Abstract
To explore the main active components and effects of cell cycle regulation mechanism of Astragali radix (AR) and Angelicae sinensis radix (ASR) on oxidative damage in vascular endothelial cells, a model of oxidative damage in human umbilical vein endothelial cells (HUVECs) induced by oxidized low-density lipoprotein (ox-LDL) treatment was developed. Based on the “knock-out/knock-in” model of the target component, cell viability, intracellular reactive oxygen species (ROS), and lactate dehydrogenase (LDH) leakage were assessed by Cell Counting Kit-8 assay, fluorescent probe 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA), and colorimetric assay, respectively, to evaluate the protective effect of the active components of AR and ASR (astragaloside IV (AS IV), astragaloside I (AS I), formononetin (FRM), calycosin (CAL), calycosin-7-O-β-D glucoside (CLG), and ferulic acid (FRA)) against oxidative damage. The cell cycle and expression of genes encoding cyclins and cyclin-dependent kinases (CDKs) were observed using flow cytometry and quantitative real-time polymerase chain reaction. The results showed that the combination of active components (ACC) significantly inhibited the decrease in cell viability as well as the increase in ROS and LDH release in HUVECs induced by ox-LDL treatment. AS IV and FRM promoted the proliferation of HUVECs but the proliferation index was decreased in the AS I and FRA groups; this inhibitory effect was counteracted by the ACC. The ACC reduced and increased the proportion of positive cells in G1 and S phases, respectively, followed by the upregulation of cyclin A (CCNA), cyclin E (CCNE), and CDK2 mRNA expression and downregulation of cyclin B (CCNB), cyclin D1 (CCND1), CDK1, CDK4, and CDK6 mRNA expression, which significantly mitigated inhibition of HUVECs proliferation induced by ox-LDL treatment. Taken together, AS IV, AS I, FRM, CAL, CLG, and FRA were the primary pharmacodynamic substances of AR and ASR that alleviated oxidative injury in HUVECs. ACC mitigated the upregulation of intracellular ROS levels and LDH release induced by ox-LDL treatment, which promoted the cell cycle procession of HUVECs by regulating the expression of genes encoding cyclins and CDKs and thus preventing oxidative damage in HUVECs.
Highlights
Atherosclerosis (AS) is a chronic immune inflammatory disease characterized by abnormal lipid deposition on the arterial walls that causes various cardiovascular and cerebrovascular disorders, and AS is a common cause of human death and disability [1]
Compared with the model group, astragaloside IV (AS IV), FRM, CAL, and calycosin-7O-β-D glucoside (CLG) significantly increased the viability of human umbilical vein endothelial cells (HUVECs)
All the active component knock-out groups effectively inhibited the decrease in HUVECs viability induced by oxidized low-density lipoprotein (ox-LDL) treatment. e viability of HUVECs in the knock-in group (ACC) increased by 83.15% compared with the model group
Summary
Atherosclerosis (AS) is a chronic immune inflammatory disease characterized by abnormal lipid deposition on the arterial walls that causes various cardiovascular and cerebrovascular disorders, and AS is a common cause of human death and disability [1]. Cardiovascular diseases, such as AS, hypertension, and heart failure, contribute to an increase in the morbidity and mortality rates worldwide [2]. Vascular endothelial cells (VECs) act as a protective barrier between the blood and vascular wall but are the primary targets of various environmental factors, such as hemodynamics, inflammatory cytokines, and oxidative stress. Accumulation of oxidized low-density lipoprotein (ox-LDL) induces oxidative stress and vascular endothelial dysfunction, promoting lipid accumulation in the subendothelial layer, atherosclerosis, and vascular stenosis [4]
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