The Neuroprotective Effect of Maltol against Oxidative Stress on Rat Retinal Neuronal Cells

PURPOSE: Maltol (3-hydroxy-2-methyl-4-pyrone), formed by the thermal degradation of starch, is found in coffee, caramelized foods, and Korean ginseng root. This study investigated whether maltol could rescue neuroretinal cells from oxidative injury in vitro. METHODS: R28 cells, which are rat embryonic precursor neuroretinal cells, were exposed to hydrogen peroxide (H2O2, 0.0 to 1.5 mM) as an oxidative stress with or without maltol (0.0 to 1.0 mM). Cell viability was monitored with the lactate dehydrogenase assay and apoptosis was examined by the terminal deoxynucleotide transferase-mediated terminal uridine deoxynucleotidyl transferase nick end-labeling (TUNEL) method. To investigate the neuroprotective mechanism of maltol, the expression and phosphorylation of nuclear factor-kappa B (NF-kappaB), extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 were evaluated by Western immunoblot analysis. RESULTS: R28 cells exposed to H2O2 were found to have decreased viability in a dose- and time-dependent manner. However, H2O2-induced cytotoxicity was decreased with the addition of maltol. When R28 cells were exposed to 1.0 mM H2O2 for 24 hours, the cytotoxicity was 60.69 ± 5.71%. However, the cytotoxicity was reduced in the presence of 1.0 mM maltol. This H2O2-induced cytotoxicity caused apoptosis of R28 cells, characterized by DNA fragmentation. Apoptosis of oxidatively-stressed R28 cells with 1.0 mM H2O2 was decreased with 1.0 mM maltol, as determined by the TUNEL method. Western blot analysis showed that treatment with maltol reduced phosphorylation of NF-kappaB, ERK, and JNK, but not p38. The neuroprotective effects of maltol seemed to be related to attenuated expression of NF-kappaB, ERK, and JNK. CONCLUSIONS: Maltol not only increased cell viability but also attenuated DNA fragmentation. The results obtained here show that maltol has neuroprotective effects against hypoxia-induced neuroretinal cell damage in R28 cells, and its effects may act through the NF-kappaB and mitogen-activated protein kinase signaling pathways.

Cytotoxicities and Anti-Fibrotic Effects of Pirfenidone and Mitomycin C on Human Fibroblasts

PURPOSE: The cytotoxicities and anti-fibrotic effects of mitomycin C and pirfenidone on human dermal fibroblast were evaluated. METHODS: Initially, 24-hour cell cultures were exposed to transforming growth factor (TGF)-beta1, different concentrations of mitomycin C, and pirfenidone solutions in order to evaluate cytotoxicity. Expressions of fibronectin, collagen type 1, alpha smooth muscle, and beta-actin were evaluated by real-time reverse transcription-polymerase chain reaction (RT-PCR) and western blot in mitomycin C solutions at concentrations of 4 microg/mL and 20 microg/mL, and in pirfenidone solutions at 250 microg/mL and 500 microg/mL. RESULTS: In comparison to cell cultures exposed to TGF-beta1 solutions, cytotoxicities were increased in solutions of mitomycin C at 4 microg/mL, 20 microg/mL, 40 microg/mL and pirfenidone at 500 microg/mL, 750 microg/mL, 1,000 microg/mL (p < 0.05, Mann Whitney U-test). The results of real-time RT-PCR show that expressions of fibronectin, collagen type 1, and alpha smooth muscle were significantly more decreased in all concentrations of mitomycin C and pirfenidone compared to those in TGF-beta1 solution. In western blot analysis, expressions of fibronectin and alpha smooth muscle were decreased in all concentrations of mitomycin C and pirfenidone compared to TGF-beta1 solution. CONCLUSIONS: Both drugs have cytotoxicities and anti-fibrotic effects, but pirfenidone was found to have less cytotoxicity and mitomycin C was found to have more anti-fibrotic effects when compared to each other.

The Role of Focal Adhesion Kinase in the TGF-beta-Induced Myofibroblast Transdifferentiation of Human Tenon's Fibroblasts

PURPOSE: To investigate the role of focal adhesion kinase (FAK) in transforming growth factor (TGF)-beta-induced myofibroblast transdifferentiation of human Tenon's fibroblasts. METHODS: Primary cultured human Tenon's fibroblasts were exposed to TGF-beta1 for up to 48 hours. The mRNA levels of FAK, alpha smooth muscle actin (alphaSMA), and beta-actin were determined by quantitative real time reverse transcription polymerase chain reaction. The protein levels of collagen type I, FAK, phospho-FAK, alphaSMA, and beta-actin were determined by Western immunoblots. After the small interfering RNA targeting FAK (siRNA(FAK)) molecules were delivered into the cells, the expressions of alphaSMA proteins were determined by Western immunoblots. RESULTS: In human Tenon's fibroblasts, TGF-beta1 significantly increased the mRNA and protein expressions of alphaSMA. However, when the action of FAK was inhibited using siRNAFAK, the TGF-beta1-induced expression of alphaSMA was attenuated. CONCLUSIONS: Our data suggest that FAK may be associated with the TGF-beta1-induced transdifferentiation of human Tenon's fibroblasts to myofibroblasts, which is the essential step of subconjunctival fibrosis.