The Efficacy of Intralesional Baicalein Injection in Rat Peyronie's Model

The Efficacy of Intralesional Baicalein Injection in Rat Peyronie's Model | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article The Efficacy of Intralesional Baicalein Injection in Rat Peyronie's Model Kadir Can Sahin, Feyyaz Irmak, Mehmet Gültekin, Ozge Gokbasi, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6752805/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 20 Oct, 2025 Read the published version in International Journal of Impotence Research → Version 1 posted 10 You are reading this latest preprint version Abstract TGF-β1 plays a fundamental role in the pathogenesis of Peyronie's disease, driving the excessive extracellular matrix accumulation and fibroblast activation characteristic of fibrosis. As in many fibrotic disorders, its action via Smad transcription factors presents a key therapeutic target. Given the notable deficiency in proven effective conservative treatments for Peyronie's disease, particularly in its acute phase, this study aimed to investigate the efficacy of Baicalein, a flavonoid known to inhibit the TGF-β1/Smad signaling pathway, thereby offering a promising therapeutic strategy. We established a rat model of Peyronie's disease and administered Baicalein intralesionally at low, moderate, and high doses. Our comprehensive analysis of histopathological and immunohistochemical parameters, including tunica albuginea thickness, fibrosis severity, and smooth muscle content, demonstrated that intralesional Baicalein dose-dependently suppresses fibrotic plaque formation, preserves crucial cavernosal smooth muscle tissue, and effectively prevents pathological increases in tunica albuginea thickness. Importantly, systemic toxicity was not detected. As the first study to investigate intralesional Baicalein for Peyronie's disease, our findings positively contribute to the literature and underscore its potential as a safe, accessible, and highly effective agent. Further in vitro and in vivo research is warranted to fully explore Baicalein's capacity to address current treatment gaps in this challenging condition. Health sciences/Medical research/Experimental models of disease Health sciences/Pathogenesis Animal Experimentation Baicalein Fibrosis Peyronie’s disease Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 INTRODUCTION Peyronie's disease (PD) is an acquired connective tissue disorder characterized by the formation of a localized fibrous plaque in the tunica albuginea (TA). The pathophysiology of PD is not yet fully understood and is still a matter of debate. Abnormal collagen accumulation in the TA secondary to repetitive penile microtrauma, rupture of elastic fibers, and remodeling of connective tissue in a fibrotic background are among the most accepted hypotheses to date [ 1 ]. Free oxygen radicals, oxidative stress, nitric oxide synthase dysfunction, autoimmunity, and certain viral infections have been the subject of various studies, considering their potential roles in the impaired tissue healing observed in PD [ 1 – 6 ]. Understanding the pathophysiology of the disease is crucial for both explaining the symptoms and managing the disease with appropriate treatment options. Recent studies have highlighted TGF-β1 as the most significant factor inducing fibrosis in the TA by affecting extracellular matrix accumulation through fibroblast stimulation [ 1 , 7 , 8 ]. While evidence supporting TGF-β1 as a cytokine crucial for tissue repair is increasing, its excessive activity is held responsible for tissue damage secondary to scarring in many serious diseases [ 9 ]. It has been reported that overexpression of TGF-β1 and activation of Smad transcription factors may be the basis of pathogenesis in PD, based on examinations of its pathophysiological mechanisms [ 10 ]. Therefore, inhibiting the TGF-β1 signaling pathway may be a promising therapeutic strategy in the treatment of PD. Given the role of TGF-β1 in PD fibrosis, targeting this pathway presents a promising therapeutic strategy. Baicalein, or 5,6,7-trihydroxyflavone, is a type of flavonoid originally derived from the roots of Scutellaria baicalensis and Scutellaria lateriflora plants [ 11 ]. Known for its anti-inflammatory and anti-thrombotic effects, Baicalein is also recognized for its efficacy as a lipoxygenase inhibitor and its ability to reduce oxidative stress at the cellular level [ 12 ]. The underlying mechanisms of Baicalein are thought to stem from its selective binding to the ATP-binding site of activin receptor-like kinase 5, a TGF-β receptor I, without causing any changes in the expression of Smad6, Smad7, TGF-β1, or TGF-β receptor I/II [ 13 ]. It exhibits an inhibitory effect on the TGF-β/Smad2/3 signaling pathway in both in vivo and in vitro fibroblasts by dose-dependent down-regulation of phosphorylated Smad2 and Smad3 levels. Baicalein does not affect Smad1, 5, and 8 in the bone morphogenetic protein signaling pathway and does not affect total Smad2 and Smad3 levels [ 13 ]. During the proliferative phase of scar formation in a mechanically loaded mouse model, Baicalein significantly suppresses the proliferation and activation of hypertrophic scar-derived fibroblasts and inhibits α-SMA expression. This leads to impaired contraction and migration abilities of hypertrophic scar-derived fibroblasts, thereby reducing collagen accumulation and alleviating hypertrophic scar formation [ 14 ]. Current European and American urology guidelines reveal a notable deficiency in proven effective conservative treatment alternatives, particularly for managing patients in the acute inflammatory phase of the disease [ 15 , 16 ]. Considering the gap in the treatment options of the PD, this study aims to investigate the efficacy of Baicalein in our rat Peyronie's model. Specifically, we aim to demonstrate its potential use as an effective agent in PD through inhibition of the TGF-β1 pathway via intralesional administration. MATERIALS AND METHODS Establishment of a Rat Model for Peyronie's Disease In the conducted animal experiment, the 3R principles were adhered to, and a power analysis was performed using G*Power version 3.1 [ 17 ], taking into account previously published animal models. A confidence interval of 95% was established, and the power analysis, conducted using one-way ANOVA, revealed that 1-β exceeded 0.5 based on the sample size determined from the literature. The procedures used and the care of animals were approved by the Institutional Animal Care and Ethics Committee (HADYEK) in Istanbul University - Cerrahpasa (E-74555795-050.01.04-794309). Thirty male albino Wistar rats, aged over 6 months and weighing at least 450 grams, were housed in a controlled environment at the laboratory. Baicalein (ab120723, Abcam, Cambridge, UK) dosage was determined from similar studies in the literature [ 18 ]. Rats were randomly assigned to six groups: six rats as the control group, six rats as the disease model group, six rats as the low-dose Baicalein (0.8 µg/L, 50 µL) group, six rats as the moderate-dose Baicalein (1.6 µg/L, 50 µL) group, and six rats as the high-dose Baicalein (3.2 µg/L, 50 µL) group. To induce the disease model, the previously described single-injection model of combined solution of 0.1 mL active recombinant TGF-β1 protein (0.01 µg/µL, ab50036, Abcam, Cambridge, UK) and 0.1 mL sodium tetradecyl sulfate (0.01 µg/µL, 3% Tromboject®, Omegapharma, Alberta, Canada) was administered intratunically as a single injection [ 19 , 20 ]. Anesthesia was induced intraperitoneally with a combination of 10 mg/kg Xylazine and 80 mg/kg Ketamine [ 21 ]. Following anesthesia, rats were placed in a supine position, and the injection area was cleaned with a 10% povidone-iodine solution. All injections were performed under 12x magnification using an operating microscope (YZ-20P5 Operation Microscope, Visionstar, Chongqing, China) with 30G − 0.8 mm − 0.5 mL syringes from the right lateral 1/3 proximal section of the penile shaft. Animals were weighed before each procedure. Following a four-week model induction period, the experiment groups received a total of three intralesional Baicalein injections, administered three weeks apart, following the described injection protocol. Intralesional injections were performed by palpation of the fibrotic area at the injection site where the experimental model was created and under 12x magnification with an operating microscope to avoid injecting the solution into the cavernous tissue or distributing it outside the tunical layers due to superficial application. The six rats in the control group received 0.2 mL sterile saline injections on the same days and using the same protocol as the disease model induction and treatment applications in the study groups. Throughout the follow-up period, rats were monitored daily for signs of distress (> 15% weight loss, impaired mobility, nutritional disorders, reduced responsiveness), which led to exclusion after consultation with the