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Asian Journal of Andrology logoLink to Asian Journal of Andrology
. 2025 Aug 5;28(1):57–63. doi: 10.4103/aja202528

Ginsenoside Rb1 mitigates diabetes-induced erectile dysfunction in rats by reducing oxidative stress and modulating the PI3K/AKT/eNOS pathway

Zong-Hai Liu 1, Wei-Bo Chen 1, Jian-Zheng Li 2, Xue-Sheng Wang 2, Lei Liu 3, Han-Wen Liu 3, Gong-Zhen Chen 2, Chao Pan 1, Ming-Zhen Yuan 1,✉
PMCID: PMC12912748  PMID: 40762464

Abstract

Erectile dysfunction is a complex and prevalent complication of diabetes, and effective treatments are lacking. Oxidative stress, fibrosis, and apoptosis are closely associated with the development of diabetes mellitus-related erectile dysfunction (DMED). Notably, ginsenoside Rb1 (Rb1) exerts antioxidant effects and displays promise in the treatment of DMED. This study evaluated the therapeutic efficacy of Rb1 in a rodent streptozotocin-induced DMED model. Thirty-two rats were randomly assigned to three groups: control group, DMED group, and DMED + Rb1 group. DMED was induced in male rats via an intraperitoneal injection of streptozotocin. After 8 weeks of Rb1 gavage, erectile function was assessed by the electrical stimulation of the cavernous nerve. In addition, western blot, quantitative real-time polymerase chain reaction, enzyme-linked immunosorbent assay, immunofluorescence, terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) staining, and Masson’s trichrome staining were performed to verify the relevant factors and protein expression. Rb1 effectively improved the erectile function of the corpus cavernosum, decreased collagen content, and increased smooth muscle content in rats with diabetes. Rb1 decreased the levels of the pro-inflammatory factors (interleukin [IL]-6, tumor necrosis factor alpha [TNF-α], and IL-1β), and increased the levels of the anti-inflammatory factors (IL-10 and IL-4). Moreover, the activities of superoxide dismutase and catalase and levels of nitric oxide (NO) were increased, whereas malondialdehyde activity was decreased. Additionally, Rb1 activated the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/endothelial nitric oxide synthase (eNOS) signaling pathway and inhibited cell apoptosis. Rb1 can improve erectile function in rats with DMED by increasing the activity of the PI3K/AKT/eNOS pathway, reducing oxidative stress, inhibiting inflammation and apoptosis, and alleviating corpus cavernosum fibrosis.

Keywords: diabetes mellitus, erectile dysfunction, ginsenoside Rb1, oxidative stress, PI3K/AKT/eNOS

INTRODUCTION

Erectile dysfunction (ED) describes the inability to reach or maintain a sufficient penile erection for sexual intercourse. Diabetes mellitus (DM) is an independent risk factor for ED.1,2 The incidence of ED is about 3.5 times greater in diabetic male compared to non-diabetic individuals. Approximately 37.5% of individuals with type 1 DM (T1DM) and 66.3% of those with type 2 DM (T2DM) experience ED.3 The high number of patients with DM-related erectile dysfunction (DMED) imposes a substantial economic burden on society, but effective treatments are still lacking. Currently, oral phosphodiesterase 5 inhibitors (PDE5is) serve as the first-line treatments for ED; however, their effectiveness in patients with DMED is often limited.4,5 Patients with DMED have displayed a high discontinuation rate of PDE5is because of factors such as ineffectiveness and psychological issues.6 Therefore, new treatments for DMED are needed.

