Antifibrotic Effects of Caffeine, Curcumin and Pirfenidone in Primary Human Keratocytes

We evaluated the small molecules (AFM) caffeine, curcumin and pirfenidone to find non-toxic concentrations reducing the transformation of activated human corneal stromal keratocytes (aCSK) to scar-inducing myofibroblasts (MYO-SF). CSK were isolated from 16 human corneas unsuitable for transplantation and expanded for three passages in control medium (0.5% FBS). Then, aCSK were exposed to concentrations of caffeine of 0-500 mu M, curcumin of 0-200 mu M, pirfenidone of 0-2.2 nM and the profibrotic cytokine TGF-beta 1 (10 ng/mL) for 48 h. Alterations in viability and gene expression were evaluated by cell viability staining (FDA/PI), real-time polymerase chain reaction (RT-PCR) and immunocytochemistry. We found that all AFMs reduced cell counts at high concentrations. The highest concentrations with no toxic effect were 100 mu M of caffeine, 20 mu M of curcumin and 1.1 nM of pirfenidone. The addition of TGF-beta 1 to the control medium effectively transformed aCSK into myofibroblasts (MYO-SF), indicated by a 10-fold increase in alpha-smooth muscle actin (SMA) expression, a 39% decrease in lumican (LUM) expression and a 98% decrease in ALDH3A1 expression (p < 0.001). The concentrations of 100 mu M of caffeine, 20/50 mu M of curcumin and 1.1 nM of pirfenidone each significantly reduced SMA expression under TGF-beta 1 stimulation (p <= 0.024). LUM and ALDH3A1 expression remained low under TGF-beta 1 stimulation, independently of AFM supplementation. Immunocytochemistry showed that 100 mu M of caffeine, 20 mu M of curcumin and 1.1 nM of pirfenidone reduce the conversion rate of aCSK to SMA(+) MYO-SF. In conclusion, in aCSK, 100 mu M of caffeine, 20 mu M of curcumin and 1.1 nM of pirfenidone significantly reduced SMA expression and MYO-SF conversion under TGF-beta 1 stimulation, with no influence on cell counts. However, the AFMs were unable to protect aCSK from characteristic marker loss.