Inhibition of 5αR2 promotes postoperative wound repair in BPH patients after TURP by alleviating fibrosis and inflammation

Abstract

Postoperative wound repair after transurethral resection of the prostate (TURP) in benign prostatic hyperplasia (BPH) patients is crucial for reducing complications and promoting recovery. Androgens, particularly dihydrotestosterone (DHT), influence prostatic development and wound healing, with type II 5α-reductase (5αR2) playing a key role in DHT synthesis. In this study, the effects of type II 5α-reductase inhibitors (5-ARIs) on postoperative healing, inflammation control, and fibrosis reduction were evaluated. A double-blinded randomized clinical trial was performed to assess 87 BPH patients treated with type II 5-ARIs (n = 47) or placebo (n = 42) over six months. The type II 5-ARIs group presented a 55.0% lower complication rate (P = 0.002), with reduced hematuria (0 vs 7.1%, P = 0.046) and catheter reintroduction (0 vs 9.5%, P = 0.025). An animal study using 12 beagles was performed, and molecular markers were analyzed via single-cell RNA sequencing, enzyme-linked immunosorbent assay (ELISA), and histology. 5αR2 inhibition accelerated urothelial regeneration, decreased inflammation, and reduced myofibroblast activation by 42.0% while increasing the expression of the urothelial marker uroplakin 3A (UPK3A) by 67.0%. Organoid experiments confirmed increased urothelial differentiation and reduced glandular epithelial expansion with type II 5-ARI treatment. These findings suggest that 5αR2 inhibition promotes TURP postoperative recovery in BPH patients by reducing inflammation, inhibiting fibrosis, and promoting wound repair. These findings support the use of type II 5-ARIs as potential adjuvant therapies for optimizing BPH patient postoperative outcomes.

INTRODUCTION

In 2023, more than 60% of benign prostatic hyperplasia (BPH) patients in China were aged 70 years or older. Across Asia, the mean prevalence of BPH has increased by 90% (range: 85.8%–170%), and the prevalence of BPH in China has increased by 99% since 2000. Among 500 million individuals aged 45 years or older, 20.33 million have BPH, and over 70% of those aged 60 years or older require surgery. In addition, 91.5% of cases occur in men aged 60 years or older, highlighting its strong correlation with aging and its healthcare burden.1,2,3 Conservatively estimated, approximately 6 million BPH patients in China will need surgical treatment, and BPH has become one of the more common diseases among men. Accelerating postoperative wound healing while minimizing postoperative complications is crucial for BPH patient recovery.4,5

Transurethral resection of the prostate (TURP) remains the gold standard surgical treatment for BPH, offering significant symptom relief by removing excess prostatic tissue obstructing the urethra.6,7,8 However, despite its widespread use, TURP is associated with notable postoperative complications, including hematuria, infection, urethral stricture, and bladder neck contracture, which can prolong recovery and negatively impact surgical outcomes.9,10 The main reason for abnormal wound healing in TURP patients is excessive postoperative inflammation and fibrosis.11,12,13,14

The traditional theory suggests that the main mechanism involved in trauma repair after TURP is the gradual migration and covering of the bladder and urethral mucosa, but new theories of trauma repair have suggested that the trans-differentiation of prostatic epithelial stem cells from residual epithelial stem cells following surgery promotes trauma repair.15,16 When this process is disrupted, persistent sterile inflammation (SI) and excessive myofibroblast activation can lead to fibrosis and stricture formation, contributing to long-term complications such as bladder neck contracture.17,18,19

Currently, there are limited pharmacological interventions available to promote TURP postoperative recovery. Moreover, the prostate is a highly androgen-sensitive organ, and androgen signaling, particularly through dihydrotestosterone (DHT), has been implicated in both prostatic development and wound healing regulation.20,21 Type II 5α-reductases (5αR2) are key enzymes responsible for converting testosterone to DHT, and their inhibition has been explored as a treatment for BPH.22

Moreover, SI has been found to be an important impediment to wound healing, characterized by significantly elevated levels of urinary inflammatory factors but negative urine microbiological cultures.5,23 The incidence of SI after BPH is as high as 19.6%,24,25 and its persistence leads to abnormal wound repair, further affecting surgical outcomes. In recent years, it has been shown that macrophages play a key role in the initiation and maintenance of SI. Following thulium laser resection of the prostate (TmLRP), increased heat shock protein 70 (HSP70), reactive oxygen species (ROS), the nod-like receptor protein 3 (NLRP3), and proinflammatory cytokines (interleukin-1 beta [IL-1β] and interleukin-18 [IL-18]) levels sustain SI, with macrophages amplifying this response via NLRP3 inflammasome activation.26 Moreover, persistent SI impairs tissue repair not only by inhibiting epithelial cell proliferation and differentiation but also by inducing macrophage-to-myofibroblast transdifferentiation, which drives excessive fibrosis and disrupts the normal wound healing process.27

This study aimed to investigate the therapeutic potential of 5αR2 inhibition in promoting wound repair and mitigating postoperative inflammation and fibrosis in BPH patients undergoing TURP. Using a combination of clinical trials, animal models, and single-cell sequencing approaches, we evaluated how type II 5-ARIs influence the healing process at both the molecular and cellular levels. By addressing the current challenges in recovery after TURP and exploring novel pharmacological approaches, this research provides insights into new strategies that may improve surgical outcomes and reduce postoperative complications in BPH patients.