responsible veterinarian. Animals were housed freely in their cages, without any restrictive procedures or deprivation-related experimental procedures. Euthanasia and Autopsy of Animals Three weeks after the final injection in the study group, all rats completing the follow-up protocol were euthanized under anesthesia by cardiac blood aspiration. The penises of the euthanized rats were clamped at the radix to determine the post-treatment curvature degree using goniometry. To avoid affecting the histopathological evaluation, artificial erection was induced by injecting saline into corpora cavernosa (CC) using a 27G needle from the contralateral side of the injection area. All rats underwent autopsy with abdominal and thoracic incisions. Subsequently, penectomy was performed, followed by bilateral orchiectomy, bilateral nephrectomy, and hepatectomy for toxicity assessment. Bilateral testes, epididymides, and vas deferens were removed en bloc and placed in Hollande's fixative, while the remaining organs were placed in 10% formaldehyde and transferred to the Medical Pathology laboratory on the same day. Histopathological and Immunohistochemical Examination of Specimens Following sufficient fixation, the length and diameter of the specimens were measured and documented with photographs. The proximal portions of the materials were marked with black tissue dye, the middle portions with green dye, and the distal portions (glans penis) with orange dye. Starting from the proximal end, the materials were serially sectioned perpendicular to their long axes at 3 mm intervals towards the distal end. Following routine processing and sectioning, the specimens were stained with Hematoxylin and Eosin (H&E), Masson-Trichrome (MTC), Reticulin, Weigert van Gieson, Alpha Smooth Muscle Actin (α-SMA), TGF-ß1, and Col1A1. To assess the treatment response, fibrosis, smooth muscle loss in the CC, chondroid differentiation, and bone metaplasia/ossification were evaluated as key parameters for efficacy. Additionally, congestion and edema were assessed to determine the level of tissue reaction. All parameters were categorized into three severity grades: absent, mild/moderate, and severe. Measurement of Tunica Albuginea Thickness To ensure standardization in the measurement of TA thickness across all specimens, the mid-section of the penile shaft, where the highest level of fibrosis was observed, was identified under microscopic examination. Subsequently, two imaginary lines were drawn to determine the level at which the measurement would be taken: one vertical line bisecting the urethra and the other horizontal line, perpendicular to the first, dividing the CC between the urethra and dorsal vein into two equal halves ( Fig. 1 ) . TA thicknesses were measured bilaterally for each case at 40x magnification using an Olympus Provis AX70 (Olympus Corporation, Shinjuku, Japan) fluorescence microscope ( Fig. 1 ) . Tissue gaps resulting from extravasation from the CC due to artificial erection post-euthanasia were excluded to calculate the net TA thickness. Determination of Fibrosis Level by Digital Area Calculation Digital images of the mid-section H&E x2 slices from all cases were obtained. Using Sketchandcalc software ( https://www.sketchandcalc.com/ ), the CC area was outlined in blue, and the fibrotic areas were outlined in green ( Fig. 2 ) . The areas of these two outlined regions were calculated using the digital area calculation software of the same application for each case, and then the ratio of these areas was determined to examine it as an indicator of treatment response. Statistical Analysis All statistical analyses were performed using SPSS version 28.0 (IBM, Armonk, USA). Quantitative data are expressed as median (minimum - maximum), and qualitative data as number (%). Categorical variables were analyzed using Chi-square test or Fisher's Exact test, as appropriate. Situations where the arithmetic means of three or more independent sample groups with non-parametric distribution conditions were different were determined using the Kruskal-Wallis test with post-hoc Bonferroni correction. For all tests, p < 0.05 was considered statistically significant. RESULTS Of the 30 rats initially enrolled in the study, four were excluded. Local infective complications led to the exclusion of one rat in the low-dose Baicalein group and one rat in the moderate-dose Baicalein group. Additionally, one rat in the high-dose Baicalein group exhibited a significant decrease in oral intake, and one rat in the disease model group sustained injuries from an inter-cage altercation and required removal from the study after veterinary evaluation. Consequently, 26 rats completed the study: six in the control group and five in each of the disease model and three treatment groups. Pre- and post-treatment weights were evaluated for all rats completing the study. Comparison of median weights revealed no statistically significant differences between groups due to treatment (Table 1) . Similarly, pre-treatment and pre-euthanasia penile curvature measurements showed no statistically significant changes across the treatment groups ( Table 1) . Statistical analysis of the parameters employed to assess treatment response revealed significant difference between the groups in fibrosis and CC smooth muscle loss (p = 0.015 and p = 0.005, respectively) ( Fig. 3 – 4 ) . Conversely, chondroid differentiation and bone metaplasia/ossification did not exhibit statistically significant intergroup differences (p = 0.118 and p = 0.206, respectively) ( Fig. 5 ) . Of the parameters evaluated to assess the tissue reaction, congestion did not show a significant difference between groups (p = 0.176), while edema was significantly different between groups (p = 0.001). The comparative analysis of the data obtained from the histopathological evaluations of the cases is compiled and presented in Table 2 . Quantitative analysis of digital measurements to assess treatment response revealed statistically significant differences between groups in both mean TA thickness and the ratio of total fibrotic area to CC area (p = 0.002 for both) ( Table 1) . Macroscopic and microscopic evaluations of the testes, kidneys, and liver, supplementary to the penectomy specimens, revealed no evidence of toxicity attributable to the experimental model or treatment protocol. Specifically, investigation of the testes showed no toxicological effects on germ cells. DISCUSSION It is known that TGF-β1 plays a fundamental role in the pathogenesis of PD through Smad-dependent pathways. Activated TGF-β1 induces Smad2/3 phosphorylation and nuclear translocation, driving the fibrotic process in PD [ 9 ]. Consequently, agents that inhibit the TGF-β1/SMAD pathway hold promise for PD treatment. Considering the inadequacy of efficacy and evidence levels in conservative treatment options [ 15 – 16 ], especially in the acute phase of PD, our study aimed to investigate the efficacy of Baicalein, a substance known to have effects on this pathway, which forms the basis of PD pathophysiology, as a potential agent that can limit fibrosis in a rat Peyronie's model. Building upon the understanding of TGF-β1's role in fibrosis, the efficacy of Baicalein treatment has been investigated in various diseases that progress with fibrosis, similar to PD [ 18 , 22 – 24 ]. Zhang et al. demonstrated that Baicalein injections reduced phosphorylated Smad2-3 levels in mice with hypertrophic scars, inhibiting hypertrophic scar formation, fibroblast proliferation, and activation [ 14 ]. Similarly, in a systemic sclerosis model, Baicalein treatment was administered at different doses to mice with systemic sclerosis, Baicalein treatment exhibited dose-dependent efficacy through B cell modulation and TGF-β1 inhibition [ 22 ]. Translating preclinical findings to clinical practice, a recent single-center retrospective study, the efficacy of oral Baicalein treatment in the acute phase of PD was investigated. In this study, in which the data of 261 Peyronie's patients were evaluated, it was shown that Baicalein reduced plaque volume, pain, and penile curvature, and positive effects on erectile dysfunction were reported [ 25 ]. To achieve targeted efficacy and minimize systemic exposure, our study employed intralesional Baicalein administration. Consistent with these previous reports, we observed a dose-dependent inhibition of fibrosis. The fact that no toxicity findings were detected in the systemic evaluation after euthanasia indicates that Baicalein can be used as a safe agent. In our histopathological examinations, it was shown that TA thickness in the disease group was significantly increased compared to the control and treatment groups. In addition, widespread fibrosis in the CC tissue adjacent to the injection area, the presence of chondroid differentiation developing in some places, and cavernosal smooth muscle cell loss were noteworthy. The described findings were more severe in the untreated rat group, and the severity