Macrophage Inactivation by Small Molecule Wedelolactone via Targeting sEH for the Treatment of LPS-Induced Acute Lung Injury

Abstract

Soluble epoxide hydrolase (sEH) plays a critical role in inflammation by modulating levels of epoxyeicosatrienoic acids (EETs) and other epoxy fatty acids (EpFAs). Here, we investigate the possible role of sEH in lipopolysaccharide (LPS)-mediated macrophage activation and acute lung injury (ALI). In this study, we found that a small molecule, wedelolactone (WED), targeted sEH and led to macrophage inactivation. Through the molecular interaction with amino acids Phe362 and Gln384, WED suppressed sEH activity to enhance levels of EETs, thus attenuating inflammation and oxidative stress by regulating glycogen synthase kinase 3beta (GSK3β)-mediated nuclear factor-kappa B (NF-κB) and nuclear factor E2-related factor 2 (Nrf2) pathways in vitro. In an LPS-stimulated ALI animal model, pharmacological sEH inhibition by WED or sEH knockout (KO) alleviated pulmonary damage, such as the increase in the alveolar wall thickness and collapse. Additionally, WED or sEH genetic KO both suppressed macrophage activation and attenuated inflammation and oxidative stress in vivo. These findings provided the broader prospects for ALI treatment by targeting sEH to alleviate inflammation and oxidative stress and suggested WED as a natural lead candidate for the development of novel synthetic sEH inhibitors.

Figure 1

WED alleviated inflammatory responses in vitro through the NF-κB pathway. (A) WED suppressed the release of LPS-induced TNF-α (p < 0.0001; df = 4, 10; F-value = 143.3) and IL-6 (p < 0.0001; df = 4, 10; F-value = 238.1) in RAW264.7 cells (mean ± SEM, n = 3, one-way ANOVA). (B) WED downregulated mRNA levels of TNF-α (p < 0.0001; df = 4, 10; F-value = 71.86), IL-6 (p < 0.0001; df = 4, 10; F-value = 147.1), COX-2 (p < 0.0001; df = 4, 10; F-value = 79.7), iNOS (p < 0.0001; df = 4, 10; F-value = 33.1), MCP-1 (p < 0.0001; df = 4, 10; F-value = 21.1), and ICAM-1 (p = 0.0019; df = 4, 10; F-value = 9.6) in LPS-induced RAW264.7 cells (mean ± SEM, n = 3, one-way ANOVA). (C) WED inactivated the NF-κB pathway (p < 0.0001; df = 4, 10; F-value = 28.2) to downregulate expression levels of its target proteins TNF-α (p < 0.0001; df = 4, 10; F-value = 29.6), IL-6 (p < 0.0001; df = 4, 10; F-value = 31.1), COX-2 (p < 0.0001; df = 4, 10; F-value = 105.5), iNOS (p < 0.0001; df = 4, 10; F-value = 41.8), and MCP-1 (p < 0.0001; df = 4, 10; F-value = 23.7) in LPS-induced RAW264.7 cells (mean ± SEM, n = 3, one-way ANOVA). (D,E) WED suppressed the translocation of p65 to the nucleus analyzed by confocal microscopy (D) and Western blot (E) (mean ± SEM, n = 3, one-way ANOVA. For p65 in nucleus, p < 0.0001; df = 4, 10; F-value = 30.3. For p65 in cytoplasm, p = 0.0015; df = 4, 10; F-value = 10.2).

Figure 2

WED alleviated the oxidative reduction in vitro through the Nrf2 pathway. (A) WED enhanced levels of GSH (p = 0.0018; df = 4, 10; F-value = 9.8) and SOD (p = 0.0002; df = 4, 10; F-value = 17.1) in LPS-induced RAW264.7 cells (mean ± SEM, n = 3, one-way ANOVA). (B) The flow cytometry demonstrated that WED reduced the LPS-induced ROS-positive cells. (C) Quantitative data of ROS-positive cells in LPS-stimulated RAW264.7 cells treated with WED (mean ± SEM; n = 3; one-way ANOVA; p < 0.0001; df = 4, 10; F-value = 24.0). (D) WED reversed the increase of mtDNA content in LPS-induced RAW264.7 cells (mean ± SEM; n = 3; one-way ANOVA; p < 0.0001; df = 4, 10; F-value = 27.6). (E) The fluorometric analysis indicated that WED suppressed the ROS production. (F) WED reversed the effect of LPS-mediated mitochondrial membrane potential. (G) WED regulated expressions of proteins Mfn1, Mfn2, Op1, Drp1, and Fis1 involved in the mitochondrial fusion and fission. (H) WED activated the Nrf2 pathway to regulate expression levels of HO-1, NQO-1, GCLC, GCLM, Nrf2, and Keap1. (I,K) WED suppressed the translocation of Nrf2 to the nucleus analyzed by Western blot (I) (mean ± SEM, n = 3, one-way ANOVA. For Nrf2 in nucleus, p < 0.0001; df = 4, 10; F-value = 38.9. For Nrf2 in cytoplasm, p = 0.0001; df = 4, 10; F-value = 18.67) and the confocal microscopy (K). (J) WED regulated mRNA levels of genes Keap1 (p < 0.0001; df = 4, 10; F-value = 202.5), HO-1 (p = 0.0002; df = 4, 10; F-value = 16.5), NQO-1 (p < 0.0001; df = 4, 10; F-value = 84.5), and Nrf2 (p < 0.0001; df = 4, 10; F-value = 30.0) involved in the Nrf2 pathway (mean ± SEM, n = 3, one-way ANOVA). (L) The luciferase assay demonstrated the activation of WED against the Nrf2 receptor (mean ± SEM; n = 5; one-way ANOVA; p < 0.0001; df = 3, 16; F-value = 65.6).

