| Therapeutic angiogenesis,also known as the "self-bypass" of hearts,is the formation of new vascular network structure on the basis of the original capillaries.Under the condition of ischemia and hypoxia,endothelial cells(EC)proliferate,dissociate from the extracellular matrix(ECM),then migrate through the vessel wall,and gradually develop from the vascular buds into a new three-dimensional vascular network system.Timely and effective recovery of blood perfusion within the ischemic tissue is of great significance,for the prevention and treatment of coronary heart disease.In view of the importance of vascular endothelial cell function,to protect the proliferation,migration and integrity of hypoxic EC,is the focus of research on angiogenesis.As a molecular switch of gene regulation,miR-126 is one of the vascular endothelial-specific miRNAs and has been shown to be an important regulator of angiogenesis in ischemic myocardium.This study was to investigate the mechanism of miR-126 targeting vascular endothelial growth factor(VEGF)signaling pathways to promote the proliferation and migration of hypoxia human umbilical vein endothelial cells(HUVEC),which participates in the regulation of Blood Activation and Qi Supplement prescription(BAQS)to promote angiogenesis.This study aims to reveal the scientific connotation of Chinese medicine to promote angiogenesis,to provide theoretical and objective basis for the BAQS clinical application for the treatment of coronary heart disease.Objective:To investigate the role of miR-126 in the proliferation and migration of HUVEC,and to explore the upstream mechanism of angiogenesis regulation at the miRNA level.To clarify the role of miR-126 in the VEGF signaling pathway to promote the proliferation and migration of HUVEC,and to reveal the miR-126 mediated downstream molecular mechanism.To investigate the effect of BAQS on the proliferation and migration of hypoxic HUVEC,and to further reveal the molecular mechanism of activating BAQS promoting angiogenesis via miR-126 targeting VEGF signaling pathway.To reveal the mechanism and compatibility of BAQS from the perspective of miRNA gene regulation.Methods:(1)HUVEC were cultured in 1%the three-gas incubator(O2,5%CO2,94%N2 and 37℃)to establish the model of hypoxia HUVEC,which were divided into hypoxia group,BAQS group,Blood Activation prescription(BA)group and Qi Supplement prescription(QS)group.Another normal control group was placed in the incubator(5%CO2,37℃)for 24h,and all groups were cultured in culture medium(0.5%Fetal bovine serum)for another 24h.BAQS group,BA group and QS group were treated with the corresponding drug serum culture medium,the hypoxia group and the normal control group were given normal control serum culture medium,for 24h.Proliferation was detected by CCK-8 method.Migration was detected by transwell insert.Apoptosis was detected by Annexin V-FITC/PI double staining combined with flow cytometry.The activity of superoxide dismutase(SOD)were determined by by WST-1 method,the content of malondialdehyde(MDA)and dehydrogenase(LDH)in supernatant of cell culture were determined by microplate method.Real-time fluorescent quantitative PCR was used to detect the mRNA levels of miR-126 and VEGF/KDR signaling pathways(2)miR-126 mimics(200nM)/miR-126 inhibitor(400nM)was transfected into HUVEC for 48 h to overexpress/inhibit miR-126 expression.The corresponding mimics/inhibitor negative control groups was established.Proliferation was detected by CCK-8 method.Migration was detected by transwell insert.Real-time quantitative PCR was used to detect the mRNA level of VEGF/KDR signaling pathways.(3)The miR-126 expression overexpressed/inhibited HUVEC were treated with BAQS serum culture medium for 24h.The cell proliferation was detected by CCK-8 method.The cell migration was detected by transwell insert.The mRNA of VEGF/KDR signal pathways key molecular was detected by real-time fluorescence quantitative PCR.Results:(1)Compared with the normal control group,the proliferation of hypoxia group was significantly decreased(P<0.01).Compared with the hypoxia group,HUVEC proliferation was significantly increased in BAQS group,BA group and QS group(P<0.01).The proliferation of HUVEC in BAQS group was higher than that in BA group and QS group,the difference was not statistically significant(P>0.05).(2)Compared with the normal control group,the levels of PKC,Ras and Raf-1 mRNA in the hypoxia group were significantly decreased(P<0.05 or P<0.01),and the difference of MEK and ERK mRNA was not significant(P>0.05).Compared with the hypoxia group,the levels of PKC,Ras and Raf-1 mRNA in BAQS group and QS group were significantly increased(P<0.05 or P<0.01),and the expression of Raf-1 mRNA in BA group was significantly increased(P<0.01).The level of MEK mRNA in BAQS group,BA group and QS group increased,and the level of ERK mRNA in BAQS group increased,the difference was not statistically significant(P>0.05).The levels of PKC and Ras mRNA in BAQS group were significantly higher than those in BA group and QS group(P<0.05 or P<0.01).The level of MEK and ERK mRNA in Huoxue Yiqi group was higher than that in BA group and QS group,the difference was not statistically significant(P>0.05).(3)Compared with the normal control group,the late phase rate and total rate of apoptosis were significantly increased in the hypoxia group(P<0.01),and the early phase rate of apoptosis in the hypoxia group was not significantly different(P>0.05).Compared with the hypoxia group,the early phase rate of apoptosis in BA,QS group was significantly decreased(P<0.05),that of BAQS group decreased,the difference was not statistically significant(P>0.05),the late phase rate and total rate of apoptosis in BAQS,BA group were significantly decreased(P<0.05 or P<0.01),the difference of QS group was not statistically significant(P>0.05).The late phase rate of apoptosis in BAQS was significantly lower than that of QS group(P>0.05).