Hypertension impairs erectile function in spontaneously hypertensive rats by suppressing adiponectin in the penile corpus cavernosum

Introduction

Erectile dysfunction (ED) is a common male sexual disorder characterized by the persistent inability to achieve or maintain an erection sufficient for satisfactory sexual performance (1). The etiology of ED is multifactorial and closely associated with metabolic conditions such as hypertension, hyperlipidemia, and diabetes, as well as psychological factors (2). Among these factors, hypertension has been recognized as an independent risk factor for ED, with a reported prevalence of up to 68% in hypertensive patients, which is approximately two- to three-fold higher than that in normotensive individuals (3). However, the precise molecular mechanisms by which hypertension induces ED remain largely unclear, resulting in the limited therapeutic efficacy of phosphodiesterase type 5 inhibitors (PDE5 inhibitors) in hypertensive patients, with an efficacy of only approximately 60–70% (4). Therefore, elucidating the underlying molecular mechanisms responsible for hypertension-related ED is highly important for the development of novel therapeutic strategies.

Adiponectin (APN) is a cardiovascular-protective adipokine with anti-inflammatory and anti-atherosclerotic properties (5,6). Circulating APN levels are markedly reduced in patients with hypertension, and hypoadiponectinemia is considered an independent risk factor for essential hypertension (7). APN activates the phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) signaling pathway and promotes the phosphorylation of endothelial nitric oxide synthase (eNOS) at the Ser1177 residue, which is a key step in eNOS-mediated nitric oxide (NO) production and endothelium-dependent vasodilation (8,9). eNOS phosphorylation and NO generation are significantly impaired in the aortas of APN-knockout mice (10), whereas lentiviral APN overexpression or treatment with recombinant APN increases NO production in a dose-dependent manner by activating the AKT/eNOS pathway in endothelial cells (11,12). Therefore, APN may improve endothelial function in the penile corpus cavernosum and subsequently ameliorate ED via the PI3K-AKT-eNOS axis.

Icariin (ICA), a major bioactive flavonoid component isolated from Epimedium species, is one of the most widely used traditional herbal medicines for the treatment of ED, and its pharmacological activity is largely attributed to the total flavonoids contained in Epimedium (13). Among these compounds, ICA is the most abundant and biologically active constituent (14). ICA has been shown to improve erectile function in spontaneously hypertensive rats (SHRs), and its potential mechanisms involve the upregulation of eNOS expression, inhibition of Rho-associated coiled-coil containing protein kinase 2 (ROCK2) activity, and modulation of posttranslational modifications of eNOS (15-17). However, the precise molecular mechanisms underlying the beneficial effects of ICA remain unclear, particularly whether ICA treatment is associated with changes in APN expression, a key regulator of endothelial function.

Serum APN levels are consistently reduced in both hypertensive patients and animal models of hypertension (18,19). Although adipose tissue accounts for the majority of circulating APN, low levels of APN are also expressed in other organs, including the kidney, heart, and liver, where APN primarily exerts local microenvironmental effects (20). However, it remains unclear whether APN is expressed in the penile corpus cavernosum, whether hypertension regulates APN expression in this tissue, and whether the beneficial effects of ICA on hypertension-induced ED may involve regulation of APN expression and downstream AKT/eNOS signaling. On the basis of these observations, this study aimed to investigate the effects of hypertension and ICA treatment on APN expression in the penile corpus cavernosum of rats and to further explore whether APN-related PI3K/AKT/eNOS signaling may be involved in the beneficial effects of ICA on hypertension-associated ED. We present this article in accordance with the ARRIVE reporting checklist (available at https://tau.amegroups.com/article/view/10.21037/tau-2026-0386/rc).

Methods

Animals and experimental groups

Twelve-week-old male Wistar-Kyoto (WKY) rats and SHRs (Charles River, China) were used in this study. Each rat was assigned a unique identification number. Randomization was performed using a strain-stratified randomization method. Briefly, WKY rats and SHRs were numbered separately and then randomly allocated within each strain using a random number table. The rats were assigned to the following six groups, with six rats in each group: WKY, WKY treated with ICA (WKY + ICA), SHR, SHR treated with ICA (SHR + ICA), SHR receiving an empty adeno-associated viral vector (SHR + NC), and SHR receiving an adeno-associated virus carrying the APN gene (SHR + AAV).

