Quercetin alleviates radiation-induced erectile dysfunction by modulating oxidative stress and apoptosis through the Nrf2/HO-1 pathway
Hongyu Liu, Huiying Yan, Yu Yao, Dahai Yu, Chenlu Wang, Yang Liu, Chaoqi Wang
TL;DR
Quercetin helps prevent radiation-induced erectile dysfunction in rats by reducing oxidative stress and protecting penile tissue through the Nrf2/HO-1 pathway.
Contribution
This study is the first to demonstrate quercetin's protective effects against radiation-induced erectile dysfunction via Nrf2/HO-1 activation in both animal models and human endothelial cells.
Findings
Quercetin improved erectile function and reduced fibrosis in radiation-exposed rats.
It reduced oxidative stress markers and apoptosis while enhancing NO/cGMP signaling.
Quercetin activated the Nrf2/HO-1 pathway in both penile tissue and human endothelial cells.
Abstract
Radiation-induced erectile dysfunction (Ri-ED) is a frequent and debilitating complication in male cancer patients undergoing pelvic radiotherapy, primarily driven by oxidative stress, endothelial injury, fibrosis, and apoptosis. Phosphodiesterase type 5 inhibitors show limited efficacy in Ri-ED because they depend on intact endothelial NO signalling. Quercetin, a naturally occurring flavonoid, possesses potent antioxidant, anti-apoptotic, and endothelial-protective properties; however, its role in Ri-ED and the underlying mechanisms remain insufficiently defined. Thirty-two male Sprague–Dawley rats were randomly assigned to four groups (n = 8): Control, radiation-exposed model, low-dose quercetin (10 mg/kg/day) and high-dose quercetin (40 mg/kg/day). A single 20 Gy pelvic irradiation was delivered, followed by oral quercetin or vehicle for 28 days. Erectile function was evaluated by…
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Figure 8- —Natural Science Foundation of Inner Mongolia Autonomous Region
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Taxonomy
TopicsEffects of Radiation Exposure · Sexual function and dysfunction studies · Menopause: Health Impacts and Treatments
Introduction
Erectile dysfunction (ED) is a common male sexual disorder characterised by the persistent inability to achieve or maintain an erection sufficient for satisfactory sexual activity. It affects more than 150 million men worldwide and is expected to increase in prevalence due to ageing populations and rising metabolic diseases [1]. Ageing itself is a well-established and independent risk factor for the development of ED, with epidemiological studies demonstrating a strong age-related increase in ED prevalence driven by progressive endothelial dysfunction, oxidative stress, and neural degeneration [2]. ED's impact extends beyond physical health, significantly affecting psychological and social functioning, which often results in the development of anxiety, depression, and reduced self-confidence [3]. The pathophysiology of ED is multifactorial, involving endothelial dysfunction, hormonal imbalance, oxidative stress, and apoptosis of penile smooth muscle and endothelial cells [4]. These mechanisms are intricately linked to underlying conditions such as cardiovascular disease, diabetes mellitus, psychological distress, and exogenous damage such as surgery or radiation exposure [5, 6].
Among these factors, radiation therapy (RT) has emerged as a significant iatrogenic cause of ED, particularly in patients undergoing pelvic or prostate cancer treatment [7, 8]. While RT is effective in tumour control, it often causes long-term damage to surrounding healthy tissues [9]. One of the most debilitating complications is radiation-induced erectile dysfunction (Ri-ED), a condition characterised by progressive fibrosis, endothelial injury, nerve damage, and accumulation of reactive oxygen species (ROS) in the corpus cavernosum [10]. Studies have demonstrated that excessive ROS impairs nitric oxide (NO) synthesis, promotes endothelial apoptosis, and accelerates cavernosal tissue remodelling, ultimately leading to loss of erectile capacity [11–13].
Currently, PDE-5 inhibitors are widely used for managing ED. However, their clinical application is often limited by treatment-related adverse effects, including headache, flushing, dyspepsia, nasal congestion, and potential cardiovascular concerns, which may reduce patient tolerance and long-term compliance [14, 15]. Thus, it is clinically significant to explore alternative therapeutic strategies that can protect or restore penile tissue structure and function, particularly in the context of radiation-induced injury.
Quercetin, a naturally occurring flavonoid abundant in fruits and vegetables, has shown considerable promise in managing diseases involving oxidative stress and inflammation, such as diabetic complications, cardiovascular diseases, and neurodegeneration [16, 17]. Its pharmacological effects are primarily mediated through the activation of antioxidant pathways (e.g., Nrf2/HO-1), inhibition of pro-apoptotic signalling(e.g., Bax/Bcl-2 ratio), suppression of inflammatory cytokines, and restoration of endothelial function [18–20]. Among these mechanisms, the nuclear factor erythroid 2–related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) pathway represents a central endogenous antioxidant defense system [21]. Under conditions of oxidative stress, Nrf2 dissociates from its cytoplasmic inhibitor Kelch-like ECH-associated protein 1 (Keap1) and translocates into the nucleus, where it binds to antioxidant response elements (AREs) and transcriptionally activates downstream cytoprotective genes, including HO-1 [22]. Activation of the Nrf2/HO-1 pathway plays a critical role in maintaining redox homeostasis, protecting endothelial cells from oxidative injury, and attenuating inflammation and fibrosis. Emerging evidence indicates that impaired Nrf2/HO-1 signalling contributes to vascular dysfunction, tissue fibrosis, and neuronal injury, all of which are key pathological features of ED [23]. Importantly, flavonoids like quercetin have been shown to upregulate endothelial nitric oxide synthase (eNOS) activity and improve vascular reactivity, further supporting their relevance in erectile physiology [24, 25].
Despite these findings, the role of quercetin in the prevention or treatment of Ri-ED remains largely unexplored. Only a few studies in other radiation- or oxidative stress–related injury models have indirectly suggested its benefits, and no comprehensive in vivo or in vitro research has systematically examined its therapeutic potential and molecular mechanisms specifically in Ri-ED. Therefore, in the present study, we investigated the protective effects of quercetin in a rat model of Ri-ED and in a radiation-induced oxidative injury cell model, with particular emphasis on the Nrf2/HO-1 signalling pathway as a central molecular target linking oxidative stress, endothelial dysfunction, and tissue remodelling.
Materials and methods
Rat grouping and establishment of the radiation-injury model
The subjects of this experiment were thirty-two 8-week-old male Sprague–Dawley rats, with an average body weight of approximately 300 g. These animals were obtained from Liaoning Changsheng Biotechnology Co., Ltd. (License No. SCXK (Liao) 2020−0001) and housed in a temperature-controlled environment (22 ± 2 °C) with 55%–80% humidity and a 12-h light/dark cycle. All animals were maintained on a standard commercial laboratory chow diet (provided by Liaoning Changsheng Biotechnology Co., Ltd.) with ad libitum access to both food and water. This study was approved by the Animal Ethics Committee of Inner Mongolia University for Nationalities (Approval No. NM-LL-2025-04-02-01). All animal procedures were conducted in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals.
To establish the Ri-ED model, rats were anesthetized via intraperitoneal injection of sodium pentobarbital (30 mg/kg) and placed in the prone position using a custom-made Plexiglas restrainer to ensure reproducible pelvic positioning. Pelvic irradiation was performed using a Siemens Gammatron cobalt-60 teletherapy unit (Siemens Medical Solutions, Germany), which emits high-energy gamma rays with a mean energy of approximately 1.25 MeV. A single irradiation dose of 20 Gy was delivered to the pelvic region at a dose rate of 2 Gy/min, resulting in a total exposure time of 10 min [9, 26–28].
