosis [40]. This method includes necrosis in the testicular tissue, organization of the germ cells, and 4 grades categorized according to hemorrhage. According to this scoring, 40 seminiferous tubules from each sample were graded from 1 to 4.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTotal Antioxidant Status / Total Oxidant Status\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTesticular tissues from the groups were homogenized in 0.9% NaCl. The supernatants were separated by centrifugation at 3000 rpm for 10 min. The parameters of oxidative stress, total antioxidant status / total oxidant status (TAS)/(TOS), were measured in the supernatants and the oxidative stress index (OSI) was determined.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTAS measurement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe antioxidant activity of the sample against the hydroxyl radical was determined by the method of Erel A. ABTS (2,2\u0026apos;-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid)) is converted to a radical by H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in an acidic medium and its antioxidants then neutralize the ABTS radicals. The absorbance of the products was determined using a BioTek Synergy HTX multimode reader (BioTek, Inc., USA) at 658 nm. \u0026nbsp;Results are expressed as mmol Trolox eq. /L [41].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTOS measurement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe method of Erel A. et al. was used to determine the total oxidant status. Briefly, this method oxidizes ferrous ions to ferric ions in the presence of various oxidative species under acidic conditions. The absorbance of the resulting-colored product was evaluated at 658 nm using the BioTek Synergy HTX multimode reader (BioTek, Inc., USA). The results are expressed as \u0026mu;mol H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e eq. /L [42].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOxidative Stress Index Measurement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOSI was defined as the ratio of TOS to TAS. Specifically, OSI = TOS (\u0026mu;mol H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e eq./L) /TAS (mmol Trolox eq./L).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern blot\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWestern blot analysis was performed to determine the levels of post-injury stress (pJNK1/2) and survival (pAKT, pERK 1/2) kinases and anti-apoptotic Bcl-xL protein in isolated testicular tissues. The western blotting was carried out as described previously [43]. Briefly, testis tissue samples were harvested from the mice after 24 hours detorsion. Tissue samples of the same group were pooled, homogenized, sonicated, and treated with protease inhibitor cocktail and phosphatase inhibitor cocktail. Total protein content was evaluated using Qubit 2.0 Fluorometer according to the manufacturer\u0026apos;s protocol (Invitrogen, Life Technologies Corporation, Carlsbad, CA, USA). Equal amounts of protein (20 \u0026micro;g) were size fractionated using any-kD Mini-Protean TGX gel electrophoresis and then transferred to a nitrocellulose membrane using the Trans-Blot TurboTransfer System (Bio-Rad, Life Sciences Research). Next, membranes were blocked in 5% nonfat milk in 50 mMol Tris-buffered saline containing 0.1% Tween (TBS-T; blocking solution) for 1 h at room temperature, were washed in 50 mMol TBS-T, and were incubated overnight with monoclonal rabbit anti-phosphorylated AKT (Thr308; 13038S; Cell Signaling), monoclonal rabbit anti-Bcl-xL (2764S; Cell Signaling), polyclonal rabbit anti-phosphorylated pERK1/2 (Thr202/Tyr204; 9101L; Cell Signaling), monoclonal rabbit anti- phosphorylated pJNK1/2 \u0026nbsp;(Thr183/Tyr185; 9255L; Cell Signaling) antibody (1:1000). The next day, membranes were washed with 50 mM TBS-T and were incubated with horseradish peroxidase-conjugated goat-anti 657 rabbit (31460; Thermo Scientific) antibody (1:2500) for 1 h at room temperature. Each blot was performed in 3 or more replicates. Protein loading was controlled with polyclonal rabbit anti-\u0026beta;-actin antibody (4967; Cell Signaling Technology). Blots were developed using Clarity Western ECL Substrate kit (Bio-Rad; Life Sciences Research) and visualized using the ChemiDoc MP System (Bio-Rad; Life Sciences Research). Blot intensities were analyzed densitometrically using ImageJ. Protein levels were normalized to the \u0026beta;-actin levels and were presented as percentage of the rosuvastatin treated mice.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSPSS software (version 26, SPSS Inc., Chicago, USA) was used for statistical analysis. The data obtained were analyzed by one-way analysis of variance (ANOVA). Differences between groups were analyzed using the least significant difference (LSD) test. All values are expressed as mean \u0026plusmn; standard deviation and *p\u0026lt;0.05 was considered statistically significant (*p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001, ****p\u0026lt;0.000). Analyses were visualized using GraphPad Prism (version 9.5.0).\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eIt was observed that the Johnsen score of the TT group decreased statistically significantly compared to the sham group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.000), (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA, \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB, \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eC, \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eD, \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eE). Acute rosuvastatin and prophylactic rosuvastatin groups increased statistically significantly compared to the TT group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eE). These results show that TT increases tissue damage and impairs spermatogenesis, as reported in the literature.