https://orcid.org/0000-0003-1665-5653
Retraction
The PLOS ONE Editors retract this article due to concerns about duplicate publication.
Following the publication of this article [1] the author requested retraction for issues involving authorship and scientific concerns. During the editorial investigation into this matter, PLOS noted that the article was previously published in [2]. In light of the duplicate publication concerns, PLOS did not investigate the merit of the authorship and scientific concerns further.
KC, BH, JF, ZH, SF, SR, HT, AMAMM, XW, YT, HX, RH, and GL agreed with the retraction. KC, JF, ZH, XW, and GL apologize for the issues with the article. JF stands by the article’s findings. XL either did not respond directly or could not be reached.
2 Dec 2024: The PLOS ONE Editors (2024) Retraction: Nesfatin-1 regulates the phenotype transition of cavernous smooth muscle cells by activating PI3K/AKT/mTOR signaling pathway to improve diabetic erectile dysfunction. PLOS ONE 19(12): e0314890. https://doi.org/10.1371/journal.pone.0314890 View retraction
Figures
Abstract
Objective
This study aims to explore the impact of Nesfatin-1 on type 2 diabetic erectile dysfunction (T2DMED) and its underlying mechanism in regulating the phenotypic switching of corpus cavernosum smooth muscle cells (CCSMCs).
Methods
Twenty-four 4-week-old male C57 wild-type mice were randomly assigned to the control group, model group, and Nesfatin-1 treatment group. Monitoring included body weight, blood glucose levels, and penile cavernous pressure (ICP). Histochemistry and Western blot analyses were conducted to assess the expressions of α-SMA, OPN, and factors related to the PI3K/AKT/mTOR signaling pathway. CCSMCs were categorized into the control group, high glucose and high oleic acid group (GO group), Nesfatin-1 treatment group (GO+N group), sildenafil positive control group (GO+S group), and PI3K inhibitor group (GO+N+E group). Changes in phenotypic markers, cell morphology, and the PI3K/AKT/mTOR signaling pathway were observed in each group.
Results
(1) Nesfatin-1 significantly ameliorated the body size, body weight, blood glucose, glucose tolerance, and insulin resistance in T2DMED mice. (2) Following Nesfatin-1 treatment, the ICP/MSBP ratio and the peak of the ICP curve demonstrated a significant increase. (3) Nesfatin-1 significantly enhanced smooth muscle and reduced collagen fibers in the corpus cavernosum. (4) Nesfatin-1 notably increased α-SMA expression and decreased OPN expression in CCSMCs. (5) Nesfatin-1 elevated PI3K, p-AKT/AKT, and p-mTOR/mTOR levels in penile cavernous tissue.
Conclusions
Nesfatin-1 not only effectively improves body weight and blood glucose levels in diabetic mice but also enhances erectile function and regulates the phenotypic switching of corpus cavernosum smooth muscle. The potential mechanism involves Nesfatin-1 activating the PI3K/AKT/mTOR signaling pathway to induce the conversion of CCSMCs to a contractile phenotype.
Citation: Chen K, Huang B, Feng J, Hu Z, Fan S, Ren S, et al. (2024) Nesfatin-1 regulates the phenotype transition of cavernous smooth muscle cells by activating PI3K/AKT/mTOR signaling pathway to improve diabetic erectile dysfunction. PLoS ONE 19(9): e0304485. https://doi.org/10.1371/journal.pone.0304485
Editor: Fábio Henrique Silva, Sao Francisco University: Universidade Sao Francisco, BRAZIL
Received: January 10, 2024; Accepted: May 14, 2024; Published: September 3, 2024
Copyright: © 2024 Chen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the manuscript and its Supporting Information files.
Funding: This paper is supported by National Natural Science Foundation of China (81860268,82201000),Ningxia Key Research and Development Project (2023BEG03021;2021BEB04034) Ningxia Natural Science Foundation (2021AAC02025), Ningxia science and technology innovation leading talent training project (2020GKLRLX06,2020GKLRLX11),Ningxia Medical University research project (XT2019017).The funders were not involved in study design, data collection and analysis, publication decisions, or manuscript preparation.
Competing interests: The authors have declared that no competing interests exist.
