To rule out potential confounding effects, only 30 adult male Sprague-Dawley rats (weight, 260±10 g; age, 13 weeks) were obtained from Shanghai Sippr-BK Laboratory Animal Co. Ltd. The rats were housed together in a room maintained at 40-60% relative humidity and 23±2˚C with 12-h light/dark cycles and ad libitum access to food and water. The rats were randomly assigned to the following five groups (n=6 per group): i) Sham treatment (epidural exposure only); ii) spinal cord transection treatment; iii) combined treatment with 0.5 mg/kg/day telmisartan (cat. no. S1738; Selleck Chemicals) administered by gavage for 14 consecutive days, plus spinal cord transection; iv) combined treatment with 3 mg/kg/day telmisartan administered by gavage for 14 consecutive days, plus spinal cord transection; and v) combined treatment with 6 mg/kg/day telmisartan administered by gavage for 14 consecutive days, plus spinal cord transection. The sample size was determined using GPower software (version 3.1.9, University of Dusseldorf, Dusseldorf, Germany). The health and behavior of the animals were monitored every 2 days. After cystometric analysis at the 3rd week or if a humane endpoint, including listlessness or cessation of eating or drinking cessation, was reached, the rats were euthanized by the intraperitoneal injection of sodium pentobarbital (200 mg/kg body weight) and decapitation. All experimental protocols were approved by the Institutional Animal Care and Use Committee of Shandong University Hospital [approval no. KYLL-2021(LW)013]. All procedures were performed in compliance with guidelines issued by the National Institutes of Health.

After 1 week of adaptation, the rats were anesthetized and fixed on an operating table in the prone position. A 2 cm median incision was made in the back skin covering the L6-S1 region. The lamina at the L6-S1 level was removed and the spinal cord was completely severed. In the Sham group, the spinal cord was only exposed and not transected. A gelatin sponge (Ethicon, Inc.) was placed between the severed ends to stop bleeding and prevent healing. Next, the muscle layer and skin layer were separately sutured. After surgery, ampicillin sodium (100 mg/kg; cat. no. S3170; Selleck Chemicals) was intramuscularly injected once a day for 5 consecutive days.

For evaluation of bladder function by cystometry, the rats were anesthetized by inhalation of 1.5-2.0% isoflurane (cat. no. R510-22; RWD Life Science Inc.) for maintenance and induction at 14 days after the operation. A midline abdominal incision was made and a catheter was inserted via the bladder dome. The catheter was connected to a dual-channel syringe pump (cat. no. HK-400A; Shenzhen Hawk Medical Instrument Co., Ltd.) and a pressure transducer in a urodynamic measurement system (Dantec Menuet). Cystometry was performed as described in a previous study (19). The following parameters were measured: Maximum cystometric capacity, residual urine volume, bladder wet weight, bladder compliance and detrusor pressure.

The rat bladder was cut from the bladder neck, and the connective tissue surrounding the bladder wall was removed. Next, the bladder tissues were fixed with 4% paraformaldehyde (cat. no. P0099; Beyotime Institute of Biotechnology) at room temperature (RT) for 24 h, embedded in paraffin and sectioned into 3-mm thick sections. For Masson staining, the paraffin-embedded sections were deparaffinized with an alcohol gradient and xylene, and then washed with distilled water. The sections were then stained with hematoxylin (cat. no. S2384; Selleck Chemicals) at RT for 5 min, washed with tap water, immersed in a 1% hydrochloric acid alcohol solution for several seconds and rinsed with running water for several minutes. Next, the sections were stained with ponceau red dye (cat. no. S4497; Selleck Chemicals) at RT for 5-10 min; after which, the tissues were rinsed with distilled water and an aqueous phosphomolybdic acid (Electron Microscopy Sciences) solution for 3-5 min. The sections were then treated with aniline blue solution (cat. no. A9540; Beijing Solarbio Science & Technology Co., Ltd.) at RT for 5 min, followed by treatment with 1% glacial acetic acid (Guangzhou Jinhuada Chemical Reagent Co., Ltd.) for 1 min. Finally, the sections were dehydrated using an alcohol gradient and xylene, and then sealed with neutral gum (cat. no. N116470-100 g, Shanghai Aladdin Biochemical Technology Co., Ltd.). Blue collagen fibers, red muscle fibers, red cellulose and red blood cells were observed under a microscope (Olympus Corporation). For H&E staining, the slides were immersed in hematoxylin at RT for 30 sec, rinsed with running water until transparent, stained with eosin at RT for 30 sec and then rinsed again with water. The slides were then air-dried at RT. Subsequently, the slides were sequentially immersed twice in 95% ethanol solution, twice in 100% ethanol, twice in a solution of 50% ethanol and 50% xylene and twice in 100% xylene. The slides were then observed under a light microscope (Olympus Corporation).

Total RNA was extracted using TRIzol^® reagent (cat. no. 15596018; Thermo Scientific, Inc.) according to the manufacturer's instructions. The amount of RNA was quantified by spectrophotometry. Subsequently, reverse transcription was performed using the Bestar™ qPCR RT kit according to the manufacturer's protocol (cat. no. 2220; DBI Bioscience), followed by qPCR that was performed by using Bestar™ qPCR MasterMix (cat. no. 2043; DBI Bioscience, Shanghai, China) on an Mx3000P qPCR instrument (Stratagene; Agilent Technologies, Inc.). The following thermocycling conditions were used for qPCR: Melting at 95˚C for 2 min; followed by 40 cycles of denaturation at 94˚C for 20 sec, annealing at 58˚C for 20 sec, elongation at 72˚C for 20 sec and 72˚C complete elongation for 5 min. The primers used for qPCR were synthesized by Sangon Biotech Co., Ltd. and are listed in Table I. mRNA expression levels were quantified using the 2^-ΔΔCq method (20) and normalized to the internal reference gene GAPDH.

