Article Figures & Data
Figures
- Fig. 1.
DMY inhibits development of arthritis in CIA rats. (A) A graphic scheme of CIA induction and DMY administration. Wistar rats were injected with the emulsion or PBS with a 1-week interval as described in the Materials and Methods. After the second injection, rats (n = 6 to 7 per group) received DMY for 5 weeks and evaluation was conducted twice a week. (B) Hind-paw swelling of rats was measured by two independent blinded observers (B.-X.X. and Q.-S.W.) from the second injection onward. On day 18, paw swelling in the CIA model reached the peak; from day 14, the value of the DMY-treated group was significantly lower than the vehicle group. The weight of rats in the group injected with DMY vs. vehicle differed significantly, especially in two groups of higher doses. Values are presented as the mean ± S.E.M. (n = 6 to 7 rats per group). *P < 0.05; **P < 0.01 vs. the vehicle-treated group; #P < 0.05; ##P < 0.01 vs. the normal group. CII, collagen II; IFA, incomplete Freund’s adjuvant.
- Fig. 2.
DMY suppresses inflammation in CIA rats. (A) Plasma cytokine levels. Cytokine levels were quantified in supernatants of peripheral blood of CIA rats isolated on day 34 by the enzyme-linked immunosorbent assay. The plasma of rats administered DMY in vivo contained significantly less IL-1β, TNF-α, and IL-17A than that of untreated CIA rats. (B) Representative joint sections stained with hematoxylin and eosin. The black boxes represent areas of magnification in the corresponding figure below. The synovial tissue of the knee joint of CIA rats displayed evident synovial hyperproliferation and inflammatory cell infiltration. While in the DMY-treated CIA groups and normal group, knee joints had slight inflammatory cell infiltration. Scale bar, 200 μm (upper panel) and 100 μm (lower panel). (C) Histologic scores for inflammation in rats treated with DMY or vehicle are shown. Results are presented as the mean ± S.E.M. (n = 6 to 7 rats per group). *P < 0.05; **P < 0.01 vs. the vehicle-treated group; #P < 0.05; ##P < 0.01 vs. the normal group. ICI, inflammatory cell infiltration; SH, synovial hyperproliferation.
- Fig. 3.
Phenotype and purity analysis of FLSs. (A) Representative images of FLSs migrated from synovial tissues on days 0, 4, 5, and 6. (B) Immunofluorescence images of FLSs stained with vimentin (green) are shown. As presented in the images, vimentin (found in connective tissue and cytoskeleton) was nearly expressed in all FLSs. Scale bar, 10 μm. (C) Fluorescence-activated cell sorting analysis of FLSs stained with FITC-conjugated CD90 antibody. Representative images are presented from three independent experiments. DAPI, 4′,6-diamidino-2-phenylindole; FITC, fluorescein isothiocyanate.
- Fig. 4.
DMY regulates IL-1β–induced FLS proliferation and apoptosis. (A) FLSs were treated with a series of concentrations of DMY for 24 and 48 hours and results showed that there was no cytotoxicity of DMY. (B) When FLSs were exposed to DMY for 24 and 48 hours with the stimulation of 10 ng/ml IL-1β, the FLSs in DMY-treated groups showed evidently lower proliferation ability than those in vehicle-treated groups. (C) FLSs were incubated with DMY (6.25, 12.5, and 25 μM) for 24 hours after stimulation of 10 ng/ml IL-1β. Cell apoptosis was determined by Annexin V/PI staining. Populations in the upper-right quadrant of the flow cytometric graph represent apoptotic cells. (D) The percentage of apoptotic cells was quantified. All values represent the mean ± S.E.M. of three independent experiments. *P < 0.05; **P < 0.01 vs. the vehicle-treated group; #P < 0.05; ##P < 0.01 vs. the normal group. OD, optical density.
- Fig. 5.
DMY inhibits migration of IL-1β–induced FLSs. (A and C) Evaluation of the Boyden chamber experiment. FLSs were added to the upper chambers of transwells. Representative images of three independent experiments are shown. Scale bar is 10 μm. (B) Cell migration was quantified by counting migrated cells in nine microscope fields. The period of migration was determined at the time of 16 hours. Values are the mean ± S.E.M. of at least three independent experiments. *P < 0.05; **P < 0.01 vs. the time point at 8 hours. (D and F) DMY dose-dependently attenuated cell migration. (E) Wound migration of FLSs derived from CIA rats. Representative photomicrographs of a scratch assay showed that the similar trend as the transwell assay. Scale bar, 25 μm. Values are the mean ± S.E.M. of at least three independent experiments. *P < 0.05; **P < 0.01 vs. the vehicle-treated group; #P < 0.05; ##P < 0.01 vs. the normal group.
- Fig. 6.
DMY regulates NF-κB signaling in FLSs. Cultured FLSs were stimulated with 10 ng/ml IL-1β. (A) DMY diminishes production of inflammatory cytokines in FLSs, especially in higher doses. mRNA levels of cytokines were normalized to β-actin, and the results are presented as the fold decrease above IL-1β treatment (no DMY treatment). *P < 0.05; **P < 0.01 vs. IL-1β stimulation. (B) Protein levels of phospho-IKK, phospho-p65, and phospho-IκB or total proteins were determined using Western blotting. With IL-1β stimulation (10 ng/ml) for the indicated time points to ensure that 15 minutes is the optimal time point for the maximal activation of NF-κB signaling. *P < 0.05; **P < 0.01 compared with the untreated control. (C) Thus, in coincubation experiments, FLSs were treated with DMY for 24 hours prior to stimulation with IL-1β for 15 minutes. #P < 0.05; ##P < 0.01 compared with IL-1β. (D) Effect of DMY on nuclear translocation of p65 in FLSs. Representative immunofluorescence photographs are shown, as the staining of p65 localization is green and nuclei stained with DAPI is blue. (E) Molecular docking of IKKα and IKKβ with DMY. All values represent the mean ± S.E.M. of three independent experiments. DAPI, 4′,6-diamidino-2-phenylindole; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