Introduction
With the improvement of living standards, the incidence of type 2 diabetes mellitus erectile dysfunction(T2DMED) has increased, which seriously affecting people’s quality of life [1, 2]. However, the current treatment of T2DMED mainly relies on strict diet and effective drug control of blood glucose, correction of dyslipidemia and control of hypertension, at the same time oral phosphodiesterase type 5 inhibitors and penile cavernous drug injection therapy are required to achieve a certain effect.Currently, there is a lack of drugs that can simultaneously control blood glucose and improve erectile dysfunction [3]. The precise mechanism underlying erectile dysfunction resulting from type 2 diabetes remains unclear. This lack of clarity is also responsible for the limited efficacy observed when simply administering drugs to manage blood glucose and address erectile dysfunction in clinical settings.
Penile erection mainly depends on dilating the cavernous artery and cavernous sinus.The contraction and relaxation of corpus cavernosal smooth muscle (CCSMs) determine the expansion of the cavernous sinus and penile erection. Therefore, maintaining the normal function of corpus cavernosal smooth muscle plays a crucial role in improving penile erection and the treatment of T2DMED. Studies have shown that the smooth muscle phenotype is an important factor in maintaining the function and structure of smooth muscle, and regulating the phenotype of corpus cavernosum smooth muscle to the contractile type can improve the occurrence of ED [4]. At present, a large number of literature confirmed hypoxia [5–7], platelet-derived growth factor [8], chronic prostatitis [9], tobacco harmful gas [10], and other external factors may lead to the transition of CCSMs phenotype to synthetic phenotype, and then lead to ED. Diabetes hyperglycemia is also an important factor leading to smooth muscle phenotype switching [11], however, it is rarely reported in T2DMED.
Nesfatin-1 is a newly discovered secretory peptide found in the hypothalamus, brain stem, and gastrointestinal tract. It is a hydrolytic fragment of the nuclear group of protein 2 (NUCB2). In recent years, as research on diabetes-related complications has advanced, the role of Nesfatin-1 as a novel peptide molecule has gained widespread recognition. It has been acknowledged for its ability to enhance glucose metabolism, reduce blood glucose levels, regulate cardiovascular function, and consequently, ameliorate diabetes complications along with associated structural and functional abnormalities in organs [12–14]. Current studies have confirmed that Nesfatin-1 can improve diabetic cardiomyopathy [15], diabetic nephropathy [16], diabetic retinopathy [17], diabetic polycystic ovary syndrome [18, 19] and other diseases,which is considered as one of the important potential target drugs for the treatment of diabetes-related complications [20]. However, there have been no reports on whether Nesfatin-1 can improve Type 2 Diabetes Mellitus Erectile Dysfunction (T2DMED) and its specific mechanism. Therefore, this study aims to investigate the potential of Nesfatin-1 to improve T2DMED and elucidate its mechanism by examining the regulation of Nesfatin-1 on the phenotype switching of cavernous smooth muscle cells. The objective is to offer new insights for the clinical treatment of T2DMED.
Materials and methods
1. Experimental materials and instruments
1.1. Experimental animals and cell culture.
Twenty-four SPF grade 4-week-old wild-type C57BL/6J male mice weighing about 20g±3g were purchased from the Laboratory Animal Center of Ningxia Medical University. (Certificate number:SCXK2020-0001).Feeding conditions: constant temperature and humidity, 12 hours of light alternating daily, free feeding and drinking, quiet environment. Corpus cavernosum smooth muscle cells (CCSMCs) (Item No. CP-M218), Purchased from Wuhan Punosai Company. The first 5 generations of cultured cells were used in the experiment.
1.2. Experimental reagents and instruments.
High-fat Diets were obtained from Research Diets, USA (60 Kcal% Fat,Lot number:D12492; Nesfatin-1 (Item No.H-003-22) purchased from Phoenix (Burlingame, CA, USA); Immunohistochemical reagents were purchased from Beijing Zhongshan Jinqiao Biotechnology Development Co.LTD.OPN, α-SMA, PI3K, AKT, p-AKT, mTOR, p-mTOR antibodies and goat anti-rabbit and mouse antibodies secondary antibodies were purchased from Affinity Biosciences. Oleic acid (Item No. HY-N1446), Esculetin (Item No. HY-NO284); Sildenafil (Item No. HY-15025, 10mM), all bought in MCE company.
2. Experimental methods
2.1 Animal experiments.
2.1.1. Animal models were established and grouped. Twenty-four 4-week-old wild-type C57BL/6J male mice weighing about 20g±3g were randomly divided into Control group, Model group, and Nesfatin-1 treatment group, with 8 mice in each group.The control group was fed with a normal diet, and the model group and Nesfatin-1 treatment group were fed with a 60% high-fat diet for 16weeks. After 16weeks, Nesfatin-1 treatment group was intraperitoneally injected with Nesfatin-1 (8nM/kg) for 4 weeks, and model group was fed with high-fat diet for 4 weeks.All animal experiments were approved by the Ethics Committee of Ningxia Medical University.
