Identification of cardioprotective agents from traditional Chinese medicine against oxidative damage

Zhou,Jian‑Ming; Xu,Zhi‑Liang; Li,Na; Zhao,Yi‑Wu; Wang,Zhen‑Zhong; Xiao,Wei

doi:10.3892/mmr.2016.5243

Identification of cardioprotective agents from traditional Chinese medicine against oxidative damage

Authors:
- Jian‑Ming Zhou
- Zhi‑Liang Xu
- Na Li
- Yi‑Wu Zhao
- Zhen‑Zhong Wang
- Wei Xiao
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Affiliations: State Key Laboratory of New‑Tech for Chinese Medicine Pharmaceutical Process, Jiangsu Kanion Modern Traditional Chinese Medicine Research Institute, Lianyungang, Jiangsu 222001, P.R. China
Published online on: May 11, 2016 https://doi.org/10.3892/mmr.2016.5243
Pages: 77-88
Copyright: © Zhou et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

Reactive oxygen species are damaging to cardiomyocytes. H9c2 cardiomyocytes are commonly used to study the cellular mechanisms and signal transduction in cardiomyocytes, and to evaluate the cardioprotective effects of drugs following oxidative damage. The present study developed a robust, automated high throughput screening (HTS) assay to identify cardioprotective agents from a traditional Chinese medicine (TCM) library using a H2O2‑induced oxidative damage model in H9c2 cells. Using this HTS format, several hits were identified as cardioprotective by detecting changes to cell viability using the cell counting kit (CCK)‑8 assay. Two TCM extracts, KY‑0520 and KY‑0538, were further investigated. The results of the present study demonstrated that treatment of oxidatively damaged cells with KY‑0520 or KY‑0538 markedly increased the cell viability and superoxide dismutase activity, decreased lactate dehydrogenase activity and malondialdehyde levels, and inhibited early growth response‑1 (Egr‑1) protein expression. The present study also demonstrated that KY‑0520 or KY‑0538 treatment protected H9c2 cells from H2O2‑induced apoptosis by altering the Bcl-2/Bax protein expression ratio, and decreasing the levels of cleaved caspase‑3. In addition, KY‑0520 and KY‑0538 reduced the phosphorylation of ERK1/2 and p38‑MAPK proteins, and inhibited the translocation of Egr‑1 from the cytoplasm to nucleus in H2O2-treated H9c2 cells. These findings suggested that oxidatively damaged H9c2 cells can be used for the identification of cardioprotective agents that reduce oxidative stress by measuring cell viabilities using CCK‑8 in an HTS format. The underlying mechanism of the cardioprotective activities of KY‑0520 and KY‑0538 may be attributed to their antioxidative activity, regulation of Egr‑1 and apoptosis‑associated proteins, and the inhibition of ERK1/2, p38-MAPK and Egr-1 signaling pathways.

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1	Galli F, Piroddi M, Annetti C, Aisa C, Floridi E and Floridi A: Oxidative stress and reactive oxygen species. Contrib Nephrol. 149:240–260. 2005. View Article : Google Scholar : PubMed/NCBI
2	Pham-Huy LA, He H and Pham-Huy C: Free radicals, antioxidants in disease and health. International journal of biomedical science: Int J Biomed Sc. 4:89–96. 2008.
3	Geronikaki AA and Gavalas AM: Antioxidants and inflammatory disease: Synthetic and natural antioxidants with anti-inflammatory activity. Comb Chem High Throughput Screen. 9:425–442. 2006. View Article : Google Scholar : PubMed/NCBI
4	Guo RF and Ward PA: Role of oxidants in lung injury during sepsis. Antioxid Redox Signal. 9:1991–2002. 2007. View Article : Google Scholar : PubMed/NCBI
5	Gilgun-Sherki Y, Melamed E and Offen D: Oxidative stress induced-neurodegenerative diseases: The need for antioxidants that penetrate the blood brain barrier. Neuropharmacology. 40:959–975. 2001. View Article : Google Scholar : PubMed/NCBI
6	Kumar SV, Saritha G and Fareedullah M: Role of antioxidants and oxidative stress in cardiovascular diseases. Ann Biol Res. 3:158–175. 2010.
