Long-term aspirin administration suppresses inflammation in diabetic cystopathy

General characteristics

The rat model of type 1 diabetes was successfully established according to the design project (Figure 1A, and Supplementary Figure 1A). The random blood glucose of diabetic rats was 3-4 times higher than that of normal rats (Figure 1B). After 10 weeks of treatment, the elevation of random blood glucose caused by diabetes was lowered by aspirin. Aspirin had hypoglycemic activity, causing an approximately 16.5% decrease in blood glucose concentrations (DCP+Aspirin vs. DCP, p < 0.001). The body weight of normal rats gradually increased, while diabetes rats developed typical symptoms such as polydipsia, polyphagia, polyuria and weight loss (Supplementary Figure 1B). After 10 weeks of treatment, aspirin improved the weight loss of diabetes rats (Figure 1C). Gross assessment preliminarily showed that the bladder wall was thicker in the DCP group, and aspirin treatment reduced bladder thickening (Figure 1D). The absolute and relative bladder weight of the DCP group were increased over those of controls by approximately 93% (Figure 1E) and 265% (Figure 1F) respectively. Aspirin treatment partially reversed this change to approximately 75% and 63% (DCP+Aspirin vs. DCP, p < 0.001).

Figure 1. STZ-induced diabetic rats intragastric administration with aspirin. (A) Schematic illustration of the experimental timeline and the chemical structure of aspirin. (B) Random blood glucose (C) Body weight. (D) Representative macroscopic findings of bladder specimens. (E) Bladder weight in the empty state. (F) Relative bladder weight (bladder weight/body weight). Data were presented as mean ± SD. N=6. (ns, no significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; NC vs DCP; DCP vs DCP+Aspirin).

Histological changes in STZ-induced diabetic rats after 10 weeks of aspirin administration

As shown in Figure 2A, H&E results showed inflammatory cell infiltration, edema, hemorrhage and capillary proliferation of mucosa and lamina propria occurred in the bladder tissue of the DCP group, consistent with previous research. Bladder wall thickness and collagen volume fraction in the DSM tissue were measured on Masson-stained images (Figure 2B). Inflammation and injury were prominently ameliorated after 10 weeks of aspirin treatment (Figure 2C). Morphometric analysis revealed bladder wall thickness (Figure 2D) and collagen volume fraction (Figure 2E) in the DCP group were up to 1.5 times compared with the NC group. After 10 weeks of aspirin treatment, the thickness of the bladder wall was reduced by 78% (DCP+Aspirin vs. DCP, p < 0.001), and the ratio of smooth muscle/collagen in the detrusor layer of the bladder was also reduced by 68% (DCP+Aspirin vs. DCP, p < 0.001). Aspirin attenuated bladder hypertrophy in STZ-induced diabetic rats.

Figure 2. The effect of aspirin on bladder injury in STZ-induced diabetic rats after 10 weeks administration. (A) H&E-stained micrographs of histological features of rat bladder sections. (B) Masson-stained micrographs. The dotted arrowheads indicate the thickness of the bladder wall. (C) Histological score based on inflammation. (D) Statistical analysis of the thickness of bladder wall. (E) Morphological evaluation was performed with measurement of bladder fibrosis and collagen content. Data were presented as mean ± SD. N=6. (***, P < 0.001; NC vs DCP; DCP vs DCP+Aspirin).

The effect of aspirin on ultrastructure of the DSM in STZ-induced diabetic rat

Morphometric analysis under the light microscope revealed a remarkable increase in the areas of the detrusor muscle and urothelium in diabetic rats (Figure 2). To take a closer look, the bladder detrusor smooth muscle cells (DSMCs) and fibroblasts were observed under transmission electron microscopy (TEM). The detrusor histology of the DCP group rats was characterized by the presence of thickened and hypertrophied microfibers, some of which were dissolved and necrotic (Figure 3A). Besides that, as shown in Figure 3B, there was severe mitochondrial damage in the DSMCs of the diabetic rat bladder, manifested as increased mitochondrial volume, decreased number of “ridges”, ruptured or even disappeared, and decreased mitochondrial matrix optical density (Figure 3C). In addition, mitochondrial vacuolar degeneration and endoplasmic reticulum swollen was observed. Aspirin repaired the damaged DSMCs by reducing mitochondrial damage.