2.1.2. Body mass, Blood glucose monitoring, and intracavernous pressure (ICP) measurement were performed. Body mass:The body mass of mice was monitored every 7 days during the same time period to analyze body mass changes.blood glucose:Oral glucose tolerance test (OGTT): After fasting for 16-18h before sampling, the mice were given 3g/kg glucose solution by gavage, and blood glucose was measured at 0min, 15mins, 30mins, 60mins, 90mins, and 120mins. The changes of blood glucose and the area under the curve (AUC) were recorded and analyzed [21]. Insulin tolerance test (ITT): After fasting for 5 hours for 2 days before sampling, the body weight was measured, and the blood glucose was measured at 0min, 15mins, 30mins, 60mins, and 120mins after intraperitoneal injection of insulin (1 IU/kg), and the AUC was analyzed.
2.1.3. Intracavernous pressure (ICP): After the mice were anesthetized with 2% pentobarbital sodium 45mg/kg and routinely sterilized. The foreskin and tunica albuginea were separated, and the subcutaneous fat and connective tissue were free until the penile cavernous nerve was obviously exposed. A platinum bipolar electrode was connected to an electrical stimulator, and the cavernous nerve was stimulated at 5V for 1min. The changes of intracavernous pressure during electrical stimulation were observed and recorded. The mean systemic blood pressure (MSBP) and the maximum intracavernous pressure (ICP) during the stimulation of the erectile nerve were measured and the ratio of ICP to MSBP was compared to evaluate the erectile response.
2.1.4. The level of glycosylated hemoglobin (HbA1c) in serum was detected by ELISA. Serum samples of mice in each group were collected, and anticoagulant was added and centrifuged. The levels of glycosylated hemoglobin (HbA1c) in serum samples of mice in each group were detected by ELISA method, and the operation steps were strictly according to the instructions in the kit.
2.1.5. Immunohistochemical staining:After dewaxing and entry, the paraffin sections were rinsed 3 times with PBS, and then the antigen was repaired with 0.01mol/L citrate buffer for 15minutes. After natural cooling for 2h, the sections were rinsed again, and the surface was covered with endogenous peroxidase blocker for 30mims. After rinsing, the sections were blocked with goat serum at room temperature for 1h, and the primary antibody was dropped and then blocked at 4°C overnight. The next day, a secondary antibody was added after rewarming for 30minutes, incubated at room temperature for 1h, and DAB was used for color development. After color development was completed, the slides were stained with hematoxylin solution for 4minutes, differentiated with 1% hydrochloric acid alcohol, flushed for 15minutes, and sealed with neutral resin after routine dehydration.
2.1.6. Animal tissues were detected by western blotting. After a freezing mill fully ground the penile tissue, the supernatant was taken, and the protein was extracted from the penile tissue of mice by the NDA/RNA/Protein Kit (Item No. R6734-01, omega Company). The protein concentration was measured according to the instructions of the BCA protein detection kit, and the loading volume was calculated. Electrophoresis at 80V voltage was followed by a 200mA electric transfer of 150mims. After blocking with 5% skim milk for 2h, the primary antibody was incubated at 4°C overnight, and the next day, the secondary antibody was incubated at room temperature for 2h. Images were acquired after exposure and quantified for analysis.
2.2. Cell experiments.
2.2.1. Culture and processing of mouse corpus cavernosum smooth muscle cells (CCSMCs). CCSMCs were routinely cultured in DMEM/F12 substrate containing 10%FBS.
2.2.2. Cell proliferation was determined by CCK-8 assay to select each drug’s treatment time and concentration. CCSMCs were routinely cultured and treated with Nesfatin-1 at concentrations of 0ng/ml, 50ng/ml, 100ng/ml, 150ng/ml, 200ng/ml, 250ng/ml and 300ng/ml. Oleic acid at Concentration of 0nM, 2.5nM, 5nM, 10nM, 15nM, 20nM. The concentration of Esculetin was 0μM, 10μM, 20μM, 30μM, 40μM; Each drug and concentration were set up 0h, 12h, 24h, 48h, 72h time gradient. Combined with the previous experiments, the concentration and time with the greatest change in cell OD were selected for further experiments.