of the findings decreased dose-dependently in the treatment groups. The presence of a dose-dependent effect was found to be consistent with the results of Baicalein treatment applied in diseases with similar mechanisms of action in the literature [ 22 ]. Although ED is known to be seen in 37.5% of men with Peyronie's disease, it is also known that many patients do not apply to a doctor due to their concerns and fears [ 26 ]. ED usually occurs as a result of TA compliance loss and cavernosal smooth muscle loss due to plaque formation and fibrosis in the penis [ 26 , 27 ]. Our study reveals that the CC tissue of the rat group in which the experimental model was created is characterized by widespread smooth muscle loss. In our treatment groups, immunohistochemical analysis showed a significant preservation of smooth muscle tissue in the CC structure, as evidenced by α-SMA staining. Applications evaluating erectile functions, such as electrostimulation and erection moment intracavernosal pressure measurement, to evaluate erection in our study groups were not used, considering that they could affect our primary endpoint, histopathological and immunohistochemical analysis results. Although the hypothesis that erectile functions will be preserved with the preservation of smooth muscle tissue in the CC structure can be put forward, evidence should be presented in this context with new studies to be designed. The literature on PD lacks standardized, objective criteria for experimental evaluation. Many parameters, such as the degree of developing penile curvature, the size of plaque formation, TA thickness, fibrosis level, collagen fiber amount and ratios, the condition of smooth muscle cells in the CC tissue, and the evaluation of elastic fibers, have been used both in the formation of experimental models and in the evaluation of the efficacy of the therapeutic agent [ 16 , 28 ]. In our study, fibrosis severity, CC smooth muscle loss degree, average TA thickness, fibrosis ratio in CC, presence of bone metaplasia, presence and prevalence of chondroid differentiation were determined as basic parameters for the evaluation of treatment efficacy. When the results of the group in which the disease model was created and the treatment groups were analyzed comparatively, fibrosis severity, average TA thickness, fibrosis ratio in CC, and edema severity were determined as significant different parameters between the groups. Taking these parameters as evaluation criteria in future studies will provide a standardized objective analysis opportunity. A review of published rat models of Peyronie's disease, widely used in research, indicates that the resulting fibrotic plaque often extends beyond the TA, inducing widespread fibrosis in the surrounding CC tissue [ 16 , 28 – 30 ]. This phenomenon may be attributed to factors such as the thinness of the rat TA, challenges in achieving optimal tunical injection, and extravasation of injected agents. In our study, despite observing increased TA thickness in the disease model group, we also noted extensive fibrosis, smooth muscle cell loss, and chondroid changes progressing to bone metaplasia in the surrounding CC tissue. Re-evaluation of published studies on rat Peyronie's models, in light of these findings, revealed a frequent discrepancy: many histopathological images, while labeled as fibrotic plaques within the TA, in fact depict more extensive fibrotic processes involving the CC tissue, despite being described as PD. The similarity of our findings to these studies raises questions about the validity of current rat Peyronie's models. Based on these observations, we suggest that these widely used models may more accurately represent a ‘diffuse penile fibrosis model,’ a distinction that warrants further discussion. This reinterpretation could have significant implications for the translation of preclinical findings to human PD treatment, necessitating a more cautious approach to the interpretation of animal model data. By demonstrating the intralesional efficacy of Baicalein in a rat Peyronie's model and representing the first instance of this administration route, our study makes a positive contribution to the literature and serves as a foundation for future research. Based on the promising results of our pilot study, we believe Baicalein, an easily accessible, low-cost, highly effective agent with non-systemic toxicity, holds high potential as a therapeutic for PD and warrants further investigation. In conclusion, intralesional Baicalein in the rat Peyronie's model suppresses fibrosis in the treatment groups, has a protective effect on the smooth muscle tissue in the CC structure, and prevents the increase in thickness in the TA observed in the course of PD without causing any systemic toxicity. In line with the results of both in vitro and in vivo studies to be designed, Baicalein has the potential to be an alternative that can address the shortcomings in PD treatment. Declarations Data availability statement: The data that support the findings of this study are available from the corresponding author, KCS, upon reasonable request. Acknowledgement: None. Competing interests statement: All authors declare that they have no competing financial interests in relation to the work described. Funding statement: This study was carried out with the support of Istanbul University – Cerrahpasa Scientific Research Projects Unit (Project number: 37567, approval date: 07.02.2024). Ethics committee approval number: E-74555795-050.01.04-794309 Author Contribution Statement: All authors read and approved the final manuscript. Conceptualization: KCS, FI, MHG, HO. Data curation: KCS, FI, OG, SK. Formal analysis: KCS. Funding acquisition: KCS, FI, MHG. Investigation: KCS, FI, MHG, OG. Methodology: SK, IG, EA, HO. Project administration: KCS, FI, MHG. Resources: KCS, FI, MHG. Supervision: IG, EA, HO. Validation: SK, OG, IG. Visualization: KCS, FI, OG. 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J Sex Med. 2017 Oct;14(10):1270-1276. doi: 10.1016/j.jsxm.2017.08.011. doi: 10.1016/j.jsxm.2017.08.011. Castiglione F, Hedlund P, Weyne E, et al. Intratunical Injection of Human Adipose Tissue-Derived Stem Cells Restores Collagen III/I Ratio in a Rat Model of Chronic Peyronie's Disease. Sex Med. 2019 Mar;7(1):94-103. doi: 10.1016/j.esxm.2018.09.003. Tables Tables 1 and 2 are available in the Supplementary Files section Additional Declarations There is NO conflict of interest to disclose. Supplementary Files Table126032025.xlsx Table226052025.xlsx Cite Share Download PDF Status: Published Journal Publication published 20 Oct, 2025 Read the published version in International Journal of Impotence Research → Version 1 posted Editorial decision: revise 07 Jul, 2025 Review # 2 received at journal 24 Jun, 2025 Reviewer # 2 agreed at journal 15 Jun, 2025 Review # 1 received at journal 02 Jun, 2025 Reviewer # 1 agreed at journal 29 May, 2025 Reviewers invited by journal 29 May, 2025 Submission checks completed at journal 28 May, 2025 First submitted to journal 27 May, 2025 Unknown event 27 May, 2025 Editor assigned by journal 26 May, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6752805","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":463760493,"identity":"2b6b6772-e7f3-4b33-94a1-cf1c24246730","order_by":0,"name":"Kadir Can Sahin","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8UlEQVRIiWNgGAWjYFACHoYDQMjYBuZUADEzcwMpWs6AtDAS1sIA0gJWBtFIQIt8+9mDhwvO2Mn2SR9++LlwXm00fztQy4+KbTi1GJzJSzg840aycRtfmrH0zG3Hc2ccZmxg7DlzG7cWhhyDwzwfmBPbeHgYpHm3HcttAGphZmzDrUW+/w1ISz1IC/Nv3jnHcucT0sJwA2TLjcMgLWzSvA01uRsIaTG4AbLlzHHjNh42M2ueYwdyNwK1HMTnF/n+HOPPPMeqZef3MD++zVNTlzvv/OGDD35U4HEYGjgMJg8QrR4I6khRPApGwSgYBSMEAACIVly39GpfPgAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-6848-9506","institution":"Istanbul University - Cerrahpasa, Cerrahpasa Faculty of Medicine","correspondingAuthor":true,"prefix":"","firstName":"Kadir","middleName":"Can","lastName":"Sahin","suffix":""},{"id":463760494,"identity":"f95d154d-7e34-4f07-adcd-808a95864ed1","order_by":1,"name":"Feyyaz Irmak","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Feyyaz","middleName":"","lastName":"Irmak","suffix":""},{"id":463760495,"identity":"4a572b82-e7bf-40ec-8f31-048b9a2583e4","order_by":2,"name":"Mehmet Gültekin","email":"","orcid":"","institution":"Istanbul University","correspondingAuthor":false,"prefix":"","firstName":"Mehmet","middleName":"","lastName":"Gültekin","suffix":""},{"id":463760496,"identity":"fc04c990-07e4-426d-aa6f-d2917b765b9e","order_by":3,"name":"Ozge Gokbasi","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Ozge","middleName":"","lastName":"Gokbasi","suffix":""},{"id":463760497,"identity":"0d9a3881-6421-4928-8f04-e988a61ca769","order_by":4,"name":"Sima Kilic","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Sima","middleName":"","lastName":"Kilic","suffix":""},{"id":463760498,"identity":"bc9e35bc-ff5c-4106-ad46-62b14c6e588b","order_by":5,"name":"Iclal