Astragaloside ? Promoted Angiogenesis Of Human Aortic Endothelial Cells After Hypoxic Injury

BackgroundAcute myocardial infarction?AMI?is mainly caused by the coronary atherosclerosis or coronary embolism and the vessel was occlusion,then the coronary blood flow perfusion was decreased,and oxygen supply insufficient,it may caused myocardial tissue necrosis andand decreased angiogenesis response accompanied with abnormal endothelial cells?ECs?.The insufficient myocardial blood flow perfusion,and the myocardial cell death and pathological remodeling,eventually leading to heart failure.Angiogenesis is crucial to restore the blood flow of ischemic tissue after vascular occlusion.Therapeutic angiogenesis is an effective alternative therapy to promote coronary collateral circulation,which can effectively restore the perfusion of ischemic tissue,and inhibit myocardial tissues remodeling,and then improve the cardiac function.Angiogenesis occurs at any time in the life of organisms which refers to the new blood vessels emergence from the existing vascular network.It is important to maintain blood and nutrition perfusion,and the angiogenesis participate in the regeneration of ischemic heart tissue or damaged peripheral blood vessels.Angiogenesis is a complex pathophysiological process that many cytokines participate in the vessel germination,maturation andfunctional angiogenesis formation.The ECs are the key factors of the progress of angiogenesis.ECs dysfunction is considered as a common risk for the majorty of cardiovascular diseases.Acute or chronic hypoxia may cause the ECs dysfunction and eventually decrease the angiogenesis.Autophagy is involved in many vascular processes,scch as angiogenesis and vascular calcification and it may produce theECs-specific exosomes and promote angiogenesis by increasing these pro-angiogenic factors expression.Nowadays,more Chinese medicine plays an important role in the treatment of various cardiovascular diseases.Astragalus membranaceus is an important regulating drug in traditional Chinese medicine.Astragalus membranaceus and its compound preparations are widely used in the treatment of cancer,immunitysystem and viral myocarditis?VMC?.Astragaloside ??AS-??is the main active ingredient of Astragalus membranaceus.It has many pharmacological effects,such as anti-aging,anti-inflammatory,anti-fibrosis,protecting ECs,promoting wound healing and improving lipid metabolism.Besides AS-? can promote mitochondrial autophagy,protect mitochondrial function and inhibit oxidative stress.We have found that AS-? can promote angiogenesis in the infarct margin of AMI mice.The effects of enalapril combined with AS-? in treating AMI mice on improving cardiac function,promoting angiogenesis and alleviating inflammation arebetter than those of enalapril alone.In this study,human aortic endothelial cells?HAECs?were cultured in vitro to investigate the protective effect of AS-? on HAECs and angiogenesis after hypoxia injury,and then to explore its underlying mechanism.ObjectiveTo study the effect of AS-? on angiogenesis of HAECs after hypoxia injury and its underlying mechanism.Methods1.Cell culture:ECM with 5%FBS was used to culture cells at 37?,5%CO₂and 21%O₂.2.hypoxia model preparation:When cells grow to 80%-90%fusion,they are placed in 8%oxygen concentration?8%O₂?to prepare hypoxia injury model.3.AS-? solution preparation:AS-? purity is more than 99%.AS-? is dissolved in DMSO to prepare storage solution to 100 mg/mL,which is packed in 10?L/branch and stored at-20?to avoid repeated freezing and thawing.4.Groups:HAECs were placed in 21%O₂ and 8%O₂ culture respectively in the pre-experiment.Cell culture supernatants were collected at different time?12h,16h,20h,24h,36h,40h,48h?.Lactate dehydrogenase?LDH?content in cell culture supernatant was detected to determine the intervention time?16h?,and drug toxicity was detected by CCK-8 to determine the therapeutic concentration of AS-??50?g/mL?.HAECs were divided into three groups:control group?C?,hypoxia group?H?,astragaloside ? treatment group?AS-??.During the modeling process,Each group of cells were cultured in low serum?1%FBS?.Group