Baicalin Protects against Thrombin-Induced Cell Injury in Human Umbilical Vein Endothelial Cells

Abstract

Thrombin plays a pivotal role in the pathogenesis of atherosclerosis. Baicalin, an active flavonoid compound, was shown to attenuate the development of atherosclerosis, but the mechanism remains elusive. In the present study, the role and mechanism of baicalin in thrombin-induced cell injury was investigated in human umbilical vein endothelial cells (HUVECs). Our results showed that baicalin significantly reduced thrombin-induced apoptosis of HUVECs. Additional experiments showed that baicalin inhibited thrombin-induced NF-κB activation and PAR-1 expression. In addition, baicalin decreased thrombin-induced PAR-1 expression by inhibiting ERK pathway. These results indicated that baicalin has protective effects on thrombin-induced cell injury in HUVECs possibly through inhibition of PAR-1 expression and its downstream NF-κB activation, which was mediated by ERK1/2 activation.

Figure 1

Effects of baicalin on cell survival and apoptosis after thrombin treatment. HUVECs were pretreated with baicalin (0, 50, 100, and 150μM) for 3 hours before exposure to thrombin (5 U/ml) for 6 hours. (a) Cell viability was determined by MTT assay. (b) Apoptosis was determined by flow cytometry. (c) Rate of apoptotic cells quantified of three independent experiments by flow cytometry was calculated. (d) Caspase-3 activity was determined by a caspase-3 assay kit. Caspase-3 activity relative to control that was set as 1. (e) Representative experiments of western blot for the cleaved caspase-3 protein expression. (f) Quantitative analysis of the ratio of cleaved caspase-3 to caspase-3. Values are means ± SD (n=3). ## P<0.01 vs. control group; ∗P<0.05; ∗∗P<0.01; ∗∗∗P<0.001 vs. thrombin group.

Figure 2

Baicalin reduced thrombin-induced NF-κB activation. Cells were treated as indicated and then were lysed for western blotting analysis. (a) Representative results of western blotting experiments to detect p-P65, P65 was used as a loading control. (b) Quantitative analysis of the ratio of p-P65 to P65. Values are means ± SD (n=3). ## P<0.01, vs. control group; ∗∗ P<0.01; ∗∗∗ P<0.001 vs. thrombin group.

Figure 3

Baicalin suppressed thrombin-induced the PAR-1 expression. Cells were treated as indicated, then total RNA was extracted for real-time RT-PCR analysis, and cells were lysed for western blotting analysis. (a) The quantitation represents the average relative ratio of PAR-1 mRNA to β-actin. (b) Representative experiments of western blot for the PAR-1 protein expression. (c) The quantitation represents the average relative ratio of PAR-1 protein to GAPDH. Values are means ± SD (n=3). ## P<0.01 vs. control group; ∗P<0.05, ∗∗P<0.01, and ∗∗∗P<0.001 vs. thrombin group.

Figure 4

Baicalin decreased thrombin-induced PAR-1 expression through inhibition of ERK1/2 phosphorylation. (a) HUVECs were pretreated with U0126 (10μM) for 3 hours before exposure to thrombin (5 U/ml). (b) HUVECs were pretreated with baicalin (50μM) for 3 hours before exposure to thrombin (5 U/ml). Representative experiments of western blot for the p-ERK1/2, ERK1/2, and PAR-1 protein expression. (A) Quantitative analysis of the ratio of p-ERK1/2 to ERK1/2, ERK1/2 was used as a loading control. (B) Quantitative analysis of the ratio of PAR-1 to GAPDH. Values are means ± SD (n=4). #P<0.05 vs. control group; ∗P<0.05; ∗∗P<0.01 vs. thrombin group.