Brain-derived neurotrophic and immunologic factors: beneficial effects of riboflavin on motor disability in murine model of multiple sclerosis

Document Type : Original Article


1 Nutrition and Metabolic Diseases Research Center, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Khuzestan, Iran

2 Department of Nutrition, Faculty of Para-Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Khuzestan, Iran

3 Health Research Institute, Diabetes Research Center, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Khuzestan, Iran

4 Physiology Research Center, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Khuzestan, Iran

5 Department of Physiology, Faculty of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Khuzestan, Iran

6 Cell and Molecular Research Center, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Khuzestan, Iran

7 Department of Immunology, Faculty of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Khuzestan, Iran

8 Infectious and Tropical Disease Research Center, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Khuzestan, Iran

9 Department of Virology, Faculty of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Khuzestan, Iran

10 Department of Vital Statistics, Faculty of Health, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Khuzestan, Iran


Objective(s): In the present study, C57BL/6 female mice (n=56) were used to explore the neuroprotective effects of riboflavin in motor disability of experimental autoimmune encephalomyelitis (EAE) as a model of multiple sclerosis.
Materials and Methods: The animals were assigned into 7 groups: sham-operated 1 (SO1), healthy mice receiving PBS (phosphate buffer saline); sham-operated 2 (SO2), healthy mice receiving PBS and riboflavin; sham treatment 1 (ST1), EAE mice receiving water; sham treatment 2 (ST2), EAE mice receiving sodium acetate buffer; treatment 1 (T1), EAE mice receiving interferon beta-1a (INFβ-1a); treatment 2 (T2), EAE mice receiving riboflavin; treatment 3 (T3), EAE mice receiving INFβ-1a and riboflavin. After EAE induction, scoring was performed based on clinical signs. Upon detecting score 0.5, riboflavin at 10 mg/kg of body weight and/or INFβ-1a at 150 IU/g of body weight administration was started for two weeks. The brain and spinal cord levels of brain-derived neurotrophic factor (BDNF), interleukin-6 (IL-6), and interleukin-17A (IL-17A) were studied using real-time PCR and ELISA methods.
Results: BDNF expression and protein levels were increased in the brain and spinal cord of the T3 group compared with the other groups (P<0.01). IL-6 and IL-17A expressions were increased in the brains of the T3 and T1 groups, respectively, compared to the other groups (P<0.01). The daily clinical score was reduced significantly by riboflavin in both effector and chronic phases of the disease compared with that of the controls (P<0.05).
Conclusion: Our findings showed that riboflavin is capable of suppressing the neurological disability mediated by BDNF and IL-6.


