An exploration of the antioxidant effects of garlic saponins in mouse-derived C2C12 myoblasts

Kang,Ji  Sook; Kim,Sung  Ok; Kim,Gi-Young; Hwang,Hye  Jin; Kim,Byung  Woo; Chang,Young-Chae; Kim,Wun-Jae; Kim,Cheol  Min; Yoo,Young  Hyun; Choi,Yung  Hyun

doi:10.3892/ijmm.2015.2398

An exploration of the antioxidant effects of garlic saponins in mouse-derived C2C12 myoblasts

Authors:
- Ji Sook Kang
- Sung Ok Kim
- Gi-Young Kim
- Hye Jin Hwang
- Byung Woo Kim
- Young-Chae Chang
- Wun-Jae Kim
- Cheol Min Kim
- Young Hyun Yoo
- Yung Hyun Choi
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Affiliations: Blue-Bio Industry RIC and Anti-Aging Research Center, Dongeui University, Busan 614-714, Republic of Korea, Department of Food Science and Biotechnology, College of Engineering, Kyungsung University, Busan 608-736, Republic of Korea, Laboratory of Immunobiology, Department of Marine Life Sciences, Jeju National University, Jeju 690-756, Republic of Korea, Research Institute of Biomedical Engineering and Department of Medicine, Catholic University of Daegu School of Medicine, Daegu 705-718, Republic of Korea, Department of Urology, Chungbuk National University College of Medicine, Cheongju 361-763, Republic of Korea, Department of Biochemistry, Busan National University College of Medicine, Yangsan 626-870, Republic of Korea, Department of Anatomy and Cell Biology, Dong-A University College of Medicine and Mitochondria Hub Regulation Center, Busan 602-714, Republic of Korea
Published online on: October 30, 2015 https://doi.org/10.3892/ijmm.2015.2398
Pages: 149-156

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Abstract

In this study, we aimed to confirm the protective effects of garlic saponins against oxidative stress-induced cellular damage and to further elucidate the underlying mechanisms in mouse-derived C2C12 myoblasts. Relative cell viability was determined by 3-(4.5-dimethylthiazol-2-yl)-2.5 diphenyltetrazolium bromide assay. Comet assay was used to measure DNA damage and oxidative stress was determined using 2',7'-dichlorofluorescein diacetate to measure intracellular reactive oxygen species (ROS) generation. Western blot analysis and small interfering RNA (siRNA)-based knockdown were used in order to investigate the possible molecular mechanisms. Our results revealed that garlic saponins prevented hydrogen peroxide (H2O2)-induced growth inhibition and exhibited scavenging activity against intracellular ROS. We also observed that garlic saponins prevented H2O2-induced comet tail formation and decreased the phosphorylation levels of γH2AX expression, suggesting that they can prevent H2O2-induced DNA damage. In addition, garlic saponins increased the levels of heme oxygenase-1 (HO-1), a potent antioxidant enzyme associated with the induction and phosphorylation of nuclear factor erythroid 2-related factor 2 (Nrf2) and the translocation of Nrf2 from the cytosol into the nucleus. However, the protective effects of garlic saponins on H2O2-induced ROS generation and growth inhibition were significantly reduced by zinc protoporphyrin Ⅸ, an HO-1 competitive inhibitor. In addition, the potential of garlic saponins to mediate HO-1 induction and protect against H2O2‑mediated growth inhibition was adversely affected by transient transfection with Nrf2-specific siRNA. Garlic saponins activated extracellular signal‑regulated kinase (ERK) signaling, whereas a specific ERK inhibitor was able to inhibit HO-1 upregulation, as well as Nrf2 induction and phosphorylation. Taken together, the findings of our study suggest that garlic saponins activate the Nrf2/HO-1 pathway by enabling ERK to contribute to the induction of phase Ⅱ antioxidant and detoxifying enzymes, including HO-1 in C2C12 cells.

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