| ObjectivesTo research the arsenic effects on cultures human umbilical vein vascular endothelial cells (HUVEC);To research the selenium effects on cultures human umbilical vein vascular endothelial cells; to research the selenium effects on HUVEC injured by arsenic.MethedsHUVEC were cultured under the condition of 37℃,5%CO2, saturation humidity. When HUVEC lied in logarithm growth we divided them into three experiment groups which were As, Se and As+Se respectively, the cells were treated with As at different concentrations (0.05,0.1,0.5,1.5,3,6,12μmol/L) for 48h in vitro, In the As2O3+Se group,all groups were treated with different content of As (0.05,0.1,0.5,1.5,.3,6,12μmol/L)and Se(0.1μmol/L) for 48h in vitro, while the group of selenium, the concentration of selenium is 0.05,0.,0.5,1,2μmol/L. The control group use 1640 culture.The morphologic changes of cells were observed by Wrigh-Giema staining and Acridine orang staining; activity of cells were measured by MTT method; the activity of SOD and GSH-px and the content of 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. The result are analysis by one way ANOVA, q-test is used in two group analysis.Results1 Effects of Arsenic on the role of HUVEC injury1.1 The effect of Arsenic on cell morphologyBy the wright-giemsa stain, the number of the cell was decreased gradually along with the increase of Arsenic concentration, the cell morphology were shrinked and changed markedly, the space between cells were augmented,cell membrane were damaged, cell nuleus were shrinked and exposed, we can see the amphiblestroid chromatin. Acridine orang staining:the nucleolus were changed markedly and collected in the edge of cells,fluorescence chips were increased.1.2 The effect of Arsenic on cell growth:Compared with the control group, the results showed that at the dose of 0.05μmol/L, the Arsenic could not inhibited the cell proliferation rate (P>0.05).At the dose of 0.01μmol/L, the Arsenic can inhibited the cell proliferation rate (P<0.01),the inhibite role was strengthened with the increasing of the Arsenic concentration.1.3 The effect of Arsenic on SOD,GSH-px activity and MDA content.Compared with the control group, the activity of SOD,GSH-px and the content of MDA changed unsignificantly at 0.05μmol/L (P>0.05); when the Arsenic concentration was 0.5μmol/L,the content of MDA increased significant (P<0.05) The activity of SOD,GSH-px were decreased,while the content of MDA increased significant y(P＜0.01), when the content of As2O3 increased.1.4 The effect of Arsenic on the content of NOS and NOS mRNAThere was no effect on NOS and NOS mRNA when the Arsenic concentration was 0.05,O.1μmol/L(P>0.05);The activity of eNOS and the expression of eNOS mRNA were down-regulated by arsenic in concentration dependent manner (P<0.01),while the activity of iNOS and the expression of eNOS mRNA were up-regulated (P<0.05 or P<0.01).1.5 The effect of Arsenic on the expression of ICAM-1 and VCAM-1 Compared with control group, there was no significant effect on the content of ICAM-1 and VCAM-1 when the Arsenic concentration was 0.05,0.1μmol/L ((P>0.05),the Aseenic at the high dose group can up-regulate the ICAM-1 and VCAM-1 (P<0.05 or P<0.01).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, the ecpression of NOS and NOSmRNA,the expression of ICAM-1 and VCAM-1 in endothelial cells, the concentration of selenium in 0.1μmol/L play an accumulate role to cell, so we determined the intervention dose of selenium was 0.1μmol/L.3 Effects of Selenium on HUVEC injuried by Arsenic3.1 Effects of Selenium on HUVEC injuried by Arsenic in cell morphology Different concentration of Arsenic and 0.01μmol/L Selenium were entered into the medium, and the HUVEC were cultured for 48 hours. The cell morphology was normal, structures were integrity and the shapes were in order in the low dose Arsenic (0.05,0.1μmol/L)+Selenium(0.1μmol/L). The number of the cell was decreased gradually, the cell morphology changed markedly, cell membrane were damaged, the cells are apoptosis, along with the concentration of Arsenic increased. Fluorescence chips can be observed by Acridine orange staining.3.2 Effects of Selenium on the changes of cell activity by Arsenic Cell proliferation was detected by MTT, the results showed that the cell proliferation rate were improved at the low dose Arsenic (0.05,O.1μmol/L)+Selenium(0.1μmol/L) (P<0.05),compared with the Arsenic group.The group at high doseArsenic+Selenium(0.1μmol/L) compared with the Arsenic group, cell proliferation rate were improved, but has no significant statistic meaning(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 ArsenicCompared with Arsenic groups, the SOD activity(Arsenic 0.05,0.5μmol/L), the GSH-Px activity(Arsenic 0.05,0.1μmol/L)increase significantly (P＜0.05),the content of MDA(Arsenic 0.05,0.1μmol/L) decreased significantly(P<0.05).3.4 Effects Selenium on the abnormal expression of NOS and NOS mRNA caused by ArsenicCompared with the Arsenic group, the activity of eNOS were increased at the Arsenic(0.05,0.1,0.5μmol/L)+Selenium group (P<0.05), the expression of eNOS mRNA were up-regulated at the Arsenic(0.05,0.5,1.5μmol/L)+Selenium (P<0.05);the activity of iNOS were decreased at the the Arsenic(0.05,0.5,1.5μmol/L)+Selenium group (P<0.05), the expression of iNOS mRNA were increased at the Arsenic (0.05,0.1,1.5μmol/L)+Selenium (P<0.05) were down-regulated (P<0.05)3.5 Effects Selenium on the abnormal expression of ICAM-1 and VCAM-1 caused by ArsenicCompared with the Arsenic group, the content of ICAM-1 and VCAM-1 decreased significantly in the Arsenic(0.05,0.1μmol/L)+Se groups (P<0.05). Along with the increasing of Arsenic content, the content of ICAM-1 and VCAM-1 changed unsignificantly compared with the Arsenic group (P> 0.05)Conclusion1,Arsenic could obviously inhibit the growth of HUVEC, and change the cell morphology markedly, even induced the cell apoptosis; appropriate dose of Selenium can be antagonistic towards the effect of Arsenic on the cell damage.2,Arsenic could decrease the activity of SOD,GSH-Px, increase the content of MDA in nutrient solution; 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,Arsenic could inhibit the expression of eNOS mRNA, decrease the activity of eNOS in nutrient solution; 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 As2O3, so that the expression and activity of eNOS and iNOS were trended to normal4,Arsenic could induce the expression of CAM, increase the content of ICAM-1 and VCAM-1; appropriate concentration of selenium could inhibit the abnormal expression of ICAM-1 and VCAM-1 owing to Arsenic,which were significantly lower than that in the culture medium of Arsenic group. |
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