Metabolic and Ischemic Stress Synergistically Drive Time-Dependent Progression of Vasculogenic Erectile Dysfunction in a Rat Model

doi:10.21203/rs.3.rs-7886871/v1

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Metabolic and Ischemic Stress Synergistically Drive Time-Dependent Progression of Vasculogenic Erectile Dysfunction in a Rat Model

https://doi.org/10.21203/rs.3.rs-7886871/v1

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Background

Erectile dysfunction (ED) is a common male sexual disorder, with the vasculogenic subtype primarily driven by endothelial injury and metabolic dysfunction. Current research often lacks a systematic, time-course analysis of its progressive pathology. We aimed to delineate the temporal dynamics of functional impairment, structural remodeling, and molecular alterations using two rat models: high-fat diet (HFD) and HFD combined with iliac artery cuff placement (HFD + Surgery), evaluating them at weeks 4, 8, 12, and 16.

Results

The HFD + Surgery group exhibited earlier onset and more severe pathological changes than the HFD group. By week 4, early functional impairment was evident, marked by downregulation of endothelial nitric oxide synthase (eNOS) and calponin. From week 8, we observed reduced erectile function (maximal ICP/MAP ratios) and nitric oxide (NO) levels, smooth muscle phenotypic transition, metabolic disturbances, and sustained inflammatory and oxidative stress activation. Fibrosis-driven structural remodeling commenced after week 12, characterized by significant increases in TGF-β1 expression and collagen deposition.

Conclusions

Our findings delineate a clear, three-stage pathological progression of vasculogenic ED: early dysfunction (weeks 4–8), phenotypic transition and metabolic disturbance (weeks 8–12), and structural remodeling (after week 12). This comprehensive analysis highlights the synergistic role of ischemia in disease progression and provides critical temporal data for identifying key mechanistic targets and optimal intervention windows.

Vasculogenic Erectile Dysfunction

Disease Progression

Time-Dependent

Rat Model

Metabolic Stress

Ischemic Stress

Erectile dysfunction (ED) is defined as the persistent inability to achieve or maintain a penile erection sufficient for satisfactory sexual performance¹. Epidemiological data indicate that up to 71% of men may experience ED at some point in their lives, which significantly impairs the quality of life for both patients and their partners ^2,3. ED is strongly associated with metabolic disorders such as dyslipidemia, obesity, diabetes, and hypertension, and is considered an early warning sign of cardiovascular disease and type 2 diabetes ^4,5. Approximately 60% to 80% of ED cases are attributed to vascular injury, primarily atherosclerosis (AS)⁶. Persistent metabolic disturbances can induce endothelial dysfunction and oxidative stress, which accelerates the progression of AS, reduces blood flow to the penile arterioles, and ultimately leads to structural remodeling and vasculogenic ED ^7–9. Despite this understanding, the time-dependent pathological evolution of ED under combined metabolic overload and impaired blood flow is still poorly defined, leaving key transition points and potential therapeutic windows uncharacterized.

To better elucidate disease progression, animal models are indispensable. However, existing models have several limitations¹⁰. Long-term high-fat or high-sucrose feeding reliably induces systemic metabolic derangement and progressive atherosclerosis, yet reported induction periods vary markedly (6–20 weeks), hindering precise identification of peak pathology and substantially increasing husbandry costs^11–13. Alternatively, iliac arterial ballooning or ligation produces immediate, high-grade stenosis but fails to replicate the gradual atherogenic process, while postoperative thrombosis and distal ischemia may complicate interpretation of hind-limb perfusion and systemic metabolism^14–16. Furthermore, most studies rely on a single tissue harvest between weeks 8 and 16, providing only a static snapshot of the disease. This precludes a continuous, dynamic evaluation of metabolic imbalance, progressive perfusion decline, escalating oxidative stress, and the eventual emergence of ED.

To address these gaps, we established a dual-hit rat model combining metabolic stress (high-fat diet) with ischemic stress (polyethylene cuff placement around the common iliac artery) as previously described^17,18. We collected blood and penile tissues at 4, 8, 12, and 16 weeks for systematic analysis of lipid profiles, inflammatory and oxidative stress markers, histological changes, and erectile function. We show that combined stress jointly drives disease onset and severity compared to metabolic stress alone. These findings highlight a synergistic interaction between the ischemic and metabolic components that jointly drive disease progression. This dynamic characterization of the disease provides an experimental foundation for future mechanistic studies and the identification of distinct intervention windows for targeted therapy.

