Journal of Food and Nutrition Research
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Journal of Food and Nutrition Research. 2015, 3(6), 384-391
DOI: 10.12691/jfnr-3-6-5
Open AccessArticle

Nitric Oxide and Inflammatory Cytokine Productions in Diabetic Rats Supplemented with Goat Milk and Soy Milk Kefir

Nurliyani1, , Eni Harmayani2 and Sunarti3

1Department of Animal Product Technology, Faculty of Animal Science, Universitas Gadjah Mada, Jl. Fauna 3 Kampus UGM, Bulaksumur, Yogyakarta 55281, Indonesia

2Faculty of Agricultural Technology, Universitas Gadjah Mada, Yogyakarta, Indonesia

3Department of Biochemistry, Faculty of Medicine, Universitas Gadjah Mada, Yogyakarta, Indonesia

Pub. Date: July 05, 2015

Cite this paper:
Nurliyani, Eni Harmayani and Sunarti. Nitric Oxide and Inflammatory Cytokine Productions in Diabetic Rats Supplemented with Goat Milk and Soy Milk Kefir. Journal of Food and Nutrition Research. 2015; 3(6):384-391. doi: 10.12691/jfnr-3-6-5


Diabetes has been known as an inflammatory diseases, and the certain of probiotics when administered in adequate amounts were reported have an anti-inflammatory properties. The purpose of this study were to evaluate the effect of administration of goat milk and soy milk kefir on immune responses including spleen lymphocyte proliferation, production of pro-inflammatory cytokine (TNF-α) and anti-inflammatory cytokine (IL-10), as well as peritoneal macrophage nitric oxide (NO) production in diabetic rats induced with streptozotocin-nicotinamide. Male Wistar rats were divided into five groups: negative control, positive control, diabetic rats fed goat milk kefir, diabetic rats fed kefir combination from goat milk and soy milk kefir and diabetic rats fed soy milk kefir. After 5 weeks treatment, the rats were sacrificed and sampled for spleen and peritoneal macrophages. The results showed that kefir combination from goat milk and soy milk kefir could increase lymphocyte proliferation and IL-10 production, as well as decreased TNF-α significantly (p<0.05) in diabetic rats. However, the effect of kefir combination showed the lowest NO production in diabetic rats compared with goat milk kefir and soy milk kefir. In conclusion, kefir combination from goat milk and soy milk may be used as biotherapeutic for type-2 diabetes mellitus by improving systemic immune responses through increasing in lympocyte proliferation and IL-10 as well as decreasing in TNF-α production.

goat milk kefir soy milk kefir lymphocyte proliferation IL-10 TNF-α Nitric oxide diabetic

