Protective Effect of Glutathione against Oxidative Stress-induced Cytotoxicity in RAW 264.7 Macrophages through Activating the Nuclear Factor Erythroid 2-Related Factor-2/Heme Oxygenase-1 Pathway.

Da Kwon,Su-Hyun Hong,Hee-Jae Cha,Cheol Park,Yung Choi,Suhkmann Kim,Hye-Jin Hwang,Gi-Young Kim,Hyesook Lee,Shin-Hyung Park,Heui-Soo Kim

doi:10.3390/antiox8040082

Abstract

Reactive oxygen species (ROS), products of oxidative stress, contribute to the initiation and progression of the pathogenesis of various diseases. Glutathione is a major antioxidant that can help prevent the process through the removal of ROS. The aim of this study was to evaluate the protective effect of glutathione on ROS-mediated DNA damage and apoptosis caused by hydrogen peroxide, H2O2, in RAW 264.7 macrophages and to investigate the role of the nuclear factor erythroid 2-related factor-2 (Nrf2)/heme oxygenase-1 (HO-1) signaling pathway. The results showed that the decrease in the survival rate of RAW 264.7 cells treated with H2O2 was due to the induction of DNA damage and apoptosis accompanied by the increased production of ROS. However, H2O2-induced cytotoxicity and ROS generation were significantly reversed by glutathione. In addition, the H2O2-induced loss of mitochondrial membrane potential was related to a decrease in adenosine triphosphate (ATP) levels, and these changes were also significantly attenuated in the presence of glutathione. These protective actions were accompanied by a increase in the expression rate of B-cell lymphoma-2 (Bcl-2)/Bcl-2-associated X protein (Bax) and poly(ADP-ribose) polymerase cleavage by the inactivation of caspase-3. Moreover, glutathione-mediated cytoprotective properties were associated with an increased activation of Nrf2 and expression of HO-1; however, the inhibition of the HO-1 function using an HO-1 specific inhibitor, zinc protoporphyrin IX, significantly weakened the cytoprotective effects of glutathione. Collectively, the results demonstrate that the exogenous administration of glutathione is able to protect RAW 264.7 cells against oxidative stress-induced mitochondria-mediated apoptosis along with the activity of the Nrf2/HO-1 signaling pathway.

Highlights

Adequate levels of reactive oxygen species (ROS) act as signaling molecules that are essential for cell growth and proliferation, the increased production of ROS can cause oxidative damage to cells [1,2]
In the present study, we investigated whether glutathione ameliorates oxidative stress-induced mitochondria-mediated apoptosis and whether the nuclear factor erythroid 2-related factor-2 (Nrf2)/heme oxygenase-1 (HO-1) signaling pathway is involved in this process in RAW 264.7 macrophages
264.7 cells treated with concentrations of 200 μM or more showed a significant decrease in cell with concentrations of 200 μm or more showed a significant decrease in cell viability, but there was no viability, but there was no significant change to the glutathione treatment up to 1 mg/mL compared significant change to the glutathione treatment up to 1 mg/mL compared with the control group

Summary

Introduction

Adequate levels of reactive oxygen species (ROS) act as signaling molecules that are essential for cell growth and proliferation, the increased production of ROS can cause oxidative damage to cells [1,2]. The accumulation of ROS beyond the antioxidant function of cells could reduce the mitochondrial membrane potential (MMP), an index of the electron transport chain performance, resulting in a compromised adenosine triphosphate (ATP) production [4,7]. Apoptogenic factors such as cytochrome c are released into the cytoplasm from the mitochondrial intermembrane space due to the loss of MMP, and the caspase cascade is activated, which could eventually trigger apoptosis. A supplementation of antioxidants has been proposed as a strategy to prevent free radical accumulation through the activity of the signal pathway corresponding to the ROS production

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