Journal List > J Korean Ophthalmol Soc > v.49(12) > 1008166

Ryu and Kim: Role of Ascorbic Acid Against the Oxidative Stress in Trabecular Meshwork Cells

Abstract

Purpose

To investigate the role of L-ascorbic acid (LAA) on the survival and its association with nitric oxide (NO) production against hydrogen peroxide-induced oxidative stress in trabecular meshwork (TM) cells.

Methods

Primarily cultured human TM cells were exposed to hydrogen peroxide; 10, 100 µM for 2 days, or 1 mM single exposure with or without co-exposure of LAA. Cellular survival and nitrite production were assessed with MTT and Griess assays, respectively. Flow cytometry using annexin/PI double staining was performed to evaluate apoptosis.

Results

Hydrogen peroxide decreased cellular survival significantly in a dose-dependent manner accompanied with decreased NO production. However cellular survival and NO production were increased significantly with co-exposure of LAA. Cellular survival and NO were highly correlated. Flow cytometric analysis revealed that LAA inhibited hydrogen peroxide-induced apoptosis.

Conclusions

LAA demonstrated a cytoprotective effect against the hydrogen peroxide-induced oxidative stress accompanied with increased NO production. The cytoprotective effect of LAA may be mediated by preserving NO in TM cells.

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Figure 1
. Effect of L-ascorbic acid on the survival of cultured trabecular meshwork cells. L-ascorbic acid increased cellular survival in a dose-dependent manner. (* p<0.05)
jkos-49-1989f1.tif
Figure 2
. Effect of hydrogen peroxide on the survival of cultured TM cells exposed to 10, 100 µM for 2 days, or 1 mM single exposure. Hydrogen peroxide decreased cellular survival significantly in both conditions. (* p<0.05)
jkos-49-1989f2.tif
Figure 3
. L-ascorbic acid increased survival of cultured trabecular meshwork cells exposed to 10, or 100 µM hydrogen peroxide. (* p<0.05)
jkos-49-1989f3.tif
Figure 4
. Comparison of the effects of 0.5 mM L-NAME, 100 µM L-ascorbic acid (LAA), or 100 µM hydrogen peroxide on the survival of cultured trabecular meshwork cells. (* p<0.05)
jkos-49-1989f4.tif
Figure 5
. Effect of L-ascorbic acid (LAA) on the production of nitric oxide in cultured trabecular meshwork cells. LAA increased nitric oxide production in a dose-dependent manner. (* p<0.05)
jkos-49-1989f5.tif
Figure 6
. Effect of hydrogen peroxide on the production of nitric oxide in cultured trabecular meshwork cells. Hydrogen peroxide decreased nitric oxide production significantly. (* p<0.05)
jkos-49-1989f6.tif
Figure 7
. Effect of L-NAME, 100 µM L-ascorbic acid (LAA), or 100 µM hydrogen peroxide on the production of nitric oxide. (* p<0.05)
jkos-49-1989f7.tif
Figure 8
. Effect of L-ascorbic acid (LAA) on the production of nitric oxide under oxidative stress induced by hydrogen peroxide. LAA abolished the inhibitory effect of hydrogrn peroxide on the NO production.
jkos-49-1989f8.tif
Figure 9
. High correlation between nitrite production and cellular survival (r=0.924).
jkos-49-1989f9.tif
Figure 10
. Annexin-PI flow cytometric analysis of the effect of L-ascorbic acid on the hydrogen peroxide-induced apoptosis in cultured trabecular meshwork cells. L-ascorbic acid (100 µM) decreased apoptosis significantly. (* p<0.05)
jkos-49-1989f10.tif
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