Model Establishment and Time-Dependent Changes in General Physiological Parameters

We established a time-course model of vasculogenic erectile dysfunction by combining a high-fat diet (HFD) with bilateral iliac artery cuff placement, imposing metabolic and ischaemic stress in male rats. Group allocation and sampling schedules at weeks 4, 8, 12, and 16 are outlined schematically (Fig. 1A), and the surgical diagram shows PE cuff placement around both common iliac arteries (Fig. 1B). Body weight increased progressively in all groups. From week 8 onward, both HFD and HFD + Surgery groups weighed significantly more than the control group (p < 0.05), with no difference between the two model groups; intra-group analysis confirmed a continuous rise over time, reflecting the persistent metabolic burden of HFD (Fig. 1C). Macroscopically, penile size in the HFD + Surgery group decreased gradually, most notably at weeks 12 and 16 (Fig. 1D). Correspondingly, the penile organ index was significantly lower than that of the control group from week 8 onward in the HFD + Surgery group, with the greatest reduction at week 16 (p < 0.05); the HFD group showed a significant decrease only at week 16 (p < 0.05). No statistical difference was detected between the two model groups at any time point. Intra-group comparisons indicated that the HFD group showed a significant decline at week 16 versus weeks 8 and 12 (p < 0.05), whereas the HFD + Surgery group displayed no further time-dependent change (Fig. 1E).

Time-Dependent Changes in Erectile Function and Iliac Artery Structure

Max ICP /MAP ratios declined continuously over time (Fig. 2A). Compared with the control group, the HFD + Surgery group showed a significant decrease starting from week 8 (P<0.05), whereas the HFD group exhibited a significant decrease only from week 12 ༈ p<0.05༉. At week 16, the ratio in the HFD + Surgery group was further lower than that in the HFD group ༈p<0.05༉. Intra-group comparisons indicated a persistent downward trend in both model groups: the HFD + Surgery group showed significant reductions from week 8 onward, with greater declines at weeks 12 and 16 ༈p<0.05༉, while the HFD group showed significant decreases at weeks 8 and 12 ༈p<0.05༉.The mating test further verified the functional impairment (Fig. 2C). From week 12 onward, intromission frequency in both model groups was significantly lower than in the control group ༈p<0.05༉and remained low through week 16. Intra-group analysis showed that the HFD + Surgery group at week 16 had significantly fewer intromissions than at weeks 4 and 8 ༈p<0.05༉, whereas the HFD group at week 16 was significantly lower than at week 8 ༈p<0.05༉.H&E staining of the common iliac artery revealed vascular changes consistent with these functional findings (Fig. 2D). The HFD group exhibited medial thickening and mild stenosis from week 8, whereas the HFD + Surgery group showed more pronounced luminal narrowing, intimal hyperplasia and medial disorganization, which became more severe at weeks 12 and 16, with obvious luminal reduction. These results collectively demonstrate that cuff-induced stenosis combined with a HFD accelerates iliac artery pathology and aggravates ED.

Figure 2 Time- dependent changes in erectile function and iliac artery structure. (A) Representative images of ICP and MAP recording at different timepoints. (B) Max ICP/MAP ratios at different timepoints. This metric provides a quantitative assessment of erectile function(n = 5). (C) The intromission frequency, a behavioral measure of mating success, recorded at the specified time points(n = 5). (D) Representative HE staining of iliac artery cross-sections at different timepoints. scale bar:200 µm(n = 3). Data were expressed as mean ± SEM. a: vs control group at the same time point, p < 0.05; b: vs HFD group at the same time point, p < 0.05; c: vs week 4 in the same group, p < 0.05; d: vs week 8 in the same group, p < 0.05; e: vs week 12 in the same group, p < 0.05.

