Effect of β-D-Mannuronic Acid (M2000) on Oxidative Stress Enzymes’ Gene Using Healthy Donor Peripheral Blood Mononuclear Cells for Evaluating the Anti-Aging Property

Author(s): Mahsa Taeb, Abdollah Jafarzadeh*, Seyed Shahabeddin Mortazavi-Jahromi, Nahid Zainodini, Mohammad Reza Mirzaei, Fahimeh Jafarnezhad-Ansariha, Zahra Aghazadeh, Abbas Mirshafiey*

Journal Name: Current Drug Discovery Technologies

Volume 16 , Issue 3 , 2019

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Graphical Abstract:


Objective: This research aimed to study the anti-aging and anti-inflammatory effects of low and high doses of the β-D-mannuronic (M2000) on gene expression of enzymes involved in oxidative stress (including SOD2, GST, GPX1, CAT, iNOS, and MPO) in peripheral blood mononuclear cells (PBMCs) of healthy donors under in vitro conditions.

Methods: The PBMCs were separated and the RNAs were then extracted and the cDNAs synthesized, and expression levels of the mentioned genes were detected by qRT-PCR.

Results: Our results indicated that the high dose of this drug could significantly reduce the expression level of the SOD2 gene compared to the lipopolysaccharide (LPS) group (p < 0.0001). Moreover, it was found that the high dose of this drug could significantly decrease the expression level of the GST gene compared to the LPS group (p < 0.0001). However, no significant reductions were observed in expression levels of the CAT and GPX1 genes compared to the LPS group. Furthermore, our data revealed that the level of iNOS and MPO gene expression was significantly reduced, in both doses of M2000, respectively, compared to the LPS group (p < 0.0001).

Conclusion: This research showed that M2000 as a novel NSAID with immunosuppressive properties could modify oxidative stress through lowering expression levels of the SOD2, GST, iNOS, and MPO genes compared to the healthy expression levels, with a probable reduction of the risk of developing inflammatory diseases related to age and aging.

Keywords: Mannuronic acid, M2000, anti-aging, oxidative stress, NSAID, aging.

