| Background & Objective Obstructive sleep apnea hypopnea syndrome(OSAHS)is an independent risk factor of various cardiovascular diseases.As one of the most important pathophysiological processes of OSAHS,intermittent hypoxia(IH)induced endothelial injury has been found the early stage of OSAHS related cardiovascular diseases.Mitochondria manifold essential roles in reactive oxygen species(ROS)generation,cell death and downstream effects on cardiovascular functions.This research aims to observe the alterations of mitochondria ultrastructure as well as the content of cytochrome c(cyt C)in mitochondria/cytoplasm of human umbilical vein endothelial cells(HUVECs)exposed to IH,and further to explore the mitochondria-dependent apoptosis pathway in IH-induced injury of HUVECs.Methods 1.HUVECs were mainly divided into two groups: IH groups were exposed to repetitive cycles of 1% O2 for 5min alternating with 21% O2 for 5min in the mini-chamber controlled by self-designed program,and those cells were treated with IH for different exposure time(8 hrs,16 hrs and 24 hrs,respectively).HUVECs in the control group were placed in an ordinary incubator(21% O2,5% CO2,37?).2.To determine apoptosis rate of HUVECs by using Annexin V-FITC/PI apoptosis detection kit.The levels of interleukin-6(IL-6),interleukin-1?(IL-1?),endothelin-1(ET-1)and soluble intercellular adhesion molecule-1(s ICAM-1)in the cultural supernatants were analyzed by enzyme linked immunosorbent assay(ELISA)and the content of nitric oxide(NO)were also measured by nitrate reductase method.3.The mitochondria and cytoplasm of HUVECs were isolated by differential centrifugation,and the content of cyt C in two separations were detected by western blot.4.To observe the mitochondria ultrastructure of control group and IH group(exposed to IH for 16 hrs)by transmission electron microscope(TEM).To determine and compare the transverse diameter of mitochondria in two groups.Results 1.Cell apoptosis rates in IH groups of 8hrs,16 hrs and 24 hrs were 6.70 ± 0.60%,8.86 ± 1.19% and 9.61 ± 1.27%,respectively.Those groups were significantly higher than control group(2.45 ± 0.80%)in time-dependent manner(p<0.05).2.The level of IL-6 in the supernatant increases with further prolonged IH duration(50.37 ± 2.85 pg/ml vs.100.50 ± 7.12 pg/ml vs.186.10 ± 12.14 pg/ml,p<0.01).Higher level of IL-6 was found in the 24-hour-IH group than in the corresponding control group(186.10 ± 12.14 pg/ml vs.146.70 ± 1.07 pg/ml,p=0.03).The content of ET-1 significantly increased with IH exposure time(16.43 ± 0.04 pg/ml vs.17.60 ± 0.18 pg/ml vs.22.43 ± 1.00 pg/ml,p<0.01).The level of IL-1? and s ICAM-1 show no significant difference between groups but increases in a time-dependent manner(IL-1?: 5.37 ± 0.52 pg/ml vs.6.036 ± 0.64 pg/ml vs.6.184 ± 0.57 pg/ml;s ICAM-1: 0.060 ± 0.003 ng/ml vs.0.066 ± 0.007 ng/ml vs.0.089 ± 0.009 ng/ml,both p>0.05).The content of NO in 24-hour-IH group shows significantly less than control group(70.42±8.26 ?mol/L vs.42.72±4.36 ?mol/L,p=0.02).The content of NO in 16-hour-IH group decreases significantly compared with that in 8-hour-IH group(29.75 ± 4.43 ?mol/L vs.68.99 ± 0.64 ?mol/L,p=0.01)while the content of NO in 24-hour-IH group shows an ascending trend with respect to that in 24-hour-IH group(p>0.05).Further analysis shows a positive correlation between cell apoptotic rate and the level of IL-6?IL-1??ET-1?s ICAM-1 in the supernatant,with correlation coefficient 0.6895,0.9036,0.6694 and 0.8187,respectively(p <0.05).3.After IH treatment for 8hrs,16 hrs and 24 hrs,the content of cyt C in cytoplasm were significantly higher than control group in a time-dependent manner(p<0.05),while the content of cyt C in mitochondria shows a gradually declined trend(p >0.05).The content of cyt C in cytoplasm rather than mitochondria shows a strongly positive correlation with cell apoptosis rate(r=0.9876,p=0.0124).Further analysis shows a positive correlationship between the content of cyt C in cytoplasm and the level of IL-1? in the supernatant(r=0.7377,p=0.0233)rather than the level of IL-6,ET-1 or s ICAM-1(p>0.05).Also,there is no correlation between the content of cyt C in mitochondria and the level of IL-6?IL-1??ET-1?s ICAM-1(p >0.05).4.Mitochondria in HUVECs treated with 16-hour-IH appears significant swelling,cristae rupture and vacuolization,compared with substantially intact ultrastructure in control group.The transverse diameter of mitochondria in IH group was significantly larger than that in control group(0.2658 ± 0.0059 ?m vs.0.2485 ± 0.0046 ?m,p=0.0250).Conclusions 1.IH induced increased cell apoptosis in a time-dependent manner,correlated with increased levels of IL-6,IL-1?,ET-1 and s ICAM-1 in the supernatant.2.Intermittent hypoxia induced cyt C changes in cytoplasm/mitochondria of HUVECs in a time-dependent manner.The content of cytoplasmic cyt C was positively correlated with apoptosis and the level of IL-1? in the supernatant.3.IH induced mitochondrial ultrastructure injuries of HUVECs,which may enhance the release of cyt C into cytoplasm,thereby activating apoptotic pathways and leading to endothelial injury. |
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