1.  Bernardes D, Oliveira-Lima OC, Silva TV, Faraco CC, Leite HR, Juliano MA, et al. Differential brain and spinal cord cytokine and BDNF levels in experimental autoimmune encephalomyelitis are modulated by prior and regular exercise. J Neuroimmunol 2013;264:24-34.
2. Kim do Y, Hao J, Liu R, Turner G, Shi FD, Rho JM. Inflammation-mediated memory dysfunction and effects of a ketogenic diet in a murine model of multiple sclerosis. PLoS One 2012;7:e35476.
3.Srivastava P, Mujtaba M, Singhal M. Gene and Cytokines expression of Multiple Sclerosis and its Therapeutic Regimen: A Systemic Review. Int J Drug Dev & Res 2012;4:55-66.
4. Erta M, Quintana A, Hidalgo J. Interleukin-6, a major cytokine in the central nervous system. Int J Biol Sci 2012;8:1254-66.
5. Azoulay D, Urshansky N, Karni A. Low and dysregulated BDNF secretion from immune cells of MS patients is related to reduced neuroprotection. J Neuroimmunol 2008;195(1-2).
6. Dhib-Jalbut S, Marks S. Interferon-beta mechanisms of action in multiple sclerosis. Neurology 2010;74 Suppl 1:S17-24.
7. Ramgolam VS, Sha Y, Jin J, Zhang X, Markovic-Plese S. IFN-beta inhibits human Th17 cell differentiation. J Immunol 2009;183:5418-27.
8.Axtell RC, de Jong BA, Boniface K, van der Voort LF, Bhat R, De Sarno P, et al. T helper type 1 and 17 cells determine efficacy of interferon-beta  in multiple sclerosis and experimental encephalomyelitis. Nat Med 2010; 16:406-12.
9.   Gallager M. The Nutrients and Their Metabolism. In: Mahan L ES, editor. Krausés Food & Nutrition Therapy. Philadelphia: Saunders; 2008. p. 74-8, 84-6.
10. Ogunleye AJ OA. The effect of riboflavin deficiency on cerebrum and cerebellum of developing rat brain. J Nutr Sci Vitaminol (Tokyo) 1989;35:193-7.
11. Cai Z, Blumbergs PC, Finnie JW, Manavis J, Thompson PD. Selective vulnerability of peripheral nerves in avian riboflavin deficiency demyelinating polyneuropathy. Vet Pathol 2009;46:88-96.
12. Johnson WD, Storts RW. Peripheral neuropathy associated with dietary riboflavin deficiency in the chicken. I. Light microscopic study. Vet Pathol 1988;25:9-16.
13. Cai Z, Finnie JW, Blumbergs PC. Avian riboflavin deficiency: an acquired tomaculous neuropathy. Vet Pathol 2006;43:780-1.
14. Wada Y, Kondo H, Itakura C. Peripheral neuropathy of dietary riboflavin deficiency in racing pigeons. J Vet Med Sci 1996;58(2):161-3.
15. Jortner BS, Cherry J, Lidsky TI, Manetto C, Shell L. Peripheral neuropathy of dietary riboflavin deficiency in chickens. J Neuropathol Exp Neurol 1987;46:544-55.
16. Cai Z, Blumbergs PC, Finnie JW, Manavis J, Thompson PD. Novel fibroblastic onion bulbs in a demyelinating avian peripheral neuropathy produced by riboflavin deficiency. Acta Neuropathol 2007;114:187-94.
17. Cai Z, Finnie JW, Blumbergs PC, Manavis J, Ghabriel MN, Thompson PD. Early paranodal myelin swellings (tomacula) in an avian riboflavin deficiency model of demyelinating neuropathy. Exp Neurol 2006;198:65-71.
18. Phillips  PH ER. Neuromalacia Associated with Low Riboflavin Diets, a Preliminary Report. Poul Sci 1983;17:463-5.
19. Phillips  PH ER. The histopathology of neuromalacia and ‘‘curled toe’’ paralysis in the chick fed low riboflavin diets. J Nutr 1983;16:451-63.
20. Wyatt RD TH, Donaldson WE, Hamilton  PB A new description of riboflavin deficiency syndrome in chickens. Poul Sci 1973;52:237-44.
21. Ghadirian P, Jain M, Ducic S, Shatenstein B, Morisset R. Nutritional factors in the aetiology of multiple sclerosis: a case-control study in Montreal, Canada. Int J Epidemiol 1998;27:845-52.
22. Mao P, Reddy PH. Is multiple sclerosis a mitochondrial disease? Biochim Biophys Acta 2010;1802:66-79.
23. Kalinowska-Lyszczarz A, Losy J. The role of neurotrophins in multiple sclerosis-pathological and clinical implications. Int J Mol Sci 2012;13:13713-25.
24. Ashoori M, Saedisomeolia A. Riboflavin (vitamin B2) and oxidative stress: a review. Br J Nutr 2014:1-7.
25. Bittner S, Afzali AM, Wiendl H, Meuth SG. Myelin oligodendrocyte glycoprotein (MOG35-55) induced experimental autoimmune encephalomyelitis (EAE) in C57BL/6 mice. J Vis Exp 2014:1-5.
26. Kafami L, Raza M, Razavi A, Mirshafiey A, Movahedian M, Khorramizadeh MR. Intermittent feeding attenuates clinical course of experimental autoimmune encephalomyelitis in C57BL/6 mice. Avicenna J Med Biotechnol 2010;2:47-52.
27. Feitoza FC VL. The response of young and adult rats to the riboflavin supplementation. Braz. arch. biol. technol. (4):855-60.
28. Liu J, Wu JQ, Yang JJ, Wei JY, Gao WN, Guo CJ. Metabolomic study on vitamins B(1), B(2), and PP supplementation to improve serum metabolic profiles in mice under acute hypoxia based on (1)H NMR analysis. Biomed Environ Sci 2010;23:312-8.
29. Soleimani M, Jameie SB, Barati M, Mehdizadeh M, Kerdari M. Effects of coenzyme Q10 on the ratio of TH1/TH2 in experimental autoimmune encephalomyelitis model of multiple sclerosis in C57BL/6. Iran Biomed J 2014;18:203-11.