The source-to-surface distance (SSD) was fixed at 80 cm, and the irradiation field was defined as a 2 cm × 2 cm square centered on the pelvic region. Accurate dose delivery was ensured through pre-irradiation calibration using a solid water phantom and an ionization chamber. To minimize off-target radiation exposure, the remainder of the animal’s body was shielded with lead-based protective materials. Rats in the control group received sham handling without irradiation [9, 26]. Each rat received a single irradiation session.
Following irradiation, erectile function was assessed using an apomorphine-induced erection test. Rats were placed in a dim and quiet isolation area and subcutaneously injected with apomorphine (100 μg/kg) into the neck skin. Erectile responses were observed for 30 min, and the occurrence and number of erections and yawns were recorded. An erection was defined as glans congestion, foreskin recession, and penile enlargement. Rats failing to meet these criteria were diagnosed as having erectile dysfunction and included in the Ri-ED model.
After confirmation of the Ri-ED model, rats were randomly assigned into four groups (n = 8 per group) and treated once daily for 28 consecutive days (Fig. 1A). The Control group received 1 mL of 0.5% carboxymethylcellulose solution (CMC-Na) orally without irradiation. The Model group received pelvic irradiation followed by an equal volume of CMC-Na. The low-dose quercetin group (Que-L) received pelvic irradiation plus quercetin (10 mg/kg/day, suspended in CMC-Na), and the high-dose quercetin group (Que-H) received pelvic irradiation plus quercetin (40 mg/kg/day).Fig. 1. Experimental design and analysis of body weight and erectile function in rats. A Schematic timeline of the experiment, including irradiation, oral quercetin administration, body weight measurements, and evaluation of erectile function at different time points. B Chemical structure of quercetin. C Body weight changes in rats from day 0 to day 28, comparing the Control, Model, Que-L (Low-dose quercetin), and Que-H (High-dose quercetin) groups (n = 8 per group). D Schematic representation of the apparatus used for evaluating erectile function in rats. E Quantitative analysis of erectile function (frequency of erections) and yawning behavior (frequency of yawns), with data expressed as event counts per rat during a 30-min observation period after apomorphine administration. Data are presented as mean ± SD and were analysed by one-way ANOVA followed by Tukey’s post hoc test. (**p < 0.01 compared with Control group; #p < 0.05 compared with Model group; ##p < 0.01 compared with Model group; &p < 0.05 compared with Que-L group)
Preparation of quercetin
Quercetin was purchased from Sigma with a purity of ≥ 98% (Fig. 1B). A 0.5% (w/v) sodium CMC-Na was prepared as the vehicle for oral administration. Quercetin was suspended in CMC-Na to achieve low-dose (10 mg/kg/day) and high-dose (40 mg/kg/day) formulations, as per the experimental design [29–31]. The suspension concentrations were calculated to allow a fixed intragastric administration volume of 10 mL/kg based on individual body weight. The quercetin suspensions were freshly prepared and thoroughly homogenized before administration to ensure uniform dosing. All solutions were filtered to ensure cleanliness before treatment. Rats were administered oral doses according to group-specific dosages, with the control group receiving an equivalent volume of physiological CMC-Na solution. Treatment commenced 24 h post-irradiation, with daily oral administration between 09:00 and 11:00 for 28 consecutive days. Based on previous animal toxicity studies, oral administration of the quercetin used in this experiment is unlikely to cause adverse reactions [32].
Erectile function tests in rats
Based on previous research techniques [33], the rats were first anaesthetised by administering 5% sodium pentobarbital via intraperitoneal injection at a dosage of 30 mg/kg. Subsequently, a 24-G needle was loaded with a 200 IU/mL heparin solution and interfaced with an MP150 physiometer (Biopac Inc.) through a PE-50 tube. During the insertion of the puncture needle, a pressure transducer was connected to the exposed left common carotid artery to monitor carotid blood pressure. At the same time, the penile skin was incised to expose the corpus cavernosum. Then, insertion of either a needle or a suitable apparatus into the penile cavernous sinus was carried out to note down the intracavernosal pressure (ICP).
The cavernous nerve was then electrically stimulated (5 V, 60 s) to induce erection, and both ICP and mean arterial pressure (MAP) were simultaneously recorded. The ICPmax/MAP ratio was subsequently calculated and compared across different groups to evaluate erectile function. This model was used to assess neurovascular erectile function through electrical stimulation of the cavernous nerve and evaluation of the ICPmax/MAP ratio, which closely reflects functional recovery in radiation-induced erectile injury.
After the erectile function assessment, rats under deep anaesthesia were euthanised by intraperitoneal administration of an overdose of sodium pentobarbital in accordance with animal welfare guidelines. Penile tissues were immediately harvested, rinsed with cold saline, and divided for histological, molecular, and biochemical analyses.
Tissue processing
After sacrifice, the penile tissues were rapidly harvested and rinsed thoroughly with phosphate-buffered saline (PBS). Samples were then fixed in 4% paraformaldehyde (PFA) at 4 °C for 24 h, followed by dehydration through a graded ethanol series, clearing in xylene, and paraffin embedding. Paraffin blocks were sectioned at a thickness of 5 μm, and these paraffin sections were used for subsequent hematoxylin–eosin (H&E) staining, Masson’s trichrome staining, and immunohistochemistry.
For immunofluorescence staining, tissues were also fixed in 4% PFA for 24 h and then cryoprotected in 30% sucrose solution at 4 °C overnight. The cryoprotected tissues were embedded in OCT compound, rapidly frozen in isopentane precooled on dry ice, and stored at − 80 °C until use. Frozen tissues were sectioned at a thickness of 5 μm using a cryostat, and these cryosections were used exclusively for immunofluorescence staining.
Histological testing and observation
Following the experiment, to evaluate the structural alterations in cavernous tissue, the prepared penile tissue sections underwent H&E staining (Solarbio, G1120) and Masson staining (Solarbio, G1340), both performed strictly according to the protocols provided by Solarbio (Beijing). Subsequently, the ImageJ software quantified the spongy tissue structure by calculating the ratio of red smooth muscle to blue collagen-positive areas in images to evaluate penile spongy tissue collagen deposition. This calculation served to highlight our investigation into how experimental variables influence tissue remodelling.
Oxidative stress detection methods
In strict accordance with the guidelines provided by Beyotime Biotechnology, penile tissue levels of superoxide dismutase (SOD, Beyotime, S0087) and malondialdehyde (MDA, Beyotime, S0131S) were determined using commercial colourimetric assay kits. After preparing the sample homogenates and centrifuging them to obtain the supernatant, reaction reagents were added in the appropriate proportion. The SOD assay is based on the principle that SOD inhibits the reduction of a chromogenic substrate driven by superoxide anions. In contrast, MDA is measured using the classical thiobarbituric acid reactive substances (TBARS) method. The solution underwent a 40-min incubation at 37 °C in the dark [34]. Following this, a microplate reader measured the absorbance at 450 nm, and the SOD activity (U/mg protein) and MDA content (nmol/mg protein) were derived from a standard curve and normalised to protein concentration.
NO (R&D Systems, KGE001), cyclic guanosine monophosphate (cGMP, R&D Systems, KGE003), and calcium (Ca^2^⁺, R&D Systems, 6255) concentrations were measured using the ELISA kit. First, the serum or tissue homogenate was centrifuged to obtain the supernatant, which was then diluted as required. Thereafter, the pre-coated plate wells were loaded with standards and samples and incubated at 37 ℃ for 90 min. After washing, the biotinylated antibody was added, followed by a 60-min incubation. After incubation with HRP-labelled streptavidin and subsequent colour development, the reaction was stopped with the supplied stop solution, and absorbance at 450 nm was measured with a microplate reader.