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eAccording to Cosentino scoring, the seminiferous tubules in the TT group did not contain all spermatogenic cells, and tissue irregularity was observed. When the TT group was compared with the sham group, the Cosentino score was statistically significantly higher (p\u0026thinsp;\u0026lt;\u0026thinsp;0.000), (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eF). When the treatment groups were analyzed, there was a non-statistically significant decrease in the Cosentino score in both the acute and prophylactic rosuvastatin groups compared to the TT group (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eF).\u003c/p\u003e\n \u003cp\u003eIn our study, it was observed that there was no statistically significant difference in sperm concentration between the TT and sham groups (data not shown). There was also no statistically significant difference between the treatment and TT groups. (data not shown). According to the sperm motility analyses of our study, when comparing the sham and TT groups, it was observed that A motility decreased statistically significantly in the injury group compared to the sham group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.000), (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB). There was also statistical significance between the acute rosuvastatin and TT groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB). A statistically significant difference was observed between TT and acute rosuvastatin groups for B motility (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC). No statistically significant difference was observed between groups for C motility (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eD). When the percentage of A, B, and C motility was considered as total motility, a statistically significant difference was observed between the sham and TT groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.000), (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA). When the TT group was compared with the acute and prophylactic rosuvastatin groups, both the acute and prophylactic groups were statistically significant compared with the TT group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA). When comparing the groups for D motility, a statistically significant difference was observed between the sham and TT groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.000), (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eE). When comparing the acute and prophylactic rosuvastatin groups with TT, a statistically significant difference was observed between the acute and prophylactic rosuvastatin groups and the TT group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eE).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eAccording to the morphological analysis of our study, there was a statistically significant difference between the sham and TT groups in the percentage of sperm with head abnormalities (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA, \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB). When comparing the acute rosuvastatin group with the TT group, there was no statistical difference in the percentage of head abnormalities. When the TT group was compared with the prophylactic rosuvastatin group, a statistically significant change in head abnormality was observed (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA). When the groups were compared for acrosome anomaly, a statistically significant difference was observed between the TT group and both the acute and prophylactic rosuvastatin groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eC, \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eD). There was no statistically significant difference between groups about neck anomaly (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eE, \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eF). When all groups were compared for the percentage of sperm with normal morphology, a statistically significant difference was observed between the sham group and the TT group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.000), (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eG, \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eH).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eWhen the groups were compared in terms of total antioxidant status (TAS), a statistically significant difference was observed between the sham and TT groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA). Statistical significance was observed when comparing the acute and prophylactic rosuvastatin groups with the TT group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 and p\u0026thinsp;\u0026lt;\u0026thinsp;0.000), (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA). When the groups were compared in terms of total oxidant status (TOS), a statistically significant difference was observed between the sham and TT groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.000), (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB). A statistically significant difference was observed between the TT group and the acute- and prophylactic rosuvastatin groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.000), (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB). When the experimental groups were compared in terms of oxidative stress index (OSI), a statistically significant difference was observed between the sham group and the TT group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.000), (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eC). When the acute rosuvastatin and prophylactic rosuvastatin groups were compared with the TT group, a statistically significant difference was observed (p\u0026thinsp;\u0026lt;\u0026thinsp;0.000), (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eC).