The synthesis of IR-61 followed a previously reported method (Wang et al., 2016). The synthesized IR-61 powder was dissolved in dimethyl sulfoxide (DMSO) to a concentration of 10 mmol in advance and then stored at −20°C for subsequent use. Before each administration, we diluted the solution by 40-fold with sterilized PBS solution and mixed it well. All the above operations were performed in the dark. Streptozotocin (STZ) was purchased from Sigma-Aldrich (United States).

The experiments were conducted on female Sprague Dawley rats (weighing 180–220 g) that were purchased from the animal facility of the Central Animal House Services of the Army Medical University (AMU, Third Military Medical University), Chongqing, China. All protocols and animal research procedures were approved by the Ethics Committee and performed in accordance with the guideline of the Animal Care and Use Committee of the Third Military Medical University. The rats were housed at room temperature and exposed to a 12-h/12-h light–dark cycle, with free access to food and water. All the rats were randomly divided into two groups: CON (control rats, n = 15) and DM (diabetic rats, n = 30). After 12-h fasting, the rats in the DM group were intraperitoneally injected with 65 mg/kg STZ that was dissolved in citrate-sodium citrate buffer (0.05 M, pH 4.0–4.5). The rats in the Control group were treated with an equal volume of vehicle (0.05 M citric acid-sodium citrate buffer, pH 4.0–4.5). Blood glucose (GLU) was measured via a glucometer (Roche Diagnostics Corporation, Indianapolis, IN, United States) using blood sampled from the tail vein 3 d and 7 d after injection. The rats with GLU levels above 16.7 mmol/L on both days were considered diabetic and selected for the subsequent experiments. The rats in the DM group were randomly divided into two groups: rats with diabetic bladder dysfunction treated with vehicle (DBD; n = 15) and rats with diabetic bladder dysfunction treated with IR-61 (DBD + IR-61; n = 15). IR-61 (1.6 mg/kg) was injected intraperitoneally once a week, and the administration duration was 10 weeks. The dose of 1.6 mg/kg showed significant protective effects on rats with DBD, which has been demonstrated in our previous experiments. Blood glucose and body weight were also measured every week.

The diabetic rats were intraperitoneally injected with IR-61 (1.6 mg/kg) and then sacrificed under anesthesia. Their hearts, livers, spleens, lungs, kidneys, and bladders were harvested for ex vivo imaging at 6 h, 1 days, 4 days, and 7 days after injection. At each time point, three diabetic rats were used for detection. Near-infrared fluorescence imaging was performed to detect the distribution of IR-61 in the organ using the Kodak FX Professional Imaging System (New Haven, CT). To detect the distribution of IR-61 in the bladder, the bladder tissues were frozen and cut into slices (8 μm). The fluorescence signals of IR-61 were detected using Leica LAS AF Lite software (Leica, Weztlar, Germany).

To determine the subcellular location of IR-61 in BSMCs, primary BSMCs were isolated from the SD rats and cultured with an adherent cultivation approach, and immunofluorescence staining with Gt anti-α-SMA antibody (1:100, ab21027, Abcam) was performed to identify the cells as previously described (Gopinath et al., 2013). The cultured BSMCs were then seeded in a 35-mm petri dish and incubated with 10 mM IR-61 for 20 min and 100 nM Mito-Tracker Green (M7514, Invitrogen) for 30 min. The cell nuclei were stained with Hoechst 33342 (C1025, Beyotime, China) and photographed with a confocal microscope (Leica TCS SP5).

