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Objective: The human umbilical vein endothelial cells(HUVECs)were used for experiment. The cells were dealed with 5.5mM,20mM,20mM/5.5mM glucose respectively. Treated cells was observed in different oxidative stress indicators(MDA and SOD). SelS,PKCαand PKCβ2 in the differences of protein and mRNA expression were also detected. To research whether the damage effection of high glucose on HUVEC was through PKC pathway and the relationship between SelS and PKC. To provide new experimental evidence for endothelial cell protection strategy.Method:1.The cells were treated with DMEM culture media containing 5.5mM(Normal group,NG),20mM(High glucose group,HG),5.5 and 20mM(Intermittent glucose group,IG,which dealt with 5.5 and 20mM glucose) and 14.5 mM mannito(lMannitol group,MG)respectively on every day.After a culture of 48 hours,7days and 14days respectively,the changes in cell morphology and the level of MDA and SOD in the supernatant of each group was measured.2.HUVECs was devided into four groups:NG,HG,IG and MG. After a culture of 2 days,7days and 14days respectively,the mRNA level of SelS,PKCαand PKCβ2 were detected in each group by real-time PCR.3.The same condition was given to four groups,after a culture of 48 hours,7days and 14days respectively,the protein level of SelS,PKCαand PKCβ2 were measured in every group by western blot.Result:1.Cell morphology:After 2 days,cells in NG and MG were observed with clear outline,most fusiform. Cytoplasmic and nucleus were visible. Cells in HG were observed with increased volume,cobblestone-like,increased cytoplasmic and small black scattered. Cells in IG were observed without clear outline,increased volume,disorganized,ill-defined with cytoplasm and nucleus,small black scattered. The changes became more serious in time-dependent manner.2.MDA content(nmol/ml):After 2 days,MDA content in HG(5.15±0.32)and IG(5.17±0.14)was higher than in NG(1.10±0.20)and MG(1.12±0.17)(P=0.004,P=0.003;P=0.002,P=0.003);After 7 days,MDA content in HG(8.44±0.44)and IG(10.86±0.36)was further higher than in NG (1.07±0.15)and MG(1.09±0.14)(P=0.008,P=0.004;P=0.003,P=0.003),the IG was higher than HG(P=0.002);After 14 days,MDA content in HG(11.92±0.38)and FG group(15.66±0.56)was still higher than in NG(1.18±0.21)and MG(1.15±0.20)(P=0.006,P=0.005;P=0.004,P=0.003),the IG was further higher than HG(P=0.003). With the time the MDA content of HG and IG in 7d and 14d were higher than in 2d, and the MDA content of 14d was higher than in 7d.(P<0.05)The level of MDA were no differences in NG and MG(P>0.05).3.SOD activity (U/ml):After 2 days,SOD activity in HG(27.76±0.40)and IG(26.45±0.49)was lower than in NG(34.41±1.05)and MG(34.32±0.98)(P=0.003,P=0.002;P=0.00,P=0.00); After 7 days,SOD activity in HG(20.27±0.52)and IG(16.29±0.38)was further lower than in NG(34.33±1.04)and MG(34.40±0.99)(P=0.006,P=0.004;P=0.003,P=0.002),the IG was lower than HG(P=0.002);After 14 days,SOD activity in HG(10.36±0.45)and IG(5.81±0.45)was still lower than in NG(34.33±1.04)and MG(34.33±0.95)(P=0.004,P=0.003;P=0.002,P=0.005), the IG was further lower than HG(P=0.001). With the time the SOD activity of HG and IG in 7 and 14 days were lower than in 2d, and the SOD activity of 14d was lower than in 7d(P<0.05). The level of SOD activity were no differences in NG and MG(P>0.05).4.After dealing with 2 days,the expression of SelS mRNA in