Figure legends
Fig. 1. Effects of MQL on the OVA-induced allergic mouse model. (A) Experimental schedule of the OVA-induced allergic mouse model. Allergic immune responses in BALB/c mice were induced by i.p injection of OVA (10 μg) with alum (1 mg) on days 7 and days 14. MQL was orally administered daily from day 7 to day 21. The level of (B) serum IgE was analyzed by ELISA. Splenocytes were seeded to 5 × 106cells/mL and cultured in the presence of OVA (100 μg/mL) for 72 h. (C) IL-4, (D) IL-5, (E) IL-13, and (F) IL-2 cytokines in culture supernatant of splenocytes were measured by ELISA. (G) Cell proliferation was measured by MTT assay, and (H) HO-1 expression was detected in splenocytes of each group by Western blot. Results are shown as mean ± SD. Asterisks (*) and (**) indicate significant differences, at p < 0.05 and p < 0.01, respectively, between the MQL-treated and non-treated groups.
Fig. 2. Immunomodulatory effects of MQL on splenocytes of BALB/c mice immunized by OVA. Allergic immune responses in BALB/c mice were induced by i.p injection of OVA (10 μg) with alum (1 mg) on days 7 and days 14. Splenocytes were seeded to 5 × 106cells/mL and cultured in the presence or absence of OVA (100 μg/mL) and MQL for 72 h. (A) IL-4, (B) IL-5, (C) IL-13, (D) IFN-γ, (E) IL-12, and (F) IL-2 cytokines in culture supernatant of splenocytes were measured by ELISA. (G) Cell proliferation was measured by MTT assay. (H) STAT1 and STAT6 phosphorylation and (I) HO-1 expression were detected by Western blot. Results are shown as mean ± SD. Asterisks (*) and (**) indicate significant differences, at p < 0.05 and p < 0.01, respectively, between the MQL-treated and non-treated groups.
Fig. 3. Effects of MQL on CD4+ T cell proliferation and HO-1 expression. CD4+ T cells isolated from splenocytes were seeded to 1 × 106 cells/mL and cultured with anti-CD3 (1 μg/mL) and anti-CD28 antibodies (1 μg/mL). CD4+ T cells were cultured in the presence or absence of MQL for 48 h. (A) IL-2 in culture supernatant of CD4+ T cells was measured by ELISA. (B) CD4+ T cell proliferation was measured by MTT assay. (C) Expression of p21 was detected by Western blot, and (D) cell cycle of CD4+ T cells was measured by flow cytometry. (E) HO-1 expression and (F) NRF2 nuclear translocation were detected in CD4+ T cell cultured in the presence or absence of MQL by Western blot. Results are shown as mean ± SD. Asterisks (*) and (**) indicate significant differences, at p < 0.05 and p < 0.01 respectively, between the MQL-treated and non-treated groups.
Fig. 4. Effects of MQL on MAP kinase signaling pathways. (A) CD4+ T cells were isolated from splenocytes and pre-treated with U0126 (5 μM), SB20350 (5 μM), or SP600125 (5 μM) for 1 h then treated with MQL for 24 h to detect HO-1 expression in CD4+ T cells. (B) To analyze MAPK phosphorylation induced by MQL, CD4+ T cells were pre-treated with MQL for 2 h before stimulation with anti-CD3 (1 μg/mL) and anti-CD28 antibodies (1 μg/mL). CD4+ T cells were treated with MQL for 2 h without stimulation, and (C) MAPK phosphorylation and (D) C-Raf phosphorylation were detected by Western blot. Data are representative of three independent experiments.
Fig. 5. Effects of MQL on ROS generation in CD4+ T cells. CD4+ T cells isolated from splenocytes were cultured in the presence or absence of MQL for 2 h. Production of reactive oxygen species in CD4+ T cells was detected by using (A) DCFH-DA and (B) MitoSoxTM Red and measured on a fluorescence plate reader or by flow cytometry. (C) CD4+ T cells isolated from splenocytes were pre-treated with NAC (1 mg/mL) for 1 h then cultured for 2 h in the presence of MQL. Phosphorylation of ERK and C-Raf were detected by Western blot. (D) CD4+ T cells were cultured with anti-CD3 (1 μg/mL) and anti-CD28 antibodies (1 μg/mL) for 24 h in the presence of NAC (1 mg/mL) and MQL. HO-1 expression in CD4+ T cells was detected by Western blot. Results are shown as mean ± SD. Asterisks (*) and (**) indicate significant differences, at p < 0.05 and p < 0.01, respectively, between the MQL-treated and non-treated groups.
Fig. 6. Effects of topical and oral administration on TMA-induced AD symptoms. (A) Experimental schedule of the TMA-induced AD-like mouse model with topical treatment of MQL. Cells from DLNs were seeded to 1 × 106 cells/mL and cultured in the presence of Con A (2 μg/mL) for 48 h. An experimental AD-like lesion was induced on the shaved flank skin of BALB/c mice by sensitization with 5% TMA (50 μL) on day 0 then treated with 2% TMA (10 μL) on days 5-14. MQL (4 and 10 mg/mL, 10 μL) and Dex. (1 mg/mL, 10 μL) were administered topically. (B) Ear swelling was measured 24 h after TMA treatment. (C) Infiltrating inflammatory cells in the ear tissues were stained using hematoxylin and eosin. The levels of (D) IgE in serum and (E) IL-13, (F) IFN-γ, and (G) IL-12 cytokines in culture supernatant of DLNs were measured by ELISA. (H) Experimental schedule of the TMA-induced AD-like mouse model with oral administration of MQL. An experimental AD-like lesion was induced on the shaved flank skin of BALB/c mice by sensitization with 5% TMA (50 μL) on day 0 then treated with 2% TMA (20 μL) on days 5, 8, 11, 14, 17, 20, 23, and 26. MQL (4 and 10 mg/kg) and Dex. (1 mg/kg) were administrated by orally from day 5 to day 26. (I) Ear swelling was measured 24 h after TMA treatment. (J) Infiltrating inflammatory cells in the ear tissues were stained using hematoxylin and eosin. The levels of (K) IgE in serum and (L) IL-13, (M) IFN-γ, and (N) IL-12 cytokines in culture supernatant of DLNs were measured by ELISA. Results are shown as mean ± SD. Asterisks (*) and (**) indicate significant differences, at p < 0.05 and p < 0.01, respectively, between the MQL-treated and non-treated groups of TMA-treated mice.