Abstract
Aim:
To investigate the influences of betulinic acid (BA), a triterpenoid isolated from birch bark, on neuroinflammatory mediators involved in the pathogenesis of multiple sclerosis and experimental autoimmune encephalomyelitis in vitro.
Methods:
Encephalitogenic T cells were prepared from draining lymph nodes and spinal cords of Dark Agouti rats 8 to 10 d after immunization with myelin basic protein (MBP) and complete Freund's adjuvant. Macrophages were isolated from the peritoneal cavity of adult untreated rats. Astrocytes were isolated from neonatal rat brains. The cells were cultured and then treated with different agents. IFN-γ, IL-17, iNOS and CXCL12 mRNA levels in the cells were analyzed with RT-PCR. iNOS and CXCL12 protein levels were detected using immunoblot. NO and ROS generation was measured using Griess reaction and flow cytometry, respectively.
Results:
In encephalitogenic T cells stimulated with MBP (10 μg/mL), addition of BA inhibited IL-17 and IFN-γ production in a dose-dependent manner. The estimated IC50 values for IL-17 and IFN γ were 11.2 and 63.8 μmol/L, respectively. When the macrophages were stimulated with LPS (10 ng/mL), addition of BA (50 μmol/L) significantly increased ROS generation, and suppressed NO generation. The astrocytes were stimulated with ConASn containing numerous inflammatory mediators, which mimicked the inflammatory milieu within CNS; addition of BA (50 μmol/L) significantly increased ROS generation, and blocked ConASn-induced increases in iNOS and CXCL12 mRNA levels, but did not affect iNOS and CXCL12 protein levels. Importantly, in both the macrophages and astrocytes, addition of BA (50 μmol/L) inhibited lipid peroxidation.
Conclusion:
Besides inhibiting encephalitogenic T cell cytokines and reducing NO generation, BA induces tissue-damaging ROS generation within CNS.
Similar content being viewed by others
Log in or create a free account to read this content
Gain free access to this article, as well as selected content from this journal and more on nature.com
or
References
Constantinescu CS, Farooqi N, O'Brien K, Gran B . Experimental autoimmune encephalomyelitis (EAE) as a model for multiple sclerosis (MS). Br J Pharmacol 2011; 164: 1079–106.
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.
Misko TP, Trotter JL, Cross AH . Mediation of inflammation by encephalitogenic cells: interferon gamma induction of nitric oxide synthase and cyclooxygenase 2. J Neuroimmunol 1995; 61: 195–204.
Hewett SJ, Misko TP, Keeling RM, Behrens MM, Choi DW, Cross AH . Murine encephalitogenic lymphoid cells induce nitric oxide synthase in primary astrocytes. J Neuroimmunol 1996; 64: 201–8.
Haider L, Fischer MT, Frischer JM, Bauer J, Höftberger R, Botond G, et al. Oxidative damage in multiple sclerosis lesions. Brain 2011; 134: 1914–24.
Cross AH, Manning PT, Stern MK, Misko TP . Evidence for the production of peroxynitrite in inflammatory CNS demyelination. J Neuroimmunol 1997; 80: 121–30.
Smith KJ, Kapoor R, Felts PA . Demyelination: the role of reactive oxygen and nitrogen species. Brain Pathol 1999; 9: 69–92.
Okuda Y, Sakoda S, Fujimur H, Yanagihara T . Aminoguanidine a selective inhibitor of the inducible nitric oxide synthase has different effects on experimental allergic encephalomyelitis in the induction and progression phase. J Neuroimmunol 1998; 8: 201–10.
Miljković D, Timotijević G, Mostarica Stojković M . Astrocytes in the tempest of multiple sclerosis. FEBS Lett 2011; 585: 3781–8.
Momcilović M, Mostarica-Stojković M, Miljković D . CXCL12 in control of neuroinflammation. Immunol Res 2012; 52: 53–63.
