Based On PLGF, The Molecular Mechanism Of Tongxinluo On Microvascular Endothelial-mediated Neuronal Protection After Cerebral Ischemia Was Explored

Cerebral vascular disease is one of the three diseases causing death,with high incidence,high morbidity,high mortality and recurrence rate characteristics.Ischemic cerebral vascular disease(ICVD)is about 55%to 80%.Therefore,the effective treatment of this disease is an important topic.The treatment of ICVD is mainly thrombolysis and neuron protection.But so far there are no acknowledgedly and effective drugs.So we must find a new treatment.With the development of traditional Chinese medicine,Tongxinluo based on the theory of "vessel"theory showed protection of BMEC integrity in ischemic area,has opened up a new treatment of ICVD.BMEC paracrine function have an important role of regulatory neuronal survival,PLGF have a significant protective effect of neurons.In this study,we investigate the expression of OGD BMEC and the molecular mechanisms of PLGF to protect neuron under the action of TongXinLuo,provide a scientific basis of Tongxinluo cured ischemic encephalopathy.Objective:In this study,under the theoretical of" intersection of Ying and Wei,vessel to pass",PLGF as an entry point,to observe the protective effect of Tongxinluo on Rats ischemic injury and the expression of PLGF;to investigate BMEC conditioned medium effects on neuronal,the expression of PLGF and the molecular mechanisms of PLGF to protect neuron;To explore the mechanism of TXL interventing BMEC in cerebral infarction surrounding neurovascular unit,To reveal the scientific connotation of "micro vascular ischemic area protection-a new target for the treatment of cerebral ischemia",for the modernization of Chinese medicine to guide treatment of cerebral vascular disease has important implications.Methods:1.The establish the middle cerebral artery occlusion model in rats,Experimental animals were randomly divided into sham operation group,model group,TXL group,positive control group and cerebral ischemia Id and 3d.The neurological score handled after awake and before drawn.We useHE staining and immunofluorescence to observe the morphological changes.2.Immunohistochemistry was used to detect PLGF expression,double immunofluorescence(PLGF + MAP-2,PLGF + CD31)analyzed the expression of PLGF in different cells;PLGF expression levels in rats homogenates and serum detected by Elisa assay,3.Primary cultured brain microvascular endothelial cells.Established OGD model.Firstly,observed the activity change of BMEC by CCK-8 in different time and at different concentrationsn of tongxinluo serum containing after injury to determined the best concentration and time.Detection of NO using silver nitrate reduction,Elisa assay detected expression of vWF and PLGF.4.To observe the influence of different conditions medium of brain BMEC under the influence of Tongxinluo containing serum on primary neuron.neuronal activity and survival detected by CCK-8,SOD activity detected by WST assay,MDA content changes detected by lipid oxidation detection kit5.Different signals pathway blockers are used,CCK-8 assay detect neuronal survival before and after PLGF intervention,apoptosis detectedby tunel assay,expression of Bax?Bcl2 and p-ERK.Results:1 Experiments in vivo(1)Nerve function score had no significant difference in Each group.Two Tongxinluo group score was significantly lower than the model group.The Tongxinluo group ratio of the two ratings was significantly decreased,relative to the 3d model group,the Tongxinluo and positive control group ratio of the two ratings was significantly decreased.(2)HE:brain and neuron of Sham group structure is regular,Model group organizational structure disturbance,neuronal karyopyknosis and stain,neurons disappeared.Compared with model group,the Tongxinluo group brain tissue lightened,increased neuronal survival.Butylphthalide group ischemic area is more serious.(3)Elisa showed that PLGF levels significantly increased in serum at 1d?and expression increased at 3d,the same with brain tissue homogenate.Relative to the sham and model group,PLGF levels significantly increased in rats serum and brain tissue homogenate with he increase of injury time.(4)Immunohistochemistry results showed PLGF is expressed mainly in neurons and endothelial cells of the ischemic penumbra and infarct center,the expression Significantly increased in vascular endothelial cells after cerebral ischemia.Double immunofluorescence labeling showed that the normal state PLGF mainly expressed in neurons,and ischemic vascular endothelial cells express a large number of PLGF.2 Experiments in vitro(1)10%tongxinluo containing serum active role significantly increased after 24h BMEC,namely is the best concentration and reaction time of TXL containing serum.(2)I-CM could decrease the activity of the normal neurons,reduced the SOD activity and increased the MDA content,this injured effect can be reversed remarkably by IT-CM.? NT-CM and IT-CM can inhibit the trend.PLGF levels significantly increased in OGD and Tonxinluo group.(3)OGD treatment causes cell death,increases neuronal apoptosis and Bax/Bcl-2 ratio.Exogenous PLGF induces a increase in Bcl-2 expression and neuronal survival.PLGF can mitigates OGD-induced decreases in neuronal survival,Bcl-2 expression,and phosphorylation of MAPK and also reduces elevated Bax level.Only inhibition of MAPK reduced the ability of PLGF to protect neurons.Conclusions:1 Tongxinluo can promote neurological recovery after ischemic injury,protect neurons and increased the expression and secretion of PLGF.Normal BMEC in released little PLGF,OGD BMEC can release more PLGF.This study confirms that Tongxinluo effective protection of cerebral ischemia be associated with PLGF.2 Tongxinluo can significantly improve the BMEC activity and secretory function.We firstly observed the paracrine function of BMEC under TXL plays an important role in protection neuronal survival and oxidative stress,and found BMEC under secrete PLGF increased reactivity after ischemia and hypoxia stimulation.3 PLGF can improve the neuronal survival rate,we firsly found that it reduced the rate of neuronal apoptosis and regulating the expression of apoptosis-related proteins,and found that this effect is associated with MAPK signaling pathway.In conclusion,to improve microvascular function mediated neuroprotective effects is Tongxinluo new target for the treatment of cerebral ischemia as the core of the neurovascular unit,this effect may be associated with and PLGF,PLGF may play a role in protecting neurons through the MAPK signaling pathway.