Gurses","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Iclal","middleName":"","lastName":"Gurses","suffix":""},{"id":463760499,"identity":"fed71c7a-b55a-4461-9643-910f11018073","order_by":6,"name":"Emre Akkus","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Emre","middleName":"","lastName":"Akkus","suffix":""},{"id":463760500,"identity":"5e20a586-a871-438c-aa3b-226db952fdef","order_by":7,"name":"Hamdi Özkara","email":"","orcid":"","institution":"Istanbul University Cerrahpasa Faculty of Medicine, Department of Urology","correspondingAuthor":false,"prefix":"","firstName":"Hamdi","middleName":"","lastName":"Özkara","suffix":""}],"badges":[],"createdAt":"2025-05-26 17:00:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6752805/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6752805/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41443-025-01197-1","type":"published","date":"2025-10-20T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":83743523,"identity":"adaadd47-9618-4853-89c0-e3ca3aea9aa5","added_by":"auto","created_at":"2025-06-02 02:36:04","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1122309,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003e Identification of the area for TA thickness measurement with two imaginary lines intersecting perpendicularly (measurement was made in the areas with red circles), H\u0026amp;E x4, \u003cstrong\u003eB and C.\u003c/strong\u003eBilateral TA thickness measurement of one subject from the control group (net TA length was calculated by subtracting the length of the red line from the length of the black line), H\u0026amp;E x40\u003c/p\u003e","description":"","filename":"Binder41.png","url":"https://assets-eu.researchsquare.com/files/rs-6752805/v1/f0b6ed53d73998e01b6c78de.png"},{"id":83743441,"identity":"a271cc92-e3dc-436a-ab8d-e2dc4cbc6c07","added_by":"auto","created_at":"2025-06-02 02:28:04","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1206883,"visible":true,"origin":"","legend":"\u003cp\u003eDetermining the ratio of the corpora cavernosa area (blue) and fibrotic area (green) of subjects, \u003cstrong\u003eA.\u003c/strong\u003e A subject from disease model group, H\u0026amp;E x2 \u003cstrong\u003eB.\u003c/strong\u003e A subject from moderate-dose Baicalein group, H\u0026amp;E x2\u003c/p\u003e","description":"","filename":"Binder42.png","url":"https://assets-eu.researchsquare.com/files/rs-6752805/v1/9a7f04edf63af349549c8561.png"},{"id":83743197,"identity":"83da1940-2791-4e72-8169-b4893043f790","added_by":"auto","created_at":"2025-06-02 02:20:04","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1556773,"visible":true,"origin":"","legend":"\u003cp\u003eFibrosis assessment, \u003cstrong\u003eA. \u003c/strong\u003eControl group, H\u0026amp;E x4, \u003cstrong\u003eB.\u003c/strong\u003e Control group, H\u0026amp;E x10, \u003cstrong\u003eC.\u003c/strong\u003e Control group,H\u0026amp;E x20, \u003cstrong\u003eD.\u003c/strong\u003e Disease model group, H\u0026amp;E x4,\u003cstrong\u003e E.\u003c/strong\u003e Disease model group, H\u0026amp;E x10, \u003cstrong\u003eF.\u003c/strong\u003e Disease model group, H\u0026amp;E x20, \u003cstrong\u003eG.\u003c/strong\u003eLow-dose Baicalein group, H\u0026amp;E x4, \u003cstrong\u003eH.\u003c/strong\u003e Low-dose Baicalein group, H\u0026amp;E x10, \u003cstrong\u003eI.\u003c/strong\u003e Low-dose Baicalein group, H\u0026amp;E x20, \u003cstrong\u003eJ.\u003c/strong\u003eModerate-dose Baicalein group, H\u0026amp;E x4, \u003cstrong\u003eK.\u003c/strong\u003e Moderate-dose Baicalein group, H\u0026amp;E x10, \u003cstrong\u003eL.\u003c/strong\u003e Moderate-dose Baicalein group, H\u0026amp;E x20, \u003cstrong\u003eM.\u003c/strong\u003eHigh-dose Baicalein group, H\u0026amp;E x4, \u003cstrong\u003eN.\u003c/strong\u003e High-dose Baicalein group, H\u0026amp;E x10, \u003cstrong\u003eO.\u003c/strong\u003e High-dose Baicalein group, H\u0026amp;E x20\u003c/p\u003e","description":"","filename":"Binder43.png","url":"https://assets-eu.researchsquare.com/files/rs-6752805/v1/4adac8349a1fd0848c84b14e.png"},{"id":83743201,"identity":"16115edd-0936-4ad0-9098-6da7ca34c44c","added_by":"auto","created_at":"2025-06-02 02:20:04","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1914862,"visible":true,"origin":"","legend":"\u003cp\u003eEvaluation of smooth muscle loss in the corpora cavernosa by α-SMA staining, x4 \u003cstrong\u003eA.\u003c/strong\u003eControl group, \u003cstrong\u003eB.\u003c/strong\u003e Severe smooth muscle loss in disease model group, \u003cstrong\u003eC.\u003c/strong\u003eLow-dose Baicalein group, \u003cstrong\u003eD.\u003c/strong\u003e Preserved smooth muscle structures in moderate-dose Baicalein group, \u003cstrong\u003eE.\u003c/strong\u003e Preserved smooth muscle structures in high-dose Baicalein group\u003c/p\u003e","description":"","filename":"Binder44.png","url":"https://assets-eu.researchsquare.com/files/rs-6752805/v1/f394293ced1b0955b7a99c7e.png"},{"id":83743202,"identity":"efebf7b4-9bed-4bcb-b919-1c09e6f10197","added_by":"auto","created_at":"2025-06-02 02:20:04","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2515490,"visible":true,"origin":"","legend":"\u003cp\u003eExamples of chondroid differentiation, bone metaplasia and ossification detected in various subjects, \u003cstrong\u003eA.\u003c/strong\u003e Chondroid differentiation in disease model group, MTC x20, \u003cstrong\u003eB.\u003c/strong\u003eChondroid differentiation in low-dose Baicalein group, MTC x20, \u003cstrong\u003eC.\u003c/strong\u003eIncreased expression of suspicious area of chondroid differentiation in TGF-ß1 staining in moderate-dose Baicalein group, MTC x20\u003cstrong\u003e D.\u003c/strong\u003e Focus of ossification in low-dose Baicalein group, MTC, x10, \u003cstrong\u003eE.\u003c/strong\u003e Area of bone metaplasia and early ossification in low-dose Baicalein group, H\u0026amp;E, x20, \u003cstrong\u003eF.\u003c/strong\u003eFocus of ossification in disease model group, H\u0026amp;E x20\u003c/p\u003e","description":"","filename":"Binder45.png","url":"https://assets-eu.researchsquare.com/files/rs-6752805/v1/1aa4bdec04805596c92fb037.png"},{"id":93996763,"identity":"941c1bef-3b34-4d3d-8c3b-0df79052d634","added_by":"auto","created_at":"2025-10-21 07:11:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":10881382,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6752805/v1/14540b27-b8b5-4a2a-9875-9ac2ac09e1ef.pdf"},{"id":83743193,"identity":"0695cb18-cfba-4ca7-bfc6-2e352a3f907a","added_by":"auto","created_at":"2025-06-02 02:20:04","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":11854,"visible":true,"origin":"","legend":"","description":"","filename":"Table126032025.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6752805/v1/7da3be3129465e0dde80db04.xlsx"},{"id":83743440,"identity":"c53de104-cd3c-44ee-82f5-3364ec010644","added_by":"auto","created_at":"2025-06-02 02:28:04","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":12339,"visible":true,"origin":"","legend":"","description":"","filename":"Table226052025.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6752805/v1/8de5b05aa0ec479e555505c4.xlsx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e conflict of interest to disclose.","formattedTitle":"The Efficacy of Intralesional Baicalein Injection in Rat Peyronie's Model","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003ePeyronie's disease (PD) is an acquired connective tissue disorder characterized by the formation of a localized fibrous plaque in the tunica albuginea (TA). The pathophysiology of PD is not yet fully understood and is still a matter of debate. Abnormal collagen accumulation in the TA secondary to repetitive penile microtrauma, rupture of elastic fibers, and remodeling of connective tissue in a fibrotic background are among the most accepted hypotheses to date [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Free oxygen radicals, oxidative stress, nitric oxide synthase dysfunction, autoimmunity, and certain viral infections have been the subject of various studies, considering their potential roles in the impaired tissue healing observed in PD [\u003cspan additionalcitationids=\"CR2 CR3 CR4 CR5\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Understanding the pathophysiology of the disease is crucial for both explaining the symptoms and managing the disease with appropriate treatment options.\u003c/p\u003e \u003cp\u003eRecent studies have highlighted TGF-β1 as the most significant factor inducing fibrosis in the TA by affecting extracellular matrix accumulation through fibroblast stimulation [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. While evidence supporting TGF-β1 as a cytokine crucial for tissue repair is increasing, its excessive activity is held responsible for tissue damage secondary to scarring in many serious diseases [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. It has been reported that overexpression of TGF-β1 and activation of Smad transcription factors may be the basis of pathogenesis in PD, based on examinations of its pathophysiological mechanisms [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Therefore, inhibiting the TGF-β1 signaling pathway may be a promising therapeutic strategy in the treatment of PD.