Morin exerts cytoprotective effects against oxidative stress in C2C12 myoblasts via the upregulation of Nrf2-dependent HO-1 expression and the activation of the ERK pathway

Lee,Moon  Hee; Han,Min  Ho; Lee,Dae-Sung; Park,Cheol; Hong,Su-Hyun; Kim,Gi-Young; Hong,Sang  Hoon; Song,Kyoung  Seob; Choi,Il-Whan; Cha,Hee-Jae; Choi,Yung  Hyun

doi:10.3892/ijmm.2016.2837

Morin exerts cytoprotective effects against oxidative stress in C2C12 myoblasts via the upregulation of Nrf2-dependent HO-1 expression and the activation of the ERK pathway

Authors:
- Moon Hee Lee
- Min Ho Han
- Dae-Sung Lee
- Cheol Park
- Su-Hyun Hong
- Gi-Young Kim
- Sang Hoon Hong
- Kyoung Seob Song
- Il-Whan Choi
- Hee-Jae Cha
- Yung Hyun Choi
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Affiliations: Department of Biochemistry, Dongeui University College of Korean Medicine, Busan 614-052, Republic of Korea, Marine Biodiversity Institute of Korea, Seocheon 325-902, Republic of Korea, Department of Molecular Biology, College of Natural Sciences and Human Ecology, Dongeui University, Busan 614-714, Republic of Korea, Laboratory of Immunobiology, Department of Marine Life Sciences, Jeju National University, Jeju 690-756, Republic of Korea, Department of Internal Medicine, Dongeui University College of Korean Medicine, Busan 614-052, Republic of Korea, Department of Physiology, Kosin University College of Medicine, Busan 602-072, Republic of Korea, Department of Microbiology, College of Medicine, Inje University, Busan 608-737, Republic of Korea, Department of Parasitology and Genetics, Kosin University College of Medicine, Busan 602-072, Republic of Korea
Published online on: December 23, 2016 https://doi.org/10.3892/ijmm.2016.2837
Pages: 399-406

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Abstract

In the present study, we investigated the cytoprotective efficacy of morin, a natural flavonoid, against oxidative stress and elucidated the underlying mechanisms in C2C12 myoblasts. Our results indicated that morin treatment prior to hydrogen peroxide (H2O2) exposure significantly increased cell viability and prevented the generation of reactive oxygen species. H2O2-induced comet-like DNA formation and γH2AX phosphorylation were also markedly suppressed by morin with a parallel inhibition of apoptosis in C2C12 myoblasts, suggesting that morin prevented H2O2-induced cellular DNA damage. Furthermore, morin markedly enhanced the expression of heme oxygenase-1 (HO-1) associated with the induction and phosphorylation of nuclear factor-erythroid 2-related factor 2 (Nrf2) and the inhibition of Kelch-like ECH-associated protein 1 (Keap1) expression. Notably, these events were eliminated by transient transfection with Nrf2‑specific small interfering RNA. Additional experiments demonstrated that the activation of the Nrf2/HO-1 pathway by morin was mediated by the extracellular signal‑regulated kinase (ERK) signaling cascade. This phenomenon was confirmed with suppressed Nrf2 phosphorylation and consequently diminished HO-1 expression in cells treated with a pharmacological inhibitor of ERK. Collectively, these results demonstrated that morin augments the cellular antioxidant defense capacity through the activation of Nrf2/HO‑1 signaling, which involves the activation of the ERK pathway, thereby protecting C2C12 myoblasts from H2O2-induced oxidative cytotoxicity.

