The Protection Effects Of Pollen Typhae Extract On The Vascular Endothelial Cell Injury Induced By Oxidized Low Density Lipoprotein

Background:As a common pathological foundation for most cardiovascular diseases,atherosclerosis is serious threaten for people's health and life.It has been indicated that injury of VEC is the precondition and the predominant mechanism in the initial stage of atherosclerosis.The oxidized low-density lipoprotein(ox-LDL),which is an independent risk factor in AS,can damage the vessel intima,upregulate the expression of endothelial adhesive molecules,induce the circulating monocytes migrating and adhering to intima,promote the macrophage to phagocytize lipid to form foam cells.The pathological processs described as above play an important role in the development of AS.Therefore,it is an key choice to prevent and treat the AS by protecting VEC.Pollen Typhae is a common used Chinese herb with long history in traditional Chinese medicine,which is the pollen of Typha angustifolia L.,T.orientalis Presl and the other plants of the genus Typha. Firstly it was included in Shen Nong's Herbal of Materia Medica. It has the effects of activating blood circulation to dissipate blood stasis and diuretic, Modern pharmacology verified that Pollen Typhae could adjust the serum contents of lipids,suppress the platelet aggregation,prevent coagulation and prevent the progression of atherosclerosis by different ways. Based on the efficacy of Pollen Typhae on AS prevention and cure,making Pollen Typhae extract act on the model of vascular endothelial cell injury induced by ox-LDL,detecting the variation of indexes such as LDH,SOD,MDA, ICAM-1,MCP-1 in the culture medium and cell,to investigate its protective effects and mechanisms.Methods:1. The human umbilical vein endothelail cells(HUVEC) were isolated from umbilical vein by perfusion and enzyme digestion method and identified by morphological observation andâ…§factor Ag immunoreactivity.2. Selected the cells in the exponential phase of growth and groups are divided on experiment as follow:different concentration of Pollen Typhae extract groups(100mg/L,75mg/L,50mg/L,25mg/L,10mg/L) and normal control group,the cell viability were determined with MTT assay after 24h,48h and 72h cultured in vitro.3. Planted the cells into 24-well culture plate randomly,divided them into 5 groups as follow:(1)normal control;(2)ox-LDL damage group:ox-LDL(eventually concentration 100mg/L);(3)low dosage Pollen Typhae extract group: Pollen Typhae extract (10mg/L)+ ox-LDL;(4)middling dosage Pollen Typhae extract group: Pollen Typhae extract (50mg/L)+ ox-LDL;(5)high dosage Pollen Typhae extract group: Pollen Typhae extract (100mg/L)+ ox-LDL.After cultivated for 24 hours,measure the content of LDH,SOD,MDA in supernatants and the expression of MCP-1 and ICAM-1 mRNA by the method of reverse transcription polymerase chain reaction (RT-PCR) in cells.Results:1. Cell culture and identify(1)morphological observation:the most cultured cells are fused after inoculation for 5 to 7 days,appearing typical'slabstone'.(2)â…§factor Ag immunoreactivity:under confocal microscopy,around 95% cellular cytoplasm appeared flavovirens fluorescence.The cells cultured in this method are proved to be HUVEC and fit for our study.2. Effect of Pollen Typhae extract on activity of HUVECsCompared with different concentration of pollen typhae extract groups(100mg/L,75mg/L,50mg/L,25mg/L,10mg/L),the OD value of the normal control group is the lowest after 24h,48h and 72h cultured in vitro(P<0.05,P<0.01,P<0.01).And all concentration groups have no significant difference compared with each other.Our data suggest that Pollen Typhae extract has no adverse reaction and has an facilitative effection on the HUVECs.3. The expression of LDH,SOD,MDA in supernatantOx-LDL induce the increase on the activity of LDH and MDA, and the decrease of SOD.The difference is significant as compared with the control group(P<0.05);Pollen Typhae extract (10,50,100mg/L) decrease the activity of LDH and MDA, and increased the activity of SOD.The difference was significant(P<0.01).4. The expression of MCP-1 and ICAM-1 mRNA in HUVECsThe expression levels of ICAM-1, MCP-1 were increased significantly in Ox-LDL stress group,the difference is significant as compared with the control group(P<0.01);while they were decreased significantly by Pollen Typhae extract groups(10,50,100mg/L) (P<0.01).Conclusion:1. Pollen Typhae extract has no adverse reaction on the HUVEC and has an facilitative effection.2. Pollen Typhae extract protects endothelial cells against lipid peroxidation insult by increasing the activity of SOD and decreasing the the activity of MDA and LDH.3. Pollen Typhae extract can inhibites the over-expression of ICAM-1 and MCP-1 mRNA and protects endothelial cells against the monocytes migrating and adhering to vessel intima.