Ischemia Regulates The Survival Of Vascular Endothelial Cells Through Autophagy Via Nonconical Signaling Pathway

Vein endothelial cells（VECs）will be damaged in varying degrees when suffering from ischemia.In severe cases,some or all of them will be necrotic,which will ultimately affect the quality of life of patients with ischemic diseases（cerebral infarction,myocardial infarction,flap ischemia,lower limb ischemia,etc.）.Autophagy plays a"double-edged sword"role in tissue cell survival:mild autophagy can decompose dysfunction organelles,provide energy for cells,and promote cell survival;excessive autophagy can cause tissue cell damage,which may be a new mechanism of Human Umbilical Vein Endothelial Cells（HUVECs）damage.Previous studies have found that ischemia-reperfusion can induce moderate autophagy and excessive autophagy in HUVECs,and moderate autophagy has a certain protective effect on short-term ischemic HUVECs.In addition,autophagy products can provide substrates for cell energy metabolism;therefore,ischemia can also lead to changes in cell energy metabolism,that is,inhibition of tricarboxylic acid cycle and enhancement of anaerobic glycolysis.Therefore,the project team put forward the scientific hypothesis that"inhibition of excessive autophagy,maintenance of autophagy balance and regulation of cell energy metabolism are conducive to reducing ischemic injury of HUVECs".Finally,it lays a preliminary theoretical foundation and provides a new therapeutic target for the prevention and treatment of ischemic necrosis of HUVECs;provides a new technical platform for the screening of new drugs for various ischemic diseases;and provides a new idea and solution for reducing the necrosis rate of ischemic diseases and improving the quality of life of patients.Section OneEstablish the ischemia model of HUVECsObjective:establish the ischemia model of HUVECs and select an appropriate ischemia time.Methods:the ischemic medium was 1 mm Na H₂PO₄,24 mm Na HCO₃,2.5 m M Ca Cl₂,118 m M Na Cl,16 m M KCl,0.5 m M sodium EDTA,20 m M sodium lactate,p H 6.8,37℃in hypoxia incubator（O₂:CO₂:N₂ 1%:5%:94%）.Results:when the ischemia time was set at 0,3,6,9 and 12 hours respectively,the cells would be broken at≥6 hours,and the extracted proteins could not be collected for Western blot detection.When the ischemia time was set at 0,1,2,3,4,5 and 6hours,the cells would be broken after more than 4 hours of ischemia treatment（hypoxia+nutritional deficiency）,and the extracted protein could not be collected for Western blot detection.There is no significant difference in apoptotic rate between ischemia group and ischemia/reperfusion group.Conclusion:3 h was the extreme ischemia time HUVECs can bear.Section TwoThe proteomics and bioinformatics analysis of the effect of ischemia on autophagy balance of HUVECsObjective:study the autophagy and energy metabolism of HUVECs under ischemia.Methods:3 h ischemia group,3 h ischemia/24 h reperfusion group and control group（n=3）were set up.Results:the protein expression was highly reproducible.The homologous protein clusters（KOGs）of ischemic HUVECs mainly focus on energy metabolism,such as C,e,F,G,h,I,and cytoskeleton Z,rather than nuclear y.The up-regulated KEGG indicated that most of the significantly different up-regulated proteins were concentrated in energy metabolism and lysosome.Autophagy signal pathway suggested that ATG5/12/16 was activated,but beclin-1/LC3-Ⅱ was not.Conclusion:ischemia does not activate the classical beclin-1/LC3-Ⅱ dependent pathway,but may activate the non classical autophagy pathway.Section ThreeVerify the effect of ischemia on autophagy balance of HUVECs and free flapsObjective:verify the effect of ischemia on autophagy balance of HUVECs and free flaps by western blot and cell viability assay.Methods:three hours ischemia group and control group（n=3）were set up.Western blot and cell activity of HUVECs and the survival area of free flaps were detected.Results:the results showed that pyruvate kinase 1 was down regulated and citrate synthase was up-regulated,which confirmed the results of proteomics.Palmitoyl COA+avidin inhibited the tricarboxylic acid cycle but increased cell activity,while pyruvate+acetate promoted the tricarboxylic acid cycle but decreased cell activity.Rapamycin could not up regulate or down regulate the cell activity of ischemic HUVECs,but the cell activity and flap survival area of high-dose chloroquine 25μM group were significantly down regulated.Conclusion:ischemic HUVECs provide energy not through glycolysis,but through the citrate synthase pathway to maintain cell survival;however,the tricarboxylic acid cycle may be a"double-edged sword".The break of autophagic homeostasis can decrease cell viability and flap survival area.