HMG-CoA reductase inhibition preserves testicular function after torsion/detorsion by modulating oxidative stress and AKT signaling

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 investigated the protective effects of the HMG-CoA reductase inhibitor rosuvastatin on TT-induced I/R injury and explored the underlying mechanisms. Male Balb/C mice (n = 28) were subjected to 720° testicular torsion for two hours, followed by 24 h of detorsion. Rosuvastatin was administered either acutely (post-torsion) or prophylactically (prior to injury). Histopathological evaluation, assessment of oxidative stress parameters, sperm motility and morphology analysis, and Western blot examination of survival and stress related signaling proteins (pAKT, pJNK1/2, pERK1/2, and Bcl-xL) were performed. Rosuvastatin treatment significantly reduced tissue damage decreased oxidative stress (as indicated by increased TAS and reduced TOS/OSI), and improved sperm motility and morphology. Both acute and prophylactic treatment regimens enhanced cell survival by increasing pAKT and Bcl-xL levels, reducing pERK1/2 activation, and modulating stress responsive JNK1/2 signaling. These findings suggest that rosuvastatin mitigates I/R induced testicular damage primarily through modulation of key intracellular pathways, particularly PI3K/AKT, and support its therapeutic potential in acute testicular injuries and related degenerative conditions.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-29283-w.

Introduction

Testicular torsion (TT) is an acute urological emergency resulting from rotation of the spermatic cord or inadequate fixation of the tunica vaginalis, which compromises blood flow to the testis and increases the risk of tissue degeneration and infertility¹. Irreversible tissue injury may occur within four to eight hours of torsion onset, leading to marked impairment of spermatogenesis². Experimental studies have shown that spermatogonia and primary spermatocytes are the most vulnerable germ cells to ischemia³. During the reperfusion phase, the generation of reactive oxygen species (ROS) can further exacerbate tissue damage⁴. Elevated ROS levels impair sperm motility, capacitation, and the acrosome reaction, and contribute to DNA fragmentation and overall reduced sperm quality. Both oxidative stress and TT have been shown to compromise sperm quality by reducing sperm concentration, motility, and viability, as well as disrupting capacitation in mice^5–8.

Oxidative stress in spermatozoa also impairs mitochondrial function and affects all cellular components^9,10. It is thought that TT not only restricts blood flow to the testis but also disrupts circulation to the epididymis³. The oxidative and hypoxic environment produced by ischemia-reperfusion (I/R) injury can affect sperm development in the testis and subsequent maturation in the epididymis, in part by altering the epididymal luminal microenvironment³. Notably, the epididymis possesses robust antioxidant enzyme activity to counteract oxidative stress and protect spermatozoa⁷. However, persistent oxidative stress increases membrane ion permeability, impairs enzyme and receptor function, disrupts sperm membrane integrity, and ultimately reduces motility and fertilizing capacity^11–14. These changes also compromise sperm-oocyte interaction¹¹. Notably, tubulin oxidation has been reported in human spermatozoa exposed to oxidative stress conditions⁹.

Although a wide range of protective agents have been explored in experimental TT models, only a few have demonstrated efficacy with minimal adverse effects^15,16. Pharmacological interventions targeting immunosuppressive, anti-apoptotic, and anti-inflammatory pathways have shown potential in preserving testicular tissue and function following TT¹⁷. In particular, antioxidants have been reported to reduce lipid peroxidation, oxidative stress, and germ cell apoptosis after testicular torsion¹⁷. Statins, through inhibition of HMG-CoA reductase, exhibit not only lipid-lowering but also direct antioxidant and anti-inflammatory effects¹⁸. Experimental studies on cerebral ischemia-reperfusion injury have shown that statins exert multiple beneficial actions, including anti-inflammatory, antioxidant, and pro-angiogenic properties^19–22.