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[1]  Tripathi, M. K . “Comforts in Quality and Production of Goat Milk” J Adv Dairy Res. 3(1), 1-2, 2015.
[2]  Alferez, M. J. M., Barrionuevo, M., Aliaga, L. L., Sanz-Sampelayo, M. R., Lisbona, F., Robles, J. C. and Campos, M. S. “Digestive utilization of goat and cow milk fat in malabsorption syndrome.” J Dairy Res. 68, 451-461, 2001.
[3]  Dierick, N. A., Decuypere, J. A. and Degeyter, I. “(2003). The combined use of whole cuphea seeds containing medium chain fatty acids and an exogenous lipase in piglet nutrition”. Archiv fur Tierernahrung 57, 49-63, 2003.
[4]  Wang, J., Wu, X., Simonavicius, N., Tian, H. and Ling, L. “Medium-chain fatty acids as ligands for orphan G proteincoupled receptor GPR84”. J. Biol. Chem. 281, 34457-34464, 2006.
[5]  Wu, F. Y. and Elsasser, T. H. “Studies on cell growth promoting activity in goat milk.” J. Chinese Agric. Chem. Society 33, 326-332, 1995.
[6]  Bergmann, R. S. D. O., Pereira, M. A., Veiga, S. M. O. M., Schneedorf, J. M., Oliveira, N. D. M. S. and Fiorin, J. E. “Microbial profile of a kefir sample preparations - grains in natura and lyophilized and fermented suspension”. Ciênc. Tecnol.. Ciênc. Tecnol. Aliment., Campinas, 30(4), 1022-1026, 2010.
[7]  Rattray, F. P. and O’Connell, M. J. “Fermented Milks Kefir”. In: Fukay JW, editor. Encyclopedia of Dairy Sciences. 2th ed. Academic Press; San Diego, USA. pp. 518-524, 2011.
[8]  Marquina, D., Santos, A., Corpas, I., Munoz, J., Zazo, J. and Peinado, J. M. “Dietary influence of kefir on microbial activities in the mouse bowel”. Lett Appl Microbiol. 35, 136-140, 2002.
[9]  Zhang, Y. and Zhang, H. “Microbiota associated with type 2 diabetes and its related complications”. Food Science and Human Wellness 2, 167-172, 2013.
[10]  Holmes, E., Li, J. V., Athanasiou, T., Ashrafian, H. and Nicholson, J. K. “Understanding the role of gut microbiome–host metabolic signal disruption in health and disease”. Trends Microbiol. 19(7), 349-359, Jul 2011.
[11]  Tisoncik, J. R., Korth, M. J., Simmons, C. P., Farrar, J., Martin, T. R. and Katze, M. G. “ Into the Eye of the Cytokine Storm” Microbiol. Mol. Biol. Rev. 76(1), 16-32 , March 2012.
[12]  Fatima, N., Shameem, M., Nabeela, Khan, P. A. and Khan, H. M. “Correlation of serum IL-4 levels in new and MDR tuberculosis patients.” AJCEM 3(1), 48-51, 2015.
[13]  Hong, E. G., Ko, H. J., Cho, Y. R., Kim, H. J., Ma, Z., Yu, T. Y., Friedline, R. H., Kurt-Jones, E., Finberg, R., Fischer, M. A., Granger, E. L., Norbury, C. C., Hauschka, S. D., William M. Philbrick, W. M., Lee, C. G., Elias, J. A. and Kim, J. K. “ Interleukin-10 prevents diet-induced insulin resistance by attenuating macrophage and cytokine response inskeletal muscle” . Diabetes 58, 2525-2535, 2009.
[14]  Pickup, J. C. and Crook, M. A. “Is type II diabetes mellitus a disease of the innate immune system?” Diabetologia 41(10), 1241-1248, Oct 1998.
[15]  Kern, P. A., Ranganathan, S., Li, C., Wood, L. and Ranganathan, G. “Adipose tissue tumor necrosis factor and interleukin-6 expression in human obesity and insulin resistance”. Am J Physiol Endocrinol Metab. 280(5), E745-E751, May 2001.
[16]  Hotamisligil, G. S., Shargill, N. S. and Spiegelman, B. M. “Adipose expression of tumor necrosis factor-alpha: direct role in obesity-linked insulin resistance”. Science 259, 87-91, 1993.
[17]  Trinchieri, G. “Interleukin-10 production by effector T cells: Th1 cells show self control”. JEM . 204 ( 2), 239-243, Feb 2007.
[18]  Cavalcanti, Y. V. N., Brelaz, M. C. A., Neves, J. K. D. A. L., Ferraz, J. C. and Valéria Pereira, V. R. A. ”Role of TNF-alpha, IFN-gamma, and IL-10 in the development of pulmonary tuberculosis. Pulmonary Medicine 2012, 1-10, 2012.
