Study On The Mechanism Of Angiogenesis In Rats With Cerebral Ischemia By MiRNA-210 Regulating HIF-VEGF-Notch Signaling Pathway

Stroke has become one of the leading causes of disability and death in patients,with ischemic stroke accounting for about 80%.After cerebral vascular occlusion,in addition to vascular proliferation in the ischemic area,improving hemispheric blood flow,restoring neuronal physiological functions,and protecting the integrity of existing microvascular structures and functions,there is an urgent need for blood flow irrigation by new blood vessels with therapeutic effect in ischemic area,so as to save damaged brain tissue and slow down the development of disease.Neovascularization is one of the important factors determining ischemic penumbra and infarct volume in ischemic stroke.Studies have shown that the process of angiogenesis is involved in a variety of genes and angiogenic factors.Micro RNAs(miRNAs)have specific gene regulation capabilities,and miRNA molecules expressed in vascular endothelial cells Promote vascular ogenesis and repair by regulating endothelial cell physiological functions.miRNA-210 is a hypoxia-specific miRNA that promotes angiogenesis by negatively regulating the target gene ephrin A3.Hypoxia-inducible factor-1α(HIF-1α),as a core regulator of angiogenesis,plays an important role in angiogenesis,mainly by up-regulating vascular endothelial growth factor(VEGF)receptors to increase vascular permeability to angiogenesis.The VEGF-Notch sigpnaling pathway is an important Pathway for angiogenesis after ischemia.The VEGF system and the downstream Notch pathway bind to their receptors,resectively,and induce cell division and proliferation to form new blood vessels.Traditional Chinese medicine attributed ischemic stroke to "stroke".It was first recorded in the "Yellow Emperor’s Internal Classics".The cerebral Palsy and the loss of function are important factors of ischemic stroke.Based on the traditional Chinese medical theory knowledge,combined with historical literature and clinical research,we believe that the basic pathogenesis of ischemic stroke is qi-blood disharmony and cerebral collateral dystrophy.The therapeutic pinciple of "Hexueshengluo Method" and "Hexueshengluofang"(HXSLF)were put forward for the treatment of ischemic stroke,which achieved good therapeutic effect in clinical treatment of ischemic stroke.Therefore,further research on the mechanism of promoting angiogenesis by hexueshengluo method is the focus of this topic,and also lays a foundation for clinical application.This paper mainly includes the following three parts:Part one Effects of HXSLF on Infarct Size,Expression of Mi RNA-210 and Ephrin A3 in Rats with Focal Cerebral IschemiaObjective: To establish a rat model of cerebral ischemia by middle cerebral artery occlusion,and to observe the effect of Xueshengluo Recipe on the area of cerebral infarction and the expression of microRNA-210 and its target gene ephrin A3 in the cerebral tissue of ischemic cortex in rats.Method: SD rats,male,weighing 250-280 g,were randomly divided into Sham group,Model group,HXSLF group,TXL group,and AST group.The model group and each drug-administered group were established with a model of focal cerebral ischemia using the Middle cerebral artery occlusion(MCAO)method.Sham group does not do ischemic treatment.Neurological function scores were performed according to the Zea-Longa 5 scoring method,and 1-3points were included in the experimental study.Each administration group started to be administered 1 day after surgery,2 times a day for 7 days.Sham group and Model group were given an equal amount of normal saline.The expression of cerebral infarct size was detected by TTC staining.The expression of ephrin A3 protein in ischemic cortex was detected by immunofluorescence staining.RT-q PCR was used to detect the gene expression levels of microRNA-210 and ephrin A3 in ischemic cortex after cerebral ischemia.Result: Compared with Sham group,the model group showed a large area of cerebral infarction(P<0.01);the expression of ephrin A3 positive cells decreased(P<0.01);the expression of miRNA-210 and ephrin A3 m RNA increased(P<0.01).Compared with Model group,the infarct size of HXSLF group,AST group and TXL group was significantly decreased(P<0.01);the expression of miRNA-210 and ephrin A3 m RNA were significantly increased(P<0.01),and