In recent years, extensive traditional Chinese medicine (TCM) research has provided evidence supporting the medicinal value of TCM extracts, leading to a significant increase in their clinical application. Ginsenoside Rb1 (hereafter termed Rb1), which belongs to the class of steroid compounds, is one of the main active ingredients in the renowned TCM ginseng. In addition, Rb1 has many pharmacological effects such as suppressing oxidation and inflammation, regulating apoptosis, improving cognition, and protecting vascular endothelial cells.7,8,9 Research illustrated that Rb1 effectively decreases the levels of reactive oxygen species (ROS) within cells, offering protection against oxidative stress (OS). This is achieved through the suppression of malondialdehyde (MDA) accumulation and the enhancement of antioxidant enzyme activities, including those of superoxide dismutase (SOD) and catalase (CAT).10,11 Meanwhile, Rb1 can also alleviate inflammation responses by suppressing the release of pro-inflammatory cytokines (including tumor necrosis factor alpha [TNF-α], interleukin [IL]-1β, and IL-6), improving the pathological state caused by inflammation, and exerting anti-inflammatory effects.12 Furthermore, relevant studies indicated that Rb1 can regulate the levels of apoptosis-related proteins, including B-cell lymphoma protein-2 (Bcl-2) and Bcl-2-associated X protein (Bax), thereby exerting antiapoptotic effects.13 Rb1 can also exert protective effects on vascular endothelial cells, thereby preventing diabetic vascular disease and related complications.14,15 Endothelial damage represents a critical pathophysiological element in DMED, and restoring endothelial function represents a key strategy for alleviating the condition.16 However, the effect of Rb1 on DMED and its potential to alleviate DMED by protecting endothelial cell function remain unclear.

The pathogenesis of DMED is multifaceted, encompassing a wide range of intricate physiological mechanisms. Long-term hyperglycemia leads to increased OS, resulting in vascular endothelial cell damage. Hence, DM is recognized as an independent risk factor contributing to the development of ED.17,18,19 NO is an effective endogenous vasodilator. Upon release, NO rapidly spreads into the corpus cavernosum (CC) and vascular smooth muscle cells, in which it activates soluble guanylate cyclase, leading to an increase in cyclic guanosine monophosphate (cGMP) accumulation. This process facilitates the relaxation of blood vessels and smooth muscle within the sponge by decreasing the concentration of calcium ions within cells, which enhances blood flow and pressure in the sponge, resulting in penile erection.20,21 However, hyperglycemia can induce OS, which impairs endothelial cell function, suppresses the activity of endothelial nitric oxide synthase (eNOS), and ultimately diminishes the production of NO.5 Emerging research has revealed that NO and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/eNOS signaling pathways are closely linked.22 Additionally, the PI3K/AKT/eNOS signaling cascade, a well-characterized pathway, serves as a critical regulator of angiogenic processes. Its activation supports various biological processes in vivo, such as the mobilization of endothelial progenitor cells, angiogenesis, and reconstruction of the vascular endothelial barrier.23 In DMED, the activity of this pathway is reduced. Therefore, increasing the activity of this pathway might effectively improve DMED.24 Thus, therapeutic approaches targeting OS reduction and modulation of the PI3K/AKT/eNOS signaling pathway may represent viable strategies for DMED treatment.

Research indicates that Rb1 can induce vasodilation through activation of the PI3K/AKT/eNOS signaling cascade.25 Moreover, Rb1 has demonstrated therapeutic potential in diabetic nephropathy, diabetic atherosclerosis, and other diseases.14,26 However, no research has investigated the therapeutic efficacy or underlying mechanisms of Rb1 in the context of DMED. Therefore, this study evaluated whether Rb1 can effectively treat DMED and examined its potential mechanisms of action.

MATERIALS AND METHODS

Animals

A total of 32 male Sprague–Dawley rats of specific pathogen-free (SPF) grade, at the age of 8 weeks, were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). All procedures involving animal subjects were reviewed and approved by the Animal Care and Use Committee of Shandong Provincial Hospital Affiliated to Shandong First Medical University (Jinan, China; Approval No. 2023-055).

DMED model

A total of 24 rats were randomly selected and fasted for 16 h prior to intraperitoneal injection with 1% streptozotocin (STZ; 60 mg kg−1; Sigma-Aldrich, St. Louis, MO, USA).27 Another 8 rats received 0.1 mol l−1 phosphate citrate buffer (pH 4.2) as blank controls (CON group). Beginning on day 3 post-STZ administration, daily blood glucose measurements were obtained via tail vein sampling. The model was considered successfully established if blood glucose levels consistently measured ≥16.7 mmol l−1 for 3 consecutive days. Following an 8-week period, erectile function was assessed using the apomorphine test.28 Finally, 16 rats with diabetes were included in the follow-up experiment (modeling failed in two rats and six rats died of hyperglycemia during feeding; Figure 1).

Figure 1.

Figure 1

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Flowchart of the experimental grouping of rats. DM: diabetes mellitus group; CON: control group; DM + Rb1: ginsenoside Rb1-treated diabetes mellitus group; STZ: streptozotocin.