Saidha S, Eckstein C, Calabresi PA . New and emerging disease modifying therapies for multiple sclerosis. Ann N Y Acad Sci 2012; 1247: 117–37.
Yogeeswari P, Sriram D . Betulinic acid and its derivatives: a review on their biological properties. Curr Med Chem 2005; 12: 657–66.
Kommera H, Kaluđerović GN, Kalbitz J, Paschke R . Lupane triterpenoids — betulin and betulinic acid derivatives induce apoptosis in tumor cells. Inv New Drugs 2011; 29: 266–72.
McCarthy KD, de Vellis J . Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue. J Cell Biol 1980; 85: 890–902.
Draper HH, Hadley M . Malondialdehyde determination as index of lipid peroxidation. Methods Enzymol 1990; 186: 421–31.
Momcilović M, Miljković Z, Popadić D, Miljković D, Mostarica-Stojković M . Kinetics of IFN-gamma and IL-17 expression and production in active experimental autoimmune encephalomyelitis in Dark Agouti rats. Neurosci Lett 2008; 447: 148–52.
Miljkovic D, Stosic-Grujicic S, Markovic M, Momcilovic M, Ramic Z, Maksimovic-Ivanic D, et al. Strain difference in susceptibility to experimental autoimmune encephalomyelitis between Albino Oxford and Dark Agouti rats correlates with disparity in production of IL-17 but not nitric oxide. J Neurosci Res 2006; 84: 379–88.
Markovic M, Miljkovic D, Momcilovic M, Popadic D, Miljkovic Z, Savic E, et al. Strain difference in susceptibility to experimental autoimmune encephalomyelitis in rats correlates with T(H)1 and T(H)17-inducing cytokine profiles. Mol Immunol 2009; 47: 141–6.
Matusevicius D, Kivisäkk P, He B, Kostulas N, Ozenci V, Fredrikson S, et al. Interleukin-17 mRNA expression in blood and CSF mononuclear cells is augmented in multiple sclerosis. Mult Scler 1999; 5: 101–4.
Kebir H, Kreymborg K, Ifergan I, Dodelet-Devillers A, Cayrol R, Bernard M, et al. Human TH17 lymphocytes promote blood-brain barrier disruption and central nervous system inflammation. Nat Med 2007; 13: 1173–5.
Tzartos JS, Friese MA, Craner MJ, Palace J, Newcombe J, Esiri MM, et al. Interleukin-17 production in central nervous system-infiltrating T cells and glial cells is associated with active disease in multiple sclerosis. Am J Pathol 2008; 172: 146–55.
Link J, Söderström M, Olsson T, Höjeberg B, Ljungdahl A, Link H . Increased transforming growth factor-beta interleukin-4 and interferon-gamma in multiple sclerosis. Ann Neurol 1994; 36: 79–86.
Panitch HS, Hirsch RL, Haley AS, Johnson KP . Exacerbations of multiple sclerosis in patients treated with gamma interferon. Lancet 1987; 18: 893–5.
Pandey MK, Sung B, Aggarwal BB . Betulinic acid suppresses STAT3 activation pathway through induction of protein tyrosine phosphatase SHP-1 in human multiple myeloma cells. Int J Cancer 2010; 127: 282–92.
Shin J, Lee HJ, Jung DB, Jung JH, Lee HJ, Lee EO, et al. Suppression of STAT3 and HIF-1 alpha mediates anti-angiogenic activity of betulinic acid in hypoxic PC-3 prostate cancer cells. PLoS One 2011; 6: e 21492.
Yang XO, Panopoulos AD, Nurieva R, Chang SH, Wang D, Watowich SS, et al. STAT3 regulates cytokine-mediated generation of inflammatory helper T cells. J Biol Chem 2007; 282: 9358–63.
Zdzisińska B, Rzeski W, Paduch R, Szuster-Ciesielska A, Kaczor J, Wejksza K, et al. Differential effect of betulin and betulinic acid on cytokine production in human whole blood cell cultures. Pol J Pharmacol 2003; 55: 235–8.