For in vivo repetitive cystometry analysis, the rats treated for 10 weeks were anesthetized (Brouwers et al., 2013; Funahashi et al., 2014; Magaldi et al., 2019), placed in a supine position on top of a thermal pad, and fixed on a small animal operating table with an elastic bandage. Then, the bladder was surgically exposed via a midline abdominal incision, and a PE-50 catheter was inserted through the apex of the bladder dome. The other end of the catheter was connected to a pressure transducer (Laborie Medical Technologies Inc., Beijing, China) via a three-way stopcock to record the intravesical pressure and a micro medicine infusion pump (Hangzhou Zeda Instruments Co., Ltd., Hangzhou, China) to infuse saline into the bladder. After the bladder was emptied, sterile room-temperature saline was infused through the micro medicine infusion pump at a rate of 0.3 ml per minute. Pumping was immediately stopped when urine was observed at the external urethra and started again when urination ended. The intravesical pressure curve was automatically recorded with a computer for 30 min. The in vivo cystometry test parameters included the urination frequency within 30 min, maximum bladder capacity (MBC, the volume of saline pumped before first urination), maximum voiding pressure (MVP, the maximum peak pressure during the actual voiding phase), residual volume (RV, manually drained and measured with a 2.5-ml syringe), voiding efficiency {VE, calculated as [(MBC-RV)/MBC] *100%}, and bladder compliance [BC, calculated as (MBC/TP-BP)]. Threshold pressure (TP) is defined as the intravesical pressure immediately before micturition. Basal pressure (BP) is defined as the minimum pressure between two micturition (Supplementary Figure S1). Five rats in each group were measured. The mean value of the three voiding cycles of each rat was used to eradicate any discrepancies. The procedure was performed three times for each rat (Wang et al., 2019).

Rats in each group (n = 5) were euthanized after filling cystometry, and the bladders of the rats were isolated and fixed using 4% paraformaldehyde solution for 48 h at room temperature. After fixation, the tissues were dehydrated, embedded in paraffin, and sliced into 5-μm-thick sections for H&E and Masson’s trichrome staining. Sections were prepared for immunofluorescence staining; these sections were deparaffinized, rehydrated, and treated with 3% hydrogen peroxide for 10 min at room temperature to eliminate the endogenous peroxidase activity. Then, the sections were placed in 0.01 M citrate buffer solution (pH 0.6), boiled (95–100°C) for 15 min to retrieve the antigen, and subsequently blocked with 1% goat serum albumin for 30 min at room temperature. After incubation with primary antibodies against α-SMA (Abcam, 1:200) and Nrf2 (CST, 1:200) overnight at 4°C, the sections were incubated with the corresponding secondary antibodies for 1 h at 37°C. The stained bladder sections were examined under a light microscope (Olympus Inc., Tokyo, Japan), and images were captured with a digital camera mounted to the microscope. All the images were analyzed using ImageJ software.

An in situ cell death detection kit, Fluorescein (11684817910, Roche Applied Science, Indianapolis, IN, United States), was used to detect the apoptosis of BSMCs according to the manufacturer’s instructions. Briefly, the bladder tissue sections in each group (n = 5) were hydrated and treated with a proteinase K working solution at 37°C for 15 min. Next, the sections were incubated with the TUNEL reaction mixture (the negative control group was only treated with 50 μl of fluorescein-labeled dUTP solution) in a dark, wet box at 37°C for 1 h. Finally, DAPI (Sigma-Aldrich) was used to stain the nuclei. The stained sections were photographed using a fluorescence microscope.

Fresh bladder tissues in each group (n = 5) were prepared into frozen slices and incubated quickly with Mito-Tracker Red, 2′,7′-dichlorodihydrofluorescein diacetate (DHE, 10 μm, Beyotime) and MitoSOX™ Red (10 μm, Invitrogen) at 37°C in the dark for 30 min to measure the relative number of active mitochondria, the in situ level of intracellular ROS, and the level of mitochondrial superoxide in the BSMCs, respectively. Quantitative analysis of the fluorescence intensity was performed using ImageJ software.

Fresh BSM tissues in each group (n = 5) trimmed into 1*1*3 mm tissue blocks were fixed in 4% glutaraldehyde overnight, fixed with 1% osmium tetroxide, dehydrated in graded ethanol and infiltrated in fresh 100% resin. Ultrathin sections cut from the blocks were stained with 5% uranyl acetate and lead citrate and then viewed by transmission electron microscopy (JEM-1400PLUS, Japan) at 100 KV.

