Curdione protects vascular endothelial cells and atherosclerosis via the regulation of DNMT1-mediated ERBB4 promoter methylation

Abstract

Atherosclerosis (AS) is initiated by the activation of the endothelial cells, which is followed by a series of events that trigger the narrowing of blood vessels and the activation of inflammation. This study aimed to investigate in vitro the roles and underlying mechanisms of curdione in AS. Human umbilical vein endothelial cells (HUVECs) were stimulated with oxidized low-density lipoprotein (ox-LDL) and then treated with curdione, after which the growth of the HUVECs and the related mechanisms were determined. HUVECs with ERBB4 overexpression were constructed to explore the role of ERBB4 in curdione-mediated AS. The interaction among ERBB4, methylation, and curdione was confirmed by chromatin immunoprecipitation (ChIP)-quantitative PCR (qPCR) and dual luciferase reporter gene assays. Both curdione and ERBB4 overexpression individually and significantly enhanced viability and proliferation while suppressing apoptosis of the ox-LDL-induced HUVECs, and the combination of curdione and ERBB4 overexpression had better effects. Compared with the ox-LDL-induced HUVECs, both curdione and ERBB4 overexpression individually decreased the levels of IL-6, IL-1β, and IL-8 (P < 0.05). They also upregulated Bax, caspase-3, E-cadherin, and F-actin while downregulating Bcl-2 and VEGF (P < 0.05). Additionally, the ERBB4 bound to the DNMT1 gene, and the curdione participated in AS via the ERBB4 gene. The study demonstrated that either curdione or ERBB4 overexpression individually may ameliorate AS development by inhibiting apoptosis, inflammation, and the EndMT of HUVECs. In addition, curdione may protect the vascular endothelial cells and AS by regulating the DNMT1-mediated ERBB4 promoter methylation.

Keywords: DNMT1; ERBB4; HUVECs; atherosclerosis; curdione; ox-LDL.

Figure 1

Effects of curdione on the growth of oxidized low-density lipoprotein (ox-LDL)-induced HUVECs. (a) The viability of HUVECs treated with ox-LDL and curdione determined by cell counting kit-8. (b) The proliferation of HUVECs treated with ox-LDL and curdione determined by EdU assay. The apoptosis of HUVECs treated with ox-LDL and curdione measured by flow cytometry (c) and TUNEL assay (d). CUR: Curdione. *P < 0.05 vs control; ^# P < 0.05 vs ox-LDL.

Figure 2

Effects of curdione on the concentrations of inflammatory cytokines, and expression of apoptosis-related, endothelial-mesenchymal transition (EndMT)-related, and DNA methylation-related proteins. (a) The mRNA expression ERBB4 in the HUVECs treated with ox-LDL and curdione using real-time quantitative PCR. The concentrations of IL-6 (b), IL-1β (c), and IL-8 (d) in the HUVECs treated with ox-LDL and curdione by ELISA. (e) The protein expression of ERBB4, apoptosis-related (Bax, Bcl-2, and caspase-3), EndMT-related (E-cadherin, F-actin, and VEGF), and DNA methylation-related proteins (DNMT1, DNMT3a, and DNMT3b) in the HUVECs treated with ox-LDL and curdione using Western blot. CUR: Curdione. *P < 0.05 vs control; ^# P < 0.05 vs ox-LDL.

Figure 3

Effects of ERBB4 on the growth of curdione regulating ox-LDL-induced HUVECs. (a) Evaluation of cell transfection efficiency through determining the expression of ERBB4 using Western blot. (b) The viability of HUVECs after OE-ERBB4 transfection via cell counting kit-8. (c) The apoptosis of HUVECs after OE-ERBB4 transfection measured by flow cytometry. (d) The proliferation of HUVECs after OE-ERBB4 transfection examined by EdU assay. (e) The apoptosis of HUVECs after OE-ERBB4 transfection measured by TUNEL assay. CUR: Curdione. *P < 0.05 vs control; ^# P < 0.05 vs ox-LDL; ^$ P < 0.05 vs ox-LDL + OE-ERBB4.

Figure 4

Potential mechanisms of ERBB4 in curdione regulating ox-LDL-induced HUVECs. The concentrations of IL-6 (a), IL-1β (b), and IL-8 (c) in HUVECs after OE-ERBB4 transfection determined by ELISA. (d) The protein expression of ERBB4, apoptosis-related (Bax, Bcl-2, and caspase-3), and EndMT-related (E-cadherin, F-actin, and VEGF) in HUVECs after OE-ERBB4 transfection detected by Western blot. CUR: Curdione. *P < 0.05 vs control; ^# P < 0.05 vs ox-LDL; ^$ P < 0.05 vs ox-LDL + OE-ERBB4.

Figure 5

Interaction between ERBB4 and DNMT1. (a) ERBB4 had the CpG island based on the MethPrimer database. (b) ChIP-qPCR was employed to detect the binding of DNMT1 to the ERBB4 promoter in HUVECs. IgG was applied as an NC. *P < 0.05 vs input; ^# P < 0.05 vs anti-DNMT1. (c) The interaction between ERBB4 and DNMT1; as well as between ERBB4 and curdione determined using dual luciferase reporter gene assay. *P < 0.05 vs si-NC; ^# P < 0.05 vs PBS.