PARTICIPANTS AND METHODS

Clinical sample size determination

To determine the appropriate sample size, we performed a power analysis, setting the significance level (α) at 0.05 and the statistical power (β) at 0.2. A preliminary experiment was conducted to estimate effect sizes on the basis of key outcome measures, such as postoperative inflammatory marker levels and complication rates. These preliminary data were then used to calculate the required sample size using validated tools, including Shiny Medsta, TrialStats, and Power and Sample Size, which apply standard formulas for comparing means and proportions. The analysis yielded an estimated range of 25.4–28.7 participants per group. To further validate this estimate, we performed sensitivity analyses to assess the robustness of our sample size under different effect size assumptions. On the basis of these findings and considering potential dropout rates and data variability, we ensured that each group included at least 28 participants to maintain sufficient statistical power for detecting clinically meaningful differences.

Clinical trial methods

In the formal randomized controlled trial (Clinical Trial Registration No. ChiCTR1900021072 [Shanghai General Hospital Urology Medical Center, Shanghai, China]), patients were randomly assigned to the treatment or placebo group at a 1:1 ratio using a computer-generated randomization sequence. This approach ensured random group allocation. To prevent researchers from predicting or influencing group assignment, the random sequence was prepared by XJW and SJX and implemented using envelopes that were sequentially numbered, opaque, and sealed. Each patient’s group was revealed only after enrollment by opening the corresponding sealed envelope, a standard technique to guard against selection bias. We did not employ stratified randomization in this single-center trial; however, we used a permuted-block design to preserve balanced group sizes while retaining randomness. The block sizes (randomly varied) were not disclosed to any investigators enrolling participants, in line with CONSORT recommendations,28 thereby further ensuring that the allocation sequence remained unpredictable and properly concealed.

Clinical sample collection

All sample collections in this study were conducted in strict accordance with the requirements of the registered clinical trial. Meanwhile, it were carried out by trained personnel in Shanghai General Hospital Urology Medical Center, which strictly adhering to Good Clinical Practice (GCP) guidelines. All participants received detailed study information, including objectives, risks, and benefits, and provided written informed consent. Confidentiality was maintained, and participants retained the right to withdraw at any time.

Ninety-five patients with BPH were initially selected. A prospective, randomized controlled trial was conducted at Shanghai General Hospital. Patients with BPH (prostate volume >40 ml and International Prostate Symptom Score [IPSS] ≥18) were recruited and randomly allocated to (1) the type II 5-ARIs group (n = 47), which received type II 5-ARI (Epristeride; Catalog #HY-107385; MedChemExpress, Monmouth Junction, NJ, USA) 2 weeks prior to and four weeks after surgery or (2) the placebo group (n = 42), which received standard postoperative care and placebo treatment, which was indistinguishable from the treatment with type II 5-ARI. Data were collected each week during the first month and every month, and abnormalities occurred in patients after surgery. The exclusion criteria included prior 5-ARI use, severe cardiovascular disease, and active infections. Patients were followed up for 6 months postoperation.

Statistical analyses

All statistical analyses were performed using SPSS software (version 26.0; IBM Corp., Armonk, NY, USA). Data distribution was assessed using the Kolmogorov–Smirnov normality test. For normally distributed data, parametric tests such as the Student’s t-test were applied. For non-normally distributed data, nonparametric tests such as the Mann–Whitney U test were used. Results were expressed as mean ± standard deviation (s.d.) unless otherwise indicated. P < 0.05 was considered statistically significant.

Animal experimental design

The animal experiments followed NIH guidelines and were approved by the Animal Care and Use Committee of Shanghai General Hospital (Approval No. AW2020104) and were conducted in Shanghai General Hospital Animal exprimenntal center. The beagles were housed in a controlled environment with proper care, anesthesia, and pain management. Postoperative monitoring was conducted to minimize distress, and humane endpoints were followed. The study adhered to the 3Rs principles (Replacement, Reduction, and Refinement) to ensure ethical treatment. On the basis of the former study and the human-like ureter structure of the dogs, the 12 beagles were split into type II 5-ARIs group and placebo group and subjected to TURP by computerized randomization algorithms. Prostate tissue was harvested at 2 weeks and 4 weeks postoperation for further analysis.

