| Objective(1)A severe secondary injury will occur on the basis of the primary injury after spinal cord injury(SCI),thus leading secondary injury and serious complications.But,the mechanism of injury is very complicated.Previous studies have found that severe spinal cord microcirculation damage occurs after spinal cord injury,but the mechanism of vascular injury and its repair is still not very clear.Therefore,the purpose of this study was to verify the changes of microvessels after spinal cord injury,and then explore the mechanism of angiogenesis based on the Notch pathway and angiogenesis factors such as VEGF,Ang-1/Tie-2 and EGFL8,and observe the changes of nerve cells after injury.This study will provide relevant theoretical basis for the treatment of SCI.(2)To explore the effects of Notch-1 and EGFL-8 proteins on the proliferation,scratches and migration of HUVEC vascular endothelial cells and Schwann cells.(3)Under the guidance of syndrome differentiation in traditional Chinese medicine(TCM),acute SCI is a syndrome of kidney deficiency and blood stasis,and it should be used to nourish the kidney and promote blood circulation.In this experiment,Bushen Huoxue Formula(BSHXF)was used for related research.In vitro,we observed the effects of BSHXF on the vascularization of vascular endothelial cells(HUVEC);in vivo,we observed the effects of BSHXF on repairing the neurological function after SCI and explored the mechanism of repairing the vascular network in the injured tissue area.Meanwhile,we further observed the effects of BSHXF on local nerve cells after SCI,to explore its effects on local vascular microcirculation and nerve cells after SCI.Method(1)Study on the destruction of microcirculation vascular network and related mechanisms after SCI.SD rats were randomly divided into sham operation group(Sham group)and model group.The BBB behavior scores were observed at different time points after SCI;HE staining was used to observe pathological changes;IF staining was used to observe the related angiogenesis;microfil contrast and micro-CT scan were used to observe the three-dimensional structure of the injury;Western blot were used to detect the expressions of related proteins such as Notch-1,VEGF,Ang-1/Tie 2 and VEGF8.Immunofluorescence was used to observe the expression of GFAP and MAP-2 proteins.(2)In vitro,the effects of Notch-1 and EGFL-8 protein on the proliferation,scratch healing,migration and vascularization of vascular endothelial cells(HUVEC),and their effects on proliferation,scratches and migration in transwell cells of Schwann cell by using MTS,cell scratch,tubeformation and Western blot.(3)Study on the effects and its underlying mechanisms of BSHXF on acute SCI through angiogenesis.In vitro,we observed the effect of BSHXF on the cytotoxicity,scratching,migration and angiogenesis of HUVEC cells,and the angiogenesis on chick embryo.In vivo,we observed the BBB score of BSHXF on SCI rats;the effect of BSHXF on repairing the local vascular network;Western blot and RT-qPCR were used to detect the effect of BSHXF on the related pathways such as Notch-1,Ang-1/Tie-2 and EGFL8 in SCI;the effects of BSHXF on oxidative stress response,glial scar and related nerve cells after SCI were detected.Results(1)The results of BBB behavioral scores indicated that SCI will cause complete paralysis of both lower limbs immediately.Meanwhile,the results of BBB behavioral scores also demonstrated that a certain degree of recovery will occur in the first and second weeks after the injury,and the slower recovery period will be entered in weeks 3 and 4.The results of angiography and IF staining suggested that the blood vessel density will be decreased after SCI.Although the injury of blood vessel will be slightly repaired,the angiogenesis of blood vessel was significantly decreased,and there was still a certain defect of the vascular network by the 28th day.The results of Western blot suggested that when the spinal cord is injured,Notch-1 will be regulated by stress and reach a peak on the third day.Although it is lower on the seventh day,it is still significantly higher than the sham group.Until the 14th after SCI,the expressions of Notch-1 will be decreased,which will be lower than those in the sham group.Correspondingly,the same trend was observed in the expressions of Notch downstream pathways including Jag-1,Hes-1,VEGF and Ang-1/Tie-2,indicating that Notch-1 regulation of VEGF and Ang-1/Tie-2 related factors involved in angiogenesis after SCI.Meanwhile,EGFL8 protein decreased in the early stage of SCI and increased on the 7th day of injury,but showed a significant decrease in the later period,suggesting that it has a certain role in promoting angiogenesis,and may be involved in angiogenesis,which were inconsistent with the changes of Notch.With the destruction of blood vessels after spinal cord injury,the numbers of neurons in the injured area were decreased,and astrocytes were activated,especially the glial scars formed due to excessive activation at a later stage may have certain effects on angiogenesis and nerve function repair.(2)Meanwhile,in vitro,we found that Notch-1 protein at a concentration of 200ng/ml could promote the proliferation,scratch healing,migration and vascularization of HUVEC cells.It can promote proliferation,scratch healing and migration of Schwann cell,which could be attenuated by its inhibitor DAPT.Similarly,the EGFL-8 at 100ng/ml could promote the proliferation,scratching and angiogenesis of vascular endothelial cells and the proliferation and migration of Schwann cells,indicating it had potential neuroprotective effects.(3)BSHXF at 7.5mg/ml can promote the proliferation,scratches healing,migration and vascularization of HUVEC,and has a certain role in promoting angiogenesis of chicken embryos.In vivo,BSHXF significantly improved BBB score.Meanwhile,angiography and fluorescent staining suggest that BSHXF can promote the repair of local vascular network.The results of western blot and RT-qPCR showed that the protein expression of Notch-1,Ang-1/Tie-2 and EGFL8 were significantly increased after intervention of BSHXF when were compared with the model group,suggesting that BSHXF regulates angiogenesis through Notch-1 participating in the repair of acute SCI.With the repair of blood vessels in the injured area,the activity of SOD and MDA in the spinal cord tissue was balanced,and the levels of oxidative stress-related proteins such as Nox-1 and HO-1 were improved;the expression of GFAP protein decreased,and the activity of astrocytes was reduced,and at the same time,neuronal cells were protected to a certain extent,thus promoting the repair of nerve function after SCI.Conelusion(1)Notch-1 involved in the mechanism of angiogenesis after SCI by regulating the expressions of VEGF,Ang-1/Tie-2 and EGFL-8.(2)Notch-1 and EGFL-8 proteins can promote the healing,migration and angiogenesis of HUVEC cells,and have a certain role in promoting angiogenes;is.Additionally,they can promote the proliferation of Schwann cells,the healing and migration of scratches,and have potential neuroprotective effects.(3)In vitro,BSHXF has the effect of promoting angiogenesis;in vivo,BSHXF can regulate VEGF,Ang-1/Tie-2 and EGFL-8 through Notch-1 to promote angiogenesis after SCI,and protect neuronal cells and inhibit the over-activation of astrocytes,thereby improving neurological function after SCI. |
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