Long-term aspirin administration suppresses inflammation in diabetic cystopathy

Abstract

Diabetic cystopathy (DCP) is one of the most common and troublesome urologic complications of diabetes mellitus, characterized by chronic low-grade inflammatory response. However, the correlation between inflammation and disease progression remains ambiguous and effective drugs interventions remain deficient. Herein, during 12-week study, 48 male Sprague-Dawley rats were randomly assigned to four groups: negative control (NC), NC treated with aspirin (NC+Aspirin), DCP, and DCP treated with aspirin (DCP+Aspirin). Type 1 diabetes mellitus was established by intraperitoneal injection of streptozotocin (65 mg/kg). After 2 weeks modeling, the rats in treatment groups received daily oral aspirin (100 mg/kg/d). After 10 weeks of treatment, aspirin ameliorated pathological weight loss and bladder weight increase in diabetic rats, accompanied by a 16.5% decrease in blood glucose concentrations. H&E, Masson, immunohistochemistry and transmission electron microscopy revealed that a dilated bladder with thickened detrusor smooth muscle (DSM) layer, inflammatory infiltration, fibrosis and ultrastructural damage were observed in diabetic rats, which were obviously ameliorated by aspirin. The dynamic investigations at 4, 7 and 10 weeks revealed inflammation gradually increased as the disease progresses. After 10 weeks of treatment, the expression of TNF-α, IL-1β, IL-6, and NF-κB has been decreased to 78%, 39.7%, 44.1%, 33.3% at mRNA level and 67.6%, 76.7%, 71.4%, 67.1% at protein level, respectively (DCP+Aspirin vs. DCP, p < 0.01). Aspirin partially restored the increased expression of inflammatory mediators in bladder DSM of diabetic rats. The study provided insight into long-term medication therapies, indicating that aspirin might serve as a potential strategy for DCP treatment.

Keywords: aspirin; bladder; detrusor smooth muscle; diabetic cystopathy; inflammatory.

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).

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).

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).

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).

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).

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).