| ?.Establishment and evaluation of chronic intermittent hypoxia model in endothelial cellsObjective: To establish and evaluate the model of chronic intermittent hypoxia in endothelial cell injury.Methods: The chamber was designed and prepared for chronic intermittent hypoxia.Its modeling capability was compared with sustained hypoxia model and Co Cl2 induced hypoxia model.The IH-induced injured HUVEC cells were maintained at 37°C at 5% CO2 in intermittent hypoxia chamber in which O2 levels were alternated between 21% for 25 min and 1% for 35 min,for a cycle of 1 hour.The sustained hypoxia model was consistently maintained by 1% O2 level.The Co Cl2 induced hypoxia model was established by add 100?mol/L Co Cl2 in the medium.Two other models were constructed which synchronize with 72 cycles of intermittent hypoxia model.The protein level of Bcl2,Bax,Cyt C in these models were tested by Western Blot.ROS levels and apoptotic endothelial cell levels in intermittent hypoxia model were detected with 2',7'-dichlorodihydro-fluorescein diacetate and TUNEL Kit.Results: We successfully designed and evaluated the chronic intermittent hypoxia cell incubator.The results of Western Blot shown that the protein levels of Bax and Cyt C in sustained hypoxia model were slight higher than the protein in Co Cl2 induced hypoxia model,but not statistical significance.Compared with NC group,the intermittent hypoxia group has a lower level of Bcl2 and higher level of Bax and Cyt C.The protein level of Bcl2,Bax,Cyt C in intermittent hypoxia group was between NC group and sustained hypoxia group.The ROS level and apoptosis cells were observed in the intermittent hypoxia model group.Conclusion: The model of chronic hypoxia endothelial cell injury was constructed by using self-prepared chronic intermittent hypoxia incubator.?.The evaluation of S1 P protecting endothelial cells injury induced by intermittent hypoxiaObjective: To evaluate the protective effect by using different concentrations of S1 P from endothelial cell injury induced by intermittent hypoxia.Methods: S1 P was prepared in 10nmol/L,100nmol/L,500nmol/L,1?mol/L concentrations.The chronic intermittent hypoxia model was constructed by using IH incubator to culture endothelial cells which managed with different S1 P concentrations.The IH condition was set to 21% O2 for 25 min and 1% for 35 min,for 72 cycles.The protein level of Bcl2,Bax,Cyt C in NC group,IH group,10nmol/L group,100nmol/L group,500nmol/L group,1?mol/L group were tested by Western Blot.Next,a specific concentration was selected according to the Western Blot results.The ROS level and apoptosis cells were observed in this specific S1 P concentration group.Results: Western Blot results shown that the level of Bcl2 in IH-endothelial cells was increased and the level of Bax,Cyt c was decreased after S1 P added.The content of Bcl2,Bax and Cyt c were influenced by different S1 P concentrations.The content of Bcl2 was increased and Bax,Cyt c was decreased compared 100nmol/L group with 10nmol/L group.While compared 500nmol/L group with 1?mol/L group,protein level of Bcl2,Bax and Cyt c were almost the same.100nmol/L group was selected according to the Western Blot results.And we found that the ROS level and apoptosis cells were reduced after managed with S1 P.Conclusion: S1 P could protect the endothelial cell injury induced by chronic intermittent hypoxia,and 100 nmol/L is available for better protection.?.Protective effect of sphingosine-1-phosphate for chronic intermittent hypoxia-induced endothelial cell injury and the underlying mechanismsObjective: To explore the protective effect of S1 P for chronic intermittent hypoxia-induced endothelial cell injury and the underlying mechanisms.Methods: HUVEC was selected to be the research object and divided into four groups: Negative Control group(NC),Chronic Intermittent Hypoxia group(CIH),Chronic Intermittent Hypoxia+PBS group(CIH+PBS),Chronic Intermittent Hypoxia+S1P group(CIH+S1P).The IH condition was set to 21% O2 for 25 min and 1% for 35 min,for 72 cycles.The concentration of S1 P was selected at 100nmol/L.The protein level of p-Akt/Akt,p-e NOS/e NOS,S1PR1,G?(i),Bcl2,Bax,Cleaved-caspase 3 and Cyt c were tested by Western Blot.The ROS level and apoptosis cells were observed.S1PR1 and G?(i)were detected by immunofluorescent techniques.In order to explore the underlying mechanisms of S1 P on endothelial cell dysfunction,the PI3 K inhibitor and S1 P Receptor 1-3 antagonists were used.The protein level of p-Akt/Akt,p-e NOS/e NOS,S1PR1,G?(i),Bcl2,Bax,Cleaved-caspase 3 and Cyt c were tested by Western Blot.The ROS level and apoptosis cells were observed.Results: Western Blot results shown that the level of p-Akt,p-e NOS,S1PR1 and G?