Antirheumatic drug leflunomide attenuates atherosclerosis by regulating lipid metabolism and endothelial dysfunction via DHODH/AMPK signaling pathway

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  Figure 1. Leflunomide exerts an anti-atherosclerotic effect in WD-fed ApoE-/- mice. (A) Schematic diagram of ApoE-/- mice treatment. (B) The body weight of ApoE-/- mice we investigated was monitored once a week for 12 weeks. n = 18 per group. (C) The TC and TG levels in the plasma of ApoE-/- mice in ND, WD, and leflunomide treatment (named Lef group) group. n = 18 per group. (D) Lipoproteins in the plasma of ApoE-/- mice in ND, WD, and Lef groups were separated by FPLC, and the TC level in the fractions was determined. (E) The plasma blood glucose (n = 18 per group), insulin (n = 8 per group), and GHbA1c (n = 8 per group) levels. (F) The IL-1β, TNFα, and IL-6 levels in the plasma. n = 8 per group. (G) Respective captured digital images of aortas. (H) Respective images of en-face aortas stained by ORO. (J) The en-face aorta lesions area was quantitatively analyzed with Image J software. n = 12 per group. (I, K) Respective images of H&E and ORO-stained aortic sinus cross sections of each group (I), and all stained sections were analyzed and quantified with Image J software (K). Scale bar = 500 μm, n = 6 per group. One-way ANOVA analysis was performed: *P < 0.05, ** P < 0.01, *** P < 0.001.

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  Figure 2. Leflunomide treatment improved lipid and glucose metabolism in WD-fed ApoE-/- mice. (A) The respective images of the H&E and ORO-stained section of the liver in the ND, WD, and leflunomide treatment (named Lef group) group ApoE-/- mice. Scale bar = 200 μm, n = 6 per group. (B) The TC and TG levels in the liver of the ND, WD, and Lef groups. n = 8 per group. (C) The plasma ALT and AST levels. n = 16 per group. (D-E) The blood glucose levels at a certain time point were detected during the OGTT and ITT and the areas under the curve (AUC) were calculated respectively. (F) Venn diagram based on RNA-Seq results from the livers of ND, WD, and Lef groups. n = 6 per group. (G) Volcano map of differentially expressed genes (DGEs) (fold change ≥ 1.5 and P < 0.01, WD vs Lef) in RNA-Seq results from the livers of WD and Lef groups. n = 6 per group. (H) KEGG enrichment analysis DGEs between WD and Lef group. n = 6 per group. (I) GSEA enrichment results show the pathways related to lipid metabolism in the RNA-Seq dataset. n = 6 per group. (J) Heatmap of lipid metabolism-related DGEs profiles based on the RNA-seq data set. P < 0.01, n = 6 per group. One-way ANOVA analysis was performed: *P <…

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  Figure 3. Leflunomide and Teriflunomide activate the AMPK pathway and alleviate lipid accumulation in FFA-stimulated AML12 cells. (A-B) Western blot analysis of the p-AMPKα, AMPKα, p-ACC, and ACC in livers of ApoE-/- mice in ND, WD, and leflunomide treatment (named Lef) group. Representative immunoblots are shown (A) and the images were quantitatively analyzed with Image J software (B), and then the ratio of p-AMPKα/AMPKα and p-ACC/ACC was calculated (B). n = 8 per group. (C-F) AML12 cells were treated with leflunomide (0, 1, 5, 20, 100 μM) (C-D) or teriflunomide (0, 1, 5, 20, 100 μM) (E-F) for 30 min (0, 1, 5, 20, 100 μM). The protein levels of p-AMPKα, AMPKα, p-ACC, and ACC were detected and the ratio of p-AMPKα/AMPKα and p-ACC/ACC was quantitatively analyzed. n = 5. (G-L) AML12 cells were treated with or without FFA (500 μM, PA: OA = 1:2) and leflunomide (0, 1, 5, 20, 100 μM) or teriflunomide (0, 1, 5, 20, 100 μM) simultaneously for 18 h. (G) The respective images of Nile red staining. Scale bar = 20 μm. n = 5. (H-K) Representative immunoblot images quantitative analysis of the ratio of p-AMPKα/AMPKα and p-ACC/ACC. n = 5. (L) Representative immunofluorescent images of SREBP1 (green) and…

