Effects Of Selenium On Human Umbilical Vein Endothelial Cells Injured By Fluorine

ObjectivesTo research the fluoride effects on cultures human umbilical vein vascular endothelial cells;To research the selenium effects on cultures human umbilical vein vascular endothelial cells;To research the selenium effects on HUVEC injured by fluride.Metheds HUVEC were cultured under the condition of 37℃,5%CO₂,saturation humidity.When HUVEC lied in logarithm growth,we divided them into 3 experiment groups which were F,Se,F+Se respectively.In the NaF group,the cells were treated with NaF at different concentrations(0,100,400,700,1000,2000μmol/L)for 48h in vitro;In the Se group,the cells were treated with Se at different concentrations(0,50,100,500,1000,2000nmol/L)for 48h in vitro;In the NaF+Se group,the cells were treated with NaF and Se for 48h in vitro.The morphologic changes of cells were observed,activity of cells were measured by MTT method; The contents of SOD,GSH-px,MDA in the culture were tested.The activity of eNOS and iNOS in the culture were tested;The expression of eNOS mRNA and iNOS mRNA was analyzed by RT-PCR.ICAM-1 and VCAM-1 productions were measured by ICAM-1 ELISA and VCAM-1 ELISA.Results1 Effects of fluoride on the role of HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS injury1.1 The effect of NaF on cell morphologyBy the wright-giemsa stain,the number of the cell was decreased gradually,the cell morphology changed markedly,when the concentration of NaF is from 400 to 2000μmol/l.Endothelial Cells were Fibroblast-like changed,the cell shrinked,the distance of cell increased.Even the high dose of NaF could induced the cell apoptosis1.2 The effect of NaF on cell growth:Cell proliferation was detected by MTT,the results showed that at the dose of 100,400μmol/L,the NaF could not inhibited the cell proliferation rate(P＞0.05).At the dose of 700-2000μmol/L,the NaF can inhibited the cell proliferation rate(P＜0.05).1.3 The determination of SOD,GSH-px,MDA.Compared with the group of control,the activity of SOD,GSH-px and the content of MDA changed unsignificantly at the low dose(P＞0.05). when the content of NaF increased,the activity of SOD,GSH-px decreased significantly(P＜0.05),but the content of MDA increased significantly(P＜0.05)1.4 The detection of the content of eNOS and iNOS The low dose of NaF had no effect on eNOS and iNOS(P＞0.05);The expression of eNOS were down-regulated by fluorin in concentration dependent manner(P＜0.05),while the activity of iNOS were upregulated (P＜0.05).The results of RT-PCR shew that the NaF had no effect on eNOS mRNA and iNOS mRNA at the dose of 100,400μmol/L(P＞0.05).But the high dose NaF could up-regulated the expression of iNOS Mrna and down-regulate the expression of eNOS mRNA by fluorin in concentration -dependent manner(P＜0.05)1.5 The detection of expression of ICAM-1 and VCAM-1 by ELISA Compared with free control group,the NaF at the high group can up-regulate the ICAM-1 and VCAM-1(P＜0.05).But the content of ICAM-1 and VCAM-1 changed unsinificantly(P＞0.05)at the low dose group.2 Effects of Selenium on human umbilical vein endothelial cell growth By studying endothelial cells Morphology,cell activity,the activity of SOD,GSH-Px in the medium and the content of MDA, endothelial cells the eNOS and iNOS expression,the ICAM-1 and VCAM-1 expression of endothelial cells,when the concentration of selenium was 100nmol/L,the cells were clearly in good condition,so we determined the intervention dose of selenium was 100 nmol/L.3 Effects of Selenium on HUVEC injuried by Fluoride3.1 Effects of Selenium on HUVEC injuried by Fluoride in cell morphology The cell morphology was normal,structures were integrity and the the shapes were in order,when the groups were F(100-700 mol/L)+Se(100nmol/L).The number of the cell was decreased gradually, the cell morphology changed markedly,when the concentration of NaF is from 2000μmol/l.Endothelial Cells were Fibroblast-like changed, the cell shrinked,the distance of cell increased.Even the high dose of NaF could induced the cell apoptosis3.2 Effects of Selenium on the changes of cell activity by Fluoride Cell proliferation was detected by MTT,the results showed that the groups were F(100-700μmol/L)+Se,the NaF could not inhibited the cell proliferation rate(P＞0.05).when the groups were F(1000-2000μmol/L)+Se,the NaF can inhibited the cell proliferation rate (P＜0.05)3.3 Effects of selenium on the activities of SOD,GSH-Px and the content of MDA in the culture medium changed by fluoride Compared with F(400-1000μmol/L)groups,the SOD,GSH-px activity increase significantly in the F+Se groups(P＜0.05),but the content of MDA decreased significantly,the activity of SOD,GSH-Px and the content of MDA changed unsignificantly at F(2000 nmol/L)+Se (P＞0.05).3.4 Effects Selenium on the abnormal expression of NOS caused by fluorideThe activity of eNOS and the expression of eNOS were down-regulated by fluorin in a concentration-dependent manner(P＜0.01),while the activity of iNOS and the expression of iNOS mRNA were upregulated(P＜0.05).Compared with the F group,the activity of eNOS and the expression of eNOS were upregulated,while the activity of iNOS and the expression of iNOS mRNA were upregulated(P＜0.05).3.5 Effects Selenium on the abnormal expression of ICAM-1 and VCAM-1 caused by fluorideCompared with F(400-1000μmol/L)groups,the content of ICAM-1 and VCAM-1 decreased significantly in the F+Se groups(P＜0.05).the content of ICAM-1 and VCAM-1 changed unsignificantly at F(2000 nmol/L)+Se(P＞0.05).Conclusion 1,NaF could obviously restrain the growth of HUVEC,and change the cell morphology markedly,even induced the cell apoptosis;Selenium can be antagonistic effect of sodium fluoride on the cell damage2,NaF could decrease the activity of SOD,GSH-px in medium,increase the content of MDA;The appropriate concentration of selenium can improve the situation so that the SOD and GSH-Px activity was significantly increased,the concentration of MDA decreased.3,NaF could inhibit the expression of eNOS mRNA,decrease the activity of eNOS in medium;and could induce the expression of iNOS mRNA, increase the activity of iNOS.Appropriate concentration of selenium could inhibit the abnormal expression of eNOS and iNOS owing to fluoride stimulus,the expression and activity of eNOS and iNOS trended to normal4,NaF could induce the expression of CAM,increase the content of ICAM-1 and VCAM-1;Appropriate concentration of se]enium could inhibit the abnormal expression of ICAM-1 and VCAM-1 owing to fluoride stimulus,which were significantly lower than that in the culture medium of fluoride group.