Figure 3. Transmission electron microscopy (TEM) of bladder DSM tissue after 10 weeks of aspirin administration. Organelles such as nucleus, mitochondria, and rough endoplasmic reticulum are seen. (A) A representative image from the bladder DSM at a magnification of ×1,000. (B) A representative image from the bladder DSM at a magnification of ×15,000. The red arrowheads indicate the swollen mitochondria. (C) Statistical analysis of the mitochondrial matrix optical density. Data were presented as mean ± SD. N=6. (***, P < 0.001; NC vs DCP; DCP vs DCP+Aspirin).

The effect of aspirin on histological and inflammation-related factor changes in STZ-induced diabetic rat

Polyuria and impaired insulin signaling induced by disorders of glucose metabolism can increase macrophage infiltration and secrete inflammatory cytokines, leading to local and systemic inflammation [14, 30]. NF-κB is an upstream mediator of cytokine transcription, which plays a key role in regulating the multiple genes involved in the inflammatory response in bladder tissue, such as TNF-α, IL-6, and IL-1β. Immunohistochemical results indicated inflammatory indicators (NC vs. DCP, p < 0.001) NF-κB, TNF-α, IL-6, and IL-1β were increased by 44.3%, 150.1%, 71.5% and 46.0% respectively in the DSM of diabetic rats (Figure 4). Interestingly, the expression of NF-κB, TNF-α, IL-6, and IL-1β in the DCP+aspirin group generally reverted toward normal levels, with a noticeable decrease (DCP vs. DCP+Aspirin, p < 0.001) to 56.5%, 65.9%, 67.2% and 66.0% of the DCP group.

Figure 4. Immunohistochemistry shows aspirin suppress DSM tissue inflammation in diabetic rats after 10 weeks administration. (A) Immunohistochemistry indicates the expression of inflammatory mediators IL-6, IL-1β, TNF-α, and NF-κB was mainly distributed in the mucosa, lamina propria and meanwhile detrusor muscle. Arrows indicate positive staining (brownish-yellow). (B) The average optical density of IL-6, IL-1β, TNF-α, and NF-κB were analyzed using Image J Analysis System. Data were presented as mean±SD. N=6. (***, P < 0.001; NC vs DCP; DCP vs DCP+Aspirin).

Hyperglycemia upregulated the inflammatory factor protein activity and mRNA level in the DSM tissue of STZ-induced diabetic rats

To explore how aspirin treatment prevented bladder injury, we evaluated mRNA and protein expression levels of pro-inflammatory cytokines at different stages of diabetes. As shown in Figure 5A, after 4 weeks of aspirin treatment, the mRNA expression levels of TNF-α, NF-κB, IL-1β, and IL-6 in the DCP group increased by 19.1% (p < 0.01), 23.3% (p < 0.001), 13.2% (p = 0.1087) and 23.6% (p < 0.001) respectively compared with the control group. After 7 weeks, TNF-α, NF-κB, IL-1β and IL-6 in the DCP group were 23.6% (p < 0.01), 29.1% (p < 0.001), 23.5% (p < 0.01) and 57.5% (p < 0.001) higher than those of the control group (Figure 5B). Statistics on 10 weeks increased (p < 0.001) by 39.1%, 70.2%, 34.5% and 111.8%, respectively (Figure 5C). To go along with this, compared with the DCP group, the mRNA expression levels of TNF-α, NF-κB, IL-1β, and IL-6 in the DCP+Aspirin group decreased to 76.7%, 75.3%, 81.9% and 71.6% after 4 weeks of aspirin treatment; 79.5%, 75.7%, 71.6% and 87.9% after 7 weeks of aspirin treatment; 78.0%, 39.7%, 44.1% and 33.3% after 10 weeks of aspirin treatment.

Figure 5. The mRNA expression levels of TNF-α, NF-κB, IL-1β and IL-6 in bladder DSM tissue of three treatment durations. (A) Statistical analysis after 4 weeks of aspirin treatment. N=3. (B) Statistical analysis after 7 weeks of aspirin treatment. N=3. (C) Statistical analysis after 10 weeks of aspirin treatment. Data were presented as mean ± SD. N=6. (ns, no significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; NC vs DCP; DCP vs DCP+Aspirin).