[19]  Van Exel, E., Gussekloo, J., de Craen, A. J., Frolich, M., Van Der Wiel, B. A., Westendorp, R. G. “Low production capacity of interleukin-10 associates with the metabolic syndrome and type 2 diabetes: the Leiden 85-Plus Study”. Diabetes 51, 1088-1092, 2002.
[20]  Pennline, K. J., Roque-Gaffney, E. and Monahan, M. “Recombinant human IL-10 prevents the onset of diabetes in the nonobese diabetic mouse”. Clin Immunol Immunopathol 71, 169-175, 1994.
[21]  Hadisaputro, S., Djokomoeljanto, R. R. J., Judiono, Soesatyo, M. H. N. E. “The effects of oral plain kefir supplementation on proinflammatory cytokine properties of the hyperglycemia Wistar rats induced by streptozotocin.” Acta Med Indones. 44, 100-104, 2012.
[22]  Stewart, A. G., Dusting, G. J., Giarracca, R. G., Harris, T., Lim, Y. and Sobey, C. G. “ Nitrite is produced by elicited but not by circulating neutrophils.” Mediat Inflamm 2, 349-356, 1993.
[23]  El-Mahmoudy, A., Shimizu, Y., Shiina, T., Matsuyama, H., Nikami , H. and Takewaki, T. “Macrophage-derived cytokine and nitric oxide profiles in type I and type II diabetes mellitus: effect of thymoquinone. ” Acta Diabetol. 42, 23-30, 2005.
[24]  Jain, S. K., Rains, J. L. and Croad, J. L. “Effect of Chromium Niacinate and Chromium Picolinate Supplementation on lipid peroxidation, TNF-α, IL-6, CRP, glycated hemoglobin, triglycerides and cholesterol levels in blood of streptozotocin-treated diabetic rats”. Free Radic Biol Med. 43(8), 1124-1131, Oct 2007.
[25]  Galen M. Pieper, G. M. “Review of alterations in endothelial nitric oxide production in diabetes. Protective role of arginine on endothelial dysfunction.” Hypertension. 31, 1047-1060, 1998.
[26]  Fang, F. C. “Mechanisms of Nitric Oxide–related Antimicrobial Activity.” J. Clin. Invest. 99 (12), 2818-2825, Jun 1997.
[27]  Kolios, G., Valatas, V. and Ward, S. G. “Nitric oxide in inflammatory bowel disease: a universal messenger in an unsolved puzzle”. Immunology 113(4), 427-437, Dec 2004.
[28]  Walker, R., Bone, A. J., Cooke, A. and Baird, J. D. “Distinct macrophage subpopulations in pancreas of prediabetic bb/e rats possible role for macrophages in pathogenesis of IDDM”. Diabetes 37:1301-304, 1988.
[29]  Kasenkas, H., Dinkci, N., Seckin, K., Kinik, O., Gonc, S. “ Antioxidant properties of kefir produced from differentcow and soy milk mixtures.” J. Agric. Sci. 17, 253‐259, 2011.
[30]  Reeves, P. G., Neilsen, F. H. and Fahey, G. C. JR. “AIN-93 purified diets for laboratory rodents: Final Report of the American Institute of Nutrition Ad Hoc Writing Committee on the Formulation of the AIN-76A Rodent Diet.” J. Nutr. 123, 1939-1951, 1993.
[31]  Ghasemi, A., Khalifi, S., Jedi, S. “Streptozotocin-nicotinamide-induced rat model of type 2 diabetes.” Acta Physiol Hung. 101, 408-420, 2014.
[32]  Jain, S., Yadav, H., Sinha, P. R., Naito, Y. and Marotta, F. “Dahi containing probiotic Lactobacillus acidophilus and Lactobacillus casei has a protective effect against Salmonella enteritidis infection in mice” Int. J. Immunopathol. Pharmacol. 21 (4), 1025-1033, 2008.
[33]  Keller, J.M., McClellan-Greena, P.D., Lee, A.M., Arendt, M.D., Maier, P.P., Segars, A. L., Whitaker, J.D., Keil, D. E., and Peden-Adams, M.M. “Mitogen-induced lymphocyte proliferation in loggerhead sea turtles: comparison of methods and effects of gender, plasma testosterone concentration, and body condition on immunity.” Vet. Immunol. Immunopathol. 103, 269-281, 2005.
[34]  Garcıa, D., Delgado, R., Ubeira, F. M. and Leiro, J. “Modulation of rat macrophage function by the Mangifera indica L. extracts Vimang and mangiferin. Int Immunopharmacol. 2, 797-806, 2002.
