Oleanolic acid controls oxidative stress and expression of the anti-inflammatory protein netrin-1 to protect against autoimmune encephalomyelitis

Abstract

Clinical and experimental studies have shown that inflammation and oxidative stress play a key role in the pathogenesis of multiple sclerosis (MS). Netrin-1 is a protein primarily known as a diffusible attractant for axon guidance. Recently, several experimental disease models have shown it as a negative guidance cue for leukocyte migration, revealing a potential anti-inflammatory role. However, its expression in experimental autoimmune encephalomyelitis (EAE), a model that mimics the chronic non-relapsing form of MS, has not been reported yet. The triterpene, oleanolic acid (OA) has proven effective in controlling the inflammatory process in EAE, therefore, its impact in oxidative damage regulation, netrin-1 expression and its relationship to disease progression deserves to be investigated. [Methods]: OA (50 mg/kg/day) or saline, were intraperitoneally administered to MOG35-55 immunized-C57/BL6 mice from immunization time to day 21. Clinical score, and netrin-1 levels and oxidative stress markers were analyzed in serum and cerebellum. [Results]: Production of reactive oxygen species (H2O2) as well as lipid and protein oxidation markers (malondialdehyde and advanced oxidation protein products) in our EAE model were significantly diminished by OA treatment (1.6, 3.4 and 2 times respectively in cerebellum; and 1.7, 2 and 6 times respectively in serum, p<0.001). High levels of superoxide anion were also detected in cerebellum sections of EAE mice by in situ DHE staining, compared to control mice, and DHE fluorescence was strongly attenuated in cerebellum samples from OA-treated EAE mice. In parallel, antioxidant parameters: ferric reducing antioxidant power (FRAP), and SOD and catalase activities were enhanced in OA-treated EAE mice (over 2.5, 7 and 6 fold, respectively both in serum and cerebellum, p<0.001). At the same time, netrin-1 levels increased in sera from 22+1 pg/ml, in untreated EAE mice, to 82+1 pg/ml in OA-treated EAE mice(p<0.001), with the concentration in cerebellum being also 2-fold higher in treated mice. Interestingly, serum and cerebellum samples from OA-treated healthy mice expressed over 5-6 and 3-fold higher netrin-1, respectively, than those from saline-treated healthy ones. [Conclusion]: This study provides new findings regarding the beneficial activity of OA in EAE. Our data suggest that OA protects, at least in part, by activating an antioxidant defence system against exacerbated oxidative stress of EAE and by inducing the anti-inflammatory protein netrin-1.