Quantitative proteomic analysis of the protective effect exerted by alliin on ox-LDL-injured HUVECs

Natural organic sulfides are predominantly found in cruciferous and liliaceous plants. Among these compounds, alliin-an organic sulfide derived from garlic-has garnered significant attention from researchers due to its potential anti-atherosclerotic properties. However, studies specifically investigating the anti-atherosclerotic effects of alliin remain limited. This study aims to elucidate the protective effects of alliin on ox-LDL-injured human umbilical vein endothelial cells (HUVECs) and their underlying mechanisms. Initially, HUVECs were exposed to 80 mg/L oxidized low-density lipoprotein (ox-LDL) for 24 h to establish an ox-LDL injury model. Subsequently, the Cell Counting Kit-8 (CCK8) assay was utilized to assess the effect of alliin on the proliferation of ox-LDL-injured cells at 12, 24 and 48 h, and the levels of total cholesterol (TC) and free cholesterol (Fch) in the HUVECs were measured according to the instructions of TC and Fch kits. Next, quantitative proteomics was then adopted to analyze the differential protein expression in cell samples from the control group (Con), the ox-LDL injury model group (Mod), and the alliin treatment group (Alliin). Among the quantified proteins, a statistical t-test with P < 0.05 was used as a threshold for significance regarding a 1.5-fold change in differential expression. Finally, functional enrichment analysis of the differential proteins in the Alliin/Mod group was performed using Gene Ontology (GO) enrichment and KEGG pathway analysis. Additionally, Western blotting was used to validate the findings. The proteomic analysis identified 6173 identified proteins, of which 5162 were quantifiable. Differential protein analysis revealed that in the Alliin/Con comparison, there were a total of 108 up-regulated proteins and 116 down-regulated proteins; in the Alliin/Mod comparison there were a total of 33 up-regulated proteins and 17 down-regulated proteins; while in the Mod/Con comparison, there were a total of 106 up-regulated proteins and 147 down-regulated proteins. GO enrichment, KEGG pathway analyses and Western blotting verification demonstrated that alliin up-regulates the expression of Low-density lipoprotein receptor (LDLR) (P < 0.05) and apolipoprotein C (ApoC) (P < 0.05) while down-regulating the expression of apolipoprotein B (ApoB) (P < 0.05) to regulate the cholesterol metabolism pathway of the ox-LDL-injured HUVECs. Our findings highlight the importance of cholesterol metabolism in alliin treatment for atherosclerosis. Alliin exerts a protective effect in the ox-LDL-induced HUVEC injury model by modulating the expression of LDLR, ApoC, and ApoB within the cholesterol metabolism pathway. These findings indicate that alliin could potentially serve as a therapeutic agent for the prevention and treatment of atherosclerosis.