\u003c/p\u003e \u003cp\u003eGiven the role of TGF-β1 in PD fibrosis, targeting this pathway presents a promising therapeutic strategy. Baicalein, or 5,6,7-trihydroxyflavone, is a type of flavonoid originally derived from the roots of Scutellaria baicalensis and Scutellaria lateriflora plants [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Known for its anti-inflammatory and anti-thrombotic effects, Baicalein is also recognized for its efficacy as a lipoxygenase inhibitor and its ability to reduce oxidative stress at the cellular level [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The underlying mechanisms of Baicalein are thought to stem from its selective binding to the ATP-binding site of activin receptor-like kinase 5, a TGF-β receptor I, without causing any changes in the expression of Smad6, Smad7, TGF-β1, or TGF-β receptor I/II [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. It exhibits an inhibitory effect on the TGF-β/Smad2/3 signaling pathway in both in vivo and in vitro fibroblasts by dose-dependent down-regulation of phosphorylated Smad2 and Smad3 levels. Baicalein does not affect Smad1, 5, and 8 in the bone morphogenetic protein signaling pathway and does not affect total Smad2 and Smad3 levels [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. During the proliferative phase of scar formation in a mechanically loaded mouse model, Baicalein significantly suppresses the proliferation and activation of hypertrophic scar-derived fibroblasts and inhibits α-SMA expression. This leads to impaired contraction and migration abilities of hypertrophic scar-derived fibroblasts, thereby reducing collagen accumulation and alleviating hypertrophic scar formation [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCurrent European and American urology guidelines reveal a notable deficiency in proven effective conservative treatment alternatives, particularly for managing patients in the acute inflammatory phase of the disease [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Considering the gap in the treatment options of the PD, this study aims to investigate the efficacy of Baicalein in our rat Peyronie's model. Specifically, we aim to demonstrate its potential use as an effective agent in PD through inhibition of the TGF-β1 pathway via intralesional administration.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eEstablishment of a Rat Model for Peyronie's Disease\u003c/h2\u003e \u003cp\u003eIn the conducted animal experiment, the 3R principles were adhered to, and a power analysis was performed using G*Power version 3.1 [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], taking into account previously published animal models. A confidence interval of 95% was established, and the power analysis, conducted using one-way ANOVA, revealed that 1-β exceeded 0.5 based on the sample size determined from the literature.\u003c/p\u003e \u003cp\u003e The procedures used and the care of animals were approved by the Institutional Animal Care and Ethics Committee (HADYEK) in Istanbul University - Cerrahpasa (E-74555795-050.01.04-794309).\u003c/p\u003e \u003cp\u003eThirty male albino Wistar rats, aged over 6 months and weighing at least 450 grams, were housed in a controlled environment at the laboratory. Baicalein (ab120723, Abcam, Cambridge, UK) dosage was determined from similar studies in the literature [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Rats were randomly assigned to six groups: six rats as the control group, six rats as the disease model group, six rats as the low-dose Baicalein (0.8 \u0026micro;g/L, 50 \u0026micro;L) group, six rats as the moderate-dose Baicalein (1.6 \u0026micro;g/L, 50 \u0026micro;L) group, and six rats as the high-dose Baicalein (3.2 \u0026micro;g/L, 50 \u0026micro;L) group.\u003c/p\u003e \u003cp\u003eTo induce the disease model, the previously described single-injection model of combined solution of 0.1 mL active recombinant TGF-β1 protein (0.01 \u0026micro;g/\u0026micro;L, ab50036, Abcam, Cambridge, UK) and 0.1 mL sodium tetradecyl sulfate (0.01 \u0026micro;g/\u0026micro;L, 3% Tromboject\u0026reg;, Omegapharma, Alberta, Canada) was administered intratunically as a single injection [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Anesthesia was induced intraperitoneally with a combination of 10 mg/kg Xylazine and 80 mg/kg Ketamine [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Following anesthesia, rats were placed in a supine position, and the injection area was cleaned with a 10% povidone-iodine solution. All injections were performed under 12x magnification using an operating microscope (YZ-20P5 Operation Microscope, Visionstar, Chongqing, China) with 30G \u0026minus;\u0026thinsp;0.8 mm \u0026minus;\u0026thinsp;0.5 mL syringes from the right lateral 1/3 proximal section of the penile shaft. Animals were weighed before each procedure.\u003c/p\u003e \u003cp\u003eFollowing a four-week model induction period, the experiment groups received a total of three intralesional Baicalein injections, administered three weeks apart, following the described injection protocol. Intralesional injections were performed by palpation of the fibrotic area at the injection site where the experimental model was created and under 12x magnification with an operating microscope to avoid injecting the solution into the cavernous tissue or distributing it outside the tunical layers due to superficial application. The six rats in the control group received 0.2 mL sterile saline injections on the same days and using the same protocol as the disease model induction and treatment applications in the study groups.\u003c/p\u003e \u003cp\u003eThroughout the follow-up period, rats were monitored daily for signs of distress (\u0026gt;\u0026thinsp;15% weight loss, impaired mobility, nutritional disorders, reduced responsiveness), which led to exclusion after consultation with the responsible veterinarian. Animals were housed freely in their cages, without any restrictive procedures or deprivation-related experimental procedures.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eEuthanasia and Autopsy of Animals\u003c/h3\u003e\n\u003cp\u003eThree weeks after the final injection in the study group, all rats completing the follow-up protocol were euthanized under anesthesia by cardiac blood aspiration. The penises of the euthanized rats were clamped at the radix to determine the post-treatment curvature degree using goniometry. To avoid affecting the histopathological evaluation, artificial erection was induced by injecting saline into corpora cavernosa (CC) using a 27G needle from the contralateral side of the injection area.\u003c/p\u003e \u003cp\u003eAll rats underwent autopsy with abdominal and thoracic incisions. Subsequently, penectomy was performed, followed by bilateral orchiectomy, bilateral nephrectomy, and hepatectomy for toxicity assessment. Bilateral testes, epididymides, and vas deferens were removed en bloc and placed in Hollande's fixative, while the remaining organs were placed in 10% formaldehyde and transferred to the Medical Pathology laboratory on the same day.\u003c/p\u003e\n\u003ch3\u003eHistopathological and Immunohistochemical Examination of Specimens\u003c/h3\u003e\n\u003cp\u003eFollowing sufficient fixation, the length and diameter of the specimens were measured and documented with photographs. The proximal portions of the materials were marked with black tissue dye, the middle portions with green dye, and the distal portions (glans penis) with orange dye. Starting from the proximal end, the materials were serially sectioned perpendicular to their long axes at 3 mm intervals towards the distal end. Following routine processing and sectioning, the specimens were stained with Hematoxylin and Eosin (H\u0026amp;E), Masson-Trichrome (MTC), Reticulin, Weigert van Gieson, Alpha Smooth Muscle Actin (α-SMA), TGF-\u0026szlig;1, and Col1A1.\u003c/p\u003e \u003cp\u003eTo assess the treatment response, fibrosis, smooth muscle loss in the CC, chondroid differentiation, and bone metaplasia/ossification were evaluated as key parameters for efficacy. Additionally, congestion and edema were assessed to determine the level of tissue reaction. All parameters were categorized into three severity grades: absent, mild/moderate, and severe.