| Part A:Emodin facilitates cholesterol efflux from THP-1 macrophages and activates PPAR-y signalingObjective:Atherosclerosis is one of the most life-threatening cardiovascular diseases. Macrophage foam cells play a key role in the development and progression of atherosclerosis. The imbalance of cholesterol intake and efflux is a major cause of the formation of foam cells. Emodin is a natural bioactive agent and has anti-inflammatory, anti-cancer, and lipid-lowering activities. Many studies have demonstrated that emodin is capable of activating peroxisome proliferator-activated receptor-γ (PPAR-γ) signaling in several biological settings. The aim of this study is to check whether emodin can promote cholesterol efflux from macrophage foam cells via activating of PPAR-y signaling.Materials and methods:Human monocyte THP-1 cells were exposed to 100 nmol/L of phorbol-12-myristate-13-acetate (PMA) for 72 h and differentiated into macrophages. Immunocytochemical measurement by flow cytometry of the macrophage biomarkers, CD14, CD11b and CD11c, was performed. PMA-differentiated THP-1 macrophages were treated with 50μg/ml of oxidized low-density lipoprotein (oxLDL) and labeled with 3H-cholesterol (0.2μ Ci/ml) for 24 h. Then the cells were incubated with 10 μg/ml of apolipoprotein A-I (apoA-I) and different concentrations of emodin (0,1,5, and 10μM) for 18 h, or 10μg/ml of apoA-I and 10 μM of emodin for 0,4,8,12,16, 18, and 24 h. For specific PPAR-y inhibition, different doses of GW-9662 (0,1,5, and 10μM), an irreversible and selective PPAR-γ inhibitor, were added to cells 1 h before the treatment with emodin and maintained in culture medium for 18 h. For small interfering RNA (siRNA) assay, PMA-differentiated THP-1 macrophages were transfected with 20,40,80 nM of human PPAR-γ siRNA or control siRNA, and incubated for 24 h. The human PPAR-γ siRNA was as follows: 5’-G-G-A-U-G-C-A-A-G-G-G-U-U-U-C-U-U-C-C-t-t-3’and 5’-G-G-A-A-G-A-A-A-C-C-C-U-U-G-C-A-U-C-C-t-t-3’. The transfected cells were treated with 50 μg/ml of oxLDL for 24 h and then stimulated with 10 μM of emodin. At the end of the incubation, cells are centrifuged and were tested for cholesterol efflux. Cholesterol efflux is presented as a percentage, calculated as 3H-cholesterol in medium/ (3H-cholesterol in medium+3H-cholesterol in cells) × 100%. Total cellular RNA was extracted, and changes in PPAR-γ mRNA expression were assessed using semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) assay. The expression changes in PPAR-γ protein were determined using Western blot analysis.Results:PMA exposure for 72 h resulted in the expression of CD14, CD11b, and CD11c on THP-1 cells. In contrast, untreated control cells did not express these macrophage biomarkers. PMA-differentiated THP-1 macrophages treated with 50μg/ml of ox-LDL for 18 h showed a significant accumulation of lipid droplets, as indicated by Oil-Red O staining. Different concentrations of emodin (1,5, and 10μM) significantly increased the PPAR-γ mRNA/β-actin ratio (1.592±0.256,2.571±0.193, and 3.591±0.218, respectively), as compared to control cells (1±0.191). The upregulation of PPAR-γ mRNA by emodin occurred after 4 h and up to 18 h, and the PPAR-γ mRNA/β-actin ratio was significantly higher than that in control cells (P<0.01). The PPAR-γ protein/β-actin ratio (1.419±0.296,2.617±0.252, and 3.417±0.296, respectively) was significantly raised in emodin (1,5, and 10 μM)-treated THP-1 macrophages when compared to control cells (1±0.162). The elevation of PPAR-γ protein began at 8 h after emodin treatment (the PPAR-γ protein/β-actin ratio of 2.169±0.173) and continued to rise at 18 h (the PPAR-γ protein/β-actin ratio of 3.812±0.265) relative to control cells (0.982±0.115) (P<0.01 for both comparisons). In the presence of apoA-I, the cholesterol efflux rate was 7.62±0.61% in ox-LDL-treated THP-1 macrophages, which was significantly higher than that in control cells (2.12±0.32%; P<0.01). Emodin at 5μM (10.74±0.93%) and 10μM (16.29±1.48%) significantly promoted apoA-I-mediated cholesterol efflux from ox-LDL-treated THP-1 macrophages. GW9662 at 5-10μM