Resveratrol improved atherosclerosis by increasing LDLR levels via the EGFR-ERK1/2 signaling pathway

Abstract

Background and aims: Atherosclerosis (AS) is a complex and chronic vascular disease and elevated low-density lipoprotein cholesterol (LDL-C) level is one of its primary causative factors. As a key surface receptor, low-density lipoprotein receptor (LDLR) plays an essential role in LDL-C clearance. Resveratrol (RSV) has emerged as a promising compound for investigating potential therapeutic targets for AS due to its ability to lower cholesterol, reduce endothelial anti-inflammatory and suppress vascular smooth muscle cell proliferation. This study explored the effects of RSV on AS through upregulating LDLR and analyzed the mechanism through a combination of in vivo and vitro experiments.

Methods: HepG2 cells were exposed to varying concentrations of RSV. The effects of RSV on LDLR expression and cholesterol uptake were analyzed by western blot, RT-qPCR and DiI-LDL uptake assay. In vivo, C57BL/6J ApoE^-/- mice were used and the experimental groups were treated with RSV, Lovastatin and Gefitinib. Plaque formation in the arteries and aortic roots was assessed by Oil Red O staining and plaque stability was evaluated using Hematoxylin-Eosin (H&E) and Elastic Van Gieson (EVG) staining. Western blot, RT-qPCR and immunohistochemical staining were employed to analyze the expression of LDLR in the livers of mice.

Results: RSV significantly enhanced the stability of LDLR mRNA and promoted LDLR protein expression. The inhibition experiments of EGFR signaling pathway (Cetuximab and Gefitinib) demonstrated that the efficacy of RSV was markedly weakened when this signaling pathway was inhibited. It indicated that RSV modulated LDLR gene expression by activating EGFR-ERK1/2 pathway. In ApoE^-/- mice, RSV notably reduced arterial plaque formation, improved plaque stability and increased hepatic LDLR expression.

Conclusion: This study elucidated the mechanism by which RSV upregulates LDLR gene expression through activating EGFR-ERK1/2 signaling pathway. In vivo experiments demonstrated its efficacy in reducing arterial plaque formation and stabilizing existing plaques. These results further indicated that RSV held potential therapeutic value for ameliorating atherosclerosis and cardiovascular diseases. Collectively, these findings provided novel theoretical support for RSV's potential role in cardiovascular therapy.

Keywords: Atherosclerosis (AS); Cardiovascular diseases; Cholesterol metabolism; Low-density lipoprotein receptor (LDLR); Resveratrol (RSV).

Fig. 1

RSV elevated DiI-LDL uptake by increasing LDLR expression. (A) After treated HepG2 cells with RSV (10, 20 and 30 µM), cell viability was measured by MTT assay. (B, C) Western blot and RT-PCR were performed to assess LDLR protein and mRNA levels, respectively. (D) DiI-LDL uptake was detected under a fluorescence microscope. (E) HepG2 cells were exposed to actinomycin D and RSV (20 µM), followed by RT-qPCR analysis to assess LDLR mRNA level. Date are expressed as mean ± SEM. Significant differences versus the control group are denoted by **P < 0.01 and ***P < 0.001

Fig. 2

RSV activated EGFR-ERK1/2 signaling pathway to promote LDLR expression. (A) HepG2 cells were exploed to RSV (10 µM, 20 µM and 30 µM). Expression levels of P-EGFR, EGFR, ERK and P-ERK were analyzed by western blot. (B) Cetuximab (5 µg/ml) and RSV (20 µM) were co-administered to HepG2 cells. Protein levels of P-EGFR, EGFR, ERK, and P-ERK were assessed using western blot analysis. (C) Dil-LDL uptake was visualized under a fluorescence microscope. Date are expressed as mean ± SEM. Significant differences versus the control group are denoted by *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 3

RSV reduced aortic plaque formation in high-fat diet-fed ApoE^-/- mice. (A, B) The presence of plaques in the aorta and aortic sinuses was assessed through Oil red O staining and the area of these plaques was quantified using Image J. Date are expressed as mean ± SEM. Significant differences versus the control group are denoted by *P < 0.05, **P < 0.01 and ***P < 0.001 (n = 6)

Fig. 4

RSV enhanced plaque stability in a high-fat diet ApoE^-/- mice. (A, C) H&E and EVG staining were performed on aortic sinus sections to assess the necrotic core area and elastic fiber content, respectively. (B) Immunofluorescent staining was performed on aortic sinus section to assess macrophage deposition. Date are expressed as mean ± SEM. Significant differences versus the control group are denoted by *P < 0.05, **P < 0.01 and ***P < 0.001 (n = 6)

Fig. 5

RSV enhanced LDLR expression by activating EGFR-ERK signaling pathway in high-fat diet-fed ApoE^-/- mice. (A) The mRNA expression of LDLR in livers was measured by RT-qPCR. (B) Western blot was performed to measure protein levels of LDLR, P-EGFR, EGFR, P-ERK and ERK in lives. (C) Immunohistochemical staining was used to analyze LDLR, P-EGFR and EGFR expression in livers. Date are expressed as mean ± SEM. Significant differences versus the control group are denoted by *P < 0.05, **P < 0.01 and ***P < 0.001 (n = 6)

Fig. 6

RSV reduced liver lipid accumulation and improved blood lipid biochemical indices. (A) Serum levels of TG, TC, LDL-C and HDL-C were measured in ApoE^-/- mice. (B) Oil red O staining was employed to detect lipid deposition in livers. Date are expressed as mean ± SEM. Significant differences versus the control group are denoted by *P < 0.05, **P < 0.01 and ***P < 0.001 (n = 6)