Time-Dependent Changes in Serum Metabolic, Hormonal, and Inflammatory Markers

To further evaluate the alterations in metabolic disorders, hormone levels, and inflammatory responses, we assessed the dynamic changes of relevant serum indicators across different time points. Serum lipid parameters changed significantly over time. In the control group, triglycerides (TG) and total cholesterol (TC) showed slight but statistically significant increases from week 12 compared with weeks 4 and 8 (p < 0.05), and low-density lipoprotein cholesterol (LDL-C) increased at week 16 (p < 0.05), while remaining within the normal physiological range. In contrast, TG in the HFD group was already significantly higher than that in the control group from week 4 (p < 0.05), whereas the HFD + Surgery group showed a significant increase from week 8 (p < 0.05). TC and LDL-C levels in both model groups were significantly elevated from week 8 onward (p < 0.05) and continued to rise thereafter. High-density lipoprotein cholesterol (HDL-C) levels in both model groups were significantly lower than those in the control group from week 8 onward (p < 0.05) and continued to decline. No significant differences in lipid parameters were observed between the two model groups at any time point (Fig. 3A–D). Serum testosterone in the control group showed a mild decrease at week 16 compared with week 4 (p < 0.05) but remained within the normal range. In the HFD + Surgery group, testosterone decreased significantly from week 8, and in the HFD group from week 12(p < 0.05), with continuous declines thereafter; no significant differences were detected between the two model groups at any time point (Fig. 3E). Nitric oxide (NO) levels in both model groups were significantly lower than those in the control group from week 8 (P < 0.05) and continued to fall; at week 16, NO in the HFD + Surgery group was significantly lower than in the HFD group (p < 0.05) (Fig. 3F). Interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) in the control group showed modest increases from week 12 compared with weeks 4 and 8 (p < 0.05). IL-6 in both model groups was significantly higher than that in the control group from week 4 (p < 0.05), and the HFD + Surgery group was consistently higher than the HFD group from week 8 onward (p < 0.05)(Fig. 3G). TNF-α in the HFD + Surgery group was significantly elevated from week 4 ༈p < 0.05༉, whereas the HFD group increased from week 8 (p < 0.05); from week 8 onward, the HFD + Surgery group remained significantly higher than the HFD group at all time points ༈p < 0.05༉(Fig. 3H). In summary, a HFD combined with vascular stenosis induces early metabolic imbalances, endothelial dysfunction, and systemic inflammation, all of which intensify in a time-dependent manner and jointly contribute to the development and progression of vascular erectile dysfunction.

Time-Dependent Changes in Penile Corpus Cavernosum Structure and Oxidative Stress Markers

To further elucidate the effects of HFD and blood flow restriction on penile tissue structure and oxidative stress levels, Masson’s trichrome staining was performed to evaluate histological changes in the corpus cavernosum at different time points. In the control group, the tissue architecture remained intact across all time points, with evenly distributed and tightly arranged smooth muscle and minimal collagen content. In contrast, both the HFD group and HFD + Surgery group exhibited a progressive decrease in smooth muscle distribution over time. Notably, the HFD + Surgery group showed more pronounced changes, indicating more severe tissue disruption and fibrosis compared to the HFD group (Fig. 4A). Quantitative analysis was consistent with the histological observations. The smooth muscle-to-collagen ratio was significantly lower in both model groups than in the control group from week 8 onward (p < 0.05), and further decreased in the HFD + Surgery group compared to the HFD group from week 12 (P < 0.05), suggesting that blood flow restriction accelerates the fibrosis process induced by HFD. Additionally, a slight decrease in this ratio was observed in the control group at week 16 compared to week 4 (P < 0.05), possibly reflecting mild age-related physiological changes (Fig. 4B).

Oxidative stress indicators showed that reduced reduced glutathione (GSH) levels were significantly lower in both model groups than in the control group starting from week 8 (P < 0.05) and continued to decline thereafter (P < 0.05); by week 16, GSH levels in the HFD + Surgery group were significantly lower than in the HFD group (P < 0.05) (Fig. 4C). Malondialdehyde (MDA), a marker of lipid peroxidation, was significantly elevated in both model groups from week 8 (P < 0.05) and exhibited a time-dependent increase thereafter (P < 0.05) (Fig. 4D). Superoxide dismutase (SOD) levels showed a significant decline in the HFD group at week 8 (P < 0.05), while the HFD + Surgery group exhibited a significant reduction as early as week 4 compared to the control group (P < 0.05), and were further decreased relative to the HFD group at week 12 (P < 0.05).Collectively, these results suggest that the combination of HFD and blood flow restriction synergistically aggravates oxidative stress and tissue fibrosis in the corpus cavernosum, with the severity of injury progressing in a time-dependent manner.