significantly antagonized the inductive effects of emodin on cholesterol efflux from THP-1 macrophages (P<0.01). The transfection of PPAR-y siRNA significantly reduced the PPAR-γ/β-actin ratio compared to control cells (P<0.01). Notably, the delivery of PPAR-γ siRNA significantly blocked the induction of cholesterol efflux by emodin.Conclusion:Taken together, our results demonstrate that emodin promotes cholesterol efflux from ox-LDL-treated THP-1 macrophages through activation of the PPAR-y signaling pathway.Part B:Emodin facilitates cholesterol efflux from THP-1 macrophages via activation of LXR-a-mediated transcription of ABCA1 and ABCG1Objective:ATP-binding cassette (ABC) transporters can utilize the energy of ATP to actively transport numerous substrates such as lipids, toxins, and drugs across the cell membrane. ABCA1 and ABCG1 are capable of transporting cholesterol to apoA-I, thus preventing the formation of macrophage foam cells. Liver X receptor alpha (LXR-a) can directly bind to the specific sites in the promoter region of ABCA1 and ABCG1 and lead to transcriptional activation. It has been documented that PPAR-y can induce the transcription of both ABCA1 and ABCG1 via a LXR-a-dependent mechanism. Given the close relationship between PPAR-y and LXR-a, this study aims to explore whether the inductive effects of emodin on cholesterol efflux are mediated through LXR-a-dependent transcription of ABCA1 and ABCG1.Materials and methods:THP-1 cells were exposed to 100 nmol/L of PMA for 72 h to differentiate into macrophages. PMA-differentiated THP-1 macrophages were treated with 50μg/ml of oxidized low-density lipoprotein (oxLDL) for 24 h, followed by incubation with 10 μg/ml of apolipoprotein A-I (apoA-I) and different concentrations of emodin (0,1,5, and 10 μM) for 18 h or 10 μg/ml of apoA-I and 10 μM of emodin for 0,4,8,12,16, 18, and 24 h. For specific PPAR-γ inhibition, different doses of GW-9662 (0,1,5, and 10μM), an irreversible and selective PPAR-γ inhibitor, were added to cells 1 h before the treatment with emodin and maintained in culture medium for 18 h. Total cellular RNA was extracted and the mRNA expression changes of LXRa, ABCA1, and ABCG1 were assessed using semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) assay. The protein expression of LXRa, ABCA1, and ABCGl was examined using Western blot analysis.Results:RT-PCR analysis revealed that emodin at 1,5, and 10 μM (1.671±0.265, 2.726±0.306, and 3.198±0.358, respectively) resulted in a significant increase in the LXR-α/β-actin ratio in ox-LDL-treated THP-1 macrophages, compared to control cells (1±0.188). Western blot analysis showed that the LXR-α/β-actin ratio was 1.571±0.199,2.169±0.266, and 2.919±0.311 in emodin (1,5, and 10μM)-treated THP-1 macrophages, respectively, which was significantly higher than that in control cells (1±0.156). When compared to control cells,10 μM of emodin significantly enhanced the PPAR-y protein expression (P<0.01). GW9662 at 5 and 10μM significantly reduced the PPAR-γ/β-actin ratio compared to THP-1 macrophages treated with emodin alone. Similarly, GW9662 pretreatment resulted in a significant decrease in the expression of LXR-α. Emodin at 5 and 10μM (1.761±0.296 and 3.266±0.334, respectively) significantly increased the ABCA1 mRNA/β-actin ratio relative to control cells (1±0.077). Similarly, emodin at 5 and 10μM (1.662±0.207 and 2.952±0.326, respectively) significantly increased the ABCGl mRNA/β-actin ratio relative to control cells (1±0.077). At the protein level, emodin treatment also significantly increased the expression of ABCA1 and ABCG1 in ox-LDL-treated THP-1 macrophages. The PPAR-y inhibitor GW9662 (5 and 10μM) was found to significantly inhibit the inductive effect of emodin on ABCA1 and ABCG1 expression.Conclusion:The present results provide first evidence for the involvement of LXRa, ABCA1, and ABCG1 in emodin-mediated induction of cholesterol efflux. PPAR-γ activation participates in the induction of LXRa, ABCA1, and ABCG1 by emodin, thus contributing to the enhancement of cholesterol efflux. |
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