The 16 rats were allocated into two groups: model group (DM) and Rb1 (DM + Rb1) group. Rb1 (high performance liquid chromatography [HPLC] ≥ 98%; Chengdu Aijicui Biotechnology Co., Ltd., Chengdu, China) was prepared by dissolving the compound in 10% dimethyl sulfoxide (DMSO) solution for use. Rats in the DM + Rb1 group received 30 mg kg−1 Rb1 via oral gavage once daily. The equal volume of sterile water was given by gavage in CON and DM groups. The gavage treatment was continued for 8 weeks. Body weight and blood glucose levels were regularly monitored before and after treatment (Figure 1).

Measurement of erectile function

Eight weeks after treatment, the function of the CC was assessed by measuring mean systemic arterial pressure (MAP) and intracavernous pressure (ICP). Anesthesia was performed with 5% sodium pentobarbital (35 mg kg−1). Subsequently, a PE-50 catheter was placed into the exposed carotid artery, and MAP was continuously monitored. After inserting 23G needles into the CC filled with heparinized saline (250 U ml−1), ICP was measured. Subsequently, the cavernous nerve was isolated and stimulated for 60 s using a bipolar stainless steel hook electrode (5 V, 25 Hz, pulse width 5 ms).29 This measurement procedure was performed in triplicate with 3-min recovery intervals. The BL-420V Pressure Sensor System (ADInstruments, Sydney, Australia) records the changes in MAP and ICP.

Measurement of OS

The level of OS in CC was measured by the enzyme-linked immunosorbent assay (ELISA) kits. Commercially available assay kits were used to measure the activities of CAT (E-BC-K031-M; Elabscience, Wuhan, China), MDA (E-BC-K025-M; Elabscience), and SOD (E-BC-K020-M; Elabscience) and the levels of NO (E-BC-K035-M; Elabscience) in CC tissues from each group. A standard curve was constructed by following the procedural guidelines outlined in the kit manual, and the optical density of the samples was measured using a microplate reader, enabling the determination of their concentrations.

Masson’s trichrome staining

The methodology employed in this study was outlined in a preceding study.30 Penile tissues were fixed overnight by immersing in 4% paraformaldehyde. Then, the penile tissue specimens were processed by embedding them in paraffin and cutting them into 5-μm-thick sections. After standard deparaffinization in xylene, the sections were progressively rehydrated in graded ethanol concentrations (ranging from 100% to 75%). According to the provided instructions, Masson’s tricolor dyeing kits (D0261-3; Nanjing Institute of Biological Engineering, Nanjing, China) were employed to evaluate the proportion of smooth muscle to collagen within CC.

Terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) staining

Paraffin-embedded sections of CC tissue were deparaffinized, rehydrated, and treated with proteinase K storage solution at 37°C for 30 min. Then, the membrane-breaking solution was applied to the tissue at room temperature for 20 min. The sections were processed using a TUNEL kit (E-CK-A322; Elabscience) according to the provided instructions. Finally, the slides were analyzed using a fluorescence microscope (Olympus Corporation, Tokyo, Japan).

Immunofluorescence staining

The CC tissues were preserved via soaking in 4% paraformaldehyde and subsequently dehydrated. The samples were cut into 5-µm-thick slices and mounted on slides. The tissue sections were incubated with goat anti-α-smooth muscle actin (anti-α-SMA; 1:1000; 19245; Cell Signaling Technology, Danvers, MA, USA) overnight at 4°C. Subsequently, the slides were rinsed with phosphate-buffered saline and subsequently incubated with an Alexa Fluor 594-labeled secondary antibody (Invitrogen, Carlsbad, CA, USA) for 1 h. Finally, the slides were analyzed using a fluorescence microscope (Olympus Corporation, Tokyo, Japan).