C were cultured in normal incubator?21%O₂?.Group H and AS-? were placed in 8%O₂ for 16h to prepare hypoxic injury model.Then group AS-? were replaced with 50?g/mL AS-? solution in 8%O₂ for another 24h.At the same time,H and C group cells were replaced with the ECM that with same volume fraction of serum was placed in each incubator for another 24h,and the cells in each group were treated at the same time after the intervention.5.Detection of experimental indicators:We detected the LDH concentration in cell supernatant,CCK-8 were used to detected the cell viability,scratch test reflected the migration ability,flow cytometry measured cell cycle,tube formation test to reflect HAECs tube formation in vitro,protein chip screening angiogenesis-related protein expression changes,qRT-PCR and WB were used to verify the protein chip results.Results1.Cell viability:Compared with group C,cell viability decreased significantly after hypoxia injury?81.12±0.72%vs 100±3.07%?and increased after AS-? treatment?85.71±2.48%??P<0.01?.2.LDH concentration:Compared with group C,LDH release increased of hypoxia injury?25.33±1.70 vs 5.33±1.25?,and LDH release decreased?18.33±1.25?after AS-? treatment.The difference was significant?P<0.01?.It suggests that AS-? could alleviate hypoxia-induced HAECs injury.3.Scratch test:Observing the scratch area of 6-well plate under microscope?40×?,it was found that the scratch area of group C almost completely healed?88.77±3.78?after 24h of scratch,the cell adherence was stronger,and the intercellular connection was tight.Compared with group C,the H group migrated less?70.97±5.65?and the cell junction was relax,the gap was wider.Compared with group H,the AS-? group cells migrated more significantly?83.05±4.67?,the cell junction was tighter and the gap was smaller.Scratch experiment indicated that AS-? could promote the migration of HAECs after hypoxia injury.4.Flow cytometry:Compared with the cells of C group,hypoxia resulted in cell cycle arrest in G0/G1 phase?P<0.01?.AS-? could promote the HAECs cells transformationinto G2/M phase after hypoxia?P<0.01?,and promote cell proliferation.5.Tube formation:Observing the matrix gel layer of 96-well plate under microscope?100×?,we found that HAECs in group C formed a complete network pipeline structure with a larger area and the number of rings was about?62.10±7.56?.Compared with group C,the H group formed loose lumen arrangement,loose and fractured intercellular connections,and the number of rings was significantly lower than that of group C?30.91±3.78?.Compared with group H,the intact tubular structure of AS-? group increased significantly,and the number of vascular rings increased significantly?48.64±4.80,P<0.001?.The tube formation experiment showed that AS-? could increase the tube formation of HAECs in vitro after hypoxia injury.6.Protein microarray:Three batches of cells in each treatment group were collected,and protein molecules with ratio??1.2 times or?0.83?between different groups were screened for further verification.It was found that after hypoxia injury,the expression of angiogenin?Ang?,VEGF,IL-6,IL-1?and RANTES decreased,while the expression of MMP-1,MMP-9,IL-4,IL-2,and IL-1?increased.After AS-? treatment,the expressions of Ang,IL-1?,IL-10 and IL-6 were up-regulated,while the expressions of MMP-9 and MMP-1 were down-regulated.7.qRT-PCR:There was no significant changes of Ang and its related genes after hypoxia injury and the treatment of AS-??P>0.05?.The experiment was repeated three times.8.WB:We detected the expression of Ang and its related proteins and autophagy related proteins,we found that there was no significant change in Ang pathway proteins.While autophagysignaling pathways were found that the expression of LC3-II and Beclin1 were down-regulated after hypoxia injury and up-regulated after treatment with AS-?,the experiment was repeated three times.conclusion1.AS-? alleviate the HAECs damage caused by hypoxia and improve the cell survival rate.2.AS-? promote HAECs proliferation,migration and angiogenesis after hypoxia injury.3.AS-? promote HAECs angiogenesis in vitro by activating autophagy pathway.