Organoid construction

Prostate tissues from 4-week-old C57BL/6 mice were aseptically dissected and minced into small fragments. Tissues were digested in a solution containing 1 mg ml⁻¹ collagenase/hyaluronidase (Catalog #07912; Stemcell Technologies, Vancouver, BC, Canada) at 37°C for 90 min with gentle shaking. The cell suspension was filtered through a 70 µm cell strainer (Catalog #352350; Corning, Corning, NY, USA), and single cells were embedded in Matrigel (Catalog #356231; Corning) and seeded as 30 µl droplets into 24-well plates. Organoids were cultured in Advanced Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12; Catalog #12634-010; Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 1× B27 (Catalog #17504044; Thermo Fisher Scientific), 1× N2 (Catalog #17502048; Thermo Fisher Scientific), 1 mmol l⁻¹ N-acetylcysteine (Catalog #A9165; Sigma-Aldrich, St. Louis, MO, USA), 50 ng ml⁻¹ epidermal growth factor (EGF; Catalog #AF-100-15; PeproTech, Cranbury, NJ, USA), and 100 ng ml⁻¹ Noggin (Catalog #250-38; PeproTech). For drug testing, organoids were treated with 10 µmol l⁻¹ epristeride, dihydrotestosterone (DHT; Catalog #D413176; Aladdin, Shanghai, China), and testosterone (T; Catalog #HY-NP181; MedChemExpress, Monmouth Junction, NJ, USA) for 1 day, 3 days, 5 days, and 7 days consecutively. Morphological changes were observed under an inverted microscope (Model IX73; Olympus, Tokyo, Japan).

Enzyme-linked immunosorbent assay (ELISA)

Urine samples and beagle prostate tissue homogenates were centrifuged (Sorvall ST 8; Thermo Fisher Scientific) at 10 000g for 15 min at 4°C. The concentrations of DHT and T were measured using commercial ELISA kits: DHT (Catalog #ab283979; Abcam, Cambridge, MA, USA) and testosterone (Catalog #ab178663; Abcam). Absorbance was measured at 450 nm using a microplate reader (Model Synergy HTX; BioTek, Winooski, VT, USA). Procedures followed manufacturer protocols, and results were calculated from standard curves.

Histology and hematoxylin and eosin (H&E) staining

Beagle prostate tissues were fixed in 4% paraformaldehyde (Catalog #BL539A; Biosharp, Hefei, China) at 4°C for 24 h. Samples were dehydrated in graded ethanol, cleared in xylene, and embedded in paraffin. Sections of 5 µm were cut using a rotary microtome (Model RM2235; Leica, Wetzlar, Germany), deparaffinized, rehydrated, and stained with hematoxylin (Catalog #G1120; Solarbio, Beijing, China) and eosin (Catalog #G1100; Solarbio). Slides were mounted with neutral resin and observed using a bright-field microscope (Model DM2500; Leica).

Immunofluorescence staining

Paraffin-embedded sections were dewaxed in xylene, rehydrated in ethanol, and subjected to antigen retrieval using 10 mmol l⁻¹ citrate buffer (pH 6.0) at 95°C for 20 min. Sections were blocked with 5% bovine serum albumin (BSA; Catalog #ST023; Beyotime, Shanghai, China) and incubated overnight at 4°C with the following primary antibodies: anti-UPK3A (1:200; Catalog #ab67286; Abcam); anti-decorin (anti-DCN; 1:100; Catalog #AF1060; R&D Systems, Minneapolis, MN, USA); anti-alpha-smooth muscle actin (anti-ACTA2; 1:500; Catalog #A5228; Sigma-Aldrich); and anti-S100 calcium-binding protein A4 (anti-S100A4; 1:200; Catalog ab41532; Abcam). Sections were then incubated with Alexa Fluor-conjugated secondary antibodies (1:400; Catalog #A11001; Invitrogen, Carlsbad, CA, USA) for 1 h at room temperature. Nuclei were counterstained with 4’,6-diamidino-2-phenylindole (DAPI; Catalog #C1002; Beyotime). Images were captured using a confocal microscope (Model LSM 880; Zeiss).

Cell counting

Quantification of cell numbers was performed by manually counting hematoxylin-stained nuclei in five randomly selected high-power fields (400×) per sample. Two blinded investigators independently conducted the counts. The mean cell number per field was used for analysis.

