Effect Of Gene Heme Oxygenase-1 On Human Umbilical Vein Endothelial Cell Apoptosis Induced By Constant High Glucose And Intermittent High Glucose

Objective:To investigate the effect of gene heme oxygenase-1(HO-1)on the apoptosis of human umbilical vein endothelial cells(HUVECs)and the expression of bax and bcl-2 induced by constant high glucose and intermittent high glucose in order to provide experimental and theoretical evidence for the gene therapy for diabetic vascular lesion .Methods:1. Cultures of untransfected HUVECs and HUVECs transfected with gene HO-1 (transfected HUVECs).2. The mRNA expression of gene HO-1 by transfected HUVECs was determined by reverse transcription polymerase chain reaction (RT-PCR).3. untransfected HUVECs and transfected HUVECs were exposed for 7 days to 5.5mmol/L D-glucose(normal glucose),20.0mmol/L D-glucose(constant high glucose)and 5.5mmol/L alternating with 20.0mmol/L D-glucose per 24 hours(intermittent high glucose)respectively . The cultures were maintained at 37°C with a gas mixture of 5% CO2–95% air. The cell viability was determined by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. The cell apoptosis was detected by the method of acridine orange (AO)/ethdium bromide (EB) staining. The mRNA expression of apoptosis related genes bax and bcl-2 was determined by RT-PCR.Results: 1. The mRNA expression level of gene HO-1 in transfected HUVECs. The mRNA expression level of gene HO-1 was significantly decreased in untransfected HUVECs while increased significantly in transfected HUVECs (P<0.05).2. The effect of constant high glucose and intermittent high glucose on HUVECs viability.After HUVECs were treated with various concentration of glucose for 7 days, by using MTT assay the cell viability decreased in both constant high glucose group and intermittent high glucose group, which were 79%(P<0.05)and 59%(P<0.001)of normal glucose group respectively, and the latter decreased more significantly(P<0.05).3. The effect of constant high glucose and intermittent high glucose on HUVECs apoptosis.After HUVECs were treated with various concentration of glucose for 7 days, by the method of acridine orange (AO)/ethdium bromide (EB) staining the apoptotic cells increased in both constant high glucose group and intermittent high glucose group, which were 1.61 folds (P<0.001) and 1.79 folds (P<0.001) of normal glucose group respectively, and the latter increased more significantly (P<0.05).4. The effect of constant high glucose and intermittent high glucose on mRNA expression of bax and bcl-2After HUVECs were treated with various concentration of glucose for 7 days, by the method of RT-PCR the mRNA expression of bcl-2 decreased significantly in both constant high glucose group and intermittent high glucose group compared with that in normal glucose group(P<0.05,P<0.001). And the mRNA expression level of bcl-2 in intermittent high glucose group was decreased more significantly than that in constant high glucose group (P<0.05). However, there was no significant difference with the mRNA expression of bax among three groups (P>0.05).5. The effect of gene HO-1 on HUVECs viabilityAfter transfected HUVECs and untransfected HUVECs were treated with various concentration of glucose for 7 days, by using MTT assay the cell viability increased in both constant high glucose/ HO-1 group and intermittent high glucose / HO-1 group. The cell viability in constant high glucose/ HO-1 group was 1.37 folds of constant high glucose group (P<0.05); The cell viability in intermittent high glucose / HO-1 group was 1.47 folds of intermittent high glucose group (P<0.05).6. The effect of gene HO-1 on HUVECs apoptosis induced by constant high glucose and intermittent high glucose.After transfected HUVECs and untransfected HUVECs were treated with various concentration of glucose for 7 days, by the method of acridine orange(AO)/ethdium bromide(EB) staining the apoptotic cells decreased significantly in both high glucose/ HO-1 group and intermittent high glucose / HO-1 group. The apoptotic cells in constant high glucose/ HO-1 group were 67% (P<0.001) of constant high glucose group. The apoptotic cells in intermittent high glucose / HO-1 group were 81% of intermittent high glucose group (P<0.001).7. The effect of gene HO-1 on mRNA expression of bax and bcl-2After transfected HUVECs and untransfected HUVECs were treated with various concentration of glucose for 7 days, by the method of RT-PCR the mRNA expression of bcl-2 increased in both constant high glucose/ HO-1 group and intermittent high glucose / HO-1 group compared with that in constant high glucose group and intermittent high glucose group respectively(P<0.05). But the mRNA expression of bax showed no significant difference (P>0.05).Conclusions:1. Both constant high glucose and intermittent high glucose could induce HUVECs apoptosis. And intermittent high glucose induced HUVECs apoptosis more significantly compared with constant high glucose. The mechanism by which may be related to the decrease of bcl-2 expression.2. The up regulation of gene HO-1 could pretect HUVECs from apoptosis induced by constant high glucose and intermittent high glucose. The mechanism by which may be related to the up regulation of bcl-2 expression.