Western blot

The expression of CC-related proteins in each group was detected by western blot. Frozen CC tissues were incubated with radioimmunoprecipitation assay (RIPA; WB3100; NCM Biotech, Suzhou, China), and total protein was extracted. Thereafter, proteinase phosphatase inhibitors (P1045; Beyotime, Shanghai, China) were added to the RIPA lysate to prevent protein degradation. The same amount of protein was separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes. The membrane was blocked with 5% nonfat milk for 1 h at the room temperature. Following blocking, the membranes were incubated with specific primary antibodies at 4°C overnight. The primary antibodies are as follows: anti-eNOS (1:1000; ab300071, Abcam, Cambridge, UK), anti-phosphorylated-eNOS (anti-p-eNOS; 1:500; AF3247; Affinity Biosciences, Changzhou, China), anti-AKT (1:2000; 10176-2-AP; Proteintech, Wuhan, China), anti-phosphorylated-AKT (1:2000; anti-p-AKT; 66444-1-Ig; Proteintech), anti-PI3K (1:500; 20584-1-AP; Proteintech), anti-phosphorylated-PI3K (anti-p-PI3K; 1:1000; AF0016; Affinity Biosciences), anti-Bax (1:1000; 50599-2-Ig; Proteintech), anti-Bcl-2 (1:1000; 26593-1-AP; Proteintech), anti-caspase 3 (1:1000; 19677-1-AP; Proteintech), and anti-β-actin (1:5000; AC038; Abclonal, Wuhan, China). Thereafter, the appropriate secondary antibodies (1:5000; SA00001-2; Proteintech) were applied for 1 h. Enhanced chemiluminescence was used to visualize the protein bands. Band intensities were quantified using ImageJ software (National Institutes of Health, Frederick, MD, USA) after normalization to β-actin expression as an internal loading control.

Quantitative real-time polymerase chain reaction (RT-qPCR)

Total RNA was extracted from the CC tissue using RNA Simple Total RNA kits (Tiangen Biotech, Beijing, China) according to the manufacturer’s protocol. RNA purity and concentration were then measured with an ultraviolet spectrophotometer. All the samples had optical density (OD) 260 nm/280 nm absorbance ratios between 1.8 and 2.0. According to the manufacturer’s instructions, first-strand cDNA was synthesized using a reverse transcription kit (Yeasen, Shanghai, China), followed by cDNA amplification through PCR. The qPCR was then conducted using SYBR Green Master Mix (Yeasen) on a QuantStudio™ 5 system (Thermo Fisher Scientific, Waltham, MA, USA). β-actin was used as an internal control to normalize the gene expression. The primers are as follows: TNF-α (forward [F], 5’-TGATCGGTCCCAACAAGGA-3’; reverse [R], 5’-TGCTTGGTGGTTTGCTACGA-3’), IL-1β (F, 5’-GGGATGATGAC GACCTGC-3’; R, 5’-CCACTTGTTGGCTTATGTT-3’), IL-4 (F, 5’-GCTCAGCCTCCAGAAGACTG-3’; R, 5’-GTCTCAGGG CTTGAGGTCTG-3’), IL-6 (F, 5’-CTCTCCGCAAGAGACTTCCA-3’; R, 5’-TCTCCTCTCCGGACTTGTGAA-3’), IL-10 (F, 5’-TCAGAA CTTCAGCCGTGCC-3’; R, 5’-GGCCTTCCTCTCTCACCTC-3’), and β-actin (F, 5’-CATGTACGTTGCTATCCAGGC-3’; R, 5’-CTCCTTAATGTCACGCACGAT-3’). Relative mRNA expression levels were calculated using the 2−ΔΔCt method.

Statistical analyses

Statistical analyses were performed using GraphPad Prism 8.0 (GraphPad Software, San Diego, CA, USA). All quantitative data are expressed as mean ± standard deviation (s.d.). For multiple group comparisons, one-way analysis of variance (ANOVA) was conducted, with Tukey’s post hoc test applied for intergroup comparisons. In this study, P < 0.05 was deemed statistically significant for all analyses.

RESULTS

Metabolic variables

Before the experiment, blood glucose levels in the DM and DM + Rb1 groups were significantly higher than those in the CON group (both P < 0.001), confirming successful model establishment. No notable difference in body weight was observed among the groups at this stage. Following the 8-week experimental period, body weight was significantly lower in the DM and DM + Rb1 groups than that in the CON group (both P < 0.001), whereas blood glucose levels remained significantly higher in the DM and DM + Rb1 groups (both P < 0.001). However, no notable difference in body weight or blood glucose levels was observed between the DM and DM + Rb1 groups (Figure 2a and 2b).

Figure 2.

Figure 2

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Comparison of (a) body weight and (b) blood glucose levels among the three groups before and after the experiment. ICP of (c) CON group, (d) DM group, and (e) DM+Rb1 group. (f) Maximum ICP/MAP ratios. Data are expressed as the mean ± standard deviation (n = 8). ***P < 0.001, the indicated value versus that in the DM group. ###P < 0.001, the indicated value versus that in the CON group. ICP: intracavernous pressure; MAP: mean systemic arterial pressure; DM: diabetes mellitus group; CON: control group; DM + Rb1: ginsenoside Rb1-treated diabetes mellitus group.

