Protective Effect And Mechanism Of Rubber Seed Oil On Human Umbilical Vein Endothelial Cells Injury

Objective:To study the protective effect and mechanism of rubber seed oil(RSO)on human umbilical vein endothelial cells(HUVECs)injury induced by oxidized low-density lipoprotein(ox-LDL),and to explore anti-atherosclerosis mechanism of RSO.Methods:1.Dissolve RSO with 0.1% volume fraction of acetone as a solvent,HUVECs were treated with 0,25,50,75,100,125,and 150μg/ml RSO for 24 h,and the cell viability was measured by CCK-8 method.The RSO concentration required for subsequent experiments was selected based on the results.2.HUVECs were treated with 0,12.5,25,50,100,150,200,250,and 300μg/ml ox-LDL for 24 h,and cell viability was measured by CCK-8 method to determine the concentration of ox-LDL suitable for inducing HUVECs injury.3.HUVECs were treated with 250μg/ml ox-LDL and 0,50,100,150μg/ml RSO for 24 h,and cell viability was measured by CCK-8 method;the concentration of interleukin(IL)-1β,IL-6,IL-10 and tumor necrosis factor-α(TNF-α)in the supernatant of cells in each group was determined by Enzyme-linked immunosorbent assay(ELISA);the intracellular ROS level was measured by reactive oxygen species(ROS)test kit;the nitric oxide(NO)concentration in the supernatant was measured by Griess method;the mRNA expression levels of endothelial nitric oxide synthase(eNOS)and Toll-like receptor 4(TLR4)were determined by quantitative real-time polymerase chain reaction(RT-qPCR);the protein expression levels of monocyte chemotactic protein-1(MCP-1),vascular cell adhesion molecule-1(VCAM-1),eNOS,TLR4,nuclear factor-κB p65(NF-κB p65)and p-NF-κB p65 were determined by Western blot(WB).Results:1.Different concentrations of RSO have no significant impact on the cell viability of HUVECs.2.The activity of HUVECs gradually decreased with the increase of ox-LDL concentration;after treatment with 250μg/ml ox-LDL,the cell viability decreased significantly,and the cell survival rate was close to 50%.Therefore,ox-LDL concentration of 250μg/ml was selected to induce injury in subsequent experiments.3.Compared with the blank control group,the model group treated with250μg/ml ox-LDL significantly decreased cell viability,significantly increased the concentrations of pro-inflammatory factors IL-1β,IL-6 and TNF-α,significantly decreased the concentration of anti-inflammatory factor IL-10,significantly increased the level of ROS,significantly decreased the concentration of NO,significantly decreased the expression of eNOS mRNA,significantly increased the expression of TLR4 mRNA,significantly decreased the expression of eNOS,significantly increased the expression of MCP-1,VCAM-1 and TLR4,and significantly increased theNF-κB p-p65/p65 ratio.Compared with the model group,the experimental group treated with250μg/ml ox-LDL+50μg/ml RSO,250μg/ml ox-LDL+100μg/ml RSO,and 250μg/ml ox-LDL+150μg/ml RSO significantly increased cell viability,significantly decreased the concentrations of pro-inflammatory factors IL-1β,IL-6 and TNF-α,significantly increased the concentration of anti-inflammatory factor IL-10,significantly decreased the level of ROS,significantly increased the concentration of NO,increased the expression of eNOS mRNA,significantly decreased the expression of TLR4 mRNA,significantly decreased the expression of MCP-1,VCAM-1 and TLR4,significantly decreased theNF-κB p-p65/p65 ratio,and the effects of RSO on HUVECs after injury was concentration dependent.Conclusions:1.25-150μg/ml RSO has no cytotoxicity to HUVECs.2.RSO can reduce the expression of pro-inflammatory factors,oxidative factors,chemokine MCP-1,and adhesion molecule VCAM-1 in injured vascular endothelial cells,and increase the secretion of anti-inflammatory and antioxidant factors,showing good anti-inflammatory and antioxidant activities.3.RSO may reduce the damage of vascular endothelial cells by inhibiti ng TLR4/NF-κB signaling pathway,thereby exerting its anti-AS effect.