Figure 3

WED attenuated the course of ALI in LPS-induced ALI mice. (A) Representative H&E staining plots. (B) Representative CD68 staining plots. (C) Representative Gr-1 staining plots. (D) WED attenuated the infiltration of proteins (p < 0.0001; df = 5, 42; F-value = 11.3), WBCs (p < 0.0001; df = 5, 42; F-value = 36.2), PMNs (p < 0.0001; df = 5, 42; F-value = 57.8), and MNs (p < 0.0001; df = 5, 42; F-value = 32.1) to the pulmonary alveoli and reduced the production of IL-6 (p < 0.0001; df = 5, 42; F-value = 31.8) and TNF-α (p < 0.0001; df = 5, 42; F-value = 41.2) and the activity of MPO (p < 0.0001; df = 5, 42; F-value = 9.8) and LDH (p < 0.0001; df = 5, 42; F-value = 27.7) in LPS-induced ALI mice (mean ± SEM, n = 8, one-way ANOVA). (E) WED reduced the production of IL-6 (p < 0.0001; df = 5, 42; F-value = 23.8) and TNF-α (p < 0.0001; df = 5, 42; F-value = 14.2) and the activity of MPO (p < 0.0001; df = 5, 42; F-value = 27.6) and LDH (p < 0.0001; df = 5, 42; F-value = 7.9) in the lung of LPS-induced ALI mice (mean ± SEM, n = 8, one-way ANOVA).

Figure 4

WED attenuated the inflammation and oxidative stress in vivo. (A) Representative TNF-α staining plots. (B) Effects of WED against inflammatory genes TNF-α (p < 0.0001; df = 5, 24; F-value = 30.5), IL-6 (p < 0.0001; df = 5, 24; F-value = 56.7), COX-2 (p < 0.0001; df = 5, 24; F-value = 12.8), iNOS (p < 0.0001; df = 5, 24; F-value = 21.0), ICAM-1 (p < 0.0001; df = 5, 24; F-value = 21.4), and MCP-1 (p < 0.0001; df = 5, 24; F-value = 25.5) in LPS-induced ALI mice (mean ± SEM, n = 5, one-way ANOVA). (C) WED suppressed the NF-κB pathway to downregulate expression levels of its target proteins IL-6, COX-2, and MCP-1. (D) Representative scanning electron microscope plots. (E) Effects of WED toward the MDA level (p < 0.0001; df = 5, 42; F-value = 13.4) and the SOD activity (p < 0.0001; df = 5, 42; F-value = 15.4) in LPS-induced ALI mice (mean ± SEM, n = 8, one-way ANOVA). (F) WED reversed the increase of the mtDNA content in LPS-induced ALI mice (mean ± SEM; n = 8; one-way ANOVA; p < 0.0001; df = 5, 24; F-value = 20.3). (G) WED regulated expressions of proteins Mfn1, Mfn2, Op1, Drp1, and Fis1 involved in the mitochondrial fusion and fission in LPS-induced ALI mice. (H) Representative Nrf2 staining plots. (I) WED activated the Nrf2 pathway to regulate expression levels of HO-1, NQO-1, GCLC, GCLM, Nrf2, and Keap1 in LPS-induced ALI mice.