Then we evaluated the expression of the inflammatory mediators aforementioned at the protein level by western blot assay. As shown in Figure 6, the results evidenced that the protein expression level of TNF-α, NF-κB, IL-1β, and IL-6 in the DSM tissue of the DCP group (p < 0.01) was remarkedly higher than that of the control group. After 4 weeks of aspirin treatment, the up-regulated rate (NC vs. DCP, p < 0.01) was 14.0%, 21.1%, 16.7% and 17.9%. After 7 weeks, the up-regulated rate (NC vs. DCP, p < 0.001) reached 22.7%, 21.1%, 21.0% and 22.9%. After 10 weeks, the up-regulated rate (NC vs. DCP, p < 0.001) was up to 50.0%, 28.2%, 38.5% and 43.3%, respectively. Correspondingly, the promotion effect of STZ on the protein expression level of TNF-α, NF-κB, IL-1β, and IL-6 in the DSM tissue of the DCP group was partially restored by aspirin administration. Aspirin postconditioning reduced inflammation to 91.0% (p < 0.05), 90.2% (p < 0.01), 94.0% (p = 0.1563) and 90.1% (p < 0.01) after 4 weeks of treatment; 7 weeks (p < 0.001) to 87.2%, 92.0%, 91.7% and 89.2%; 10 weeks (p < 0.001) to 67.6%, 76.7%, 71.4% and 67.1%. To summarize, the levels of inflammatory cytokines increased gradually with the course of the disease. Aspirin exerts a therapeutic mitigative effect on inflammation formation, which is consistent with the mRNA expression level. The pharmacological inhibition of aspirin may be one of the main mechanisms for inhibiting the release of inflammatory mediators in DSM of diabetic rats.

Figure 6. The protein expression levels of TNF-α, NF-κB, IL-1β and IL-6 in bladder DSM tissue of three treatment durations. (A, B) Western blot bands and statistical analysis after 4 weeks of aspirin treatment. N=3. (C, D) Western blot bands and statistical analysis after 7 weeks of aspirin treatment. N=3. (E, F) Western blot bands and statistical analysis after 10 weeks of aspirin treatment. Data were presented as mean ± SD. N=6. (ns, no significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; NC vs DCP; DCP vs DCP+Aspirin).

Animals

48 adult male Sprague–Dawley rats (average weight approximately 220±10 g) were purchased from Beijing HFK Bioscience Co., Ltd. All rats were housed with a standard 12-hour light-dark cycle at a temperature of 22±2° C and humidity of 40%-60%. Rats were given free access to standard food and water. Throughout the research, we spare no effort to alleviate the suffering of animals and use as few animals as possible.

Induction of diabetes and treatment groups

In this 12-week study, a total of 48 rats were randomly divided into 4 groups of 12 each by simple randomization method using Excel software: negative control (NC), NC treated with aspirin (NC+Aspirin), DCP, and DCP treated with aspirin (DCP+Aspirin). Rats in model groups were intraperitoneally injected with STZ (65 mg/kg) to establish a type 1 diabetic rat model [6, 39, 40]. Rats in the control group were intraperitoneally injected with the same dose of citric acid-sodium citrate buffer for the control treatment. After 3 days, blood samples were collected from the tail vein to measure the random blood glucose. A blood glucose level above 16.7 mM confirms the presence of diabetes. Aspirin was dissolved in sterile water and prepared into a concentration of 20 mg/mL. 2 weeks after modeling, the rats in treatment groups received daily aspirin (100 mg/kg/d) by gavage, served with the equal volume control irrigation sterile water (5mL/kg/d) [26, 29, 41]. Subsequently, 12 rats in each group were divided into 3 subgroups according to treatment duration: 3 rats in the 4W group, 3 rats in the 7W group and the remaining 6 rats in the 10W group. After treatment, animals were euthanized in carbon dioxide tanks and bladder specimens were collected.

Morphological evaluation

The body weights were recorded daily until the ending point. After 10 weeks of administration, random blood glucose was measured before execution. And then we did the gross morphology observation. After macroscopic examination, bladders were rapidly dissected to drain the urine and then weigh the bladder in an empty state. Subsequently, the bladders were opened at the dorsal side of the bladder neck along with the midline towards the dome and then divided longitudinally into muscle strips. DSM strips (5-7 mm long and 2-3 mm wide) from the urinary bladder dome were collected. Half of them removed the bladder mucosa under a stereomicroscope and preserved in liquid nitrogen. The other half of the urinary bladder was placed in a 4% paraformaldehyde solution or 2.5% glutaraldehyde solution for histological.

