Hyaluronic acid and chondroitin sulfate, alone or in combination, efficiently counteract induced bladder cell damage and inflammation

Abstract

Interstitial cystitis and/or bladder pain syndrome (IC/BPS) are characterized by discomfort, abdominal pain, and pelvic pain, and they are often associated with chronic diseases. Pathological conditions related to IC/BPS can occur due to a defect in the integrity of the bladder lining. This defect has been ascribed to damage to the glycosaminoglycan (GAG) layer of the urinary epithelium. In addition, the incipient cascade of inflammation events might prompt extracellular matrix degradation. Several medical devices based on GAG instillation were proposed to re-establish epithelial integrity by GAGs binding to proteoglycans or interacting with structural urothelium. However, to date, only in vitro studies have investigated the GAG, hyaluronic acid (HA). In the present study, TNFα treatment was used to mimic IC/BPS-induced damage in bladder cells in an in vitro model. Highly purified fermentative HA and pharmaceutical grade bovine chondroitin sulfate (CSb), alone or in combination, were evaluated for the ability to counteract bladder cell damage. We evaluated NF-κB with western blots, and we analyzed interleukin 6 and 8 expression at the transcriptional and protein levels with quantitative RT-PCR, western blotting, and ELISA. We also evaluated the expression of an antibacterial peptide, human β-defensin-2. We confirmed our results in a 3D bladder epithelium model. Our results demonstrated that inflammatory status was reduced in the presence of HA, CSb, and the combination of both (HA/CSb 1.6%/2% w/v). This result suggested that these GAGs might be suitable for treating IC/BPS. All the assayed biomarkers showed that HA/CSb treatment modulated cells towards a more physiological status. Finally, we compared two commercial products suggested for the IC/BPS treatments and found that the product with more Ca++, showed enhanced anti-inflammatory activity and provided superior mucoadhesivity.

Fig 1. Schematic drawing of the signaling pathway related to the inflammation response to a TNFα insult, in the in vitro IC/BPS model.

Fig 2. Representative GAG effects on IL-6 and IL-8 gene expression in the 2D bladder model after a TNF-α challenge.

qRT-PCR results show the effects of 4-h treatments with CSb at 0.6% and 2% (w/v) concentrations, HA at 0.6% and 1% (w/v) concentrations, and a fixed HA/CSb formulation on bladder cells challenged with TNF-α. Data are the means ± SD of triplicate assays. *p<0.01 compared to TNF-α alone; §p<0.01 CSb compared to HA.

Fig 3. GAG effects on NF-κB expression in a 2D bladder model challenged with TNF-α.

Western blot analyses show NF-κB expression in the presence of (A) single HA and CSb treatments and (B) the HA/CSb formulation. Data are the means ± SD. *p<0.01 compared to TNF-α alone; §p<0.01 for HA (1% w/v) compared to CSb (A).

Fig 4. GAG effects on IL-6 and IL-8 protein expression in the 2D bladder model after a TNF-α challenge.

ELISA results show the effects of CSb at 0.6% and 2% (w/v), HA at 0.6% and 1% (w/v), and a fixed HA/CSb formulation on bladder cells challenged with TNF-α. Data are the means ± SD. *p <0.01 compared to TNF-α treatment alone; §p<0.01 CSb 0.6% compared to 0.6% HA.

Fig 5. hBD-2 protein expression in epithelial bladder cells stimulated with GAGs.

ELISA assays show hBD-2 expression after 72h of GAG treatment. Data are the means ± SD. *p<0.01 compared to control.

Fig 6. Effects of the HA/CSb formulation on a 3D bladder model challenged with TNF-α.

(A) qRT-PCR results show HA/CSb effects on IL-6 and IL-8 gene expression. (B) Western blots show HA/CSb effects on NF-κB protein expression. (C) ELISA results show HA/CSb effects on IL-6 and IL-8 cytokine production after 48 h. Data are the means ± SD. *p<0.01 compared to TNF-α alone.

Fig 7. Effects of the HA/CSb formulation on ZO-1 expression in a human bladder 3D cell model challenged with TNF-α.

Immunofluorescence image shows ZO-1 (red) expression after 48 h of HA/CSb treatment. Nuclei are shown in blue.

Fig 8. Comparison of two commercial medical devices for HA/CSb instillation.

(A) Flow curves in the range of 1–1000 s^-1 were performed at 37°C; η0 values are shown for IALURIL and INSTILLAMED. (B) The mucoadhesion indexes (Δ%) of IALURIL and INSTILLAMED is plotted as a function of the shear rate (a) and Δ% measured at a shear rate of 9.1 s^-1 according to previously reported method [10].

Fig 9. IALURIL and INSTILLAMED effects on cytokine expression in an inflammation model challenged with TNF-α.

ELISA results show reductions in (A) IL-6 and (B) IL-8 protein expression with IALURIL and INSTILLAMED treatment compared to TNF-α alone.