| Chapter.1 Increasing intracellular ceramide was concerned with the apoptosis of human umbilical vein endothelial cells induced by high D-glucoseObjectiveTo investigate the effect of high D-glucose on the apoptosis of HUVECs, and on the levels of ceramide and acid sphingomyelinase, thus to evaluate the role of the ceremide signal pathway in the high D-glucose-induced apoptosis in human vascular endothelial cells (HUVECs).Methods①HUVECs were incubated with 5mM, 30mM D-glucose respectively for variously time( 4,8,12,16,24h) to choose the appropriate incubating time.Then HUVECs were divided into several groups, that were incubated with D-glucose alone in a series of concentrations (5,10,20,30mM) ,30mM D-glucose+2μM Desipramine , and 25mM L-glucose respectively for 16h. Cellular ceramide (cell membrane fraction and cytosol plus organelle fraction) was detected by liquid chromatography/ion spray ionization tandem mass Spectrometry (LC/MS/ MS). ②HUVECs were incubated with 5mM, 30mM D-glucose respectively for variously time(0.5,1,2,4,8,12h) to choose the appropriate incubating time. Then HUVECs were incubated with D-glucose alone in a series of concentrations(5, 10,20,30mM) ,30mM D-glucose+2μM Desipramine , and 25mM L-glucose respectively for 2h. Intracellular acidic sphingomyelinase (A-Smase) activity was determined using the fluorescent substrate BODIPY C12- sphingomyelin and HPLC.③HUVECs were incubated with 5mM, 30mM D-glucose , and with 30mM D-glucose+2μM Desipramine, 30mM D-glucose+2μM Desipramine+50μM C2-CER, 25mM L-glucose respectively for 24 and 48h. Apoptosis of HUVECs was observed by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling(TUNEL) assay, and quantitated by flow cytometry using Annexin-FITC/ propidiumiodid (PI)staining.④HUVECs were incubated with a series of concentrations of exogenous C2-CER(25,50μM) and with A-Smase (0.01,0.1,1U/ml) for 24,48h. Apoptosis of HUVEC was observed by TUNEL assay, and quantitated by flow cytometry using Annexin-FITC/PI staining.Results①The intracellular ceramide increased in a concentration-dependent manner after treating with high D-glucose , and it got to the peak after 16h (P < 0.01). But the contents of membranous ceramide had no change. ②The intracellular A-SMase activity increased in a concentration -dependent manner after treating with high D-glucose, and it achieved the peak after being treated for 2h (P < 0.01) .③Desiparmine could inhibit both increasing of the intracellular ceramide and A-SMase activity (P < 0.05) . High L-glucose had no effect on the level of intracellular ceramide and A-SMase activity.④High D-glucose, but not high L-glucose ,could induce the apoptosis of HUVECs after 48h, which could be inhibited by Desiparmine,a inhibitor of A-SMase. Exogenous ceramide could reverse the inhibition of Desiparmine (P<0.05) .⑤The increase of intracellular ceramide correlated positively with the concentration of D-glucose (P=0.000) . The increase of intracellular ceramide correlated positively with the apoptosis of HUVECs (P=0.000).⑥The apoptosis rate of HUVECs increased significantly by incubating with 50μM exogenous C2-CER and 1U/ml A-Smase for 48h(P<0.05).ConclusionIncreased cellular ceramide, which got by lysosomal acidic SMase,was concerned with the apoptosis of HUVECs induced by high D-glucose . Chapter.2 Ceramide induced apoptosis in HUVECs through triggering downstream signal pathway of JNK/SAPKObjectiveTo investigate the effects of high D-glucose and exogenous ceramide on the protein expression of p-JNK, p-c-Jun, bax, bcl-2, thus to study whether the cell apoptosis of HUVECs induced by high D-glucose was coursed by triggering the downstream JNK/SAPK signal pathway.Methods①HUVECs were incubated for 48h with D-glucose alone(5mM, 30mM respectively), 30mM D-glucose+25μM SP600125, and 50μM C2-CER+25μM SP600125. Cell apoptosis was observed by TUNEL assay, and quantitated by flow cytometry using Annexin-FITC/ PI staining.②HUVECs were incubated with 5mM, 30mM D-glucose and 50μM C2-CER respectively for various time(6h, 12h, 24h). Then HUVECs were incubated for 12h with 30mM D-glucose +2μM Desipramine. The protein expression of p-JNK and p-c-Jun of HUVECs was tested with Western blot.