Rb1 treatment improves erectile function in rats with DMED

The erectile function of rats in each group was quantitatively assessed by measuring the maximum ICP/MAP following various therapeutic interventions. Relative to CON group, the DM group exhibited significantly reduced maximum ICP and ICP/MAP ratios (both P < 0.001). After Rb1 treatment, maximum ICP and maximum ICP/MAP were significantly higher in the DM + Rb1 group than those in the DM group (P = 0.008; Figure 2c–2f). The current experimental results confirm successful induction of DMED through STZ induction, while demonstrating the therapeutic potential of Rb1 in ameliorating ED in this animal model.

Rb1 alleviates fibrosis in the CC of rats with DMED

The proportion of collagen fibers relative to muscle fibers was evaluated using Masson’s trichrome staining, and α-SMA expression was detected by immunofluorescence analysis. The results demonstrated a statistically significant elevation in the collagen-to-muscle fiber ratio in DM rats compared to CON rats (Figure 3a). Concurrently, immunofluorescence analysis revealed a marked reduction in α-SMA expression in the DM group relative to that in the CON group (Figure 3b). These results indicated that rats with DM exhibited more severe CC fibrosis than normal rats. However, after Rb1 treatment, a significant attenuation of fibrotic changes was observed in rats with diabetes, as evidenced by a marked reduction in the collagen fiber to muscle fiber ratio and the concomitant upregulation of α-SMA. These findings collectively suggest that Rb1 treatment effectively ameliorated tissue fibrosis in the diabetic model.

Figure 3.

Figure 3

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Rb1 treatment increases the smooth muscle content of the CC. (a) Masson’s trichrome staining of penile tissues in the CON, DM, and DM+Rb1 groups (n = 3 rats per group) after different treatments. Smooth muscle stained red, whereas collagen stained blue. (b) Representative immunofluorescence images of the α-SMA-positive CC in the CON, DM, and DM + Rb1 groups (n = 3 rats per group). CC: corpus cavernosum; α-SMA: α-smooth muscle actin; DM: diabetes mellitus group; CON: control group; DM + Rb1: ginsenoside Rb1-treated diabetes mellitus group; DAPI: 4’-6-diamidino-2-phenylindole.

Rb1 decreases inflammatory responses in the CC tissue

The expression of inflammatory markers in the CC was measured using RT-qPCR. Quantitative analysis revealed that DM rats demonstrated significantly elevated expression of pro-inflammatory cytokines (IL-6, P < 0.001; TNF-α, P < 0.001; and IL-1β, P = 0.0004) coupled with markedly reduced anti-inflammatory cytokine levels (IL-10, P < 0.001; and IL-4, P < 0.001) in CC tissue compared to CON group. Treatment with ginsenoside Rb1 effectively rebalanced CC cytokine profiles in diabetic rats, reducing pro-inflammatory mediators (IL-6, P = 0.0004; TNF-α, P = 0.0003; and IL-1β, P = 0.0023) and elevating anti-inflammatory factors (IL-10, P = 0.0001; and IL-4, P = 0.0008; Figure 4a–4e) relative to DM group. Collectively, these data demonstrate that Rb1 exerts anti-inflammatory effects in the CC of rats with DM, effectively modulating cytokine balance.

Figure 4.

Figure 4

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Rb1 alleviates the inflammatory response and OS in rats with diabetes. Expression of (a) TNF-α, (b) IL-1β, (c) IL-6, (d) IL-4, and (e) IL-10 in the CC in each group. Data are presented as the mean ± standard deviation (n = 3 rats per group). **P < 0.01, ***P < 0.001, the indicated value versus that in the DM group. DM: diabetes mellitus group; CON: control group; DM + Rb1: ginsenoside Rb1-treated diabetes mellitus group; IL-1β: interleukin-1β; TNF-α: tumor necrosis factor alpha; OS: oxidative stress; CC: corpus cavernosum.

