Introduction
Interstitial cystitis/bladder pain syndrome (IC/BPS) is a chronic, noninfectious bladder disorder of unknown aetiology, characterised by urothelial barrier dysfunction, oxidative stress, and inflammation [1-2]. Current therapies are often ineffective, highlighting the need for novel models and therapeutic approaches. We aimed to establish a human in vitro IC/BPS model to explore molecular mechanisms driving disease and evaluate the therapeutic potential of the human amniotic membrane (hAM), known for its regenerative, anti-inflammatory and immunomodulatory properties [3–6].

Methods

Normal human urothelial cells (SV-HUC1) were exposed to glucose oxidase (GO; 20 mU/ml) for 3 hours to induce peak oxidative stress, or for 24 hours to model prolonged oxidative stress and inflammation [7]. To assess the therapeutic effect of hAM, cells were incubated with hAM during an additional 24-hour exposure to GO. mRNA expression was evaluated by qPCR, while protein levels were assessed using ELISA, Western blotting, and immunofluorescence. Statistical significance was determined using ANOVA. The preparation of hAM followed the recommendations of COST Action CA17111 [8].

Results
GO treatment induced a transient NRF2-mediated antioxidant response, with peak expression of NQO1 and SOD2 at 3 h, which declined by 24 h. Prolonged exposure led to activation of the JAK1/STAT1 pathway and enrichment of type I/II interferon signalling, accompanied by increased expression of selected interferon-stimulated genes and pro-inflammatory cytokines such as IL6 and CXCL8. hAM treatment significantly reduced the expression and secretion of inflammatory mediators, decreased phosphorylated JAK1 levels, restored SOD2 expression, and upregulated the epithelial differentiation marker KRT13 and the tight junctional proteins, suggesting improved urothelial barrier function.

Conclusion
Our in vitro IC/BPS model recapitulates key pathological hallmarks of the disease, including transient NRF2-driven antioxidant activation and sustained JAK1/STAT1-mediated inflammatory signalling. Treatment with hAM attenuates oxidative stress and pro-inflammatory responses while promoting epithelial differentiation and junctional integrity, underscoring its potential as a multi-target therapeutic strategy for IC/BPS.

References

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8. Janev A, et al. Front Bioeng Biotechnol. 2023;11:1258753.