Lu Q, Xia N, Xu H, Guo L, Wenzel P, Daiber A, et al. Betulinic acid protects against cerebral ischemia-reperfusion injury in mice by reducing oxidative and nitrosative stress. Nitric Oxide 2011; 24: 132–8.
Nader MA, Baraka HN . Effect of betulinic acid on neutrophil recruitment and inflammatory mediator expression in lipopolysaccharide-induced lung inflammation in rats. Eur J Pharm Sci 2012; 46: 106–13.
Yun Y, Han S, Park E, Yim D, Lee S, Lee CK, et al. Immunomodulatory activity of betulinic acid by producing pro-inflammatory cytokines and activation of macrophages. Arch Pharm Res 2003; 26: 1087–95.
van Horssen J, Witte ME, Schreibelt G, de Vries HE . Radical changes in multiple sclerosis pathogenesis. Biochim Biophys Acta 2011; 1812: 141–50.
Gonsette RE . Oxidative stress and excitotoxicity: a therapeutic issue in multiple sclerosis? Mult Scler 2008; 14: 22–34.
Erickson MA, Dohi K, Banks WA . Neuroinflammation: a common pathway in CNS diseases as mediated at the blood-brain barrier. Neuroimmunomodulation 2012; 19: 121–30.
Martin-Banderas L, Holgado MA, Venero JL, Alvarez-Fuentes J, Fernández-Arévalo M . Nanostructures for drug delivery to the brain. Curr Med Chem 2011; 18: 5303–21.
Pareek TK, Belkadi A, Kesavapany S, Zaremba A, Loh SL, Bai L, et al. Triterpenoid modulation of IL-17 and Nrf-2 expression ameliorates neuroinflammation and promotes remyelination in autoimmune encephalomyelitis. Sci Rep 2011; 1: 201.
Kommera H, Kaluderović GN, Kalbitz J, Dräger B, Paschke R . Small structural changes of pentacyclic lupane type triterpenoid derivatives lead to significant differences in their anticancer properties. Eur J Med Chem 2010; 45: 3346–53.
Kommera H, Kaluderović GN, Dittrich S, Kalbitz J, Dräger B, Mueller T, et al. Carbamate derivatives of betulinic acid and betulin with selective cytotoxic activity. Bioorg Med Chem Lett 2010; 20: 3409–12.
Lowe DB, Storkus WJ . Chronic inflammation and immunologic-based constraints in malignant disease. Immunotherapy 2011; 3: 1265–74.
Acknowledgements
This work was supported by the Ministry of Education and Science of the Republic of Serbia (173035, 175038, and 173013). The authors would like to thank BioSolutions Halle GmbH for betulinic acid.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Blaževski, J., Petković, F., Momčilović, M. et al. Betulinic acid regulates generation of neuroinflammatory mediators responsible for tissue destruction in multiple sclerosis in vitro. Acta Pharmacol Sin 34, 424–431 (2013). https://doi.org/10.1038/aps.2012.181
Received:
Accepted:
Published:
Issue date:
DOI: https://doi.org/10.1038/aps.2012.181
Keywords
This article is cited by
-
Evaluation of betulinic acid effects on pain, memory, anxiety, catalepsy, and oxidative stress in animal model of Parkinson’s disease
Metabolic Brain Disease (2023)
-
Computational investigations of physicochemical, pharmacokinetic, toxicological properties and molecular docking of betulinic acid, a constituent of Corypha taliera (Roxb.) with Phospholipase A2 (PLA2)
BMC Complementary and Alternative Medicine (2018)
-
Betulinic Acid Induces Apoptosis in Differentiated PC12 Cells Via ROS-Mediated Mitochondrial Pathway
Neurochemical Research (2017)
-
Highly lipophilic 3-epi-betulinic acid derivatives as potent and selective TGR5 agonists with improved cellular efficacy
Acta Pharmacologica Sinica (2014)
