Experimental Study Of Astragaloside Ⅳ In Promoting Angiogenesis And Myocardial Protection After Myocardial Infarction Via Mediating PTEN/PI3K/Akt Signaling Pathway

Aim:In this study,acute myocardial infarction rat model and human umbilical vein endothelial cells(HUVEC)were employed to observe the effects of astragaloside IV(AS-IV)on angiogenesis and cardioprotection after myocardial infarction.Meanwhile,the relationship between PTEN/PI3K/Akt signaling pathway and cardioprotection/angiogenesis induced by AS-IV was investigated in order to clarify the mechanism.Methods:1.In vivo study60 male Sprague Dawley rats survived from the surgery were randomly divided into 4 groups:sham operation group(n = 15),myocardial infarction model group(n = 15),AS-IV low dose group(AS-IV 20mg/kg,n = 15)and AS-IV high dose group(AS-IV50mg/kg,n = 15).The sham group and model group were given the same amount of normal saline.The intervention was carried out for 2 weeks.After 2 weeks of intervention,cardiac function indexes were observed by echocardiography.The gross structure of heart was observed with naked eye.The area of myocardial infarction was observed by TTC staining.The pathological changes of infarction and the degree of fibrosis were observed by H-E staining and Masson staining.TUNEL method was used to observe the apoptosis of the marginal zone of infarcts;electron microscopy was used to evaluate the ultrastructural changes of mitochondria,myofilament and endoplasmic reticulum in myocardial tissue;Expression of PTEN and other proteins were measured by WB procedure.2.In vitro study(1)The effect of different concentrations of AS-IV on the proliferation of HUVECs was detected by CCK-8 assay.The effect of different concentrations of AS-IV on the formation of HUVECs was observed by the cavity formation assay of Matrigel tube.Western Blot Method to detect angiogenesis related factors and PTEN/PI3K/Akt pathway in different concentrations of AS-IV intervention;based on this,the best concentration of AS-IV intervention HUVEC pro-angiogenesis was screened out;(2)Construction of overexpression PTEN lentiviral vector was assisted by Shanghai Genechem company,and the infected procedure and related efficiency testing were carried out;(3)After transfection of PTEN gene in HUVEC cells by lentivirus,HUVECs were intervened by the optimal drug concentration(80μmol/L),and were divided into Control group(serum-free medium),AS-Ⅳ group(AS-Ⅳ 80μmol/L+serum-free medium),AS-Ⅳ + Lv-GFP group(AS-Ⅳ80μmol/L + GFP-only empty lentiviral vector + serum-free medium)and AS-Ⅳ+Lv-PTEN Group(AS-Ⅳ 80μmol/L+PTEN lentiviral vector+serum-free medium).Then CCK-8 method was used to detect the proliferation of HUVECs under different conditions.At the same time,Matrigel tube cavity formation experiment was used to observe the different formation of HUVECs under different conditions.To clarify the intrinsic link between AS-Ⅳpro-angiogenesis and PTEN/PI3K/Akt signaling pathway,the Western Blot method was used to detect the expression of downstream factors of PTEN gene after AS-Ⅳ intervention and PTEN lentiviral vector infection.Results:1.A total of 60 rats with successful resuscitation after myocardial infarction were included in the study,with a total success rate of 60%,and survival rate in the sham group was 100%.Model rats’ state was poor,then rats gradually recovered after the intervention in varying degrees;2.Compared with the model group,the AS-Ⅳ treatment group could improve cardiac contractile and diastolic function,LVEF and LVFS were recovered(P<0.01),and the extent of cardiac chamber dilatation was improved with a dose-dependent manner;3.Compared with the model group,AS-Ⅳ treatment group could significantly reduce the myocardial infarct size(P<0.01).Compared with the low-dose group,infarct size in AS-Ⅳ high dose group was further reduced(P<0.01);4.The degree of myocardial cell lesions,fibrosis and collagen deposition in AS-Ⅳ treatment group were less than those in myocardial infarction model group,and there was a small amount of inflammatory cell infiltration;compared with low-dose group,the degree of lesion and fibrosis levels in AS-Ⅳ high-dose group had been further improved;5.Compared with the model group,the anti-apoptotic factor Bcl-2 was significantly up-regulated and the pro-apoptotic factor Bax was significantly down-regulated in the AS-Ⅳ treated group(P<0.01);Compared with the low dose group,the anti-apoptotic ability of AS-Ⅳhigh-dose group was further improved(P<0.01);TUNEL immunofluorescence also suggested that apoptosis of myocardium cell in AS-IV treatment group was lighter than that in model group;6.Compared with model group,mitochondria and myofilament damage of myocardial cells in AS-IV treatment group were significantly improved,mitochondria swelling and fusion number were significantly reduced,and myofilaments were arranged in an orderly manner.In high-dose treatment group,the improvement was more significant than the low-dose treatment group;7.Compared with the model group,the number of neovascularization in the AS-IV treatment groups were significantly higher(P<0.01);the number of neovascularization in the AS-IV high-dose AS-IV group were slightly higher than that in the low-dose AS-IV group(P<0.01);8.Compared with the model group,the expression of PTEN was significantly down-regulated and the downstream factors PI3K/Akt were significantly up-regulated in the AS-IV treated group(P<0.01);9.AS-IV could promote the proliferation of HUVEC cells,on this basis,AS-IV also had the ability to promote the formation of HUVEC lumen,and expression of PTEN/PI3K/Akt signaling pathway and VEGF were also enhanced;The concentration of 80μmol/L AS-IV is the optimal concentration of HUVEC to promote its proliferation and lumen formation;10.Using lentivirus as vector for overexpressing the gene of interest(PTEN)is a suitable experimental method;The optimum conditions were as follows:MOI =10,containing 5μg/ml Polybrene,while adding Enhanced Infection Solution and virus co-culture 72h;11.Overexpression of PTEN inhibited the activation of PTEN/PI3K/Akt pathway by AS-IV,which attenuated cell proliferation and lumen formation,suggesting that AS-IV could upregulate PI3K/Akt pathway by downregulating expression of PTEN and phosphorylation of Akt,mediating angiogenesis.Conclusion:AS-IV could inhibit the level of PTEN,and then activated PI3K/Akt pathway and upregulated the expression of VEGF,inducing angiogenesis and cardioprotection after myocardial infarction.