Sample collection and pretreatment for single-cell RNA sequencing (scRNA-seq)

Fresh mouse prostate tissues were digested in a solution of 1 mg ml⁻¹ Collagenase IV (Catalog #17104019; Thermo Fisher Scientific), 0.5 mg ml⁻¹ Dispase II (Catalog #04942078001; Roche, Mannheim, Germany), and 0.1 mg ml⁻¹ DNase I (Catalog #DN25; Sigma-Aldrich) at 37°C for 45 min with intermittent pipetting. The cell suspension was filtered through a 40 µm strainer (Catalog #352340; Corning) and washed in 0.04% BSA in phosphate-buffered saline (PBS), and viability was assessed with trypan blue (Catalog #C0040; Beyotime). Only samples with >85.0% viability were used for sequencing.

Single-cell RNA sequencing

Libraries were constructed using the Chromium Single Cell 3’ Reagent Kits version 3 (10×; Catalog #1000075; Genomics, Pleasanton, CA, USA) per manufacturer protocol. Sequencing was carried out on an Illumina NovaSeq 6000 (Illumina, San Diego, CA, USA), targeting 50 000 reads per cell. Library quality and concentration were assessed using a Bioanalyzer (Model 2100; Agilent Technologies, Santa Clara, CA, USA) and Qubit (Thermo Fisher Scientific).

Bioinformatic analysis

Raw data were processed using Cell Ranger version 6.1.1 (10×; Genomics) for demultiplexing, alignment to the UU_Cfam_GSD_1.0 German Shepherd reference genome, and gene-barcode matrix generation. Downstream analysis was performed using the Seurat package (version 4.1.1; R Foundation, Vienna, Austria), including normalization, clustering, and differential expression. Cell types were identified using known markers. Functional enrichment analysis was conducted using clusterProfiler (version 4.6.2) and enrichR, and visualizations were created using ggplot2 and UMAP in R (version 4.5.1; R Foundation for Statistical Computing, Vienna, Austria).

RESULTS

Inhibiting 5αR2 improved the postoperative outcomes of BPH patients

According to the data collected from 87 patients after regular use of a placebo or type II 5-ARI to reduce DHT levels in our hospital, the baseline data of the two groups were consistent (Supplementary Table 1). Postoperative follow-up data revealed that patients in the type II 5-ARIs group had significantly better IPSS scores than did the patients in the placebo group did during the first 6 months after the operation (P = 0.006). The perioperative IPSS, quality of life (QoL), maximum urinary flow rate (Qmax), and postvoid residual urine volume (PVR) did not significantly differ between the two groups (all P > 0.05; Supplementary Table 2), and there was a total of 1 complicating event in the patients with lower DHT levels in the perioperative period, which was bladder neck contracture at 153 days postoperatively, whereas there were a total of 11 complicating events in the patients in the placebo group, with 3 hematuria events, 4 catheter reintroductions, 2 cases of urethral stricture (at 86 days and 121 days, respectively), and 2 cases of bladder neck contracture (151 days and 174 days, respectively; Supplementary Table 3), and the type II 5-ARIs group had a 55.0% lower complication rate (P = 0.002). Hematuria (P = 0.046) and catheter reintroduction (P = 0.025) were significantly less common in the treatment group (Figure 1). On the basis of the above results, reducing DHT levels with type II 5-ARI in patients with BPH can improve postoperative outcomes and reduce the incidence of postoperative complications to some extent.

Supplementary Table 1.

Preoperative baseline data for patients in the type II 5α-reductase inhibitors and placebo groups

Characteristic	Type II 5-ARIs group (n=47)	Placebo group (n=42)	P
Age (year)	69.3±8.4	67.9±7.7	0.42
Qmax (ml s−1)	7.8±4.1	8.2±3.7	0.63
PVR (ml)	100.6±39.3	93.1±34.8	0.35
PV (ml)	59.2±13.1	64.2±9.3	0.13
IPSS	21.3±4.2	20.8±3.9	0.56
QoL	4.8±0.9	4.6±1.0	0.32
BOOI (cm H2O)	53.2±24.5	51.9±16.1	0.77
Operation time (min)	89.59±17.97	90.98±19.05	0.73

Data are expressed as the mean±s.d.. Qmax: maximum urinary flow rate; PVR: post-void residual urine volume; PV: prostate volume; IPSS: International Prostate Symptom Score; QoL: quality of life score; BOOI: Bladder Outlet Obstruction Index; 5-ARI: 5α-reductase inhibitor; s.d.: standard deviation

Supplementary Table 2.