③HUVECs were incubated with 5mM, 30mM D-glucose and 50μM C2-CER respectively for variously time(6h, 12h, 24h) . Then HUVECs were incubated for 24h with 30mM D-glucose+2μM Desipramine ,30mM D-glucose+25μM SP600125, and 50μM C2-CER+25μM SP600125 respectively. The protein expression of bax and bcl-2 were tested with Western blot.④HUVECs were incubated for 16h with D-glucose alone(5mM, 30mM respectively) and 30mM D-glucose+25μM SP600125. Cellular ceramide was measured with LC/MS/ MS.⑤HUVECs were incubated for 2h with D-glucose(5mM, 30mM respectively) and 30mM D-glucose +25μM SP600125. Intracellular acidic sphingomyelinase (A-Smase) activity was determined using the fluorescent substrate BODIPY C12- sphingomyelin and HPLC.Results①SP600125, a specific inhibitor of JNK could prevent the apoptosis of HUVECs induced by high D-glucose and exogenous ceramide (P < 0.05) .②High D-glucose and exogenous ceramide could up-regulate the expression of protein p-JNK and p-c-Jun, which got to the peak after 12h (P < 0.05 ) . The effect of high D-glucose could be inhibited by Desipramine, a inhibitor of A- SMase (P < 0.05) .③The protein expression of bax could be up-regulated by both high D-glucose and exogenous ceramide, and it achieved the peak after 24h (P < 0.05) . This effect could be inhibited significantly by SP600125 (P < 0.05) . Desipramine could inhibit the increasing expression of protein bax induced by high D-glucose (P < 0.05) .④The protein expression of bcl-2 could be down-regulated by incubating with high D-glucose and exogenous ceramide, which was lower significantly after 24h (P<0.05). Desipramine could inhibit the decreasing of expression of bcl-2 that induced by high D-glucose, while SP600125 could inhibit the same effects induced by both high D-glucose and exogenous ceramide(P<0.05).⑤SP600125 had no effect on increasing of A-SMase activity and cellular ceramide induced by high D-glucose.ConclusionJNK/SAPK signal pathway could be triggered by ceramide. There was a cascade of 'stimuli of high D-glucose- increasing production of ceramide- activation of JNK/SAPK- changes of expression of bax and bcl-2- cell apoptosis', which concerned with HUVECs apoptosis induced by high D-glucose. Chapter.3 Effect of pioglitazone on high D-glucose-induced apoptosis in human umbilical vein endothelial cells and the mechanisms involvedObjectiveTo investigate the effect and the involved mechanisms of pioglitazone on the apoptosis of human umbilical vein endothelial cells (HUVECs) induced by high D-glucose.Methods①HUVECs were incubated with 5mM, 30mM D-glucose ,30mM D-glucose+ pioglitazone (0.1,l,10μM) ,30mM D-glucose+10μM pioglitazone + 50μM exogenous C2-ceramide and 30mM D-glucose+10μM pioglitazone+ 1U/ml exogenous acidic sphingomyelinase (A-Smase ) respectively for 48h. Apoptosis of HUVECs was observed by TUNEL assay, and quantitated by flow cytometry using Annexin-FITC/propidiumiodid(PI)staining.②HUVECs were incubated with 5mM, 30mM D-glucose ,30mM D-glucose+ pioglitazone (0.1,1,10μM) respectively for 2h. Intracellular A-Smase activity was determined using the fluorescent substrate BODIPY C12- sphingomyelin and HPLC.③HUVECs were incubated with 5mM, 30mM D-glucose ,30mM D-glucose+ pioglitazone (0.1,1,10μM) respectively for 16h. Intracellularceramide was detected by LC/MS/ MS.Results①High D-glucose increased the apoptosis in HUVECs after 48h. Pioglitazone could inhibit the high D-glucose-induced apoptosis in manner of concentration-dependent in HUVECs (P < 0.05) .Exogenous ceramide and A-SMase could reverse the inhibition of pioglitazone (P < 0.05) .②High D-glucose caused the increase in the intracellular A-SMase activity and ceramide. Pioglitazone could inhibit both increase of intracellular A-SMase activity and ceramide (P < 0.05 ) .ConclusionPioglitazone attenuated high D-glucose-induced HUVECs apoptosis through its inhibition on intracellular acidic sphingomyelinase and ceramide. |
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