Study On The Protective Effect Of Borneol Combined With Astragaloside IV And Panax Notoginseng Saponins On Neurovascular Unit After Cerebral Ischemia/reperfusion Injury

Objective: To investigate the effects of borneol combined with Astragaloside Ⅳ(AST Ⅳ)and Panax notoginseng saponins(PNS)on cerebral ischemia reperfusion injury(CIRI)rats brain tissue damage,the structural changes of components in neurovascular unit(neurons,astrocytes and microvessels),the major component-specific proteins--Glialfibrillary acidic protein(GFAP),Neuron specific nuclear protein(Neur N)),Laminin(LN),Endothelial barrier antigen(EBA),and related functional proteins--αII-Spectrin(αII-SP),Aquaporin 4(AQP-4),matrix metalloproteinase 9(Matrix metalloproteinase-9,MMP-9),and vascular endothelial growth factor(VEGF)expression.And to explore its protective mechanism of Notch signaling pathway against neurovascular units after CIRI in rats.Methods: The adult male Sprague-Dawley(SD)rats were randomly divided into Sham Operation Group(SOG),Model Group(MG),Borneol Group(BG),AST Ⅳ Group(AG),PNS Group(PG),AST Ⅳ + PNS Group(APG),Borneol + AST Ⅳ + PNS Low-dose Group(LDG),Borneol + AST Ⅳ + PNS High-dose Group(HDG)and Edaravone Group(EG).The EG was intraperitoneally injected and the other groups were administered by intragastric administration,twice a day.A rat model of CIRI was established by blocking the right middle cerebral artery with wire bolt.After 2h of ischemia and 22 h of reperfusion,neurological function scores were scored,cerebral infarct volume were observed by TTC staining,and pathological changes of ischemic cortex in brain were observed by HE staining.The protein expressions of GFAP,Neu N,LN and EBA in ischemic cortex of brain were detected by Immunohistochemistry.The expression of GFAP,neurofilament 200(NF-200)and LN in cerebral ischemic cortex of brain were detected by fluorescent three-label method.The expressions of proteins such as αII SP,AQP-4,MMP-9,VEGF,Notch1,intracellular domain of Notch(NICD),Hes1 and Delta-like ligand 4(DLL4)in ischemic cortex of brain were detected by western blotting.Results: After 2h of ischemia and 22 h after reperfusion,in the MG,neurological deficits,right cerebral infarction,and nerve cell injury occurred.Each drug could significantly reduce the score of nerve function defect,cerebral infarct volume and the rate of cell damage(P<0.05,0.01),and the effect of borneol + AST Ⅳ + PNS was better than that of single drug and AST Ⅳ + PNS(P<0.05,0.01).After 2h of ischemia and 22 h after reperfusion,immunohistochemical results showed that Neu N,LN,and EBA positive cells in the MG were significantly reduced(P<0.01),and GFAP positive cells were significantly increased(P<0.01);Borneol,PNS,AST Ⅳ,AST Ⅳ + PNS and Borneol + AST Ⅳ + PNS could significantly enhance the expression of Neu N,LN,EBA protein(P<0.05,0.01),significantly reduce the expression of GFAP protein(P<0.05,0.01),and the effect of Borneol + AST Ⅳ + PNS was better than that of single drug and AST Ⅳ + PNS(P<0.05,0.01).Fluorescence three-labeling results showed that NF-200 and LN positive cells in the MG were significantly reduced(P<0.01),and GFAP positive cells were significantly increased(P<0.01);Borneol,AST Ⅳ,PNS,AST Ⅳ + PNS and Borneol + AST Ⅳ + PNS could significantly reduce the expression of GFAP protein(P<0.05,0.01),and the effect of Borneol + AST Ⅳ + PNS was better than that of single drug and AST Ⅳ + PNS(P<0.05,0.01).Borneol,AST Ⅳ + PNS and Borneol + AST Ⅳ + PNS could significantly enhance the expression of LN proteins(P<0.05,0.01),and the effect of Borneol + AST Ⅳ + PNS was better than that of single drug and AST Ⅳ + PNS(P<0.05,0.01).Borneol +AST Ⅳ + PNS could significantly enhance the expression of NF-200proteins(P<0.01),and the effect of Borneol + AST Ⅳ + PNS was better than that of AST Ⅳ + PNS(P<0.05).Westrn blotting results showed that the expression of AQP-4,and MMP-9 protein in the ischemic cortex of the MG rats were significantly increased(P<0.05),and the expression level of αII SP protein was significantly decreased(P<0.05),while VEGF,NICD,Notch1,Hes1 and DLL4 protein expression did not change significantly(P>0.05).Borneol + AST Ⅳ + PNS could significantly increase the expression of αII SP,VEGF,NICD,Notch1,Hes1 and DLL4 protein(P<0.01),and the effect of Borneol + AST Ⅳ + PNS was better than that of single drug and AST Ⅳ + PNS(P<0.05,0.01).Borneol + AST Ⅳ + PNS could significantly down-regulate the expression of AQP-4 and MMP-9proteins(P<0.01),and the effect of Borneol + AST Ⅳ + PNS was better than that of single drug and AST Ⅳ + PNS(P<0.05,0.01).Conclusions: 1.Borneol combined with AST Ⅳ and PNS could protect the brain after CIRI in rats.It could improve neurological deficits,reduce the rate of nerve cell damage and the volume of cerebral infarction.And the effect of Borneol + AST Ⅳ + PNS was better than that of single drug and AST Ⅳ + PNS.2.Borneol compatible with AST Ⅳ and PNS had brain protective effect after CIRI in rats,which could reduce the damage of neurons and microvascular basement membranes,inhibit the over-activation of astrocytes,reduce cerebral edema.And the effect of Borneol + AST Ⅳ +PNS was better than that of single drug and AST Ⅳ + PNS.The three drugs could maintain the structure of neurovascular unit,and the effect of Borneol+ AST Ⅳ + PNS was better than that of AST Ⅳ + PNS.3.Borneol compatible with AST Ⅳ and PNS had a protective effect on brain after CIRI in rats.Furthermore,the effect of three-drug combination was better than that of single drug and AST Ⅳ + PNS.The mechanism may be related to the activation of the Notch signaling pathway,up-regulation of the expression of NICD,Notch1,Hes1,VEGF and DLL4,thereby exerting protective effects on ischemic brain tissue.