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eIn this study, when the sham and TT groups were compared for pAKT expression, a statistically significant difference was observed between the two groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.000), (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA). In the acute and prophylactic rosuvastatin groups, pAKT expression was significantly increased compared to the TT group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA). When the expression of Bcl-xL, an anti-apoptotic gene, was compared between groups, a statistically significant difference was observed between the sham and TT groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.000), (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB). Acute and prophylactic use of rosuvastatin caused an increase in Bcl-xL expression along with an increase in pAKT expression (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB). In this study, when groups were compared for pERK1 expression, a statistically significant difference was observed between sham and TT groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eC). When the acute and prophylactic rosuvastatin groups were compared with the TT group, a statistically significant difference was observed between both the prophylactic- and acute rosuvastatin groups and the TT group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.000), (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eC). pERK2 expression showed a statistically significant difference between the sham and TT groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eD). A statistically significant decrease was observed when the acute and prophylactic rosuvastatin groups were compared with the TT group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.000), (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eD).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eIn this study, when the groups were compared in terms of pJNK1 expression, a statistically significant difference was observed between the sham and TT groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.000), (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eE). When the acute and prophylactic rosuvastatin groups were compared with the TT group, a statistically significant difference was observed (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eE). When groups were compared for pJNK2 expression, a statistically significant difference was observed between sham and TT groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.000), (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eF). When comparing acute- and prophylactic rosuvastatin with the TT group, statistically significant differences were observed (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 and p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eF).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eBased on previous findings showing that HMG-CoA reductase inhibitor rosuvastatin reduces ishemic injury following cerebral ischemia by regulating signaling pathways such as survival kinases, we examined the role of rosuvastatin in tissue protection, functionality and associated signaling pathways after TT [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Torsion at 720\u0026deg; for 2 hours is sufficient to disrupt the seminiferous epithelium and germ cell-specific degeneration [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. In our study, the Johnsen score was used to monitor the effect of I/R injury on spermatogenesis [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. According to the Johnsen score result of our study, it was observed that prophylactic- and acute statin administration alleviated the damage caused by TT, which were associated with increased activation of AKT signaling and antiapoptotic Bcl-xL.\u003c/p\u003e \u003cp\u003eTo assess the effect of torsion-induced I/R injury on testicular tissue pathology through necrosis, the Cosentino scoring system was used to score the irregularity, vacuolation, and coagulation in the seminiferous tubules between 1 and 4 [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. In our study, it was observed that statins suppressed hypoxia-induced necrosis and ROS increase caused by TT damage in testicular tissue by exerting antioxidant properties. Consistent with the literature, statin treatment significantly improved histological criteria in the I/R injured groups [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Both prophylactic and acute administration of rosuvastatin reduced testicular dysfunction and histopathological deterioration in injured testicular tissue.\u003c/p\u003e \u003cp\u003eAs in previous studies, this study used epididymal sperm samples [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Spermatogenesis in mice takes approximately 30\u0026ndash;35 days [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Therefore, it is thought that the sperm count in the epididymis may not have been much affected by TT damage. Although there was no statistically significant difference between the TT group and the treatment groups in this study, it is thought that statins may protect epididymal sperm concentration through antioxidant effects in the long term after torsion.\u003c/p\u003e \u003cp\u003eOxidative stress impairs sperm motility and capacitation; plasma membrane lipids and flagellum proteins are oxidized and unable to perform their functions [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Increased levels of S-glutathionylation and protein oxidation and decreased percentage of motile spermatozoa with oxidative stress suggest that key proteins associated with the motility mechanism may be affected by oxidative stress-related protein modifications [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. These results suggest that increased lipid peroxidation due to oxidative stress is suppressed by rosuvastatin treatment and that statins may have an antioxidant effect in the epididymis.