Protective Effects Of Astragaloside Ⅳ On Diastolic Function Of Rat Thoracic Aortic Rings Impaired By Microvesicles Derived From Hypoxia/reoxygenation-treated HUVECs

Cardiovascular diseases endanger human health seriously,the occurrence of myocardial infarction,which is the most critical situation in cardiovascular diseases.The infarct area is usually given immediate reperfusion to restore the blood supply for treatment.However,tissue damage is even worsened after ischemia-reperfusion of the myocardial tissue,we define this irreversible pathological condition as ischemia/reperfusion（I/R）injury.Vascular endothelial dysfunction is an important part of myocardial ischemia/reperfusion injury,vascular endothelial cells are closely linked to form a barrier between blood and vascular smooth muscle while secreting many active substances such as Nitric Oxide（NO）.When ischemia/reperfusion happened,vascular endothelial function disrupted and lost the ability to regulate NO and other endothelial factors.These processes may further aggravate ischemia/reperfusion injury.Microvesicles（MVs）are small vesicles of 100-1000 nm diameters released from stimulated or apoptotic cells membranes under various pathological conditions.MVs released by injured endothelial cells under the condition of I/R may carry ROS.The generation of ROS is also considered to take an important part in the complex and delicate mechanism of I/R injury.MVs,as a marker and functional vesicle may in return play a critical role in the pathogenesis of vascularendothelialdysfunctionandcardiomyocyteinjury.Establish hypoxia/reoxygenation（H/R）model in vitro to mimic I/R injury in vivo,which can induce HUVECs releasing H/R-EMVs.H/R-EMVs increased the apoptosis rate of H9c2 cells significantly and impaired the diastolic function of thoracic aorta rings in vitro by enhancing oxidative stress.Astragalus membranaceus,which is a Chinese traditional medicine,has long been used for the management of various cardiovascular diseases.Astragalus total saponins is the active site,Astragaloside IV（AST）is a major effective constituent isolated from Astragalus total saponins.AST could relax cardiovascular smooth muscle and protect the ischemic-hypoxic myocardium.Our previous studies have shown that AST could antagonize oxidative stress caused by H₂O₂ in H9c2 by preventing ROS production.Moreover,clinical application and published studies demonstrate its beneficial effects of AST on regulation of endothelial dysfunction.However,it remains unknown if AST could prevent vascular endothelial dysfunction which impaired by H/R-EMVs.In the previous study,the H/R-EMVs were used to injury the endothelial diastolic function of rat thoracic aortic rings.The present research investigated whether and how AST ameliorates relaxation of rat thoracic aortic rings impaired by microvesicles derived from hypoxia/reoxygenation-treated HUVECs.Objective:To investigate the effects of AST on endothelial diastolic function of thoracic aortic rings of rats injured by endothelial microvesicles derived from H/R-treated HUVECs and to explore the underlying mechanisms.Methods:1.Preparation of H/R injury model in HUVECsHUVECs were cultured with high glucose DMEM comprising 10%FBS for 24h.When the cell fusion reached about 80%,the original medium was replaced by oxygen-deficient solution.Put HUVECS into hypoxic chamber and then,a mixture of 95%N₂-5%CO₂ was bubbled into the hypoxic chamber at a flow rate of 20 L/min.HUVECs were exposured to the oxygen-deficient condition in the hypoxic chamber for 12 h and then to normoxia for 4 h at 37?C.The viability of HUVECS was tested by MTT.H/R injury model that mimic I/R injury was established.2.Two-step centrifugal extraction,protein quantification and transmission electron microscopy observation of H/R-EMVsHypoxic buffer was collected and centrifuged at 2700 g,4?C for 20 min to remove cell debris.Remaining supernatant was collected in ultracentrifuge tubes and subjected to a centrifugation at 33000 rpm for 148 min to pellet H/R-EMVs.The pellet was resuspended in D-Hank’s and kept at-20?C for subsequent experiments.20μL of H/R-EMVs was dropped onto the copper grid and allowed to stand for 2min at room temperature.After the H/R-EMVs suspension was fully immersed in the copper mesh,the filter paper removed excess liquid.Incubate 40μL of 2%phosphotungstic acid staining solution and let stand for 2 min at room temperature.The incandescent lamp was irradiated with the copper mesh and dried,and the ultrastructure of the H/R-EMVs was observed under a transmission electron microscope.3.Preparation of rat thoracic aortic rings with intact endothelium and experimental groupingWistar male rats were selected and the body weight was in the range of240²60 g.The neck was dislocated and the chest was quickly opened to obtain the thoracic aorta.The thoracic aorta was placed in a petri dish containing 4?C K-H solution.The connective tissue of the thoracic aorta was carefully removed using a microscopic ophthalmic scissors and cut into a 3⁴ mm thoracic aortic ring.Vascular loops were incubated in DMEM containing 10%FBS.H/R-EMVs group:thoracic aortic rings of rats were incubated in culture medium and treated with H/R-EMVs in a final concentration of 10μg/ml;different doses of AST groups:thoracic aortic rings of rats were treated with 