Immunohistochemical tests
To perform antigen retrieval, place the sections in citrate buffer (pH 6.0), heat the solution at 95 °C for 20 min, and cool it to room temperature. Rinse the sections three times with PBS (pH 7.4) for 5 min each. Apply primary antibodies: α-SMA (Sigma-Aldrich, A5228, 1:200), Bax (Abcam, ab32503, 1:150), Bcl-2 (Abcam, ab141523, 1:200). Incubate sections in a wet box at 4 °C overnight (16 h), then wash three times with PBS to remove unbound antibodies.
Secondary antibody staining: Incubate HRP—labeled anti—mouse/rabbit IgG (EnVision + System, Dako) at RT for 30 min. Wash with PBS, then perform DAB colour development for 3–5 min, observing under a microscope. Counterstain nuclei with hematoxylin, differentiate with ethanol hydrochloride, and counterstain with tap water.
An Olympus BX53 light microscope was used to capture images. Positive staining appeared as brownish-yellow granules with blue nuclei. Through ImageJ analysis of the area-integrated optical density (IOD) of positive signals, angiogenesis, fibrosis, and marker expression (α-SMA, Bax/Bcl-2) were assessed.
Immunofluorescence detection methods
Rat mid-penis tissues were first fixed and then dehydrated in a 30% sucrose solution for 2 days. After OCT embedding, 5 μm frozen sections were prepared. The slides were baked at 37 °C for 30 min, followed by washing with PBS for 5 min. Tissue sections were then permeabilized with 0.3% Triton X-100 for 15 min and washed again with PBS. Blocking was performed using 5% bovine serum albumin (BSA) for 1 h at room temperature. Subsequently, the sections were incubated overnight at 4 °C with primary antibodies against CD31 (Abcam, ab28364, 1:100), eNOS (BD Biosciences, AB_1645410, 1:200), NF (Abcam, ab9035, 1:500), nNOS (BD Biosciences, AB_397700, 1:200), Nrf2 (Proteintech, 80593-1-RR, 1:250), and HO-1 (Proteintech, 66743-1-Ig, 1:250).
After primary antibody incubation, the sections were incubated with species-specific Alexa Fluor–conjugated secondary antibodies according to the host species of the primary antibodies, including goat anti-rabbit IgG (H + L), Alexa Fluor 488 (Invitrogen, A-11008, 1:500), and goat anti-mouse IgG (H + L), Alexa Fluor 594 (Invitrogen, A-11005, 1:500). Cell nuclei were counterstained with DAPI.
Immunofluorescence images were captured using a confocal microscope (Zeiss LSM 880) under identical acquisition settings for all groups. The distribution and expression of the target antigens were observed to evaluate changes in angiogenesis, fibrosis, and related pathological factors.
Immunofluorescence signals were quantified using ImageJ software by measuring the mean fluorescence intensity (MFI) of target proteins within manually defined regions of interest (ROIs) in the corpus cavernosum. For each section, three randomly selected fields were analysed at identical magnification after background subtraction. Fluorescence intensities were normalized to DAPI or expressed relative to the Control group. All analyses were performed in a blinded manner.
Western blot analysis of signalling pathways
After penile tissue had been isolated, the samples were immediately placed on ice and homogenized in RIPA lysis buffer (Beyotime, China) supplemented with protease and phosphatase inhibitor cocktails. The tissue lysates were incubated on ice for 30 min and then centrifuged at 12,000 × g for 15 min at 4 °C. The supernatants containing total protein were collected for subsequent analysis.
Protein concentrations were determined using a bicinchoninic acid (BCA) protein assay kit according to the manufacturer’s instructions. Equal amounts of protein (30–40 μg per lane) were separated by 10% SDS–PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% nonfat dry milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) at room temperature for 60 min.
The membranes were then incubated overnight at 4 °C with primary antibodies against Nrf2 (Abcam, ab62352, 1:1000), HO-1 (Abcam, ab305290, 1:1000), and β-actin (Abcam, ab8226, 1:5000). After washing with TBST, the membranes were incubated with the corresponding horseradish peroxidase–conjugated secondary antibody (Proteintech, SA00001, 1:1000) for 90 min at room temperature.
Protein bands were visualized using an enhanced chemiluminescence (ECL) detection kit (Thermo Fisher Scientific), and band intensities were quantified using ImageJ software (NIH, USA). Target protein expression levels were normalized to β-actin.
Cell culture
Human umbilical vein endothelial cells (HUVECs) were purchased from Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd. (cat. no. DFSC-EC-01). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco), 1% penicillin–streptomycin (100 U/mL penicillin, 100 μg/mL streptomycin), and two mM L-glutamine. Cultures were maintained in a humidified incubator at 37 °C with 5% CO₂ and 95% air. Cells at passages 3–6 were used for all experiments. Before each assay, cells were seeded into the appropriate culture plates at the designated density and allowed to adhere overnight. Only cultures with 70–80% confluence and normal morphology were used for further experiments.
Cell counting Kit-8 (CCK-8) assay for cell viability
Cell viability was assessed using a CCK-8 (C0038, Beyotime, China) according to the manufacturer’s instructions.
Radiation dose–response experiment: Endothelial cells were seeded into 96-well plates at 5 × 10^3^ cells/well in 100 μL of complete medium and incubated overnight. Cells were then exposed to a single radiation dose of 0, 2, 4, 6, 8, or 10 Gy using the same irradiation system as in the animal experiments. After irradiation, the cells were returned to the incubator, and cell viability was measured at 24 h and 48 h.
Quercetin dose–response experiment: Under non-irradiated conditions, cells were treated with quercetin at final concentrations of 0, 0.01, 0.5, 1, 5, 10, or 20 μM for 24 h or 48 h. Quercetin was dissolved in DMSO, and the final DMSO concentration did not exceed 0.1% in any of the groups. Cell viability was then determined by CCK-8 assay.
Radiation-injury protection experiment: To evaluate the protective effect of quercetin against radiation-induced injury, cells were first exposed to a single 6 Gy dose and then immediately incubated in medium containing quercetin (1, 5, or 10 μM) for 48 h. The following groups were included: Control (no irradiation, no quercetin), IR (6 Gy + vehicle), and IR + Que (6 Gy + quercetin at the indicated concentrations).
At each indicated time point, 10 μL of CCK-8 solution was added to each well and incubated for two hours at 37 °C. Absorbance at 450 nm was measured using a microplate reader, and cell viability was expressed as a percentage of the untreated control group.
Intracellular ROS fluorescence staining
Intracellular ROS were detected using a dihydroethidium (DHE) fluorescent probe (ROS assay kit, S0064S, Beyotime, China). Endothelial cells were seeded on glass coverslips placed in 24-well plates and divided into four groups: Control, Que, IR, and IR + Que. Cells in the IR and IR + Que groups were exposed to a single 6 Gy dose of radiation, after which they were cultured for 48 h in medium with (IR + Que, 10 μM quercetin) or without quercetin (IR). The Que group received quercetin (10 μM) without irradiation, whereas the Control group received neither irradiation nor quercetin.
48 h after irradiation, the medium was removed, and the cells were washed twice with pre-warmed PBS. DHE working solution (10 μM in serum-free medium) was then added, and cells were incubated at 37 °C for 30 min in the dark. After incubation, cells were washed with PBS, fixed with 4% paraformaldehyde for 15 min, and counterstained with DAPI (1 μg/mL) for 5 min to visualise nuclei. Fluorescence images were acquired using a fluorescence microscope with identical exposure settings for all groups, where ROS signals appeared red and DAPI-stained nuclei appeared blue. The mean fluorescence intensity of ROS in each group was quantified using ImageJ software and expressed as a fold of the Control group.
