Figure 3.

Leonurine does not impair immune responses in the periphery of EAE mice. MNCs were isolated from the DLN of naive mice, vehicle‐treated EAE mice and leonurine‐treated EAE mice on day 19 post immunization. (A) Cells were analysed for expression of CD4 and CD8 in the lymphocyte gate by flow cytometry. The percentages and absolute numbers of CD4⁺ and CD8⁺ cells in the DLN were shown (n = 5). (B) The pathogenic IFN‐γ‐ and IL‐17‐producing CD4⁺ T cells were analysed by flow cytometry, Treg cells were analysed for expression of Foxp3 in the CD4⁺ gate by flow cytometry, and the percentages and absolute numbers in the DLN were shown (n = 5). (C, D) MNCs were isolated from the DLN and spleen of vehicle‐ and leonurine‐treated EAE mice on day 15 post immunization and then stimulated with MOG _35‐55 (5, 10 μg/mL) for 72 h ex vivo. Cell proliferation was examined by [³H] thymidine incorporation, and the levels of cytokine IFN‐γ, IL‐4, IL‐10, and IL‐17 were analysed by ELISA (n = 4). (E) DLN cells and splenocytes from vehicle‐ and leonurine‐treated EAE mice on day 15 post immunization were transferred into sublethally irradiated mice. Mice were monitored and scored daily (n = 5). Statistical significance indicated as *P < 0.05