Figure 1.
Treatment with EGCG suppresses inflammatory arthritis in IL-1RaKO mice.
IL-1RaKO mice were immunized with 100 µg of Cll in CFA to induce arthritis. The mice were intraperitoneally injected with saline or EGCG (40 mg//kg) three times per week for 2.5 weeks. (A) Disease severity was recorded using the mean arthritis score ± SD (left) and arthritis incidence (right). (B) Tissue sections from the joints of each mouse were stained with H&E, toluidine blue and safranin O. (C) Tissue sections from the joints from the IL-1RaKO mice treated with EGCG (n = 10) or untreated (n = 10) were stained with TRAP. The histopathologic score of osteoclast formation is shown in the right graph. (D) TRAP staining for identification of osteoclasts. Osteoclast precursors were cultured in the presence of EGCG with M-CSF and RANKL. TRAP+ cells containing three or more nuclei were scored as osteoclast. TRAP+ cells were counted three times by blind scoring. (E) The levels of IgG2a or CII-specific IgG2a were measured in serum obtained from both groups of mice by ELISA. (F) T cell proliferative responses were determined in a mixed lymphocyte reaction by a [3H]thymidine incorporation assay Data represent the mean ± SD of three independent experiments or representative of more than three independent experiments (*P<0.05, **P<0.01 and ***P<0.001).
Figure 2.
Expression of inflammatory cytokines and molecules associated with mTOR signaling.
(A–B) The joint tissues were obtained from CIA and EGCG injected mice and were stained with the anti-IL-1β, anti–IL-6, anti-TNF-α, anti-IL-17, anti-RANK, anti-VEGF (A), anti-mTOR, anti-HIF-1α, anti-STAT3 or isotype control Abs (B). Data are representative of three independent experiments.
Figure 3.
The anti arthritic effect of EGCG is dependent on the inhibition of IL-1β signaling and IL-17 expression.
(A) Spleen tissues from each mouse were analyzed for CD4, IL-17 (green), IL-1R (green or blue), IL-1β (red) and DAPI by confocal microscopy.(B) Splenocytes were stained with CD4-PerCP, IL-17-FITC, and IL-1R-PE using flowcytometry. (C) CD4+CD25+Foxp3+ T cells and CD4+IL-17+ T cells using Abs specific for CD4 (red), CD25 (blue), Foxp3 (green) and IL-17 (red). (D) Splenocyteswere stained with CD4-PerCP, CD25-APC, Foxp3-PE and IL-17-FITC.(E) The expression of Foxp3, IL-17, IL-10 and TGF-β were determined by real-time PCR. Data represent the mean ± SD of three independent experiments or representative of more than three independent experiments (*P<0.05 and **P<0.01).
Figure 4.
EGCG decreased signaling of associated Th17 differentiation.
(A) Spleen tissues from each mouse were stained for p-STAT3 (S727) (red), p-STAT3 (Y705) (red) and CD4 (green).(B) For flowcytometry analysis, splenocytes were stained with CD4-PerCP,p-STAT3705-PE or p-STAT3727-PE. (C) Spleen tissues from each mouse were stained for CD4 (white), IL-17 (green), mTOR (red), HIF-1α (red) and DAPI. Data are representative of more than three independent experiments (*P<0.05 and **P<0.01).
Figure 5.
EGCG decreases the frequency of Th17 cells via inhibition mTOR, HIF-1α and STAT3.
(A) The expression levels of IL-17, CCL6, RUNX, AHR, Foxp3 and IL-10 by real-time PCR in murine splenocytes. (B) IL-17 and TNF-α were measured in the culture supernatant by ELISA. (C) The expression levels of Glut-1, LDHα, MCT4, GPI and HIF-1α by real-time PCR. (D) Expression of p-STAT3, HIF-1α and mTOR in splenocytes was examined by western blot. Data represent the mean ± SD of three independent experiments or representative of more than three independent experiments (*P<0.05, **P<0.01 and ***P<0.001).