Effect of daphnetin on cytokines secretion in cell culture supernatant.

<p>The levels of cytokines IL-2 (A), IFN-γ (B), IL-4 (C) and IL-6 (D) were analyzed after culturing the splenocytes in the presence of different concentration daphnetin and ConA (5 µg/mL) for 24 h. The cytokine levels were quantified by ELISA. The experiments were performed in triplicates and the data were present as means ± SD. ^##P<0.01 vs. Control group. *<i>P</i><0.05 or **<i>P</i><0.01 vs. ConA group.</p

Cell cycle arrest of splenocytes lymphocytes by daphnetin.

<p>Cells were harvested and DNA contents were stained with propidium iodide for flow cytometric analysis. The results were from three independent experiments and presented as mean ± SD. ^##<i>P</i><0.01 vs. Control group; **<i>P</i><0.01 vs. ConA group.</p

Effect of daphnetin treatment on nuclear translocation of NFAT induced by ConA.

<p>Mouse CD3 T cells were pretreated with different concentrations of daphnetin (4, 8, 16 µg/mL) for 1 h and then stimulated with ConA for another 30 min. NFAT localization was analyzed by immunocytochemistry and confocal microscopy.</p

Effects of daphnetin on cell immunity of DNFB-treated mice evaluated by DTH.

<p>The DTH reaction was evaluated by the increase in the ear patch weight (8-mm punches) between the left and right ear was measured 24 h after the second challenge. ^##<i>P</i><0.01 vs. Control group. **<i>P</i><0.01 vs. DTH group.</p

Effect of daphnetin on [Ca²⁺]i -related signaling pathway in mouse T cells.

<p>Mouse CD3 T cells were treated with daphnetin (4, 8, 16 µg/mL) for 0.5 h and then stimulated with ConA (5 µg/mL) for 0.5 h. Representative western blots showed protein expression of CaM, CaN, CaMKII and P-CaMKII. Data were presented as means ± SD of three independent experiments. ^##<i>P</i><0.01 vs. Control group; **<i>P</i><0.01 vs. ConA group.</p

Effect of daphnetin on mouse splenocytes viability and proliferation.

<p>(A) Chemical structure of daphnetin used in the study. (B) Effect of daphnetin on the viability of mouse splenocytes. The cells were treated with daphnetin (0–64 µg/mL) for 48 h. The cell viability was determined by MTT assay. (C) Effect of daphnetin on the ConA induced mouse splenocytes proliferation. Splenocytes cultured with fisetin (4, 8, 16 µg/mL) combined with ConA (5 µg/mL) for 24 h. Cell proliferation was assessed by MTT assay. Data are presented as means ± SD of three independent experiments. Significant differences from control group were indicated by *<i>P</i><0.05 and **<i>P</i><0.01.</p

Flowcytometric evaluation of the effect of daphnetin on CD4 and CD8.

<p>(A) Control group, (B) DTH group, (C) CTX group, (D) Daphnetin 5 mg/kg group, (E) Daphnetin 10 mg/kg group, (F) Daphnetin 20 mg/kg group. The results were from three independent experiments and presented as mean ± SD. ^##<i>P</i><0.01 vs. Control group. *<i>P</i><0.05 or **<i>P</i><0.01 vs. ConA group.</p

Effects of different concentration of daphnetin on the [Ca²⁺]i in mouse T cells.

<p>T Cells were pretreated with 20 µM Fluo-3-AM and incubated in the presence of daphnetin (4, 8, 16 µg/mL) for 30 min at 37°C and measured at 37°C using a Confocal Laser Microscope. The fluorescence intensity in ConA group was increased compared with the control group, and daphnetin could diminish the fluorescence intensity. The results were from three independent experiments and presented as mean ± SD. ^##<i>P</i><0.01 vs. Control group; **<i>P</i><0.01 vs. ConA group.</p

Daphnetin reduced ear swelling and leukocytes infiltration.

<p>Histological changes in right ear of mice at 24 h following elicitation with DNFB. (A) Control group: microphotograph showed normal structure of the ear; (B) DTH group: microphotograph showed histopathologic changes (edema, infiltration of inflammatory cells) of the ear; (C) CTX group; Daphnetin group (D: 5 mg/kg; E; 10 mg/kg; F: 20 mg/kg): microphotograph showing decreased histopathologic changes of the ear.</p