The study included six experimental groups, with six rats in each group, resulting in a total sample size of 36 rats. The sample size estimation was based on the primary outcome measure and was performed using one-way analysis of variance (ANOVA), with a two-sided α level of 0.05 and a statistical power of 0.80. The calculation indicated that, under a design with six experimental groups and a total sample size of 36 rats, the study was able to detect a large between-group effect, with a Cohen’s f of 0.65. Preliminary experimental results indicated marked differences in the primary outcome measure between the model and intervention groups, suggesting a large expected effect size. In addition, previous similar animal studies have commonly used five or more animals per group (16,17,21-23). Therefore, a sample size of six rats per group was considered appropriate for the preliminary mechanistic validation conducted in the present study.

The rats in the WKY + ICA and SHR + ICA groups received ICA (10 mg/kg/day) by gavage for four weeks, whereas those in the WKY and SHR groups received an equal volume of vehicle [normal saline containing 2% dimethyl sulfoxide (DMSO)] for the same duration. At week 3, rats in the SHR + NC group were injected intracavernosally with an empty adeno-associated viral vector, whereas those in the SHR + AAV group were injected intracavernosally with an AAV carrying the APN gene (4.31×10¹³ vg/mL; GOSV5004403; GeneChem Co., Ltd., Shanghai, China). All experimental procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (NIH) and were approved by the Animal Ethics Committee of Southwest Medical University (approval No. SWMU20250140).

Measurement of maximal intracavernous pressure (ICPmax)/mean arterial pressure (MAP)

The rats were initially placed in an induction chamber and exposed to 3–5% isoflurane with an oxygen flow of 1–2 L/min until the righting reflex was lost. Thereafter, anesthesia was maintained with 1.5–2.0% isoflurane delivered through a face mask, and the depth of anesthesia was confirmed by the absence of pedal withdrawal. A heparinized 24G catheter was introduced into the left common carotid artery, while a 26G catheter was gently advanced into the penile corpus cavernosum. Both catheters were connected to a pressure recording device for continuous monitoring of MAP and ICPmax. Electrical stimulation of the cavernous nerve (3 V/5 V, 12 Hz, 5 ms pulse width, 30 s duration, and 3 min intervals) (24) was applied, and the values of ICPmax and MAP were obtained using a BL-420S data acquisition system (TECHMAN Technology, Chengdu, China). At the end of the recording, anesthesia was deepened to 5% isoflurane for 3–5 min to achieve euthanasia. Approximately 1–2 mL of carotid arterial blood was collected, and penile tissues were harvested. After being rinsed in ice-cold saline, the penis was separated into four parts. In the proximal region, the urethra, dorsal vein, and fascia were carefully removed with microforceps, and the remaining tissue was flash-frozen at −80 °C for Western blotting and NO assessment. The distal parts were either fixed in 4% paraformaldehyde or embedded in optimal cutting temperature (OCT) compound for immunohistochemistry and immunofluorescence processing.

Measurement of serum testosterone (T) levels

Serum T concentrations were determined using a commercial ELISA kit specific for rat samples (BPE30610; Langton Biotech, Shanghai, China) following the manufacturer’s protocol. Optical density (OD) values were read at 450 nm using a microplate reader. A standard curve was generated from the serial standard wells, and T levels in individual samples were calculated according to their corresponding OD values.

Determination of NO levels in penile corpus cavernosum

Penile corpus cavernosum tissues were homogenized on ice, and NO levels were quantified using a colorimetric NO assay kit (S0021S; Beyotime Biotechnology, Shanghai, China) according to the manufacturer’s instructions. The absorbance was measured at 540 nm using a microplate reader. A standard curve was constructed, and the NO concentration in each sample was calculated from the standard curve based on the corresponding OD values.

Western blot analysis of protein expression in the penile corpus cavernosum

Fresh penile corpus cavernosum was homogenized and lysed in radioimmunoprecipitation assay (RIPA) buffer (Beyotime), followed by centrifugation to collect the supernatant. Total protein content was determined using a BCA assay kit (Beyotime). The samples were denatured by boiling in loading buffer, separated by SDS-polyacrylamide gel electrophoresis, and subsequently transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with rapid blocking buffer (Epizyme, Shanghai, China) at room temperature for 30 min and incubated overnight at 4 °C with the following primary antibodies: anti-APN (1:1000, ab181281, Abcam), anti-PI3 kinase p85 (1:1000, 4292, Cell Signaling Technology), anti-phospho-PI3 kinase p85/p55 (1:1000, 4228, Cell Signaling Technology), anti-AKT (pan) (1:1000, 4691, Cell Signaling Technology), anti-phospho-AKT (Ser473) (1:1000, 4060, Cell Signaling Technology), anti-eNOS (1:2000, ab300071, Abcam), anti-phospho-eNOS (1:1000, 28939-1-AP, Proteintech), and anti-β-actin (1:2000, ab8227, Abcam). After being washed, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1.5 h at room temperature. Immunoreactive bands were visualized using an enhanced chemiluminescence detection kit (Oriscience Biotechnology Co., Ltd.), and band intensities were quantified using ImageJ software (version 1.8.0; NIH, USA).

