| Background and Objective:GBS is an autoimmune disease and an acute inflammatory disorder that afflicts the peripheral nervous system. EAN has been developed as an animal model of GBS.Phenotypically polarized macrophages generally appear either in a pro-inflammatory M1 mode, i.e., classically activated, or in an anti-inflammatory M2, alternatively activated form【2-3】. RAD001,a potent inhibitor for mammalian target of rapamycin.To date, RAD001 has also exerted a protective effect via the beneficial regulation of some autoimmune diseases【4-10】.Various drugs have been tested recently as treatment options for EAN. However, the therapeutic potential of RAD001 for EAN is unknown. We hypothesized that RAD001 may exert anti-inflammatory and neuroprotective effects in a EAN. Thus, this study was designed to explore the prophylactic and therapeutic effects and mechanisms of RAD001 in EAN rats.Methods:First of all,induction and clinical evaluation of EAN was finished.For preventive treatment, RAD001 solution was administered(1mg/kg/day) for 16 consecutive days post-immunization(dpi). For therapeutic treatment, the same dose of RAD001 solution was administered daily from the day when the first clinical signs were observed. Control animals received the same volume of the vehicle solution.Electromyography(EMG) recordings were displayed at day 16post-immunization. At the peak of disease, the sciatic nerves were harvested.To evaluate the extent of mononuclear cell(MNC) infiltration and demyelination, serial transverse sections were stained with hematoxylin-eosin(H&E) and luxol fast blue(LFB).Then,RT-PCR,lymphocyte proliferation assay,flow cytometry and western blot analysis was performed and ELISA was used to analyze the levels of cytokines.GraphPad Prism 5.0 software was used for statistical analysis.Results:1. Effects of preventative and therapeutic RAD001 treatment on EAN rats The severity of EAN, the onset of clinical signs were reduced in both treatment groups. Preventative RAD001 treatment delayed the onset and decreased the severity of the clinical course. While, for the therapeutic RAD001 treatment, the clinical scores reduced from day 10 to day 16. More extensive inflammatory infiltration was seen in the control group. The maximal mean histological scores were markedly higher in the control group. The MNCV and CMAP amplitude was higher and the CMAP latency values were shorter in both treatment groups.2. Effects of RAD001 on levels of p-P70S6 K,p-4E-BP1 and p-Akt The local expression of p-P70S6 K and p-4E-BP1 was reduced and the local expression of p-Akt was increased in both treatment groups( p < 0.05).Both treatment groups significantly reduced m RNA levels of p-P70S6 K and p-4E-BP1( p< 0.05) and increased m RNA levels of p-Akt( p < 0.05).3. Effects of RAD001 on T cell proliferative responses For P0 stimulation, significant reductions in T cell proliferation were observed in both the preventative and therapeutic groups. there were also differences between the three groups without antigen stimulation.4. RAD001 promotes M2 macrophage polarization in EAN rats In sciatic nerves harvested on dpi 16, both RAD001 treatment groups displayed elevated local expression of M2 macrophages and reduced expression of M1 macrophages compared to the control group(p < 0.05). Additionally, both the RAD001-preventive and RAD001-therapeutic groups showed elevated expression of M2 macrophages in EAN spleens compared to the control group(p < 0.05).5. Effects of RAD001 treatment on cytokine profiles The production of pro-inflammatory cytokines was significantly reduced in the treatment groups(p <0.05).while,anti-inflammatory cytokines was up-regulated.Conclusions:We have demonstrated that RAD001 attenuates EAN through an Akt-mediated phenotypic shift in macrophages. The anti-inflammatory effects of RAD001 in EAN, and possibly its human counterpart, GBS, may improve the environmental milieu and indirectly exert neuroprotective effects. RAD001 thus can be a candidate for alleviating polyneuritic disease. |
PDF Full Text Request