the expression of HXSLF group and TXL group ephrin A3 positive cells decreased significantly(P<0.01).Compared with TXL group,the infarct size of HXSLF group and AST group decreased more significantly(P<0.01),and the expression of miRNA-210 increased more significantly(P<0.01).the expression of ephrin A3 in HXSLF group increased more significantly(P<0.01),and the expression of ephrin A3 positive cells decreased more significantly(P<0.01).Conclusion:1.HXSLF can significantly reduce the cerebral infarction area of MCAO rats and improve the blood supply in the ischemic area.2.HXSLF can promote the expression of mirna-210 in the cortex of MCAO rats in the ischemic area,inhibit the expression of the target gene ephrin A3,and play an anti-ischemic role.Part two Effects of HXSLF on Proliferation,Tube Formation,Mi RNA-210 and Ephrin A3 in HUVECsObjective: To observe the proliferative capacity,tube formation ability and expression of miRNA-210 and ephrin A3 in HUVECs under hypoxic conditions,and to explore the angiogenesis effect of Xueshengluo Recipe.Method: Human umbilical vein endothelial cells(HUVECs)were cultured in DMEM medium and 10% fetal bovine serum,and cultured at 37 ° C in a 5%CO 2 incubator.It is divided into NC group,OGD group,HXSLF group,HXSLF inhibitor group,NC group,OGD group,AST group,TXL group.The above anoxic conditions are 1% O2,94% N2,5% CO2.Cell viability assay was performed by MTT assay;angiogenic structure formation ability was detected;flow cytometry was used to detect the expression of ephrin A3 in cells;RT-q PCR was used to detect the expression level of miRNA-210 in cells.Result: Compared with NC group,the proliferation of OGD group cells was significantly decreased(P<0.01);the angiogenic structure was decreased,and the lumen-like structure could not be formed(P<0.01);the expression of ephrin A3 protein was decreased(P<0.01);and the expression of microRNA-210 increased(P<0.01).Compared with OGD group,HXSLF group and AST group could significantly stimulate cell proliferation and viability(P<0.01);angiogenic structural capacity was significantly enhanced(P<0.01);ephrin A3 protein expression was significantly decreased(P<0.01);and increase the expression of microRNA-210(P<0.01).Compared with HXSLF inhibitor group,the proliferation and angiogenic structure of HXSLF group were significantly increased(P<0.01);the expression level of ephrin A3 protein was significantly decreased(P<0.01);the expression of miRNA-210 was increased(P<0.01).Compared with TXL group,AST group had slightly lower cell proliferation and angiogenic structure(P<0.01);ephrin A3 protein expression decreased(P<0.01);miRNA-210 expression increased,there was significant difference(P<0.01).Conclusion:1.HXSLF can increase the activity of HUVECs cells after hypoxia and promote cell proliferation.2.HXSLF can increase the vascularity of HUVECs cells after hypoxia and promote angiogenesis.3.HXSLF can significantly up-regulate the expression level of HIFVEGF-Notch signaling pathway molecules.Part three Effect of HXSLF on HIF-VEGF-Notch Signaling Pathway in HUVECsObjective: By detecting the expression of HIF-1α,VEGF,VEGFR2,DLL4 and Notch1 in HIF-VEGF-Notch signaling pathway in HUVECs,the mechanism of action of Heshengluo Recipe on angiogenesis was explored.Method: The culture conditions and groupings are the same as above.The expression levels of HIF-1α,VEGF,VEGFR2,DLL4 and Notch1 protein were detected by Western blot.The expression levels of HIF-1α,VEGF,VEGFR2,DLL4 and Notch1 m RNA were detected by Real-time PCR.Result: Compared with NC group,the expression of HIF-1α,VEGF,VEGFR2,DLL4,Notch1 protein and m RNA in OGD group was increased(P<0.01).Compared with OGD group,the expression of HXSLF group,AST group HIF-1α,VEGF,VEGFR2,DLL4,Notch1 protein and m RNA was significantly increased(P<0.01).Compared with HXSLF inhibitor group,HXSLF group HIF-1α,VEGF,VEGFR2,DLL4,Notch1 protein and m RNA expression were increased(P<0.01).Compared with TXL group,the expression of AST group HIF-1α,VEGF,VEGFR2,DLL4,Notch1 protein and m RNA was significantly increased(P<0.01).Conclusion:1.HXSLF can significantly up-regulate the expression level of HIFVEGF-Notch signaling pathway molecules.2.HXSLF is involved in the regulation of angiogenesis after ischemic stroke by regulating the expression level of mirna-210 and the level of HIFVEGF-Notch signaling pathway.