HG(0.80±0.03)and IG(0.87±0.05)was higher than in NG(0.71±0.02)and MG(0.72±0.03)(P=0.01,P=0.002;P=0.006,P=0.001)and the IG was higher than HG(P=0.03);After 7 days,the expression of SelS mRNA in HG(0.95±0.08)and IG(1.09±0.03)was further higher than in NG(0.82±0.04)and MG(0.82±0.02)(P=0.01,P=0.00;P=0.002,P=0.004),the IG was higher than HG(P=0.01);After 14 days,the expression of SelS mRNA in HG(1.07±0.03)and IG(1.16±0.02)was still higher than in NG(0.91±0.03)and MG(0.90±0.03)(P=0.008,P=0.004;P=0.003,P=0.003),the IG was further higher than HG(P=0.003). With the time in 7d and 14d,the expression of SelS mRNA in HG and IG were higher than in 2d(P<0.05), the 14d was highest (P<0.05).5.After dealing with 2 days, the expression of SelS protein in HG(0.51±0.02)and IG(0.58±0.04)was higher than in NG(0.37±0.05) and MG(0.36±0.03)(P=0.02,P=0.00;P=0.00,P=0.00)and the IG was higher than HG(P=0.04);After 7 days, the expression of SelS protein in HG(0.63±0.02)and IG(0.75±0.03)was further higher than in NG(0.53±0.05) and MG(0.53±0.02)(P=0.006,P=0.004;P=0.003,P=0.002),the IG was higher than HG(P=0.002), After 14 days, the expression of SelS protein in HG(0.85±0.03)and IG(1.01±0.04)was still higher than in NG(0.56±0.05)and MG(0.55±0.04)((P=0.004,P=0.003;P=0.002,P=0.005),the IG was further higher than HG(P=0.003). With the time in 7d and 14d, the expression of SelS mRNA and protein were increased in HG and IG higher than 2d (P<0.05), and the 14d was higher than HG (P<0.05).6.The expression of PKCαmRNA and protein among HG,IG, NG and MG were no differences(P>0.05).7.After dealing with 2days,the expression of PKCβ2 mRNA in HG(1.05±0.03)and IG(1.12±0.03)was higher than in NG(0.97±0.05)and MG(0.97±0.05)(P=0.03,P=0.00;P=0.003,P=0.003);after 7 days,the expression of PKCβ2 mRNA in HG(1.11±0.02)and IG(1.21±0.01)was further higher than in NG(0.95±0.07)and MG(0.96±0.05)(P=0.00,P=0.008;P=0.004;P=0.003),the IG was higher than HG(P=0.03);after 14 days,the expression of PKCβ2 mRNA in HG(1.18±0.03)and IG(1.28±0.04)was still higher than in NG(0.97±0.07)and MG(0.95±0.03)(P=0.01,P=0.00;P=0.002,P=0.004),the IG was further higher than HG(P=0.02). With the time in 7d and 14d,the expression of PKCβ2 mRNA in HG and IG were higher than in 2d(P<0.05), the 14d was highest (P<0.05).8.After dealing with 2 days,the expression of PKCβ2 protein in HG(0.56±0.03)and IG(0.65±0.04)was higher than in NG(0.47±0.04)and MG(0.48±0.06)(P=0.04,P=0.00;P=0.003,P=0.002)and the IG was higher than HG(P=0.03);after 7 days, the expression of PKCβ2 protein in HG(0.74±0.03)and IG(0.83±0.02)was further higher than in NG(0.49±0.03)and MG(0.48±0.07)(P=0.006,P=0.005;P=0.003,P=0.004),the IG was higher than HG(P=0.03);after 14 days,the expression of PKCβ2 protein in HG(0.85±0.03)and IG(1.01±0.05)was still higher than in NG(0.48±0.06)and MG(0.48±0.04)(P=0.01,P=0.002;P=0.002,P=0.004), the IG was further higher than HG(P=0.03). With the time in 7d and 14d, the expression of PKCβ2 protein were increased in HG and IG higher than 2d (P<0.05), and the 14d was higher than HG (P<0.05).Conclusion:1.Bothe constant and intermittent High glucose induce the oxidative stress to injure the endothelial,the effect of intermittent high glucose was much significant.2.High glucose induced damage in HUVECs may be through the PKC pathway. High glucose injured HUVECs with the increasing expression of SelS mRNA and protein.
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