Inhibition Of Yincha Compound Components On Injury Of Human Umbilical Vein Endothelial Cells Induced By Oxidized Low Density Lipoprotein

ObjectivesThis study was designed to obtain the optimum extraction process route of tea polyphenols,explore the dosage of Yincha compound ingredients in vitro experiments, use cell morphologyand apoptosis to research the inhibition of Yincha Compound on ox-LDL-induced apoptosisin human umbilical vein endothelial cells, research the inhibition of Yincha Compound onox-LDL-induced inflammatory damage in human umbilical vein endothelial cells on proteinsand RNA level.MethodsExtract tea polyphenols with water, detect in the content of tea polyphenols in extract waterby ferrous tartrate method, use the response surface method to design four factors and threelevels experiment to thus to optimize the extraction process base on the results of single factorexperiment, use HPLC method to detect the content of EGCG, EGC and caffeine in teapolyphenols; detect the influence of by different drug different dosage on cell viability by theCCK-8assay; acridine orange staining method observed the morphological changes ofhuman umbilical vein endothelial cells, Annexin V-FITC/PI flow cytometry detect theapoptosis rate of human umbilical vein endothelial cells; detect the protein expression levelsof VCAM-1, ICAM-1, MCAP-1, E-selectin inflammatory cytokines by enzyme linkedimmunosorbent assay; detect the mRNA expression levels of VCAM-1, ICAM-1, MCP-1,E-selectin, NF-κB p50, NF-κB p65by real-time quantitative polymerase chain reaction.Results1. The results of response surface optimization show: when solid-liquid ratio1:20,temperature60℃, time60min, extract3times, the maximum extraction rate of tea polyphenols close to32%; the tea water extract contains41.98%EGCG、8.51%EGC、0.13%caffeine；2. CCK-8results indicate different concentrations of tea polyphenols, extract of ginkgobiloba, Yincha compound and simvastatin show varying degrees of effect on cell viability, thelower concentration of drug, the higher cell viability, and ultimately select tea polyphenols20μg/ml, extract of ginkgo biloba20μg/ml, Yincha compound20μg/ml, simvastatin6.25μg/ml;3. Acridine orange staining results show: the model group ox-LDL group had smaller roundcell nucleus shrinkage, chromatin condensation phenomena; normal control group cellmorphology is intact, cell growth state is exuberant; after pretreat with tea polyphenols orextract of gingko biloba pretreatment, some cells show a small round, cell nucleus shrinkage,chromatin condensation phenomena; Yincha group cell morphology is complete, good growth state, better than tea polyphenols and extract of ginkgo biloba. Results of flowcytometry further confirm the morphological test results.4. Elisa and qRT-PCR assay detect results show that the important atherosclerosis-relatedinflammatory cytokines VCAM-1, ICAM-1, MCAP-1, E-selectin, NF-κB p50, NF-κB p65expression have a large expression in ox-LDL group, normal control cells expressing lowamounts, between both groups there is a significant difference (P <0.05); tea polyphenolsinhibit the expression of inflammatory cytokines stronger than extract of ginkgo biloba,Yincha compound is significantly stronger than medication alone group (P <0.05).Conclusions1. Response surface optimization of water extraction results are satisfactory, tea polyphenolsyield near30%, tea polyphenols EGCG content reaches up to40%, the content of caffeine isless than1%, has a certain reference value for the practical application;2. ox-LDL successfully induced apoptosis in human umbilical vein endothelial cells, Yinchacompound ingredients can inhibit ox-LDL-induced human umbilical vein endothelial cellsmorphology changes and apoptosis;3. Yincha compound ingredients can reduce the expression of atherosclerosis development-related major cytokines, the effect of inhibit damage is better than tea polypheols and extractof ginkgo biloba, and the compound plays a major role in the composition is tea polyphenols.