10,20,40,60 mg/L AST co-incubated with 10μg/ml H/R-EMVs respectively;control group were treated with the same volume of D-Hank’s solution.Each group was incubated at 37?C and 5%CO₂ for 4 h.4.Diastolic function of rat thoracic aortic rings caused by AChThe aortic rings were suspended in a pre-set 10 mL K-H bath,37?C,95%O₂-5%CO₂,connected to an explant tissue perfusion device and a tension sensor.Aortic rings were allowed to equilibrate for 60 min under a basal resting tension of2.0 g.After the equilibration,aortic rings were treated with phenylephrine(PE,10^-6mol/L)to test their contractility,and then washed for several times with Krebs solution until the tension was restored to a stable level around baseline.When they arrived at the stable maximal contractile response which were usually taken for 20min,the relaxations of aortic rings response to ACh in concentrations of 10^-910^-6mol/L were examined to detect the endothelium-dependent or independent relaxation.5.Measurement of NO in thoracic aortic rings of ratsThe NO production of thoracic aortic rings of rats was tested by using Griess reagent.6.Western blot for detecting the expression of p-eNOS/t-eNOS,p-Akt/t-Akt and p-ERK1/2/ERK1/2The expression of total endothelial NO synthase（t-eNOS）and phosphorylated eNOS（p-eNOS,Ser-1177）,total serine/threonine kinase（t-Akt）and phosphorylated Akt（p-Akt,Ser-473）,extracellular regulated protein kinases（ERK1/2）and phosphorylated extracellular regulated protein kinases（p-ERK1/2,Thr202/Tyr204）of thoracic aortic rings of rats was measured by Western blot.Results:1.H/R was used to bulid the injury model of HUVECsAfter exposuring to the oxygen-deficient condition in the hypoxic chamber for12 h and then to normoxia for 4 h at 37?C.The viability of HUVECs in H/R group was significantly decreased comparing with control group（74.16%±5.11%vs100%±0%,P<0.05）.With stable experimental results and modest decrease of cell viability,H12/R4 was adaptive for establishment of H/R model on HUVECs.2.Isolation,protein quantification and microstructure analysis of H/R-EMVsH/R-EMVs can be observed at the bottom of the ultracentrifuge tubes as white little sedimentation after ultracentrifuge.To quantify the protein content of H/R-MVs,a BCA protein assay kit was used.The protein content of H/R-MVs was 0.31±0.02μg/μL.The isolated H/R-EMVs were subjected to transmission electron microscopy revealing small,rounded vesicles of 100¹000 nm surrounded by a bilayered membrane,without cell debris and other organelle structures.3.Effects of AST on ACh-induced vasorelaxation of rat thoracic aortic rings impaired by H/R-EMVsCompared with control,the relaxation of aortic rings induced by 10^-910^-6mol/L cumulative increments of ACh was impaired by H/R-EMVs.H/R-EMVs decreased the relaxation of rat thoracic aortic rings significantly（66.07%±1.78%vs99.67%±5.57%,P<0.01）.Compared with H/R-EMVs group,the relaxation of aortic rings induced by cumulative increments of ACh was increased by AST at the concentrations of 20,40 and 60 mg/L respectively（Emax:73.92%±0.61%,85.95%±2.21%,95.51%±4.46%vs 66.07%±1.78%,P<0.01）in a concentration-dependent manner.There was no significant difference between 10mg/L AST and H/R-EMVs group（64.93%±0.91%）.4.Effects of AST on NO production in thoracic aortic rings of rats impaired by H/R-EMVsCompared with control,H/R-EMVs reduced NO production in aortic rings significantly（56.17±4.31 vs 84.02±5.12μmol/L,P<0.01）.Compared with H/R-EMVs group,AST at the concentrations of 20,40,60 mg/L increased the NO production in aortic rings significantly in a concentration-dependent manner（62.11±3.91,73.37±2.99,81.91±4.15 vs 56.17±4.31μmol/L,P<0.05,P<0.01）.There was no significant difference between 10 mg/L AST and H/R-EMVs group（54.78±3.11μmol/L）.5.Effects of AST on expressions of p-eNOS/t-eNOS,p-Akt/t-Akt,p-ERK1/2/ERK1/2 in thoracic aortic rings of rats impaired by H/R-EMVsCompared with control,H/R-EMVs decreased the expressions of p–eNOS,p-Akt,p-ERK1/2 and did not affect the expression of t-eNOS,t-Akt and ERK1/2 of aortic rings.Compared with H/R-EMVs group,there was no significant difference between 40 mg/L AST group and H/R-EMVs group on the expressions of t-eNOS,t-Akt and ERK1/2,but the expressions of p-eNOS,p-Akt and p-ERK1/2 were increased in the 40 mg/L AST group significantly（P<0.01）.The ratio of p-eNOS/t-ENOS,p-Akt/t-Akt and p-ERK1/2/ERK1/2 were increased.Conclusion:1.Stable H12/R4 injury model of HUVECs was established.2.H/R-EMVs were generated from HUVECs via H12/R4 treatment and displayed a spherical shape under transmission electron microscopy.And the protein content of H/R-EMVs was measured at 0.31±0.02μg/μL.3.ACh induced endothelial-dependent diastolization and NO production of rat thoracic aortic rings was damaged by 10μg/mL H/R-EMVs.4.ACh caused endothelium-dependent diastolization and NO production of rat thoracic aortic rings that damaged by H/R-EMVs was significantly increased by AST at the concentration of 20,40,60 mg/L in a concentration-dependent manner.5.The expressions of p-eNOS,p-Akt and p-ERK1/2 in thoracic aortic rings of rats were increased by AST at the concentration of 40 mg/L.The expressions of t-eNOS,t-Akt and ERK1/2 were not altered by AST.The protective effects of AST on thoracic aortic rings of rats impaired by H/R-EMVs may owing to eNOS/Akt and ERK1/2 signal pathways.