There is increasing evidence that statin therapy can activate the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway^23,24, which is crucial for cell survival, growth, and proliferation²⁵. Activation of AKT promotes cell survival by inhibiting pro-apoptotic proteins and suppressing apoptotic cascades^26–28. Phosphorylated AKT (pAKT) is particularly important in ischemic injury, where it reduces oxidative stress, inflammation, and apoptosis²⁹. Among available statins, rosuvastatin is one of the most potent and is considered safe based on its HMG-CoA reductase inhibition profile³⁰. It is metabolized in the body, and its strong efficacy is attributed to the presence of active metabolites³⁰. Its strong efficacy is partly attributed to its active metabolites, and no toxicity has been reported in mice at a range of doses³¹.

Given this background, we investigated whether rosuvastatin could protect testicular tissue and preserve function following testicular torsion. We hypothesized that rosuvastatin confers tissue protection, at least in part, through enhanced phosphorylation of AKT and modulation of survival pathways. To test this, mice were subjected to two hours of 720-degree torsion followed by 24 h of detorsion, with rosuvastatin administered either acutely or prophylactically. Our results demonstrate that rosuvastatin significantly attenuated testicular torsion-induced damage in both sperm and testicular tissue. Treatment was associated with increased expression of pAKT, Bcl-xL, and pJNK1/2, as well as reduced pERK1/2 levels. Collectively, these findings indicate that the protective effects of rosuvastatin are mediated by modulation of key molecular signaling pathways, most notably PI3K/AKT.

Materials and methods

Animal experimental design and procedures

Ethical approval and animal care

All procedures were conducted in accordance with the National Institutes of Health (NIH) guidelines for the care and use of laboratory animals. Ethical approval was obtained from the Istanbul Medipol University Animal Experiments Local Ethics Committee (IMU HADYEK; 25/10/2021, E-38828770-772.02.02-5440), and all necessary permissions were granted by local authorities. Animals were housed at the Istanbul Medipol University Medical Research Center (MEDITAM) under standard laboratory conditions with a 12-hour light/dark cycle (lights on at 7:00 a.m.).

Experimental design and groups

Adult male Balb/C mice (8–12 weeks old, 20–25 g; n = 28) were randomly assigned to four groups (n = 7 per group): (i) Sham, (ii) Torsion/detorsion (TT) control, (iii) Acute rosuvastatin (20 mg/kg; Crestor, AstraZeneca), administered as a single intragastric dose immediately after detorsion, and (iv) Prophylactic rosuvastatin (20 mg/kg/day), administered via oral gavage for 15 consecutive days prior to torsion induction³².

Drug administration

In the prophylactic group, rosuvastatin was given by oral gavage at 20 mg/kg/day for 15 days, with the last dose administered 24 h before surgery^33–35. In the acute group, a single 20 mg/kg dose was administered immediately after detorsion. Rosuvastatin tablets (Crestor, AstraZeneca) were freshly dissolved in distilled water prior to each administration, following established protocols for rodent studies using oral gavage with distilled water as vehicle^36,37.

Anesthesia and surgical procedures

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 h^38,39. Following the ischemic period, the testis was detorsioned by rotating it 720° counterclockwise, repositioned in the scrotum, and the incision was sutured^40,41. Mice were returned to their cages and allowed to recover for 24 h to permit reperfusion.

Euthanasia and tissue collection

At the end of the experiment (24 h after detorsion), all animals were euthanized under deep anesthesia via an overdose of ketamine (50 mg/kg) and xylazine (10 mg/kg). All anesthesia and euthanasia procedures complied with the American Veterinary Medical Association (AVMA) Guidelines for the Euthanasia of Animals (2020). No outdated or non-standard agents (e.g., chloral hydrate, ether, or chloroform) were used. All experimental protocols were reported in accordance with the ARRIVE guidelines (PLoS Biol 8(6), e1000412, 2010).

For tissue analysis, one testis from each animal was fixed in 10% neutral buffered formalin (NBF) for histopathological evaluation, while the contralateral testis was snap-frozen and stored at − 80 °C for Western blot analysis. All rosuvastatin doses were based on previous studies, and drug solutions were freshly prepared in distilled water using an ultrasonic bath (Bandelin Sonorex RK 52, Berlin, Germany)³².

