Protective Effect Of Alliin On Ox-LDL Injuriedmodel Of Human Umbilical Vein Endothelial Cells

Objective:To investigate the protective effect of alliin on oxidized low-density lipoprotein(ox-LDL)injuried model established in human umbilical vein endothelial cells(HUVEC),and the relevant targets and signaling pathways involved in the protective mechanism.Method:The effect of alliin at different concentrations on the proliferation viability of HUVEC was examined using the MTT kit.To make a HUVEC injuried model:after 80μg/mL ox-LDL induced HUVEC for 24h.The cell damage was observedthrough Oil Red O staining.The effect of different concentrations of alliin on the proliferation viability of HUVEC was detected by using MTT kit.LDH of cell supernatant was assayed by using lactate dehydrogenase(LDH)kit.The effect of alliin on caspase 3,BAX expression in HUVEC injuried model was detected by using Western blotting.Enzyme-linked immunosorbent assay(ELISA)was used to measure the inflammatory factors IL-6,IL-1β,TNF-α,MCP-1,VCAM-1,ICAM-1.The key compounds of alliin for the treatment of atherosclerosis along with their targets and pathways of action were analysed through network pharmacology.The contents of malondialdehyde(MDA),superoxide dismutase(SOD)and reactive oxygen species(ROS)were measured in each group.The key target eNOS protein levels were detected by Western blotting.Results:After 24h and 48h treatment,different concentrations of alliin did not affect the proliferation activity of normal HUVEC cells.80μg/mL ox-LDL induced a large number of red-stained particles in HUVEC cells for 24 hours,indicating that the HUVEC injury model was successfully established;After 24h of 50～100μg/mL alliin treatment,it significantly promoted the proliferation of injured HUVECcells(P<0.05);Amino acid can significantly reduce the expression of LDH in the HUVEC injury model(P<0.01);Alliin significantly down-regulates the expression of caspase 3 and BAX protein in the HUVEC injury model(P<0.05),and can effectively reduce IL-6,IL-6,and BAX in the HUVEC injury model.The levels of IL-1β,TNF-α,MCP-1,VCAM-1,ICAM-1 inflammatory factors(P<0.01),suggesting that alliin has an anti-inflammatory effect.With the help of network pharmacology analysis method,the neighbor targets affected by the potential targets of alliinine anti-AS are also included in the network analysis.The results showed that 13 network hub nodes proteins such as eNOS,SIRT1,IL-6,etc.,suggesting that these nodes may be garlic A key target for the treatment of AS.The results showed that alliin treatment could increase the level of SOD in injured cells,reduce the content of MDA in cell culture medium,and inhibit lipid peroxidation and oxidative stress injury of endothelial cells.The difference is statistically significant(P<0.01).Finally,it was detected by Western blotting that alliin pretreated HUVEC can up-regulate the expression of eNOS protein(P<0.05),verifying the results of network pharmacology.Conclusion:Alliin exerts anti-inflammatory and antioxidant activity and these effects might be related to the eNOS protein expression upregulation as well as protecting the ox-LDL-induced HUVEC injuring.