2.2.3. Cellular immunofluorescence staining experiments. CCSMCs were cultured on cover slides. When the density reached 90%, it was fixed in 4% paraformaldehyde for 30mins, translatated with 0.3% Triton X-100 for 20mins, sealed with 5% BSA for 1h, the primary antibody was incubated at 4°C overnight, and the secondary antibody was incubated with fluorescein labeled at room temperature for 2h, and then sealed with DAPI-containing tablets.
2.2.4. Western blot was used to detect the expression of corresponding proteins in CCSMCs. The grouped treated cells were fully lysed with RIPA lysate for 30mims, adherent cells were treated with a cell scraper, centrifuged, and the supernatant was removed for determination of protein concentration by BCA and fixed with Buffer. Electrophoresis was performed at 80V with a 200mA electrotransfer and blocked with skim milk. The primary antibody was incubated at 4°C overnight, and the secondary antibody was incubated for 2h. After exposure, images were acquired and quantified for analysis.
3. Statistical methods
Graph Pad Prism 9.0 software was used to analyze the experimental results. Data were expressed as mean± standard deviation (mean±SD). One-way variance analysis was used for comparison of means between groups, and the Dunnett test was used for pairwise comparison of homogeneity of variance. P<0.05 was considered statistically significant.
Results
1. The type 2 diabetes mellitus erectile dysfunction (T2DMED) mouse model was induced through a 16-week high-fat feeding protocol
The exogenous administration of Nesfatin-1 demonstrated an improvement in the body weight and glucose metabolism indices of the T2DMED mice
After 16 weeks of high-fat feeding, the mice became fatter, weight increased significantly, and fasting blood glucose increased.The results of oral glucose tolerance test (OGTT) and insulin tolerance test (ITT) showed that blood glucose was significantly higher than that of normal control group at 15, 30, 60 and 120 minutes. Exogenous give Nesfatin-1 4 weeks after treatment, body size, body weight in mice, significantly reduced fasting glucose (Fig 1A–1C) (Body mass:Control group: 31.56±1.6g; Model group: 57.0±2.2g; Nesfatin-1 group: 41.1±2.2g. Fasting blood glucose: Control group:5.0±0.5; Model group:9.4±1.5; Nesfatin-1 group:5.7±0.2). Glycated hemoglobin (HbA1c) levels compared with model group significantly reduced (Control group: 4.9μ±1.4mol/ml; Model group: 11.1μ±3.1mol/ml; Nesfatin-1 group: 3.2μ±0.5mol/ml). OGTT and TII results of mice treated with Nesfatin-1 were significantly improved.The oral glucose tolerance test (OGTT) and insulin tolerance test (ITT) showed that blood glucose levels were significantly higher at 15, 30, 60, and 120 minutes compared to the control group. After 4 weeks of Nesfatin-1 treatment, the OGTT and ITT results in the treatment group significantly decreased (Fig 1D–1E), with statistically significant differences (P<0.05). Additionally, the level of glycated hemoglobin (HbA1c) after Nesfatin-1 treatment was significantly lower than the model group (P<0.05) (Fig 1C) (Control group: 4.9μ±1.4mol/ml; Model group: 11.1μ±3.1mol/ml; Nesfatin-1 group: 3.2μ±0.5mol/ml). with statistically significant differences (P<0.05).
a.Changes in the mouse body size; b.Changes in mice body mass; c.Changes of fasting blood glocuse in each group of mice; d.Changes of HbA1c in each group of mice; e.Changes in OGTT value and AUC in each group of mice; f.Changes in ITT value and AUC in each group of mice. (OGTT:Oral glucose tolerance test; ITT:Insulin tolerance test;AUC:area under the curve; n = 5 per group, *P<0.05).
2. T2DMED mice, subjected to a high-fat diet for 16 weeks, exhibited the development of erectile dysfunction
Notably, the exogenous administration of Nesfatin-1 significantly ameliorated erectile dysfunction in T2DMED mice
The peak value of the ICP curve in normal mice was 155±13.1cmH2O, and the mean value of ICP/MSBP was 0.90±0.05, while the peak value of ICP curve in T2DMED mice was significantly reduced to 80±22cmH2O, and the mean value of ICP/MSBP was 0.49±0.13. The difference was statistically significant (P < 0.05). After Nesfatin-1 treatment, the peak value of ICP curve in mice increased significantly and the ICP curve became steeper (Fig 2A). The average peak value of the curve was 154.4±16.1cmH2O, and As the average of ICP/MSBP was 0.9±0.04. Further statistical comparison of ICP/MSBP showed statistical differences among the three group (Fig 2B) (P<0.05).
a.ICP curves in mice. b.Changes in the ratio of ICP/MSBP of mice in each group. (ICP:Intracaversal pressure; MSBP:Systemic blood pressure (MSBP);n = 4 per group *P<0.05).