Sperm analysis

The epididymis ipsilateral to the torsion/detorsion (T/D) testis was used for sperm function analysis. Following dissection with fine scissors, the epididymis was placed in a Petri dish containing RPMI medium and incubated at 37 °C for 10 min to facilitate semen release. The resulting semen was transferred to a new Petri dish containing 5 mL RPMI 1640 (Sigma-Aldrich, Munich, Germany)⁴².

Sperm concentration and motility

Sperm concentration and motility were assessed using a Makler Counting Chamber (Sefi Medical Instruments LTT, Haifa, Israel). For concentration, sperm cells were counted within one hundred squares of the chamber grid, and results were expressed as million sperm per milliliter. Motility was classified into four categories: progressively motile (A motility), slow progressive (B motility), motile but non-progressive (C motility), and immotile (D motility), and reported as percentages (%)⁴³.

Sperm morphology

To evaluate sperm morphology, Diff-3 staining was performed. Ten microliters of sperm suspension was placed on a positively charged glass slide and air-dried. After staining, slides were examined by light microscopy at 100× magnification under immersion oil. For each animal, 100 spermatozoa were evaluated. Morphology was classified according to Kruger’s strict criteria⁴⁴, with abnormalities further categorized as head, acrosome, neck, or tail defects. Results are reported as percentages.

Histopathological analyses

Histopathological evaluation of testicular tissue was performed according to established protocols^16,45. After euthanasia, testes were collected and fixed in 10% neutral buffered formalin (NBF) for histological analysis. Paraffin-embedded sections were stained with hematoxylin and eosin (Bio-Optica Mayer’s Hematoxylin and Eosin Y Plus) according to the manufacturer’s instructions to assess overall tissue architecture.

Assessment of spermatogenesis and tissue damage

Spermatogenesis was quantified using Johnsen’s scoring system, which assigns a score from 1 to 10 based on the presence and organization of germ cell layers⁴⁶. For each animal, fifty seminiferous tubule cross-sections were evaluated.

Testicular damage and necrosis were assessed using the Cosentino scoring method⁴⁰, which grades germ cell disorganization, necrosis, and intratubular hemorrhage on a scale from 1 to 4. Forty tubule cross-sections per sample were analyzed⁴⁷.

Total antioxidant status and total oxidant status (TAS/TOS) analysis

Testicular tissues from each group were homogenized in 0.9% NaCl solution. The homogenates were centrifuged at 3000 rpm for 10 min, and the supernatants were collected for biochemical analysis. Total Antioxidant Status (TAS) and Total Oxidant Status (TOS) were measured in these supernatants, and the Oxidative Stress Index (OSI) was subsequently calculated.

TAS measurement

TAS was measured according to the method described by Erel⁴⁸. This assay quantifies the antioxidant capacity of the samples against hydroxyl radicals. In brief, ABTS (2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) is converted into a radical cation by hydrogen peroxide (H₂O₂) under acidic conditions, and the degree of neutralization by sample antioxidants is measured. Absorbance was read at 658 nm using a BioTek Synergy HTX multimode reader (BioTek, Inc., USA). Results are expressed as mmol Trolox equivalents per liter (mmol Trolox eq./L).

TOS measurement

TOS was determined using the method established by Erel⁴⁹. In this assay, ferrous ions are oxidized to ferric ions in the presence of various oxidants under acidic conditions, forming a color complex measured at 658 nm using the same spectrophotometer. Results are given as µmol hydrogen peroxide equivalents per liter (µmol H₂O₂ eq./L).

Oxidative stress index calculation

The Oxidative Stress Index (OSI) was calculated as the ratio of TOS to TAS using the following formula:

Western blot analysis

Western blotting was performed to assess the expression levels of post-injury stress kinases (pJNK1/2), survival kinases (pAKT, pERK1/2, and their total forms), and the anti-apoptotic protein Bcl-xL in testicular tissues, following established protocols⁵⁰. Equal amounts of protein (20 µg per sample) were loaded per lane. For each signaling protein, both phosphorylated and total forms (e.g., pAKT and total AKT; pERK1/2 and total ERK1/2; pJNK1/2 and total JNK1/2) were probed to allow accurate normalization. At least three biological replicates (n = 3 per group) were included, and each sample was analyzed in technical duplicates or triplicates for reproducibility. Densitometric analysis was performed using ImageJ software.

