| ObjectivesAtherosclerosis (AS) is a special kind of vessel wall's chronic inflammation when it reacts to scleratheroma factors.From the beginning of AS ,there are various cytokines, mediators of inflammation, fatty substances and fabric components increase,when the number of them gets to a certain level, the heart and cerebrovascular disease emerges. Cell adhesion molecules(CAMs) is a kind of glycoprotein which can make adhesive attraction, accommodate the combination between cells ,cells and extracellular matrix.the expression level of CAMs in atherosclerotic plaque and the progress of atherosclerosis are at equal pace, and it is usually regarded as one of the facts that take part in the morbility of AS. Homocysteine(Hcy) is a kind of sulfur containing amino acid,with high level,it can induce the cell adhesion of white blood corpuscle and emigration from blood vessel endothelium, it has been proved as one of the independent risk factors which can lead to AS.Tea polyphenols(TP) is a kind of phenolics complex extracted from green tea, it has many pharmacological actions,and shows extensive biological activity. Plenty of inwestigations indicate that,TP has some effects such as anti-oxidation, cardiovascular disease-prevention and so on.To study the mechanism of CAMs increasing on HUVEC(Human umbilical vein endothelial cells) and further promote atherosclerosis by Hcy,in the present study,we applied Hcy to HUVEC, and followed by detection of cell proliferation, the changes of CAMs such as ICAM-1 and E-selectin mRNA and proteins,as well as MDA,NO,NOS and GSH-PX ,based on the results we can study that whether TP can restrain the expression of CAMs produced by Hcy, and the results can supply support for the measures of reducing the incidence of atherosclerosis and the further abroad application of tea polyphenols.MethodsHUVEC were cultured under the condition of 37℃, 5%CO2, saturation humidity. HUVEC lied in logarithm growth period were divided into control group and 3 experimental groups which were tea polypenols, Hcy, and Hcy+tea polyphenols respectively. The final concentration of tea polyphenols and Hcy in medium were 25mg/L and 5mmol/L, respectively, and cells with equal volume of PBS as control. After 24 hours administered with tea polyphenols and/or Hcy, cell viability was determined by methyl thiazolyl tetrazolium(MTT) assay, the expression of ICAM-1 and E-selectin were analyzed using sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE), Western blotting and immuno- cytochemistry, the mRNA levels of ICAM-1 and E-selectin were detected by reverse transcription polymerase chain reaction(RT-PCR) and determine the level of Maleic Dialdehyde(MDA), NitricOxide(NO),NOS and glutathione peroxidase (GSH-PX)..Results1. The effect of tea polyphenols and Hcy on cell morphology:It can be seen that the cell of the control and tea polyphenols groups were round, structures were integrity and the shapes were in order. Abnormal cells were induced by Hcy. The morphology of the cells treated with both Hcy and tea polyphenols were almost normol, but was still different with control.2. The effect of tea polyphenols and Hcy on cell growth:HUVEC were cultured with Hcy and/or tea polyphenols. After 24 hours, cell proliferation was detected by MTT and the results showed that Hcy inhibited the cell proliferation rate by36.0%(P<0.05), and tea polyphenols decreased the effects of Hcy on HUVEC(7.9%, P<0.05), but it could not inhibite normol HUVEC (4.9%, P >0.05).3. The detection of expression of ICAM-1 and E-selectin proteins by SDS PAGE and Western blot:The results of SDS PAGE and Western blot showed that Hcy markedly, increased ICAM-1 and E-selectin protein expression by 30 % and 34 % compared with control(P<0.05), respectively. Otherwise, tea polyphenols could down-regulate the expression of ICAM-1 and E-selectin by 16% and 30%(P<0.05) respectively.4. The detection of expression of ICAM-1 and E-selectin proteins by immunocytochemistry:The results of immunocytochemistry analysis showed that the expressions of ICAM-1 and E-selectin proteins mostly existed on the cell surface and in the extracellular matrix.The coloration of cells in the group of control was light; The effects of Hcy on HUVEC changed the cell morphology, decreased the number markedly and the coloration of cells was heavier than that in control.Compared with the group of control,the expression of ICAM-1 and E-selectin in group of Hcy increased by 30% and 37% (P<0.05)respectively;but tea polyphenols could inhibit the effects of Hcy,decreased the expression of ICAM-1 and E-selectin by 17% and 24%(P<0.05).5. The results of RT-PCR:Hcy could up-regulate the ICAM-1 mRNA level of HUVEC by 67% (P<0.01) The changes of and E-selectin mRNA levels of HUVEC exposed to Hcy was markedly:417% (P<0.01) .tea polyphenols could down-regulate the expression of ICAM-1 and E-selectin by 37% and 44%(P<0.01) respectively.6. The determination of MDA,NO,NOS and GSH-PX:Compared with the group of control, Hcy could up-regulate the content of MDA (P<0.05); decreased the content of NO(P<0.05),the activity of NOS(P<0.05), but it had no effect on GSH-PX(P>0.05) in medium. Tea polyphenols could reverse these effects but had no effect on GSH-PX (P>0.05) .Conclusions1. Hcy in given dose could obviously restrain the growth of HUVEC,and change the cell morphology markedly.2. The expression of ICAM-1 and E-selectin were increased induced by Hcy related to the changes of the levels of their mRNA.3. The expression of ICAM-1 and E-selectin were increased induced by Hcy related to the changes of the protein levels.4. Tea polyphenols inhibited the effects of Hcy induced changes on HUVEC morphology.5. Tea polyphenols could reverse the changes of the expression of ICAM-1 and E-selectin induced by Hcy on HUVEC.6. Hcy can increase the contents of malon dialdehyde (MDA) in medium, decrease the content of nitric oxide (NO) and the activity of NOS;TP can reverse these effects; however, neither Hcy nor TP has effect on the activity of GSH-PX in medium。...
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