Trimetazidine Protects Against Atherosclerosis by Changing Energy Charge and Oxidative Stress

Abstract

BACKGROUND This study investigated the effect and the possible mechanism of trimetazidine in atherosclerosis. MATERIAL AND METHODS We established an atherosclerotic rat model by high-fat diet and vitamin D injection. Rats were separated into 3 different groups: control, atherosclerosis, and trimetazidine (n=10). The aortic artery was isolated and its morphological features were examined by hematoxylin and eosin (HE) staining. Serum low-density lipoprotein cholesterol (LDL-c), total cholesterol (TC), and triglycerides (TG) were analyzed using an automatic biochemical analyzer. Human aortic smooth muscle cells (HASMCs) were cultured and divided into 5 groups: no treatment, H₂O₂ treatment only, trimetazidine preincubation before H₂O₂ treatment, oxidized low-density lipoprotein (oxLDL) treatment only, and trimetazidine preincubation before oxLDL treatment. HASMCs proliferation was tested using the Cell Counting Kit-8. Reactive oxygen species (ROS) and malondialdehyde (MDA) levels, superoxide dismutase (SOD) activity of the aortic artery, and HASMCs were measured using commercially available kits. RESULTS HE staining assay showed that trimetazidine suppressed the progression of atherosclerosis and reduced foam cell formation in the aortic artery without affecting serum lipid levels. HASMCs proliferation assay revealed that trimetazidine alleviated the inhibitory effect of H₂O₂ on HASMCs proliferation and inhibited oxLDL-induced proliferation of HASMCs. Moreover, trimetazidine ameliorated ROS up-regulation elicited by H₂O₂ or oxLDL in HASMCs. Additionally, trimetazidine restored SOD activity and reduced MDA content of HASMCs. CONCLUSIONS Trimetazidine suppressed the progression of atherosclerosis by enhancing energy value, decreasing ROS level of aortic artery, modulating HASMCs proliferation, and reducing oxidative stress in HASMCs.

Figure 1

Effect of trimetazidine on the development of atherosclerosis in rats fed HFD. HE staining of cross-sections of rat aortic roots in each group (A: Control. B: Atherosclerosis. C: Trimetazidine. Scale bar: 200 μm). Trimetazidine markedly inhibited the development of atherosclerosis in aortic arteries (n=10 per group). (D) Error bars show serum lipid level in each group. Trimetazidine had no significant effect on serum lipid profile. (E) Rat body weights in 3 groups were all increased at the end of the experiment. Body weights in the atherosclerosis group were significantly increased compared to the control group at each time point, while trimetazidine had no effect on the body weight changes compared with the atherosclerosis group. Data are mean ± standard deviation. n=10, * P<0.05. NS indicates no significance vs. atheroscerosis group.

Figure 2

Changes in energy charge in atherosclerostic aortas after trimetazidine treatment. (A) The chromatograms of ATP, ADP, and AMP. The peak times of ATP, ADP, and AMP were 4.25, 4.88, and 7.09 min, respectively, in the chromatogram of a standard sample. (B) Energy charge value of rat aortic artery measured by high-performance liquid chromatography. The fomula [EC=ATP+0.5ADP/(ATP+ADP+AMP)] was used to measure the energy charge level. Energy charge values in the atherosclerotic group decreased remarkably compared to the control group, * P<0.05. Pretreatment with trimetazidine produced much more energy charge value than in the atherosclerotic group, ** P<0.05. n=10, error bars indicate that data are expressed as the mean ± standard deviation.

Figure 3

Effects of trimetazidine on HASMCs proliferation treated by oxLDL or H₂O₂. (A) Cell viability detected by CCK-8 assay. HASMCs were treated by different concentrations of trimetazidine (0, 0.1, 1, 10, 50, and 100 μM). Trimetazidine at 50 μM significantly affected cell survival. (B) HASMCs were treated with different concentrations of trimetazidine (0.1, 1, and 10μM) and 80 μg/ml oxLDL. (C) Cells were pretreated for 12 h with or without trimetazidine, then treated with H₂O₂ (100 μM) for 2 h or oxLDL (80 μg/ml) for 24 h. H₂O₂ (100 μM) inhibited HASMCs proliferation. Trimetazidine pretreatment alleviated the inhibitory effect of H₂O₂ on HASMCs proliferation. Trimetazidine had an anti-proliferation effect on HASMCs against oxLDL. n=3. * P<0.05 vs. control. ^# P<0.05 vs. oxLDL. ** P<0.05 vs. H₂O₂.

Figure 4

Effect of trimetazidine on ROS level in aorta of rats and HASMCs. (A) ROS level of rat aortic artery in atherosclerosis group increased remarkably compared to the control group, * P<0.05. Trimetazidine treatment reduced ROS production compared to the atherosclerosis group, n= 10 per group, ** P<0.05. (B) Ox-LDL and H₂O₂ induced ROS generation in HASMCs and the effect of trimetazidine. Cells were pretreated for 12 h with or without trimetazidine (10 μM), then treated with H₂O₂ (100 μM) for 2 h or ox-LDL (80 μg/ml) for 24 h. Both H₂O₂ and oxLDL induced ROS production. Cells pretreated with trimetazidine before H₂O₂ or oxLDL stimulation produced much lower ROS levels than cells incubated with only H₂O₂ or oxLDL, respectively. Treatment with trimetazidine only had no effect on the ROS level. DCHF-DA is a dye that can be oxidized by ROS to the fluorescent compound DCF, as monitored by fluorescence microplate spectrophotometry. ROS level is quantified by the fluorescence intensity of DCF. Data are shown as mean ±SD and are expressed as DCF fluorescence intensities compared with unstimulated cells. n=3. * P<0.05 vs. control. ** P<0.05 vs. H₂O₂. ^# P<0.05 vs. ox-LDL. NS indicates no significance vs. control.

Figure 5

Effect of trimetazidine on superoxide dismutase (SOD) activity and malondialdehyde (MDA) level. (A) Trimetazidine restored SOD activity of HASMCs, which inhibited by H₂O₂ and oxLDL. Both H₂O₂ and oxLDL decreased SOD activity. Cells pretreated with trimetazidine before H₂O₂ or oxLDL stimulation had increased SOD activity than cells incubated with only H₂O₂ or oxLDL, respectively. Trimetazidine alone had no effect on SOD activity. n=3. * P<0.05 vs. control. ** P<0.05 vs. H₂O₂. ^# P<0.05 vs. ox-LDL. NS indicates no significance vs. control. (B) Trimetazidine reduced MDA content of HASMCs stimulated by H₂O₂ and oxLDL. Both H₂O₂ and oxLDL increased MDA content. Trimetazidine pretreatment reduced MDA level stimulated by H₂O₂ or oxLDL. Trimetazidine alone had no effect on the MDA level. n=3. * P<0.05 vs. control. ** P<0.05 vs. H₂O₂. ^# P<0.05 vs. ox-LDL. NS indicates no significant difference vs. control.