The Effects Of Different Leech Extracts On Endothelial Cells Coagulation Factors And Endothelial Dysfuction Molecules

Cardiovascular disease,currently the leading cause of death and illness in developed countries,will soon become the preeminent health problem worldwide.Atherosclerosis,a progressive disease characterized by the accumulation of lipids and fibrous elements in the large arteries,constitutes the single most important contributor to this growing burden of cardiovascular disease.Leech is a traditional Chinese medicine and exhibits effects in promoting blood circulation and remove blood stasis.Its decoctum,oral liquids and other formulations are widely applied in the prevention and treatment of cardiovascular diseases.Bleeding adverse reactions,which was often,seriously threaten patients' health.In this process,we speculate that some of the components of the leech may exert an effect on the coagulation mechanism of the body,so that the coagulation activity changes,resulting in bleeding adverse reactions.Endothelial cells play a pivotal role in blood clotting.Therefore,in this paper,leeches as raw materials,were hydrolyzed by enzymes in order to obtain a preparation method which not change the mRNA expression of coagulation-related factors in endothelial cells but can reduce bleeding adverse reactions during inhibiting atherosclerosis.We hope to find a new oral preparation which is safe and clinically available.The thesis is mainly about the followings:1 Preparation for leech extract under different conditionsIn this study,leeches are the material,were hydrolyzed with different enzymes.Then the pepsin hydrolysate of leech extract and double enzymes hydrolysate of leech extract were transformated by Lactobacillus.Finally,8 different leech extracts were obtained.2 The effect of different leech extracts on endothelial cells coagulation related factorsRT-PCR were applied to study the coagulation-related factor mRNA expression in EA.hy926 cells after different leeches extracts treated.The results showed that LP could significantly promote the expression of coagulation-related factor mRNA in EA.hy926 endothelial cells in the concentration range of 0-50 ?g/ml.PHL and MRS-1 had no effect on the expression of coagulation-related factor mRNA in EA.hy926 cells.PTHL can significantly decrease the expression of coagulation-related factor mRNA in EA.hy926 cells.CEHL can promote the factor PAI and PS mRNA expression,but it can inhibit the expression of vWF,tPA,TFPI and TM.MRS can promote the expression of PAI,vWF,tPA and TM in EA.hy926 cells,but not on PS and TFPI expression.MRS-3 can promote the expression of PAI,vWF,tPA,PS and TM in EA.hy926 cells,but no significant effect on TFPI mRNA expression.In the above results,only PHL and MRS-1 had no significant effect on the expression of coagulation-associated factor in EA.hy926 cells,and MRS-1 was the control group,so the effect of PHL on endothelial dysfunction was explored.3 Protective effect of PHL on endothelial cell dysfunctionFirstly,we use the concentration of 10 ng/mL of TNF-a,to induce vascular endothelial cell 12h to establish the endothelial dysfunction model.We adopt the method of RT-PCR to detect the mRNA expression of ET-1,MCP-1,IL-6and iNOS on endothelial cells.Results indicate that,compared with the control group,modelgroup has significantly higher levels IL-6 and MCP-1(#p<0.05).Endothelial dysfunction model is established successfully.Results indicate that:50?g/mL PHL can decrease the expression of MCP-1,IL-6,ET-1 and iNOS quantity(*p<0.05,**p<0.01).Thus PHL have the protection of endothelial cell dysfunction.The expression of PAI,vWF,tPA,PS,TFPI and TM in EA.hy926 endothelial cells was not significantly affected by PHL,and 50?g/ml PHL can suppresse TNF-a induced overexpression of MCP-1,IL-6,ET-1 and iNOS.Thus PHL have the protection of endothelial cell dysfunction.It provides an experimental basis for the development of new oral preparations for the prevention and prevention of atherosclerosis in the clinic,which can be used for a long time.