| Objectives:The cerebrovascular disease is common disease,frequently encountered disease harm for the humanity life and health,prevention and cure of the cerebrovascular disease has been an important topic of our country health work and a medical research.HUANG JIAO granula was the effective preparation treating cerebral apoplexy,which was developed on the foundation of many year clinical experiences summarize in the Wuhan first hospital.This topic aimed directly at the pathology physiology mechanism after cerebral ischemia,under the Chinese medicine basic theory instruction,cut-in point of nerve cell apoptosis,through in vivo and in vitro cell culture experiment,Observed HUANG JIAO granula affect endogenous apoptosis way of caspase dependence mitochondrium and apoptosis way of non-caspase dependence AIF,which penetrating approached the mechanism of protecting brain,intended to penetrating explore the drug efficacy and the action mechanism of traditional Chinese medicine compound prescription.Methods:1.Invivo experiment:Fetched several clean level Wistar rats, referencing literature adopted improvement way of longa coil bolt manufacture model of cerebrum median artery embolism animal.According to the weight the rats had been divided into six groups at random:The sham-operation control group(i.e.sham-operation group),the mold control group(i.e.model group),Nimodipine(i.e.western medicine group),HUANG JIAO granula 10g/kg,5g/kg,2.5g/kg group(i.e.large, medium and small group).The first part:observed HUANG JIAO granula effect on the neurologic impairment and brain tissue morphologic change of rat focal cerebral infarction,determined moisture capacity and infarct volume of brain tissue of cerebral infarction rat.The second part: used the methods of normal position apoptosis and immunohistochemistry to detect expression of nerve cell bcl-2,bax and Caspase-3 masculine cell of brain organization,and observed HUANG JIAO granula affect endogenous apoptosis way of caspase dependence mitochondrium of rat focal cerebral infarction.2.In vitro cell culture experiment:the third part,adopted the method of "the blood serum Pharmacology",utilized HUANG JIAO granula medicine serum interfere in hypoxia ischemic in vitro model of common tool PC-12 cell of differentiation similar neuron in the nerve research,again observed change of AIF on apoptosis way,carried on the comparison with western medicine Buflomedil Hydrochloride,inquired into specific molecular mechanism of HUANG JIAO granula protecting hypoxia ischemic cell anti-apoptosis way I.e.anti-apoptosis way.Results:1.Focal cerebral infarction rat model group had bad general condition,decreased food-intake,become emaciated gradually,color pattern were gloomy bloom,didn't depilate,hemiparalysis symptom was obvious,displayed the left side body abandons obviously at foreleg,walks assumed at the counter clock to follow tail.HUANG JIAO granula and Nimodipine group had good general condition,after treatment 3 days treatment group and the model group symptom had the varying degree to restore;the treatment group symptom improvement was obvious,especially large dose treatment group was good.2.Cerebral infarction rat brain tissue moisture capacity and infarct volume change:the sham-operation group brain tissue moisture capacity had not remarkable changes and seen the infarct focus;the model group brain tissue moisture capacity obviously increased,had seen the infarct focus;But Nimodipine group and HUANG JIAO granula different dosage treatment group's brain tissue moisture capacity obviously reduced,and infarct volume obviously was lower than the model group,had significant deviation comparing with the model group(P＜0.01).Under the light microscope,the sham-operation group cerebral cortex neuron shape structure was obviously normal; ischemia side cerebral cortex neuron of the model group rats showed ischemic change,massive neuron was denatured necrosis,appeared the vacuole,displayed the triangle for the cell body reduction,displayed endochylema eosin dyeing,the karyopyknosis anachromasis,nucleolus vanishing and so on;the seahorse CA1 area nerve cell arrange rarefaction and disorder,had the varying degree lost phenomenon of neuron.Ischemia side cerebral cortex nerve cell of HUANG JIAO granula,Nimodipine group also assumed ischemia change,but part of the partial cells was pyknosis, light anachromasis degree,the cell haven't the reduction,the cell environment crevice had not obvious change.3.The experimental and focal cerebral infarction rat brain organization bcl-2,bax masculine nerve cell's comparison:The masculine cell number of apoptosis and bax of rat model group obviously was higher than the normal group;the masculine cell number of bcl-2 obviously was lower than the normal group(P＜0.05 or P＜0.01);The traditional Chinese medicine large,medium and small dose and the western medicine group masculine cell number of apoptosis and bax was lower than the model group,the masculine cell number of bcl-2 obviously was higher than the model group(P＜0.05 or P＜0.01);Traditional Chinese medicine large dose group masculine cell number of apoptosis,bax and bcl-2 had non-significance difference with the western medicine group (P＞0.05).4.The experimental and focal cerebral infarction rat cranial nerve cell Caspase-3 masculine cell's comparison:the Caspase-3 protein masculine cell number of the model group increased obviously than the sham-operation group(P＜0.01),HUANG JIAO granula large and medium dose and the western medicine group might reduce the number of Bcl-2 protein masculine cell of cerebral ischemia leading,which had significant difference comparison with the model group(P＜0.05 or P＜0.01),large dose group had remarkable difference.5.Utilized HUANG JIAO granula medicine serum interfere in hypoxia ischemic in vitro model of PC-12 cell of differentiation similar neuron,after hypoxia ischemic 24 hours,then ventilation blood-supply 1 hours,the control group still might observe AIF nucleus obviously shifting,Utilized HUANG JIAO granula and buflomedil hydrochloride group mightn't observe AIF nucleus shifting; buflomedil hydrochloride and HUANG JIAO granula medicine serum might prevent AIF activating and releasing on hypoxia ischemic then ventilation blood-supply 1 hours and 3 hours.Conclusion:Acute stage's effective treatment was the key link of influencing prognosis,which aimed directly at clinical syndrome primarily characteristic of sthenia syndrome in acute stage,the methods of Rouse mind arresting convulsion,eliminating phlegm muddy stagnant blood,TONG DAO FU SHI were being suitable effective remedial measure. HUANG JIAO granula composed of cornu bubali and RADIX ET RHIZOMA RHEI could improve infarct volume,reduce hydro-cephalus,and improve nerve function damage degree of the experimental cerebral infarction rat.On the endogenous apoptosis way of caspase dependence mitochondrium,HUANG JIAO granula elevated the specific value of Bcl-2/Bax,and urged Bax-Bcl-2 hetero-dimerization,through up-regulating the express of Bcl-2 protein being induced by cerebral ischemia,and down regulating the express of Bax protein;actived suppression Caspase-3 activation,thus direct repression apoptosis of ischemic neuron,reduced the brain damage reason of cerebral ischemia,educed brain protective function.At the same time, in the research of AIF mediated caspase non-dependence signal passage, we discovered that HUANG JIAO granula could prevent effectively AIF activation release,caused cell unable to enter the apoptosis way of non-caspase mediated AIF.The above results had provided the experiment basis of HUANG JIAO granula treating cerebral infarction,which were possibly one of its treatment cerebral arterial thrombosis molecular mechanisms.
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