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1	Szczesny B, Olah G, Walker DK, Volpi E, Rasmussen BB, Szabo C and Mitra S: Deficiency in repair of the mitochondrial genome sensitizes proliferating myoblasts to oxidative damage. PLoS One. 8:e752012013. View Article : Google Scholar : PubMed/NCBI
2	Miller CJ, Gounder SS, Kannan S, Goutam K, Muthusamy VR, Firpo MA, Symons JD, Paine R III, Hoidal JR and Rajasekaran NS: Disruption of Nrf2/ARE signaling impairs antioxidant mechanisms and promotes cell degradation pathways in aged skeletal muscle. Biochim Biophys Acta. 1822:1038–1050. 2012. View Article : Google Scholar : PubMed/NCBI
3	Ji LL, Gomez-Cabrera MC and Vina J: Exercise and hormesis: Activation of cellular antioxidant signaling pathway. Ann NY Acad Sci. 1067:425–435. 2006. View Article : Google Scholar : PubMed/NCBI
4	Powers SK and Jackson MJ: Exercise-induced oxidative stress: Cellular mechanisms and impact on muscle force production. Physiol Rev. 88:1243–1276. 2008. View Article : Google Scholar : PubMed/NCBI
5	Song W, Kwak HB and Lawler JM: Exercise training attenuates age-induced changes in apoptotic signaling in rat skeletal muscle. Antioxid Redox Signal. 8:517–528. 2006. View Article : Google Scholar : PubMed/NCBI
6	Harasym J and Oledzki R: Effect of fruit and vegetable antioxidants on total antioxidant capacity of blood plasma. Nutrition. 30:511–517. 2014. View Article : Google Scholar : PubMed/NCBI
7	Guo W, Kong E and Meydani M: Dietary polyphenols, inflammation, and cancer. Nutr Cancer. 61:807–810. 2009. View Article : Google Scholar
8	Landete JM: Dietary intake of natural antioxidants: Vitamins and polyphenols. Crit Rev Food Sci Nutr. 53:706–721. 2013. View Article : Google Scholar : PubMed/NCBI
9	Hardigree AA and Epler JL: Comparative mutagenesis of plant flavonoids in microbial systems. Mutat Res. 58:231–239. 1978. View Article : Google Scholar : PubMed/NCBI
10	Wu TW, Zeng LH, Wu J and Fung KP: Morin: A wood pigment that protects three types of human cells in the cardiovascular system against oxyradical damage. Biochem Pharmacol. 47:1099–1103. 1994. View Article : Google Scholar : PubMed/NCBI
11	Kitagawa S, Sakamoto H and Tano H: Inhibitory effects of flavonoids on free radical-induced hemolysis and their oxidative effects on hemoglobin. Chem Pharm Bull (Tokyo). 52:999–1001. 2004. View Article : Google Scholar
12	Liu YH, Mo SL, Bi HC, Hu BF, Li CG, Wang YT, Huang L, Huang M, Duan W, Liu JP, et al: Regulation of human pregnane X receptor and its target gene cytochrome P450 3A4 by Chinese herbal compounds and a molecular docking study. Xenobiotica. 41:259–280. 2011. View Article : Google Scholar
13	Yang SH, Choi HG, Lim SJ, Lee MG and Kim SH: Effects of morin on the pharmacokinetics of etoposide in 7,12-dimethylbenz[a] anthracene-induced mammary tumors in female Sprague-Dawley rats. Oncol Rep. 29:1215–1223. 2013.
14	Al Numair KS, Chandramohan G, Alsaif MA and Baskar AA: Protective effect of morin on cardiac mitochondrial function during isoproterenol-induced myocardial infarction in male Wistar rats. Redox Rep. 17:14–21. 2012. View Article : Google Scholar : PubMed/NCBI
15	Al-Numair KS, Chandramohan G, Alsaif MA, Veeramani C and El Newehy AS: Morin, a flavonoid, on lipid peroxidation and antioxidant status in experimental myocardial ischemic rats. Afr J Tradit Complement Altern Medicines. 11:14–20. 2014. View Article : Google Scholar
16	Kim J, Kim JS and Park E: Cytotoxic and anti-inflammatory effects of onion peel extract on lipopolysaccharide stimulated human colon carcinoma cells. Food Chem Toxicol. 62:199–204. 2013. View Article : Google Scholar : PubMed/NCBI
17	Nandhakumar R, Salini K and Niranjali Devaraj S: Morin augments anticarcinogenic and antiproliferative efficacy against 7,12-dimethylbenz(a)-anthracene induced experimental mammary carcinogenesis. Mol Cell Biochem. 364:79–92. 2012. View Article : Google Scholar : PubMed/NCBI
18	Park JY, Kang KA, Kim KC, Cha JW, Kim EH and Hyun JW: Morin induces heme oxygenase-1 via ERK-Nrf2 signaling pathway. J Cancer Prev. 18:249–256. 2013. View Article : Google Scholar
19	Zhang Y and Gordon GB: A strategy for cancer prevention: Stimulation of the Nrf2-ARE signaling pathway. Mol Cancer Ther. 3:885–893. 2004.PubMed/NCBI
20	Ishii T, Itoh K, Takahashi S, Sato H, Yanagawa T, Katoh Y, Bannai S and Yamamoto M: Transcription factor Nrf2 coordinately regulates a group of oxidative stress-inducible genes in macrophages. J Biol Chem. 275:16023–16029. 2000. View Article : Google Scholar : PubMed/NCBI
21	Niture SK, Khatri R and Jaiswal AK: Regulation of Nrf2-an update. Free Radic Biol Med. 66:36–44. 2014. View Article : Google Scholar
22	Kobayashi M and Yamamoto M: Molecular mechanisms activating the Nrf2-Keap1 pathway of antioxidant gene regulation. Antioxid Redox Signal. 7:385–394. 2005. View Article : Google Scholar : PubMed/NCBI
