Inhibition of Rho Kinase Induces Antioxidative Molecules and Suppresses Reactive Oxidative Species in Trabecular Meshwork Cells

Abstract

Purpose: To investigate the effect of rho kinase inhibitors on oxidative stress in trabecular meshwork (TM) cells.

Methods: TM cells were isolated from the eyes of cynomolgus monkeys. Y-27632 and menadione were used to inhibit rho kinase and induce production of reactive oxygen species (ROS), respectively. The cynomolgus monkey array and 12,613 probes were used in DNA microarray analysis, and the affected genes were categorized using gene ontology analysis. The mRNA levels of the target genes were confirmed by real-time RT-PCR. Intracellular oxidative stress was detected using a fluorescent reagent sensitive to ROS. Cell viability was assessed by the WST-8 assay.

Results: Gene ontology analysis revealed upregulation of genes involved in antioxidant activity, and upregulation of catalase was confirmed by real-time RT-PCR after 30 min treatment with Y-27632. Production of ROS was increased by menadione, and the effect was partly suppressed by pretreatment with Y-27632. At a lower dose of menadione, Y-27632 stimulated TM cells and significantly increased their viability following menadione treatment compared to control cells.

Conclusion: Using microarray analysis, Y-27632 was shown to upregulate antioxidative genes including catalase and partially reduce ROS production and cell death by oxidative stress caused by menadione.

Figure 1

(a) Quantitative PCR analysis of catalase mRNA. The TM cells were treated with 25 μM Y-27632 for 30 min. The relative expression level of catalase of samples treated with Y-27632 was compared to that of the control sample using the comparative C_T method (ΔΔC_T method). The 18S ribosomal RNA was used as an endogenous control. Data are shown as mean ± SE from six independent experiments. ^∗P < 0.05 compared with control by Wilcoxon rank sum test. (b) The effects of Y-27632 on the intracellular production of reactive oxygen species (ROS). The TM cells were treated with or without 25 μM Y-27632 for 30 min, followed by 100 μM menadione stimulated for 1 h. ROS were detected by CellROX reagent, and the fluorescence of the TM cells were measured by cell sorter SH800. Data are shown as mean ± SE from five independent experiments. ^∗∗P < 0.01 and ^∗P < 0.05 compared with control by the Wilcoxon rank sum test (a) and Tukey Kramer HSD test (b).

Figure 2

(a) The effect of Y-27632 on oxidative stress-induced cell death. The TM cells were treated with or without 25 μM Y-27632 for 30 min, followed by menadione stimulation of the cells for 24 h. Cell viabilities were shown as relative value compared with the control. Data are shown as the mean ± SE from six independent experiments. ^∗P < 0.05 compared with control by Wilcoxon rank sum test. (b) The effect of Y-27632 on extracellular antioxidative activity. The xanthine-oxidase-induced superoxide production was assessed using a superoxide-sensitive luminescent reagent. Data are shown as the mean ± SE from six independent experiments. ^∗P < 0.05 compared with the control by Dunnett's test. Nac: n-acetyl cysteine.