Time-Dependent Molecular Mechanisms of Penile Dysfunction and Fibrosis

To further investigate the molecular mechanisms by which HFD combined with blood flow restriction impairs penile function and promotes tissue fibrosis, we examined the expression levels of key functional proteins using Western blot analysis, including endothelial nitric oxide synthase (ENOS), the contractile phenotype marker calponin, the synthetic phenotype marker osteopontin (OPN), and the fibrosis-related factor transforming growth factor-β(TGF-β) (Fig. 5A). In the control group, the expression levels of calponin, OPN, and TGF-βexhibited mild fluctuations over time (p < 0.05), potentially associated with physiological aging. In contrast, both model groups showed more pronounced and pathological trends. Specifically, ENOS expression was significantly reduced in the HFD + Surgery group as early as week 4 (p < 0.05), while the HFD group displayed a significant decline beginning at week 12 p < 0.05). Moreover, ENOS expression in the HFD + Surgery group was significantly lower than that in the HFD group from week 8 onward (p < 0.05), suggesting that blood flow restriction exacerbates HFD-induced endothelial dysfunction (Fig. 5B). TGF-β expression exhibited a time-dependent increase, with significantly higher levels in the HFD + Surgery group starting at week 8 compared to both the control and HFD groups (p < 0.05), whereas the HFD group showed a significant elevation at week 12 compared to the control ( p < 0.05) (Fig. 5C), indicating earlier and more sustained activation of fibrotic pathways under combined intervention. Smooth muscle phenotype-related markers also demonstrated dynamic temporal changes. The synthetic marker OPN progressively increased in both model groups, with the HFD + Surgery group showing significantly higher levels than both the control and HFD groups from week 8 onward (p < 0.05); the HFD group showed a significant increase compared to the control from week 12 (p < 0.05) (Fig. 5D). Conversely, the contractile marker calponin exhibited a gradual decline over time. The HFD + Surgery group showed significantly lower levels than the control as early as week 4 (p < 0.05) and was consistently lower than the HFD group at all subsequent time points (p < 0.05) (Fig. 5E).To further confirm the functional impairment of endothelial and smooth muscle components in penile tissue, dual immunofluorescence staining was performed to assess the expression of platelet endothelial cell adhesion molecule-1 (CD31) and alpha-smooth muscle actin (α-SMA) (Fig. 5F). The expression of CD31 and α-SMA in the corpus cavernosum of the control group remained stable across all time points, with consistent fluorescence intensity, whereas both model groups exhibited a gradual decline in the fluorescence intensity of both markers over time. Quantitative analysis confirmed this trend. CD31 fluorescence intensity was significantly decreased in the HFD + Surgery group compared to both the control and HFD groups from week 8 (p < 0.05), and reduced in the HFD group starting at week 8 (p < 0.05) (Fig. 5G). α-SMA fluorescence intensity was significantly lower in the HFD + Surgery group than in both the control and HFD groups from week 8 (p < 0.05), and the HFD group also showed a significant decline at week 12 compared to the control (p < 0.05) (Fig. 5H). Collectively, these findings suggest that HFD combined with blood flow restriction synergistically impairs endothelial and smooth muscle function, promotes a phenotypic shift in smooth muscle cells, and activates profibrotic signaling, ultimately exacerbating structural and functional deterioration of penile tissue.

VED is the most prevalent subtype of ED; its hallmark is insufficient penile perfusion, typically caused by flow-limiting lesions such as atherosclerosis ^4,19–21. Existing animal models usually rely on a single trigger—e.g., high-fat feeding or cavernous nerve injury—thus failing to replicate the clinical scenario in which metabolic derangement and arterial insufficiency act synergistically ^22,23. In this study, we established a composite VED model by combining a HFD with iliac artery cuff placement to couple systemic metabolic stress with a localized ischemic insult. By sampling at 4, 8, 12 and 16 weeks, we dynamically traced the time-dependent transition from functional impairment to structural remodeling, delineating stage-specific pathological features. This time-dependent approach provides a framework for identifying critical disease nodes and for developing targeted therapeutic interventions.

