Original Article

Betulinic acid regulates generation of neuroinflammatory mediators responsible for tissue destruction in multiple sclerosis in vitro

Authors: Jana Blaževski, Filip Petković, Miljana Momčilović, Reinhard Paschke, Goran N Kaluđerović, Marija Mostarica Stojković, Djordje Miljković
DOI: 10.1038/aps.2012.181

Abstract

Jana BLAŽEVSKI1, #, Filip PETKOVIĆ1, #, Miljana MOMČILOVIĆ1, Reinhard PASCHKE2, Goran N KALUĐEROVIĆ2, 3, Marija MOSTARICA STOJKOVIĆ4, Djordje MILJKOVIĆ1, *
1Department of Immunology, Institute for Biological Research “Siniša Stanković”, University of Belgrade, Serbia; 2 Biozentrum, Martin-Luther-Universität Halle-Wittenberg, Weinbergweg 22, 06120 Halle (Saale), Germany; 3Institut für Chemie, Martin-Luther-Universität Halle-Wittenberg, Kurt-Mothes Str 2, 06120 Halle (Saale), Germany; 4Institute for Microbiology and Immunology, School of Medicine, University of Belgrade, Serbia

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.


Keywords: betulinic acid; experimental autoimmune encephalomyelitis; multiple sclerosis; neuroinflammation; T cell; macrophage; astrocyte; cytokines; NO; ROS; CXCL12

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.
# The two authors contributed equally to this work.
* To whom correspondence should be addressed.
E-mail georgije_zw@yahoo.com
Received 2012-07-03 Accepted 2012-12-05
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