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  Figure 4. Leflunomide alleviates lipid accumulation in FFA-stimulated AML12 cells via the AMPK pathway. (A-B) AML12 cells were transfected with control siRNA (si-control) or AMPKα siRNA (si-AMPKα) for 24 h, and then treated with or without leflunomide (100 μM) or teriflunomide (100 μM) for 30 min. The representative immunoblots of p-AMPKα, AMPKα, p-ACC, and ACC were shown and the p-ACC/ACC ratio was calculated. n = 5. (C-E) AML12 cells were first transfected with si-control or si-AMPKα for 24 h and then were treated with FFA and leflunomide (100 μM) or teriflunomide (100 μM) simultaneously for 18 h. (C) The Nile red stain was performed and the respective images were shown. Scale bar = 20 μm. (D-E) The representative immunoblots of p-AMPKα, AMPKα, p-ACC, and ACC were shown, and the p-ACC/ACC ratio was calculated. n = 5. (F) Representative immunofluorescent images of SREBP1 (green) and the cell nucleus stained with DAPI (blue). n = 5. One-way ANOVA analysis was performed: *P < 0.05, ** P < 0.01, *** P < 0.001.

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  Figure 5. The effects of leflunomide and teriflunomide on lipid accumulation and AMPK activation in AML12 cells were mediated by inhibiting DHODH. (A-B) The AML12 cells were first transfected with empty vector pCDNA3.1 or plasmid pCDNA3.1-mDHODH for 24h, and then cells were treated with leflunomide (100 μM) or teriflunomide (100 μM) for 30 min. (C-F) After transfected with empty vector pCDNA3.1 or plasmid pCDNA3.1-mDHODH for 24 h, the AML12 cells were treated with FFA (500 μM) and leflunomide (100 μM) or teriflunomide (100 μM) simultaneously for 18 h. (A-B & D-E) The representative immunoblots of p-AMPKα, AMPKα, p-ACC, and ACC were shown and the ratio of p-ACC/ACC and p-AMPKα/AMPKα were analyzed and calculated. n = 5. (C) Nile red staining was performed and the respective images were shown. Scale bar = 20 μm. (F) The immunofluorescent staining was performed with specific SREBP1 antibody (green) and DAPI (blue) for cell nucleus staining. One-way ANOVA analysis was performed: *P < 0.05, ** P < 0.01, *** P < 0.001.

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  Figure 6. Leflunomide and teriflunomide alleviate PA-induced endothelial dysfunction via the DHODH/AMPK/eNOS pathway. (A-C) HUVECs with PA (500 μM) and leflunomide (0, 1, 5, 20, 100 μM) or teriflunomide (0, 1, 5, 20, 100 μM) for 18 h. (A) The NO production in the HUVECs with PA (500 μM) and leflunomide or teriflunomide (0, 1, 5, 20, 100 μM) for 18 h. n = 5. (B-C) The representative immunoblots of p-eNOS, eNOS, p-AMPKα, and AMPKα were shown, and the ratio of p-eNOS/eNOS and p-AMPKα/AMPKα were analyzed. n = 5. (D-F) HUVCEs were first transfected with si-control or si-AMPKα for 24 h and then were treated with PA (500 μM) and leflunomide (100 μM) or teriflunomide (100 μM) simultaneously for 18 h. (D) The NO production level was determined. n = 5. (E-F) The protein expression levels of p-eNOS, eNOS, p-AMPKα, and AMPKα were detected and the relative eNOS protein expression was quantified. n = 5. (G) The HUVECs were transfected with empty vector pCDNA3.1 or plasmid pCDNA3.1-DHODH, and then the mRNA levels of DHODH in HUVECs were detected using qRT-PCR. (H-J) HUVECs were first transfected with empty vector pCDNA3.1 or plasmid pCDNA3.1-DHODH, and then they were treated with PA (500 μM) and…