H&E, Masson and immunohistochemical staining

The sections were stained with H&E and Masson’s trichrome, then examined by light microscope. For HE staining, sections were randomly selected and scored based on inflammation and infiltration level of the tissue, the severity of edema and hemorrhage. As shown in Supplementary Table 1 [42], the scale ranged from 0 to 5 points indicating light to severe according to the inflammation. The thickness of the bladder wall and the ratio of smooth muscle to collagen within the bladder DSM layer wall were measured by using Masson trichrome-stained images.

For immunohistochemistry (IHC) staining, the tissue sections were incubated with TNF-α (1:100), IL-1β (1:100), NF-κB (1:250), IL-6 (1:100) and β-actin (1:500) primary antibody at 4° C overnight. We observed the five visual fields of every section under a low-power microscope, taking the average value as the observation result of each section. The average optical density was measured by using the IHC Profiler plugin to analyze the images of the sections in Image J (NIH, Bethesda, MD, USA). To minimize the variability between images, the density was normalized to that of an unstained area, and the exposure time and microscope settings were fixed throughout the acquisition.

Transmission electron microscopy

The bladder specimens were quickly sliced into 1 mm³ and immersed in 2.5% glutaraldehyde solution for 48 h. Subsequently, postfixation was performed with 1% osmium tetroxide for 2 h, and dehydration followed by impregnation with epoxy resin was performed. The ultrathin sections were made at 70~80nm and stained with 2% uranyl acetate and Reynolds lead citrate solution. The sections were observed under a transmission electron microscope (H-7700; Hitachi High-Technologies, Tokyo, Japan).

Quantitative reverse transcription-polymerase chain reaction (qRT-PCR)

The total RNA extraction from DSM tissues was performed using RNAiso Plus Kit (Takara-Bio, Shiga, Japan) according to the manufacturer’s instructions. Reverse transcription of total RNA was performed using PrimeScript RT Master Mix (Takara-Bio, Shiga, Japan). Quantitative reverse-transcription PCR was performed using TB Green Premix Ex TaqTM (Takara-Bio, Shiga, Japan) on an ABI PRISM7500 sequence detection system. The PCR conditions were 95° C for 30 s followed by 40 cycles of 95° C for 5 s and 60° C for 30 s. All of the reactions were run three times, and mRNA expression was normalized relative to GAPDH. Supplementary Figure 2 showed the stability of GAPDH expression. The primer sequences are shown in Table 1.

Western blot assay

DSM tissues were extracted with RIPA lysis buffer (Solarbio, Beijing, China). Protein concentrations were determined using BCA Protein Quantification Kit (Yeasen Biotech Co., Ltd). The protein sample (20 μg) was loaded onto 12% SDS-PAGE gel for electrophoretic separation and then transferred to a nitrocellulose membrane. The membrane was blocked with Tris-buffered saline with Tween 20 (TBST) containing 5% bovine serum albumin (BSA) for 1.5 h. After shaking slowly at room temperature for 1.5 h, the membrane was incubated with primary rabbit antibody against TNF-α (1:500; Abcam Cat# ab205587, RRID: AB_2889389), IL-1β(1:1000; Novus Cat# NB600-633, RRID: AB_10001060), NF-κB (1:1000; Abcam Cat# ab32360, RRID: AB_776748), and β-actin (1:5000; Abcam Cat# ab8227, RRID: AB_2305186) and with primary mouse antibody against IL-6 (1:1000; Abcam Cat# ab9324, RRID: AB_307175) on a 4° C shaking table overnight. Take out the membrane the next day and clean the membrane 3 times with TBST for 5 minutes each time. Then the membrane was slowly incubated with the secondary antibody (1:5000; Abcam) at room temperature for 1.5h. Subsequently, the PierceTM ECL Western Blotting Substrate Kit (Thermo Fisher Scientific, USA) was used to detect the protein expression. Supplementary Figure 3 showed the original protein bands.

Statistical analysis

Data were further analyzed with GraphPad Prism 9.0.0 software (San Diego, CA, USA) and were expressed as the mean ± SD. The histological score was assessed using a Kruskal-Wallis test followed by Dunn’s multiple comparisons test. The other data were analyzed with ANOVA followed by the Tukey’ s multiple comparisons test. All images were processed and analyzed using dedicated software (Image Pro 6.0). All microscopic sections were randomly selected and reviewed by two pathologists, who were blinded to the study design. Percentage change of the DCP group from the NC group or percentage change of the DCP+Aspirin group from the DCP group. n=the number of strips or PCR samples and N=the number of rats. P < 0.05 was considered statistically significant.

Data availability statement

The data generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.