Fig 1.
4'-HW abrogated LPS-induced production level of NO and PGE2 through inhibition of iNOS and COX-2 expression in RAW 264.7 macrophages.
(A) The chemical structure of 4'-HW. (B) Cytotoxicity in RAW 264.7 macrophages treated with 4'-HW (0.5, 1.5 and 15 μM) for 24 h. (C, D, E, F) Cells were treated with 0.5, 5 and 15 μM 4'-HW for 1h prior to the addition of LPS (100 ng/mL) for an 24 h incubation. NO and PGE2 levels were determined with Griess regent and a commercial kit, respectively. The protein level of iNOS and COX-2 was determined by Western blot analysis using specific antibodies. The immune-reactive bands were quantified using the NIH Image J software. (G and H) Cells were pretreated with 4'-HW for 1 h prior to the addition of LPS (100 ng/mL), and then cells were further incubated for 4 h. The mRNA levels of iNOS and COX-2 were determined by quantitative real-time PCR. The data shown represent the mean ± SD of three independent experiments. #p<0.05 vs the control group; *p<0.05 vs the LPS-treated group.
Fig 2.
4'-HW suppressed LPS-induced expression of pro-inflammatory cytokines in RAW 264.7 macrophages.
(A) The cells were treated with 0.5, 5 and 15 μM 4'-HW for 1 h prior to LPS stimulation (100 ng/mL, 4 h). The transcripts of TNF-α, IL-6 and IL-1β were determined by quantitative real-time PCR. (B) Cells were pretreated with 4'-HW for 1 h prior to stimulation with LPS (100 ng/mL) for 12 h, the Supernatant levels of TNF-α, IL-6 and IL-1β, determined by ELISA. The data shown represent the mean ± SD of three independent experiments. #p<0.05 vs the control group; *p<0.05 vs the LPS-treated group.
Fig 3.
Effect of 4'-HW on LPS-induced NF-κB activation in RAW 264.7 macrophages.
(A) Cells were treated with 0.5, 5 and 15 μM 4'-HW for 1 h prior to stimulation with LPS (100 ng/mL, 0.5 h). Cell lysates were analyzed by western blot. (B) Cells were pretreated with 15 μM 4'-HW 1 h and then with LPS (100 ng/mL) for 0.5 h. p65 localization was assessed under a fluorescence microscope as described in Materials and Methods. (C) Cells were treated with 0.5, 5 and 15 μM 4'-HW for 1 h prior to stimulation with LPS (100 ng/mL, 0.5 h). Nuclear and cytoplasmic proteins were obtained and analyzed by western blot. (D) THP-1 cells stably transfected with an NF-κB-luciferase reporter were treated with different concentrations of 4'-HW for 1 h prior to LPS stimulation (100 ng/mL, 4 h). The induced luciferase activity was assayed as described under Materials and Methods. The data shown represent the mean ± SD of three independent experiments. #p<0.05 vs the control group; *p<0.05 vs the LPS-treated group.
Fig 4.
Effects of 4'-HW on LPS-induced expression of IκB-α, IKK-α/β, p-TAK1, MyD88 and the interaction of TAb1 with TAK1 in RAW 264.7 macrophages.
(A, B) Cells were treated with 0.5, 5 and 15 μM 4'-HW for 1 h prior to LPS stimulation (100 ng/mL, 15 min). Cell lysates were analyzed by western blot. (C) The effect of 4'-HW on the interaction of TAK1 and TAB1 in LPS stimulated cells. Cells were exposed to 4'-HW (15 μM) for 1 h, then stimulated with LPS (100 ng/mL) for 15 min. Immunoprecipitation assays were conducted to evaluate the binding of endogenous TAK1 and TAB1. The immune-reactive bands were quantified using the NIH Image J software. The data shown represent the mean ± SD of three independent experiments. #p 0.05 vs the control group; *p<0.05 vs the LPS-treated group.
Fig 5.
Effect of 4'-HW on LPS-induced activation of (A) MAPKs and (B) AKT in RAW 264.7 macrophages.
Cells were treated with 0.5, 5 and 15 μM 4'-HW for 1h prior to LPS stimulation (100ng/mL, 0.5 h). Cell lysates were analyzed by western blot. The immune-reactive bands were quantified using the NIH Image J software. The data shown represent the mean ± SD of three independent experiments. #p<0.05 vs the control group; *p<0.05 vs the LPS-treated group.
Fig 6.
Effects of 4'-HW on LPS-induced ROS production.
RAW 264.7 macrophages cells were pretreated with 0.5, 5 and 15 μM 4'-HW for 1 h and then stimulated with 100 ng/mL LPS for an additional 24 h. The ROS production was assayed as described under Materials and Methods. The data shown represent the mean ± SD of three independent experiments. #p<0.05 vs the control group; *p<0.05 vs the LPS-treated group.
Fig 7.
Effect of 4'-HW on LPS-induced acute lung injury in mice.
Mice (n = 6 per group) were injected with 4'-HW or vehicle for 12 h and 1 h respectively before LPS injection (3 mg/kg, Intratracheal injection). (A) Lungs from each experimental group were processed for histological evaluation at 6 h after the LPS challenge: (a) control, (b) LPS, (c) LPS + DEX (5 mg/kg), (d) LPS + 4'-HW (20 mg/kg), (e) LPS + 4'-HW (10 mg/kg). (B) The BALF was collected 6 h after LPS challenge to measure the number of total cells. (C) The BALF was collected 6 h after LPS challenge to measure the concentration of total proteins. (D) Effect of 4'-HW on the MPO activity in lung homogenates. (E) the levels of TNF-α, IL-6 and IL-1β in total RNA from lung tissue as determined by quantitative real-time PCR. (F) The BALF levels of TNF-α, IL-6 and IL-1β, determined by ELISA. #p<0.05 vs the control group; *p<0.05 vs the LPS-treated group.
Fig 8.
Proposed signaling mechanism for the effects of 4'-HW on LPS-induced inflammation in RAW 264.7 macrophages.
4'-HW exerts its anti-inflammatory activity on RAW 264.7 macrophages by inhibiting ROS production, TAK1/IKK/NF-κB signaling pathway and reducing the phosphorylation of NF-κB, MAPKs and PI3/Akt signaling pathways.