Immunofluorescence staining of cellular Nrf2 and HO-1
To assess the cellular localisation and expression of Nrf2 and HO-1, endothelial cells were seeded on glass coverslips and treated as described in Sect. 2.11, with four groups: Control, Que, IR, and IR + Que. At 48 h after irradiation, the culture medium was removed, and the cells were washed with PBS. They were then fixed with 4% paraformaldehyde for 15 min at room temperature. After fixation, cells were permeabilised with 0.3% Triton X-100 in PBS for 10 min at room temperature, followed by blocking with 5% BSA for one h.
After blocking, cells were incubated overnight at 4 °C with primary antibodies diluted in BSA blocking buffer: anti-Nrf2 antibody (Proteintech, 80593-1-RR, 1:250) or anti-HO-1 antibody (Proteintech, 66743-1-Ig, 1:250). The following day, cells were washed three times with PBS (5 min each) and then incubated in the dark at room temperature for one h with the corresponding fluorescent secondary antibodies: Alexa Fluor 594 conjugated goat anti-rabbit IgG for Nrf2 (1:500, Proteintech) and Alexa Fluor 488 conjugated goat anti-mouse IgG for HO-1 (1:500, Proteintech).
After secondary antibody incubation, the cells were washed with PBS, counterstained with DAPI (1 μg/mL) for 5 min to visualise the nuclei, rewashed with PBS, and then mounted using an anti-fade mounting medium. Fluorescence images were captured using a fluorescence microscope (scale bar = 50 μm). The mean fluorescence intensity of Nrf2 or HO-1 in each group was quantified using ImageJ software and normalised to the Control group, consistent with the quantitative data shown in Fig. 7.
Statistical analyses
For quantitative image analysis, three sections at comparable anatomical levels were selected from each animal, and five non-overlapping high-power fields per section were captured under identical microscope settings. The mean value of these fields was taken as one biological replicate (n) for each animal. All quantitative data were analysed using ImageJ software with uniform exposure and threshold parameters across all groups.
Statistical analyses were performed using SPSS 22.0 software. All data are presented as mean ± standard deviation (SD). Data normality was assessed using the Shapiro–Wilk test, and all datasets satisfied the assumption of normal distribution (p > 0.05). Group differences were evaluated by one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test, and a p < 0.05 was considered statistically significant.
Results
Quercetin significantly improves erectile function in radiation-injured rats
During the 28-day experimental period, rats in the Model group exhibited a progressive decline in body weight compared with the Control group. Quercetin treatment attenuated radiation-induced weight loss in both the Que-L and Que-H groups, with the high-dose group showing a more pronounced improvement. However, body weights in the quercetin-treated groups remained lower than those of the Control group throughout the observation period (Fig. 1C).
Behavioural assessment demonstrated that the frequency of erections was significantly reduced in the Model group compared with the Control group, while quercetin treatment dose-dependently improved erectile responses. The Que-H group exhibited a markedly higher erection frequency than the Model group, approaching Control levels, whereas yawning frequency did not differ significantly among groups, indicating that central dopaminergic responsiveness was not affected by irradiation or quercetin treatment (Fig. 1D and E).
Electrophysiological evaluation further confirmed radiation-induced erectile dysfunction (Fig. 2A). As shown in Fig. 2B, the maximum ICPmax/MAP ratio was significantly reduced in the Model group (0.25 ± 0.05) compared with the Control group (0.77 ± 0.06, p < 0.01). Quercetin treatment significantly partially restored the ICPmax/MAP ratio in both the Que-L group (0.43 ± 0.06, p < 0.05 vs. Model) and the Que-H group (0.58 ± 0.06, p < 0.01 vs. Model). Notably, the ICPmax/MAP ratio in the Que-H group was significantly higher than that in the Que-L group (p < 0.05), approaching but not fully reaching Control levels.Fig. 2. Erectile function assessment and pathological staining analysis in different rat groups. A Representative raw recordings of penile ICP and MAP under 5 V electrical stimulation in rats. B, C Quantitative analysis of erectile function levels in different groups (n = 8 per group). D Quantification of the smooth muscle/collagen ratio in each group based on Masson’s trichrome staining; E Quantification of the α-SMA–positive area in each group based on immunohistochemical staining. F Representative photomicrographs of H&E staining showing the general histological architecture of penile tissue in each group. G Representative photomicrographs of Masson’s trichrome staining, in which collagen fibers are stained blue and smooth muscle fibers are stained red, illustrating radiation-induced collagen deposition and smooth muscle loss in the corpus cavernosum. H Representative photomicrographs of α-SMA immunohistochemical staining, showing changes in smooth muscle cell marker expression in penile tissue. Arrows indicate areas of smooth muscle loss, collagen deposition, or altered α-SMA expression, as appropriate. Scale bar = 100 μm. (**p < 0.01 compared with Control group; #p < 0.05 compared with Model group; ##p < 0.01 compared with Model group; &p < 0.05 compared with Que-L group. ICP intracavernous pressure, MAP mean arterial pressure)
Consistently, analysis of the area under the curve of ICP/MAP (AUC/MAP) revealed a similar pattern (Fig. 2C). Pelvic irradiation markedly decreased AUC/MAP in the Model group (0.26 ± 0.05) compared with the Control group (0.77 ± 0.07, p < 0.01). Quercetin treatment significantly partially reversed this reduction in a dose-dependent manner, with AUC/MAP values of 0.46 ± 0.04 in the Que-L group (p < 0.05 vs. Model) and 0.58 ± 0.05 in the Que-H group (p < 0.01 vs. Model). Furthermore, the AUC/MAP in the Que-H group was significantly higher than that in the Que-L group (p < 0.05), indicating superior recovery of overall erectile capacity.
Collectively, these electrophysiological findings demonstrate that quercetin significantly improves erectile function in radiation-injured rats by partially restoring both peak erectile response and overall erectile performance in a dose-dependent manner.
Quercetin significantly improved the pathological damage of the penile tissues in radiation-injured rats
The successful establishment of the disease model was confirmed by H&E staining (Fig. 2F). In the Control group, the corpus cavernosum exhibited an intact architecture, characterised by well-organised trabeculae and regularly arranged smooth muscle cells. In contrast, the Model group showed marked structural disruption, including thinning and fragmentation of trabeculae, disorganisation and loss of smooth muscle bundles, vascular congestion, partial endothelial disruption, and focal inflammatory cell infiltration accompanied by nuclear pyknosis. Compared with the Model group, both Que-L and Que-H treatments partially restored cavernosal architecture, improved smooth muscle morphology, and attenuated vascular and inflammatory alterations, which was consistent with the observed improvement in erectile function.
Masson’s trichrome staining further demonstrated radiation-induced fibrotic remodelling of the corpus cavernosum (Fig. 2D, G). Quantitative analysis revealed that the smooth muscle–to–collagen ratio in the Model group was significantly reduced compared with the Control group (0.10 ± 0.03 vs. 0.27 ± 0.04, p < 0.01), indicating excessive collagen deposition. Quercetin treatment significantly increased this ratio in both the Que-L group (0.17 ± 0.03, p < 0.05 vs. Model) and the Que-H group (0.26 ± 0.04, p < 0.01 vs. Model). Notably, the value in the Que-H group was close to that of the Control group, suggesting a pronounced antifibrotic effect.
