Fig 1.
Treatment of MRL/lpr mice with astilbin ameliorates disease development associated with lupus.
A. Representative lymph nodes and spleen from MRL/lpr mice treated with astilbin or CTX starting at 8 weeks of age (left panel). The lymph nodes and spleen from treated mice were weighed and graphed as mean ± SEM (right panel). B. Total number of CD3+ T and CD19+ B cells in the lymph nodes (LN) and spleen. The absolute cell number was obtained by multiplying the total splenocyte or lymph node cell number by the percentage of CD3+ T and CD19+ B cells. C. H&E-stained paraffin wax-embedded kidney sections from treated MRL/lpr mice at 20 wk. Bar equals to 50 μm. Representative images shown from n = 5–10 samples were later scored, with results displayed in Table 1. D. Body mass for each individual mouse measured over time graphed as mean ± SEM from 8 to 20 weeks of age. E. H&E-stained kidney sections at 20 wk from MRL/lpr mice treated with astilbin starting from 12 weeks of age. Bar equals to 50 μm. The data show mean ± SEM, n ≥ 6 mice per group as described in Materials and methods. * p < 0.05, ** p< 0.01, *** p < 0.001 versus vehicle controls.
Table 1.
Effects of astilbin treatment on renal histological scores.
Fig 2.
Astilbin treatment reduces production of autoantibodies and complement C3 deposits.
MRL/lpr mice were treated with astilbin or CTX starting at 8 weeks of age. Sera were collected from the mice at 20 or 30 wk. A. ANA titers at 20 wk. ANA titers are evaluated by scoring serum dilutions (from 1:20 to 1:320) and a higher titer is defined as detectable in more dilution. Each circle indicates the ANA titer of an individual mouse and black circles mean positive response. B. Anti-dsDNA Ab levels in sera at 20 wk. n ≥ 6 per group as described in Materials and methods. C. Anti-dsDNA Ab levels in sera at 30 wk. n = 3 mice per group except astilbin treatment group (20 mg/kg, n = 4). The serum levels of anti-dsDNA antibodies were determined by ELISA. The data show mean ± SEM. * p < 0.05, ** p< 0.01 versus vehicle controls. D. C3 deposits in the kidney sections from treated MRL/lpr mice at 20 wk. C3 deposits were detected by immunefluorescence microscopy. Bar equals to 1 μm.
Fig 3.
Astilbin treatment reduces proinflammatory cytokine production.
MRL/lpr mice were treated with astilbin (10, 20, 40 mg/kg) starting at 8 weeks or astilbin (20 mg/kg) at 12 weeks of age. Sera were collected at 20 wk and serum cytokine levels were determined by CBA assay. A. IFN-γ. B. IL17A. C. IL-1β. D. TNF-′. E. IL-6. The data show mean ± SEM, n ≥ 6 mice per group as described in Materials and methods. * p < 0.05, ** p< 0.01, *** p < 0.001 versus vehicle controls.
Fig 4.
Astilbin treatment decreases the percentage of CD44hiCD62Llo activated CD4+ T cells and several cytokine mRNA expression.
MRL/lpr mice were treated with astilbin (10, 20, 40 mg/kg) starting at 8 weeks or astilbin (20 mg/kg) at 12 weeks of age. Flow cytometry analysis of whole splenocytes stained with CD3e-percp-cy5.5, CD44-FITC, CD62L-PE, CD4–APC was performed at 20 wk. A. Representative flow cytometry dot plots of live cell events gated on CD3+ CD4+ cells. B. Graphs show the percentage of CD44hiCD62Llo CD3+ CD4+ T cells in the spleens from the mice treated as indicated. The mRNA expression of IFN- (C), TNF-′ (D) and IL17A (E) in the spleens from the treated mice was examined by real-time PCR. β-actin was used as loading control. The data show mean ± SEM of six mice. * p < 0.05,** p< 0.01, *** p < 0.001 versus vehicle controls.
Fig 5.
Astilbin decreases the mitochondrial membrane potential in activated T cells.
Splenocytes were isolated from 12-week-old naive MRL/lpr mice and stimulated without (-) or with (+) anti-CD3 and anti-CD28 antibodies (1 μg/ml for each) for 24 h. These cells were incubated with various concentrations of astilbin or resveratrol (50 M) for 24h and then stained with JC-1. Flow cytometry analysis was performed. Data were representative of thee independent experiments (n ≥ 3 mice).
Fig 6.
Astilbin treatment decreases the percentage of CD19-B220-CD138+ plasma cells and BAFF mRNA expression.
MRL/lpr mice were treated with astilbin (10, 20, 40 mg/kg) starting at 8 weeks or astilbin (20 mg/kg) at 12 weeks of age. Flow cytometry analysis of whole splenocytes stained with CD19-FITC, CD138-PE, B220–APC was performed at 20 wk. A. Representative flow cytometry dot plots of live cell events gated on CD19- cells. B. Graphs show the percentage of CD19-B220-CD138+ plasma cells in the spleens from the mice treated as indicated. C. The mRNA expression of BAFF in the spleens from the treated mice was examined by real-time PCR. β-actin was used as loading control. The data show mean ± SEM of six mice. *p < 0.05, *** p < 0.001 versus vehicle controls.
Fig 7.
Astilbin treatment decreases CD80 and CD86 expression on spleen B cells from MRL/lpr mice.
MRL/lpr mice were treated with astilbin starting at 8 or 12 weeks of age. At 20 wk, splenocytes were isolated from the mice and stimulated with LPS (1 μg/ml) or PBS for 24 h. Left panel, representative histograms comparing CD80 (A) and CD86 (B) expression gated for CD19+ cells. Right panel, cumulative data of flow cytometry for CD80 (A) and CD86 (B) expression gated for CD19+ cells. MFI: mean fluorescence intensity. Gray line represents isotype control staining. The data show mean ± SEM, n = 3, 3, 4 mice for vehicle, astilbin, CTX treatment, respectively. * p < 0.05,** p< 0.01.
Fig 8.
Astilbin downregulates CD80 and CD86 expression on activated B cells.
Splenocytes were isolated from naive MRL/lpr mice and stimulated with LPS (1 μg/ml) in the presence of various concentrations of astilbin for 24 h. Left panel, representative histograms comparing CD80 (A) and CD86 (B) expression gated for CD19+ cells. Right panel, cumulative data of flow cytometry for CD80 (A) and CD86 (B) expression gated for CD19+ cells. MFI: mean fluorescence intensity. Gray line represents isotype control staining. The data show mean ± SEM of at least three mice. ** p < 0.01,** * p< 0.005.