Table 1.
Primer sequences for quantitative PCR.
Figure 1.
Astragaloside IV attenuated EAE progression.
A, ASI treatment given at 20/kg/day i.p. from one day before MOG35–55 immunization and continued for 14 days significantly reduced mean clinical score of EAE mice. Inset denoted average clinical score daily of EAE mice received MPD (20 mg/kg/day, i.p. from day 8 to day 10 post-immunization), ASI or no treatment. B, ASI treatment prevented against body weight loss of EAE mice. MPD, methylprednisolone; ASI, astragaloside IV. All data are presented as mean±standard error of the mean (n = 17). *, p<0.05; **, p<0.01; ***, p<0.001.
Figure 2.
Astragaloside IV reduced demyelination and neuroinflammation.
A, B, C, ASI lessened demyelination of EAE mice stained by LFB and prevented infiltration of monocytes into spinal cords of EAE mice. Insets denoted nuclei of monocytes stained by cresyl echt violet in anterior median fissure of spinal cords. D, E, F, ASI decreased activation of astrocytes labeled by GFAP in dentate gyrus of EAE mice. Insets indicated enlarged astrocytes in control, EAE and EAE+ASI mice. G, H, I, ASI inhibited activation of microglial cells labeled by Iba I in dentate gyrus of EAE mice. Insets indicated enlarged microglia in control, EAE and EAE+ASI mice. Error bars in LFB staining denoted 70 µm, while those in Iba I staining represented 50 µm. ASI, astragaloside IV.
Figure 3.
Astragaloside IV regulated BBB permeability and ROS levels.
A, ASI prevented BBB leakage of EAE mice measured by Evan’s blue dye infiltration; B, ASI decreased in vivo ROS level of EAE mice analyzed by DHE fluorescent intensity; C, D, ASI resumed down-regulated anti-oxidative enzymes, GSH-Px and T-SOD in EAE mice; E, F, ASI prevented increase of iNOS and MDA in EAE mice. All data are presented as mean±standard error of the mean. n = 5 in each group. *, p<0.05; **, p<0.01; ***, p<0.001. ASI, astragaloside IV.
Figure 4.
Effects of astragaloside IV on mRNA expressions of neuroinflammatory biomarkers and anti-oxidative enzymes.
A, ASI decreased GFAP mRNA expression in cortices of EAE mice. B, ASI reduced CD11b mRNA expression in cortices of EAE mice. C, D, ASI did not change mRNA expressions of Sod1 and Gpx1 in cortices of EAE mice. All data are presented as mean±standard error of the mean. n = 5 in each group. *, p<0.05; **, p<0.01. ASI, astragaloside IV.
Figure 5.
Effects of astragaloside IV on splenic RORγt, T-bet and Foxp3 mRNA expressions of EAE mice.
All data are presented as mean±standard error of the mean. n = 5 in each group. *, p<0.05; **, p<0.01. ASI, astragaloside IV.
Figure 6.
Effects of astragaloside IV on cytokine profile in CNS of EAE mice.
All data are presented as mean±standard error of the mean and compared with EAE group. n = 5 in each group. *, p<0.05.
Figure 7.
Effects of astragaloside IV on apoptotic proteins.
A, western blots of GFAP, Bcl-2, Bax, p53, p-tau in cortices of EAE mice treated with astragaloside IV. B-E, gray intensity analysis of GFAP, Bcl-2, Bax, p53 and p-tau. All data are presented as mean±standard error of the mean and compared with EAE group. n = 5 for each group. *, p<0.05; **, p<0.01; ***, p<0.001.
Figure 8.
Astragaloside IV reduced oxidative stress in SH-SY5Y cells and decreased iNOS expression in BV-2 cells upon IFNγ stimulation.
A, SH-SY5Y treated with ASI (50 µM) for 24 hr reduced cellular DCF fluorescence. B, SH-SY5Y treated with different doses of ASI for 24 hr inhibited cellular DCF fluorescent intensity (n = 5). C, SH-SH5Y cells were pretreated with ASI for 1 hr followed by challenge of H2O2 (100 µM) for 24 hr. D, BV-2 cells were pretreated with ASI for 1 hr followed by stimulation of IFNγ (100 ng/ml) for 24 hr. ASI, astragaloside IV.
Figure 9.
Effects of astragaloside IV on proliferation and cytokine secretion of CD4+ T cells.
A, ASI did not prevent proliferation of CD4+ T cells isolated from EAE mice. B, ASI inhibited TNFα secretion from CD4+ T cells isolated from EAE mice. C, ASI reduced IFNγ production of CD4+ T cells isolated from EAE mice. D. ASI decreased IL17 secretion from CD4+ T cells isolated from EAE mice. Control CD4+ T cells were isolated from spleens of normal C57 BL/6 mice. EAE mice used for CD4+ T cell isolation were induced with MOG35–55 for 21 days. ASI, astragaloside IV. All data are presented as mean±standard error of the mean and compared with EAE group. n = 5 for each group. *, p<0.05; ***, p<0.001.
Figure 10.
Demonstration of pathways of astragaloside IV to counterbalance oxidative stress in EAE mice.
On one hand, ASI enhanced antioxidative defense system and thus elevated ROS degradation capability of CNS. On the other hand, ASI inhibited T-cell infiltration, BBB leakage and gliosis in CNS, thus, reduced ROS generation. ASI regulated ROS generation and degradation and thus decreased its damage to neurons. Meanwhile, ASI could also influence the balance of pro- and anti-apoptotic proteins of Bcl-2 family and make the balance move towards anti-apoptotic direction. ASI, astragaloside IV.