3. Nesfatin-1 increases the smooth muscle content of T2DMED mice
Masson staining was used to observe the changes of in smooth muscle and collagen fibers in the corpus cavernosum. We found that the smooth muscle fibers in T2DMED mice were significantly less than those in the control group, and the content of collagen fibers was significantly increased. After 4 weeks of Nesfatin-1 treatment, the smooth muscle increased and the collagen fibers decreased (Fig 3A and 3B), with statistically significant differences (P<0.05).
a. Masson staining results in each group of mice. (up bar: 200μm; down bar:100μm); b.Changes in the ratio of smooth muscle fiber to collagen fiber content in mice. (n = 4 per group *P<0.05).
4. Nesfatin-1 regulates the corpus cavernosal smooth muscle phenotype in T2DMED mice
Immunohistochemistry showed that α-SMA, a marker of smooth muscle contraction phenotype, significantly decreased in T2DMED mice and increased significantly after Nesfatin-1 treatment. However, the synthetic phenotypic marker OPN was significantly increased in the model group and decreased significantly by Nesfatin-1 treatment (Fig 4A and 4B). Further quantitative analysis of α-SMA and OPN by Western blot showed that the change trend was consistent with the results of immunohistochemistry (Fig 4C and 4D).
a.Immunohistochemical micrographs and quantitative analyses of α-SMA in mice; b.Immunohistochemical micrographs and quantitative analyses of OPN in mice; (up bar: 200μm; down bar: 100μm); c.The level ofα-SMA and OPN in penile cavernous tissue of mice was determined by Western blot; d.Quantitative analysis of α-SMA and OPN by Western blot. (n = 4 per group *P<0.05).
5. Nesfatin-1 regulates the PI3K/AKT/mTOR signaling pathway
The protein level PI3K, p-AKT/AKT and p-mTOR/mTOR in the penile cavernous tissue of mice was remarkably decreased in T2DMED(P<0.05). Compared to the model group, the level of PI3K, p-AKT/AKT and p-mTOR/mTOR in penile cavernous tissue was remarkably increased in the treatment group(P<0.05). Western blot results and quantitative analysis of gray values of bands showed that the PI3K was inhibited in T2DMED mice, and the phosphorylation levels of its downstream factors AKT and mTOR were also significantly decreased, but the total AKT and mTOR did not change. After Nesfatin-1 treatment, PI3K was significantly activated in the treatment group, and its downstream p-Akt and p-mtor were also significantly increased(P<0.05), but there was no significant difference in the total AKT and mTOR levels(Fig 5A and 5B).
a.The level of PI3K,p-AKT,AKT, p-mTOR and mTOR in the penile cavernous tissue of mice was determined by Western blot. b.Quantitative analysis of PI3K,p-AKT,AKT, p-mTOR and mTOR in each group(n = 4 per group *P<0.05).
6. The cell model was established through co-culture with high glucose and high oleic acid
Sildenafil, Nesfatin-1, and Esculetin were administered to observe their effects on the morphology and quantity of CCSMCs
CCSMCs were detected by CCK-8 experiment according to the corresponding drug concentration gradient and time gradient, and the corresponding optical density (OD) value of each hole measured at 450nm was determined. The results showed that the concentration of Acid oil was 20mM; The concentration of Nesfatin-1 at 150ng/mL; Esculetin at 40μM and treated separately for 48 hours had the greatest effect on OD values (Fig 6A). Therefore, we selected the drug concentration and treatment time for subsequent experiments.
a.DO values of oleic acid, nesfatin-1, and Esculetin in cck-8 assay. b.Micrographs of spongy smooth muscle cells in each group after 48 hours of treatment. (magnification:400X).
