The Effective Constituent Of Angelica Sinensis Inhibits Atherosclerosis By Regulating The Level Of Autophagy In HUVECs

Objective:To study the role and mechanism of the effective constituent of Angelica sinensis to regulate the autophagic flux level of human umbilical vein endothelial cells(HUVECs)for anti-atherosclerosis(AS).Methods:1.The content of Ferulic Acid(FA)different parts were compared: The method of High performance liquid chromatography(HPLC)was used to determine the content of FA in Angelica head,Angelica body and Angelica tail.The Column: Diamonsil C18(4.6 mm×200 mm,5 ?m),mobile phase: acetonitrile-0.1% phosphoric acid solution(17:83),the flow rate: 1.0 mL/min;sample size: 20 ?L;column temperature: 37?;the detection wavelength: 316 nm.2.Culture and identification of HUVECs: Collagenase digestion method was used to isolate HUVECs,light microscope and immunohistochemical method were used to identified the HUVECs.3.Effect of oxidized low density lipoprotein(ox-LDL)on autophagy and inflammation of HUVECs: Different concentrations of ox-LDL(0mg/L?20m g/L?40mg/L?60mg/L?80mg/L)were treated at different time(0h?12h?24h?48h)for HUVECs,Western Blot was used to detect the expression of autophagy marker proteins L C3 II,p62 and inflammatory related proteins MCP-1,VCAM-1 and LOX-1.The formation of autophagosome in HUVECs was observed by transmission electron microscope(TEM).4.The effective part of angelica sinensis(organic acid)and FA were used to regulate the autophagy and anti-inflammatory of HUVECs: After treatment of HUVECs with the effective part of angelica sinensis(organic acid),FA and ox-LDL,Western Blot was used to detect the expression of autophagy marker proteins LC3 II,p62 and inflammatory related proteins MCP-1,VCAM-1,LOX-1;TEM observed the formation of autophagy in HUVECs.Results: 1.The content of FA in Angelica tail is highest,followed by the Angelica head;angelica body has the lowest.2.Isolated HUVECs presented elliptic or long fusiforml and arranged like pavement stones under the light microscope;endothelial cell specific markers,Weibel-Palade were observed in the cytoplasm under TEM;correlation antigens ofhuman VIII factor were positive.3.The Western Blot results showed that ox-LDL increased the expression of MCP-1,VCAM-1,LOX-1,LC3 II,and p62 in a dose dependent manner(0mg/L,20mg/L,40mg/L,60mg/L and 80mg/L),and expressed obviously at 24h;the results of TEM showed that the number of autophagosome was the highest when ox-LDL concentration was 40 mg/L.4.The effective part of angelica sinensis(organic acid)and FA can increase the expression of LC3 II and reduce the expression of p62,MCP-1,VCAM-1,LOX-1;the results of TEM showed that the effective part of angelica sinensis(organic acid)promoted the degradation of organelles in autophagic lysosomes.Conclusion: The effective part of angelica sinensis(organic acid)may play a role in preventing AS by enhancing the autophagic flux and reducing the inflammatory to protect endothelial cells,and the main component of this function may be FA.