Figure 5

sEH served as the direct target of WED in anti-inflammation and antioxidation. (A) Identification of the cellular target of WED using the pull-down technology on the basis of WED-coupled Sepharose 6B beads and LC–MS/MS analysis. (B) The LC-MS/MS plot of sEH. (C) The binding protein was detected by Western blot. (D) The IP-MS analysis indicated the interaction of WED with sEH. (E) CETSA and DARTS results. (F) Quantitative data of CETSA and DARTS (mean ± SEM; n = 3; one-way ANOVA; p < 0.0001; df = 5, 12; F-value = 100.2). (G) The MST result of WED with sEH (mean ± SEM, n = 3). (H) The scheme of Bio-WED. (I) The colocation of WED with sEH detected by the fluorescence microscope. (J) The CYP/sEH-mediated AA metabolism pathway. (K) The binding capability of WED with sEH detected by the fluorescence-based binding assay (mean ± SEM, n = 3). (L) The inhibitory effect of WED against the human sEH activity detected by the system of human recombinant sEH-mediated hydrolysis of the substrate PHOME (mean ± SEM, n = 3). (M) WED suppressed the sEH activity (14,15-EET/14,15-DHET, p < 0.0001; df = 4, 10; F-value = 55.89) analyzed by levels of 14,15-EET (p = 0.0003; df = 4, 10; F-value = 15.1) and its corresponding diol (p = 0.0034; df = 4, 10; F-value = 8.2) in LPS-mediated RAW264.7 cells (mean ± SEM, n = 3, one-way ANOVA).

Figure 6

sEH knockdown and rescue abolished anti-inflammatory and antioxidant effects of WED in vitro. (A) sEH knockdown abolished the effects of WED toward inflammatory and antioxidant genes TNF-α (p = 0.0028; df = 1, 8; F-value = 18.0), IL-6 (p < 0.0001; df = 1, 8; F-value = 355.1), COX-2 (p = 0.0058; df = 1, 8; F-value = 13.9), iNOS (p < 0.0001; df = 1, 8; F-value = 133.8), NQO-1 (p = 0.0037; df = 1, 8; F-value = 16.4), Nrf2 (p = 0.0194; df = 1, 8; F-value = 8.5), and Keap1 (p = 0.0006; df = 1, 8; F-value = 30.0) in LPS-induced RAW264.7 cells (mean ± SEM, n = 3, two-way ANOVA). (B) sEH knockdown abolished the effects of WED toward the NF-κB and Nrf2 pathways measured by Western blot. (C) sEH rescue weakened the effects of WED toward inflammatory and antioxidant genes TNF-α (p = 0.0006; df = 1, 8; F-value = 29.7), IL-6 (p = 0.0921; df = 1, 8; F-value = 3.7), COX-2 (p = 0.0489; df = 1, 8; F-value = 5.4), iNOS (p = 0.4830; df = 1, 8; F-value = 0.5), NQO-1 (p = 0.6083; df = 1, 8; F-value = 0.3), HO-1 (p = 0.8389; df = 1, 8; F-value = 0.04), and Nrf2 (p = 0.8812; df = 1, 8; F-value = 0.02) in LPS-induced RAW264.7 cells (mean ± SEM, n = 3, two-way ANOVA). (D) sEH rescue weakened the effects of WED toward the NF-κB and Nrf2 pathways measured by Western blot.

Figure 7

Phe362 and Gln384 played roles in the binding of WED with sEH. (A) The RMSD plot analyzed by molecular dynamics. (B) Effect of WED against the volume of the pocket. (C) The trajectories of Phe362, Ile363, Ser374, and Asn378 with WED. (D) The trajectories of Tyr363, Gln384, Gln502, and Met503 with WED. (E) The interactions of WED with sEH through hydrogen bonds of Phe362 and Gln384. (F) Phe362Ala and Gln384Ala mutations abolished the binding of WED with sEH. (G) Phe362Ala and Gln384Ala mutations abolished the anti-inflammatory [IL-6, p = 0.0014, df = (2, 12), F-value = 12.0; TNF-α, p < 0.0001, df = (2, 12), F-value = 37.4; COX-2, p = 0.0005, df = (2, 12), F-value = 15.1] and antioxidative effects [HO-1, p = 0.0001, df = (2, 12), F-value = 20.6; Nrf2, p < 0.0001, df = (2, 12), F-value = 30.5] of WED in LPS-stimulated RAW264.7 cells (mean ± SEM, n = 3, two-way ANOVA).