CCSMCs were divided into groups according to the drug concentration and treatment time selected by CCK-8 assay, and pictures of each group were taken by inverted microscope camera system. CCSMCs treated with high glucose and high oleic acid were round in shape and decreased in number. After Nesfatin-1 and Sildenafi treatment, most of the cells returned to spindle shape and their number increased significantly. There was no significant difference in cell morphology and number between Nesfatin-1 and Sildenafi treatment groups. However, after treatment with PI3K/AKT inhibitor Esculetin, the morphology of CCSMCs returned to the main round shape and the number of CCSMCS decreased significantly. (Fig 6B)
7. Through in vitro cell experiments, it has been confirmed that Nesfatin-1 regulates the transformation of CCSMCs into a contractile type, with the PI3K/AKT/mTOR signaling pathway playing a crucial role in this process
Immunofluorescence indicated that α-SMA expression decreased significantly in GO group, while OPN expression increased significantly and its shape changed from spindle to round. After Nesfatin-1 and Sildenafi treatment, α-SMA increased significantly, OPN decreased significantly, and cell morphology returned to a spindle shape. However, α-SMA expression was not significantly increased and OPN expression was not significantly decreased in the GO+N+E group, showing a trend similar to that in the GO group (Fig 7A and 7B). We quantitatively analyzed α-SMA and OPN of cells in each group by Western blot, and the change trend was consistent with the immunofluorescence results (Fig 7C and 7D). We quantitatively analyzed the PI3K/AKT/mTOR signaling pathway expression of CCSMCs in each group by Western blot. The results indicated that the expressions of p-AKT/AKT and p-mTOR/mTOR were significantly increased in CCSMCs treated after Nesfatin-1 and Sildenafi treatment (P<0.05). However, the expressions of p-AKT/AKT and p-mTOR/mTOR in the GO+N+E group were significantly decreased compared with those in GO+N and GO+S groups (P<0.05), showing a trend similar to that in the GO group (Fig 7E and 7F).
a. α-SMA expression in CCSMCs by immunofluorescence (Bar 50μm); b:OPN expression in CCSMCs by immunofluorescence.(Bar 50μm); c.The level ofα-SMA and OPN in penile cavernous tissue of mice were determined by Western blot; d.Quantitative analysis of α-SMA,OPN in each group;e.The level of PI3K,p-AKT,AKT, p-mTOR and mTOR in the penile cavernous tissue of mice was determined by Western blot;f.Quantitative analysis of PI3K,p-AKT,AKT, p-mTOR and mTOR in each group.(n = 4 per group *P<0.05).
Discussion
Currently, more than half of diabetes patients worldwide are suffering from erectile dysfunction (ED), and diabetes is gradually recognized as an important cause of ED [21, 22] The clinical treatment of diabetes-related erectile dysfunction mainly relies on basic therapies such as dietary control, hypoglycemic medication, combined with oral phosphodiesterase type 5 inhibitors (tadalafil/sildenafil, etc.) and penile cavernosal injection to improve erectile dysfunction However, in clinical treatment, there are still a considerable number of patients who do not respond well to these medications [23], and the lack of both can improve diabetes and can improve ED drugs. At present, the specific mechanism of erectile dysfunction caused by type 2 diabetes mellitus (T2DM) remains unclear, which is also the reason the poor effect of simply drugs to control blood glucose and treating erectile dysfunction with drugs is not satisfactory in clinical practice. Therefore, it is urgent to explore the mechanism of T2DMED, explore new drugs and new targets for improving T2DMED, and provide a new treatment idea for T2DMED patients.We through the establishment of T2DMED mouse model and exogenous Neisfatin ‐ 1 treatment. We found that the body weight, blood glucose, insulin resistance and erectile function of the treated mice were significantly decreased, and the expression of contractile phenotypic marker α-SMA was increased, while the synthetic phenotypic marker OPN was decreased, the corpus cavernosum smooth muscle fibers were increased, and PI3K/AKT/mTOR signaling pathway was activated.We established an in vitro model of spongy smooth muscle cultured with high oleic acid and high glucose and treated it with Nesfatin 1. We found that the CCSMCs phenotype changed to contractile after treatment, and the PI3K/AKT/mTOR signaling pathway was activated. After we added Esculetin to inhibit PI3K/AKT signaling pathway, the expression of α-SMA was significantly decreased and the expression of OPN was increased in corpus cavernosum smooth muscle cells. The effect of Nesfatin-1 on improving the phenotype switching of CCSMCs was significantly inhibited, which further demonstrated that Nesfatin-1 regulated the phenotype switching of CCSMCS through PI3K/AKT/mTOR signaling pathway.
Some studies have reported the use of the streptozotocin (STZ) injection method to induce diabetic erectile dysfunction in animal models, where the destruction of pancreatic islet cells leads to diabetes [24–27]. However, this approach does not align with the clinical epidemiological characteristics, given that type 2 diabetes mellitus is more prevalent in practical medical settings.In our research, we adopted a well-recognized modeling technique by continuously feeding mice with a high-fat diet for 16 weeks to establish a mouse model that closely mimics the disease characteristics and metabolic status of type 2 diabetes mellitus. Evaluation of relevant indicators of glucose metabolism in the model group revealed a significant increase in body weight, body type, and blood glucose values compared to the control group, showing statistically significant differences (P<0.05).Subsequently, we measured the intracavernous pressure (ICP) in the mice. The ICP of the mice in the model group decreased, manifesting the typical characteristics of the Type 2 Diabetes Mellitus Erectile Dysfunction (T2DMED) model. This outcome indicated that mice with type 2 diabetes mellitus induced solely by a high-fat diet would exhibit an erectile dysfunction phenotype, affirming the successful establishment of the T2DMED model.