For quantitative analysis, phosphorylated protein bands were first normalized to their respective total protein bands, then the resulting ratios were further normalized to β-actin as a loading control. For Bcl-xL, band intensities were normalized directly to β-actin. Results are presented as mean ± SD from at least three independent experiments (n = 3 per group) and expressed as a percentage relative to the rosuvastatin-treated group.

Briefly, testes were collected 24 h after detorsion. Tissues from each group were pooled, homogenized, sonicated, and treated with protease and phosphatase inhibitor cocktails. Total protein concentrations were measured using a Qubit 2.0 Fluorometer (Invitrogen, USA) according to the manufacturer’s protocol. Equal protein amounts (20 µg) were separated using Any kD Mini-PROTEAN TGX gel electrophoresis (Bio-Rad) and transferred onto nitrocellulose membranes with the Trans-Blot Turbo Transfer System (Bio-Rad).

Membranes were blocked with 5% non-fat milk in Tris-buffered saline containing 0.1% Tween-20 (TBS-T) for 1 h at room temperature, washed, and then incubated overnight at 4 °C with the following primary antibodies (1:1000): monoclonal rabbit anti-phospho-AKT (Thr308; 13038 S), monoclonal rabbit anti-total AKT (9272 S), monoclonal rabbit anti-Bcl-xL (2764 S), polyclonal rabbit anti-phospho-ERK1/2 (Thr202/Tyr204; 9101 L), polyclonal rabbit anti-total ERK1/2 (9102 S), monoclonal rabbit anti-phospho-JNK1/2 (Thr183/Tyr185; 9255 L), and monoclonal rabbit anti-total JNK1/2 (9252 S) (all from Cell Signaling Technology).

After primary incubation, membranes were washed and incubated for 1 h at room temperature with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (31460; Thermo Scientific) at 1:2500 dilution. Protein loading was verified using a polyclonal rabbit anti-β-actin antibody (4967; Cell Signaling Technology). Bands were visualized using the Clarity Western ECL Substrate Kit (Bio-Rad) and detected using a ChemiDoc MP Imaging System (Bio-Rad). Densitometric analysis was performed using ImageJ as described above.

Statistical analysis

Statistical analyses and data visualization were performed using SPSS software (version 26; SPSS Inc., Chicago, USA) and GraphPad Prism (version 9.5.0; GraphPad Software, USA). Data distribution was assessed using the Shapiro–Wilk test, which is suitable for small sample sizes. Homogeneity of variances was also tested prior to applying parametric analyses.

For datasets that did not meet the assumption of normality (p < 0.05), the non-parametric Kruskal–Wallis test was employed for group comparisons to ensure consistency across all variables. For data meeting both normality and homogeneity assumptions, one-way analysis of variance (ANOVA) was applied. Post hoc comparisons were performed using the least significant difference (LSD) test. All data are presented as mean ± standard deviation (SD). A p-value of less than 0.05 was considered statistically significant. The level of statistical significance is indicated as follows: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Results

A significant decrease in the Johnsen score was observed in the TT group compared to the sham group (p < 0.001; Fig. 1A–E), indicating marked testicular damage and impaired spermatogenesis. Both acute and prophylactic rosuvastatin treatments significantly increased the Johnsen score relative to the TT group (p < 0.001; Fig. 1E), reflecting partial restoration of spermatogenesis.

Fig. 1.

Histopathological effects of rosuvastatin on testicular tissue after torsion/detorsion (TT) injury. (A–D) Representative H&E-stained sections of seminiferous tubules from the sham-operated group (A), TT control group (B), acute rosuvastatin group (C), and prophylactic rosuvastatin group (D), all acquired at 40× magnification (scale bar = 50 μm). (E) Johnsen scores indicating spermatogenesis status across groups. (F) Cosentino scores assessing tissue damage and necrosis. Data are presented as mean ± SD (n = 7/group). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, not significant. Comparisons are made versus the TT group unless otherwise indicated.

