| Theoretical discussion and clinical application of stroke treatment based on "san jiao ci di" therapyFrom TCM classics that generations of physicians understanding of stroke,mainly expounds the Chinese medicine for stroke disease and the understanding of the etiology and pathogenesis,based on the "Yang" stroke "in the initial treatment" the theoretical basis,especially based on Yang vulva in ontology structure to explain the pathogenesis of stroke,and on the basis of the "san jiao ci di" treatment,and clinical basis an attached.The second part experimental studyBy constructing Sprague-Dawley(SD)in ischemia-reperfusion injury rats model,thus to observe "the san jiao ci di" treatment for transient cerebral ischemia reperfusion injury in rats to protect nerve vascular unit,and discuss the Wnt/β-catenin signaling pathway in "san jiao ci di" treatment for the role of ischemia reperfusion injury in rats and the mechanism research,enrich cerebral ischemic injury treatment based on the combination of disease and syndrome differentiation of new ideas and new methods,and the treatment can lay a solid foundation for better application to clinical,at the same time and provide a scientific basis for the rational use of drugs in the Chinese medicine treatment of cerebral ischemic injury,It can be divided into the following four experiments.Experiment 1 Establishing and staging cerebral ischemia reperfusion injury model in SD ratsObjective: To construct and replicate the model of cerebral ischemia reperfusion injury,and to observe and evaluate the stability of the model and the possible staging nodes of acute and chronic stages of cerebral ischemia injury.Methods: Thirty SPF male SD rats were randomly divided into 3 groups: Normal group,Sham group and cerebral ischemia-reperfusion injury group(I/R).The Middle cerebral artery occlusion(MCAO)method was used to establish the focal cerebral ischemia-reperfusion injury model.2,3,5-triphenyltetrazolium chloride(TTC)staining was performed 24 hours after cerebral ischemia reperfusion,and the infarct size was calculated according to the TTC staining.Pathological changes of cerebral ischemia in each group were evaluated by hematoxylin-eosin(HE)staining after reperfusion for 24 h and 3d.The changes of general condition,body weight,survival rate and neurological deficit score were observed at different time points.Results: Compared with the normal group and the sham group,the general condition,body weight,survival rate,neurological deficit score,cerebral infarct area and ischemic pathology of the rats in the cerebral ischemia-reperfusion injury group were significantly changed.Compared with reperfusion ischemia at 24 h,the proliferation of capillaries was obvious at 3d after reperfusion.Conclusion: The model of cerebral ischemia/reperfusion injury in rats with right middle cerebral artery obstruction was successfully established by Longa line embolization method,and the time node of the acute stage of cerebral ischemia/reperfusion injury in rats might be 3 days after reperfusion injury.Experiment 2 Protective effects of different doses of Yanghua decoction on acute cerebral ischemia reperfusion injuryObjective: To investigate the protective effect of Yanghua Decoction on acute cerebral ischemia/reperfusion injury by establishing cerebral ischemia/reperfusion injury model.Methods: Sixty-two SPF male SD rats were randomly divided into 5 groups: Sham operation group(Sham),cerebral ischemia reperfusion injury group(I/R),low-dose Yanghua decoction group(L group),medium-dose Yanghua decoction group(M group),and high-dose Yanghua decoction group(H group).Cerebral ischemia/reperfusion injury was established by MCAO.The cerebral index,TTC staining and HE staining were performed 3 days after 2h reperfusion in cerebral ischemia.The cerebral infarct area was calculated according to TTC staining.The changes of neurological deficit scores in each group were observed at different time points.Results: Compared with I/R group,the middle and high dose of yanghua decoction can significantly reduce the cerebral infarction area and cerebral index,significantly reduce the pathological changes of ischemic disease,and significantly change the neurological deficit score of model animals.Conclusion: Yanghua decoction has a certain protective effect on cerebral ischemia-reperfusion injury in acute stage,and high dose of Yanghua decoction(24g/kg/d)is the best therapeutic dose for cerebral ischemia-reperfusion injury.Experiment 3 Study on the protective effect and mechanism of "Sanjiao cidi therapy" on the neurovascular units of acute cerebral ischemia reperfusion injuryObjective: this study through the establishment of animal model of cerebral ischemia-reperfusion injury to explore "the sanjiao cidi treatment" on acute cerebral ischemia-reperfusion injury in rats nerve vascular unit protection,observe the Wnt/ β-catenin signaling pathway in rats with acute phase changes in cerebral ischemia reperfusion injury,and observe "the sanjiao cidi treatment" to regulate the Wnt/β-catenin signaling pathway.Methods:A total of 150 SPF male SD rats were randomly