We delved deeper into understanding the mechanism behind Type 2 Diabetes Mellitus Erectile Dysfunction (T2DMED). Penile erection is mainly regulated by the cavernous sinus of the penis, which is mainly composed of cavernous endothelial cells and smooth muscle cells. When the cavernous sinus is congested, it leads to the expansion of the cavernous body of the penis and produces an important mechanism of erection [28].We further explored the mechanism of T2DMED. At present, a large number of studies have reported that the dysfunction of cavernous endothelial cells leads to the occurrence of ED [29], but they have neglected that cavernous smooth muscle is the main tissue component responsible for controlling blood flow into the corpus cavernosa and causing erectile tissue. The regulation of cavernous smooth muscle phenotype is an important factor affecting cavernous function.The phenotypes of cavernous smooth muscle cells (CCSMCs) can be categorized into contractile and synthetic types. Contractile CCSMCs exhibit considerable plasticity, allowing them to undergo phenotypic regulation from a contractile state to a synthetic state in response to local environmental stimul [30]. The characteristic of smooth muscle cell transition to the synthetic phenotype is an increase in expressions of extracellular matrix and type I collagen, while the expressions of contractile cytoskeletal proteins (such as α-SMA, SM22α, etc.) decrease and the expressions of synthetic cytoskeletal proteins (such as OPN, tenascin-C, etc. [31]). The main component of contractile smooth muscle cells is myofilament, which is an important factor in maintaining smooth muscle contraction function. The synthetic phenotype has a strong ability to synthesize extracellular matrix, manifesting as excessive proliferation and fibrosis of smooth muscle cells, which can cause contractile dysfunction..Our study found that type 2 diabetes mellitus leads to a significant reduction of α-SMA and a significant increase of OPN in corpus cavernosum smooth muscle cells, which is consistent with the transformation of corpus cavernosum smooth muscle cells into synthetic cells, which may be the mechanism of type 2 diabetes mellitus leading to ED.
In recent years, the significant role of Nesfatin-1 in regulating blood glucose and improving diabetes-related complications has been widely reported [14, 32]. Previous studies have found that Nesfatin-1 can improve obesity-related type 2 diabetes and ameliorate atherosclerosis in the cardiovascular system [33].
Therefore, we speculate that Nesfatin-1 may improve T2DMED while improving type 2 diabetes. Therefore, we hypothesize that Nesfatin-1 may not only improve type 2 diabetes but also alleviate T2DMED. Thus, we attempted to investigate the role of Nesfatin-1 in improving T2DMED and its specific mechanism using the T2DMED mouse model. Through exogenous administration of Nesfatin-1 to the model mice, we observed a significant reduction in blood glucose, body weight, and body size, coupled with improvements in diabetes-related characteristics, effectively correcting metabolic disorders in mice. Even more surprisingly, we found a significant improvement in erectile dysfunction in mice treated with Nesfatin-1, confirming the potential of Nesfatin-1 as a therapeutic option for T2DMED.
