Fig 1.
Echinatin inhibits LPS-induced inflammatory responses in MH-S cells.
(A) The chemical structure of echinatin. (B) MH-S cells were treated with different concentrations of echinatin for 24 h. The cell viability of these MH-S cells was measured by CCK-8 assay. After echinatin pretreatment for 2 h, the MH-S cells were cultured with LPS (5 μg/mL) for an additional 24 h. (C) The release of NO was measured by Griess reagent. (D) The effect of echinatin on PGE2 production was measured by ELISA kit. The gene expression of iNOS (E) and COX-2 (F) in MH-S cells was measured by qRT-PCR using GAPDH as the internal control. The protein levels of iNOS (G) and COX-2 (H) were measured by Western blotting. GAPDH is used as an internal reference. Data are represented as mean ± SD. # p < 0.05, ## p < 0.01, ### p < 0.001, #### p < 0.0001 vs. the control group. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. the LPS group.
Fig 2.
Echinatin suppresses LPS-induced inflammatory response in RAW264.7 cells.
(A) RAW264.7 cells were treated with different concentrations of echinatin for 24 h. Next, the cell viability of these RAW264.7 cells was detected by CCK-8 assay. After echinatin pretreatment for 2 h, RAW264.7 cells were incubated in the presence of LPS (0.5 μg/mL) for an additional 24 h. The release of NO (B) was measured using the Griess reagent. (C) The effect of echinatin on PGE2 production in LPS-induced RAW264.7 cells. The gene expression of iNOS (D) and COX-2 (E) was measured by qRT-PCR using GAPDH as the internal control. The protein levels of iNOS (F) and COX-2 (G) were detected by Western blotting. Data are represented as mean ± SD. # p < 0.05, ## p < 0.01, ### p < 0.001, #### p < 0.0001 vs. the control group. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. the LPS group.
Fig 3.
Echinatin decreases the mRNA levels and release of IL-1β and IL-6 in LPS-activated RAW264.7 cells.
Cells were pre-treated with different concentrations of echinatin or curcumin for 2 h and treated with LPS (0.5 μg/mL) for an additional 24 h. (A) The mRNA levels of IL-1β. (B) The mRNA levels of IL-6. The release of IL-1β (C) and IL-6 (D) in LPS-stimulated RAW264.7 cells were measured using ELISA kits. Data are represented as mean ± SD, n = 3. # p < 0.05, ## p < 0.01, ### p < 0.001, #### p < 0.0001 vs. the control group. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. the LPS group.
Fig 4.
Echinatin blocks the activation of NF-κB signaling pathway in LPS-stimulated RAW264.7 cells.
RAW264.7 cells were incubated with echinatin (3, 10, and 30 μM) or BAY (5 μM) for 2 h, followed by LPS (0.5 μg/mL) stimulation for an additional 5 min. The levels of p-TAK1 (A), p-IKK (C), and p-IκBα (E), total protein levels of TAK1 (A), IKK (C), and IκBα (G), as well as GAPDH and tubulin were detected by Western blot analysis. The quantitative analysis of the ratio of p-TAK1 (B), p-IKK (D), p-IκBα (F), and IκBα (H) normalized to the loading control is indicated. RAW264.7 cells were pre-treated with echinatin or BAY for 2 h, followed by LPS-stimulating for an additional 30 min. (I) The cytoplasmic and nuclear fraction technology was used to measure the distribution of p65. GAPDH was used as a cytoplasmic loading control, whereas PARP1 was used as nuclear loading control. (J) The quantitative analysis of the ratio of p65 normalized to GAPDH or PARP1 is shown. Data are represented as mean ± SD. n = 3. # p < 0.05, ## p < 0.01, ### p < 0.001, #### p < 0.0001 vs. the control group. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. the LPS group.
Fig 5.
Echinatin represses the phosphorylation of MAPK in LPS-induced RAW264.7 cells.
After treatment with echinatin or PD98059 (20 μM), SP600125 (20 μM) and SB203580 (20 μM) for 2 h, the cells were incubated with LPS (0.5 μg/mL) for an additional 30 min. The phosphorylated and total ERK (A), JNK (C) and p38 (E) were detected by Western blotting. The statistical results of the ratio of p-ERK (B), p-JNK (D) and p-p38 (F) normalized to the loading control are indicated. Data are represented as mean ± SD. n = 3. # p < 0.05, ## p < 0.01, ### p < 0.001, #### p < 0.0001 vs. the control group. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. the LPS group.
Fig 6.
Echinatin activates Nrf2-HO-1 anti-oxidant signaling in LPS-induced RAW264.7 cells.
RAW264.7 cells were treated with echinatin or curcumin for 1 h, followed by LPS (0.5 μg/mL) stimulation for an additional 8 h. (A) The protein levels of Keap1, Nrf2, and HO-1 were detected by Western blot assay. The quantitative results of the ratio of Keap1 (B), Nrf2 (C), and HO-1 (D) normalized to the β-actin are presented. (E) The cytoplasmic and nuclear fraction technology was used to measure the distribution of Nrf2. GAPDH was used as a cytoplasmic loading control, whereas PARP1 was used as a nuclear loading control. (F) The quantitative analysis of the ratio of Nrf2 normalized to GAPDH or PARP1 is shown. Data are represented as mean ± SD. n = 3. # p < 0.05, ## p < 0.01, ### p < 0.001, #### p < 0.0001 vs. the control group. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. the LPS group.
Fig 7.
Echinatin directly interacts with TAK1 and Keap1.
The effects of echinatin on the thermal stability of TAK1 (A) and Keap1 (B) were detected by CETSA and Western blotting. The top panel shows the quantification of the thermal stability of TAK1 and Keap1 in the range of 45°C to 63°C. The optical densities of TAK1 and Keap1 were normalized to those obtained at 45°C. (C) The 3D representation of echinatin binding to TAK1 (PDB: 4O91) protein. (D) The 3D representation of the interaction of echinatin at the active site of Keap1 (PDB: 6LRZ). Data are represented as mean ± SD. n = 3. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.
Table 1.
Molecular docking between echinatin with Keap1 and TAK1.
Fig 8.
Echinatin ameliorates LPS-induced ALI in mice.
Mice were administrated with echinatin (10, 20, and 50 mg/kg) or DEX (0.1 mg/kg), or vehicle for 2 h, followed by the tail vein injection of LPS (10 mg/kg) for another 12 h. (A) H&E staining was used to access pathological changes observed in lung tissues. Scale bar = 100 μm (×200) and 50 μm (×400). The black arrow replaces alveolar septa widening and the blue arrow replaces neutrophil infiltration. (B) Score of the lung injury. Effects of echinatin on the serum levels of IL-1β (C) and IL-6 (D) in LPS-stimulated ALI mice were analyzed by ELISA. Data are represented as mean ± SD, n = 6. # p < 0.05, ## p < 0.01, ### p < 0.001, #### p < 0.0001 vs. the control group. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. the LPS group.
Fig 9.
The molecular mechanism of action of echinatin exerting anti-inflammatory effects in macrophages.