McCord JM. Oxygen-derived free radicals in postischemic tissue injury. N Engl J Med 1985; 312(3): 159-63.
Pham-Huy LA, He H, Pham-Huy C. Free radicals, antioxidants in disease and health. Int J Biomed Sci IJBS 2008; 4(2): 89-96.
Sohal RS, Weindruch R. Oxidative stress, caloric restriction, and aging. Science 1996; 273(5271): 59-63.
Finkel T, Holbrook NJ. Oxidants, oxidative stress and the biology of ageing. Nature 2000; 408(6809): 239-47.
Halliwell B. Free radicals and antioxidants: Updating a personal view. Nutr Rev 2012; 70(5): 257-65.
Babich PS, Skvortsov AN, Rusconi P, et al. Non-hepatic tumors change the activity of genes encoding copper trafficking proteins in the liver. Cancer Biol Ther 2013; 14(7): 614-24.
Augustin W, Wiswedel I, Noack H, Reinheckel T, Reichelt O. Role of endogenous and exogenous antioxidants in the defence against functional damage and lipid peroxidation in rat liver mitochondria. Mol Cell Biochem 1997; 174(1-2): 199-205.
Fusco D, Colloca G, Monaco MRL, Cesari M. Effects of antioxidant supplementation on the aging process. Clin Interv Aging 2007; 2(3): 377-87.
Al‐Gayyar MM, Eissa LA, Rabie AM, El‐Gayar AM. Measurements of oxidative stress status and antioxidant activity in chronic leukaemia patients. J Pharm Pharmacol 2007; 59(3): 409-17.
Basha PS, Rani AU. Cadmium-induced antioxidant defense mechanism in freshwater teleost Oreochromis mossambicus (Tilapia). Ecotoxicol Environ Saf 2003; 56(2): 218-21.
Becuwe P, Ennen M, Klotz R, Barbieux C, Grandemange S. Manganese superoxide dismutase in breast cancer: From molecular mechanisms of gene regulation to biological and clinical significance. Free Radic Biol Med 2014; 77: 139-51.
Liu R, Liu IY, Bi X, et al. Reversal of age-related learning deficits and brain oxidative stress in mice with superoxide dismutase/catalase mimetics. Proc Natl Acad Sci USA 2003; 100(14): 8526-31.
Cho S-G, Lee YH, Park H-S, et al. Glutathione S-transferase mu modulates the stress-activated signals by suppressing apoptosis signal-regulating kinase 1. J Biol Chem 2001; 276(16): 12749-55.
Fanò G, Mecocci P, Vecchiet J, et al. Age and sex influence on oxidative damage and functional status in human skeletal muscle. J Muscle Res Cell Motil 2001; 22(4): 345-51.
Chelikani P, Fita I, Loewen P. Diversity of structures and properties among catalases. Cell Mol Life Sci 2004; 61(2): 192-208.
Palomero J, Galán AI. Muñoz MaE, Tuñón MaJ, González-Gallego J, Jiménez R. Effects of aging on the susceptibility to the toxic effects of cyclosporin A in rats. Changes in liver glutathione and antioxidant enzymes. Free Radic Biol Med 2001; 30(8): 836-45.
Rotruck JT, Pope AL, Ganther H, Swanson A, Hafeman DG, Hoekstra W. Selenium: Biochemical role as a component of glutathione peroxidase. Science 1973; 179(4073): 588-90.
Wijnen L, Monteba-van Heuvel M, Pearson P, Khan PM. Assignment of a gene for glutathione peroxidase (GPX1) to human chromosome 3. Cytogenet Cell Genet 1978; 22(1-6): 232-5.
Bala K, Tripathy B, Sharma D. Neuroprotective and anti-ageing effects of curcumin in aged rat brain regions. Biogerontology 2006; 7(2): 81-9.
Karpuzoglu E, Ahmed SA. Estrogen regulation of nitric oxide and inducible nitric oxide synthase (iNOS) in immune cells: implications for immunity, autoimmune diseases, and apoptosis. Nitric Oxide 2006; 15(3): 177-86.
Johnson K, Ledbetter S, Nussbaum R, Rovera G, Ledbetter D. Localization of myeloperoxidase to the long arm of human chromosome 17: Relationship to the 15; 17 translocation of acute promyelocytic leukemia. Oncogene 1987; 1(3): 319-22.
Ferrini M, Wang C, Swerdloff RS, Hikim APS, Rajfer J, Gonzalez-Cadavid NF. Aging-related increased expression of inducible nitric oxide synthase and cytotoxicity markers in rat hypothalamic regions associated with male reproductive function. Neuroendocrinology 2001; 74(1): 1-11.
Sidiropoulos PI, Hatemi G, Song I-H, et al. Evidence-based recommendations for the management of ankylosing spondylitis: systematic literature search of the 3E Initiative in Rheumatology involving a broad panel of experts and practising rheumatologists. Rheumatology (Oxford) 2008; 47(3): 355-61.
Mirshafiey A, Taeb M, Mortazavi-Jahromi SS, et al. Introduction of β-d-mannuronic acid (M2000) as a novel NSAID with immunosuppressive property based on COX-1/COX-2 activity and gene expression. Pharmacol Rep 2017; 69(5): 1067-72.
Fattahi MJ, Abdollahi M, Agha Mohammadi A, et al. Preclinical assessment of β-d-mannuronic acid (M2000) as a non-steroidal anti-inflammatory drug. Immunopharmacol Immunotoxicol 2015; 37(6): 535-40.
Harman D. The biologic clock: The mitochondria? J Am Geriatr Soc 1972; 20(4): 145-7.
Hamilton ML, Van Remmen H, Drake JA, et al. Does oxidative damage to DNA increase with age? Proc Natl Acad Sci USA 2001; 98(18): 10469-74.
Stadtman ER. Protein oxidation in aging and age‐related diseases. Ann N Y Acad Sci 2001; 928(1): 22-38.
Moskovitz J, Bar-Noy S, Williams WM, Requena J, Berlett BS, Stadtman ER. Methionine sulfoxide reductase (MsrA) is a regulator of antioxidant defense and lifespan in mammals. Proc Natl Acad Sci USA 2001; 98(23): 12920-5.
Lavrovsky Y, Chatterjee B, Clark R, Roy A. Role of redox-regulated transcription factors in inflammation, aging and age-related diseases. Exp Gerontol 2000; 35(5): 521-32.
