Study On The Anti-atherosclerosis Mechanism Of The Effective Fractions Of Angelica Sinensis Via TLR4/NF-κB Signaling Pathway

Objective:To investigate the role of effective fractions of Angelica sinensis（organic acids）on the expression of TLR4 and its signaling pathways NF-κB,LOX-1,VCAM-1,MCP-1 and IL-6 in human umbilical vein endothelial cells,and to explore the anti-Atherosclerosis mechanism of the effective fractions of Angelica sinensis（organic acids）.Methods:1.The isolation,culture of human umbilical vein endothelial cells（HUVECs）and establishment of endothelial injury model:HUVECs were isolated by type I pro-enzymatic digestion,and human VIII factor-associated antigen was used for cell identification.HUVECs were cultured with DMEM/F12 medium,at 37°C,5%CO₂ and passaged.HUVECs were injured by LPS（Lipopolysaccharide）at different concentrations,namely 0,0.1,0.5,1,5,and 10μg/mL,which caused damage to HUVECs.The effects of different concentrations of LPS on the morphology of endothelial cells were observed under an inverted microscope.The expression of LPS-specific ligand TLR4 was detected by immunofluorescence,and the appropriate concentration of LPS in the endothelial injury model was determined.2.Study on the effects of Angelica sinensis to damaged endothelium and its mechanism:HUVECs were randomly divided into five groups,including normal group,model group,high,medium,and low concentrations of effective fractions of Angelica sinensis（organic acids）,used（LPS 0.1μg/mL）induced HUVECs injury,activated TLR4 and downstream signaling pathways NF-κB,LOX-1,VCAM-1,MCP-1 and IL-6,and intervened with the effective fractions of Angelica sinensis at different concentrations for 24 hours,then the expression of TLR4 and NF-κB protein were detected by Immunofluorescence.The expressions of LOX-1,VCAM-1 and MCP-1 were tested respectively by Western blot.And the content of human IL-6 in cell supernatant were detected by ELISA.3.The effect of sodium ferulate on injured endothelium:The HUVECs were induced by LPS（0.1μg/mL）for activating TLR4 and its downstream signals NF-κB,LOX-1,VCAM-1,MCP-1,and IL-6.The injury HUVECs were interfered with different concentrations of sodium ferulate standard,and were used after 24 hours.Immunofluorescence was used to detect the expression of TLR4 and NF-κB protein.Western blot was used to detect the expression of LOX-1,VCAM-1 and MCP-1 protein respectively.The content of human IL-6 in the supernatant was detected by ELISA.Results:1.The HUVECs isolated by type I collagenase digestion.when they were grown to a confluent state showed a single-layered"paving stone"arrangement.The VIII factor-associated antigen was detected by immunofluorescence,demonstrating that the digestion cells was HUVECs.Different concentrations of LPS stimulated HUVECs for 12 hours（12h）and 24h,and the damage of HUVECs aggravated with the increase of LPS concentration.The expression of LPS specific receptor TLR4protein was significantly higher in 24h group than in 12h group.2.HUVECs were stimulated by LPS for 24 hours.Compared with the blank control group,the expressions of TLR4 and its downstream signaling molecules NF-Κb,LOX-1,VCAM-1,MCP-1,and the level of IL-6 were significantly increased in the model group.After intervention with effective fraction（organic acids）of Angelica sinensis,the expressions of TLR4,NF-κB,LOX-1,VCAM-1,MCP-1 and the level of IL-6 were significantly decreased in each treatment group.3.After treatment with sodium ferulate,the expressions of TLR4,NF-κB,LOX-1,VCAM-1,MCP-1 and the level of IL-6 were significantly decreased in each treatment group.Conclusion:The effective fraction（organic acids）of Angelica sinensis can down-regulate the expression of TLR4 protein induced by LPS,inhibit the activation of downstream signaling pathway NF-κB,and decrease the expression of LOX-1,VCAM-1,MCP-1 and IL-6.The roles of the effective fraction（organic acids）of Angelica sinensis would prevent inflammation and lipid-mediated pathological processes of atherosclerosis.Ferulic acid,the main component of organic acids of Angelica sinensis,plays a major role in this process.