Paeoniflorin suppresses the apoptosis and inflammation of human coronary artery endothelial cells induced by oxidized low-density lipoprotein by regulating the Wnt/β-catenin pathway

Abstract

Context: Paeoniflorin (PF) contributes to improving coronary artery disease (CAD).

Objective: This study clarified the efficiency of PF in CAD and the molecular mechanism.

Materials and methods: Human coronary artery endothelial cells (HCAECs) were treated with oxidized low-density lipoprotein (ox-LDL; 20, 40, 80 and 160 μg/mL) and PF (0.05, 0.1 0.2 and 0.4 mM). To study cell phenotypes, HCAECs were treated with 80 μg/mL ox-LDL with or without 0.1 mM PF for 24 h, and cell viability and apoptosis were evaluated using the methyl thiazolyl tetrazolium (MTT) assay and flow cytometry, respectively. In addition, inflammatory cytokines levels were measured by enzyme-linked immunosorbent assay (ELISA). Western blot evaluated the Wnt/β-catenin pathway-related factors.

Results: ox-LDL and PF (0.2 and 0.4 mM) suppressed cell viability in a dose-dependent manner. The IC₅₀ value of PF was 722.9 nM. PF facilitated cell viability (115.76%), inhibited apoptosis (46.28%), reduced IL-6 (63.43%) and IL-8 (66.70%) levels and increased IL-10 levels (181.15%) of ox-LDL-treated HCAECs. Additionally, PF inactivated the Wnt/β-catenin pathway, and XAV939 treatment further promoted cell viability (120.54%), suppressed apoptosis (56.92%), reduced the levels of IL-6 (76.16%) and IL-8 (86.82%) and increased the IL-10 levels (120.22%) of ox-LDL-induced HCAECs after PF treatment. Moreover, PF alleviated plaque lesions of the aorta and aorta root and serum lipid of ApoE^-/- mice with a high-fat diet.

Discussion and conclusions: This study first revealed that PF inhibited ox-LDL-induced HCAECs apoptosis and inflammation via the Wnt/β-catenin pathway and alleviated CAD, suggesting the potential of PF as a drug for CAD treatment.

Keywords: Coronary artery disease; blood lipid; interleukin; plaque lesion; proliferation.

Figure 1.

Effects of ox-LDL and PF on cell viability. (A) Chemical structure of PF; (B) the HCAECs were treated with 0, 20, 40, 80 and 160 μg/mL ox-LDL, and cell viability was examined using the MTT assay; (C) the HCAECs were treated with 0, 0.05, 0.1, 0.2 and 0.4 mM PF, and cell viability was assessed by the MTT assay; (D) the IC₅₀ value of PF and (E) cell viability was evaluated by the MTT assay after treatment with 80 μg/mL ox-LDL and 0.1 mM PF. *p < 0.05, **p < 0.01 and ***p < 0.001.

Figure 2.

PF promotes cell viability and inhibits apoptosis and inflammation induced by ox-LDL. (A) Cell viability was analysed using the MTT assay; (B) cell apoptosis was analysed using flow cytometry; (C) the Bax and Bcl2 levels were examined using western blot and (D) the levels of IL-6, IL-8 and IL-10 were measured by ELISA. *p < 0.05 and **p < 0.01.

Figure 3.

PF suppresses the activation of the Wnt/β-catenin pathway. The expression of Wnt/β-catenin pathway-related factors β-catenin, c-myc, cyclin D1 and E-cadherin was examined using western blot. GAPDH was the internal control. **p < 0.01.

Figure 4.

PF suppresses apoptosis and inflammation via regulating the Wnt/β-catenin pathway. (A) The protein levels of β-catenin, c-myc, cyclin D1 and E-cadherin were assessed using western blots; (B) MTT assay was conducted to assess cell viability; (C) flow cytometry was performed to analyse apoptosis; (D) the Bax and Bcl2 levels were examined using western blots, and (E) the levels of IL-6, IL-8 and IL-10 were determined by ELISA. *p < 0.05 and **p < 0.01.

Figure 5.

Effects of PF on the formation of atherosclerosis in ApoE^−/− mice. (A) The whole aorta was stained using the Oil red O, and the plaque areas (%) were quantified; (B) the lesion areas (%) of the aortic roots from mice of each group were examined using the Oil red O staining assay and quantified; and (C) the levels of cholesterol, triglycerides, LDL-cholesterol and HDL-cholesterol were detected using the specific kits. (D) The macrophage marker F4/80 was detected in aortic roots of mice using IF staining assay. (E) The body weight of mice was evaluated every two weeks. **p < 0.01 and ***p < 0.001.