Rb1 enhances the activity of the PI3K/AKT/eNOS signaling pathway and reduces OS

This study was designed to systematically investigate the therapeutic potential and underlying mechanisms of the PI3K/AKT/eNOS pathway in ameliorating DMED in a rat model. Integrated analysis of western blot and ELISA demonstrated significant alterations in both the PI3K/AKT/eNOS pathway and antioxidant capacity in DMED rats. Specifically, the protein ratios of p-PI3K/PI3K, p-AKT/AKT, and p-eNOS/eNOS in CC tissues were markedly lower in the DM group than those in the CON group (p-PI3K/PI3K, P = 0.0009; p-AKT/AKT, P = 0.0002; and p-eNOS/eNOS, P = 0.0005). Concurrently, a significant impairment of antioxidant defense systems was observed, as evidenced by substantially decreased activities of SOD and CAT in the DM group relative to those in the CON group (SOD, P < 0.001; and CAT, P < 0.001). Furthermore, MDA activity was notably increased (P = 0.0003), and NO content was notably decreased (P < 0.001). These findings suggested a decrease in the activity of the PI3K/AKT/eNOS pathway together with elevated OS and impaired NO production in rats with diabetes. After Rb1 treatment, the p-PI3K/PI3K, p-Akt/AKT, and p-eNOS/eNOS protein ratios were significantly increased compared with those in the DM group (p-PI3K/PI3K, P = 0.0029; p-AKT/AKT, P = 0.0105; and p-eNOS/eNOS, P = 0.0118; Figure 5a–5d). Rb1 administration significantly enhanced antioxidant enzyme activities (SOD, P = 0.0008; and CAT, P = 0.0005; Figure 5e and 5f) while reducing MDA activity (P = 0.0029; Figure 5g). Additionally, the NO level was significantly increased (P < 0.001; Figure 5h). The results suggest that Rb1 exerts significant antioxidant effects, which might be mediated by regulating the PI3K/AKT/eNOS signaling pathway.

Figure 5.

Figure 5

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Modulation of the PI3K/AKT/eNOS signaling pathway by Rb1. (a) Representative western blots of p-PI3K, PI3K, p-AKT, AKT, p-eNOS, and eNOS. Semiquantitative analysis of (b) PI3K, (c) AKT, and (d) eNOS and their phosphorylation levels. The activities of (e) SOD, (f) MDA, and (g) CAT and (h) NO content in the penile tissues of rats in each group. Data are presented as the mean ± standard deviation (n = 3 rats per group). *P < 0.05, **P < 0.01, ***P < 0.001, the indicated value versus that in the DM group. PI3K: phosphatidylinositol 3-kinase; p-PI3K: phosphorylated PI3K; AKT: protein kinase B; p-AKT: phosphorylated AKT; eNOS: endothelial nitric oxide synthase; p-eNOS: phosphorylated eNOS; SOD: superoxide dismutase; MDA: malondialdehyde; CAT: catalase; NO: nitric oxide; DM: diabetes mellitus group; CON: control group; DM + Rb1: ginsenoside Rb1-treated diabetes mellitus group.

Rb1 suppresses apoptosis in the CC of rats with DMED

To explore the antiapoptotic effects of Rb1 in the CC, we quantitatively analyzed key apoptosis-regulatory proteins using western blot analysis. The DM rats demonstrated significant apoptosis activation, evidenced by a reduction in Bcl-2/Bax ratio (P = 0.0001) and elevation in cleaved caspase 3 (c-caspase 3; P < 0.001) relative to CON group. Following Rb1 administration, the ratio of Bcl-2/Bax was significantly increased (P = 0.0014; Figure 6a and 6b), whereas the expression of c-caspase 3 was markedly decreased (P < 0.001; Figure 6d). The CC tissues of each group were further analyzed by TUNEL staining. In the DM group, the area of positive apoptotic signals was notably enlarged. After Rb1 treatment, the positive apoptotic area was significantly decreased (Figure 6c). The results demonstrate that DM significantly induces apoptosis in penile cavernous cells, whereas Rb1 effectively counteracts this process by exerting an anti-apoptotic effect within CC tissue.

Figure 6.

Figure 6

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Expression of apoptosis-related proteins after Rb1 treatment. (a) Representative western blots of Bax, Bcl-2, and c-caspase 3. Semiquantitative analysis of the (b) Bcl-2/Bax ratio and (d) c-caspase 3 expression. (c) Representative images of TUNEL staining for each group. Data are presented as the mean ± standard deviation (n = 3 rats per group). **P < 0.01, ***P < 0.001, the indicated value versus that in the DM group. DM: diabetes mellitus group; CON: control group; DM + Rb1: ginsenoside Rb1-treated diabetes mellitus group; c-caspase 3: cleaved caspase 3; Bcl-2: B-cell lymphoma protein-2; Bax: Bcl-2-associated X; TUNEL: terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling.

