Study Of Therapeutic Effect And Underlying Mechanisms Of Dimethyl Fumarate On Experimental Autoimmune Neuritis

Background: Guillain-Barré syndrome(GBS) is an acute, post-infectious, immune-mediated, demyelinating disease of peripheral nerves and nerve roots. It is currently the most frequent cause of acute flaccid paralysis worldwide and constitutes one of the serious emergencies in neurology. Currently, intravenous immunoglobulin and plasma exchange are proven effective treatments for GBS. However, despite these treatment options, up to 20% of patients remain severely disabled and approximately 5% die. Experimental autoimmune neuritis(EAN), classic animal model of GBS, effectively mimics clinical, histopathological, and electrophysiological features of GBS and is widely applied for exploring new strategies for treating GBS. Dimethyl fumarate(DMF), a carbohydrate belonging to fumaric acid esters, exhibits various biological activities, including multiple immunomodulatory and neuroprotective effects. It has been approved by Food and Drug Administration for the treatment of relapsing-remitting multiple sclerosis. However, the effects and underlying mechanisms of DMF in EAN are unclear. The aim of this study is to investigate the potential effect of DMF in EAN and elucidate the underlying mechanism.Methods: EAN was induced by injecting an inoculum containing dissolved P0 peptide 180-199 in Lewis rats. Animals were then randomly assigned to three groups: preventative, therapeutic and control group. Clinical signs of EAN were scored and quantified using double-blind method. At the peak of disease(i.e., day 16 post-immunization), electromyographic(EMG) recordings of the sciatic nerves were made to assess the extent of peripheral nerve injury recording the amplitude and latency of compound muscle action potential and motor nerve conduction velocity. At the climax of the clinical course of EAN(i.e., day 16 post-immunization), sciatic nerves were harvested for histopathological assessment to evaluate the extent of inflammatory cells infiltration and demyelination, for fluorescent immunohistochemistry to evaluate the polarization of macrophages, for western bolt analysis to explore the pathway of DMF in EAN. Meanwhile, spleens were removed and splenic mononuclear cells were isolated to analyze antigen-specific lymphocyte proliferation, to explore the polarization of macrophages by flow cytometry and evaluate the cytokine profile by real-time quantitative polymerase chain reaction(RT-PCR) and enzyme-linked immuno sorbent assay(ELISA). Immortalized murine macrophage cell line RAW 264.7 was induced to M1 type and co-cultured with DMF to investigate its direct effect on macrophage polarization by immunocytochemistry and RT-PCR. The Mann-Whitney U test was used to compare differences between control group and treatment groups where appropriate.Results: DMF treatment attenuated clinical severity and reduced neurological deficits with ameliorating inflammatory cells infiltration, and demyelination of sciatic nerves. In addition, DMF treatment decreased the level of proinflammatory M1 macrophages while increased the number of anti-inflammatory M2 macrophages in the sciatic nerves of EAN rats detected by immunohistochemistry. The same trend of macrophages polarization can be detected by flow cytometry in splenic MNCs. In RAW264.7, a shift in macrophage polarization from M1 to M2 phenotype was demonstrated to be depended on DMF application. In sciatic nerves, DMF treatment elevated the level of the antioxidant transcription factor nuclear factor erythroid-derived 2-related factor 2(Nrf2) and its target gene hemoxygenase-1(HO-1) detected by western blot analysis and immunohistochemistry, indicating the protective effects of DMF in EAN was associated with the activation of Nrf2-HO-1 pathway. Moreover, DMF improved inflammatory milieu in spleens of EAN rats, detected by ELISA and RT-PCR.Conclusions: Taken together, our data demonstrated that DMF can effectively suppressed clinical severity and peripheral nerve injury in EAN rats. The mechanism involved altering the balance of M1/M2 macrophages, activating the anti-oxidant pathway Nrf2-HO-1, and attenuating inflammation milieu. All the results indicated that DMF could be a potent candidate for treatment of inflammatory neuropathies in patients.