30. Xu Q, Ming Z, Dart AM, Du XJ. Optimizing dosage of ketamine and xylazine in murine echocardiography. Clin Exp Pharmacol Physiol 2007;34:499-507.
31. Datta SC, Opp MR. Lipopolysaccharide-induced increases in cytokines in discrete mouse brain regions are detectable using Luminex xMAP technology. J Neurosci Methods 2008;175:119-24.
32. Ogonovszky H, Berkes I, Kumagai S, Kaneko T, Tahara S, Goto S, et al. The effects of moderate-, strenuous- and over-training on oxidative stress markers, DNA repair, and memory, in rat brain. Neurochem Int 2005;46:635-40.
33. Nosrat IV, Margolskee RF, Nosrat CA. Targeted taste cell-specific overexpression of brain-derived neurotrophic factor in adult taste buds elevates phosphorylated TrkB protein levels in taste cells, increases taste bud size, and promotes gustatory innervation. J Biol Chem 2012;287:16791-800.
34. Pfaffl MW. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 2001;29:e45.
35. Zhu W FE, Begum F, Vora P, Au K, Gong Y, MacNeil B, Pillai P, Namaka M. The role of dorsal root ganglia activation and brain-derive neurotrophic factor in multiple sclerosis. J Cell Mol Med 2012;16:1856–65.
36. Mieno MN, Yamaguchi T, Ohashi Y. Alternative statistical methods for estimating efficacy of interferon beta-1b for multiple sclerosis clinical trials. BMC Med Res Methodol 2011;11:80.
37. Naghashpour M, Majdinasab N, Shakerinejad G, Kouchak M, Haghighizadeh MH, Jarvandi F, et al. Riboflavin supplementation to patients with multiple sclerosis does not improve disability status nor is riboflavin supplementation correlated to homocysteine. Int J Vitam Nutr Res 2013; 83:281-90.
38. Cicek G, Schiltz E, Hess D, Staiger J, Brandsch R. Analysis of mitochondrial antigens reveals inner membrane succinate dehydrogenase flavoprotein subunit as autoantigen to antibodies in anti-M7 sera. Clin Exp Immunol 2002; 128:83-7.
39. Reese D, Shivapour ET, Wahls TL, Dudley-Javoroski SD, Shields R. Neuromuscular electrical stimulation and dietary interventions to reduce oxidative stress in a secondary progressive multiple sclerosis patient leads
to marked gains in function: a case report. Cases J 2009; 2:7601.
40. Hamamcioglu K, Reder AT. Interferon-beta regulates cytokines and BDNF: greater effect in relapsing than in progressive multiple sclerosis. Mult Scler 2007;13: 459-70.
41. El-behi M, Rostami A, Ciric B. Current views on the roles of Th1 and Th17 cells in experimental autoimmune encephalomyelitis. J Neuroimmune Pharmacol 2010; 5:189-97.
42. De Santi L, Annunziata P, Sessa E, Bramanti P. Brain-derived neurotrophic factor and TrkB receptor in experimental autoimmune encephalomyelitis and multiple sclerosis. J Neurol Sci 2009; 287:17-26.
43. Pezet S, Malcangio M, McMahon SB. BDNF: a neuromodulator in nociceptive pathways? Brain Res Brain Res Rev 2002; 40:240-9.
44. Cho HJ, Kim JK, Zhou XF, Rush RA. Increased brain-derived neurotrophic factor immunoreactivity in rat dorsal root ganglia and spinal cord following peripheral inflammation. Brain Res 1997; 764:269-72.
45. Benveniste EN, Whitaker JN, Gibbs DA, Sparacio SM, Butler JL. Human B cell growth factor enhances proliferation and glial fibrillary acidic protein gene expression in rat astrocytes. Int Immunol 1989;1:219-28.
46. Selmaj KW, Farooq M, Norton WT, Raine CS, Brosnan CF. Proliferation of astrocytes in vitro in response to cytokines. A primary role for tumor necrosis factor. J Immunol 1990;144:129-35.
47. Levison SW, Jiang FJ, Stoltzfus OK, Ducceschi MH. IL-6-type cytokines enhance epidermal growth factor-stimulated astrocyte proliferation. Glia 2000;32:328-37.
48. Frei K, Malipiero UV, Leist TP, Zinkernagel RM, Schwab ME, Fontana A. On the cellular source and function of interleukin 6 produced in the central nervous system in viral diseases. Eur J Immunol 1989;19:689-94.
49. Murphy PG, Borthwick LA, Altares M, Gauldie J, Kaplan D, Richardson PM. Reciprocal actions of interleukin-6 and brain-derived neurotrophic factor on rat and mouse primary sensory neurons. Eur J Neurosci 2000;12:1891-9.
50. Nakafuku M, Satoh T, Kaziro Y. Differentiation factors, including nerve growth factor, fibroblast growth factor, and interleukin-6, induce an accumulation of an active Ras.GTP complex in rat pheochromocytoma PC12 cells. J Biol Chem 1992;267:19448-54.
51. Satoh T, Nakamura S, Taga T, Matsuda T, Hirano T, Kishimoto T, et al. Induction of neuronal differentiation in PC12 cells by B-cell stimulatory factor 2/interleukin 6. Mol Cell Biol 1988; 8:3546-9.
52. Galligan CL, Pennell LM, Murooka TT, Baig E, Majchrzak-Kita B, Rahbar R, et al. Interferon-beta is a key regulator of proinflammatory events in experimental autoimmune encephalomyelitis. Mult Scler 2010; 16:1458-73.