Hypothesis / aims of study
Painful bladder syndrome/Interstitial cystitis (PBS/IC) is a chronic debilitating inflammatory condition of the urinary bladder of complicated poorly-understood pathophysiology accompanied by daytime and/or night-time urinary frequency and urinary urgency, in the absence of bacterial infection. The main cause of PBS/IC is thought to be a persistent bladder inflammation by the deficiency of the glycosaminoglycan covering the urothelium surface that results in the leaky urothelium and subsequent activation of neurogenic inflammation. The use of medicinal honey for wound management has been supported through clinical studies highlighting its antimicrobial activities and anti-inflammatory properties. Our previous studies showed that 4% of Manuka honey (MH) can protect the monolayer primary human urothelial cells viability and inhibited pro-inflammatory cytokines release. The objectives of this study were to determine the effect of MH on the human urothelial-fibroblast cells' co-culture model system permeability and pro-inflammatory cytokines production induced by tumor necrosis factor-alpha (TNF-α).
Study design, materials and methods
200 μl of the fibroblast cells (FB) (3x10⁶ cells/ml) were seeded on the basal side of the transwell insert membrane for 2 hours to adhere. After that, 300 μl (3 × 10⁶ cells/ml) of the primary human urothelial cells (HUC) were seeded in the apical compartment of the transwell insert. Cells were co‐cultured for 5 days and media was changed every day with HUC medium supplemented with fetal calf serum (FCS, 5% v/v) and Ca2+ (2 mM). The physical barrier was monitored every day (Pre-treatment) by transepithelial electrical resistance (TEER) using an epithelial voltohmmeter (EVOM). On day 5, 20 µl of TNF-α (10 ng/ml) were added to the apical chamber and incubated for 24 h with/without 20 µl of 4% MH pre-treatment. The TEER values will be measured after treatment (post-treatment) and supernatants were collected for cytokine expression assay (Human IL-6 and IL-8 DuoSet, R&D system) following the manufacturer's instructions. Samples were diluted at 1:10 (IL-6) and 1:5 (IL-8) in sample buffer and run in duplicate (n=6).
Interpretation of results
In the present study, we found that HUC supplemented with FCS (0.5% v/v) and Ca2+ (2 mM) showed significantly increased TEER value compared to un-supplemented similarly to previous studies (1), and TNF-α showed significantly decreased TEER value of epithelial cells (2, 3). TNF-α is a major cytokine secreted by activated mast cells. Mast cells-derived TNF-α has been shown to induce IL-6 and IL-8 release from urothelial cells and significantly increased in urine from PBS/IC patients. Our data suggest that urothelial cells are responsive to inflammatory stimuli (TNF-α) by releasing IL-6 and IL-8 and may participate in the pathology of inflammatory urinary tract disease including PBS/IC. As novel findings, this is the first study of the beneficial effects of Manuka honey on human urothelial cells and human fibroblast cells co-culture model system induced by TNF-α related to an in vitro model of PBS/IC and inflammation diseases. Further studies, which take these into animal models, will need to be undertaken.