The Role Of [Gly14]-humanin In High Glucose-induced Apoptosis In Human Umbilical Vein Endothelial Cells

Objectives:Hyperglycemia damages vascular endothelial cells,causes endothelial dysfunction and leads to the occurrence and development of vascular complications.Calcium(Ca2+)is an important messenger of apoptosis in endothelial cells.In human umbilical vein endothelial cells(HUVECs),increased activity of store-operated channels(SOCs)can mediate apoptosis induced by high glucose.The purpose of this study was to investigate the Calcium-related mechanism of apoptosis induced by high glucose in HUVECs and to explore whether the protective effect of Humanin(HNG)on HUVECs is related to Ca2+and SOCs.Methods:Human umbilical vein endothelial cells(HUVECs)were cultured in vitro,and HUVECs were induced by 30 mM glucose for 72 hours to construct apoptotic model.30 mM mannitol was used as osmotic pressure control and 1 uM HNG was used as drug intervention.Western blot was used to detect the expression levels of caspase-3,cleaved caspase-3,BAX and BCL-2 in HUVECs.The wide-field model of total internal reflection fluorescence microscopy(TIRFM)was used to measure the levels of Calcium ion released by endoplasmic reticulum calcium pool and extracellular calcium influx in HUVECs.Calcium ion levels were detected by confocal microscopy when we overexpress or knock down STIM1/ORAI1 in HUVECs.Results:1.Western Blot results showed that high-glucose treatment had no effect on the expression of full-length caspase-3 protein.High glucose increased the expression of cleaved caspase-3 and BAX protein,reduced the expression of bcl-2 protein.HNG reduced the levels of cleaved caspase-3 and BAX protein increased by high glucose treatment,and increased the levels of bcl-2 protein decreased by high glucose treatment.Treatment with only Mannitol or HNG had no significant effect on expression of caspase-3,cleaved caspase-3,BAX,and bcl-2 protein levels.2.Total internal reflection fluorescence microscopy showed that high glucose treatment had no significant effect on area(total cytoplasmic high calcium),△F(amplitude of calcium release),t1(velocity of calcium release)and t2(opening time of calcium release channels)during the release of ER calcium pool.3.In the process of extracellular calcium influx,high glucose treatment increased area(total cytoplasmic high calcium),prolonged t2(opening time of extracellular calcium influx channels),and had no significant effect on △F(amplitude of extracellular calcium influx)and t1(velocity of extracellular calcium influx);HNG intervention could reduce area and shorten t2;treatment with only mannitol or HNG had no effect on area,AF,t1 and t2 in the process of endoplasmic reticulum calcium pool release and extracellular calcium influx.4.Only overexpression of STIM1 increased AF(the magnitude of extracellular calcium influx),area(the total amount of cytoplasmic high calcium),but had no significant effect on t1(the velocity of extracellular calcium influx)and t2(the opening time of extracellular calcium influx channels);treatment with high glucose when overexpression of STIM1 increased △F,area and further increased t2 compared with overexpression of STIM1 alone,but had no significant effect on t1;HNG intervention decreased area and t2,had no significant effect on t1.5.Only overexpression of ORAI1 had no significant effect on △F(the magnitude of extracellular calcium influx),t1(the velocity of extracellular calcium influx),t2(the opening time of extracellular calcium influx channels)and area(the total amount of cytoplasmic high calcium);overexpression of ORAI1 increased area and prolonged t2,but had no significant effect on △F and t1;HNG could reduce area and t2 caused by treatment with high glucose and overexpression of ORAI1,had no significant effect on △F and t1.6.Treatment with high glucose and Knock-down of STIM1 resulted in the decrease of area(the total amount of cytoplasmic high calcium),△F(amplitude of external calcium influx),but had no significant effect on t1(velocity of external calcium influx)and t2(opening time of external calcium influx channels);HNG had no significant effect on external calcium influx when treating with high glucose and knock-down of STIM1.7.Treatment with high glucose and knock-down of ORAI1 resulted in the decrease of area(the total amount of cytoplasmic high calcium)and △F(amplitude of exocalcium influx),but had no significant effect on t1(velocity of exocalcium influx)and t2(opening time of exocalcium influx channels);HNG had no significant effect on external calcium influx when treating with high glucose and knock-down of ORAI1.Conclusion:HNG can inhibit the apoptosis induced by high glucose in HUVECs,and play a protective role in cells by reducing extracellular calcium influx,which may be related to shortening the opening time of STIM1/ORAI1 channel.