\u003c/p\u003e\n\u003ch3\u003eMeasurement of Tunica Albuginea Thickness\u003c/h3\u003e\n\u003cp\u003eTo ensure standardization in the measurement of TA thickness across all specimens, the mid-section of the penile shaft, where the highest level of fibrosis was observed, was identified under microscopic examination. Subsequently, two imaginary lines were drawn to determine the level at which the measurement would be taken: one vertical line bisecting the urethra and the other horizontal line, perpendicular to the first, dividing the CC between the urethra and dorsal vein into two equal halves \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. TA thicknesses were measured bilaterally for each case at 40x magnification using an Olympus Provis AX70 (Olympus Corporation, Shinjuku, Japan) fluorescence microscope \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. Tissue gaps resulting from extravasation from the CC due to artificial erection post-euthanasia were excluded to calculate the net TA thickness.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eDetermination of Fibrosis Level by Digital Area Calculation\u003c/h3\u003e\n\u003cp\u003eDigital images of the mid-section H\u0026amp;E x2 slices from all cases were obtained. Using Sketchandcalc software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.sketchandcalc.com/\u003c/span\u003e\u003cspan address=\"https://www.sketchandcalc.com/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), the CC area was outlined in blue, and the fibrotic areas were outlined in green \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. The areas of these two outlined regions were calculated using the digital area calculation software of the same application for each case, and then the ratio of these areas was determined to examine it as an indicator of treatment response.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eAll statistical analyses were performed using SPSS version 28.0 (IBM, Armonk, USA). Quantitative data are expressed as median (minimum - maximum), and qualitative data as number (%). Categorical variables were analyzed using Chi-square test or Fisher's Exact test, as appropriate. Situations where the arithmetic means of three or more independent sample groups with non-parametric distribution conditions were different were determined using the Kruskal-Wallis test with post-hoc Bonferroni correction. For all tests, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cp\u003eOf the 30 rats initially enrolled in the study, four were excluded. Local infective complications led to the exclusion of one rat in the low-dose Baicalein group and one rat in the moderate-dose Baicalein group. Additionally, one rat in the high-dose Baicalein group exhibited a significant decrease in oral intake, and one rat in the disease model group sustained injuries from an inter-cage altercation and required removal from the study after veterinary evaluation. Consequently, 26 rats completed the study: six in the control group and five in each of the disease model and three treatment groups.\u003c/p\u003e \u003cp\u003ePre- and post-treatment weights were evaluated for all rats completing the study. Comparison of median weights revealed no statistically significant differences between groups due to treatment \u003cb\u003e(Table\u0026nbsp;1)\u003c/b\u003e. Similarly, pre-treatment and pre-euthanasia penile curvature measurements showed no statistically significant changes across the treatment groups (\u003cb\u003eTable\u0026nbsp;1)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eStatistical analysis of the parameters employed to assess treatment response revealed significant difference between the groups in fibrosis and CC smooth muscle loss (p\u0026thinsp;=\u0026thinsp;0.015 and p\u0026thinsp;=\u0026thinsp;0.005, respectively) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. Conversely, chondroid differentiation and bone metaplasia/ossification did not exhibit statistically significant intergroup differences (p\u0026thinsp;=\u0026thinsp;0.118 and p\u0026thinsp;=\u0026thinsp;0.206, respectively) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. Of the parameters evaluated to assess the tissue reaction, congestion did not show a significant difference between groups (p\u0026thinsp;=\u0026thinsp;0.176), while edema was significantly different between groups (p\u0026thinsp;=\u0026thinsp;0.001). The comparative analysis of the data obtained from the histopathological evaluations of the cases is compiled and presented in \u003cb\u003eTable\u0026nbsp;2\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eQuantitative analysis of digital measurements to assess treatment response revealed statistically significant differences between groups in both mean TA thickness and the ratio of total fibrotic area to CC area (p\u0026thinsp;=\u0026thinsp;0.002 for both) (\u003cb\u003eTable\u0026nbsp;1)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eMacroscopic and microscopic evaluations of the testes, kidneys, and liver, supplementary to the penectomy specimens, revealed no evidence of toxicity attributable to the experimental model or treatment protocol. Specifically, investigation of the testes showed no toxicological effects on germ cells.\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eIt is known that TGF-β1 plays a fundamental role in the pathogenesis of PD through Smad-dependent pathways. Activated TGF-β1 induces Smad2/3 phosphorylation and nuclear translocation, driving the fibrotic process in PD [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Consequently, agents that inhibit the TGF-β1/SMAD pathway hold promise for PD treatment. Considering the inadequacy of efficacy and evidence levels in conservative treatment options [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], especially in the acute phase of PD, our study aimed to investigate the efficacy of Baicalein, a substance known to have effects on this pathway, which forms the basis of PD pathophysiology, as a potential agent that can limit fibrosis in a rat Peyronie's model.\u003c/p\u003e \u003cp\u003eBuilding upon the understanding of TGF-β1's role in fibrosis, the efficacy of Baicalein treatment has been investigated in various diseases that progress with fibrosis, similar to PD [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Zhang et al. demonstrated that Baicalein injections reduced phosphorylated Smad2-3 levels in mice with hypertrophic scars, inhibiting hypertrophic scar formation, fibroblast proliferation, and activation [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Similarly, in a systemic sclerosis model, Baicalein treatment was administered at different doses to mice with systemic sclerosis, Baicalein treatment exhibited dose-dependent efficacy through B cell modulation and TGF-β1 inhibition [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTranslating preclinical findings to clinical practice, a recent single-center retrospective study, the efficacy of oral Baicalein treatment in the acute phase of PD was investigated. In this study, in which the data of 261 Peyronie's patients were evaluated, it was shown that Baicalein reduced plaque volume, pain, and penile curvature, and positive effects on erectile dysfunction were reported [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. To achieve targeted efficacy and minimize systemic exposure, our study employed intralesional Baicalein administration. Consistent with these previous reports, we observed a dose-dependent inhibition of fibrosis. The fact that no toxicity findings were detected in the systemic evaluation after euthanasia indicates that Baicalein can be used as a safe agent.\u003c/p\u003e \u003cp\u003eIn our histopathological examinations, it was shown that TA thickness in the disease group was significantly increased compared to the control and treatment groups. In addition, widespread fibrosis in the CC tissue adjacent to the injection area, the presence of chondroid differentiation developing in some places, and cavernosal smooth muscle cell loss were noteworthy. The described findings were more severe in the untreated rat group, and the severity of the findings decreased dose-dependently in the treatment groups. The presence of a dose-dependent effect was found to be consistent with the results of Baicalein treatment applied in diseases with similar mechanisms of action in the literature [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAlthough ED is known to be seen in 37.5% of men with Peyronie's disease, it is also known that many patients do not apply to a doctor due to their concerns and fears [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. ED usually occurs as a result of TA compliance loss and cavernosal smooth muscle loss due to plaque formation and fibrosis in the penis [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Our study reveals that the CC tissue of the rat group in which the experimental model was created is characterized by widespread smooth muscle loss. In our treatment groups, immunohistochemical analysis showed a significant preservation of smooth muscle tissue in the CC structure, as evidenced by α-SMA staining. Applications evaluating erectile functions, such as electrostimulation and erection moment intracavernosal pressure measurement, to evaluate erection in our study groups were not used, considering that they could affect our primary endpoint, histopathological and immunohistochemical analysis results. Although the hypothesis that erectile functions will be preserved with the preservation of smooth muscle tissue in the CC structure can be put forward, evidence should be presented in this context with new studies to be designed.