To further evaluate smooth muscle content and integrity, α-smooth muscle actin (α-SMA) immunohistochemical staining was performed (Fig. 2E, H). Quantitative analysis showed that α-SMA expression was markedly decreased in the Model group compared with the Control group (0.03 ± 0.01 vs. 0.10 ± 0.01, p < 0.01), indicating severe smooth muscle loss following irradiation. Quercetin treatment significantly partially restored α-SMA expression in both the Que-L group (0.06 ± 0.01, p < 0.05 vs. Model) and the Que-H group (0.08 ± 0.01, p < 0.01 vs. Model). The higher α-SMA level observed in the Que-H group suggests a dose-dependent protective effect of quercetin on cavernosal smooth muscle preservation.
Collectively, these histological and quantitative findings indicate that quercetin mitigates radiation-induced fibrosis and smooth muscle loss in the corpus cavernosum, thereby contributing to the preservation of penile tissue structure and tissue architectural integrity.
Quercetin's improvement of erection may be related to vascular endothelial function and neuroprotection
Immunofluorescence analysis demonstrated that pelvic irradiation caused marked endothelial injury in the corpus cavernosum. Quantitative analysis showed that CD31 expression was significantly reduced in the Model group (0.036 ± 0.007) compared with the Control group (0.075 ± 0.008, p < 0.05). Quercetin treatment significantly increased CD31 levels in both the Que-L group (0.048 ± 0.007, p < 0.05 vs. Model) and the Que-H group (0.052 ± 0.006, p < 0.05 vs. Model) (Fig. 3A, B). Although CD31 expression was numerically higher in the Que-H group than in the Que-L group, no statistically significant difference was observed between the two doses, and CD31 levels in the Que-H group remained lower than those in the Control group, indicating partial recovery.Fig. 3. Expression analysis of endothelial and neuronal markers in the penile tissues of different rat groups. A Immunofluorescence staining assessing the expression of CD31 (red) and eNOS (green) in the corpus cavernosum of the penis in different groups. DAPI staining (blue) marks the total cell nuclei, and the merged images show co-localisation of the markers (n = 8 per group). Scale bar = 50 μm. B Quantification of CD31 meanfluorescence density and C eNOS mean fluorescence density in the penile tissue, with statistical analysis showing a significant decrease in the model group compared to the control group, and partial recovery in the Que-L and Que-H groups. D Immunofluorescence staining assessing the expression of NF (red) and nNOS (green) in the dorsal nerve of the penis. DAPI staining (blue) marks the cell nuclei, and the merged images show co-localisation of the markers. Scale bar = 50 μm. E, F Statistical analysis of the mean fluorescence density of NF and nNOS. Data are presented as mean ± SD and were analysed by one-way ANOVA followed by Tukey’s post hoc test. (*p < 0.05 compared with Control group; **p < 0.01 compared with Control group; #p < 0.05 compared with Model group)
A similar pattern was observed for eNOS expression. Compared with the Control group (0.075 ± 0.011), eNOS levels were significantly decreased in the Model group (0.034 ± 0.003, p < 0.05). Quercetin administration significantly partially restored eNOS expression in both the Que-L group (0.052 ± 0.003, p < 0.05 vs. Model) and the Que-H group (0.054 ± 0.007, p < 0.05 vs. Model) (Fig. 3A, C). Although the Que-H group showed slightly higher eNOS expression than the Que-L group, this difference did not reach statistical significance, and eNOS levels in the Que-H group did not fully return to Control values.
Neuronal integrity was further evaluated by NF immunofluorescence staining. Quantitative analysis revealed that NF expression was significantly reduced in the Model group (0.042 ± 0.002) compared with the Control group (0.066 ± 0.003, p < 0.05). Quercetin treatment significantly increased NF expression in both the Que-L group (0.057 ± 0.009, p < 0.05 vs. Model) and the Que-H group (0.060 ± 0.006, p < 0.05 vs. Model) (Fig. 3D, E). Although NF expression was higher in the Que-H group than in the Que-L group, no statistically significant difference was detected between the two doses, and NF levels in the Que-H group remained below those of the Control group.
nNOS expression was also markedly impaired following irradiation. Quantitative analysis showed that nNOS levels in the Model group (0.027 ± 0.004) were significantly lower than those in the Control group (0.075 ± 0.008, p < 0.01). Quercetin treatment significantly partially restored nNOS expression in both the Que-L group (0.048 ± 0.004, p < 0.05 vs. Model) and the Que-H group (0.061 ± 0.001, p < 0.01 vs. Model) (Fig. 3D, F). Notably, nNOS expression in the Que-H group was significantly higher than that in the Que-L group (p < 0.05), although it remained lower than Control levels. These findings indicate that high-dose quercetin exerts a stronger protective effect on neuronal nitric oxide signalling, while overall recovery remains partial.
Collectively, these results demonstrate that quercetin significantly attenuates radiation-induced endothelial and neuronal damage in penile tissue. The protective effects are characterised by partial restoration of vascular and neural markers, with a significant dose-dependent enhancement observed specifically for nNOS expression.
Quercetin improves penile oxidative stress in radiation-injured rats
Quercetin markedly alleviated radiation-induced oxidative stress in penile tissues (Fig. 4A–G). ROS-positive area increased from 0.2 ± 0.2% in the Control group to 4.2 ± 0.1% in the Model group (p < 0.01), and was reduced by quercetin to 2.2 ± 0.4% in Que-L (p < 0.05 vs. Model) and 0.9 ± 0.5% in Que-H (p < 0.01 vs. Model; p < 0.05 vs. Que-L) (Fig. 4B).Fig. 4. Analysis of oxidative stress and related biochemical markers in different groups. A Representative immunofluorescence images showing ROS (red) and DAPI (blue)staining in the control, model, and treatment groups (Que-L and Que-H). The merged images highlight ROS-positive areas. Scale bar = 20 μm. B Quantification of ROS-positive area (% of total area) based on ImageJ analysis, showing a significant increase in the Model group compared with the Control group and a marked reduction in the Que-L and Que-H groups. C, D ELISA detection of SOD activity and MDA content in penile tissue homogenates. E–G ELISA detection of NO production, cGMP production, and Ca.^2^⁺ concentration in penile tissue. Data are presented as mean ± SD and were analysed by one-way ANOVA followed by Tukey’s post hoc test (n = 8 per group). (*p < 0.05 compared with Control group; **p < 0.01 compared with Control group; #p < 0.05 compared with Model group; ##p < 0.01 compared with Model group; &p < 0.05 compared with Que-L group)
Consistently, SOD activity decreased to 186 ± 43 U/mg protein in the Model group compared with 566 ± 16 U/mg protein in Controls (p < 0.05), and was restored to 299 ± 62 U/mg protein in Que-L (p < 0.05 vs. Model) and 438 ± 51 U/mg protein in Que-H (p < 0.01 vs. Model; p < 0.05 vs. Que-L). MDA increased to 5.78 ± 0.31 nmol/mg protein in the Model group versus 2.00 ± 0.48 nmol/mg protein in Controls (p < 0.01), and was reduced to 4.18 ± 0.33 nmol/mg protein in Que-L (p < 0.05 vs. Model) and 3.00 ± 0.32 nmol/mg protein in Que-H (p < 0.01 vs. Model; p < 0.05 vs. Que-L) (Fig. 4C, D).
