Study On The Promotion Of Angiogenesis After Acute Cerebral Infarction And The Regulation Mechanism Of MiR-210 By Kudiezi Injection

In the acute ischemic stroke,the formation of heat poisoning is a turning point in the sudden change and aggravation of the condition.The characteristics of fire poisoning,on the one hand,is violent and cruel,which can trigger the initiation of a series of damage factors and jujure the brain network.Previous studies have confirmed that this process is consistent with the ischemic cascade injury with the inflammatory reaction as the central link;On the other hand,the rapid increase in the number of fire poisons further damages the integrity of the body and deteriorates the quality of the body.Current studies have demonstrated that ischemic cascade damage can lead to structural and functional destruction of neurovascular units and inhibit angiogenesis.Promoting angiogenesis after ischemic stroke is important for restoring oxygen supply in ischemic tissue and improving neurological function after acute ischemic stroke.miR-210 is a currently recognized hypoxia-specific miRNA,which has a significant role in promoting ischemia-induced angiogenesis,and miR-210 can also regulate the expression of vascular endothelial growth factor(VEGF)to mediate angiogenesis.Kuddiezi injection has the effect of clearing away heat,detoxifying meridians,and protecting the brain from acute ischemic stroke.However,could it regulate miR-210-mediated VEGF/PI3K/Akt pathway and promote angiogenesis?It is not yet clear.In this study,the middle cerebral artery occlusion(MCAO)rat model was used to observe the effect of Kudiezi injection on angiogenesis after 3 days of reperfusion in acute ischemic stroke;and then,using miR-210 agonist(miR-210 agomir)as a positive control to observe the effect of Kudiezi injection on the regulation of miR-210 and its effect on miR-210-mediated angiogenic signal pathway,and to clarify the mechanism of Kudiezi injection in regulating angiogenesis after acute ischemic stroke,and to explain the key links and targets for the treatment of acute ischemic stroke with Kudiezi injection,to provide scientific basis for the clinical use of drugs from the molecular level,and further enrich the overall view of treatment of traditional Chinese medicine with cultivating pathogens.[Objective]To establish rat MCAO model and explore the effect of Kudiezi injection on angiogenesis after ischemia and reperfusion in rats for 3 days.Through miR-210 mediation of VEGF/PI3K/Akt pathway,find its role and target;From the perspective of angiogenesis,we tried to deepen the protective mechanism of Kudiezi injection on neurovascular units,and enrich evil spirits to support the overall view of Chinese medicine treatment.[Method]1.Study 1:Male Sprague-Dawley rats,8-week-old,were established by a line embolization method to determine the left middle cerebral artery occlusion model,the Zea Longa score determines whether the model is successful.The experiments were randomly divided into 3 groups:Sham,MCAO,and KDZ.At 3 days after MCAO,Garcia scored 18 points for neurological deficits in rats;TTC staining was used to measure the volume of cerebral infarction;immunofluorescence was used to detect the expression of vWF,which is a microvascular endothelial cell specific marker in ischemic cortex;Western blot was used to detect the expression of VEGF protein in ischemic cortex.2.Study 2:male Sprague-Dawley rats,8-week-old,were established by a line embolization method to determine the left middle cerebral artery occlusion model,the Zea Longa score determines whether the model is successful.The experiment was randomly divided into 6 groups:sham group(Sham),model group(MCAO),Kudiezi injection group(MCAO+KDZ),agomir negative control group(MCAO+miR-ctl),miR-210 agomir group(MCAO+miR-210)and miR-210 agomir were treated with Kudiezi injection(MCAO+miR-210+KDZ).At 3 days after MCAO,Garcia was evaluated for neurological impairment at 18 points;TTC staining was used to measure the volume of cerebral infarction;Real Time PCR was used to detect miR-210 content in ischemic cortex;immunofluorescence was used to detect the vWF in the ischemia cortex;Western blot analysis was used to detect the expression of VEGF,VEGFR2,PI3K and Akt in the ischemic cortex.[Result]1.Study 1:After 3 days of MCAO,Garcia scores showed that the neurological deficits in the MCAO+KDZ group were significantly better than those in the MCAO group(P<0.05);TTC staining showed that the MCAO+KDZ group had a significantly smaller infarct volume than the MCAO group(P<0.05);vWF-labeled immunofluorescence showed that MCAO and MCAO+KDZ groups all exhibited a certain degree of endothelial cell proliferation and increased microvessel density(P<0.05),and MCAO+KDZ group significantly increased endothelial cell proliferation compared with MCAO group(P<0.05).Western blot showed that the expression of VEGF in MCAO+KDZ was significantly increased compared with MCAO group(P<0.05).2.Study 2:3 days after MCAO,Real Time PCR showed that the miR-210 content in MCAO+KDZ,MCAO+miR-210,MCAO+miR-210+KDZ showed an upward trend compared with Sham group,however,there was no significant difference in the level of miR-210 between MCAO+miR-210,MCAO+miR-210+KDZ(P>0.05).Both Garcia score and TTC staining showed that compared with MCAO group,the neurological scores of MCAO+miR-210,MCAO+miR-210+KDZ group were significantly higher(P<0.05),while there was no significant difference between MCAO+miR-210 and MCAO+miR-210+KDZ groups(P>0.05);Immunofluorescent staining with vWF showed that the microvessel density was significantly increased in the MCAO+miR-210+KDZ group compared with the Sham group(P<0.05);Western blot showed that compared with MCAO group,the expression of VEGF/VEGFR2/PI3K/Akt protein was significantly increased in MCAO+miR-210 and MCAO+miR-210+KDZ groups,and the expression of VEGFR2 and PI3K protein was more pronounced in MCAO+miR-210+KDZ(P<0.05).However,there was no significant difference in VEGF and Akt protein between MCAO+miR-210,MCAO+miR-210+KDZ(P>0.05).[Conclusion]Kudiezi injection can improve the neurological function,decrease the volume of cerebral infarction and promote the proliferation of vascular endothelial cells in rats after acute ischemic stroke.Its molecular mechanism may be related to its regulation of miR-210-mediated VEGF/PI3K/Akt signaling pathway.