Immunohistochemistry

Penile corpus cavernosum tissues were fixed in 4% paraformaldehyde, dehydrated, embedded in paraffin, sectioned, dewaxed, and subsequently blocked. The sections were incubated overnight at 4 °C with a rabbit anti-rat APN antibody (1:2000; ab181281; Abcam). After being rinsed with phosphate-buffered saline (PBS), the slides were treated with HRP-conjugated goat anti-rabbit IgG (1:100; Beyotime, China) for 30 min at 37 °C. Images were acquired using a digital slide scanner (Ningbo Kangfeng Bioinformation Technology Co., Ltd., China). The average optical density (AOD) of the immunoreactive areas was quantified using ImageJ software (NIH, USA).

Immunofluorescence

Frozen penile corpus cavernosum tissues were sectioned and counterstained with 4',6-diamidino-2-phenylindole (DAPI), and fluorescence images were acquired using a fluorescence microscope (Olympus Corporation, Japan). Green fluorescent protein (GFP) signals visualized under blue excitation indicated successful AAV transduction in penile tissue. The transduction efficiency was calculated as the percentage of GFP-positive nuclei relative to total DAPI-stained nuclei using ImageJ software (GFP/DAPI ×100%).

Statistical analysis

All data are presented as the mean ± standard deviation (x ± SD). Statistical analyses were performed using GraphPad Prism version 9.5 (GraphPad Software, San Diego, CA, USA). Before group comparisons, data normality was assessed using the Shapiro-Wilk test, and homogeneity of variance was assessed using the Brown-Forsythe test. When the data met the assumptions of normality and homogeneity of variance, differences among groups were analyzed using one-way ANOVA, followed by Tukey’s post hoc multiple comparisons test. A P value <0.05 was considered statistically significant.

Results

Body weight, MAP, NO, and serum T levels

There were no significant differences in body weight or serum T levels among the experimental groups. MAP was markedly higher in the SHR group than in the WKY group (P<0.05). The NO level in the SHR group was significantly lower than that in the WKY group (P<0.05). Notably, compared with those in the SHR group, the NO concentrations in both the SHR + ICA group and the SHR + AAV group increased (P<0.05) (Table 1).

ICPmax/MAP ratio

Under both 3-V and 5-V cavernous nerve stimulation, the ICPmax/MAP ratio in the WKY group was significantly higher than that in the SHR group (P<0.05). The ratio in the SHR group was markedly lower than that in the SHR + ICA and SHR + AAV groups (P<0.05). No significant difference was detected between the WKY and WKY + ICA groups (Figure 1).

Figure 1 Erectile function in different experimental groups. (A) Representative recordings of ICPmax and MAP during cavernous nerve stimulation at 3 V and 5 V. (B) Quantitative analysis of the ICPmax/MAP ratio among groups. The ratio in the SHR group was significantly lower than that in the SHR + ICA and SHR + AAV groups. *, P<0.05, SHR vs. WKY; ^#, P<0.05, SHR + Icariin vs. SHR; ^, P<0.05, SHR + AAV vs. SHR. AAV, adeno-associated virus; ICA, icariin; ICP, intracavernous pressure; ICPmax, maximal intracavernous pressure; MAP, mean arterial pressure; NC, negative control; SHR, spontaneously hypertensive rat; WKY, Wistar-Kyoto rat.

Immunofluorescence staining of the penile corpus cavernosum

The cell nuclei in all groups were stained blue with DAPI. GFP signals were detected only in the SHR + NC and SHR + AAV groups and were predominantly localized to the endothelial cell membrane. The transduction efficiency, calculated as the percentage of GFP-positive cells over total nuclei (GFP/DAPI ×100%), was 83.2±2.15% in the SHR + NC group and 84.2±4.02% in the SHR + AAV group, whereas no GFP signal was observed in the remaining four groups (Figure 2).

Figure 2 AAV transduction in the penile corpus cavernosum of each group. (A) Representative immunofluorescence images showing AAV transduction in the penile corpus cavernosum. GFP-positive cells indicate successful AAV transduction (scale bars =50 µm). (B) Quantification of AAV transduction efficiency in each group. *, P<0.05 vs. SHR for the SHR + NC group; ^#, P<0.05 vs. SHR for the SHR + AAV group. AAV, adeno-associated virus; DAPI, 4',6-diamidino-2-phenylindole; GFP, green fluorescent protein; HE, hematoxylin and eosin; NC, negative control; SHR, spontaneously hypertensive rat; WKY, Wistar-Kyoto rat.