Figure 8

GSK3β is the downstream key pathway of sEH in the anti-inflammatory and antioxidant effects of WED. (A) sEH knockdown suppressed the GSK3β activity. (B) sEH rescue activated the GSK3β activity. (C) Inhibition of sEH by WED suppressed the GSK3β activity. (D) Inhibition of GSK3β by LiCl abolished effects of WED toward inflammatory and antioxidant genes TNF-α (p = 0.0316; df = 1, 8; F-value = 6.8), IL-6 (p = 0.0006; df = 1, 8; F-value = 29.8), COX-2 (p < 0.0001; df = 1, 8; F-value = 52.5), iNOS (p = 0.0001; df = 1, 8; F-value = 50.2), HO-1 (p = 0.0097; df = 1, 8; F-value = 11.4), NQO-1 (p = 0.0005; df = 1, 8; F-value = 32.2), and Nrf2 (p < 0.0001; df = 1, 8; F-value = 55.7) in vitro (mean ± SEM, n = 3, two-way ANOVA). (E) Inhibition of GSK3β by LiCl abolished effects of WED toward the NF-κB (p = 0.0154; df = 1, 8; F-value = 9.4) and Nrf2 (p < 0.0001; df = 1, 8; F-value = 69.8) pathways measured by Western blot (mean ± SEM, n = 3, two-way ANOVA).

Figure 9

WED suppressed the sEH activity to allow the inhibition of GSK3β in vivo. (A) Heat map of sEH substrates (e.g., 8,9-EET, 11,12-EET, and 14,15-EET) and their corresponding diols (e.g., 8,9-DHET, 11,12-DHET, and 14,15-DHET). (B) WED reduced the ratio of 8,9-DHET/8,9-EET (p < 0.0001; df = 5, 30; F-value = 20.5), 11,12-DHET/11,12-EET (p < 0.0001; df = 5, 30; F-value = 37.5), and 14,15-DHET/14,15-EET (p < 0.0001; df = 5, 30; F-value = 22.9) in vivo to suppress the sEH activity (mean ± SEM, n = 6, one-way ANOVA). (C) Inhibition of sEH by WED led to the inactivation of the GSK3β activity (p < 0.0001; df = 5, 12; F-value = 25.3) in LPS-induced ALI mice (mean ± SEM, n = 3, one-way ANOVA).

Figure 10

sEH genetic KO abolished the pulmonary protective effect of WED. (A) Genotyping and confirmation of sEH knockout mice. (B,C) Representative plots of H&E (B) and CD68 and Gr-1 (C) staining in LPS-induced ALI Ephx2^+/+ and Ephx2^–/– mice treated with WED. (D) Measurement of pulmonary MPO (p < 0.0001; df = 1, 20; F-value = 32.7), TNF-α (p = 0.0323; df = 1, 20; F-value = 5.3), and IL-6 (p = 0.0002; df = 1, 20; F-value = 20.5) from LPS-induced ALI Ephx2^+/+ and Ephx2^–/– mice treated with WED (mean ± SEM, n = 6, two-way ANOVA). (E) Ephx2 KO abolished the effect of WED on the inhibition of GSK3β (p = 0.0019; df = 1, 8; F-value = 20.4) in LPS-induced ALI (mean ± SEM, n = 3, two-way ANOVA).

Figure 11

sEH genetic KO abolished the anti-inflammatory and antioxidant effects of WED. (A) Representative TNF-α staining plots in LPS-induced ALI Ephx2^+/+ and Ephx2^–/–mice treated with WED. (B) Ephx2 KO abolished the effect of WED on expressions of COX-2 and p-p65/p65 in LPS-induced ALI. (C) Ephx2 KO abolished the effect of WED on mRNA levels of COX-2 (p = 0.0267; df = 1, 16; F-value = 6.0), iNOS (p = 0.0033; df = 1, 16; F-value = 11.9), TNF-α (p = 0.0058; df = 1, 16; F-value = 10.1), IL-6 (p = 0.0003; df = 1, 16; F-value = 21.6), and ICAM-1 (p = 0.0842; df = 1, 16; F-value = 3.4) in LPS-induced ALI (mean ± SEM, n = 5, two-way ANOVA). (D) Representative HO-1 and Nrf2 staining plots in LPS-induced ALI Ephx2^+/+ and Ephx2^–/– mice treated with WED. (E) Measurement of pulmonary MDA (p = 0.0012; df = 1, 20; F-value = 14.3), GSH (p = 0.0008; df = 1, 20; F-value = 15.5), and SOD (p = 0.0012; df = 1, 20; F-value = 14.4) from LPS-induced ALI Ephx2^+/+ and Ephx2^–/– mice treated with WED (mean ± SEM, n = 6, two-way ANOVA). (F) Ephx2 KO abolished the effect of WED on expressions of Mfn1, Drp1, HO-1, and Nrf2 in LPS-induced ALI. (G) Ephx2 KO abolished the effect of WED on mRNA levels of NQO-1 (p = 0.0047; df = 1, 16; F-value = 10.8) and Nrf2 (p = 0.0041; df = 1, 16; F-value = 11.2) in LPS-induced ALI (mean ± SEM, n = 5, two-way ANOVA).