In the pathogenesis of VED, the interplay between metabolic stress and regional hypoperfusion is considered pivotal for triggering functional decline and tissue remodeling. In our study, the HFD + Surgery group showed a significant drop in MAX ICP /MAP as early as week 8, with a further reduction versus the HFD group by week 16, indicating that flow restriction is a primary driver of early functional loss under metabolic overload ^24,25. The decrease in intromission frequency lagged behind the ICP change, underscoring the lower sensitivity of behavioral readouts. From week 12 onward, the HFD + Surgery rats also exhibited a progressive reduction in penile size and organ index, reaching a nadir at week 16, consistent with concomitant structural impairment. Histology confirmed luminal narrowing of the iliac artery beginning at week 8, providing an anatomical basis for perfusion deficits, whereas comparable lesions in the HFD group emerged only after week 12. Collectively, these findings demonstrate that regional blood-flow restriction accelerates both functional deterioration and structural damage, highlighting the value of the HFD + Surgery model for capturing the early trajectory of VED.

Hyperlipidemia is well-recognized as a major risk factor for ED ^1,26. Consistent with this, HFD feeding in both experimental groups induced pronounced dyslipidaemia between weeks 4 and 8, as evidenced by increased body weight, TG, TC, and LDL-C, along with reduced HDL-C. This aligns with clinical observations wherein the LDL/HDL ratio and absolute HDL-C have been independent predictors of VED²⁷. And abnormal LDL-C has been associated with ED, partly mediated through testosterone deficiency ²⁸. As anticipated, serum testosterone declined markedly from week 8 onward in both models, supporting the notion that disturbed lipid metabolism compromises steroidogenesis^29,30. Low testosterone, in turn, diminishes eNOS activity, promotes oxidative stress and fibrosis, and impairs endothelial and smooth muscle function, thereby creating a vicious cycle that exacerbates ED progression ³¹.The pro-inflammatory milieu was more pronounced in the HFD + Surgery group: IL-6 and TNF-α rose significantly from week 4, suggesting that local hypoperfusion amplifies diet-induced systemic inflammation. TNF-α activates the NF-κB pathway, up-regulating endothelial adhesion molecules and fostering endothelial dysfunction and atherosclerosis ³². IL-6 increases chemokines such as monocyte chemoattractant protein-1 (MCP-1), enhancing monocyte recruitment and vascular inflammation ^33,34. This systemic inflammatory state, amplified by ischemia, creates a vicious cycle that exacerbates oxidative stress in penile tissue.

Consistent with enhanced oxidative injury, the HFD + Surgery cohort displayed an earlier and more severe oxidative response: SOD activity declined from week 4 and was significantly lower than in the HFD group by week 12, while GSH fell and MDA rose from week 8, with the gap between groups widening over time. SOD and GSH are key endogenous antioxidants—SOD converts superoxide to H₂O₂, and GSH, together with peroxidases, removes downstream peroxides—jointly preserving redox homeostasis and vascular integrity ^35,36. The concomitant rise in MDA, a marker of lipid peroxidation, reflects ongoing cellular injury ^37,38. These changes collectively heighten oxidative burden in the corpus cavernosum, a central step in ED pathogenesis. Furthermore, NO levels dropped from week 8 in both models and were further reduced in the HFD + Surgery group by week 16. This is critical because metabolic and ischemic stress, via excessive ROS, restrict NO synthesis and bioavailability, thereby aggravating endothelial dysfunction and propelling VED progression ³⁹.