(4)Compared with the normal control group,SOD activity in hypoxia group decreased significantly,MDA and LDH contents were significantly increased(P<0.01 or P<0.05).Compared with the hypoxia group,the activity of SOD in BAQS group was significantly increased(P<0.01),MDA content in BAQS group and QS group was significantly decreased(P<0.01).The LDH content of BAQS group,BA group,QS group were significantly decreased(P<0.01 or P<0.05).The activity of SOD in BAQS group was significantly higher than that in BA group and QS group(P<0.01).The MDA content of BAQS group was significantly lower than that of BA group and QS group.The LDH content of BAQS group was significantly lower than that of BA group(P<0.05),and there was no significant difference between BAQS group and QS group(P>0.05).(5)Compared with the normal control group,the migration of HUVEC in hypoxia group was significantly decreased(P<0.01).Compared with the hypoxia group,HUVEC migration was significantly increased in BAQS group,BA group and QS group(P<0.01).The migration of BAQS group was significantly higher than that in BA group(P<0.01).There was no significant difference between BAQS group and QS group(P>0.05).(6)Compared with the normal control group,FAK,p38,MAPKAPK and HSP27 mRNA were significantly decreased in the hypoxia group(P<0.01).Compared with the hypoxia group,the levels of FAK and p38 mRNA in BAQS group,BS group and QS group were significantly increased(P<0.05 or P<0.01).MAPKAPK mRNA in BAQS group and QS group were significantly increased(P<0.05 or P<0.01),and the MAPKAPK mRNA in BA group was not significantly different(P>0.05).The level of HSP27 mRNA in BAQS group was significantly increased(P<0.05).There was no significant difference in HSP27 mRNA level between BA group and QS group(P>0.05).(7)Compared with the normal control group,VEGF mRNA was significantly up-regulated in hypoxia group(P<0.05).Compared with the hypoxia group,the level of VEGF mRNA in BAQS group was significantly increased(P<0.01).The level of VEGF mRNA in BAQS group was significantly higher than that in BA group and QS group(P<0.05).Compared with the normal control group,the KDR mRNA of the hypoxia group was significantly decreased(P<0.01).Compared with the hypoxia group,the KDR mRNA level of BAQS group,BA group and QS group increased,the difference was not statistically significant(P>0.05).(8)Compared with the normal control group,the levels of miR-126 in hypoxia group and drug group were significantly decreased(P<0.05 or P<0.01).Compared with the hypoxia group,the level of miR-126 in BAQS group and BA group was down-regulated(P<0.05),but the difference was not statistically significant(P>0.05).(9)Compared with mimics negative control group(mimics NC group),the proliferation and migration of miR-126 mimics group(miR-126 overexpression group)were significantly decreased(P<0.01).Compared with the negative control group(inhibitor NC group),the proliferation and migration of miR-126 inhibitor group(miR-126 expression-inhibited)were significantly increased(P<0.01 or P<0.05).(10)Compared with mimics NC group,the levels of miR-126 mRNA in miR-126 mimics group were significantly increased,whereas VEGF,KDR and PKC mRNA were decreased(P<0.05 or P<0.01).Compared with inhibitor NC group,the levels of miR-126 mRNA in miR-126 inhibitor group were significantly decreased,whereas VEGF,KDR,PKC and p38 mRNA were significantly increased(P<0.05 or P<0.01).(11)Compared with mimics NC+normal control serum group,the proliferation and migration of miR-126 mimics+normal control serum group were significantly decreased(P<0.01).Compared with miR-126 mimics+normal control serum group,the proliferation and migration of miR-126 mimics+BAQS serum group were significantly increased(P<0.01).(12)The levels of VEGF,KDR,PKC and p38 mRNA were significantly increased after transfection with miR-126 inhibitor(P<0.05 or P<0.01).Compared with the miR-126 inhibitor+normal control serum group,BAQS significantly increased the levels of VEGF,KDR,PKC,p38 MRNA level(P<0.05 or P<0.01).Conclusions:(1)BAQS and its disassembled prescriptions can promote the proliferation and migration and reduce the apoptosis of hypoxia HUVEC,through downregulating miR-126 mRNA expression,upregulating VEGF,KDR and its downstream signaling pathways including PKC-Ras-Raf-1,FAK,p38-MAPKAPK-HSP27,and reducing oxidative damage to improve the antioxidant capacity of endothelial cells.This may be one of the mechanisms of BAQS to promote therapeutic angiogenesis.The effect of BAQS was superior to BA and QS in promoting proliferation and migration;BA and QS have synergistic effects.(2)miR-126 can significantly inhibit the proliferation and migration of HUVEC,therefore miR-126 can negatively regulate angiogenesis.The mechanism may be related to the inhibitory effects of miR-126 on VEGF,KDR and its downstream signaling pathways.(3)BAQS can improve the proliferation and migration of HUVEC inhibited by miR-126,via reducing the inhibitory effect of miR-126 on VEGF,KDR,PKC and p38.It is believed that cellular mechanisms of BAQS promoting angiogenesis may be related to its regulation on miR-126 targeting VEGF signaling pathways to promote endothelial cell proliferation and migration subsequently. |
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