| Objective:Diabetes vascular complication is the major cause of mortality and mutilation in the type 2 diabetic patients. And the damage of endothelial cells is the start step of diabetes vascular complication. High glucose is the sign of metabolize turbulence in the diabetic patients, and is the risk of diabetes vascular complication. Many studies in vivo have approved that high glucose may destroy endothelial cells, but the former studies were on constant high glucose. Normally, the level of blood glucose changes within a limited range, however, the inter-and intra-day glucose variability is relatively high in diabetic patients. Many clinic studies have demonstrated that the insulin treatment, with the purpose of decreasing the glucose variability, could reduce the card vascular incident significantly,it suggest that glucose fluctuation may be involved in the development of chronic vascular complication in diabetes .We compared the effects of intermittent with constant high glucose media on apoptosis and the production of NO and ET-1 in cultured human umbilical vein endothelial cell strain(HUVEC) so as to explore the pathological effects of intermittent hyperglycemia on diabetes vascular complication.Methods:Human umbilical vein endothelial cells were cultured to confluence and were exposed for 72h to 5.55mmol/L glucose normal control,25mmol/L glucose constant high glucose and 5.55mmol/L alternating with 25mmol/L glucose intermittent high glucose on a daily basis.The cell apoptosis rate and the associated gene expression(Bax and Bcl-2) were detected by flow cytometry; the production of NO was measured by nitrate reduction test, and the production of ET-1 was measured by radioimmunoassay (RIA).Results:(1) After HUVECs were exposed to constant high glucose and intermittent high glucose for 72h,it enhanced the apoptosis rate significantly.Compared with cells exposed to normal glucose,the apoptosis rate were increased to ( 8.10±0.10 ) %( P <0.001)and ( 10.20±0.20 ) %( P<0.001)respectively. Furthermore there was a significantly increase of the apoptosis rate in intermittent high glucose compared with it in constant high glucose(P<0.001).(2) After HUVECs were exposed to constant high glucose and intermittent glucose for 72h,it enhanced the expression of Bax(expressed by the fluorimetric index , FI) significantly.Compared with cells exposed to normal glucose ,the expression of Bax were increased to 1.13±0.021 (P<0.001) and 1.34±0.020(P<0.001)respectively. Furthermore there was a significantly increase of the expression of Bax in intermittent high glucose compared with it in constant high glucose(P<0.001).(3) After HUVECs were exposed to constant high glucose and intermittent glucose for 72h,it reduced the expression of Bcl-2 (expressed by the fluorimetric index , FI ) expression of significantly .Compared with cells exposed to normal glucose , the expression of Bcl-2 were reduced to 0.90±0.059,(P<0.001) and 0.79±0.062(P<0.001)respectively. Furthermore there was a significantly decrease of the expression of Bcl-2 in intermittent high glucose compared with it in constant high glucose(P<0.001).(4) After HUVECs were exposed to constant high glucose and intermittent glucose for 72h, it reduced the production of NO significantly. Compared with cells exposed to normal glucose,the production of NO were reduced to(13.50±2.00)μmol/L( P <0.001),(18.10±3.70)μmol/L(P<0.001)respectively. Furthermore there was a significantly decrease of the production of NO in intermittent high glucose compared with it in constant high glucose(P<0.001).(5) After HUVECs were exposed to constant high glucose and intermittent glucose for 72h, it enhanced the production of ET-1 significantly.Compared with cells exposed to normal glucose ,the production of ET-1 were increased to (2.70±0.50) pg/μg.Protein( P <0.001) and (3.50±0.50) pg/μg.Protein( P<0.001)respectively. Furthermore there was a significantly increase of the production of ET-1 in intermittent high glucose compared with it in constant high glucose(P<0.001). Conclusion:The intermittent high glucose group had increased ET-1, Bax and the apoptosis rate compared with constant glucose group;however, both the expression of Bcl-2 and the production of NO in intermittent high glucose group were significantly lower than those in constant high glucose group. Our data suggest that glucose fluctuation may be involved in the damage of endothelial cells, and then facilitating the development of chronic vascular complication in diabetes by affecting the apoptosis and the production of NO and ET-1. |
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