Cosentino scoring revealed that seminiferous tubules in the TT group exhibited loss of spermatogenic cell populations and structural disorganization, with significantly higher scores than the sham group (p < 0.001; Fig. 1F). While Cosentino scores decreased in both rosuvastatin-treated groups, this reduction did not reach statistical significance compared to the TT group (Fig. 1F).

Sperm concentration analysis showed no statistically significant differences between the TT and sham groups, or between the TT group and either treatment group (Supplementary Fig. 1). In terms of motility, A-type (rapid progressive) motility was significantly reduced in the TT group compared to sham (p < 0.001; Fig. 2B). Acute rosuvastatin treatment significantly improved A motility relative to TT (p < 0.05; Fig. 2B). Similarly, B-type (slow progressive) motility was higher in the acute rosuvastatin group compared to TT (p < 0.05; Fig. 2C). No significant group differences were found for C-type (non-progressive) motility (Fig. 2D).

Fig. 2.

Effects of rosuvastatin on sperm motility parameters after torsion/detorsion (TT) injury. (A) Total motility (%), calculated as the sum of rapid progressive (A motility), slow progressive (B motility), and non-progressive (C motility) sperm. (B) Rapid progressive motility (%). (C) Slow progressive motility (%). (D) Non-progressive motility (%). (E) Immotility (%). Data are presented as mean ± SD (n = 7/group). Results are expressed as a percentage of total sperm counted. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, not significant. Comparisons are made versus the TT group unless otherwise indicated.

When total motility (A + B + C) was evaluated, the TT group showed a significant reduction compared to sham (p < 0.001; Fig. 2A). Both acute and prophylactic rosuvastatin treatments significantly improved total motility compared to TT (p < 0.01 for both; Fig. 2A). For D-type (immotile) sperm, the TT group showed a significant increase compared to sham (p < 0.001; Fig. 2E), while both rosuvastatin treatment groups significantly reduced D motility relative to TT (p < 0.01; Fig. 2E).

Morphological analysis showed a significant increase in sperm head abnormalities in the TT group compared to sham (p < 0.01; Fig. 3A and B). Head abnormalities were not significantly reduced by acute rosuvastatin treatment, but the prophylactic group showed a significant improvement compared to TT (p < 0.05; Fig. 3A). For acrosomal defects, both acute and prophylactic rosuvastatin significantly reduced abnormalities relative to TT (p < 0.05; Fig. 3C and D). No significant group differences were observed for neck anomalies (Fig. 3E and F). The percentage of sperm with normal morphology was markedly lower in the TT group than in sham (p < 0.001; Fig. 3G and H).

Fig. 3.

Effects of rosuvastatin on sperm morphology parameters after torsion/detorsion (TT) injury. (A) Percentage of sperm with head abnormalities. (B) Representative images of sperm with head abnormalities. (C) Percentage of sperm with acrosome abnormalities. (D) Representative images of sperm with acrosome abnormalities. (E) Percentage of sperm with neck abnormalities. (F) Representative images of sperm with neck abnormalities. (G) Percentage of sperm with normal morphology. (H) Representative images of sperm with normal morphology. All images were obtained from Diff-3-stained preparations using light microscopy (Nikon) at 100× magnification (scale bar = 10 μm). Data are presented as mean ± SD (n = 7/group). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, not significant. Comparisons are made versus the TT group unless otherwise indicated.

Total Antioxidant Status (TAS) was significantly reduced in the TT group compared to sham (p < 0.01; Fig. 4A). Both acute and prophylactic rosuvastatin treatments significantly increased TAS levels relative to TT (p < 0.001 for both; Fig. 4A). Conversely, Total Oxidant Status (TOS) was significantly higher in the TT group than in sham (p < 0.001; Fig. 4B), but both rosuvastatin treatments significantly reduced TOS compared to TT (p < 0.001; Fig. 4B). Similarly, the Oxidative Stress Index (OSI) was elevated in the TT group versus sham (p < 0.001; Fig. 4C) and was significantly decreased by both rosuvastatin regimens (p < 0.001 for both; Fig. 4C).