23	Pi J, Bai Y, Reece JM, Williams J, Liu D, Freeman ML, Fahl WE, Shugar D, Liu J and Qu W: Molecular mechanism of human Nrf2 activation and degradation: Role of sequential phosphorylation by protein kinase CK2. Free Radic Biol Med. 42:1797–1806. 2007. View Article : Google Scholar : PubMed/NCBI
24	Apopa PL, He X and Ma Q: Phosphorylation of Nrf2 in the transcription activation domain by casein kinase 2 (CK2) is critical for the nuclear translocation and transcription activation function of Nrf2 in IMR-32 neuroblastoma cells. J Biochem Mol Toxicol. 22:63–76. 2008. View Article : Google Scholar : PubMed/NCBI
25	Eom SA, Kim DW, Shin MJ, Ahn EH, Chung SY, Sohn EJ, Jo HS, Jeon SJ, Kim DS, Kwon HY, et al: Protective effects of PEP-1-catalase on stress-induced cellular toxicity and MPTP-induced Parkinson's disease. BMB Rep. 48:395–400. 2015. View Article : Google Scholar :
26	Park MH and Han JS: Padina arborescens extract protects high glucose-induced apoptosis in pancreatic β cells by reducing oxidative stress. Nutr Res Pract. 8:494–500. 2014. View Article : Google Scholar : PubMed/NCBI
27	Jeong MH, Yang K, Lee CG, Jeong DH, Park YS, Choi YJ, Kim JS, Oh SJ, Jeong SK and Jo WS: In vitro genotoxicity assessment of a novel resveratrol analogue, HS-1793. Toxicol Res. 30:211–220. 2014. View Article : Google Scholar : PubMed/NCBI
28	Jiang R, Teng Y, Huang Y, Gu J, Ma L, Li M and Zhou Y: Preeclampsia serum-induced collagen I expression and intracellular calcium levels in arterial smooth muscle cells are mediated by the PLC-γ1 pathway. Exp Mol Med. 46:e1152014. View Article : Google Scholar
29	Rogakou EP, Pilch DR, Orr AH, Ivanova VS and Bonner WM: DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139. J Biol Chem. 273:5858–5868. 1998. View Article : Google Scholar : PubMed/NCBI
30	Kregel KC and Zhang HJ: An integrated view of oxidative stress in aging: Basic mechanisms, functional effects, and pathological considerations. Am J Physiol Regul Integr Comp Physiol. 292:R18–R36. 2007. View Article : Google Scholar
31	Finkel T: Signal transduction by reactive oxygen species. J Cell Biol. 194:7–15. 2011. View Article : Google Scholar : PubMed/NCBI
32	Li M and Fukagawa NK: Age-related changes in redox signaling and VSMC function. Antioxid Redox Signal. 12:641–655. 2010. View Article : Google Scholar :
33	Safdar A, deBeer J and Tarnopolsky MA: Dysfunctional Nrf2-Keap1 redox signaling in skeletal muscle of the sedentary old. Free Radic Biol Med. 49:1487–1493. 2010. View Article : Google Scholar : PubMed/NCBI
34	Rangasamy T, Guo J, Mitzner WA, Roman J, Singh A, Fryer AD, Yamamoto M, Kensler TW, Tuder RM, Georas SN, et al: Disruption of Nrf2 enhances susceptibility to severe airway inflammation and asthma in mice. J Exp Med. 202:47–59. 2005. View Article : Google Scholar : PubMed/NCBI
35	Reddy NM, Kleeberger SR, Kensler TW, Yamamoto M, Hassoun PM and Reddy SP: Disruption of Nrf2 impairs the resolution of hyperoxia-induced acute lung injury and inflammation in mice. J Immunol. 182:7264–7271. 2009. View Article : Google Scholar : PubMed/NCBI
36	Katori M, Anselmo DM, Busuttil RW and Kupiec-Weglinski JW: A novel strategy against ischemia and reperfusion injury: Cytoprotection with heme oxygenase system. Transpl Immunol. 9:227–233. 2002. View Article : Google Scholar : PubMed/NCBI
37	Motterlini R and Foresti R: Heme oxygenase-1 as a target for drug discovery. Antioxid Redox Signal. 20:1810–1826. 2014. View Article : Google Scholar
38	Bryan HK, Olayanju A, Goldring CE and Park BK: The Nrf2 cell defence pathway: Keap1-dependent and -independent mechanisms of regulation. Biochem Pharmacol. 85:705–717. 2013. View Article : Google Scholar
39	Chen HH, Chen YT, Huang YW, Tsai HJ and Kuo CC: 4-Ketopinoresinol, a novel naturally occurring ARE activator, induces the Nrf2/HO-1 axis and protects against oxidative stress-induced cell injury via activation of PI3K/AKT signaling. Free Radic Biol Med. 52:1054–1066. 2012. View Article : Google Scholar : PubMed/NCBI
40	Sun Z, Huang Z and Zhang DD: Phosphorylation of Nrf2 at multiple sites by MAP kinases has a limited contribution in modulating the Nrf2-dependent antioxidant response. PLoS One. 4:e65882009. View Article : Google Scholar : PubMed/NCBI
41	Shen G, Hebbar V, Nair S, Xu C, Li W, Lin W, Keum YS, Han J, Gallo MA and Kong AN: Regulation of Nrf2 transactivation domain activity. The differential effects of mitogen-activated protein kinase cascades and synergistic stimulatory effect of Raf and CREB-binding protein. J Biol Chem. 279:23052–23060. 2004. View Article : Google Scholar : PubMed/NCBI
42	Nguyen T, Sherratt PJ, Huang HC, Yang CS and Pickett CB: Increased protein stability as a mechanism that enhances Nrf2-mediated transcriptional activation of the antioxidant response element. Degradation of Nrf2 by the 26 S proteasome. J Biol Chem. 278:4536–4541. 2003. View Article : Google Scholar