[35]  Zykova, S. N., Jenssen, T. G., Berdal, M., Olsen, R., Myklebust, R. and Seljelid, R. “Altered cytokine and nitric oxide secretion in vitro by macrophages from diabetic type II–like db/db mice. Diabetes 49, 1451-1458, 2000.
[36]  Titheradge, M. A. “Nitric Oxide Protocols.” Humana Press Inc., Totowa, New Jersey. 1998.
[37]  Warif, N. M. A., Ayob, A. A., Razali, W. M W., Budin, S. B., Zainalabidin, S. and Mohamed, J. “Effects of Hibiscus sabdariffa Linn. aqueous extract on oxidative stress and T lymphocyte.population in spleen of streptozotocin-induced diabetic rats.” Jurnal Sains Kesihatan Malaysia 12 (1), 15-21, 2014.
[38]  Maher M Al-Enazi. “Combined therapy of rutin and silymarin has more protective effects on streptozotocin-induced oxidative stress in rats.” JAPS. 4 (1), 21- 28, Jan 2014.
[39]  Lee, S. I., Kim, J. S., Oh, S. H., Park, K. Y., Lee, H. G. and Kim, S. D. “Antihyperglycemic effect of Fomitopsis pinicola extracts in streptozotocin-induced diabetic rats.” J Med Food. 11(3), 518-24, Sept 2008.
[40]  Muller, Y. D., Golshayan, D., Ehirchiou, D., Wyss, J. C., Giovannoni, L., Maier, R., Serre-Beiner, V., Yung, G. P., Morel, P., Buhler, L. H. & Seebach, J. D. “Immunosuppressive effects of streptozotocin-induced diabetes result in absolute lymphopenia and relative increase of T regulatory cells.” Diabetes 60(9), 2331-2340, 2011.
[41]  Gaulton, G. N., Schwartz, J. L., Eardley, D. D. “Assessment of the diabetogenic drugs alloxan and streptozotocin as models for the study of immune defects in diabetic mice.” Diabetologia 28, 769-775, 1985.
[42]  Martinez-Villaluenga, C., Torino, M. I., Martín, V., Arroyo, R., Garcia-Mora, P., Pedrola, I. E., Vidal-Valverde, C., Rodriguez, J. M. and Frias, J. “Multifunctional properties of soy milk fermented by Enterococcus faecium strains isolated from raw soy milk.” J Agric Food Chem. 60(41), 10235-10244, Oct 2012.
[43]  Kullisaar, T., Songisepp, E., Mikelsaar, M., Zilmer, K., Vihalemm, T. and Zilmer, M. “Antioxidative probiotic fermented goats' milk decreases oxidative stress-mediated atherogenicity in human subjects.” Br J Nutr. 90(2), 449-456, Aug 2003.
[44]  Jain, S., Yadav, H. and Sinha, P. R. “Probiotic dahi containing Lactobacillus casei protects against Salmonella enteritidis infection and modulates immune response in mice.” J Med Food. 12(3), 576-583, Jun 2009.
[45]  Maroof, H., Hassan, Z. M., Mobarez, A. M. and Mohamadabadi, M. A. “Lactobacillus acidophilus could modulate the immune response against breast cancer in murine model. J Clin Immunol., 1-12, Jun 2012.
[46]  Rubinstein, R., Genaro, A. M., Motta, A., Cremaschi, G. and Wald, M. R. Impaired immune responses in streptozotocin-induced type I diabetes in mice. Involvement of high glucose. Clin Exp Immunol. 154, 235-246, 2008.
[47]  Salih, S. F. “Lymphocytes apoptosis in type 2 diabetes mellitus.” Iraqi J. Comm. Med. 4, 330-332, Oct 2012.
[48]  Gomes, A. C., Bueno, A. A., de Souza, R. G. M. and João Felipe Mota. Gut microbiota, probiotics and diabetes.” J. Nutr. 13(60), 1-13, 2014.
[49]  Lee, C.C. and Liu, S. “Role of inflammatory cytokines in type 2 di abetes.” Review of Endocrinology, 19-21, Feb 2008.
[50]  Carasi, P., Racedo, S. M., Serradell, M. A., Jacquot, C. and Urdaci, M. C. “Impact of kefir derived lactobacillus kefiri on the mucosal immune response and gut microbiota.” J Immunol Res. 1-12, 2015.
[51]  Hashem, R. M., Mahmoud, M. F., El-Moselhy, M. A. and Soliman, H. M. “Interleukin-10 to tumor necrosis factor-alpha ratio is a predictive biomarker in nonalcoholic fatty liver disease: interleukin-10 to tumor necrosis factor-alpha ratio in steatohepatitis.” Eur J Gastroenterol Hepatol. 20(10), 995-1001, Oct 2008.