Yen J-H, Chen C-J, Tsai W-C, et al. Manganese superoxide dismutase and cytochrome P450 1A1 genes polymorphisms in rheumatoid arthritis in Taiwan. Hum Immunol 2003; 64(3): 366-73.
Lucena MI, Andrade RJ, Martínez C, et al. Glutathione S‐transferase m1 and t1 null genotypes increase susceptibility to idiosyncratic drug‐induced liver injury. Hepatology 2008; 48(2): 588-96.
Bekris LM, Shephard C, Peterson M, et al. Glutathione-s-transferase M1 and T1 polymorphisms and associations with type 1 diabetes age-at-onset. Autoimmunity 2005; 38(8): 567-75.
Gavalas NG, Akhtar S, Gawkrodger DJ, Watson PF, Weetman AP, Kemp EH. Analysis of allelic variants in the catalase gene in patients with the skin depigmenting disorder vitiligo. Biochem Biophys Res Commun 2006; 345(4): 1586-91.
Keng T, Privalle CT, Gilkeson GS, Weinberg JB. Peroxynitrite formation and decreased catalase activity in autoimmune MRL-lpr/lpr mice. Mol Med 2000; 6(9): 779.
Wong PS-Y, Eiserich JP, Reddy S, Lopez CL, Cross CE, van der Vliet A. Inactivation of glutathione S-transferases by nitric oxide-derived oxidants: Exploring a role for tyrosine nitration. Arch Biochem Biophys 2001; 394(2): 216-28.
Tanaka Y, Tran POT, Harmon J, Robertson RP. A role for glutathione peroxidase in protecting pancreatic β cells against oxidative stress in a model of glucose toxicity. Proc Natl Acad Sci USA 2002; 99(19): 12363-8.
Jang YY, Song JH, Shin YK, Han ES, Lee CS. Protective effect of boldine on oxidative mitochondrial damage in streptozotocin-induced diabetic rats. Pharmacol Res 2000; 42(4): 361-71.
St Clair E, Wilkinson WE, Lang T, et al. Increased expression of blood mononuclear cell nitric oxide synthase type 2 in rheumatoid arthritis patients. J Exp Med 1996; 184(3): 1173-8.
Van’t Hof R, Hocking L, Wright P, Ralston S. Nitric oxide is a mediator of apoptosis in the rheumatoid joint. Rheumatology (Oxford) 2000; 39(9): 1004-8.
Kolb H, Kolb-Bachofen V. Nitric oxide in autoimmune disease: Cytotoxic or regulatory mediator? Immunol Today 1998; 19(12): 556-61.
Lau D, Baldus S. Myeloperoxidase and its contributory role in inflammatory vascular disease. Pharmacol Ther 2006; 111(1): 16-26.
Choi D-K, Pennathur S, Perier C, et al. Ablation of the inflammatory enzyme myeloperoxidase mitigates features of Parkinson’s disease in mice. J Neurosci 2005; 25(28): 6594-600.
Green PS, Mendez AJ, Jacob JS, et al. Neuronal expression of myeloperoxidase is increased in Alzheimer’s disease. J Neurochem 2004; 90(3): 724-33.
Mirshafiey A, Matsuo H, Nakane S, Rehm BH, Koh C-S, Miyoshi S. Novel immunosuppressive therapy by M2000 in experimental multiple sclerosis. Immunopharmacol Immunotoxicol 2005; 27(2): 255-65.
Mirshafiey A, Rehm B, Abhari RS, Borzooy Z, Sotoude M, Razavi A. Production of M2000 (β-d-mannuronic acid) and its therapeutic effect on experimental nephritis. Environ Toxicol Pharmacol 2007; 24(1): 60-6.
Mirshafiey A, Cuzzocrea S, Rehm B, Mazzon E, Saadat F, Sotoude M. Treatment of experimental arthritis with M2000, a novel designed non‐steroidal anti‐inflammatory drug. Scand J Immunol 2005; 61(5): 435-41.
Mirshafiey A, Rehm B, Sotoude M, Razavi A, Abhari RS, Borzooy Z. Therapeutic approach by a novel designed anti-inflammatory drug, M2000, in experimental immune complex glomerulonephritis. Immunopharmacol Immunotoxicol 2007; 29(1): 49-61.
Mirshafiey A, Cuzzocrea S, Rehm B, Matsuo H. M2000: A revolution in pharmacology. Med Sci Monit 2005; 11(8): PI53-63.
Farahani MM, Motevaseli E, Maghsood F, Heidari-Kharaji M, Mirshafiey A. Anti-inflammatory property of β-D-Mannuronic acid (M2000) on expression and activity of matrix metalloproteinase-2 and-9 through CD147 molecule in phorbol myristate acetate-differentiated THP-1 cells. Iran J Allergy Asthma Immunol 2017; 16(5): 443.
Barati A, Jamshidi AR, Ahmadi H, Aghazadeh Z, Mirshafiey A. Effects of β-D-mannuronic acid, as a novel non-steroidal anti-inflammatory medication within immunosuppressive properties, on IL17, RORγt, IL4 and GATA3 gene expressions in rheumatoid arthritis patients. Drug Des Devel Ther 2017; 11: 1027.
Aletaha S, Haddad L, Roozbehkia M, et al. M2000 (β‐D‐mannuronic acid) as a novel antagonist for blocking the TLR2 and TLR4 downstream signalling pathway. Scand J Immunol 2017; 85(2): 122-9.
Ahmadi H, Jamshidi AR, Mahmoudi M, et al. The potent inhibitory effect of β-D-mannuronic acid (M2000) as a novel NSAID with immunosuppressive property on anti-cyclic citrullinated peptide antibodies, rheumatoid factor and anti-dsDNA antibodies in patients with rheumatoid arthritis. Curr Drug Discov Technol 2017; 14(3): 206-14.
Fattahi MJ, Jamshidi AR, Mahmoudi M, et al. evaluation of the efficacy and safety of β-d-mannuronic acid in patients with ankylosing spondylitis: A 12-week randomized, placebo-controlled, phase I/II clinical trial. Int Immunopharmacol 2018; 54: 112-7.
Hosseini S, Abdollahi M, Azizi G, et al. Anti-aging effects of M2000 (β-D-mannuronic acid) as a novel immunosuppressive drug on the enzymatic and non-enzymatic oxidative stress parameters in an experimental model. J Basic Clin Physiol Pharmacol 2017; 28(3): 249-55.

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Year: 2019
Page: [265 - 271]
Pages: 7
DOI: 10.2174/1570163815666180515122834
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