Fig. 4.

Effects of rosuvastatin on total antioxidant status (TAS), total oxidant status (TOS), and oxidative stress index (OSI) after torsion/detorsion (TT) injury. (A) TAS levels (mmol Trolox equivalents/L). (B) TOS levels (µmol H₂O₂ equivalents/L). (C) OSI, calculated as the ratio of TOS to TAS [OSI = TOS (µmol H₂O₂ eq./L)/TAS (mmol Trolox eq./L)]. Data are presented as mean ± SD (n = 7/group). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, not significant. Comparisons are made versus the TT group unless otherwise indicated.

Phosphorylated AKT (pAKT) expression was significantly lower in the TT group compared to sham (p < 0.001; Fig. 5A), but both acute and prophylactic rosuvastatin treatments restored pAKT levels (p < 0.001 for both). The anti-apoptotic protein Bcl-xL was also reduced in TT versus sham (p < 0.001; Fig. 5D), and both treatment groups showed significantly higher Bcl-xL levels compared to TT (p < 0.05), mirroring the pattern seen with pAKT. Phosphorylated ERK1 (pERK1) expression did not differ between sham and TT groups (ns; Fig. 5B), but both rosuvastatin regimens significantly decreased pERK1 compared to TT (p < 0.01 and p < 0.001, respectively). Similarly, pERK2 levels were unchanged between sham and TT (ns; Fig. 5C), yet both treatments led to a marked reduction in pERK2 compared to TT (both p < 0.001).

Fig. 5.

Effects of rosuvastatin on survival and stress-related signaling proteins in testicular tissue after torsion/detorsion (TT) injury. Shown are expression levels (% of control) for phosphorylated AKT (pAKT; 60 kDa), Bcl-xL (30 kDa), phosphorylated ERK1 (pERK1; 42 kDa), phosphorylated ERK2 (pERK2; 44 kDa), phosphorylated JNK1 (pJNK1; 46 kDa), and phosphorylated JNK2 (pJNK2; 54 kDa). Representative western blot bands are displayed for each protein, including both phosphorylated and total forms of AKT (60 kDa), ERK1/2 (44/42 kDa), and JNK1/2 (54/46 kDa), as well as Bcl-xL (30 kDa). β-actin (45 kDa) was used as the loading control. For densitometric analysis, phosphorylated protein band intensities were first normalized to their respective total protein bands and then to β-actin; Bcl-xL was normalized directly to β-actin. Data are presented as mean ± SD from n = 3 or more independent experiments per group. Statistical significance is indicated as follows: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, not significant. Comparisons are made versus the TT group unless otherwise indicated.

Phosphorylated JNK1 (pJNK1) levels were significantly higher in the TT group compared to sham (p < 0.01; Fig. 5E). Acute rosuvastatin treatment did not significantly affect pJNK1 expression versus TT (ns), while prophylactic rosuvastatin led to a further significant increase (p < 0.05). Similarly, phosphorylated JNK2 (pJNK2) was elevated in the TT group compared to sham (p < 0.01; Fig. 5F). Acute rosuvastatin further increased pJNK2 compared to TT (p < 0.05), but prophylactic treatment showed no significant difference from the TT group (ns).

Discussion

Building on previous studies showing that HMG-CoA reductase inhibitors like rosuvastatin can reduce ischemic injury, especially in models of cerebral ischemia, by modulating survival signaling pathways, we aimed to explore whether rosuvastatin could also protect testicular tissue after TT-induced ischemia/reperfusion (I/R) injury¹⁵. Severe testicular torsion, such as 720° for 2 h, is known to cause substantial disruption of the seminiferous epithelium and marked germ cell loss^38,51. In this study, we used the Johnsen score to quantify spermatogenic impairment following I/R injury⁴⁴. Our results indicate that both prophylactic and acute administration of rosuvastatin effectively reduced TT-induced testicular damage. These protective effects were accompanied by increased activation of the AKT pathway and upregulation of the anti-apoptotic protein Bcl-xL.

