Table 1.
Specific primers used in PCR analysis.
Figure 1.
Proliferation of MOG-reactive T cells was significantly inhibited by Plumbagin.
A, Chemical structure of Plumbagin. B, Splenocytes were isolated 18 days postimmunization from vehicle-treated and PL-treated EAE mice, and examined ex vivo for proliferation by MTS in the presence (MOG) or absence (Med) of MOG peptide. C, Proliferation as measured by MTS in splenocytes from EAE mice stimulated in vitro by MOG peptide with or without PL. Data are presented as mean ± SD of triplicates. *p<0.05; **p<0.01.
Figure 2.
Amelioration of EAE by PL treatment.
A&B, Clinical scores of EAE in mice. PL (2 mg/kg) or vehicle control was administered i.p. daily in EAE mice, started from day 7 postimmunization (A) or day3 before immunization (B). Data are expressed as mean ± SD and represent three independent experiments.
Figure 3.
Histology and immunohistochemical staining of spinal cord tissue.
Spinal cord sections obtained from normal control mice, EAE mice or PL-treated mice at day 18 postimmunization (treatment protocol) were analyzed by Luxol Fast Blue staining for demyelination, H&E staining for inflammation, and Immunohistochemical staining (IH) for CD4+ T cell infiltration. ×25 or ×400. Data presented are representative of three independent experiments. CD4+ T cell infiltrates stained by immunohistochemistry were enumerated microscopically in normal mice (10.33±3.51), EAE mice (128.67±7.02) and PL-treated mice (34.33±3.51), respectively. Data are expressed as mean ± SD. p<0.001 for EAE mice versus normal mice, p<0.01 for PL-treated mice versus EAE mice.
Figure 4.
Cytokine and other molecule profiling of MOG-reactive T-cells in response to PL treatment.
A, Splenocytes from PL-treated mice (open bars) or EAE mice (solid bars) were challenged with MOG peptide, and culture supernatants were collected at 48 h for cytokine measurement by ELISA. Data are presented as mean ± SD (pg/ml). B, Splenocytes isolated from EAE mice were stimulated with MOG peptide in the presence or absence of PL (0.5 µM) for 24 h. mRNA expression of selected genes was measured by RT-PCR. C, Splenocytes isolated from EAE mice and PL-treated mice were stimulated with MOG peptide for 24 h. mRNA expression of stated genes was measured by real-time PCR.
Figure 5.
Inhibition of Th1 and Th17 cells by PL through suppressing JAK/STAT pathway.
Splenocytes isolated from EAE mice or PL-treated mice were re-stimulated with MOG peptide for 24 h. CD4+ T-cells were purified and proteins subjected to electrophoresis and immunoblotting. Membranes were probed with antibodies to JAK, STATs or their phosphorylated form, and other proteins as indicated. A, Molecules related to Th1 differentiation; B, Molecules related to Th17 differentiation; C, JAK family.
Figure 6.
Inhibition of NF-κB signaling pathway by PL.
CD4+ T-cells from PL-treated mice, EAE mice and adjuvant control mice were cultured in the absence or presence of MOG peptide for 24 h, extracted proteins were subjected to electrophoresis and analyzed by immunoblotting for phosphorylated and total p65 and IκBα.