7	Venardos KM, Perkins A, Headrick J and Kaye DM: Myocardial ischemia-reperfusion injury, antioxidant enzyme systems, and selenium: A review. Cur Med Chem. 14:1539–1549. 2007. View Article : Google Scholar
8	Zhao ZQ: Oxidative stress-elicited myocardial apoptosis during reperfusion. Curr Opin Pharmacol. 4:159–165. 2004. View Article : Google Scholar : PubMed/NCBI
9	Saini HK, Machackova J and Dhalla NS: Role of reactive oxygen species in ischemic preconditioning of subcellular organelles in the heart. Antioxid Redox Signal. 6:393–404. 2004. View Article : Google Scholar : PubMed/NCBI
10	Zima AV and Blatter LA: Redox regulation of cardiac calcium channels and transporters. Cardiovasc Res. 71:310–321. 2006. View Article : Google Scholar : PubMed/NCBI
11	Cesselli D, Jakoniuk I, Barlucchi L, Beltrami AP, Hintze TH, Nadal-Ginard B, Kajstura J, Leri A and Anversa P: Oxidative stress-mediated cardiac cell death is a major determinant of ventricular dysfunction and failure in dog dilated cardiomyopathy. Circ Res. 89:279–286. 2001. View Article : Google Scholar : PubMed/NCBI
12	Kwon SH, Pimentel DR, Remondino A, Sawyer DB and Colucci WS: H₂O₂ regulates cardiac myocyte phenotype via concentration-dependent activation of distinct kinase pathways. J Mol Cell Cardiol. 35:615–621. 2003. View Article : Google Scholar : PubMed/NCBI
13	Rayment NB, Haven AJ, Madden B, Murday A, Trickey R, Shipley M, Davies MJ and Katz DR: Myocyte loss in chronic heart failure. J Pathol. 188:213–219. 1999. View Article : Google Scholar : PubMed/NCBI
14	Giordano FJ: Oxygen, oxidative stress, hypoxia, and heart failure. J Clin Invest. 115:500–508. 2005. View Article : Google Scholar : PubMed/NCBI
15	Kimes BW and Brandt BL: Properties of a clonal muscle cell line from rat heart. Exp Cell Res. 98:367–381. 1976. View Article : Google Scholar : PubMed/NCBI
16	Su CY, Chong KY, Edelstein K, Lille S, Khardori R and Lai CC: Constitutive hsp70 attenuates hydrogen peroxide-induced membrane lipid peroxidation. Biochem Biophys Res Commun. 265:279–284. 1999. View Article : Google Scholar : PubMed/NCBI
17	Park ES, Kang JC, Kang DH, Jang YC, Yi KY, Chung HJ, Park JS, Kim B, Feng ZP and Shin HS: 5-AIQ inhibits H₂O₂-induced apoptosis through reactive oxygen species scavenging and Akt/GSK-3β signaling pathway in H9c2 cardiomyocytes. Toxicol Appl Pharmacol. 268:90–98. 2013. View Article : Google Scholar : PubMed/NCBI
18	Aggeli IK, Gaitanaki C and Beis I: Involvement of JNKs and p38-MAPK/MSK1 pathways in H₂O₂-induced upregulation of heme oxygenase-1 mRNA in H9c2 cells. Cell Signal. 18:1801–1812. 2006. View Article : Google Scholar : PubMed/NCBI
19	Tanaka H, Sakurai K, Takahashi K and Fujimoto Y: Requirement of intracellular free thiols for hydrogen peroxide-induced hypertrophy in cardiomyocytes. J Cell Biochem. 89:944–955. 2003. View Article : Google Scholar : PubMed/NCBI
20	Qu S, Zhu H, Wei X, Zhang C, Jiang L, Liu Y, Luo Q and Xiao X: Oxidative stress-mediated up-regulation of myocardial ischemic preconditioning up-regulated protein 1 gene expression in H9c2 cardiomyocytes is regulated by cyclic AMP-response element binding protein. Radic Biol Med. 49:580–586. 2010. View Article : Google Scholar
21	Law CH, Li JM, Chou HC, Chen YH and Chan HL: Hyaluronic acid-dependent protection in H9C2 cardiomyocytes: A cell model of heart ischemia-reperfusion injury and treatment. Toxicology. 303:54–71. 2013. View Article : Google Scholar
