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. 2024 Jul 2;20(10):3725–3741. doi: 10.7150/ijbs.93465

Figure 6.

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 leflunomide (100 μM) or teriflunomide (100 μM) for 18 h. (I) The NO production in the cells was detected. (H-J) The protein levels of p-eNOS, eNOS, p-AMPKα, and AMPKα were detected and the relative expression of p-eNOS and the ratio of p-AMPKα/AMPKα were analyzed. n = 5. One-way ANOVA analysis was performed: *P < 0.05, ** P < 0.01, *** P < 0.001.