Astragaloside IV(As-Ⅳ) Stimulate Angiogenesis To Treat Myocardial Infarction Through Hif-1α Signaling

Ischemic heart disease, which is caused by blood vessels’ severe stenosis or evenobstructed completely, is detrimental to our health. The rapid establishment of effectivecirculation is an effective treatment for ischemic heart disease, which could reversemyocardial salvage, maximize the ability of "pumping" to protect the ischemic heart andreduce the incidence of mortality. Recently, in view of traumatic caused by percutaneouscoronary intervention, the surgery of coronary artery bypass graft (GABG) and othertreatment, especially in the presence of diffuse lesions, therapeutic revascularization hasbecome the new areas of research direction. There are a large number of clinical and basicexperiments in improving the efficacy of therapeutic revascularization now, but itsstability, security and operability, cost-efficacy of the drug problem are placed in front ofthe researcher.The patients of myocardial infarction (MI)’s most common and earliest syndromes isthe Qi deficiency of heart. The scholar thinks, almost all of the patients with myocardialinfarction have different degrees of qi deficiency, the qi deficiency of heart is important for low cardiac function. Astragaloside IV(As-IV), one of the major active constituents oftraditional Chinese medicine-Astragalus, has been commonly used to treat Myocardialinfarction (MI). And in vitro experimental research proved that As-IV can enhance theproliferation of endothelial cells and the formation of the lumen in vitro by activatingHif-1α through PI3K-AKT signaling pathways. Our experiment based on the method ofTonifying Qi theory and learning from the modern medicine, is planed to research whetherAs-IV can raise the expression of Hif-1α to promote angiogenesis and vessel mature, andthus promote the recovery of myocardial infarction.Part1The effect of As-IV mycardial infarction in mice1.ObjectiveStudy the effect of As-IV on the mice with mycardial infarction.2.Method(1)We establish myocardial infarction model in mice by permanent coronary arteryligation. Daily intraperitoneally injected As-IV. After21days used ultrasonic testing todetect the changes of cardiac function in mice.(2) Masson stainning method to detect the area of myocardial fibrosis in mice aftermyocardial infarction.(3) CD31and α-SMA fluorescent staining were used to observe the density of newblood vessels in the the peri-infarct myocardial tissue.(4) Lectin fluorescent staining to observe the infusion of new blood vessels in the theperi-infarct myocardial tissue.(5) Western blot to detect the Hif-1α and VEGF protein expression changes inmyocardial tissue.3.Results(1) As-IV’s effect on heart Function after MI: By echocardiography we detectedthat As-IV treatment significantly improved ejection fraction percentage（61±2.7%）andfractional shortening percentage（44±.3.2%）（P<0.01); Fibrotic area was also significantlyreduced in As-IV treated mice （7.8±2.4%） compared with vehicle treated mice(17.5±2.3%）（P<0.01).(2)As-IV’s effect on new blood vessel growth and mature in the infarct borderzone: The numbers of capillaries (CD31+)（214±21.3/mm2) and of small arterioles in theperi-infarct area (CD31and α-SNA+)(7±2.1/mm2) were significantly increased by As-IVin comparison with vehicle treated (P<0.01). What’s more,we also detected themyocardial blood perfusion (revealed by using lectin+area) were improved in mice givenAs-IV(0.39±0.11×104/pixels）(P<0.01).(3)As-IV’s effect on Hif-1α and VEGF protein expression in the infarct borderzone：Hif-1a and VEGF levels were also significantly increased inAs-IV treated mice inthe infarct border (P<0.01).4.Conclusion(1) As-IV improved cardiac Function after MI.(2) As-IV improved cardiac function by enhancing blood vessel growth in the infarctborder zone.(3) As-IV increased Hif-1a and VEGF protein expression in the infarct border zone.Part2The effect of As-IV on Maintain cell activity and the angiogenicpotential of human umbilical endothelial vein cells (HUVECs) in hypoxiccondition1.ObjectiveStudy the effect of As-IV on maintaining cell vatility and the angiogenic potential ofHUVECs under hypoxic condition.2.Method(1) MTT test to detect different concentration As-IV’s effects on maintaining thehuman vein endothelial cell activity under hypoxia conditions.