\u003c/p\u003e \u003cp\u003eThis study observed a statistically significant decrease in the percentage of morphological abnormalities due to TT injury with statin treatment. The decrease in head and acrosome abnormalities in the TT group with statin treatment suggests that statins exert antioxidant effects via plasmalogens in the sperm membrane. In agreement with previous studies, our results suggest that the ischemia/reperfusion process may damage epididymal stored spermatozoa and that prophylactic and acute statin use may improve sperm motility and morphology.\u003c/p\u003e \u003cp\u003eTotal antioxidant status (TAS) and total oxidant status (TOS) provide a comprehensive measure of oxidative stress [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. This study investigated the effects of statins on total oxidant and antioxidant levels in testicular torsion. These findings confirm that statins affect TAS (Total Antioxidant Status), TOS (Total Oxidant Status), and OSI (Oxidative Stress Index) levels in testicular torsion. These results highlight the potential effects of statins in reducing oxidative stress, in line with the literature.\u003c/p\u003e \u003cp\u003eStudies have shown that the suppressing effect of statins on ischemia is likely to be related to PI3K/AKT pathways [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. In testicular tissue, increased AKT phosphorylation has been shown to prevent oxidative damage and apoptosis caused by testicular I/R injury and to support the survival of testicular spermatogonium [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. AKT supports cell survival by protecting mitochondrial integrity, inhibiting Cytochrome C release and changes in mitochondrial membrane potential in a caspase-independent way [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. It is thought that rosuvastatin, a potent statin, promotes glycolysis by increasing pAKT expression and preserves mitochondrial membrane potential [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBcl-xL also supports the survival of growth factor-deprived cells by maintaining mitochondrial integrity and function [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. pAKT-expressing cells are more metabolically active and depend on external energy sources to maintain high levels of metabolism. On the other hand, cells expressing Bcl-xL can survive despite limited external sources of nutrients [\u003cspan additionalcitationids=\"CR54\" citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Rosuvastatin can affect the energy metabolism of cells, reducing metabolic activity and helping them to develop metabolic adaptations to survive. These findings suggest that rosuvastatin may enable cells to survive in a low-energy state.\u003c/p\u003e \u003cp\u003eIt is known that there are important cross-talks between the Raf-MEK-ERK signaling pathway and the Phosphoinositol 3-Kinase (PI3K) and AKT signaling pathways at the level of Raf1 [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. In our study, decreased ERK1/2 expression is thought to be caused by inhibition of Ras by pAKT, whose expression is increased to high levels by statins. Activated AKT inhibits apoptosis by maintaining mitochondrial integrity. In addition, it is thought that statins increase pAKT expression at high levels and may cause inhibition of pERK1/2 together with inhibition of Ras in the ERK1/2 pathway. These findings are in line with previous studies [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eExtracellular stressors or pro-inflammatory cytokines stimulate the protein kinase/c-Jun N-terminal kinase (SAP/JNK) signaling pathway [\u003cspan additionalcitationids=\"CR60\" citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. The JNK pathway mediates an immediate early response to a wide range of cellular abnormalities or stressors [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. These findings suggest that increased JNK activation with statin treatment may play an important role in the regulation of intracellular ROS levels [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. The JNK signaling pathway also plays an important role in controlling apoptosis, and statin treatment has been observed to increase pJNK1/2 expression and activation of this pathway.\u003c/p\u003e \u003cp\u003eThese findings suggest that statin treatment induces protective mechanisms and supports cell survival by exerting different effects on intracellular signaling pathways. In this study, rosuvastatin was used either preventively (prophylactically, for 15 days before the torsion) or therapeutically (acutely, immediately after the torsion). The results of this study suggest that the protective effects of both methods are still important. However, more studies are needed to understand the whole situation.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eIn conclusion, this study shows that prophylactic- and acute statin administration has preventive and curative effects against I/R damage caused by testicular torsion. In our study, when the damage caused by testicular torsion was examined histopathologically, acute and prophylactic statin use was observed to have protective effects on testicular tissue and spermatogenesis.