(i)in CIH+S1P group was increased.The S1 P influenced group significantly higher the Bcl2 protein and lower the Bax,Cleaved-caspase 3 protein and the Cyt c protein level than the CIH group and the CIH+PBS group.The addition of S1 P is beneficial for the elimination the IH-induced increase in ROS levels and reduced the number of TUNEL-positive cells.After PI3 K inhibitor was added in CIH+S1P group,the level of p-Akt,p-e NOS,Bcl2 was decreased,the level of Bax,Cyt c was increased.The ROS level and apoptosis cells were deteriorated.After S1 P Receptor 1 antagonist was added in CIH+S1P group,the level of p-Akt,p-e NOS,Bcl2 was decreased and Bax,Cyt c has the opposite trend.The ROS level and apoptosis cells were increasd.In the meantime,there is no difference between the S1 P Receptor 2-3 groups and the CIH+S1P group on the results of Western Blot,ROS and apoptosis cells.Conclusion: The protective effect of S1 P on IH-induced endothelial dysfunction as the result of activation of the S1PR1/Akt/e NOS signaling pathway.?.Establishment and evaluation of the S1 P microbubblesObjective: To establish and evaluate the S1 P microbubbles.Methods: The S1P-MBs were prepared through the rotary evaporation and sonication process.Size,morphology,mean particle size,zeta potential were all characterized by different precise instruments.The particle size and zeta potential were detected and compared a week later.Compared the protein level of Bcl2,Bax,Cyt c in CIH+S1P-MBs group with NC+S1P-MBs to observe if S1P-MBs influences the cell.Different ultrasonic intensities(0.1W/cm2?0.3W/cm2?0.5W/cm2?1.0W/cm2)were used to interfere S1P-MBs and MBs groups.The protein level of Bcl2,Bax and Cyt c were tested by Western Blot.Then,a specific ultrasonic intensity was selected according to the Western Blot results.Results: The S1 P has no effect on the average of MBs.The average number diameter are 1.46±5.02 ?m and zeta potential for S1P-MBs is-30.6±2.23 m V.The similar results were founded a week later.Western Blot results shown that the level of Bcl2,Bax and Cyt C with S1P-MBs was the same in NC group and IH group without ultrasonic intervention.Under the condition of 0.1W/cm2,0.3W/cm2 ultrasonic intensity,the MBs and S1P-MBs were not completely broken.The MBs and S1P-MBs were almost completely broken.in the case of 0.5W/cm2 ultrasonic irradiation.In the 1.0W/cm2 ultrasonic irradiation,the level of Bcl2 were decreased,and the level of Bax,Cyt C were increased significantly.Conclusion: The S1P-MBs we established were stable and uniform.The best ultrasonic intensity to S1P-MBs was 1 MHz,0.5 W/cm2,40 s.?.Protective effect of S1 P microbubbles for chronic intermittent hypoxia-induced endothelial cell injury and the underlying mechanismsObjective: To explore the protective effect of S1P-MBs for chronic intermittent hypoxia-induced endothelial cell injury and the underlying mechanisms.Methods: HUVEC was selected to be the research object and divided into six groups: Negative Control group(NC),normal cells incubated with normal MBs(MBs),normal cells incubated with S1P-MBs(S1P-MBs),CIH-induced cells(CIH),CIH-induced cells incubated with normal MBs(CIH+MBs),CIH-induced cells incubated with S1P-MBs(CIH+S1P-MBs).The IH condition was set the same as above.The same content was added between S1P-MBs and normal MBs.All groups were followed by the same ultrasound exposure.The protein level of p-Akt/Akt,p-e NOS/e NOS,S1PR1,G?(i),Bcl2,Bax,Cleaved-caspase 3 and Cyt c were tested by Western Blot.The ROS level and apoptosis cells were observed.In order to explore the underlying mechanisms of S1P-MBs on endothelial cell dysfunction,the endothelial cells were divided into eight groups: Negative Control group(NC),Chronic intermittent hypoxia group(CIH),Chronic intermittent hypoxia+ PI3 K inhibitor group(CIH+LY294002),Chronic intermittent hypoxia+PI3K inhibitor+S1P group(CIH+LY294002+S1P),Chronic intermittent hypoxia+PI3K inhibitor+S1P-MBs group(CIH+LY294002+S1P-MBs),Chronic intermittent hypoxia+S1PR1 antagonist group(CIH+W146),Chronic intermittent hypoxia+S1PR1 antagonist+S1P group(CIH+W146+S1P),Chronic intermittent hypoxia+S1PR1 antagonist+S1P-MBs group(IH+W146+S1P-MBs).The protein levels of p-e NOS/e NOS,p-Akt/Akt,S1PR1,G?(i),Bcl2,Bax,Cyt C were assessed according to the Western Blot.Results: Western Blot results shown that the level of p-Akt,p-e NOS,S1PR1 and G?(i)in S1P-MBs group was increased.The S1 P influenced group significantly higher the Bcl2 protein and lower the Bax,Cleaved-caspase 3 protein and the Cyt c protein level than the CIH group and the CIH+MBs group.The ROS level was decreased and the number of TUNEL-positive cells reduced.After PI3 K inhibitor was added in CIH+S1P group and CIH+S1P-MBs group,the increasing level of S1PR1 and G?(i)was presented,the level of p-Akt,p-e NOS was decreased,the level of Bcl2,Bax and Cyt c has little difference compared with CIH group.After S1 P Receptor 1 antagonist was added in CIH+S1P group and CIH+S1P-MBs group,the level of p-Akt,p-e NOS,Bcl2,Bax,Cyt c has little variation compared with CIH.The protein level of G?(i)in CIH+W146+S1P-MBs group was higher than in CIH+W146+S1P group.The protein level of G?(i)could be enhanced by S1P-MBs,but this result did not offer S1P-MBs have the extra protection,compared with S1 P,under W146 interfered.Conclusion: The protective effect of S1P-MBs,which has the same function like S1 P,on IH-induced endothelial dysfunction as the result of activation of the S1PR1/Akt/e NOS signaling pathway. |
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