The Study On The Effects Of Panax Notoginseng Against Cerebral Ischemia-reperfusion Injury Based On Neurovascular Unit

Objective: To study the intervention effect of Panax Notoginseng(PN)on ischemic brain injury by observing the effect of Panax Notoginseng on neurovascular unit(NVU)in acute phase of cerebral ischemia-reperfusion injury(CIRI).Methods:(1)SPF SD rats were randomly divided into Sham group,CIRI group and PN group(PN,50 mg/kg?100 mg/kg?150 mg/kg).Except the sham group,the rats were modeled for middle cerebral artery occlusion(MCAO)in the other groups,which were ischemia for 2 h and reperfusion for 7d.The PN groups were given different doses of PN solution by gavage in cerebral ischemia reperfusion injury respectively.However,the Sham group and CIRI group were given equivalent normal saline.The neurological deficits of rats were evaluated with Zea Longa method,the percentages of cerebral infarction volume were calculated by TTC staining,and the ultrastructural changes of neurovascular unit in ischemic penumbra were observed by transmission electron microscopy(TEM).(2)After being purified and amplified in vitro,the cell morphology of neurons,astrocytes and microvascular endothelial cells were observed and detected by immunofluorescence technology.The Transwell pools were used to construct the co-culture systems to simulate the NVU,which were randomly divided into Control group,OGD/R group and PN drug serum(PNDS)group.The PNDS group was also divided into 3 different dose groups of corresponding serum drug concentration according to the PN group with different doses.Oxygen-glucose deprivation(OGD)models were established during OGD/R group and PNDS group.However,the Control group did not accept acupuncture of OGD.The OGD/R group and Control group were replaced with DMEM sugar-containing medium containing 10% blank serum,but the PNDS groups were replaced with DMEM sugar-containing medium containing 10% different concentrations of PN when they had completed OGD.They needed to be reoxygenation for 24 h.The growth states of the cells were observed by inverted microscope,the permeability of the co-culture models were determined by 4-hour permeation experiment,and the apoptosis rates of the cells were detected by flow cytometry.Results:(1)1)Compared with the Sham group,the scores of neurological defect and the percentages of cerebral infarction volume in the CIRI group were significantly increased(P<0.01).And the cells of target tissue were badly destroyed under the TEM.2)PN significantly reduced the neurobehavioral scores and the percentages of infarct volume of rats(P<0.01),as well as the ultrastructural damage of cells caused by ischemia-reperfusion injury.(2)3 kinds of high purity primary cells were successfully cultured and the co-cultured models in vitro were established.1)The round cell body and the strong refraction were shown in the neurons of Control group,also,the axons were thick and long and interwoven in a network.Meanwhile,the liquid level in the pool did not change significantly after 4h.2)Compared with the Control group,the OGD/R group showed neuronal deformation,poor refractive index and loose intercellular connections.Brain microvascular endothelial cells and astrocytes shrunk.Some of them shed.The apoptosis rates of neurons and microvascular endothelial cells in the OGD/R group increased significantly(P<0.01),and the liquid level decreased significantly in the 4h infiltration experiment(P<0.01).3)Compared with the OGD/R group,it was found that neurons,brain microvascular endothelial cells and astrocytes in the group of PNDS had no obvious contraction,the intercellular contact were still relatively close,and the cell growth state were significantly improved.After the interventions of Panax Notoginseng drug serum,the apoptosis rates of neurons and microvascular endothelial cells were decreased(P<0.01),as well as the infiltrations of 4h(P<0.01).(3)The volume of cerebral infarction in rats was dose-dependent under the action of PN,while the efficacies were stabilized at 100 mg/kg for neurological score,leakage test and apoptosis.Conclusion:(1)Panax Notoginseng can significantly improve the neural deficit scores,reduce the volume of cerebral infarction and cell damage in rats after cerebral ischemia.(2)It has an overall protective effect on neurovascular unit by inhibiting the apoptosis of neurons and microvascular endothelial cells,and reducing the permeability of blood brain barrier.(3)100mg/kg may be a better drug dose for cerebral ischemia-reperfusion injury in rats.