Radiation also impaired NO/cGMP signalling and Ca^2^⁺ homeostasis. NO decreased to 0.46 ± 0.03 in the Model group from 1.00 ± 0.00 in Controls (p < 0.05) and increased to 0.72 ± 0.07 in Que-L and 0.80 ± 0.02 in Que-H (both p < 0.05 vs. Model). cGMP decreased to 0.24 ± 0.04 in the Model group (p < 0.05 vs. Control) and increased to 0.42 ± 0.08 in Que-L (p < 0.05 vs. Model) and 0.60 ± 0.06 in Que-H (p < 0.01 vs. Model; p < 0.05 vs. Que-L). Ca^2^⁺ increased to 1.52 ± 0.07 μg/mg protein in the Model group from 0.49 ± 0.10 μg/mg protein in Controls (p < 0.05) and was reduced to 1.08 ± 0.12 in Que-L and 1.00 ± 0.23 in Que-H (both p < 0.05 vs. Model), with no significant difference between the two doses (Fig. 4E–G).
Overall, these findings indicate that quercetin mitigates radiation-triggered oxidative injury and partially restores redox balance and NO/cGMP-related erectile signalling in penile tissue.
Quercetin ameliorates the level of penile apoptosis in radiation-injured rats
Immunohistochemical analysis indicated that apoptosis-related proteins were predominantly altered in the corpus cavernosum, particularly within regions corresponding to the vascular endothelium and surrounding smooth muscle. Following irradiation, the Model group showed a marked increase in Bax (2.73 ± 0.30, fold of Control) and a pronounced decrease in Bcl-2 (0.27 ± 0.05, fold of Control) relative to the Control group (p < 0.01), indicating enhanced apoptotic signalling (Fig. 5A–C). Quercetin significantly partially reversed these changes, as evidenced by reduced Bax in the Que-L group (1.98 ± 0.29, fold of Control) and Que-H group (1.28 ± 0.16, fold of Control), together with partially restored Bcl-2 in the Que-L group (0.45 ± 0.10, fold of Control) and Que-H group (0.65 ± 0.10, fold of Control) (p < 0.05 or p < 0.01 vs. Model). A stronger anti-apoptotic shift was observed in the Que-H group than in the Que-L group (p < 0.05), supporting a dose-related protective effect.Fig. 5. Quercetin attenuates apoptosis in the penile tissues of the radiation-induced ED model. A Immunohistochemical staining showing the expression of Bax and Bcl-2 in the corpus cavernosum of each group, with arrows indicating positively stained cells (scale bar = 20 μm). B, C Quantitative analysis of Bax and Bcl-2 mean density in the corpus cavernosum of each group, expressed as a fold of the Control group, based on immunohistochemical staining and ImageJ analysis. D TUNEL fluorescence staining results and apoptosis index analysis in different groups (TUNEL, green; DAPI, blue; scale bar = 20 μm). Data are presented as mean ± SD and were analysed by one-way ANOVA followed by Tukey’s post hoc test (n = 8 per group). (**p < 0.01 compared with Control group; #p < 0.05 compared with Model group; ##p < 0.01 compared with Model group)
Consistently, TUNEL staining showed a marked elevation of apoptotic index in the Model group (0.59 ± 0.05) compared with the Control group (0.13 ± 0.02; p < 0.01) (Fig. 5D, E). Quercetin significantly reduced TUNEL positivity in both the Que-L group (0.36 ± 0.10) and the Que-H group (0.21 ± 0.08) (p < 0.05 or p < 0.01 vs. Model), with a greater reduction in the Que-H group than in the Que-L group (p < 0.05). Collectively, these findings demonstrate that quercetin mitigates radiation-induced apoptosis in penile tissue by shifting the Bax/Bcl-2 balance toward cell survival and reducing DNA fragmentation.
Quercetin mitigates radiation-induced erectile dysfunction via Nrf2/HO-1- mediated modulation of oxidative stress and apoptosis
As illustrated in Fig. 6A–D, the Model group showed significantly reduced positive expression of Nrf2 (2.2 ± 0.4%) and HO-1 (2.4 ± 0.3%) compared with the Control group (5.8 ± 0.6% and 6.3 ± 0.3%, respectively; p < 0.01). Quercetin treatment partially restored Nrf2 and HO-1 expression, with partial recovery in the Que-L group (Nrf2: 3.5 ± 0.5%; HO-1: 3.6 ± 0.2%) and a more pronounced increase in the Que-H group (Nrf2: 4.8 ± 0.6%; HO-1: 5.0 ± 0.5%) (p < 0.05 or p < 0.01 vs. Model). DAPI (blue) stained nuclei, and merged images confirmed the co-localisation of Nrf2 (red) and HO-1 (green). Quantitative fluorescence analysis further supported the marked reduction of Nrf2/HO-1 signals after irradiation and their partial restoration following quercetin administration.Fig. 6. Expression and localisation analysis of Nrf2/HO-1 in the penile tissues of different rat groups. A Immunofluorescence staining images showing the expression of Nrf2 (red) in the control, model, and treatment groups (Que-L and Que-H). DAPI staining (blue) marks the cell nuclei, and merged images show the localisation of Nrf2 relative to the nuclei (DAPI). Scale bar = 20 μm. B Quantification of Nrf2-positive areas, showing a significant decrease in the model group compared to the control group, and partial recovery in the Que-L and Que-H groups. C Immunofluorescence staining images showing the expression of HO-1 (green) in the control, model, and treatment groups (Que-L and Que-H). DAPI staining (blue) marks the cell nuclei, and merged images show the localisation of HO-1 relative to the nuclei (DAPI). Scale bar = 20 μm. D Quantification of HO-1-positive areas, showing a significant decrease in the model group compared to the control group, and partial recovery in the Que-L and Que-H groups. E Western blot analysis was used to detect the expression levels of Nrf2 and HO-1 in penile tissues, with β-actin serving as the loading control. F, G Quantification of Nrf2 and HO-1 protein expression levels normalised to β-actin. Data are presented as mean ± SD and were analysed by one-way ANOVA followed by Tukey’s post hoc test (n = 8 per group). (*p < 0.05 compared with Control group; **p < 0.01 compared with Control group; #p < 0.05 compared with Model group; ##p < 0.01 compared with Model group; &p < 0.05 compared with Que-L group)
Western blot analysis (Fig. 6E) revealed a marked downregulation of Nrf2 and HO-1 in the Model group compared with the Control group (p < 0.01). Densitometric quantification (fold of control) further showed that Nrf2 decreased from Control (1.01 ± 0.01) to Model (0.33 ± 0.01), and increased following quercetin treatment in a dose-dependent manner, with higher levels in Que-L (0.56 ± 0.05) and Que-H (0.75 ± 0.07) (Fig. 6F). A similar trend was observed for HO-1, which declined from Control (1.00 ± 0.01) to Model (0.25 ± 0.04) and rose after quercetin administration in Que-L (0.43 ± 0.04) and Que-H (0.58 ± 0.10) (Fig. 6G). Consistently, quercetin significantly elevated Nrf2 and HO-1 levels relative to the Model group (p < 0.01), with a more pronounced effect in the Que-H group than in the Que-L group (p < 0.05). Together, these findings support the involvement of Nrf2/HO-1 signalling in the protective effects of quercetin against radiation-induced erectile dysfunction (Fig. 6F, G).
