Protective Effects of Scutellarin on Human Cardiac Microvascular Endothelial Cells against Hypoxia-Reoxygenation Injury and Its Possible Target-Related Proteins

Abstract

Scutellarin (SCU) is one of the main components of traditional Chinese medicine plant Erigeron breviscapus (Vant.) Hand.-Mazz. In this paper, we studied the protective effects of SCU on human cardiac microvascular endothelial cells (HCMECs) against hypoxia-reoxygenation (HR) injury and its possible target-related proteins. Results of MTT assay showed that pretreatment of SCU at doses of 1, 5, and 10 μM for 2 h could significantly inhibit the decrease in cell viability of HCMECs induced by HR injury. Subcellular fractions of cells treated with vehicle control, 1 μM SCU, HR injury, or 1 μM SCU + HR injury were separated by ultracentrifugation. The protein expression profiles of cytoplasm and membrane/nuclei fractions were checked using protein two-dimensional electrophoresis (2-DE). Proteins differentially expressed between control and SCU-treated group, control and HR group, or HR and SCU + HR group were identified using mass spectrometry (MS/MS). Possible interaction network of these target-related proteins was predicted using bioinformatic analysis. The influence of SCU on the expression levels of these proteins was confirmed using Western blotting assay. The results indicated that proteins such as p27BBP protein (EIF6), heat shock 60 kDa protein 1 (HSPD1), and chaperonin containing TCP1 subunit 6A isoform (CCT6A) might play important roles in the effects of SCU.

Figure 1

Effects of SCU on cell viability of HCMECs under normal condition or HR treatment. (a) Chemical structure of SCU. (b) Results of MTT assay of cell viability of cells treated with SCU at different concentrations or vehicle control for 24 h. ^∗ p < 0.05, compared with control. (c) Results of MTT assay of cell viability of cells treated with SCU at different concentrations or vehicle control for 48 h. ^∗ p < 0.05, compared with control. (d) Results of MTT assay of cell viability of cells treated with vehicle control, HR treatment, and HR treatment with 2 h pretreatment of SCU at different concentrations. ^# p < 0.05, compared to HR group. Data are presented as mean ± SD, n = 3 independent experiments.

Figure 2

The proteome maps (2-DE images) of cytoplasm fraction of cells of control group, SCU group, HR group, and SCU + HR group. (a) Representative group of gels of nine replicate groups of gels, which were gotten by triplicate electrophoresis of samples from three independent experiments. Differentially expressed spots were shown by the arrows. (b) The expanded region of differentially expressed protein spots. The proteins within the circles were the differentially expressed proteins. Comparison was made between control group and HR group, control group and SCU group, and HR group and SCU + HR group.

Figure 3

The proteome maps (2-DE images) of nuclei/membrane fraction of cells of control group, SCU group, HR group, and SCU + HR group. (a) Representative group of gels of nine replicate groups of gels, which were gotten by triplicate electrophoresis of samples from three independent experiments. Differentially expressed spots were shown by the arrows. (b) The expanded region of differentially expressed protein spots. The proteins within the circles were the differentially expressed proteins. Comparison was made between control group and HR group, control group and SCU group, and HR group and SCU + HR group.

Figure 4

The result of the MALDI-TOF MS/MS analysis of spot7 cut from the 2-DE gels of nuclei/membrane fraction. It was identified to be human eukaryotic translation elongation factor 1 gamma by protein database search. (a) Peptide mass fingerprint of tryptic digest of the spot. ∗ unique peptides further identified by MS/MS. (b) MS/MS profile of the peptide with a mass of 11241.68 Da. (c) MS/MS profile of the peptide with a mass of 1347.77 Da.

Figure 5

Protein-protein interaction network obtained from bioinformatic analysis based on STRING database. Possible target-related proteins of SCU were shown as round balls in red while possible proteins involved in HR injury were shown as round balls in green. Possible interactions between proteins were shown in line with different colors, as indicated in the figure.

Figure 6

Results of Western blotting assay of protein levels of EIF6, HSPD1, and CCT6A in cells of control group, SCU group, HR group, and SCU + HR group shown were representative results of three independent experiments.