Fig 1.
Schematic drawing of the signaling pathway related to the inflammation response to a TNFα insult, in the in vitro IC/BPS model.
Table 1.
Molecular weights (MW), dispersity (Mw/Mn), intrinsic viscosity (IV), and recovery, based on the refractive index (RI), of HA, CSb, and HA/CSb formulations after thermal treatment.
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.