22	Diestel A, Drescher C, Miera O, Berger F and Schmitt KR: Hypothermia protects H9c2 cardiomyocytes from H₂O₂ induced apoptosis. Cryobiology. 62:53–61. 2011. View Article : Google Scholar
23	Eguchi M, Liu Y, Shin EJ and Sweeney G: Leptin protects H9c2 rat cardiomyocytes from H₂O₂-induced apoptosis. FEBS J. 275:3136–3144. 2008. View Article : Google Scholar : PubMed/NCBI
24	Park ES, Kang JC, Jang YC, Park JS, Jang SY, Kim DE, Kim B and Shin HS: Cardioprotective effects of rhamnetin in H9c2 cardiomyoblast cells under H₂O₂-induced apoptosis. J Ethnopharmacol. 153:552–560. 2014. View Article : Google Scholar : PubMed/NCBI
25	Zhang JH, Chung TD and Oldenburg KR: A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J Biomol Screen. 4:67–73. 1999. View Article : Google Scholar
26	Aggeli IK, Beis I and Gaitanaki C: ERKs and JNKs mediate hydrogen peroxide-induced Egr-1 expression and nuclear accumulation in H9c2 cells. Physiol Res. 59:443–454. 2010.
27	Ryter SW, Kim HP, Hoetzel A, Park JW, Nakahira K, Wang X and Choi AM: Mechanisms of cell death in oxidative stress. Antioxid Redox Signal. 9:49–89. 2007. View Article : Google Scholar
28	Clerk A, Michael A and Sugden PH: Stimulation of multiple mitogen-activated protein kinase sub-families by oxidative stress and phosphorylation of the small heat shock protein, HSP25/27, in neonatal ventricular myocytes. Biochem J. 333:581–589. 1998. View Article : Google Scholar : PubMed/NCBI
29	Di Meo S, Venditti P and De Leo T: Tissue protection against oxidative stress. Experientia. 52:786–794. 1996. View Article : Google Scholar : PubMed/NCBI
30	Bogoyevitch MA: Signalling via stress-activated mitogen-activated protein kinases in the cardiovascular system. Cardiovasc Res. 45:826–842. 2000. View Article : Google Scholar : PubMed/NCBI
31	Ravingerová T, Barancík M and Strnisková M: Mitogen-activated protein kinases: A new therapeutic target in cardiac pathology. Mol Cell Biochem. 247:127–138. 2003. View Article : Google Scholar : PubMed/NCBI
32	Wang Y: Mitogen-activated protein kinases in heart development and diseases. Circulation. 116:1413–1423. 2007. View Article : Google Scholar : PubMed/NCBI
33	Gao Z, Huang K and Xu H: Protective effects of flavonoids in the roots of Scutellaria baicalensis Georgi against hydrogen peroxide-induced oxidative stress in HS-SY5Y cells. Pharmacol Res. 43:173–178. 2001. View Article : Google Scholar : PubMed/NCBI
34	Park C, So HS, Shin CH, Baek SH, Moon BS, Shin SH, Lee HS, Lee DW and Park R: Quercetin protects the hydrogen peroxide-induced apoptosis via inhibition of mitochondrial dysfunction in H9c2 cardiomyoblast cells. Biochem Pharmacol. 66:1287–1295. 2003. View Article : Google Scholar : PubMed/NCBI
35	Anestopoulos I, Kavo A, Tentes I, Kortsaris A, Panayiotidis M, Lazou A and Pappa A: Silibinin protects H9c2 cardiac cells from oxidative stress and inhibits phenylephrine-induced hypertrophy: Potential mechanisms. J Nutr Biochem. 24:586–594. 2013. View Article : Google Scholar
36	Maiwulanjiang M, Chen J, Xin G, Gong AG, Miernisha A, Du CY, Lau KM, Lee PS, Chen J, Dong TT, et al: The volatile oil of Nardostachyos Radix et Rhizoma inhibits the oxidative stress-induced cell injury via reactive oxygen species scavenging and Akt activation in H9c2 cardiomyocyte. J Ethnopharmacol. 153:491–498. 2014. View Article : Google Scholar : PubMed/NCBI
37	Chen B, Mao R, Wang H and She JX: Cell line and drug-dependent effect of ERBB3 on cancer cell proliferation, chemosensitivity, and multidrug actions. Int J High Throughput Screen. 1:49–55. 2010.