DISCUSSION

Over recent decades, extensive preclinical investigations on pharmacological interventions for DMED have been conducted. Several rodent models have been employed in diabetes research, which are categorized into genetic (e.g., Zucker diabetic fatty rats for obesity-associated T2DM, and BioBreeding rats for autoimmune T1DM) and induced types (e.g., STZ-treated rats mimicking β-cell-deficient T1DM or diet-modified T2DM).31 The STZ-induced DMED rat model has been widely adopted in experimental urology research because of its cost-effectiveness, technical accessibility, and robust validation as a standardized preclinical paradigm for erectile pathophysiology investigations.32,33 This chemically induced model features three major advantages: (1) faithful representation of T1DM pathophysiology, (2) technical simplicity requiring minimal specialized equipment, and (3) rapid expedited model readiness. Through standardized STZ administration, the DMED model achieves full pathological manifestation within 8 weeks, as evidenced by sustained hyperglycemia and impaired erectile responses in cavernous nerve stimulation tests.

Recent research on TCM has emphasized its potential in managing diabetes and its associated complications. TCM, including its extracts, has received growing attention because of its minimal toxic and side effects while significantly improving symptoms and complementing the shortcomings of Western medicine.34 Ginseng offers protective effects against DM-induced ED. Ginsenosides have been found to enhance erectile function in rats with DMED.35 However, the specific active components of ginsenosides remain unclear. Rb1, a primary active compound in ginseng, exhibits antioxidant, antiapoptotic, and anti-inflammatory properties. Thus, Rb1 can be used to treat various complications of diabetes.36,37 Considering the close association between these biological effects and DMED, we hypothesized that Rb1 might enhance erectile function in rats with STZ-induced diabetes. This study explored the underlying molecular mechanism of its action. The results revealed that Rb1 significantly improved erectile function in rats with DMED by inhibiting penile tissue fibrosis, OS, inflammatory responses, and apoptosis. In addition, the mechanism by which Rb1 improves erectile function in DMED rats might involve enhancing the activity of the PI3K/AKT/eNOS pathway.

Notably, intragastric administration was employed in this preclinical investigation to better mimic the oral administration route used in clinical practice. Experimental rodents received a daily dose of 30 mg kg−1 via precision gavage. On the basis of body surface area normalization according to the U.S. Food and Drug Administration (FDA) guidelines,38 the human equivalent dose was calculated as 4.8 mg kg−1. For a standard 70-kg adult, this translates to 336 mg daily.

Inflammation-mediated endothelial dysfunction represents a core pathogenic mechanism in ED, which can result in reduced NO synthesis.19 This study found that the expression of the pro-inflammatory cytokines IL-6, TNF-α, and IL-1β was significantly elevated in the penile tissue of rats with diabetes, whereas the expression of the anti-inflammatory cytokines IL-10 and IL-4 was markedly reduced. Rb1 exerted significant anti-inflammatory effects in CC tissue, as evidenced by the marked downregulation of pro-inflammatory cytokines and substantial attenuation of inflammatory responses. OS is primarily characterized by the accumulation of ROS, which damage endothelial function, decrease NO bioavailability, and contribute to vascular dysfunction. These processes are central to the development and progression of ED.39 ROS can react with NO to produce reactive nitrogen species, thereby causing oxidative damage to DNA and destroying lipids and proteins.40 Furthermore, SOD, CAT, and MDA are the important indicators of OS. SOD is an important antioxidant among metalloenzymes, and it is involved in the elimination of oxygen-free radicals.41 CAT safeguards the cells from oxidative damage by decomposing hydrogen peroxide within cells.42 Moreover, MDA levels represent an indicator of lipid peroxidation in the body and reflect the severity of OS-induced damage.43 The current investigation revealed significant OS alterations in the penile tissue of diabetic rats, characterized by substantial reduction in the activities of antioxidant enzymes, including SOD and CAT, along with a concomitant elevation of MDA activity compared with the findings in CON rats. Following Rb1 treatment, the activities of SOD and CAT increased, whereas MDA activity decreased, suggesting that Rb1 provides protection against OS in DMED. Additionally, the levels of NO in the CC tissue of diabetic rats were notably decreased, whereas Rb1 enhanced NO production, potentially resulting in improvements in OS and inflammatory responses.