\u003c/p\u003e \u003cp\u003eThe literature on PD lacks standardized, objective criteria for experimental evaluation. Many parameters, such as the degree of developing penile curvature, the size of plaque formation, TA thickness, fibrosis level, collagen fiber amount and ratios, the condition of smooth muscle cells in the CC tissue, and the evaluation of elastic fibers, have been used both in the formation of experimental models and in the evaluation of the efficacy of the therapeutic agent [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. In our study, fibrosis severity, CC smooth muscle loss degree, average TA thickness, fibrosis ratio in CC, presence of bone metaplasia, presence and prevalence of chondroid differentiation were determined as basic parameters for the evaluation of treatment efficacy. When the results of the group in which the disease model was created and the treatment groups were analyzed comparatively, fibrosis severity, average TA thickness, fibrosis ratio in CC, and edema severity were determined as significant different parameters between the groups. Taking these parameters as evaluation criteria in future studies will provide a standardized objective analysis opportunity.\u003c/p\u003e \u003cp\u003eA review of published rat models of Peyronie's disease, widely used in research, indicates that the resulting fibrotic plaque often extends beyond the TA, inducing widespread fibrosis in the surrounding CC tissue [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan additionalcitationids=\"CR29\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. This phenomenon may be attributed to factors such as the thinness of the rat TA, challenges in achieving optimal tunical injection, and extravasation of injected agents. In our study, despite observing increased TA thickness in the disease model group, we also noted extensive fibrosis, smooth muscle cell loss, and chondroid changes progressing to bone metaplasia in the surrounding CC tissue. Re-evaluation of published studies on rat Peyronie's models, in light of these findings, revealed a frequent discrepancy: many histopathological images, while labeled as fibrotic plaques within the TA, in fact depict more extensive fibrotic processes involving the CC tissue, despite being described as PD. The similarity of our findings to these studies raises questions about the validity of current rat Peyronie's models. Based on these observations, we suggest that these widely used models may more accurately represent a \u0026lsquo;diffuse penile fibrosis model,\u0026rsquo; a distinction that warrants further discussion. This reinterpretation could have significant implications for the translation of preclinical findings to human PD treatment, necessitating a more cautious approach to the interpretation of animal model data.\u003c/p\u003e \u003cp\u003eBy demonstrating the intralesional efficacy of Baicalein in a rat Peyronie's model and representing the first instance of this administration route, our study makes a positive contribution to the literature and serves as a foundation for future research. Based on the promising results of our pilot study, we believe Baicalein, an easily accessible, low-cost, highly effective agent with non-systemic toxicity, holds high potential as a therapeutic for PD and warrants further investigation.\u003c/p\u003e \u003cp\u003eIn conclusion, intralesional Baicalein in the rat Peyronie's model suppresses fibrosis in the treatment groups, has a protective effect on the smooth muscle tissue in the CC structure, and prevents the increase in thickness in the TA observed in the course of PD without causing any systemic toxicity. In line with the results of both in vitro and in vivo studies to be designed, Baicalein has the potential to be an alternative that can address the shortcomings in PD treatment.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability statement:\u0026nbsp;\u003c/strong\u003eThe data that support the findings of this study are available from the corresponding author, KCS, upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgement:\u003c/strong\u003e None.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests statement:\u003c/strong\u003e All authors declare that they have no competing financial interests in relation to the work described.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding statement:\u0026nbsp;\u003c/strong\u003eThis study was carried out with the support of Istanbul University \u0026ndash; Cerrahpasa Scientific Research Projects Unit (Project number: 37567, approval date: 07.02.2024).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics committee approval number:\u0026nbsp;\u003c/strong\u003eE-74555795-050.01.04-794309\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contribution Statement:\u0026nbsp;\u003c/strong\u003eAll authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConceptualization:\u003c/strong\u003e KCS, FI, MHG, HO. \u003cstrong\u003eData curation:\u003c/strong\u003e KCS, FI, OG, SK. \u003cstrong\u003eFormal analysis:\u003c/strong\u003e KCS. \u003cstrong\u003eFunding acquisition:\u003c/strong\u003e KCS, FI, MHG. \u003cstrong\u003eInvestigation:\u003c/strong\u003e KCS, FI, MHG, OG. \u003cstrong\u003eMethodology:\u003c/strong\u003e SK, IG, EA, HO. \u003cstrong\u003eProject administration:\u003c/strong\u003e KCS, FI, MHG. \u003cstrong\u003eResources:\u003c/strong\u003e KCS, FI, MHG. \u003cstrong\u003eSupervision:\u003c/strong\u003e IG, EA, HO. \u003cstrong\u003eValidation:\u003c/strong\u003e SK, OG, IG. \u003cstrong\u003eVisualization:\u003c/strong\u003e KCS, FI, OG. \u003cstrong\u003eWriting \u0026ndash; original draft:\u003c/strong\u003e KCS. \u003cstrong\u003eWriting \u0026ndash; review \u0026amp; editing:\u0026nbsp;\u003c/strong\u003eMHG, EA, HO.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eDevine CJJ, Somers KD, Jordan GH, et al. Proposal: trauma as the cause of the Peyronie\u0026rsquo;s lesion. J Urol. 1997;157:285-290. doi: 10.1016/s0022-5347(01)65361-8.\u003c/li\u003e\n \u003cli\u003eSikka S, Hellstrom W. Role of oxidative stress and antioxidants in Peyronie\u0026rsquo;s disease. Int J Impot Res. 2002;14:353-360. doi: 10.1038/sj.ijir.3900880.\u003c/li\u003e\n \u003cli\u003eVernet D, Ferrini MG, Valente EG, et al. Effect of nitric oxide on the differentiation of fibroblasts into myofibroblasts in the Peyronie\u0026rsquo;s fibrotic plaque and in its rat model. Nitric Oxide. 2002;7:262-776. doi: 10.1016/s1089-8603(02)00124-6.\u003c/li\u003e\n \u003cli\u003eGonzalez-Cadavid NF, Rajfer J. Mechanisms of disease: new insights into the cellular and molecular pathology of Peyronie\u0026rsquo;s disease. Nat Clin Pract Urol. 2005;2:291-297. doi: 10.1038/ncpuro0201.\u003c/li\u003e\n \u003cli\u003eGonzalez-Cadavid NF, Rajfer J Experimental models of Peyronie\u0026apos;s disease. Implications for new therapies. J Sex Med. 2009 Feb;6(2):303-13. doi: 10.1111/j.1743-6109.2008.01104.x.\u003c/li\u003e\n \u003cli\u003eHerati AS, Pastuszak AW. The genetic basis of Peyronie disease: a review. Sex Med Rev. 2016;4:85-94. doi: 10.1016/j.sxmr.2015.10.002.\u003c/li\u003e\n \u003cli\u003eEl-Sakka AI, Hassan MU, Nunes L, et al. Histological and ultrastructural alterations in an animal model of Peyronie\u0026rsquo;s disease. Br J Urol. 1998;181:445\u0026ndash;52. doi: 10.1046/j.1464-410x.1998.00529.x.\u003c/li\u003e\n \u003cli\u003eEl-Sakka AI, Hassoba HM, Chui RM, et al. An animal model of Peyronie\u0026rsquo;s-like condition associated with an increase of transforming growth factor beta mRNA and protein expression. J Urol 1997;158:2284\u0026ndash;90. doi: 10.1016/s0022-5347(01)68236-3.\u003c/li\u003e\n \u003cli\u003eBorder WA, Ruoslahti E. Transforming growth factor-b in disease. The dark side of tissue repair. J Clin Invest 1992;90:1\u0026ndash;7. doi: 10.1172/JCI115821.\u003c/li\u003e\n \u003cli\u003eJang JH, Ryu JK, Suh JK. Activin receptor-like kinase 5 inhibitor attenuates fibrosis in fibroblasts derived from Peyronie\u0026rsquo;s plaque. Korean J Urol. 2012;53:44\u0026ndash;9. doi: 10.4111/kju.2012.53.1.44.\u003c/li\u003e\n \u003cli\u003eMatsumoto T. Phytochemistry Research Progress. 1\u003csup\u003est\u0026nbsp;\u003c/sup\u003eedition. Nova Science Publishers Inc. May 2013. ISBN: \u0026lrm; 978-1604562323\u003c/li\u003e\n \u003cli\u003eDeschamps JD, Kenyon VA, Holman TR. Baicalein is a potent in vitro inhibitor against both reticulocyte 15-human and platelet 12-human lipoxygenases. Bioorg Med Chem. 2006 Jun 15;14(12):4295-301. doi: 10.1016/j.bmc.2006.01.057.\u003c/li\u003e\n \u003cli\u003eMiyazono K, Kamiya Y, Morikawa M. Bone morphogenetic protein receptors and signal transduction, J. Biochem. 147 (1) (2010) 35\u0026ndash;51. doi: 10.1093/jb/mvp148.