In further mechanistic exploration, our investigation revealed that Nesfatin-1 treatment in T2DMED mice resulted in an increase in the number of smooth muscle fibers, a significant upregulation in the expression of the contractile phenotype marker, and a substantial improvement in the contractility of the penile cavernosum. These findings suggest that Nesfatin-1 has the capacity to reduce α-SMA and increase OPN, inducing a transformation in the synthetic phenotype of corpus cavernosum smooth muscle towards the contractile phenotype. Consequently, this restoration of the normal contractile function of corpus cavernosum smooth muscle is indicative of the therapeutic potential of Nesfatin-1 in T2DMED.Studies [34] have pointed out that impaired NO activity released by cavernous endothelial cells is an important cause of erectile dysfunction, and excessive damage of NO to cavernous endothelial cells under pathological conditions is the main mechanism of ED induced by NO-CGMP pathway. cGMP acts as a second messenger to regulate Ca2+ channels, inhibit calcium influx, reduce intracellular calcium concentration, and thus induce relaxation of corpus cavernosum smooth muscle, which is one of the mechanisms of cGMP regulating erectile function. Usually in the diabetic state, NO bioactivity is impaired and the formation of cGMP is limited, resulting in the failure of normal relaxation of corpus cavernosum smooth muscle, which may be one of the reasons for the difficulty in routine treatment of ED in diabetic patients [35]. However, other data suggest that it is at least partially independent of the NO-cgmp pathway [36, 37]. In another study, experimentalshad combined drugs with different NO-binding sites in rat and rabbit models of erectile dysfunction similarly stimulated cGMP production and induced relaxation of cavernous smooth muscle and penile erection independent of NO [38, 39]. This suggests that there are other pathways besides the NO-cGMP pathway that regulate the contraction and relaxation of cavernous smooth muscle. Therefore, exploring a new regulatory pathway is of great significance to improve the clinical treatment of diabetic erectile dysfunction. As a downstream regulator of the classical PI3K/AKT signaling pathway, mTOR has been widely recognized to participate in the regulation of glucose metabolism and energy metabolism. In our previous study, we found that the phosphorylation level of mTOR was significantly inhibited in type 2 diabetic mice induced by HFD.Mu Y et al. came to the same conclusion as us [40]. Similarly, in the context of a high-fat diet-induced model Michelet X et al. also found reduced expression of the mTOR signaling pathway [41].We used PI3K/AKT/mTOR signaling pathway to further explore its possible mechanism in regulating the phenotypic switching of corpus cavernosum smooth muscle. We found that regardless of the inhibition of PI3K/AKT/mTOR signaling pathway and the conversion of cavernous smooth muscle phenotype to synthetic phenotype in the model mice, the PI3K/AKT/mTOR signaling pathway was significantly activated and the expression of contractile phenotype markers was significantly increased after Nesfatin-1 treatment. In a study on phenotypic transformation of vascular smooth muscle, Shui-Bo Zhu [40] et al. demonstrated that activation of PI3K/AKT signaling pathway would promote the transformation of smooth muscle to contractile phenotype and improve smooth muscle function. This is consistent with our conclusion.
To validate our findings, we conducted in vitro cell experiments. We cultured cavernous smooth muscle cells in a high-glucose and high-oleic acid environment to simulate the microenvironment of diabetic metabolic disorder. Our observations revealed that corpus cavernosum smooth muscle cells exhibited a synthetic phenotype consistent with those from type 2 diabetic mice when exposed to a culture environment with high oleic acid and high glucose. After Nesfatin-1 intervention, there was a transformation from a synthetic to a contractile type.
Upon further exploration of the mechanism, we observed that the PI3K/AKT/mTOR signaling pathway was significantly inhibited. In spongy smooth muscle cells cultured with high oleic acid and high glucose, α-SMA expression was significantly decreased, and OPN expression was significantly increased. Following Nesfatin-1 treatment, p-AKT and p-mTOR significantly increased, activating the PI3K/AKT/mTOR signaling pathway. Simultaneously, α-SMA expression significantly increased, and OPN expression significantly decreased.
To further confirm the role of the signaling pathway, we introduced Esculetin to Nesfatin-1 treated cells to directly inhibit the PI3K/AKT/mTOR signaling pathway. We found that Nesfatin-1 significantly inhibited the improvement of phenotype switching in cavernous smooth muscle cells. This further substantiates that Nesfatin-1 regulates the phenotypic switching of CCSMCs through the PI3K/AKT/mTOR signaling pathway.
In recent years, there has been a growing body of research on the role of the star protein Nesfatin-1. Most of these studies have focused on the significant impact of Nesfatin-1 on improving diabetes and obesity [38]. Currently, there is a lack of research investigating the role of Nesfatin-1 in the treatment of type 2 diabetes-related erectile dysfunction (T2DMED). Erectile dysfunction, as a complication of type 2 diabetes, is associated with type 2 diabetes but also has independent pathological mechanisms. Whether Nesfatin-1 can ameliorate T2DMED and the mechanisms involved present crucial scientific questions.This study employed a high-fat feeding approach to establish a type 2 diabetes mouse model, confirming the occurrence of T2DMED. Leveraging cell experiments to simulate the glucose and lipid metabolism microenvironment of corpus cavernosum smooth muscle cells in type 2 diabetes, Nesfatin-1 was introduced for intervention and investigation. The findings demonstrate that Nesfatin-1 not only improves T2DMED but also plays a crucial role in the phenotypic transformation of corpus cavernosum smooth muscle cells.
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