| Objective: This study discussed the neurovascular unit protection and related mechanism of aromatic resuscitation drug including L-Borneolum,Borneolum,Borneolum Syntheticum,Styrax,Benzoinum,inorder to explain the scientific connotation that aromatic resuscitation drug has the effect of inducing resuscitation and restoring consciousness,and lay an experimental foundation for resuscitation drugs used in clinic by comparing the pharmacodynamical effects of five resuscitation durgs.Methods:(1)The permanent focal middle cerebral artery occlusion(p MCAO)rat model was made by suture-occluded method,and the anesthetic recovery time of model rat was recorded.Neurological defict testing was performed with Longa's Scale.(2)The brain tissue water content,brain index and brain edema rate of pMCAO rat was measured by wet-dry weighting method.(3)The Cerebral infarction was observed by TTC-staining,and the cerebral infarction rate was calculated.(4)Cellular morphology of cerebral ischemia tissue was observed by HE-staining and denatured cell index was calculated.(5)Serum content of VEGF,NGF,IL-1? and TNF-? of each group was detected by ELISA.(6)The expression of Wnt3 a,?-catenin,Dsh1,LEF1,GSK-3?,APC Claudin-5,Bax,Bcl-2,Caspase-3 mRNA and protein of ischemic penumbra tissue was detected by RT-PCR,Western-blotting and immunohistochemical method.(7)Ultrastructure of neuron and BBB in ischemic penumbra of pMCAO rat was observed by transmission electron microscope.Results:(1)L-Borneolum,Borneolum,Borneolum Syntheticum can significantly prolong the anesthetic recovery time of model rats(P<0.01).Styrax and Benzoinum have no obvious effect on anesthetic recovery time.L-Borneolum and Benzoinum can reduce the neurological score of model rats significantly 24 h after the surgery(P<0.05).Borneolum and Borneolum Syntheticum have a tendency to improve the neurological defict.(2)L-Borneolum,Borneolum Syntheticum,Styrax and Benzoinum can reduce the ischemia brain water content,delta water content,delta brain index(P<0.01);L-Borneolum,Borneolum Syntheticum and Benzoinum can remarkably reduce the brain edema rate(P<0.01).(3)Results of TTC-staining showed that,a large,pale,infarcted region was observed in pMCAO model group,resuscitation durgs can decrease cerebral infarction size in different degrees.L-Borneolum,Borneolum Syntheticum,Styrax and Benzoinum can significantly reduce the cerebral infarction rate of model rate(P<0.01).(4)Results of TTC-staining showed that,significant change occurred in ischemic penumbra of pMCAO model group.Theses changes were expressed in necrosis and liquidation of nervous tissue,cribriform focos was formed.The boundary between the focos and surrounding tissue was clear.Resuscitation drugs can improve the histopathology of ischemic penumbra in different degrees.L-Borneolum,Borneolum Syntheticum,Styrax and Benzoinum can significantly reduce DCI of neruon(P<0.05 or P<0.01).(5)All of the five resuscitation drugs can significantly increase serum levels of VEGF,and decrease serum levels of TNF-? in pMCAO model rats(P<0.05 or P<0.01).All of the five resuscitation durgs had no effects on NGF and IL-1?.(6)In general,resuscitation durgs have a trend of increasing the expression of Wnt3 a protein,?-catenin mRNA,Dsh1 mRNA,GSK-3? mRNA and protein,Claudin5 mRNA and protein,Bax mRNA and protein,Bcl-2 mRNA and protein.Resuscitation durgs have a trend of decreasing the expression of Wnt3 a mRNA,?-catenin protein,LEF1 mRNA,APC mRNA and protein,Caspase-3 mRNA and protein,and can reduce the ratio of Bax and Bcl-2 expression.(7)The results of transmission electron microscope showd that,nucleus of neurons dissolved,double karyotheca was incoherent,organelle dissolved and disappeared,cytoplasm was blanker compared to the sham group.Capillaries walls were thicker and the space between endothelial cells,basilar membrane and gliocyte was wider in the model group compared to the sham group.after establishing models.Resuscitation drugs can improve the neuron and BBB ultrastructure of model rat at different levels.Conclusions: The resuscitation drugs can improve neurological function,decrease brain water content and brain infarction rate,alleviate the nerve cell damage of pMCAO rats,which may be the main basis of pharmacodynamics that resuscitation durgs express the effect of inducing resuscitation and restoring consciousness.The strong-to-weak sequence of five resuscitation durgs on pharmacodynamics was L-Borneolum,Borneolum Syntheticum,Benzoinum,Styrax,Borneolum.It suggests that L-Borneolum is the first choice to treat cerebral ischemia among the resuscitation durgs,and then Borneolum Syntheticum and Benzoinum are the second choice.The protective mechanism of resuscitation drugs on NVU was related to the regulation of Wnt/?-catenin signaling pathway,anti-inflammation,antiapoptosis,promoting micrangium regeneration and protecting BBB,which which may be the main biological mechanism that resuscitation durgs express the effect of inducing resuscitation and restoring consciousness.The results of cluster analysis and correspondence analysis suggest that the difference of resuscitation durgs on mechanism of protective effects on neurovascular unit may have a relationship with the difference of cold and heat properties.The anti-apoptosis effect of L-Borneolum,Borneolum Syntheticum may have a relationship with their cold property,and the angiogenesis effect and BBB protection may have a relationship with their heat property.This study dicussed the cerebral protection and mechanism of resuscitation drugs with the key point of NVU,and the research idea conformed to the holism of TCM.The study adopted some molecular biology methods,such as RT-PCR,Western-blotting,and immunohistochemical staining,and combined optical imaging techniques such as transmission electron microscope,so the method was integrative innovation.In addition,the results of this study lay an experimental foundation for resuscitation drugs used in clinic effectively and efficiently. |
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