\u003c/li\u003e\n \u003cli\u003eZhang YF, Zhou SZ, Cheng XY, et al. Baicalein attenuates hypertrophic scar formation via inhibition of the transforming growth factor-\u0026beta;/Smad2/3 signalling pathway. Br. J. Dermatol. 174 (1) (2016) 120\u0026ndash;130. doi: 10.1111/bjd.14108.\u003c/li\u003e\n \u003cli\u003eSalonia A, Bettocchi C, Capogrosso P, et al. EAU Guidelines on Sexual and Reproductive Health 2024, EAU Guidelines Office, Arnhem, The Netherlands, ISBN 978-94-92671-23-3.\u003c/li\u003e\n \u003cli\u003eNehra A, Alterowitz R, Culkin DJ, et al. Peyronie\u0026rsquo;s Disease: AUA Guideline. J Urol. 2015 Sep;194(3):745-53. doi: 10.1016/j.juro.2015.05.098.\u003c/li\u003e\n \u003cli\u003eFaul F, Erdfelder E, Buchner E, et al. Statistical power analyses using G*Power 3.1: Tests for correlation and regression analyses. Behavior Research Methods. 2009; 41,1149-1160. doi: 10.3758/BRM.41.4.1149.\u003c/li\u003e\n \u003cli\u003eBai H, Yuan R, Zhang Z, et al. Intra-articular Injection of Baicalein Inhibits Cartilage Catabolism and NLRP3 Inflammasome Signaling in a Posttraumatic OA Model. Oxid Med Cell Longev. 2021 Sep 2:2021:6116890. doi: 10.1155/2021/6116890.\u003c/li\u003e\n \u003cli\u003eChung E, Garcia F, De Young L, et al. A comparative study of the efficacy of intralesional verapamil versus normal saline injection in a novel Peyronie disease animal model: assessment of immunohistopathological changes and erectile function outcome. J Urol. 2013 Jan;189(1):380-4. doi: 10.1016/j.juro.2012.08.191.\u003c/li\u003e\n \u003cli\u003eGarcia F, De Young L, Chung E, et al. A durable novel rat model for Peyronie\u0026rsquo;s disease. J Urol. 2012;187(4):e682-e683. Doi: 10.1016/j.juro.2012.02.1586\u003c/li\u003e\n \u003cli\u003eTsukamoto A, Niino N, Sakamoto M, et al. The validity of anesthetic protocols for the surgical procedure of castration in rats. Exp. Anim. 2018;67(3), 329\u0026ndash;336. doi: 10.1538/expanim.18-0003.\u003c/li\u003e\n \u003cli\u003ePeng B, Hu Q, He R, et al. Baicalein alleviates fibrosis and inflammation in systemic sclerosis by regulating B-cell abnormalities. BMC Complement Med Ther. 2023 Feb 21;23(1):62. doi: 10.1186/s12906-023-03885-1.\u003c/li\u003e\n \u003cli\u003eKong EKC, Huang Y, Sanderson JE, et al. Baicalein and Wogonin inhibit collagen deposition in SHR and WKY cardiac fibroblast cultures. BMB Rep. 43 (4) (2010) 297\u0026ndash;303. doi: 10.5483/bmbrep.2010.43.4.297.\u003c/li\u003e\n \u003cli\u003eLi B, Chen K, Qian N, et al. Baicalein alleviates osteoarthritis by protecting subchondral bone, inhibiting angiogenesis and synovial proliferation. J Cell Mol Med. 2021 Jun;25(11):5283-5294. doi: 10.1111/jcmm.16538.\u003c/li\u003e\n \u003cli\u003eLi WJ, Bao J, Zheng DC, et al. Treatments of Peyronie\u0026apos;s disease with Scutellaria baicalensis and surgery according to the disease course: a single-center retrospective study of 261 patients. Ann Palliat Med. 2021;10(3):2979-2989. doi: 10.21037/apm-20-2389.\u003c/li\u003e\n \u003cli\u003ePaulis G, Romano G, Paulis A. Prevalence, psychological impact, and risk factors of erectile dysfunction in patients with Peyronie\u0026rsquo;s disease: a retrospective analysis of 309 cases. Res Rep Urol 2016;8:95-103. doi: 10.2147/RRU.S109319.\u003c/li\u003e\n \u003cli\u003eWespes E. Smooth muscle pathology and erectile dysfunction. Int J Impot Res. 2002;14 Suppl 1:S17-S21. doi: 10.1038/sj.ijir.3900792.\u003c/li\u003e\n \u003cli\u003eAntoniassi T, Facio Junior FN, Spessoto LCF, et al. Anti-fibrotic effect of mycophenolate mofetil on Peyronie\u0026apos;s disease experimentally induced with TGF-\u0026beta;. Int J Impot Res. 2020 Mar;32(2):201-206. doi: 10.1038/s41443-019-0138-7. doi: 10.1038/s41443-019-0138-7.\u003c/li\u003e\n \u003cli\u003eLin H, Liu C, Wang R. Effect of Penile Traction and Vacuum Erectile Device for Peyronie\u0026apos;s Disease in an Animal Model. J Sex Med. 2017 Oct;14(10):1270-1276. doi: 10.1016/j.jsxm.2017.08.011. doi: 10.1016/j.jsxm.2017.08.011.\u003c/li\u003e\n \u003cli\u003eCastiglione F, Hedlund P, Weyne E, et al. Intratunical Injection of Human Adipose Tissue-Derived Stem Cells Restores Collagen III/I Ratio in a Rat Model of Chronic Peyronie\u0026apos;s Disease. Sex Med. 2019 Mar;7(1):94-103. doi: 10.1016/j.esxm.2018.09.003.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 and 2 are available in the Supplementary Files section\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"international-journal-of-impotence-research","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"ijir","sideBox":"Learn more about [International Journal of Impotence Research](http://www.nature.com/ijir/)","snPcode":"41443","submissionUrl":"https://mts-ijir.nature.com/cgi-bin/main.plex","title":"International Journal of Impotence Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Animal Experimentation, Baicalein, Fibrosis, Peyronie’s disease","lastPublishedDoi":"10.21203/rs.3.rs-6752805/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6752805/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTGF-β1 plays a fundamental role in the pathogenesis of Peyronie's disease, driving the excessive extracellular matrix accumulation and fibroblast activation characteristic of fibrosis. As in many fibrotic disorders, its action via Smad transcription factors presents a key therapeutic target. Given the notable deficiency in proven effective conservative treatments for Peyronie's disease, particularly in its acute phase, this study aimed to investigate the efficacy of Baicalein, a flavonoid known to inhibit the TGF-β1/Smad signaling pathway, thereby offering a promising therapeutic strategy.\u003c/p\u003e \u003cp\u003eWe established a rat model of Peyronie's disease and administered Baicalein intralesionally at low, moderate, and high doses. Our comprehensive analysis of histopathological and immunohistochemical parameters, including tunica albuginea thickness, fibrosis severity, and smooth muscle content, demonstrated that intralesional Baicalein dose-dependently suppresses fibrotic plaque formation, preserves crucial cavernosal smooth muscle tissue, and effectively prevents pathological increases in tunica albuginea thickness. Importantly, systemic toxicity was not detected. As the first study to investigate intralesional Baicalein for Peyronie's disease, our findings positively contribute to the literature and underscore its potential as a safe, accessible, and highly effective agent. Further in vitro and in vivo research is warranted to fully explore Baicalein's capacity to address current treatment gaps in this challenging condition.\u003c/p\u003e","manuscriptTitle":"The Efficacy of Intralesional Baicalein Injection in Rat Peyronie's Model","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-02 02:19:59","doi":"10.21203/rs.3.rs-6752805/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2025-07-07T11:53:36+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-06-24T11:54:47+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-06-15T12:50:14+00:00","index":2,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-06-02T10:51:26+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-05-29T16:52:37+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2025-05-29T16:34:57+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-05-28T11:20:07+00:00","index":"","fulltext":""},{"type":"submitted","content":"International Journal of Impotence Research","date":"2025-05-27T15:46:42+00:00","index":"","fulltext":""},{"type":"checksFailed","content":"","date":"2025-05-27T15:32:13+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-05-26T16:56:24+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"international-journal-of-impotence-research","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"ijir","sideBox":"Learn more about [International Journal of Impotence Research](http://www.nature.com/ijir/)","snPcode":"41443","submissionUrl":"https://mts-ijir.nature.com/cgi-bin/main.plex","title":"International Journal of Impotence Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"4d24ba92-0e68-4470-963e-425f78bfe28c","owner":[],"postedDate":"June 2nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":49244657,"name":"Health sciences/Medical research/Experimental models of disease"},{"id":49244658,"name":"Health sciences/Pathogenesis"}],"tags":[],"updatedAt":"2025-10-21T07:10:54+00:00","versionOfRecord":{"articleIdentity":"rs-6752805","link":"https://doi.org/10.1038/s41443-025-01197-1","journal":{"identity":"international-journal-of-impotence-research","isVorOnly":false,"title":"International Journal of Impotence Research"},"publishedOn":"2025-10-20 04:00:00","publishedOnDateReadable":"October 20th, 2025"},"versionCreatedAt":"2025-06-02 02:19:59","video":"","vorDoi":"10.1038/s41443-025-01197-1","vorDoiUrl":"https://doi.org/10.1038/s41443-025-01197-1","workflowStages":[]},"version":"v1","identity":"rs-6752805","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6752805","identity":"rs-6752805","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}