Experimental Autoimmune Neuritis Nerve Demyelination Is Attenuated by Blocking JAK2/STAT3 Signaling Pathway in Rats

Abstract

Background: Guillain‒Barré syndrome (GBS) is an immune-mediated peripheral neuropathy in which inflammatory cells and cytokines participate. The JAK-STAT signaling pathway is a major pathway involved in cytokine signal transduction, but the role of this pathway in GBS is not clear. AG490 is a tyrosine kinase inhibitor that specifically inhibits JAK2 activity and downregulates STAT3 phosphorylation. The aim of this study was to investigate the function of the JAK2/STAT3 pathway in a rat model of experimental autoimmune neuritis (EAN).

Methods: Lewis rats were divided into three groups: the control, the EAN, and the AG490 groups. The EAN and AG490 groups were immunized with P2 peptide to create the EAN models, while the control group received an equal volume of vehicle solution without P2 peptide. Starting from Day 5 post-immunization (PI), the AG490 group was administered AG490 (10 mg/kg) every other day, while the control and EAN groups received an equal volume of vehicle solution without AG490. All rats were weighed and evaluated according to the EAN function score (1-10) by two investigators. Rats were sacrificed on Day 16 PI, and the sciatic nerves were examined by light microscopy, indirect immunohistochemistry, and western blotting.

Results: AG490-treated rats had improved clinical scores compared with those of EAN rats. Hematoxylin and eosin (H&E) and CD45 staining showed significant inflammatory infiltration of the sciatic nerve in the EAN group compared with the control group, and demonstrated reduced inflammatory infiltration in the AG490 group. Luxol fast blue (LFB) staining showed a reduction of myelin loss in the AG490 group compared with the EAN group. The levels of TGF-β1, IFN-γ, and IL-6 increased in the EAN group and showed a significant decrease in rats treated with AG490. The JAK2-STAT3 signaling pathway was activated in EAN rats, and the AG490 group showed decreased expression levels of JAK2, p-JAK2, and p-STAT3 compared with those of the EAN group. Immunofluorescence also showed a decrease in the levels of p-JAK2 and p-STAT3 in the sciatic nerve of EAN rats.

Conclusions: The JAK2/STAT3 signaling pathway is involved in the pathogenesis of EAN, and inhibition of this pathway can reduce the inflammatory response in EAN rats. Despite the limitations in extrapolating EAN findings to human GBS, this study provided new insights into the pathogenesis and potential therapeutic targets of human GBS.

Keywords: AG490; JAK‐STAT signaling pathway; experimental autoimmune neuritis; treatment.

FIGURE 1

Clinical scores of rats in each group. AG490 was administered every other day from Day 5 to Day 15 post‐immunization (PI). Clinical scores of rats in EAN and AG490 groups increased after immunization. AG490 treatment significantly ameliorated severity in EAN from day 12 PI. Mean ± SD, n=10/group. ns, no significant difference, * p < 0.05, ** p < 0.01, AG490 versus EAN.

FIGURE 2

AG490 attenuated the histologic signs in EAN. The sciatic nerves were harvested at day 16 PI in each group for histologic examinations. (A) Representational photomicrographs of H&E staining and CD45 staining in sciatic nerve of each group. Inflammatory cells were indicated by arrows in each group. (B) The quantitative analysis of inflammatory cell infiltration per FOV. (C) The quantitative analysis of CD45‐positive cells per FOV. (D) Representational photomicrographs of LFB staining in the sciatic nerve of each group. Demyelinated lesions in EAN group were indicated by arrows. (E) Relative quantitative analysis of LFB‐positive area ratio per FOV (normalized to control group). Lower LFB‐positive area ratio indicated severe demyelination in sciatic nerves. FOV, field of view. Scale bars: 50 µm. Mean ± SD, n = 6/group. ### p < 0.001, Control versus EAN. *** p < 0.001, AG490 versus EAN.

FIGURE 3

Western blotting showed the protein levels in the sciatic nerves. (A, B) AG490 treatment reduced the levels of TGF‐β1, IFN‐γ, and IL‐6 in EAN rats. (C–F) The levels of JAK2, p‐JAK2, and p‐STAT3 were decreased after treatment with AG490 but the level of STAT3 showed no significant difference. Mean ± SD, n = 5/group. # p < 0.05, ## p < 0.01, ### p < 0.001, Control versus EAN. * p < 0.05, ** p < 0.01, ns, no significant difference, AG490 versus EAN.

FIGURE 4

Effect of AG490 on the expression of p‐JAK2 in the sciatic nerves. (A) Representational photomicrographs of p‐JAK2 in the sciatic nerve of each group. p‐JAK2 positive cells were indicated by arrows. (B) The quantitative analysis of p‐JAK2 positive cells per FOV. FOV, field of view. Scale bars: 50 µm. Mean ± SD, n = 9/group. ### p < 0.001, Control versus EAN. *** p < 0.001, AG490 versus EAN.

FIGURE 5

Effect of AG490 on the expression of p‐STAT3 in the sciatic nerves. (A) Representational photomicrographs of p‐STAT3 in the sciatic nerve of each group. p‐STAT3 positive cells were indicated by arrows. (B) The quantitative analysis of p‐STAT3 positive cells per FOV. FOV, field of view. Scale bars: 50 µm. Mean ± SD, n = 9/group. ### p < 0.001, Control versus EAN. *** p < 0.001, AG490 versus EAN.