(2) Flow cytometry to detect As-IV’s effects on cell cycle and apoptosis underhypoxia conditions.(3) Plane lumen in vitro experiment to detect As-IV’s effects on endothelial cell’s lumen formation ability; Three-dimensional lumen formation based on Magnetic beadsexperiment to detect As-IV’s effects on endothelial cell lumen formation ability underhypoxia conditions.(4) Western blot to detect As-IV’s effects on endothelial cells’ Hif-1α and VEGFprotein expression under hypoxic condition.3.Results(1)Different concentration of As-IV’s effects on maintaining HUVECs’ vatilityunder hypoxic conditions: As-IV significantly stimulated cell proliferation in theconcentration range of0.1to1μM under hypoxic conditions, whereas0.5u M As-IVdemonstrated the optimal effect (P<0.01).(2)As-IV’s effects on cell cycle and apoptosis under hypoxic conditions：0.5u MAs-IV significantly promoted the proliferation in hypoxic condition (P<0.01). Also, As-IVsignificantly decreased apoptotic cell compared with control group(15.4%)(P<0.01).(3)As-IV’s effects on endothelial cell’s lumen formation ability：Compared withthe hypoxic control group As-IV significantly increased the number of thesprouts(7±1.7/beads) under the hypoxic condition(P<0.01). Moreover, HUVECs werecultured in a Matrigel Basement Membrane Matrix, and the number of endothelialnetwork（18±3.1/field)(P<0.01) by counting network branches and lengths was muchmore(P<0.01). However, As-IV has no obvious effect on the viability and angiogenicpotential of HUVECs under the common condition.(4)As-IV’s effects on endothelial cells’ Hif-1α and VEGF protein expressionunder hypoxic condition: Western blot analysis showed an increase in the level of Hif-1αand VEGF in As-IV treated but not HUVECs in control group(P<0.01).4.Conclusion(1) As-IV could enhance the proliferation and inhibit the apoptosis of HUVECs nhypoxic condition.(2) As-IV could increases the angiogenic potential of HUVECs in hypoxiccondition.(3) As-IV increased Hif-1a and VEGF protein expression under hypoxic condition. Part3The effect of As-IV on rat ischemic cardiomyocytes throughHif-1α signaling1.ObjectiveStudy the effect of As-IV on rats ischemic cardiomyocytes through Hif-1α signaling.2.Method(1) Flow cytometry to detect the effects of As-IV’s and Hif-1α siRNA on ratsischemic cardiomyocytes’ apoptosis.(2) Elisa to detect the effects of As-IV’s and Hif-1α siRNA on the release of LDH ofrats ischemic cardiomyocytes.(3) QRT-PCR and Western blot to detect As-IV’s effects on rats ischemiccardiomyocytes’ Hif-1α and VEGF mRNA and protein expression.3.Result(1)The effect of As-IV and Hif-1α siRNA post-treatment on the apoptotic indexof ischemia cardiomyocytes：Treatment withAs-IV significantly decreased the apoptoticindex(17.8±2.8%)(P<0.01). Compared with the stimulate ischemia (SI)+consiRNA+As-IV group(15.4%), SI+Hif-1α siRNA+As-IV treatment (35.6±3.4%) did havea significant impact on increasing cell apoptotic index（P<0.01).(2)The effects of As-IV’s and Hif-1α siRNA on the release of LDH of ratsischemic cardiomyocytes: Treatment with As-IV significantly decreased the releasementof LDH(P<0.01). Compared with the SI+con siRNA+As-IV group, SI+Hif-1α siRNA+As-IV treatment did have a significant impact on increasing the releasement of LDH（P<0.01).(3) The effects of As-IV and Hif-1α siRNA on Hif-1α and VEGF mRNAexpression of ischemic cardiomyocytes: Treatment with As-IV significantly increasedHif-1α and VEGF mRNA expression(P<0.01).4.Conclusion(1) Treatment with As-IV could decreased the apoptotic index of ischemiccardiomyocytes. However, the protective effect of As-IV was abolished by Hif-1α siRNA. (2) Treatment with As-IV could decreased the release of LDH of ischemiccardiomyocytes. However, the protective effect of As-IV was abolished by Hif-1α siRNA.(3) Treatment with As-IV could increased Hif-1α and VEGF mRNA expression.However, the effect of As-IV was abolished by Hif-1α siRNA.