Bladder tissues in each group (n = 5) that were denuded of mucosal and adventitial layers were homogenized and lyzed on ice for 30 min with RIPA buffer (Beyotime Biotechnology, China) containing a cocktail of protease and phosphatase inhibitors (Thermo Scientific, IL, United States). After centrifugation (15,000 g at 4°C for 15 min), the supernatants were collected, and the protein concentration was measured using a BCA protein assay kit (Beyotime Biotechnology, China). Samples containing equivalent amounts of proteins (20 mg) were separated by electrophoresis on 10% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, United States)that were blocked with western blocking buffer (Beyotime Biotechnology, China). Then, the PVDF membranes were probed with primary antibodies at 4°C overnight, followed by incubation with horseradish peroxidase-conjugated secondary antibodies (CST, 1:1,000) for 1 h at room temperature. The band intensities were visualized and analyzed using an enhanced chemiluminescence detection system (Bio-Rad Laboratories) and ImageJ software (Bethesda, MD, United States). The primary antibodies used were ɑ-smooth muscle actin (α-SMA) (Abcam, 1:1,000), cleaved-Caspase 3 (Abcam, 1:1,000), Bax (1:1,000, Abcam), Bcl-2 (1:1,000, Abcam), cleaved-Caspase 9 (Abcam, 1:1,000), Cytochrome C (Abcam, 1:1,000), Nrf2 (CST, 1:1,000), Keap1 (CST, 1:1,000), SOD-1 (Abcam, 1:1,000), SOD-2 (Affinity, 1:1,000), HO-1 (CST, 1:1,000), GPX-1 (CST, 1:1,000), β-actin (1:1,000, Proteintech) and GAPDH (1:1,000, Beyotime Biotechnology, China).

All the data are presented as the mean ± SD. The statistical analyses were performed using one-way or two-way analysis of variance to determine statistical significance using SPSS 26.0 (SPSS Inc., Chicago, IL, United States). p < 0.05 was considered statistically significant.

Some vital organs were harvested from the diabetic rats for ex vivo imaging. We observed clearly stronger NIR fluorescence in the bladder than in other organs, such as the heart, liver, spleen, lung, and kidney. The results illustrated that IR-61 could highly accumulate in the bladder, as proven by the NIR fluorescence intensity of the bladder, which was significantly higher than that of other organs 6 h, 1 days, 4 days, and 7 days after intraperitoneal injection (Figure 1A). Imaging on 7 days after injection showed that there was no significant accumulation of IR-61 in the bladder, which is why we chose one injection per week as the frequency of administration.

To determine the tissue distribution of IR-61, fresh bladders were frozen and sectioned for histological analysis 1 day after IR-61 injection. Fluorescence microscopy showed that IR-61 was mostly localized in the smooth muscle layer of the bladder (Figure 1C and Supplementary Figure S2). Consequently, exploring the role of IR-61 in BSMCs was the focus of this study.

To define the subcellular location of IR-61, we isolated and cultured primary BSMCs from 8-week-old SD rats. The cells were identified by immunofluorescence staining of α-SMA, which is a specific marker of smooth muscle cells (SMCs) (Supplementary Figure S3). The targeted accumulation of IR-61 in the mitochondria of BSMCs was confirmed by its colocalization with MitoTracker Green (Figure 1D and Supplementary Figure S4). These results support the claim that IR-61 is a mitochondria-targeting NIR dye, which is consistent with the previous study.

Supplementary Figures S5A,B describe the changes in the body weight and blood glucose levels of the rats in the three groups. Compared with the control group, the DBD group exhibited remarkably reduced body weight (p < 0.0001) and increased blood glucose levels (p < 0.0001) at week 10. No significant differences in these two parameters were observed between the DBD and DBD + IR-61 groups (Table 1). The results described above indicated that IR-61 did not change the blood glucose levels or body weights of the diabetic rats.

The bladder weight was significantly increased in the DBD and DBD+IR-61 groups compared with the control group (Supplementary Figure S5C). However, the bladder weight was still significantly lower in the DBD+IR-61 group than in the DBD group. Since the development of the animals during the 10 weeks experiment differed, we further evaluated the ratio of the bladder weight to the body weight of the rats. Our results indicated that the ratio was significantly higher in the DBD and DBD + IR-61 groups than in the Control group (p < 0.0001, p < 0.001, respectively Supplementary Figure S5D). There was no difference between the DBD group and DBD + IR-61 group.

The representative cystometry curves of each group are presented in Figure 2A. The analysis results of the curves revealed that the rats in the DBD group had significantly decreased MVP, urination frequency, and VE (vs. Control p < 0.0001, p < 0.0001, p < 0.05, respectively) but increased MBC, RV, and BC (vs. Control p < 0.0001 for each), suggesting typical DBD in the decompensation phase (Figures 2B–G); however, IR-61 treatment remarkably increased the MVP, urination frequency, and VE (vs. DBD group p < 0.01, p < 0.05, p < 0.05, respectively) but significantly decreased the MBC, RV, and BC (vs. DBD group p < 0.001, p < 0.0001, p < 0.0001, respectively). These data confirmed that IR-61 protected the voiding function of the diabetic rats.