divided into 6 groups,which were Sham operation group(Sham),cerebral ischemia reperfusion injury group(I/R),Yanghua decoction treatment group(YHT),Fuyang prescription treatment group(FYF),"cidi treatment" group(CDZ)and positive control group(PCG).The cerebral ischemia-reperfusion injury was established by MCAO.TTC staining was performed to evaluate the cerebral infarct size in each group 3 days of reperfusion.TUNEL staining was used to evaluate apoptosis in the ischemic penumbra of each group after 3 days of reperfusion.After 3 days of reperfusion,Nissl staining was used to evaluate the number of intact cells in the ischemic penumbra of each experimental group.After 3 days of reperfusion,the submicroscopic structure of cerebral ischemia cortex was observed by transmission electron microscope.Immunohistochemical staining IgG was used to evaluate the changes in BBB permeability after 3 days of reperfusion.The expressions of MMP-3,MMP-9,BDNF,VEGF and Wnt/β-catenin signaling pathway related proteins and genes were detected by Western blot and qPCR after acute cerebral ischemia reperfusion in each group.Results: Compared with I/R group,sanjiao cidi treatment" can significantly reduce the cerebral infarction area,reduce the apoptosis of cells in the ischemic penumbra,and promote the survival of nerve cells in the area.transmission electron microscopy and immunohistochemical IgG results showed that "sanjiao cidi" treatment could improve the permeability of blood-brain barrier structurally and functionally.Western blot and qPCR results showed that"sanjiao cidi treatment" significantly down-regulated the expression of MMP-3 and MMP-9 proteins and genes,and simultaneously up-regulated the expression of VEGF and BDNF proteins and genes.The results also indicated that "sanjiao cidi treatment"could activate the Wnt/β-catenin signaling pathway.Conclusion: "sanjiao cidi treatment" has protective effect on neurovascular units in acute cerebral ischemia-reperfusion injury model,which may be related to the down-regulation of MMP-3 and MMP-9 expression,the up-regulation of VEGF and BDNF expression,and may be related to the activation of Wnt/β-catenin pathway.Experiment 4 Study on the protective effect and mechanism of "sanjiao cidi treatment" on neurovascular units after cerebral ischemia reperfusion injury in convalescent periodObjective: this study through the establishment of animal model of cerebral ischemia-reperfusion injury in rats to explore "sanjiao cidi treatment" for nerve recovery stage of cerebral ischemia reperfusion injury of vascular cell protection,observe the Wnt/beta-catenin signaling pathway in convalescence of cerebral ischemia-reperfusion injury in rats,and to observe "sanjiao cidi treatment" to regulate Wnt/beta-catenin signaling pathway.Methods:A total of 150 SPF male SD rats were randomly divided into 6 groups,which were Sham operation group(Sham),cerebral ischemia reperfusion injury group(I/R),Yanghua decoction treatment group(YHT),Fuyang prescription treatment group(FYF),"sanjiao cidi treatment" group(CDZ)and positive control group(PCG).The cerebral ischemia-reperfusion injury was established by MCAO.TTC staining was performed to evaluate the cerebral infarct area in each group after reperfusion for7 d.TUNEL staining was used to evaluate the apoptosis of cells in the ischemic penumbra of each group after 7 days reperfusion.Nissl staining was used to evaluate the number of intact cells in the ischemic penumbra of each experimental group 7 days after reperfusion.After 7 days of reperfusion,the submicroscopic structure of cerebral ischemia cortex was observed by transmission electron microscope.Immunohistochemical staining IgG was used to evaluate the changes of BBB permeability 7 days after reperfusion.Western blot and qPCR were used to detect the expressions of MMP-3,MMP-9,BDNF,VEGF and Wnt/β-catenin signaling pathway related proteins and genes in each group after cerebral ischemia reperfusion in the recovery period.Results: Compared with I/R group,"sanjiao cidi treatment" could significantly reduce the cerebral infarct area,reduce the apoptosis of ischemic penumbra,and promote the survival of nerve cells in this area.transmission electron microscopy and immunohistochemical IgG results showed that "sanjiao cidi" treatment could improve the permeability of blood-brain barrier structurally and functionally.Western blot and qPCR results showed that "sanjiao cidi treatment" significantly down-regulated the expression of MMP-3 and MMP-9 proteins and genes,while up-regulated the expression of VEGF and BDNF proteins and genes.The results also indicated that "sanjiao cidi treatment" could activate the Wnt/β-catenin signaling pathway.Conclusion: "sanjiao cidi treatment" has a protective effect on neurovascular units of cerebral ischemia-reperfusion injury model in nerve recovery stage of cerebral ischemia reperfusion injury,which may be related to the down-regulation of MMP-3 and MMP-9expression,the up-regulation of VEGF and BDNF expression,and may be related to the activation of Wnt/β-catenin pathway. |
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