Quercetin directly protects endothelial cells against radiation-induced oxidative injury via Nrf2/HO-1 activation and promotion of Nrf2 nuclear translocation
To further determine whether the protective effects of quercetin on Ri-ED are associated with its direct regulation of endothelial cells, we established an in vitro model of radiation-induced endothelial injury. CCK-8 assays showed a dose-dependent decline in cell viability at 24 h and 48 h after irradiation, with a more pronounced reduction at higher doses and at 48 h. A dose of 6 Gy was therefore selected for subsequent experiments, as it induced a clear reduction in viability while still preserving an experimental window to evaluate the cytoprotective effects of quercetin (Fig. 7A, B). In non-irradiated cells, quercetin over the tested concentration range exerted minimal cytotoxicity, and cell viability at 5–10 μM remained comparable to the control group; thus, 10 μM quercetin was chosen as the working concentration for the following experiments (Fig. 7C, D). At 48 h after 6 Gy irradiation, endothelial cell viability decreased compared with the control group, whereas co-treatment with quercetin partially restored cell viability (Fig. 7E).Fig. 7. In vitro validation of the protective effects of quercetin against radiation-induced injury in endothelial cells. A, B Cell viability of endothelial cells at 24 h and 48 h after exposure to increasing radiation doses (0–10 Gy), assessed by CCK-8 assay and expressed as a percentage of the non-irradiated control. C, D Cell viability at 24 h and 48 h in non-irradiated cells treated with increasing concentrations of quercetin, showing the range of doses with minimal cytotoxicity. E Effects of radiation and selected quercetin concentrations on endothelial cell viability at 48 h (Control, IR, and IR + Que at the indicated doses). F Representative immunofluorescence images of intracellular ROS (red) and nuclei (DAPI, blue) in the Control, Que, IR and IR + Que groups (scale bar = 50 μm). G Quantification of ROS fluorescence intensity (fold of Control) showing radiation-induced ROS accumulation and its attenuation by quercetin. H Representative immunofluorescence images of Nrf2 (red) and nuclei (DAPI, blue) in each group (scale bar = 50 μm). I Quantification of Nrf2 fluorescence intensity (fold of Control). J Representative immunofluorescence images of HO-1 (green) and nuclei (DAPI, blue) in each group (scale bar = 50 μm). K Quantification of HO-1 fluorescence intensity (fold of Control). Data are presented as mean ± SD and were analysed by one-way ANOVA followed by Tukey’s post hoc test (n = 3 independent experiments). (*p < 0.05 compared with Control group; **p < 0.01 compared with Control group; ##p < 0.01 compared with IR group)
Consistent with the in vivo findings, immunofluorescence staining showed that irradiation induced a marked accumulation of intracellular ROS, which was attenuated by quercetin co-treatment (Fig. 7F, G). Quantification (fold of Control) demonstrated that ROS fluorescence intensity was significantly increased in the IR group (2.98 ± 0.44; p < 0.01 vs. Control) and significantly reduced by quercetin co-treatment (IR + Que: 1.78 ± 0.18; p < 0.01 vs. IR), whereas quercetin alone had little effect on basal ROS levels (1.03 ± 0.14) (Fig. 7G).
Moreover, irradiation significantly decreased the fluorescence intensity of Nrf2 and its downstream effector HO-1, whereas quercetin partially restored their expression in irradiated endothelial cells (Fig. 7H–K). Specifically, Nrf2 fluorescence intensity (fold of Control) was reduced after irradiation (0.54 ± 0.05; p < 0.01 vs. Control) and was significantly partially restored with quercetin co-treatment (0.85 ± 0.13; p < 0.01 vs. IR), while quercetin alone remained close to baseline (0.96 ± 0.12) (Fig. 7I). A similar pattern was observed for HO-1: irradiation reduced HO-1 expression (0.54 ± 0.07; p < 0.01 vs. Control), and quercetin co-treatment significantly partially restored it (0.85 ± 0.09; p < 0.01 vs. IR); quercetin alone showed no obvious deviation from baseline (1.11 ± 0.14) (Fig. 7K). In parallel, merged images suggested enhanced nuclear localisation of Nrf2 under irradiation with quercetin co-treatment, supporting activation of an Nrf2-dependent antioxidant transcriptional program and subsequent up-regulation of HO-1.
Together, these in vitro data indicate that quercetin directly mitigates radiation-induced oxidative injury in endothelial cells by reducing ROS accumulation and partially restoring the Nrf2/HO-1 axis at both expression and subcellular localisation levels, providing mechanistic support for the endothelial-protective effects observed in the Ri-ED rat model.
Discussion
ED is a common complication in cancer patients, arising from both the malignancy itself and its treatment modalities [7, 8, 35]. Cancer-related ED often results from psychological stress, systemic inflammation, hormonal disturbances, or direct tumour compression of neurovascular structures [36]. In contrast, Ri-ED, especially in prostate and rectal cancer patients, is predominantly caused by ionising radiation, which directly damages the penile endothelium, smooth muscle, and cavernous nerves. This leads to oxidative stress, endothelial dysfunction, apoptosis, and fibrosis, ultimately impairing erectile function. Epidemiological data indicate that more than 50% of prostate cancer patients develop Ri-ED within 5 years post-radiotherapy, with the risk increasing with age [37]. Additionally, up to 31.8% of rectal cancer patients may experience severe ED 1 year after radiotherapy [38]. These findings emphasise the need to differentiate between cancer-induced ED and Ri-ED, as they involve distinct pathophysiological mechanisms and require targeted therapeutic approaches.
Radiation damage typically causes tissue remodelling, which is characterised by increased collagen deposition, reduction of smooth muscle, and destruction of the vascular structure [39, 40]. Therefore, some scholars believe that when choosing the therapeutic irradiation for prostate cancer, high-dose-rate prostate brachytherapy should be preferred, which may reduce early treatment-related damage to the patient in the early stage of the treatment [41]. For patients on long-term therapy, strategies must be devised to prevent and alleviate Ri-ED. In this study, we found that quercetin significantly enhanced the recovery of erectile function in rats with a radiation injury model, and also improved the cavernous smooth muscle/collagen ratio and α-SMA expression level. This suggests that quercetin may enhance vasodilatory capacity and tissue elasticity by restoring cavernosal smooth muscle content and reducing fibrosis. Likely associated with its antioxidant and anti-inflammatory properties, quercetin's mechanism of action may relieve radiation-induced penile fibrosis by inhibiting collagen synthesis and minimising extracellular matrix deposition, as strongly suggested by the remarkable decrease in the degree of fibrosis in treated rats. Similar antifibrotic effects have been reported in DMED and nerve damage-related ED [42], suggesting that quercetin may be able to attenuate fibrosis and improve erectile function under a variety of pathological conditions.
Radiation injury impairs penile erectile function through several mechanisms, among which oxidative stress plays a central pathological role. Excessive generation of ROS directly disrupts vascular homeostasis by damaging endothelial cells, degrading NO, and activating pro-inflammatory and apoptotic signalling pathways [43]. These effects collectively compromise endothelial integrity, reduce penile blood flow, and impair the smooth muscle relaxation essential for erection [39]. In the present study, we observed that quercetin significantly attenuated oxidative stress in rats with Ri-ED. Specifically, quercetin reduced ROS levels, increased SOD activity, and lowered MDA content—a marker of lipid peroxidation. These antioxidant effects contributed to the restoration of NO and cGMP levels, along with the normalisation of Ca^2^⁺ homeostasis.