Persistent metabolic stress, inflammation and oxidative imbalance progressively drive structural remodelling and functional decline of the corpus cavernosum. eNOS is the rate-limiting enzyme for NO synthesis—was already markedly reduced in the HFD + Surgery group at week 4, preceding the change in the HFD group and mirroring the fall in circulating NO, indicating that local hypoperfusion accelerates diet-induced endothelial dysfunction ^39,40. The subsequent decrease in CD31, a key regulator of endothelial adhesion and barrier integrity, from week 8 signaled overt endothelial disruption^41,42. Smooth-muscle injury evolved in parallel with the endothelial defect. The contractile marker calponin declined from week 4 and remained consistently lower than in HFD animals, denoting early loss of cavernosal smooth-muscle contractility. Conversely, the synthetic marker OPN rose significantly from week 8, and α-SMA immunofluorescence fell over the same period, together indicating a shift from a contractile to a synthetic phenotype and progressive cytoskeletal disintegration. Such phenotype switching—triggered by sustained lipid overload and ischaemic stress—reduces contractile capacity, increases cytokine release and extracellular-matrix (ECM) production, and thereby initiates fibrotic signalling cascades ^43,44. TGF-β, a master profibrotic cytokine, was up-regulated in the HFD + Surgery group from week 8, earlier and to a greater extent than in HFD animals. Consistent with this rise, Masson’s trichrome staining showed reduced smooth muscle and increased collagen from week 8 in both models, with a significantly lower smooth-muscle-to-collagen ratio in the HFD + Surgery group from week 12, confirming that hypoperfusion exacerbates diet-induced tissue remodelling. By stimulating fibroblast activation and promoting ECM deposition and collagen cross-linking, TGF-β sustains fibrotic progression and culminates in irreversible structural and functional impairment ^45,46. In summary, our data reveal that VED evolves through a temporally coordinated cascade: At week 4, simultaneous down-regulation of eNOS and calponin marks the onset of endothelial and smooth muscle functional loss. From week 8, decreased CD31 and α-SMA along with rising OPN denote endothelial disintegration and smooth muscle phenotype conversion, which accelerates local structural remodeling. By week 12, pronounced TGF-β activation drives overt fibrosis. The tight synchrony between endothelial and smooth muscle injury, and their collective progression toward fibrosis, underpins the relentless worsening of VED.

Our model offers several distinctive advantages.First, the inclusion of four scheduled observation points allows stage-specific capture of disease evolution and precise definition of pathological turning points and therapeutic windows. Second, the protocol interrogates multiple, inter-linked mechanisms—metabolic disturbance, systemic inflammation, haemodynamic impairment, oxidative stress and tissue remodelling, thereby charting the continuum from reversible functional loss to irreversible structural damage. Third, by combining metabolic overload with local vascular stenosis, the model replicates the clinical scenario of metabolic syndrome complicated by vascular disease, which enhances translational relevance. Collectively, these features deepen our understanding of VED progression and supply a robust platform for timing clinical interventions, identifying therapeutic targets and pursuing mechanistic studies.This study has two principal limitations. First, the observation period was comparatively short and may not encompass the transition to late, irreversible pathology. Second, neurogenic, psychogenic and other non-vascular contributors were not assessed. Extending the study duration, integrating interventional or omics-based screens to uncover early driver pathways and biomarkers, and evaluating the model in cardiovascular comorbidity settings will further broaden its experimental utility.

This study, which combines a HFD with iliac artery cuff–induced flow restriction, systematically maps the temporal progression of VED from systemic metabolic derangement to local structural remodelling. Our findings identify pivotal chronological nodes and delineate the underlying mechanistic cascade, thereby providing a solid experimental foundation for mechanistic elucidation, biomarker discovery and rational timing of therapeutic intervention in VED.

animal experiments

Eight-week-old male Sprague–Dawley were acquired from SPF (Beijing) Biotechnology Co.,Ltd. All animal experiments were conducted in accordance with the guidelines approved by the Animal Ethics and Welfare Committee of Beijing University of Chinese Medicine (BUCM-2024101202-4030). The rats housed at 22–24°C under a 12 h light–dark cycle, and had access to food and water. After 1 week of acclimation, they were stratified by body weight and randomly assigned to three groups (n = 20/group). The control group was provided with a normal diet and underwent sham exposure of both common iliac arteries. The HFD group was provided with a HFD and underwent sham exposure. The HFD + Surgery group was provided with a HFD and had fixed-diameter polyethylene cuffs placed around both common iliac arteries. HFD is composed of 45% kcal% Fat in rodent diet (Changzhou SYSE. Bio-tec Co.Ltd, Changzhou, China).The experiment lasted 16 weeks; five rats per group were euthanised at weeks 4, 8, 12, and 16 to establish the time course of ED. At each endpoint, all animals were euthanised under inhaled isoflurane anaesthesia. Serum and tissue samples were collected, photographed, and weighed to calculate organ indices; specimens were then fixed in 4% paraformaldehyde or snap-frozen in liquid nitrogen for subsequent analyses.