\u003c/p\u003e \u003cp\u003eIn testicular torsion, statin treatment activates the PI3K/AKT pathway and is thought to increase pAKT phosphorylation, which plays a role in regulating cell survival, growth, and division. Rosuvastatin may also focus on a different mechanism that promotes survival in an energy-limited environment rather than mitochondrial membrane integrity via Bcl-xL. In addition, increased pAKT expression by rosuvastatin is thought to reduce the expression of pERK1/2. The stress-activated JNK pathway also plays an important role in the control of apoptosis, and statin treatment is thought to increase pJNK1/2 expression and activate this pathway. These findings suggest that statin treatment induces protective mechanisms and promotes cell survival by different effects on intracellular signaling pathways. Further studies are needed to better understand the potential protective effects of statins and to use them more effectively.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding Statement:\u003c/strong\u003e The Istanbul Medipol University BAP Commission funded this thesis (2022/32) and supported by the Turkish Academy of Sciences (TUBA).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u0026nbsp;\u003c/strong\u003eB.Y. and E.K. conceived and designed the study. B.Y., O.B., N.S., U.V.U. and M.C.B. performed the experiments and collected data. Z.B., N.A., and I.K. contributed to data analysis and interpretation. B.Y. drafted the manuscript. E.K. supervised the study and critically revised the manuscript. All authors read and approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest Statement:\u003c/strong\u003e The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statements:\u003c/strong\u003e The data underlying this article are available in the article and in its online supplementary material.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eYecies, T., Bandari, J., Schneck, F. \u0026amp; Cannon, G. 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Neurobiol.\u003c/em\u003e \u003cb\u003e54\u003c/b\u003e, 3492\u0026ndash;3505. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s12035-016-9926-y\u003c/span\u003e\u003cspan address=\"10.1007/s12035-016-9926-y\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2017).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"HMG Co-A reductase, testicular torsion, cell signaling, sperm motility","lastPublishedDoi":"10.21203/rs.3.rs-6611379/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6611379/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTesticular torsion (TT) is a urological emergency that results in ischemia/reperfusion (I/R) injury, leading to oxidative stress, cellular apoptosis, and impaired spermatogenesis. This study aimed to investigate the protective effects of the HMG-CoA reductase inhibitor rosuvastatin on TT-induced I/R injury and elucidate the underlying mechanisms. Male Balb/C mice (n\u0026thinsp;=\u0026thinsp;28) were subjected to 720\u0026deg; testicular torsion for two hours, followed by 24 hours of detorsion. Rosuvastatin was administered either acutely post-torsion or prophylactically prior to injury. Histopathological analysis, oxidative stress parameters, sperm motility and morphology assessments, as well as western blot analysis of survival-related signaling proteins (pAKT, pJNK, pERK1/2, and Bcl-xL), were performed. Rosuvastatin treatment significantly reduced tissue damage decreased oxidative stress (as evidenced by increased TAS and reduced TOS/OSI), and improved sperm motility and morphology. Both treatment regimens enhanced cell survival by increasing pAKT and Bcl-xL levels and decreasing pERK1/2 activation, while also activating stress-responsive JNK1/2 signaling. These findings suggest that rosuvastatin mitigates I/R-induced testicular damage through modulation of key intracellular signaling pathways, notably PI3K/AKT, and supports its therapeutic potential in acute testicular injuries and related degenerative conditions.\u003c/p\u003e","manuscriptTitle":"HMG-CoA Reductase Inhibition Protects Testis and Sperm Quality from Testicular Ishemia and Reperfusion via Activation of Antioxidant Status and AKT Signaling","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-03 09:07:34","doi":"10.21203/rs.3.rs-6611379/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-08-11T12:09:59+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-06T19:12:39+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"67933225166471207514377444437152249139","date":"2025-07-15T21:22:03+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-01T20:39:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"158000349674090242994485492939760297762","date":"2025-06-18T14:36:11+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-05-30T18:01:44+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-05-30T17:57:18+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-05-09T07:38:59+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-05-08T09:03:38+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-05-07T10:56:29+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d60feb19-403b-46b8-90bb-c229319436a2","owner":[],"postedDate":"June 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":49415681,"name":"Biological sciences/Cell biology"},{"id":49415682,"name":"Health sciences/Biomarkers"},{"id":49415683,"name":"Health sciences/Medical research"},{"id":49415684,"name":"Health sciences/Molecular medicine"},{"id":49415685,"name":"Health sciences/Pathogenesis"},{"id":49415686,"name":"Health sciences/Risk factors"},{"id":49415687,"name":"Health sciences/Signs and symptoms"},{"id":49415688,"name":"Health sciences/Urology"}],"tags":[],"updatedAt":"2025-11-24T16:05:36+00:00","versionOfRecord":{"articleIdentity":"rs-6611379","link":"https://doi.org/10.1038/s41598-025-29283-w","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-11-22 15:57:47","publishedOnDateReadable":"November 22nd, 2025"},"versionCreatedAt":"2025-06-03 09:07:34","video":"","vorDoi":"10.1038/s41598-025-29283-w","vorDoiUrl":"https://doi.org/10.1038/s41598-025-29283-w","workflowStages":[]},"version":"v1","identity":"rs-6611379","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6611379","identity":"rs-6611379","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}