Immunohistochemistry

APN expression was predominantly observed in the cytoplasm and plasma membrane of vascular endothelial cells in the penile corpus cavernosum, with weaker staining detected in smooth muscle cells. The APN staining intensity in the SHR group was significantly lower than that in the WKY group (P<0.05). In contrast, APN expression markedly increased in both the SHR + ICA group and the SHR + AAV group compared with that in the SHR group (P<0.05) (Figure 3).

Figure 3 Immunohistochemical analysis of APN expression in the penile corpus cavernosum. (A) Representative immunohistochemical staining for APN in the penile corpus cavernosum of each group (scale bars =100 µm). (B) Quantitative analysis of APN expression among the groups. *, P<0.05, SHR vs. WKY; ^#, P<0.05, SHR + Icariin vs. SHR; ^, P<0.05, SHR + AAV vs. SHR. AAV, adeno-associated virus; APN, adiponectin; NC, negative control; SHR, spontaneously hypertensive rat; WKY, Wistar-Kyoto rat.

Western blot analysis

The protein levels of APN, phosphorylated PI3K (p-PI3K), PI3K, p-PI3K/PI3K, AKT, phosphorylated AKT (p-AKT), p-AKT/AKT, eNOS, phosphorylated eNOS (p-eNOS), and p-eNOS/eNOS in the penile corpus cavernosum were significantly higher in the WKY group than in the SHR group (P<0.05). Conversely, the expression of these proteins in the SHR group was markedly lower than that in the SHR + ICA and SHR + AAV groups (P<0.05) (Figures 4,5).

Figure 4 Western blot analysis of APN and PI3K/AKT signaling in the penile corpus cavernosum. (A) Representative Western blots showing the expression of APN, p-PI3K, PI3K, AKT, and p-AKT in each group. (B) Quantitative analysis of protein expression. The expression levels in the WKY group were significantly higher than those in the SHR group, whereas compared with those in the SHR + ICA and SHR + AAV groups, the expression in the SHR group was markedly lower (P<0.05). *, P<0.05, SHR vs. WKY; ^#, P<0.05, SHR + Icariin vs. SHR; ^, P<0.05, SHR + AAV vs. SHR. AAV, adeno-associated virus; AKT, protein kinase B; APN, adiponectin; ICA, icariin; NC, negative control; p-AKT, phosphorylated AKT; p-PI3K, phosphorylated PI3K; PI3K, phosphoinositide 3-kinase; SHR, spontaneously hypertensive rat; WKY, Wistar-Kyoto rat.

Figure 5 Western blot analysis of eNOS signaling in the penile corpus cavernosum. (A) Representative Western blots showing the expression of eNOS and p-eNOS in each group. (B) Quantitative analysis of protein expression. The expression levels in the WKY group were significantly higher than those in the SHR group, whereas compared with those in the SHR + ICA and SHR + AAV groups, the expression in the SHR group was markedly lower (P<0.05). *, P<0.05, SHR vs. WKY; ^#, P<0.05, SHR + Icariin vs. SHR; ^, P<0.05, SHR + AAV vs. SHR. AAV, adeno-associated virus; eNOS, endothelial nitric oxide synthase; ICA, icariin; NC, negative control; p-eNOS, phosphorylated eNOS; SHR, spontaneously hypertensive rat; WKY, Wistar-Kyoto rat.

Discussion

APN is expressed in various cell types, including adipocytes, cardiomyocytes, endothelial cells, and skeletal muscle cells (25) and exerts multiple vasculoprotective effects, such as anti-inflammatory action, anti-atherosclerotic activity, and endothelium-dependent vasodilation (5). This study demonstrated that APN is predominantly expressed in the cytoplasm and plasma membrane of vascular endothelial cells in the rat penile corpus cavernosum. Compared with the WKY group, SHRs exhibited significantly reduced APN expression, impaired PI3K/AKT/eNOS signaling activity, decreased NO production, and lower ICPmax/MAP ratios. Under hypertensive conditions, chronic inflammatory responses and oxidative stress are markedly increased (26). Proinflammatory cytokines [e.g., tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6)], together with oxidative stress, contribute to the downregulation of APN expression (27-29). Effective control of hypertension suppresses proinflammatory cytokines (e.g., TNF-α and IL-6) and oxidative stress, thereby promoting the upregulation of APN expression (26,30,31). These findings suggest that hypertension may downregulate APN expression in endothelial cells of the penile corpus cavernosum through mechanisms involving oxidative stress and proinflammatory cytokines, thereby suppressing the PI3K/AKT/eNOS signaling pathway, reducing NO production, and inducing endothelial dysfunction, ultimately leading to ED.