Postoperative follow-up data for patients in the type II 5α-reductase inhibitors and placebo groups

Parameter	Preoperative	1 month	6 month
IPSS
Type II 5-ARIs group	21.3±4.2	8.4±3.7	4.6±1.7
Placebo group	20.8±3.9	8.8±3.4	5.5±1.3
P	0.56	0.692	0.006
QoL
Type II 5-ARIs group	4.8±0.9	1.7±1.4	1.2±0.9
Placebo group	4.6±1.0	1.6±1.5	1.1±1.1
P	0.32	0.74	0.64
Qmax (ml s−1)
Type II 5-ARIs group	7.8±4.1	16.5±2.1	22.8±3.2
Placebo group	8.2±3.7	15.9±1.8	21.9±2.7
P	0.63	0.15	0.16
PVR (ml)
Type II 5-ARIs group	100.6±39.3	22.4±14.2	10.8±7.9
Placebo group	93.1±34.8	29.6±20.3	12.3±9.5
P	0.35	0.053	0.42

*Statistically significant (P<0.05). Data are expressed as the mean±s.d.. Qmax: maximum urinary flow rate; PVR: post-void residual urine volume; IPSS: International Prostate Symptom Score; QoL: quality of life score; 5-ARI: 5α-reductase inhibitor

Supplementary Table 3.

Postoperative complications in patients in the type II 5α-reductase inhibitors and placebo groups

Complications	Type II 5-ARIs group (total=47)	Placebo group (total=42)	P
Perioperative
Hematuria	0	3	0.046*
Recatheterization	0	4	0.025*
Urinary tract infection	0	0	-
Follow-up
Urethral stricture	0	2	0.139
Bladder neck contracture	1	2	0.511
Ree-operation rate	0	0	-

*Statistically significant (P<0.05). Data are expressed as the number of complications observed (incident numbers). -: no value; 5-ARI: 5α-reductase inhibitor

Figure 1.

Postoperative complication rates in the type II 5-ARI and placebo groups. Comparison of postoperative complication rates between the type II 5-ARIs group and the placebo group. The type II 5-ARIs group presented a significantly lower overall complication rate, including reduced incidences of hematuria and catheter reintroduction (**P<0.01). 5-ARI: 5α-reductase inhibitor.

Inhibiting 5αR2 can reduce postoperative inflammatory mediator levels by decreasing DHT concentrations

Measurements of urinary inflammatory factor (tumor necrosis factor-alpha [TNF-α] and IL-1β) levels preoperatively and at 1 day, 3 days, 5 days, and 7 days postoperation revealed that 5αR2 inhibition significantly reduced inflammation in the postoperative period. The TNF-α and IL-1β levels were significantly lower in the 5αR2 inhibition group (P < 0.01), suggesting a protective effect against postsurgical inflammatory responses (Figure 2a and 2b).

Figure 2.

Effects of 5αR2 on postoperative inflammatory and fibrosis biomarker levels. (a) Changes in urinary TNF-α levels at different postoperative time points. (b) Changes in urinary IL-1β levels postoperatively. (c) Reduction in the intraprostatic DHT concentration at 2 weeks and 4 weeks postsurgery. Immunofluorescence staining for (d) CD68 and (e) F4-80, indicating reduced macrophage infiltration in the type II 5-ARIs group compared with the placebo group (scale bar: overview images [left] = 2000 µm; magnified images [right] = 100 µm). Single-cell sequencing data on TGF-β relating genes expression in the (f) type II 5-ARIs and (g) placebo groups. ^**P < 0.01; ^***P < 0.001. 5αR2: type II 5α-reductase; TNF-α: tumor necrosis factor alpha; IL-1β: interleukin-1 beta; DHT: dihydrotestosterone; CD68: cluster of differentiation 68 (macrophage marker); F4-80: murine macrophage surface marker; TGF-β: transforming growth factor beta; scRNA-seq: single-cell RNA sequencing.

To explore the underlying mechanism, we measured DHT levels in prostate tissue homogenates before surgery and at 2 weeks and 4 weeks after surgery. The ELISA results revealed that DHT levels were significantly reduced by 48.0% at 2 weeks and 63.0% at 4 weeks (both P < 0.001) in the 5αR2 inhibition group, which was correlated with reduced inflammatory marker expression (Figure 2c). Immunofluorescence analysis further confirmed a significant decrease in macrophage infiltration (CD68⁺ and F4-80⁺ cells) in the 5αR2 inhibition group compared with the Placebo group, confirming the link between DHT level reduction and inflammation suppression (Figure 2d and 2e).

Furthermore, single-cell sequencing analysis revealed that 5αR2 inhibition was associated with the downregulation of key fibrosis-related genes, including TGF-β and its receptors, in 2-week postoperative samples (Figure 2f and 2g). These findings suggest that by lowering DHT levels, 5αR2 inhibition not only reduces inflammation and macrophage infiltration but also mitigates fibrotic progression. Thus, 5αR2 inhibition limits testosterone conversion to DHT, leading to reduced macrophage activity, decreased inflammation levels, and a reduced degree of fibrosis postoperation, supporting its potential as a therapeutic strategy for promoting wound healing and reducing complications after prostate surgery.