38	Li H, Deng Z, Liu R, Loewen S and Tsao R: Carotenoid compositions of coloured tomato cultivars and contribution to antioxidant activities and protection against H₂O₂-induced cell death in H9c2. Food Chem. 136:878–888. 2013. View Article : Google Scholar
39	Woo SM, Min KJ, Kim S, Park JW, Kim DE, Chun KS, Kim YH, Lee TJ, Kim SH, Choi YH, et al: Silibinin induces apoptosis of HT29 colon carcinoma cells through early growth response-1 (EGR-1)-mediated non-steroidal anti-inflammatory drug-activated gene-1 (NAG-1) up-regulation. Chem Biol Interact. 211:36–43. 2014. View Article : Google Scholar : PubMed/NCBI
40	O'Donnell-Tormey J, Nathan CF, Lanks K, DeBoer CJ and de la Harpe J: Secretion of pyruvate. An antioxidant defense of mammalian cells. J Exp Med. 165:500–514. 1987. View Article : Google Scholar : PubMed/NCBI
41	Holleman AF: Notice sur l'action de l'eau oxygénée sur les acides α-cétoniques et sur les dicétones 1. 2. Recl Trav Chim Pays-Bas Belg. 23:169–172. 1904.In French. View Article : Google Scholar
42	Dorn GW II: Apoptotic and non-apoptotic programmed cardiomyocyte death in ventricular remodelling. Cardiovasc Res. 81:465–473. 2009. View Article : Google Scholar :
43	Shih PH, Yeh CT and Yen GC: Anthocyanins induce the activation of phase II enzymes through the antioxidant response element pathway against oxidative stress-induced apoptosis. J Agric Food Chem. 55:9427–9435. 2007. View Article : Google Scholar : PubMed/NCBI
44	Sukhatme VP, Cao XM, Chang LC, Tsai-Morris CH, Stamenkovich D, Ferreira PC, Cohen DR, Edwards SA, Shows TB, Curran T, et al: A zinc finger-encoding gene coregulated with c-fos during growth and differentiation, and after cellular depolarization. Cell. 53:37–43. 1988. View Article : Google Scholar : PubMed/NCBI
45	Yan SF, Fujita T, Lu J, Okada K, Shan Zou Y, Mackman N, Pinsky DJ and Stern DM: Egr-1, a master switch coordinating upregulation of divergent gene families underlying ischemic stress. Nat Med. 6:1355–1361. 2000. View Article : Google Scholar : PubMed/NCBI
46	Gaggioli C, Deckert M, Robert G, Abbe P, Batoz M, Ehrengruber MU, Ortonne JP, Ballotti R and Tartare-Deckert S: HGF induces fibronectin matrix synthesis in melanoma cells through MAP kinase-dependent signaling pathway and induction of Egr-1. Oncogene. 24:1423–1433. 2005. View Article : Google Scholar
47	Guha M, O'Connell MA, Pawlinski R, Hollis A, McGovern P, Yan SF, Stern D and Mackman N: Lipopolysaccharide activation of the MEK-ERK1/2 pathway in human monocytic cells mediates tissue factor and tumor necrosis factor alpha expression by inducing Elk-1 phosphorylation and Egr-1 expression. Blood. 98:1429–1439. 2001. View Article : Google Scholar : PubMed/NCBI
48	Hjoberg J, Le L, Imrich A, Subramaniam V, Mathew SI, Vallone J, Haley KJ, Green FH, Shore SA and Silverman ES: Induction of early growth-response factor 1 by platelet-derived growth factor in human airway smooth muscle. Am J Physiol Lung Cell Mol Physiol. 286:L817–L825. 2004. View Article : Google Scholar : PubMed/NCBI
49	Li CJ, Ning W, Matthay MA, Feghali-Bostwick CA and Choi AM: MAPK pathway mediates EGR-1-HSP70-dependent cigarette smoke-induced chemokine production. Am J Physiol Lung Cell Mol Physiol. 292:L1297–L1303. 2007. View Article : Google Scholar : PubMed/NCBI
50	Kaufmann K and Thiel G: Epidermal growth factor and thrombin induced proliferation of immortalized human keratinocytes is coupled to the synthesis of Egr-1, a zinc finger transcriptional regulator. J Cell Biochem. 85:381–391. 2002. View Article : Google Scholar : PubMed/NCBI
51	Rössler OG and Thiel G: Thrombin induces Egr-1 expression in fibroblasts involving elevation of the intracellular Ca²⁺ concentration, phosphorylation of ERK and activation of ternary complex factor. Mol Biol. 10:402009.