Freshly harvested bladders were homogenized in RIPA lysis buffer (cat. no. P0013C; Beyotime Institute of Biotechnology) and the amount of soluble protein in each homogenate was quantified using a BCA Protein Assay kit (cat. no. 23225; Thermo Fisher Scientific, Inc.). Subsequently, soluble protein (30 µg) was separated by 10% SDS-PAGE and transferred nitrocellulose membranes. Following blocking with 5% skimmed milk for 1 h at RT, the membranes were incubated overnight at 4˚C with the following primary rabbit antibodies: Anti-bFGF (1:1,000; cat. no. F3393; Sigma-Aldrich; Merck KGaA), anti-TGF-β1 (1:1,000; cat. no. 3711; Cell Signaling Technology, Inc.), anti-α-SMA (1:1,000; cat. no. 19245; Cell Signaling Technology, Inc.), anti-Collagen I (1:1,000; cat. no. ab255809; Abcam), anti-Collagen III (1:1,000; cat. no. ab7778; Abcam) and anti-GAPDH (1:1,000; cat. no. 5174; Cell Signaling Technology, Inc.). The membranes were then incubated with a horseradish peroxidase-labeled goat anti-rabbit antibody (1:2,000; cat. no. SA00001-2; ProteinTech Group, Inc.) at RT for 2 h. The protein bands were visualized with a chemiluminescent development agent (Chemistar™ High-sig ECL Western Blotting Substrate; Tanon Science & Technology Co., Ltd.). Protein expression was semi-quantified using GAPDH as the loading control by Image J software (2.0; National Institutes of Health).

The paraffin-embedded sections were prepared according to the protocol described in the ‘Masson and H&E staining’ section. After deparaffinization and hydration, the paraffin-embedded sections were placed in a microwave for antigen retrieval at 120˚C for 20 min in an autoclave, followed by washing with xylene and gradual rehydration in graded ethanol. Then, the sections were blocked with 3% H₂O₂ at RT for 15 min, and then further blocked with PBS containing 3% BSA (cat. no. ST023; Beyotime Institute of Biotechnology) at RT for 30 min. Next, the sections were incubated overnight at 4˚C with the following rabbit antibodies: Anti-rat α-SMA (1:150; cat. no. 41550; Signalway Antibody LLC), anti-rat Collagen I (1:200; cat. no. ab270993; Abcam) and anti-rat Collagen III (1:100; cat. no. ab7778; Abcam). Subsequently, the sections were incubated with a horseradish peroxidase-labeled goat anti-rabbit secondary antibody (1:4,000; cat. no. ab205718; Abcam) for 1 h at RT. The sections were developed using a DAB reagent kit (cat. no. DAB-1031; Fuzhou Maixin Biotech Co., Ltd.) for ~15 min, and then counterstained with hematoxylin for 4 min at RT. After three washes with PBS, the sections were mounted onto slides with neutral gum and then observed under a CX43 light microscope (Olympus Corporation). Brown particles were regarded as positive staining. The number of positive cells per high power field was counted using Image J software (2.0; National Institutes of Health).

All statistical analyses were performed using GraphPad Prism software (version 9.00; GraphPad Software, Inc.). All experiments were repeated 3 times and data are presented as the mean ± standard deviation. Comparisons among multiple groups were analyzed using one-way ANOVA followed by Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

After spinal cord transection, the cystometry test results were altered; rats in the model group displayed significantly higher bladder compliance, increased maximum cystometric capacity, increased residual urine volume, increased bladder wet weight and decreased detrusor pressure compared with that in the sham group (Fig. 1). After treatment with telmisartan, the values of the aforementioned parameters were significantly recovered, and the degree of recovery increased in a dose-dependent manner.

The antifibrotic effect of telmisartan was evaluated b(1)y comparing changes in the content of collagen fibers and smooth muscle fibers in the bladder wall as determined by Masson and H&E staining. The Masson staining results demonstrated that the bladder walls of rats that received spinal cord transection had disordered fibrous connective tissue, a distorted layered structure, increased thickness, reduced lamina propria, smooth muscle hypertrophy and increased numbers of intermuscular fibers compared with the sham group (Fig. 2A). Moreover, the H&E staining results showed that the bladder detrusor cells in the sham group had a long spindle shape and were uniformly distributed, structurally tight and arranged in parallel (Fig. 2B). Compared with the sham group, rats that received spinal cord transection showed thickened bladder propria, hypertrophic and disordered detrusor cells, decreased numbers of muscle cells and increased amounts of intermuscular connective tissue. Telmisartan treatment relieved bladder tissue fibrosis and detrusor cell hypertrophy in rats that received spinal cord transection.

Western blotting and RT-qPCR were used to investigate changes in fibrosis-related gene expression. The results showed significantly increased expression levels of bFGF, TGF-β1, Collagen I, Collagen III and α-SMA expression in the smooth muscle cells of rat bladder tissue after spinal cord transection compared with those in the sham group (Fig. 3). Furthermore, telmisartan reversed the effects of spinal cord transection on the expression of fiber-related genes to various degrees. The IHC results showed the expression of target proteins in tissue in situ. The results demonstrated that the expression of Collagen I, Collagen III and α-SMA was significantly increased after spinal cord transection compared with that in the sham group, whereas telmisartan treatment limited these effects (Fig. 4).