Effects And Mechanisms Of Effective Components Group Of Xiaoshuantongluo Prescription (XECG) On Ischemic Cerebrovascular Diseases

Stroke is one of the leading causes of death and long-term disability in the population,and it is consequently a major public health-care burden in the worldwide. There are many risks and multiplicity of molecular and cellular events involved in the complex disease-stroke.It is manifested that the drugs targeted to a single molecule, even some specific cell,such as many neuroprotective agents,are disappointing in the stroke treatment.So the combined and cock-tail therapies direct at different targets simultaneously are advised in recent years,and some of them are under clinic trials.The damage is observed not only at the neuronal structure and function,but also in the glials and blood vessels of the brain after acute and chronic ischemic brain injury. Now,the whole brain protection rather than neuroprotection is advised in the stroke prevention and treatment field,which is based on the achievements in the pathophysiologic research and the frustration in the neuroprotective agents.In the Traditional Chinese Medicines,it is upholded and emphasized that the integrated regulation and the activation and mobilization of endogenic compensation and guarding systems for the defense against all sorts of etiological factors.The Xiaoshuantongluo prescription(XSTL),a fufang activating blood circulation and removing stasis,has been used in the clinic for stroke treatment for many years,but the material foundation and exact mechanisms about the XSTL is not yet known.Based on the experiences in the field of Traditional Chinese Medicine modemization research,and the systematic model thouroughout the TCM,Professor Du Guanhua proposed the Effective Components Group model for fufang research.According to the ECG research model,we have made some progress on the research of XSTL,a prescription used frequently for the stroke treatment and acts on acroparalysis and other symptoms arising from apoplexy.And the effects and mechanisms of the Effective Components Group of Xiaoshuantongluo(XECG) against ischemic cerebral injury will be researched and discussed in this thesis. 1.The screening and determination of XECGBased on the knowledge about the pathophysiology of ischemic cerebral injury,the hypoxic endothelial cell injury model,the aortic rings relaxation model,the hypoxic neuron injury model,the DPPH clearance and the oxidative brain mitochondria injury model were selected for the evaluation of 16 components extracted from XSTL.XECG and the extraction artwork were determined based on the results of screening and the needs for scale manufacture.2.The effects of XECG on the ischemic brain diseases1) The effects of XECG on the blood stasis and brain circulation dysfunction induced by Dextran-500After the injection of Dextran-500 and evans-blue in the ICR mice,the visible blood stasis signs were observed,and the cerebral blood flow decreased 31.6±10.8%from the baseline.The signs of blood stasis were relieved obviously in the XECG treatment group, and the dysfunction of brain circulation was also ameliorated after XECG treatment.2) The effects of XECG on the acute ischemic cerebral injury in ratsThe occlusion of middle cerebral artery(MCAO) is the major reason of ischemic stroke in clinic.After the left middle cerebral artery was occlused for 24h,visible performance dysfunction was observed in the rats,and the infarction zone could be measured in the ipsilateral cerebral sphere by TTC staining.The performance dysfunction was relieved and the infarction zone was reduced in the XECG pretreatment group.The activity of MPO increased abnormally after MCAO and the increased MPO activity was reduced in the XECG pretreatment group,especially in the high dose group. The excessive production of NO and activation of iNOS was found be inhibited by the XECG pretreatment for 7days as well.24h after MCAO,the excessive expression of the proapoptotic protein Bax was measured by western blotting analysis,and the balance of Bcl-2 family was broken.In the XECG pretreatment group,the Bax expression was inhibited and the Bcl-2/Bax balance was rescued.All of the above results suggested that XECG has beneficial effects on the acute ischemic cerebral injury,and the involved mechanisms may include the inhibition of excessive activation of iNOS and inflammatory events,and the excessive expression of Bax. 3) The effects of XECG on the chronic hypoperfusion brain injury in the ratsAfter the bilateral common carotid arteries ligation for 4weeks,the damage and loss of neuron in the brain,especially in the hippocampus,could be observed.And astrocytic reactions and white matter damage had also been examined.In Kluver-Barrera stained sections,the rarefaction and vacuolization of the white matter could be observed.In the morris water maze test,the impairment of learning and memory was improved by the treatment with XECG.At the same time,the results of the pathological analysis suggested the neuronal damage and loss in the hippocampus was ameliorated in the XECG treatment groups,and white matter damage was relieved by the XECG treatment as well.4) The effects of XECG on the cerebrovascular reactivity after cerebral ischemiaIt is manifested that the cerebrovascular reactivity depressed after cerebral ischemia, and the cerebrovascular reactivity was related to the prognosis of stroke in the basic and clinic research.The cerebrovascular reactivity is defined as change of cerebral blood flow(CBF) after the application of the inhibitor of carbonic anhydrase,acetazolamide, expressed in percentage of baseline flow.In the aortic tone test and cerebrovascular reactivity assay,the vascular reactivity decreased significantly after the bilateral common carotid arteries occlusion.The vascular reactivity was improved in the XECG treatment groups,and the increase of CBF was from 2.1±1.8%in the vehicle treatment group to 21.4±8.4%in the XECG high dose group after acetazolamide injection.Post-mortem Cerebral Angiograms suggested that the posterior cerebral artery(PCA),posterior communicating artery(PComA),and basilar artery in XECG treatment were larger than those in the vehicle treatment group.3.The mechanisms involved in the XECG effects on the ischemic brain injury1) Effects of XECG on the angiogenesis after cerebral ischemiaAngiogenesis is very important to the brain reorganization and recovery after brain ischemia,and it is correlated with longer survival in ischemic stroke patients.In our study,the perimeter,density of capillaries,and endothelial proliferation was measured with immunohistochemistry assay.The results showed that the increase of density of capillaries and enlarge of vessel perimeter was significant in the XECG treatment group, and XECG could promote endothelial proliferation.It suggested that XECG could promote angiogenesis after brain ischemia. 2) Mechanisms of angiogenesis in the ischemic brainThe effects of Traditional Chinese Medicine arises from the direct actions to the related molecular targets,but also the improvement of microenvironment,the maintenance of homeostasis,and the mobilization of endogenous compensate and recovery systems.The improvement of molecular and cellular microenvironment plays key roles in the brain reorganization,including angiogenesis and neurogenesis,after cerebral ischemia.In our study,the blood viscosity,brain NO content,iNOS activity abnormally increased in the vehicle treatment group,and improvement could be observed in the XECG treatment group.The expression of major angiogenic molecules,such as VEGF,Tie-2 and Flk,were enhanced in the XECG treatment group as well.These results suggested XECG can improve the vascular niche and pro-angiogenesis after brain ischemia.3) Effects and mechanisms of XECG and some compounds on the hypoxic endothelial cell injuryThe increase of HIF level and many molecular events are similar after hypoxic exposure or cobalt chloride treatment,so cobalt chloride is used to mimic hypoxic injury in many cellular models.In our study,obviously endothelial cell damage was observed after cobalt chloride treatment for 24 hours,and the damage was attenuated in the groups pretreated with some compounds from XECG,especially Astragaloside(Ast),Hyperoside (Hyp),Quercitin(Que),Natoginsenoside R1(NR1).Furthermore,the endothelial cell apoptosis induced by cobalt chloride was inhibited significantly by these compounds,and the mechanisms may involve the inhibition of excessive cellular ROS generation and the over-expression of Bax and caspase-3.In general,XECG treatment can improve the performance dysfunction after acute stroke,the impairment of learning and memory induced by chronic brain ischemia.The mechanisms may involve the improvement of cerebrovascular reaction and homeostasis, pro-angiogenesis and endothelial cell protection.