[52]  Kang, J. W., Choi, S. C., Cho, M. C., Kim, H. J., Kim, J. H., Lim, J. S., Kim, S. H., Han, J. Y. and Yoon, D. Y. “A proinflammatory cytokine interleukin-32β promotes the production of an anti-inflammatory cytokine interleukin-10.” Immunology . 128(1 Pt 2), e532–e540, Sept 2009.
[53]  Hsieh, F. C., Lee, C. L., Chai, C. Y., Chen, W. T., Lu, Y. C. and Wu, C. S. “Oral administration of Lactobacillus reuteri GMNL-263 improves insulin resistance and ameliorates hepatic steatosis in high fructose-fed rats.” J Nutr Metab. 10(35), 1-14, 2013.
[54]  Lescheid, D. W. “Probiotics as regulators of inflammation”. Funct. Food Health Dis. 4(7), 299-311, 2014.
[55]  Radhika, P., Annapurna, A. and Rao, S. N. “Immunostimulant, cerebroprotective & nootropic activities of Andrographis paniculata leaves extract in normal & type 2 diabetic rats”. Indian J Med Res. 135, 636-641, May 2012.
[56]  Thomas, C. M. and Versalovic, J. “Probiotics-host communication: Modulation of signaling pathways in the intestine”. Gut Microbes, 1(3), 148-163, 2010.
[57]  Aritrina, P. “The effect of kefir combination from goat milk and soy milk on plasma tumor necrosis factor alpha (TNF-α) and inducible nitric oxide gene expression of hepar in rats induced with Streptozotocin and nicotineamide.” Thesis. Faculty of Medicine, Universitas Gadjah Mada., Indonesia. 2014.
[58]  Foley, E. and O’Farrell, P. H.“Nitric oxide contributes to induction of innate immune responses to Gram-negative bacteria in Drosophila”. Genes & Development 17, 115-125, 2003.
[59]  Ibiza, S., and Serrador, J. M. “The role of nitric oxide in the regulation of adaptive immune responses” Immunología 27 (3), 103-117, 2008.
[60]  Bogdan, C. “Nitric oxide and the immune response”. Nat. Immunol. 2(10), 907-916, Oct 2001.
[61]  Chang, P. V., Hoa, L., Offermanns, S. and Medzhitov, R. The microbial metabolite butyrate regulates intestinal macrophage function via histone deacetylase inhibition. Proc Natl Acad Sci USA, 111(6), 2247-2252, 2014.
[62]  Takeuchi, O., Sato, S., Horiuchi, T., Hoshino, K., Takeda, K., Dong, Z., Modlin, R. L. and Akira, S. “Role of toll-like receptor 1 in mediating immune response to microbial lipoproteins.” J. Immunol. 169, 10-14, 2002.
[63]  Villena, J. and Kitazawa, H. “Modulation of intestinal TLR4-inflammatory signaling pathways by probiotic microorganisms: lessons learned from Lactobacillus jensenii TL2937.” Frontiers in Immunology, 4, 1-12, Jan 2014.
[64]  Stempelj, M., Kedinger, M., Augenlicht, L. and Klampfer, L. “Essential role of the JAK/STAT1 signaling pathway in the expression of inducible nitric-oxide synthase in intestinal epithelial cells and its regulation by butyrate.” J Biol Chem., 282, 9797-9804, March 2007.
[65]  Nathan, C. and Xie, Q. W. 1994. “Regulation of biosynthesis of nitric oxide.” J. Biol. Chem. 269, 13725-13728, 1994.
[66]  Itoh, T., Miyake, Y., Onda, A., Kubo, J., Ando, M., Tsukamasa, Y. and Takahata, M. “Immunomodulatory effects of heat-killed Enterococcus faecalis TH10 on murine macrophage cells” Microbiology Open, 1(4), 373-380, 2012.
[67]  Bermudez-Brito, M., Plaza-Díaz, J., Muñoz-Quezada, S., Gómez-Llorente, C. and Gil, A. “Probiotic mechanisms of action. Ann Nutr Metab., 61, 160-174, 2012.
[68]  Hassa, P. O., Haenni, S. S., Buerki, C., Meier, N. I., S. Lane, W. S., Owen, H., Gersbach, M., Imhof , R. and Hottiger, M. O. “Acetylation of poly(ADP-ribose) polymerase-1 by p300/CREB-binding protein regulates coactivation of NF-kappa B-dependent transcription.” J Biol Chem., 280, 40450-4046, Dec 2005.
[69]  Kassan, M., Choi, S. K., Galán, M., Bishop, A., Umezawa K., Trebak, M., Belmadani, S. and Matrougui , K. “Enhanced NF-κB activity impairs vascular function through PARP-1-, SP-1-, and COX-2-dependent mechanisms in type 2 diabetes. ” Diabetes, 62(6), 2078-87, Jun 2013.