Rosuvastatin protects against oxidized low‑density lipoprotein‑induced endothelial cell injury of atherosclerosis in vitro

Abstract

Atherosclerosis‑induced cardiovascular diseases (CVDs) are accompanied by substantial morbidity and mortality. The loss and injury of endothelial cells is the primary cause of atherosclerosis. Rosuvastatin is an alternative agent used to reduce the risk of cardiovascular disease. Subsequently, the present study aimed to investigate the protective effects of rosuvastatin on oxidized‑low‑density lipoprotein (ox‑LDL)‑induced human umbilical vein endothelial cell (HUVEC) injury. The viability of ox‑LDL‑cultured HUVECs with or without rosuvastatin (0.01, 0.1 and 1 µmol/l) pretreatment, and pretreatment at different time points (3, 6, 12 and 24 h) was determined using an MTT assay. Morphological changes and the extent of apoptosis were detected; the anti‑oxidase activity, including superoxide dismutase (SOD) and catalase (CAT), was examined, and the contents of malondiahdehyde (MDA) and nitric oxide (NO) were measured. The phosphorylation levels of endothelial nitric oxide synthase (eNOS), protein kinase B (Akt) and phosphoinositide 3 kinase (PI3K) were detected using western blot analysis. The results demonstrated that pretreatment with 0.01‑1 µmol/l rosuvastatin decreased cell apoptosis caused by ox‑LDL. Notably, pretreatment with 1 µmol/l rosuvastatin for >12 h increased cell viability. Additionally, DAPI staining revealed that rosuvastatin inhibited HUVEC apoptosis. Rosuvastatin treatment also resulted in increased SOD and CAT activities and decreased MDA content in ox‑LDL‑stimulated HUVECs. Furthermore, pretreatment with 0.01‑1 µmol/l rosuvastatin significantly increased` the NO content compared with HUVECs treated with ox‑LDL alone. Western blot analyses demonstrated that rosuvastatin upregulated the phosphorylation of eNOS, Akt and PI3K. These findings indicated that rosuvastatin could protect HUVECs against ox‑LDL‑induced injury through its anti‑oxidant effect and its ability to upregulate the expression of vascular endotheliocyte‑protecting factors.

Keywords: rosuvastatin; atherosclerosis; endothelial dysfunction; nitric oxide synthase.

Figure 1.

Effects of rosuvastatin on cell viability. (A) HUVECs were treated with different concentrations of rosuvastatin (0.01, 0.1, 1 and 10 µmol/l) for 48 h. (B) HUVECs were pretreated with different concentrations of rosuvastatin for 24 h and treated with ox-LDL (200 µg/ml) for a further 24 h. (C) HUVECs were pretreated with 1 µmol/l rosuvastatin for various times (3, 6, 12 and 24 h) and then cultured with ox-LDL. Values are expressed as the mean ± standard deviation from three independent experiments. **P<0.01 vs. the control group; ^#P<0.05 and ^##P<0.01 vs. the ox-LDL group. HUVECs, human umbilical vein endothelial cells; ox-LDL, oxidized low-density lipoprotein.

Figure 2.

Effects of rosuvastatin on HUVEC morphology. HUVECs were pretreated with different concentrations of rosuvastatin for 24 h and then exposed to ox-LDL (200 µg/ml) for a further 24 h. The morphology of HUVECs was observed under a light microscope (magnification, ×100; scale bar=100 µm). (A) Control group; (B) ox-LDL-induced group; (C) 0.01 µmol/l rosuvastatin pretreated group; (D) 0.1 µmol/l rosuvastatin pretreated group; (E) 1 µmol/l rosuvastatin pretreated group. HUVECs, human umbilical vein endothelial cells; ox-LDL, oxidized low-density lipoprotein.

Figure 3.