Compared with the SHR group, the SHR + AAV group showed markedly increased APN expression, restored PI3K/AKT/eNOS signaling, elevated NO production, and improved ICPmax/MAP ratios. AAV-mediated APN overexpression effectively reversed APN downregulation in the corpus cavernosum of SHRs, restored PI3K/AKT/eNOS signaling, and ameliorated ED. Upregulation of APN in the penile corpus cavernosum may ameliorate ED via activation of the PI3K/AKT/eNOS pathway, highlighting its potential as a therapeutic target for hypertension-associated ED. Compared with the SHR group, the SHR + ICA group showed markedly increased APN expression, restored PI3K/AKT/eNOS signaling, elevated NO production, and improved ICPmax/MAP ratios. These findings indicate that ICA ameliorates ED in SHRs and may increase APN expression in the penile corpus cavernosum and activate the PI3K/AKT/eNOS pathway. Peroxisome proliferator-activated receptor gamma (PPARγ) agonists are classical APN upregulators that increase circulating APN levels via transcriptional activation of the APN gene, thereby improving endothelial function (32). However, prolonged use of these agents is associated with adverse effects, including fluid retention, weight gain, and osteoporosis, which may be attributed to the broad regulatory actions of PPARγ in multiple tissues, such as increased renal sodium reabsorption, enhanced adipogenesis, and suppressed osteoblast differentiation (33). ICA improved erectile function without apparent severe adverse effects and may increase APN expression in the penile corpus cavernosum, highlighting its translational potential in APN-related therapeutic strategies.

This study demonstrated that APN is predominantly expressed in the cytoplasm and plasma membrane of vascular endothelial cells in the rat penile corpus cavernosum. Moreover, APN expression was significantly downregulated in the corpus cavernosum of SHRs, suggesting that APN may be involved in the local pathophysiology of hypertension-related ED. SHRs showed decreased APN expression in the penile corpus cavernosum, along with impaired PI3K/AKT/eNOS signaling, reduced NO production, and ED compared with WKY rats. AAV-mediated APN overexpression reversed these changes, indicating that APN regulates erectile function via the PI3K/AKT/eNOS pathway. Moreover, ICA treatment increased the ICPmax/MAP ratio and may increase APN expression in the penile corpus cavernosum and activate PI3K/AKT/eNOS signaling, suggesting that APN-related PI3K/AKT/eNOS signaling may be involved in its beneficial effects on hypertension-associated ED.

This study has some limitations, and future investigations incorporating ICA combined with APN knockdown or inhibition are warranted to further clarify whether the effects of ICA are dependent on APN. ICA treatment was associated with increased APN expression and activation of PI3K/AKT/eNOS signaling in the penile corpus cavernosum of SHRs; however, the precise relationship between ICA treatment and APN expression remains unclear. Moreover, the generalizability of these findings to other types of hypertension-related ED models, such as renovascular hypertension models, remains to be further validated.

Conclusions

APN is predominantly expressed in vascular endothelial cells of the rat penile corpus cavernosum and is downregulated under hypertensive conditions. AAV-mediated APN overexpression improved erectile function in SHRs, possibly through activation of the PI3K/AKT/eNOS signaling pathway and increased NO production. ICA improved erectile function in SHRs and may increase APN expression and enhance PI3K/AKT/eNOS signaling, suggesting that APN-related signaling may be involved in its beneficial effects on hypertension-associated ED.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the ARRIVE reporting checklist. Available at https://tau.amegroups.com/article/view/10.21037/tau-2026-0386/rc

Data Sharing Statement: Available at https://tau.amegroups.com/article/view/10.21037/tau-2026-0386/dss

Peer Review File: Available at https://tau.amegroups.com/article/view/10.21037/tau-2026-0386/prf

Funding: This study was supported by the 2024 China National Health Commission Research Project (No. WKZX2024ED1018) and 2025 Research Projects of Sichuan Medical Association (No. S20250084).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tau.amegroups.com/article/view/10.21037/tau-2026-0386/coif). All authors report institutional funding support from the 2024 China National Health Commission Research Project (No. WKZX2024ED1018) and 2025 Research Projects of Sichuan Medical Association (No. S20250084) for the present manuscript. The authors have no other conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All animal experimental procedures were performed under a project license (approval No. SWMU20250140) granted by the Animal Ethics Committee of Southwest Medical University, in compliance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (NIH) for the care and use of animals.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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