Inhibiting 5αR2 changes the expression and distribution of fibrosis-driving cells

After analyzing the Beagle single-cell sequencing data, we generated a cell fractionation map (Figure 3a) to assess the impact of 5αR2 inhibition on fibrosis-related cell populations. The results revealed a 42.0% reduction in ACTA2⁺ and S100A4⁺ myofibroblasts, which are key drivers of fibrotic tissue remodeling through excessive extracellular matrix (ECM) deposition. Since myofibroblasts are central to fibrosis progression because they secrete collagen and other ECM components, their reduction suggests that 5αR2 inhibition directly limits fibrotic activity, thereby promoting a more controlled and balanced wound healing process (Figure 3b and 3c).

Figure 3.

scRNA-seq analysis of fibrotic and urothelial markers. (a) Single-cell clustering results from prostate tissue samples. Distribution of fibrosis-promoting cells (ACTA2- and S100A4-expressing) and antifibrotic cells (DCN-expressing) in the (b) type II 5-ARIs and (c) placebo groups, respectively. Expression levels of target fibroblast genes in samples from the (d) type II 5-ARIs and (e) placebo groups (red frame, cells that inhibit fibrosis; blue frame, cells that promote fibrosis). scRNA-seq: single-cell RNA sequencing; ACTA2: alpha smooth muscle actin gene; S100A4: S100 calcium binding protein A4; DCN: decorin; 5-ARIs: 5-alpha reductase inhibitors.

Interestingly, although DCN, an antifibrotic marker, was more abundant at the protein level, its gene expression was not significantly upregulated (Figure 3d and 3e). This finding implies that while 5αR2 inhibition inhibits fibrosis, it does so primarily by suppressing profibrotic signaling rather than by activating antifibrotic pathways. Additionally, the downregulation of TGF-β and its receptor genes (Figure 2f and 2g) suggests that 5αR2 inhibition mitigates fibrosis by disrupting excessive TGF-β signaling, a key pathway known to drive myofibroblast differentiation and ECM overproduction.

5αR2 inhibition reduces postoperative fibrosis through decreased coexpression of ACTA2 and DCN with S100A4 in the prostate

Further analysis of gene coexpression patterns revealed that S100A4 was coexpressed with DCN and ACTA2 to varying degrees in both groups, indicating potential differences in fibrotic activity. Quantification of these coexpressing cells revealed a significant reduction in the 5αR2 inhibition group. Specifically, among the S100A4⁺ cells, only 37.4% (3554 cells) also expressed DCN in the 5αR2 inhibition group, whereas 63.2% (3928 cells) expressed DCN in the placebo group, indicating a shift away from fibroblast activation and matrix remodeling (Figure 4a). Similarly, 22.9% (1942 cells) of the ACTA2⁺ cells also expressed S100A4 in the 5αR2 inhibition group, which was significantly lower than the 41.8% (1047 cells) in the placebo group, suggesting a reduction in myofibroblast differentiation and fibrotic activity (Figure 4b).

Figure 4.

Coexpression analysis of fibrosis and urothelial markers. (a) Co-expression of DCN and S100A4 in fibroblast populations. (b) Co-expression of ACTA2 and S100A4 in myofibroblasts. Immunohistochemistry analysis of (c) DCN and (d) α-SMA expression, demonstrating reduced fibrotic activity in the type II 5-ARIs group. DCN: decorin; S100A4: S100 calcium binding protein A4; ACTA2: alpha smooth muscle actin gene; α-SMA: alpha smooth muscle actin.

Additionally, DCN protein levels were greater in the 5αR2 inhibition group than those in the placebo group, despite the absence of significant upregulation at the transcript level (Figure 4c). This finding aligns with our earlier findings that DHT levels were reduced by 48.0% at 2 weeks and 63.0% at 4 weeks (both P < 0.001; Figure 2c), which was correlated with a 42.0% decrease in the percentage of ACTA2⁺ and S100A4⁺ myofibroblasts, as confirmed by histological staining (Figure 4d).

These results suggest that 5αR2 inhibition reduces fibrosis by limiting myofibroblast activation rather than directly increasing DCN expression, resulting in a less fibrotic microenvironment postoperation. This effect may be driven by DHT suppression and reduced inflammation, highlighting the role of 5αR2 inhibition in modulating wound healing dynamics and fibrosis resolution after prostate surgery.