Iliac artery cuff surgery

Reference for establishing the vascular ED model¹⁷. Rats were anesthetized inhalation of isoflurane, secured in a supine position on a surgical board, and the surgical site was shaved and disinfected. A midline longitudinal abdominal skin incision was made to expose the abdominal muscle layer, followed by dissection of adipose tissue until the common iliac arteries were exposed. Two polyethylene tubes (PE-160, Becton Dickinson, Franklin Lakes, NJ), each 5 mm in length with an inner diameter of 1.14 mm and an outer diameter of 1.57 mm, were loosely placed around each common iliac artery. In the control and HFD group, the common iliac arteries were exposed following identical procedures, but no polyethylene tubes were placed. Following the surgical intervention, the abdominal muscle layer and skin were closed in layers using 3 − 0 sterile silk sutures. Rats were returned to their cages after full recovery from anesthesia.

Mating Test

48 and 4 h before testing, oestrus was induced in female rats by subcutaneous injections of oestradiol benzoate (10 µg / 100 g body weight) and progesterone (0.5 mg / 100 g body weight), respectively ⁴⁷. Each male was allowed to acclimate to the observation cage for 5 min and was then paired 1:1 with an oestrous female. Tests were conducted under dim light and minimal noise for 30 min. Male sexual behaviour—including following/chasing, grasping, mounting, intromission and ejaculation—was recorded manually^48,49.

Measurement of ICP/MAP

Rats were anaesthetised with inhaled isoflurane. A heparinised saline-filled PE-50 catheter was retrogradely advanced into the carotid artery for continuous monitoring of mean arterial pressure (MAP). Through a midline abdominal incision, the pelvic ganglion and cavernous nerve were exposed, and the cavernous nerve was stimulated with a protective bipolar electrode. A heparinised 25-G butterfly needle was inserted horizontally into the corpus cavernosum and connected to a BL-425I data-acquisition system (Chengdu Taimeng Software Co., China). Electrical stimulation was delivered as a continuous square wave (5 ms pulse width, 20 Hz, 10 V) for 60 s, with ≥ 5 min between stimulations. Intracavernosal pressure (ICP) and MAP were recorded simultaneously, and the maximal ICP/MAP ratio (Max ICP/MAP) was calculated from the peak ICP obtained during each stimulation.

Measurement of Serum Markers

TG, TC, HDL-C, LDL-C, NO were quantified with commercial colorimetric kits according to the manufacturers’ instructions. Serum testosterone, IL-6 and TNF-α were determined using enzyme-linked immunosorbent assay (ELISA) kits (CLBA458Ge; ILBA33Ra; MM-0190R1), following the protocols supplied by the respective kit providers.

Measurement of Oxidative Stress Markers

SOD, MDA, GSHlevels in rat penile tissue were quantified with commercial colorimetric assay kits, following the manufacturers’ instructions.

Masson’s trichrome staining

Rat penile tissue samples were fixed in 4% paraformaldehyde and embedded in paraffin. Masson’s trichrome staining was then performed. The proportion of red-stained muscle fibers to blue-stained collagen fibers was used to evaluate fibrosis in the penile tissue. Three samples were randomly selected per group, and three fields were randomly chosen from each sample. ImageJ was used for image analysis.

Hematoxylin–Eosin Staining of the Common Iliac Arteries

Iliac artery tissue samples were fixed in 4% paraformaldehyde, embedded in paraffin, and stained with hematoxylin and eosin (H&E). Sections were examined under a light microscope to evaluate luminal stenosis.