52	Lohoff M, Giaisi M, Köhler R, Casper B, Krammer PH and Li-Weber M: Early growth response protein-1 (Egr-1) is preferentially expressed in T helper type 2 (Th2) cells and is involved in acute transcription of the Th2 cytokine interleukin-4. J Biol Chem. 285:1643–1652. 2010. View Article : Google Scholar :
53	Gashler A and Sukhatme VP: Early growth response protein 1 (Egr-1): Prototype of a zinc-finger family of transcription factors. Prog Nucleic Acid Res Mol Biol. 50:191–224. 1995. View Article : Google Scholar : PubMed/NCBI
54	Liu C, Rangnekar VM, Adamson E and Mercola D: Suppression of growth and transformation and induction of apoptosis by EGR-1. Cancer Gene Ther. 5:3–28. 1998.PubMed/NCBI
55	Thiel G and Cibelli G: Regulation of life and death by the zinc finger transcription factor Egr-1. J Cell Physiol. 193:287–292. 2002. View Article : Google Scholar : PubMed/NCBI
56	Bhindi R, Fahmy RG, McMahon AC, Khachigian LM and Lowe HC: Intracoronary delivery of DNAzymes targeting human EGR-1 reduces infarct size following myocardial ischaemia reperfusion. J Pathol. 227:157–164. 2012. View Article : Google Scholar : PubMed/NCBI
57	Kasneci A, Kemeny-Suss NM, Komarova SV and Chalifour LE: Egr-1 negatively regulates calsequestrin expression and calcium dynamics in ventricular cells. Cardiovasc Res. 1:695–702. 2009.
58	Zins K, Pomyje J, Hofer E, Abraham D, Lucas T and Aharinejad S: Egr-1 upregulates Siva-1 expression and induces cardiac fibroblast apoptosis. Int J Mol Sci. 15:1538–1553. 2014. View Article : Google Scholar : PubMed/NCBI
59	Aebert H, Cornelius T, Ehr T, Holmer SR, Birnbaum DE, Riegger GA and Schunkert H: Expression of immediate early genes after cardioplegic arrest and reperfusion. Ann Thorac Surg. 63:1669–1675. 1997. View Article : Google Scholar : PubMed/NCBI
60	Chen CA, Chen TS and Chen HC: Extracellular signal-regulated kinase plays a proapoptotic role in podocytes after reactive oxygen species treatment and inhibition of integrin-extracellular matrix interaction. Exp Biol Med (Maywood). 237:777–783. 2012. View Article : Google Scholar
61	Hartney T, Birari R, Venkataraman S, Villegas L, Martinez M, Black SM, Stenmark KR and Nozik-Grayck E: Xanthine oxidase-derived ROS upregulate Egr-1 via ERK1/2 in PA smooth muscle cells; model to test impact of extracellular ROS in chronic hypoxia. PLoS One. 6:e275312011. View Article : Google Scholar : PubMed/NCBI
62	Iyoda T, Zhang F, Sun L, Hao F, Schmitz-Peiffer C, Xu X and Cui MZ: Lysophosphatidic acid induces early growth response-1 (Egr-1) protein expression via protein kinase Cδ-regulated extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) activation in vascular smooth muscle cells. J Biol Chem. 287:22635–22642. 2012. View Article : Google Scholar : PubMed/NCBI
63	Wang C, Dostanic S, Servant N and Chalifour LE: Egr-1 negatively regulates expression of the sodium-calcium exchanger-1 in cardiomyocytes in vitro and in vivo. Cardiovasc Res. 65:187–194. 2005. View Article : Google Scholar