Excessive OS can also impair erectile function by inducing apoptosis in penile tissue cells.44 In the present investigation, apoptotic activity was systematically evaluated through western blot and TUNEL staining. In comparison to CON rats, diabetic rats exhibited elevated expression of proapoptotic factors such as Bax and c-caspase 3, alongside reduced expression of the antiapoptotic factor Bcl-2. Nonetheless, Rb1 suppressed the expression of proapoptotic factors and enhanced that of antiapoptotic factors, thereby exerting antiapoptotic effects.

Accumulating evidence indicates that CC fibrotic remodeling constitutes a hallmark pathological feature of DMED.45 The fibrotic progression in DMED exhibits distinct histopathological changes, characterized by progressive depletion of CC smooth muscle cells accompanied by the excessive deposition of collagen fibers.46 Additionally, chronic inflammation and OS also play significant roles in promoting fibrosis.47,48 In the present study, Masson’s trichrome staining and α-SMA immunofluorescence staining demonstrated significant structural changes in the CC tissue of DMED rats, including an increase in collagen fiber content and a decrease in smooth muscle fiber content compared with the findings in normal rats. Following Rb1 treatment, the collagen fiber content in the CC tissue was reduced, whereas the smooth muscle fiber content was increased.

This study also found that Rb1 improves DMED by activating the PI3K/AKT/eNOS signaling pathway. NO plays an essential role in DMED. NO is produced by non-adrenergic, non-cholinergic nerves and endothelial cells. It facilitates the relaxation of CC vascular smooth muscle by activating the NO/cGMP signaling pathway, which promotes penile erection.49 However, DM-induced OS can suppress eNOS activity and reduce NO synthesis, thereby decreasing cGMP levels in the CC and contributing to ED.50 A previous study suggested that DMED is strongly associated with dysfunction in the PI3K/AKT/eNOS pathway.51 Other studies confirmed that STZ-induced DMED can be effectively ameliorated through suppressing excess OS and increasing PI3K/AKT/eNOS pathway activity.52 Our findings revealed reduced p-PI3K/PI3K, p-AKT/AKT, and p-eNOS/eNOS ratios in rats with diabetes compared with those in normal rats. However, Rb1 treatment significantly increased these protein ratios. The outcomes of this study are in agreement with previous literature, supporting the therapeutic potential of Rb1 in ameliorating ED by regulating the expression of PI3K/AKT/eNOS signaling pathway proteins.

The present investigation provided compelling evidence that Rb1 exerts therapeutic effects on erectile function in rats with DMED by activating the PI3K/AKT/eNOS pathway and reducing OS in the CC. In addition, Rb1 can inhibit apoptosis in the CC and reduce tissue fibrosis. These findings collectively establish a robust preclinical foundation for the development of novel therapeutic strategies targeting DMED while providing critical mechanistic insights for future pharmacological interventions.

Despite these significant findings, several methodological limitations in the current investigation warrant careful consideration. Rb1 can improve ED induced by STZ in rats with diabetes, but the underlying mechanism requires further investigation. Notably, a STZ-induced T1DM rat model was implemented in the present investigation. Clinically, T2DM is the predominant etiology underlying DMED, accounting for approximately 80%–90% of clinical cases. Whereas the majority of patients with diabetes receive routine pharmacological interventions or insulin therapy for glycemic management, the rodent diabetes model employed in this investigation failed to replicate this clinically relevant condition. Although most preclinical DMED studies employed STZ-induced hyperglycemic models, the therapeutic efficacy of Rb1 against T2DM-mediated ED, along with post-glycemic control treatment outcomes in rats, requires systematic validation through rigorously designed experiments using established T2DM animal models. Moreover, this study did not establish a concentration gradient for the drug, necessitating further experiments to determine the optimal concentration range.

AUTHOR CONTRIBUTIONS

ZHL designed and conceived this work and provided a major contribution to manuscript writing. WBC participated in the design of the experiment. JZL and GZC participated in the animal experiments. XSW and CP were responsible for obtaining the experimental data. LL and HWL were responsible for analyzing the experimental data. MZY was responsible for revising the manuscript. All authors read and approved the final manuscript.

COMPETING INTERESTS

All authors declare no competing interests.

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