The bladder wall thickness was measured using H&E images (Figure 3A), and the ratio of smooth muscle to collagen in the full layer and smooth muscle layer was determined by Masson’s trichrome staining (Figure 3C). Morphometric analysis of bladder tissues revealed that the bladder wall thickness and the smooth muscle/collagen ratio in the full layer and smooth muscle layer were significantly decreased (p < 0.01, p < 0.01, p < 0.05, respectively) in the rats in the DBD group, but IR-61 treatment prevented the decrease of bladder wall thickness and the smooth muscle to collagen ratio (p < 0.05 for each) (Figures 3B,D,E); these results indicated that IR-61 could effectively alleviate the bladder tissue damage in the diabetic rats.

In addition, we detected the expression of α-SMA in bladder tissue by WB and immunofluorescence staining and found that the expression of α-SMA was decreased in the DBD group (vs. Control p < 0.05) and increased in the DBD + IR-61 group (vs. DBD group p < 0.05), which also indicated that IR-61 could prevent the decreased smooth muscle content caused by DM.

We examined whether IR-61 had any protective effects on the number of apoptotic BSMCs. TUNEL staining demonstrated that the number of apoptotic cells in the bladder was markedly increased in the DBD group compared with the Control and DBD + IR-61 groups (p < 0.001 for each, Figures 4A,B). Moreover, Western blotting showed that the protein level of cleaved Caspase-3 was clearly increased in the DBD group and markedly decreased in the DBD + IR-61 group compared with the control group (p < 0.001 for each, Figures 4C,D). These data illustrated that IR-61 ameliorated the apoptosis of BSMCs.

To determine whether the apoptosis of BMSCs and the anti-apoptotic effect of IR-61 in diabetic rats are related to mitochondria, we conducted Western blot assays on proteins associated with mitochondrial apoptosis pathways, such as Bcl-2, BAX, Cytochrome C, and cleaved Caspase-9 (Figures 4E–I). The results indicated that the protein levels of BAX, Cytochrome C, and cleaved Caspase-9 were significantly increased (p < 0.01 for each) and the protein levels of Bcl-2 were decreased (p < 0.05, p < 0.001, p < 0.05, p < 0.05, respectively) in the DBD group compared with the Control group, but IR-61 treatment prevented these changes. All these data demonstrated that IR-61 inhibited mitochondrial-related cellular apoptosis in the bladder tissue of the diabetic rats.

Frozen bladder tissue sections were stained with dihydroethidium (DHE) (Figure 5A) and MitoSOX (Figure 5C) and used to measure the cellular and mitochondrial superoxide levels. The results of analyzing the DHE and MitoSOX images showed that both the cellular and mitochondrial superoxide levels were clearly increased by DBD (p < 0.05 for each) and suppressed by IR-61 treatment (p < 0.05 for each); these results revealed the antioxidative effect of IR-61 (Figures 5B,D). Moreover, the fluorescence staining of MitoTracker Red (Figure 5E) proved that IR-61 preserved mitochondrial function, which was significantly reduced in bladder tissues of the DBD group (p < 0.0001, Figure 5F). Transmission electron microscopy (TEM) images of BSM showed that the BSMCs in the rats in the DBD group exhibited increased mitochondrial calcification and degeneration, whereas IR-61 attenuated the damage to BSMC mitochondria observed in the diabetic rats (Figure 5G); these results showed that IR-61 had a protective effect on the mitochondria of BSMCs.

As shown in Figure 6, we explored whether the antioxidative stress effect of IR-61 is due to the activation of Nrf2. Representative original blots of the protein levels in the bladders of the three groups were examined (Figure 6A). Compared with those in the control group, the protein levels of Nrf2, HO-1, GPX-1, SOD-1, and SOD-2 were decreased in the DBD group (p < 0.05, p < 0.05, p < 0.01, p < 0.001, p < 0.01, respectively, Figures 6B,D–G), while the protein levels of Keap1 were increased (p < 0.0001, Figure 6C). However, IR-61 treatment significantly prevented the DM-induced reduction in Nrf2, HO-1, SOD-1, SOD-2 and suppressed the increase of Keap-1 observed in the bladders in the DBD + IR-61 group, which indicated that the protective effect of IR-61 may be related to the activation of Nrf2.