Immunofluorescence

Penile tissue paraffin sections (5 µm) were deparaffinised in xylene, rehydrated through graded ethanol, incubated overnight at 4°C with rabbit anti-CD31 (1 : 100, 11265-1-AP, Proteintech) and mouse anti-α-SMA (1 : 200, 67735-1-Ig, Proteintech), and then treated for 1 h at room temperature with Alexa Fluor 594–conjugated goat anti-rabbit IgG (CD31, red) and Alexa Fluor 488–conjugated goat anti-mouse IgG (α-SMA, green). After nuclear counter-staining with DAPI, sections were imaged under a confocal microscope (×400), and mean fluorescence intensities of CD31 and α-SMA were quantified in three random fields per section using ImageJ.

Western blotting

Total protein from rat penile tissue was extracted with RIPA lysis buffer containing protease and phosphatase inhibitors (Beijing LABLEAD Trading Co., Ltd., China). Protein concentration was determined with a BCA kit (Beijing LABLEAD Trading Co., Ltd., China). Equal amounts of protein (30 µg per lane) were separated by SDS-PAGE and transferred to PVDF membranes. Membranes were blocked 1X TBST containing 5% non-fat milk for 3 h at room temperature. They were incubated overnight at 4°C with the primary antibodies: ENOS (1: 500, 27120-1-AP, Proteintech), osteopontin (1: 1000, CY5333, Abways), TGF-β (1: 1000, 26155-1-AP, Proteintech), calponin (1: 1000, CY5204, Abways) and β-actin (1: 5000, 66009-1-Ig, Proteintech). After three washes with 1X TBST, membranes were incubated with goat anti-rabbit IgG or goat anti-mouse IgG secondary antibodies at room temperature for 1 hour. Protein bands were visualised using an enhanced chemiluminescence (ECL) kit (Beijing LABLEAD Trading Co., Ltd., China) and quantified by densitometry with ImageJ software.

Statistical analysis

Statistical analysis was performed with GraphPad Prism 10.1.2 (GraphPad Software, USA). Data are presented as mean ± SEM. Group differences were assessed by one-way ANOVA followed by Tukey’s multiple-comparison test. Statistical significance was determined at p < 0.05

Data availability

All uncropped original blots and raw data are included in the manuscript as Supplementary Information and Supplementary Data 1.

Competing interests

The authors declare no competing interests.

Funding

This study was funded by the National Natural Science Foundation of China (82174389), Zhangzhou Pien Tze Huang Pharmaceutical Co., Ltd. (YF2017-004-2024-08), and the High level Key Discipline of National Administration of Traditional Chinese Medicine - Traditional Chinese constitutional medicine (No.zyyzdxk-2023251).

Author Contribution

Y.Z. and J.Z. contributed equally to this work. Y.Z. was responsible for the study's conceptualization and animal model establishment. J.Z. conducted a portion of the animal experiments and helped with the manuscript's writing. Y.Z., S.H., and X.Z. collected the functional and physiological data. Y.Y., W.S., and H.M. handled the biochemical analysis and data curation. L.Z., M.X., and T.W. carried out the histological and morphological experiments. M.S., L.L., and Y.Z. acquired the funding, managed the project, and were responsible for the final manuscript's review and editing.

Acknowledgements

Not applicable

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Metabolic and Ischemic Stress Synergistically Drive Time-Dependent Progression of Vasculogenic Erectile Dysfunction in a Rat Model

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Version 1

Abstract

Background

Results

Conclusions

Figures

Introduction

Result

Model Establishment and Time-Dependent Changes in General Physiological Parameters

Time-Dependent Changes in Erectile Function and Iliac Artery Structure

Time-Dependent Changes in Serum Metabolic, Hormonal, and Inflammatory Markers

Time-Dependent Changes in Penile Corpus Cavernosum Structure and Oxidative Stress Markers

Time-Dependent Molecular Mechanisms of Penile Dysfunction and Fibrosis

Discussion

Conclusion

Materials and Methods

animal experiments

Iliac artery cuff surgery

Mating Test

Measurement of ICP/MAP

Measurement of Serum Markers

Measurement of Oxidative Stress Markers

Masson’s trichrome staining

Hematoxylin–Eosin Staining of the Common Iliac Arteries

Immunofluorescence

Western blotting

Statistical analysis

Data availability

Declarations

Competing interests

Funding

Author Contribution

Acknowledgements

References

Additional Declarations

Supplementary Files

Status:

Version 1