Effect of rosuvastatin on apoptosis in HUVECs treated with ox-LDL. HUVECs were pretreated with different concentrations of rosuvastatin for 24 h and then exposed to ox-LDL (200 µg/ml) for a further 24 h. Cell injury was evaluated by DAPI staining (magnification, ×100; scale bar =100 µm). (A) Control group; (B) ox-LDL-induced group; (C) 0.01 µmol/l rosuvastatin pretreated group; (D) 0.1 µmol/l rosuvastatin pretreated group and (E) 1 µmol/l rosuvastatin pretreated group. HUVECs, human umbilical vein endothelial cells; ox-LDL, oxidized low-density lipoprotein.

Figure 4.

Effect of rosuvastatin on the levels of NO and oxidative stress in HUVECs treated with ox-LDL. (A) Culture supernatants of HUVECs were collected to detect the NO content using an NO assay kit (Microwell plate method) and the absorbance was measured at 570 nm. HUVECs were collected for ultrasonic cell disruption in an ice bath to detect the activity of (B) SOD using a SOD assay kit. (C) The activity of CAT was determined with a Catalase assay kit and (D) MDA content was assessed using an MDA assay kit. Values are expressed as the mean ± standard deviation from three independent experiments. *P<0.05 and **P<0.01 vs. the control group. ^#P<0.05 and ^##P<0.01 vs. the ox-LDL group. CAT, catalase; SOD, superoxide dismutase; MDA, malondialdehyde; NO, nitric oxide; HUVECs, human umbilical vein endothelial cells; ox-LDL, oxidized low-density lipoprotein.

Figure 5.

Effect of rosuvastatin on the phosphorylation of eNOS in HUVECs treated with ox-LDL. (A) The effect of 0.01–1 µmol/l rosuvastatin on the phosphorylation of eNOS. (B) The phosphorylation level of eNOS with 0.01–1 µmol/l rosuvastatin pretreatment was assessed using densitometric analysis. (C) The effect of 1 µmol/l rosuvastatin treatment at various time points on the phosphorylation of eNOS. (D) The phosphorylation level of eNOS with 1 µmol/l rosuvastatin pretreatment at various time points was conducted using densitometric analysis. The phosphorylation of eNOS was expressed as the ratio of p-eNOS to total eNOS, the subjacent band is target band of eNOS and were used to calculate the ratio of p-eNOS expression. GAPDH was presented as a loading control. The densitometric analysis values are expressed as the mean ± standard deviation from three independent experiments. **P<0.01 vs. the control group; ^##P<0.01 vs. the ox-LDL group. HUVECs, human umbilical vein endothelial cells; ox-LDL, oxidized low-density lipoprotein; p-eNOS, phosphorylated endothelial nitric oxide synthase.

Figure 6.

Effect of rosuvastatin on the phosphorylation of the PI3K/Akt signaling pathway mediators. (A) The effect of rosuvastatin on the phosphorylation of PI3K/Akt. The phosphorylation of (B) PI3K/(C) Akt was conducted by densitometric analysis, as well as expressed as the ratio of p-PI3K to total PI3K and p-Akt to total Akt. GAPDH was used as a loading control. The densitometric analysis values are expressed as the mean ± standard deviation from three independent experiments. **P<0.01 vs. the control group; ^##P<0.01 vs. the ox-LDL group. Akt, protein kinase B; PI3K, phosphoinositide 3 kinase; con, control; ox-LDL, oxidized-low-density lipoprotein; p-, phosphorylated.

Figure 7.

Effect of rosuvastatin on the expression of Bcl-2/Bax. (A) The effect of rosuvastatin on expression of Bcl-2/Bax. (B) The ratio of Bcl-2/Bax was assessed using densitometric analysis. GAPDH was presented as a loading control. Densitometric analysis values are expressed as the mean ± standard deviation from three independent experiments. **P<0.01 vs. the control group; ^##P<0.01 vs. the ox-LDL group. Bcl-2, B-cell lymphoma-2; con, control; ox-LDL, oxidized-low-density lipoprotein.