Fig 1: Evodiamine enhances apo A1-mediated ChE from THP-1 macrophages and increases ABCA1 protein level. (a) Differentiated THP-1 cells were loaded with [3H]-cholesterol together with the indicated treatments for 24 h. On the next day, the cells were washed twice with PBS and incubated with the same compounds [solvent vehicle control (Veh; ≤0.1% DMSO), evodiamine (1–20 μM), and the PPARγ agonist pioglitazone (10 μM) as positive control] with or without 10 µg/mL apo A1. Extracellular as well as intracellular radioactivities were quantified with scintillation counter. Differentiated THP-1-derived macrophages were treated with solvent vehicle control (Veh; ≤0.1% DMSO), evodiamine (10 μM), and the PPARγ agonist pioglitazone (10 μM) as positive control. After 24 h incubation, the cells were lysed and 20 μg protein was resolved via SDS-PAGE. Immunodetection was performed with antibodies against the indicated proteins, ABCA1 (b), ABCG1 (c), and SR-B1 (d), and visualized by chemiluminescence detection. All experiments were performed at least three times and data are presented as means ± S.D. vs. solvent vehicle control, *p < 0.05, **p < 0.01, ***p < 0.001, n.s. no significance (ANOVA/Bonferroni).
Fig 2: Effect of evodiamine on ABCA1 transcription and the degradation rate of ABCA1 protein. (a) Differentiated THP-1 macrophages were incubated with 10 μM evodiamine or 10 μM pioglitazone as positive control for 24 h. Total RNA was extracted and ABCA1 mRNA expression levels were quantified by qRT-PCR. (b) 293 T cells were transfected with a luciferase reporter construct driven by the human ABCA1 promoter as described in the Materials and Methods section. After transfection, cells were treated with 10 μM Evodiamine or 1 μM T0901317 as positive control for 24 h. (c) Differentiated THP-1 macrophages were incubated for 24 h with (black circle) or without (Veh; white circle) evodiamine (10 μM) and lysed after addition of cycloheximide (CHX; 140 μM) at different time points (0, 10, 20, 40 min). Western blot analysis shows the decline of ABCA1 protein level with cycloheximide in the presence and absence of evodiamine. All data are means ± S.D. (n = 3) vs. solvent vehicle control (DMSO), *p < 0.05, **p < 0.01, n.s. no significance (ANOVA/Bonferroni).
Fig 3: Interactions between polyclonal anti-ABCA1 antibody, evodiamine and ABCA1 protein (ABCA1 peptide from Abcam, #ab125995, covering C-terminal domain 2085–2245aa). (a) Interaction of polyclonal anti-ABCA1 antibody with immobilized ABCA1 on CM5 sensor chip as assessed by Biacore 3000. The interaction affinity is calculated with association constant (KA) equal to 1.18e6M and dissociation constant (KD) equal to 4.57e-7M (antibody concentrations from bottom to top are 4.25e-8, 8.5e-8,1.75e-7, 3.5e-7 and 7e-7M). (b) Interaction affinity between evodiamine and ABCA1. Evodiamine interacts with an association constant (KA) equal to 1,23e6M and a dissociation constant (KD) equal to 8,1e-7M (evodiamine concentrations from bottom to top are 2.5e-7, 5e-7 and 1e-6M). There is a bulk effect with Evodiamine at 1e-6M. This explains the higher signal (RU) in regard to the two lower concentrations. (c) Interaction of retinal dehydogenase-1 (ALDHA1) with immobilized ABCA1 peptide. The two proteins interact with a KA equal to 1.25e6M and KD of 8.03e-7M (ALDHA1 concentrations from bottom to top are; 6e-8, 1.25e-7, 2.5e-7, 5e-7M and 1e-6M).
Supplier Page from Abcam for Recombinant Human ABCA1 protein (His-DHFR)