The Study On The Mechanism Of Promoting Angiogenesis Of Shexiang Tongxin Dropping Pills Based On TGF-β1/ALK1/Smad1/5 Signaling Pathway

Objectives:In this study,we cultured human umbilical vein endothelial cells under the condition of ischemia and hypoxia in vitro and created a rat model of AMI to observe the effect of Shexiang Tongxin dripping pills(STDP)on promoting angiogenesis after myocardial infarction in rats,and to explore the related mechanism.Methods:Experiment 1: HUVECs were cultured for 24 hours with a serum-free medium in an anoxic incubator(1%O2;5%CO2;94%N2)to establish a model of ischemic and hypoxic endothelial cells.Different concentrations(0%,5%,10%,20%,40%)of drug-containing serum of Shexiang Tongxin dripping pills were added for further culture of HUVECs for 24 h.MTT assay was used to detect cell proliferation.Cell migration ability was detected by Transwell chamber assay.The lumen formation ability of cells was detected by Matrigel matrix gel method.The expressions of TGF-β1,VEGFA,p-Smad1/5 and Smad1/5 proteins were detected by Western Blot.Experiment 2: A total of 90 male SD rats were selected and 70 of them were randomly selected to make a rat model of acute myocardial infarction by ligation of the left coronary artery.After successful modeling,48 rats were randomly divided into model group,STDP low-dose group,STDP medium-dose group and STDP high-dose group.The remaining 20 rats were randomly divided into blank group and sham ope.ration group with 10 rats in each group.The operation procedure of the sham group was the same as that of the model group,only thundering but not ligating the coronary artery.On the third day after surgery,rats in each group were gavaged intragastrically.The blank group,sham operation group and model group were gavaged with normal saline of the same volume in each STDP dose group.The STDP low-dose,medium-dose and high-dose groups were given corresponding doses of water solution of Shexiang Tongxin dropping pills by gastric administration for 14 consecutive days once a day,respectively.The general condition of rats was observed.Two-dimensional ultrasound measured the cardiac function of rats.TTC staining was used to determine the myocardial infarction area in rats.HE staining was used to observe the pathological morphology of myocardium in rats.The apoptosis of myocardial cells in rats was detected by TUNEL fluorescence assay.CD31 immunofluorescence was used to detect the neovascularization at the edge of myocardial infarction.Serum IL-12 levels were detected by ELISA.The expressions of TGF-β1,VEGFA,p-Smad1/5 and Smad1/5 proteins were detected by Western Blot.The mRNA expressions of TGF-β1,Smad1,Smad5,ALK1 and Id1 were detected by RT-PCR.Results:Experiment 1:1.The serum containing Shexiang Tongxin dripping pills could promote the proliferation,migration and lumen formation of HUVECs,and the promotion ability was strongest when the concentration of serum containing Shexiang Tongxin dripping pills reached 20%.2.Western Blot results showed that the serum containing Shexiang Tongxin dripping pills could promote the expression of TGF-β1,VEGFA,and p-Smad1/5 protein in HUVECs,and the promoting ability was the strongest when the serum concentration reached 20%.Experiment 2:1.Cardiac function test results showed that compared with model group,cardiac LVIDs and LVIDs of STDP medium-dose and high-dose groups were significantly decreased(P<0.01),while LVFS and LVEF were significantly increased(P<0.01).2.TTC staining results showed that myocardium in model group was thinned and myocardial infarction was obvious.Compared with the model group,the area of myocardial infarction in STDP medium and high dose groups was significantly reduced(P<0.01).3.The results of HE staining showed that the myocardial fibers in STDP medium and high dose groups were partially dissolved and broken,the intercellular stroma was slightly wider and the transverse lines were clearer,and the myocardial injury was relieved compared with that in the model group.4.TUNEL test results showed that compared with model group,the apoptosis level of myocardial cells in STDP medium-dose and high-dose groups decreased.5.CD31 immunofluorescence test results showed that compared with model group,CD31 fluorescence expression and MVD were significantly increased in STDP medium-dose and high-dose groups(P<0.01).6.ELISA results showed that compared with model group,serum IL-12 level in STDP low-dose group was decreased(P<0.05);The level of IL-12 in STDP medium-dose and high-dose groups was significantly decreased(P<0.01).7.Western Blot results showed that compared with model group,the protein expressions of TGF-β1,VEGFA and p-Smad1/5 in medium-dose and high-dose group were increased(P<0.01).8.RT-PCR results showed that compared with model group,the mRNA expressions of TGF-β1,Smad1,Smad5,ALK1 and Id1 in medium-dose and high-dose group were increased(P<0.01).Conclusion:1.The serum containing Shexiang Tongxin dripping pills could promote the proliferation,migration and lumen formation of human umbilical vein endothelial cells,and increase the expression of TGF-β1,VEGFA and p-Smad1/5 protein;2.Shexiang Tongxin Drop Pill can reduce the infarct area of acute myocardial infarction model rats,improve the heart function,reduce the pathological injury of myocardium,reduce the level of serum IL-12;3.Shexiang Tongxin dripping pills can promote angiogenesis in acute myocardial infarction model rats,and the mechanism may be related to the up-regulation of protein and gene expression related to TGF-β1/ALK1/Smad1/5 signaling pathway,and the up-regulation of protein expression of VEGFA.