Department of Ophthalmology, RWTH Aachen University, 52074 Aachen, Germany.
Cornea Bank Aachen, RWTH Aachen University, 52074 Aachen, Germany.
Int J Mol Sci. 2023 Jan 11;24(2):1461. doi: 10.3390/ijms24021461.
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 μM, curcumin of 0−200 μM, pirfenidone of 0−2.2 nM and the profibrotic cytokine TGF-β1 (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 µM of caffeine, 20 µM of curcumin and 1.1 nM of pirfenidone. The addition of TGF-β1 to the control medium effectively transformed aCSK into myofibroblasts (MYO-SF), indicated by a 10-fold increase in α-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 µM of caffeine, 20/50 µM of curcumin and 1.1 nM of pirfenidone each significantly reduced SMA expression under TGF-β1 stimulation (p ≤ 0.024). LUM and ALDH3A1 expression remained low under TGF-β1 stimulation, independently of AFM supplementation. Immunocytochemistry showed that 100 µM of caffeine, 20 µM of curcumin and 1.1 nM of pirfenidone reduce the conversion rate of aCSK to SMA+ MYO-SF. In conclusion, in aCSK, 100 µM of caffeine, 20 µM of curcumin and 1.1 nM of pirfenidone significantly reduced SMA expression and MYO-SF conversion under TGF-β1 stimulation, with no influence on cell counts. However, the AFMs were unable to protect aCSK from characteristic marker loss.
我们评估了小分子（AFM）咖啡因、姜黄素和吡非尼酮，以寻找无毒浓度，减少激活的人角膜基质成纤维细胞（aCSK）向致瘢痕的肌成纤维细胞（MYO-SF）的转化。CSK 从 16 个不适合移植的人角膜中分离出来，并在对照培养基（0.5% FBS）中培养三代。然后，将 aCSK 暴露于 0-500 μM 的咖啡因、0-200 μM 的姜黄素、0-2.2 nM 的吡非尼酮和致纤维化细胞因子 TGF-β1（10 ng/mL）48 小时。通过细胞活力染色（FDA/PI）、实时聚合酶链反应（RT-PCR）和免疫细胞化学评估活力和基因表达的变化。我们发现所有 AFM 在高浓度下均降低细胞计数。无毒性作用的最高浓度分别为 100 μM 咖啡因、20 μM 姜黄素和 1.1 nM 吡非尼酮。在对照培养基中添加 TGF-β1 可有效将 aCSK 转化为肌成纤维细胞（MYO-SF），α-平滑肌肌动蛋白（SMA）表达增加 10 倍，亮氨酸丰富糖蛋白（LUM）表达降低 39%，ALDH3A1 表达降低 98%（p<0.001）。在 TGF-β1 刺激下，100 μM 咖啡因、20/50 μM 姜黄素和 1.1 nM 吡非尼酮的浓度均显著降低 SMA 表达（p≤0.024）。在 TGF-β1 刺激下，LUM 和 ALDH3A1 表达仍然较低，与 AFM 补充无关。免疫细胞化学显示，100 μM 咖啡因、20 μM 姜黄素和 1.1 nM 吡非尼酮可降低 aCSK 向 SMA+MYO-SF 的转化率。总之，在 aCSK 中，100 μM 咖啡因、20 μM 姜黄素和 1.1 nM 吡非尼酮可显著降低 TGF-β1 刺激下的 SMA 表达和 MYO-SF 转化，而对细胞计数无影响。然而，AFM 无法保护 aCSK 免受特征标志物丢失。