Importantly, quercetin also upregulated the expression of CD31 and eNOS, which are markers of endothelial health, and partially restored neuronal markers such as nNOS and NF. Concomitantly, Masson and α-SMA staining revealed that quercetin attenuated radiation-induced collagen over-deposition and preserved (or partially restored) well-organised cavernosal smooth muscle compared with the model group, in which collagen replacement of smooth muscle was prominent. These findings suggest that quercetin improves endothelial function and neurovascular integrity by scavenging ROS and enhancing the endogenous antioxidant defence system, thereby maintaining NO/cGMP-mediated vasodilation [44]. Importantly, accumulating evidence indicates that activation of the Nrf2/HO-1 pathway plays a critical role in preserving NO/cGMP signalling and endothelial function in erectile dysfunction models. Engin et al. demonstrated that pharmacological activation of Nrf2 significantly improved diabetic erectile dysfunction by suppressing oxidative stress–induced endothelial injury and restoring NO bioavailability and cGMP signaling [45]. Similarly, Song et al. reported that activation of the Nrf2/HO-1 pathway ameliorated erectile dysfunction in cavernous nerve injury models by reducing oxidative stress–mediated neural damage and preserving downstream NO/cGMP signaling [46]. These findings provide strong mechanistic support for the involvement of the Nrf2/HO-1 axis in regulating NO/cGMP-dependent erectile function and are consistent with our observations in the Ri-ED model. The preservation of endothelial (CD31, eNOS) and neuronal (nNOS, NF) markers, together with reduced fibrosis and better-maintained smooth muscle architecture, is in line with the improved erectile function observed in the quercetin-treated rats, as reflected by higher ICPmax/MAP values and better erectile responses. This mechanism aligns with prior reports showing that oxidative stress impairs endothelial function and that eNOS dysfunction is a hallmark of Ri-ED [47]. Moreover, studies on space radiation-induced endothelial dysfunction have similarly shown that NOx and cGMP levels decrease significantly, supporting the relevance of ROS in radiation-related penile injury [48]. Thus, the antioxidant, anti-fibrotic, and endothelial-protective effects of quercetin are particularly significant in this pathological context, highlighting its therapeutic promise in preserving endothelial integrity and erectile function following radiation exposure [49].
Moreover, the attenuation of oxidative stress may further reduce endothelial cellapoptosis, thus indirectly improving erectile function. The current study showed that quercetin downregulated the pro-apoptotic protein Bax, upregulated the anti-apoptotic protein Bcl-2, and reduced the proportion of TUNEL-positive cells, thereby regulating apoptosis. Immunohistochemical analysis indicated that these changes in Bcl-2 and Bax expression were predominantly observed in the penile corpus cavernosum, particularly within endothelial and smooth muscle cell regions—tissues that are crucial for maintaining erectile function and are highly susceptible to radiation damage. This suggests that quercetin inhibits apoptosis and protects penile tissue from radiation damage by regulating the Bcl-2/Bax balance. Similar mechanisms have been reported in the cardioprotective effects of quercetin, suggesting that its anti-apoptotic properties may be tissue-broad-spectrum [50]. Similar to previous findings, quercetin was also able to inhibit mitochondrial apoptosis in both cardiac and hepatic injury models [13].To our knowledge, this is the first report of quercetin modulating apoptosis in radiation-injured penile tissue, suggesting its potential role in preserving cavernous smooth muscle integrity and supporting erectile function.
Remarkably, Nrf2, a pivotal transcription factor that governs the antioxidant response, triggers the upregulation of phase II detoxification enzymes, such as HO-1, thereby fortifying the cell's resilience against oxidative stress [51]. Our research demonstrated that quercetin remarkably enhanced the expression levels of Nrf2 and HO-1, indicating its potential to mediate antioxidant and anti-apoptotic activities via the Nrf2/HO-1 signalling pathway, thus validating its antioxidant mechanism. Nrf2 and HO-1 are the primary regulators of cellular redox homeostasis, and several studies have demonstrated that their activation is linked to the improvement of DMED and chronic inflammation, further supporting the potential value of the Nrf2/HO-1 pathway in the treatment of Ri-ED [23]. Consistent with these in vivo findings, our endothelial cell experiments further confirmed that quercetin upregulated Nrf2 and HO-1 expression under radiation-induced oxidative stress, strengthening the mechanistic link between this pathway and its protective effects. Our findings are in strong agreement with previous studies on the protective effects of quercetin and other flavonoids in models of oxidative stress-related diseases, including diabetes, cardiovascular disorders, and neurogenic ED [52, 53]. It has been demonstrated that quercetin can upregulate Nrf2 and HO-1 expression, inhibit the pro-apoptotic protein Bax, and enhance the expression of the anti-apoptotic protein Bcl-2, thereby exerting antioxidant, anti-apoptotic, and neurovascular protective effects [54, 55]. Building on this foundation, our study further shows that quercetin not only exerts anti-fibrotic, anti-apoptotic and neurovascular protective effects in a Ri-ED rat model, but also directly protects endothelial cells from radiation-induced oxidative injury by activating the Nrf2/HO-1 pathway and reducing ROS accumulation. These integrated in vivo and in vitro data link the observed functional and structural improvements to specific antioxidant and anti-apoptotic mechanisms, thereby strengthening the mechanistic basis and experimental evidence for the broad therapeutic potential of quercetin in oxidative stress–related forms of ED.
In conclusion, this study provides compelling evidence that quercetin exerts protective effects against Ri-ED by alleviating corpus cavernosum fibrosis, restoring vascular endothelial function, reducing oxidative stress, and inhibiting apoptosis (Fig. 8). These therapeutic actions are primarily mediated through the activation of the Nrf2/HO-1 signalling pathway, which plays a central role in redox homeostasis and cellular defence mechanisms [56–58]. However, several limitations of this study should be acknowledged. The experiments were conducted in a rat model, which cannot fully recapitulate the complex pathophysiology and comorbidities of human Ri-ED, thereby limiting direct clinical extrapolation. Only a single radiation regimen and treatment schedule were used. Although two doses of quercetin (10 and 40 mg/kg) were selected based on previous in vivo reports and our pilot data on efficacy and tolerability, a systematic dose–response analysis was not performed [29]. In addition, although quercetin was administered for 28 consecutive days without apparent adverse effects, such as abnormal behavior or significant body weight loss, a comprehensive and time-dependent toxicity assessment was not conducted. Therefore, potential short-term or long-term organ-specific toxicities cannot be fully excluded and warrant further systematic toxicological and safety evaluation prior to clinical translation. In addition, pharmacokinetic profiling was not conducted in this model, so the optimal human dose, the appropriate route of administration (e.g., oral and improved formulations), and the exposure required to protect penile tissue remain to be defined. Ultimately, our mechanistic investigation primarily focused on the Nrf2/HO-1 axis. At the same time, other potentially important pathways, such as inflammatory signalling and eNOS-mediated vasodilation, were not assessed and should be explored in future studies.Fig. 8. Mechanistic scheme showing how quercetin alleviates radiation-induced erectile dysfunction by modulating oxidative stress and apoptosis via activation of the Nrf2/HO-1 pathway
Despite its limitations, this study provides meaningful translational insight. Quercetin, a naturally derived flavonoid with a well-established safety profile, exhibits potent antioxidant, anti-apoptotic, and anti-fibrotic effects, suggesting its value as an adjunct rather than a replacement for current therapies in patients undergoing pelvic radiotherapy. As PDE-5 inhibitors remain the first-line treatment for ED but often perform poorly in the presence of severe vascular and neurogenic damage, targeting upstream oxidative stress, endothelial dysfunction, and fibrosis with quercetin may enhance their efficacy, allowing for additive or synergistic benefits. Future studies should therefore compare quercetin with PDE-5 inhibitors and test combination regimens in Ri-ED models, followed by early-phase clinical trials to evaluate optimised dosing, safety and erectile outcomes in cancer survivors with radiation-related sexual dysfunction.
Conclusion
Quercetin improved erectile function and reduced penile tissue damage in a rat model of Ri-ED, while alleviating oxidative stress, apoptosis, and fibrosis, and preserving endothelial, smooth muscle, and neurovascular markers. In vivo and in vitro findings consistently indicate that quercetin confers partial protection against radiation-induced injury, at least in part, via Nrf2/HO-1–mediated modulation of oxidative stress and apoptosis, supporting its further evaluation as a potential adjunctive natural compound for Ri-ED.