Fasudil ameliorates disease progression in experimental autoimmune encephalomyelitis, acting possibly through antiinflammatory effect

Abstract

Aim: The purpose of this investigation was to further explore the mechanism(s) underlying the amelioration in EAE caused by Fasudil, particularly focusing on anti-inflammatory effect.

Methods: We induced a chronic-progressive experimental autoimmune encephalomyelitis (EAE) in B6 mice immunized with myelin oligodendrocyte glycoprotein(35-55) and performed Fasudil intervention in early and late stages of the disease.

Results: The administration of Fasudil (40 mg/kg, i.p) had a therapeutic effect in delaying the onset and ameliorating the severity of EAE, accompanied by the improvement in myelination and the decrease in inflammatory cells in spinal cords. Fasudil inhibited TLR-4, p-NF-kB/p65, and inflammatory cytokines (IL-1β, IL-6, and TNF-α) and enhanced IL-10 production in spinal cords. The ratio of arginase/iNOS was enhanced mainly in the spinal cords of EAE mice treated with Fasudil, reflecting a shift toward the M2 (antiinflammation) macrophage/microglia phenotype. The administration of Fasudil also induced the upregulation of CB2 receptor in spinal cords, but did not significantly trigger CB1 receptor. Levels of neurotrophic factors NGF, BDNF, and GDNF in the CNS were not altered by Fasudil.

Conclusion: Fasudil ameliorates disease progression in EAE, acting possibly through antiinflammatory pathway.

Figure 1

Fasudil delays onset and ameliorates severity of EAE. Fasudil was injected (intraperitoneally) at 40 mg/kg/day every other day on day 3 p.i. (Fasudil early treatment, n = 23) or at onset of clinical symptoms of EAE (Fasudil late treatment, n = 23). The injection of normal saline was set up as control (EAE control, n = 26) in a similar manner. (A) clinical score of EAE mice treated with Fasudil early or late treatment. (B) body weight of EAE mice treated with Fasudil early and late treatment. *P < 0.05; **P < 0.01.

Figure 2

Fasudil inhibits inflammation and improves myelination in spinal cord. On day 21 p.i., spinal cords were used for luxol fast blue, H&E and immunohistochemistry staining. (A) Demyelination stained with luxol fast blue. Total white matter in luxol fast blue was manually outlined, and pixel area (%) of demyelination in total white matter was calculated by Image‐Pro Plus software. (B) Inflammation stained with H&E stain. Color photograph in H&E stain was automatically converted to black and white, and a number of inflammatory foci (>20 mononuclear cells/focus) in whole spinal cord were calculated by Image‐Pro Plus software. (C) CD4 T cells, CD68 macrophage/microglia and GFAP astrocytes. Quantitative results are analyzed for 6 mice in each group and are representative of three experiments with similar results. *P < 0.05, **P < 0.01.

Figure 3

Fasudil inhibits inflammatory molecules. On day 28 p.i., brains and spinal cords were collected, and proteins were extracted by protein extraction kit. (A) Representative bands of Western blot for TLR‐4, p‐NF‐kB/p65, and AP‐1 in brains and spinal cords by Western blot. Quantitative analysis of TLR‐4, p‐NF‐KB/p65, and AP‐1 was performed from 5 to 6 mice in each group. (B) The levels of IL‐1β, IL‐6, TNF‐α, and IL‐10 in spinal cords by ELISA. Quantitative analysis was performed from 5 to 6 mice in each group. *P < 0.05; **P < 0.01, ***P < 0.001.

Figure 4

Expression and location of p‐NF‐kb/65 in spinal cord. On day 28 p.i., slices from spinal cords were stained with anti‐p‐NF‐kB/65, anti‐GFAP, and anti‐CD11b. In EAE mice, p‐NF‐kB/p65 was observed in both white matter and gray matter. Double immunohistochemistry showed that astrocytes, but not microglia, overlap with p‐NF‐kB/p65.

Figure 5

Fasudil shifts M1 to M2 phenotype. On day 28 p.i., brains and spinal cords were collected, and proteins were extracted for Western blot. (A) Representative bands of Western blot for iNOS and arginase in brains and spinal cords, (B) quantitative analysis of iNOS, (C) quantitative analysis of arginase, and (D) quantitative analysis of arginase/iNOS ratio. *P < 0.05.

Figure 6

Fasudil enhances the expression of CB ₂R in brains and spinal cords. On day 28 p.i., brains and spinal cords were collected, and proteins were extracted for Western blot. (A) Representative bands of Western blot for CB ₁R and CB ₂R in brains and spinal cords, (B) quantitative analysis of CB ₁R, and (C) quantitative analysis of CB ₂R. Quantitative analysis was performed from 5 to 6 mice in each group. *P < 0.05.