Regulation of Autophagy by the Cdk1/p53/p21 Feedback Loop in an Interstitial Cystitis/Bladder Pain Syndrome Cell Model: Implications for Inflammatory Response.

Abstract

Introduction and hypothesis: The Cdk1/p53/p21 feedback loop could play an important role in maintaining a consistently high urinary tract epithelial permeability. This study is aimed at elucidating whether this feedback loop is associated with the inadequate autophagic response and the heightened inflammatory response in interstitial cystitis/bladder pain syndrome (IC/BPS).

Methods: To construct the IC/BPS cell model, HTB4 cells were first treated with 1% H₂O₂ for 1 h, followed by a 24-h exposure to TNFα (10 ng/ml). The protein expression levels of LC3-I/II and Beclin 1 were assessed using Western blot analysis. Autophagosome formation was visualized using monodansylcadaverine (MDC) staining. The secretion levels of inflammatory cytokines, including IL-6, IL-8, and TNFα, were quantified using enzyme-linked immunosorbent assay. Additionally, malondialdehyde (MDA) levels and the activities of antioxidant enzymes (GSH and SOD2) were measured.

Results: Compared with normal urothelial HTB4 cells, the number of autophagosomes and the levels of LC3-I/II and Beclin 1 are significantly increased in IC/BPS cells. These cells also exhibit markedly higher levels of inflammatory markers (TNFα, IL-6, IL-8) and MDA, alongside notably reduced levels of the antioxidants GSH and SOD2. Compared with IC/BPS cells or IC/BPS cells co-transfected with p21 siRNA and Cdk1 control, the IC/BPS cells co-transfected with p21 siRNA and Cdk1 inhibitor exhibit a significant increase in terms of autophagosome numbers and the LC3-I/II level. Additionally, the levels of inflammatory factors are significantly decreased in the IC/BPS cells co-transfected with p21 siRNA and Cdk1 inhibitor.

Conclusion: In IC/BPS, the Cdk1/p53/p21 feedback loop could regulate the level of autophagy and inflammatory response. This discovery identifies promising new therapeutic targets for IC/BPS, which may pave the way for innovative clinical approaches to this complex disorder.