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. 2014 Mar;23(1):93-103.
doi: 10.5607/en.2014.23.1.93. Epub 2014 Mar 27.

Glutathione protects brain endothelial cells from hydrogen peroxide-induced oxidative stress by increasing nrf2 expression

Juhyun Song  1 , So Mang Kang  2 , Won Taek Lee  1 , Kyung Ah Park  1 , Kyoung Min Lee  3 , Jong Eun Lee  2
Affiliations

Glutathione protects brain endothelial cells from hydrogen peroxide-induced oxidative stress by increasing nrf2 expression

Juhyun Song et al. Exp Neurobiol. 2014 Mar.

Abstract

Glutathione (GSH) protects cells against oxidative stress by playing an antioxidant role. Protecting brain endothelial cells under oxidative stress is key to treating cerebrovascular diseases and neurodegenerative diseases including Alzheimer's disease and Huntington's disease. In present study, we investigated the protective effect of GSH on brain endothelial cells against hydrogen peroxide (H2O2). We showed that GSH attenuates H2O2-induced production of nitric oxide (NO), reactive oxygen species (ROS), and 8-Oxo-2'-deoxyguanosine (8-OHdG), an oxidized form of deoxiguanosine. GSH also prevents H2O2-induced reduction of tight junction proteins. Finally, GSH increases the level of nuclear factor erythroid 2-related factor 2 (Nrf2) and activates Nrf2-mediated signaling pathways. Thus, GSH is a promising target to protect brain endothelial cells in conditions of brain injury and disease.

Keywords: Reactive oxygen species (ROS); apoptosis; glutathione (GSH); hydrogen peroxide (H2O2); murine brain endothelial cells (bEnd.3 cells); nuclear factor erythroid 2-related factor 2 (Nrf2).

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Figures

Fig. 1
Fig. 1
The effect of GSH H2O2-induced cell death. bEDN.3 cells were treated with H2O2 (500 µM) and/or GSH (1 mM or 10 mM) for 24 hr. (A) Cytotoxicity was determined by the release of LDH into the culture media. GSH reduced H2O2's cytotoxicity. (B) H2O2-induced nitrite production was measured by using Griess reagent. GSH reduced H2O2-induced nitrite production. Data are expressed as mean±S.E.M. (#p < 0.1, *p < 0.05, **p < 0.001). (C) 8-OHdG levels were measured by immunocytochemistry. 8-OHdG-positive cells (green color) were increased in the H2O2 (500 µM) treatment group compared to the normal control (NC) group. GSH decreased 8-OHdG levels in bEND.3 cells under H2O2-induced oxidative stress. Scale bar: 600 µm, 8-Oxo-2'-deoxyguanosine (8-OHdG): green, 4', 6-diamidino-2-phenylindole (DAPI): blue.
Fig. 2
Fig. 2
The effect of GSH on H2O2-induced ROS generation. H2O2 (500 µM) and/or GSH (10 mM) were treated in bEND.3 cells for 24 hr. ROS levels were measured using DCF-DA. ROS level in bEND.3 cells was increased in the H2O2 (500 µM) treatment group compared with normal control (NC) group. Also, ROS level was not change in the GSH (10 mM) treatment group compared with NC group. GSH decreased the H2O2-induced increase of DCF-DA-positive cells (green). Scale bar: 600 µm, 2', 7'-dichlorodihydrofluorescein diacetate (DCF-DA): green, 4', 6-diamidino-2-phenylindole (DAPI): blue.
Fig. 3
Fig. 3
The effect of GSH H2O2-induced decrease in tight junction proteins. H2O2 (500 µM) and/or GSH (10 mM) were treated in bEND.3 cells for 24 hr. The level of Claudin 5, a tight junction protein, was evaluated by immunocytochemistry. GSH attenuated H2O2-induced decrease in the number of Claudin 5-positive cells (green). Scale bar: 600 µm, Claudin 5: green, 4', 6-diamidino-2-phenylindole (DAPI): blue.
Fig. 4
Fig. 4
The effect of GSH on Nrf2 signaling. H2O2 (500 µM) and/or GSH (10 mM) were treated in bEND.3 cells for 24 hr. The protein level of Nrf2, ERK, phospho-ERK was evaluated by using Western blot analysis. (A) The phospho-ERK protein levels decreased by H2O2 treatment. GSH treatment increased the level of phospho-ERK. Bar graph showed the quantification of phosphor-ERK/ERK protein in all groups. (B) Nrf2 protein levels decreased by H2O2 treatment. GSH increased the basal level of Nrf2 proteins. GSH co-treatment increased Nrf2 protein levels compared to H2O2 treatment. β-actin was used as an internal control (mean±S.E.M., n=3) (#p<0.1, *p<0.05, **p<0.001).
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