Inhibiting the function of 5αR2 accelerates the postoperative repair process by promoting urothelial regeneration

Analysis of the beagle prostate samples revealed that 5αR2 inhibition significantly promoted epithelial regeneration post-TURP. At 2 weeks, the 5αR2 inhibition group presented 3–4 layers of regenerated urothelium with minimal polarity issues, whereas the placebo group presented limited epithelial coverage and persistent inflammation. By four weeks, the 5αR2 inhibition group presented a nearly normal urethral epithelium (6–7 layers with organized umbrella cells), with the number of layers of organized umbrella cells significantly exceeding that in the placebo group (3–4 layers with occasional umbrella cells; Figure 5a). Specifically, epithelial cell counts per 100 µm were greater in the 5αR2 inhibition group than those in the placebo group at both 2 weeks (mean ± s.d.: 31.00 ± 8.96 vs 11.33 ± 3.79, P < 0.01) and 4 weeks (mean ± s.d.: 64.33 ± 6.03 vs 35.67 ± 4.04, P < 0.01), indicating enhanced wound repair (Figure 5b).

Figure 5.

Urothelial regeneration and prostate organoid assay. (a) Histological comparison of urethral epithelial regeneration in the type II 5-ARIs and placebo groups at 2 weeks and 4 weeks postsurgery. (b) Quantification of regenerated urothelial layers, showing a significant increase in the number of layers in the type II 5-ARIs group. Single-cell distribution of urothelial and glandular markers in (c) type II 5-ARIs group and (d) placebo group, respectively. Expression of urothelial and glandular genes in targeted epithelial cells in the (e) type II 5-ARIs group and (f) placebo group, respectively (red frame, targeted epithelial cells). (g) Immunofluorescence staining for KRT18, UPK3A, and NKX3-1 in postoperative 4-week beagle prostate samples of different treatments. Scale bars = 100 µm. (h) Morphological analysis of mouse prostate organoids following different drug treatments. Scale bars = 150 µm. (i) Immunofluorescence staining for the 7-days different treatments organoid slides showing increased urothelial differentiation and reduced glandular epithelial expansion following type II 5-ARIs treatment. Scale bars = 20 µm. ^***P<0.001. CK18: cytokeratin 18; UPK3A: uroplakin 3A; NKX3-1: NK3 homeobox 1; AR: androgen receptor; 5-ARI: 5-alpha reductase inhibitor; T: testosterone; DHT: dihydrotestosterone; NC: negative control.

Single-cell sequencing revealed that 5αR2 inhibition increased UPK3A⁺ urothelial cells while reducing NKX3-1⁺ glandular cells, suggesting a shift toward urothelial differentiation and away from glandular proliferation (Figure 5c and 5d). Similarly, UPK3A gene expression was upregulated, whereas NKX3-1 expression was decreased in the 5αR2 inhibition group (Figure 5e and 5f). Immunofluorescence staining confirmed that the 5αR2 inhibition group presented greater urothelial coverage, whereas the placebo group presented a predominance of glandular cells and less urothelial regeneration (Figure 5g). Prostate organoid cultures further supported these findings, as 5αR2 inhibition increased urothelial differentiation (marked by UPK3A and CK18 upregulation) while reducing glandular epithelial proliferation. The addition of Y-27632, combined with testosterone and 5αR2 inhibition, suppressed dihydrotestosterone conversion, further enhancing urothelial differentiation (Figure 5h and 5i). Together, these findings demonstrate that 5αR2 inhibition accelerates urothelial regeneration, limits glandular expansion, and promotes wound repair by restoring urethral integrity, making it a promising strategy for improving postoperative recovery.

DISCUSSION

The aging of society has led to an increase in the total number of patients with BPH requiring surgical treatment, and the optimization of various minimally invasive surgeries has greatly reduced the risk of BPH surgery and accelerated the recovery of patients.29 However, the issue of postoperative wound repair in patients with BPH has not received enough attention, and the repair mechanism and regulatory factors are poorly understood.30,31 Our study provides compelling evidence that 5αR2 inhibition can effectively enhance postoperative wound healing by reducing inflammation, modulating fibrotic responses, and promoting urothelial repair.

The prior experimental results of the group indicated that the process of BPH postoperative trauma repair was not bladder mucosal epithelial migration and covering, as described in traditional theory, but rather the transdifferentiation of prostatic basal stem cells at the site of the prostate trauma. Our findings suggest that 5αR2 inhibition promotes urothelial regeneration following TURP, as evidenced by a significant increase in UPK3A-positive urothelial cells and a concurrent reduction in NKX3-1-expressing glandular epithelial cells in the type II 5-ARIs group. Single-cell sequencing analysis revealed a 67.0% increase in UPK3A⁺ cell populations, indicating a marked shift toward expression of the urothelial phenotype. Although the precise cellular origin of these regenerating urothelial cells remains to be fully elucidated, the observed phenotype shift implies enhanced epithelial remodeling under reduced DHT conditions. This enhancement is particularly relevant in the context of TURP, where incomplete urothelial re-epithelialization increases the risk of postoperative fibrosis, infection, and recurrent stricture formation.