To assess the severity of torsion-induced I/R injury and testicular necrosis, we used the Cosentino scoring system, which evaluates irregularity, vacuolization, and coagulative changes in seminiferous tubules^47,52,53. In our study, rosuvastatin treatment lessened TT-induced tissue damage by reducing hypoxia-driven necrosis and limiting ROS accumulation, likely due to its antioxidant effects. Consistent with prior studies, statin administration significantly improved histopathological outcomes in I/R-injured testicular tissue¹⁷. Both prophylactic and acute rosuvastatin treatments effectively preserved testicular function and histological integrity.

As in previous studies, we used epididymal sperm samples to assess sperm parameters⁴. While spermatogenesis in mice takes about 30–35 days to complete⁵⁴, acute histopathological changes, such as germ cell apoptosis, vacuolization, inflammation, and lower Johnsen scores, can occur as early as 24 h after ischemia/reperfusion (I/R) injury. Prior studies have reported pronounced germ cell apoptosis and reduced spermatogenic gene expression within 24 h of torsion and reperfusion⁵⁵, and similar rapid changes in germ cell survival and inflammation have been observed^56,57. Because our analysis was performed just 24 h after detorsion, it is likely that epididymal sperm concentration was not yet significantly altered by TT-induced damage. Although no statistically significant differences in sperm counts were observed between the TT and treatment groups, rosuvastatin may still help preserve epididymal sperm counts over longer periods, potentially via its antioxidant effects.

Oxidative stress impairs sperm motility and capacitation by promoting lipid peroxidation in the plasma membrane and oxidative modification of flagellar proteins, ultimately disrupting their function⁵⁸. Increased S-glutathionylation and protein oxidation, together with reduced sperm motility under oxidative conditions, indicate that motility-related proteins are especially sensitive to oxidative stress⁹. Our findings suggest that rosuvastatin helps counteract lipid peroxidation and provides antioxidant benefits in the epididymal environment.

Rosuvastatin treatment also led to a notable reduction in sperm morphological abnormalities after TT injury. In particular, fewer head and acrosomal defects were seen, suggesting that rosuvastatin may preserve sperm membrane integrity, possibly through antioxidant mechanisms involving plasmalogens. Consistent with earlier reports, our results support the idea that I/R injury impairs epididymal spermatozoa, and that both prophylactic and acute statin administration can improve sperm motility and morphology by mitigating oxidative stress.

Total Antioxidant Status (TAS) and Total Oxidant Status (TOS) reliably indicate oxidative stress by reflecting the tissue balance between oxidant and antioxidant systems^48,49,59. In this study, rosuvastatin significantly improved TAS, reduced TOS, and lowered the Oxidative Stress Index (OSI) in the setting of testicular torsion. These findings further reinforce the antioxidant potential of statins in protecting against oxidative stress, in agreement with existing literature.

Previous research has shown that the protective effects of statins in ischemic injury are mainly mediated by the PI3K/AKT signaling pathway⁶⁰. In testicular tissue, increased AKT phosphorylation helps prevent oxidative damage and apoptosis, and supports the survival of spermatogonia after I/R injury⁶¹. AKT achieves this protection by preserving mitochondrial integrity, preventing cytochrome c release, and maintaining mitochondrial membrane potential in a caspase-independent manner⁶². Rosuvastatin, as a potent HMG-CoA reductase inhibitor, has been reported to boost glycolytic activity through increased pAKT expression, helping maintain mitochondrial function and cellular energy balance during oxidative stress⁶³.

Bcl-xL is another key player in cell survival under metabolic stress, as it helps maintain mitochondrial integrity even when nutrients are limited⁶³. While pAKT-expressing cells depend on external energy sources for their high metabolic activity, Bcl-xL-expressing cells can remain viable even when energy supply is low^63–65. Rosuvastatin may promote metabolic flexibility by both reducing the overall energy demands of the cell and supporting adaptive survival mechanisms under low-energy conditions. These findings suggest that rosuvastatin helps cells better cope with ischemic stress by making their energy metabolism more adaptable.

