Figure 1.
Inhibition of type-I collagen and fibronectin expression in dermal fibroblasts by kynurenines.
Type-I Collagen and fibronectin expression at the mRNA and protein level in cultured fibroblasts treated with increasing concentrations (6.25, 12.5, 25, 50, 100, and 150 μg/ml) of KynA, Kyn, L-Kyn or D-Kyn. A & B: Relative type-I collagen and fibronectin mRNA expression in treated fibroblasts, respectively. GAPDH was used as the reference gene. C: Evaluation of type-I collagen and fibronectin expression at the protein level using Western blotting. D & E: The Mean±SEM ratio of type-I collagen and fibronectin density to β-actin at the protein level, respectively. β-actin was used as protein loading control.
Figure 2.
Stimulatory effect of kynurenines on MMP1 expression.
A: Dermal fibroblasts were treated with increasing doses (6.25, 12.5, 25, 50, 100, and 150 μg/ml) of KynA, Kyn, L-Kyn or D-Kyn. Following 24 hours of treatment cells were collected, and MMP1 expression was determined by Q-PCR after RNA extraction and cDNA synthesis. B: Evaluation of MMP1 expression at the protein level by Western blotting after 48 hours of treatment. C: The Mean±SEM ratio of MMP1 to β-actin density at the protein level. β-actin and GAPDH were used as loading controls for western blotting and Q-PCR, respectively.
Figure 3.
Stimulatory effect of kynurenines on MMP3 secretion by fibroblasts.
A: Evaluation of MMP3 mRNA expression in fibroblasts treated with increasing doses (6.25, 12.5, 25, 50, 100, and 150 μg/ml) of KynA, Kyn, L-Kyn or D-Kyn following 24 hours of treatment. GAPDH was used as loading control for Q-PCR. B: Evaluation of MMP3 presence in the fibroblast conditioned medium using Western blotting after 48 hours of treatment. C: The Mean±SEM ratio of MMP3 density at the protein level.
Figure 4.
Stimulatory effect of Kynurenines on MMP activity and Kynurenines lasting effect on MMP1 expression.
A: The effect of kynurenines of MMP activity. To determine MMP activity in the fibroblasts conditioned medium, cells were treated with 100 μg/ml of KynA, Kyn, L-Kyn or D-Kyn for 48 hours and MMP activity was evaluated using SensoLyte Plus 520 generic MMP Assay Kit (** P-value<0.001, n = 4). B: Kynurenines lasting effect on the MMP1 expression. To determine the lasting effect of kynurenines on MMP1 expression, fibroblasts were treated with KynA or Kyn (100 μg/ml) for 48 hours. The medium was replaced and cells were harvested immediately, 12, 24, and 48 hours after treatment removal. The MMP1 expression in dermal fibroblasts was evaluated using western blotting. C: MMP1/β-actin expression ratio was calculated in treated fibroblasts. Data is mean±SEM of 4 independent experiments (* P-value<0.05 and ** P-value<0.01, n = 4).
Figure 5.
Effect of kynurenines on fibroblast and keratinocyte proliferation rate and viability.
A & B: To determine the effect of different kynurenines on dermal cell proliferation rate, fibroblast and keratinocytes were treated with KynA or Kyn (100 μg/ml). Cells were harvested and total cell number was counted after 36, 72 and 108 hours of incubation (* P-value<0.05 and ** P-value<0.01, n = 6). C: Determination of cellular viability by FACs analysis using live/dead, viability/cytotoxicity assay kit. Fibroblasts and keratinocytes were either cultured in DMEM+2% FBS or DMEM+2% FBS supplemented with increasing concentrations of KynA or Kyn (50 and 150 μg/ml). The viability of cells was determined by FACS analysis following 3 days of incubation.
Figure 6.
Effect of kynurenines on fibroblast and keratinocyte migration.
A & B: Images of human fibroblasts and keratinocytes taken immediately, 12 and 24 h after addition of KynA or Kyn (100 μg/ml) in an in vitro wound scratch assay, respectively. C: Reduction of fibroblast migration in the presence of KynA after 12 and 24 hours of treatment. D: Enhancement of keratinocyte migration in the presence of KynA and Kyn. Cell migration was evaluated by manually counting the total number of cells migrated from the edges of the wound into the denuded area (* P-value<0.05 and ** P-value<0.01, n = 4).
Figure 7.
Clinical appearance and histological evaluation of wounds in rabbit ear model.
A: The clinical appearance of wounds that either received nothing (Control), cream only (cream) or cream containing Kyn or KynA on day 35 post wounding. B: Tissue samples were subjected to H&E staining to determine the dermal and epidermal hypertrophy. Scar elevation index (C) and epidermal thickness index (D) was evaluated quantitatively. Uninjured rabbit ear skin was used as the normal sample (* P-value<0.05 and ** P-value<0.01, n = 4).
Figure 8.
Effect of Kyn and KynA topical application on collagen deposition, tissue cellularity and ECM expression.
A: Evaluation of collagen deposition in tissue samples using Masson's Trichrome staining at day 35 post-wounding. In this staining collagen fibers are stained blue, keratin and muscle fibers are stained red, and cell cytoplasm and nuclei are stained light pink and dark brown, respectively. B: Quantification and statistical analysis of tissue cellularity. Q-PCR analysis of relative MMP1 (C), type-I collagen (D) and fibronectin (E) mRNA expression in tissue samples (* P-value<0.05 and ** P-value<0.01, n = 4).