At the molecular level, androgen signaling via DHT has been shown to suppress urothelial differentiation while maintaining prostatic glandular characteristics. We suggested that type II 5-ARI can alleviate the androgenic suppression of urothelial differentiation by inhibiting 5αR, thereby enhancing postsurgical epithelial integrity. Furthermore, organoid models confirmed that type II 5-ARI promote UPK3A expression, further supporting their role in accelerating urothelial regeneration.

In addition to epithelial regeneration, 5αR2 inhibition was associated with a significant reduction in macrophage infiltration and polarization toward the proinflammatory M1 phenotype. Immunofluorescence staining revealed a marked decrease in CD68⁺ and F4-80⁺ macrophages in the type II 5-ARIs group, consistent with reductions in TNF-α and IL-1β levels postoperation. These findings suggest that 5αR2 inhibition attenuates the sterile inflammatory response by modulating macrophage behavior, potentially by reducing DHT-driven nuclear factor kappa light chain enhancer of activated B cells (NF-κB) activation, which has been implicated in macrophage-mediated proinflammatory signaling. Given that prolonged macrophage activation contributes to excessive fibroblast proliferation and matrix deposition, this anti-inflammatory effect likely plays a central role in preventing fibrosis and stricture formation.

The accelerating effect of a nonspecific 5-ARI, finasteride, on post-TURP wound repair has likewise been demonstrated.32 A previous study has shown that finasteride promotes epithelialization post-TURP.33

Our results extend previous findings by demonstrating enhanced urothelial differentiation following treatment with type II 5-ARIs. While recent theories have proposed that basal stem or progenitor cells may contribute to urothelial regeneration after prostate surgery, the specific role of 5αR2 inhibition in this process has not been fully elucidated or previously reported in the literature. Type II 5-ARIs selectively inhibit intraprostatic 5αR2, thereby reducing local DHT synthesis while minimizing systemic side effects on the cardiovascular and respiratory systems.34,35,36 Given these properties, type II 5-ARIs may facilitate epithelial remodeling, potentially through indirect effects on local progenitor cell dynamics. However, further investigation is needed to determine whether this includes true trans-differentiation events or alternative mechanisms of epithelial regeneration.

While our study highlights the therapeutic potential of 5αR2 inhibition in BPH postoperative care, several limitations exist. The six-month follow-up may not capture long-term complications such as fibrosis or stricture recurrence, necessitating extended studies. Our single-center trial with a limited sample size may affect generalizability, and validation in larger, multicenter cohorts is needed. Additionally, while we observed enhanced urothelial differentiation and reduced inflammation, the underlying signaling pathways involved remain unclear. Future research should identify downstream effectors of DHT suppression and assess the systemic effects of prolonged 5αR2 inhibition, including hormonal and metabolic consequences.

CONCLUSION

This study provides compelling evidence that the inhibition of type II 5α-reductase enhances postoperative wound repair in BPH patients undergoing TURP by promoting urothelial regeneration, reducing inflammation, and attenuating fibrosis. Using a combination of data from clinical trials, animal models, and single-cell transcriptomic analysis, we demonstrated that 5αR2 inhibition promotes epithelial recovery, suppresses macrophage-mediated SI, and limits excessive fibroblast activation, thereby reducing the incidence of postoperative complications such as hematuria, catheter reintroduction, and bladder neck contracture.

Nevertheless, additional research is needed to evaluate the long-term safety and efficacy of this strategy. In particular, mechanistic studies exploring the downstream molecular pathways influenced by 5αR2 inhibition, especially those regulating immune cell behavior and fibrotic remodeling, could further clarify its role in reshaping the wound healing microenvironment. Although our data suggest increased urothelial differentiation under DHT suppression, further investigations are warranted to determine whether this phenomenon reflects direct effects on progenitor cell fate or secondary responses to an improved inflammatory milieu.

From a clinical standpoint, incorporating 5αR2 inhibitors into perioperative management protocols holds promise as a novel adjunctive therapy for BPH surgery. Future prospective studies focusing on optimal timing, patient stratification, and combination strategies will be critical for validating and translating these findings into broader clinical practice. If substantiated in larger, multicenter trials, this approach may ultimately lead to improved healing outcomes and reduced complication rates in patients undergoing TURP.

AUTHOR CONTRIBUTIONS

CHZ and WHW performed all experiments and drafted the manuscript. SYJ conducted data analysis and prepared the figures. YFJ and BMH supervised the entire research process and provided clinical samples and data. SJX and XJW secured funding and critically revised the manuscript. All authors read and approved the final manuscript.

COMPETING INTERESTS

All authors declare no competing interests.