Crosstalk between the Raf–MEK–ERK and PI3K–AKT signaling pathways is well documented, especially at the level of Raf1^66,67. In our study, statin treatment selectively reduced ERK1/2 phosphorylation in the treatment groups compared to the control group. This reduction may be due to increased pAKT levels, since AKT can inhibit Ras activity upstream of ERK signaling. Given that activated AKT helps maintain mitochondrial integrity and suppress apoptosis, the concurrent drop in pERK1/2 may reflect AKT-mediated suppression of Ras-dependent pathways. This observation aligns with previous reports on reciprocal regulation between these survival pathways⁶⁸.

The SAPK/JNK pathway is quickly activated by extracellular stressors and inflammatory cytokines, acting as a key regulator of the cellular stress response^69–72. In this study, pJNK1/2 expression was higher in both the control and treatment groups relative to sham. However, only prophylactic rosuvastatin significantly increased JNK1, while acute rosuvastatin had a greater effect on JNK2. These findings suggest that JNK activation may have a context-dependent role in modulating intracellular ROS and supporting cellular adaptation under ischemic stress⁷³. Although JNK is often linked to pro-apoptotic signaling, its effects are highly dependent on timing and context^74,75. In I/R injury models, low or transient JNK activation has actually been associated with cytoprotective effects, including modulation of Akt signaling and enhanced antioxidant defenses^74,75. Statins have also been reported to trigger JNK phosphorylation as part of a hormetic, adaptive response under oxidative stress^76–78. Thus, the increased pJNK1/2 levels seen here, especially after statin treatment, likely represent a controlled stress adaptation that promotes cell survival, rather than apoptosis.

Overall, our results show that rosuvastatin protects testicular tissue by modulating several key intracellular signaling pathways, including pAKT, pERK1/2, and pJNK1/2. These effects were most pronounced when comparing the treatment groups to controls. Both prophylactic (before injury) and acute (after injury) administration of rosuvastatin proved beneficial, suggesting that statins could have both preventive and therapeutic value in cases of testicular ischemia/reperfusion injury. Importantly, our acute treatment protocol supports the idea that a single high-dose statin could be a practical emergency intervention for conditions like testicular torsion. However, more studies are needed to clarify the exact molecular mechanisms involved and to determine the long-term outcomes of statin use in testicular pathology.

Conclusion

This study shows that both prophylactic and acute administration of rosuvastatin provide significant protection against ischemia/reperfusion injury caused by testicular torsion. Histopathological analysis revealed that rosuvastatin preserved testicular structure and supported spermatogenesis in both treatment models, highlighting its potential to minimize tissue damage after torsional injury.

Mechanistically, rosuvastatin’s protective effects seem to be driven primarily by activation of the PI3K/AKT pathway, as indicated by increased pAKT levels, which promote cell survival and resistance to apoptosis. Higher Bcl-xL expression suggests that rosuvastatin may also help cells adapt to energy stress by supporting mitochondrial function. At the same time, reductions in pERK1/2 and variable increases in JNK1/2 activation, especially in the treatment groups, point to context-dependent signaling changes that may contribute to an adaptive, cytoprotective response rather than straightforward pro-apoptotic or proliferative effects.

Taken together, these results suggest that rosuvastatin acts through multiple protective pathways, with the most pronounced molecular benefits seen in the treated groups versus controls. Future studies should clarify the long-term benefits of statin therapy, explore other molecular mechanisms involved, and evaluate its real-world potential as a preventive or emergency treatment in acute urological conditions like testicular torsion.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

B.Y. and E.K. conceived and designed the study. B.Y., O.B., N.Ş., Ü.V.Ü. and M.Ç.B. performed the experiments and collected data. Z.B., N.A., and İK. 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.

Funding

The Istanbul Medipol University BAP Commission funded this thesis (2022/32) and supported by the Turkish Academy of Sciences (TUBA).

Data availability

The data underlying this article are available in the article and in its online supplementary material.

Declarations

Competing interests

The authors declare no competing interests.