| Object:To investigate the effects that Extracts from Ginkgo Biloba (EGB) , improve neurological deficits,inhibits neuronal cell apoptosis and expression of Bcl-2 and Bax after cerebral ischemia in diabetic rats. Mainly to observe the amount of the nerve cell occurrence apoptosis and the apoptosis related to Bcl-2, Bax proteinum expression content of influence.Indirectly study EGB's brain protection function and possibility of protection mechanism, for acute ischemic cerebrovascular disease of clinical treatment in order to provide theoretical foundation and experimental basis.Methods:Selection of the 36 experiment and health male Wistar big rats, the quality is 200-250gs.The methods of application alloxan have preparate the diabetes big rat models, cavity injected dose is 120/100mg/kg for 2days. 72 hours later,the rats are empressed of takeing tail vein's blood about 20μl, with empty the stomach blood sugar≥16.7 mmol/L,the sucessful molds of diabetes big rats are to be succeced . After 6 weeks later, with the adoption of reforming line bolt 's method , Wistar diabetic rats were subjected to middle cerebral artery permanency occlusion molds.Model after the establishment, 36 rats are processed the neurologic impairment scores , the 1-3 score ratsare taken as the research objects. Correspond the model rats are randomly complemented so that the experiment animals amount is constant.Then the Wistar model rats are randomly divided into two groups: ischemic control group, and EGB treat group. Each rats In EGB treat group was intraperitoneal injected with EGB(Ginaton) 50mg/kg per day after occlusion, ischemic control group was administered the equatable sodium Chloride in stead of drug. Two groups are divided into 3 teams according to 3 days,7 days and 14 days,each team was composed of 6 model rats.According to the time observation point , the models were killed at surgery 3 days,7 days and 14days, the rats were fastly broken a head and taken the brain, exposed the heart, injected sodium chloridea 100ml through the left ventricles and affused 4% paraform (4℃,pH7.4) 150ml. Take the anterior fontanelle as the center, the coronal plane±1 mm, the brain slice was sliced , and placed in 4% paraform to fix. Routine dehydrate, transparent, immerse in wax, entrapment, the rats brain's coronal plane paraffin section was sliced continuously to make 6μm thick every 100μm.Choose the same lay and border brain slice, respective observation in ischemia peripheral area was adopted, in order to contrast two groups the amount of nerve cell apoptotis by TUNEL staining counting method and immunohisto chemistry staining were performed to examine proto-oncogene B-cell lymphoma/leukemia gene-2(bcl-2) and proto-oncogene Bc1-associated x protein(bax) expression variety circumstance in the effects of this EGB treatment.Results:Though the comparison of two groups, The counts of TUNEL masccline apoptosis cell was increased significantly in the ischemic control group compared with the EGB treat groups (P<0.01), statistics has the difference. The expressing of Bcl-2 significantly increased,Bax significantly decreased in the EGB treat groups compared with the control group. Comparation of the two groups ,the ratio of Bcl-2/Bax protein was increased significantly in EGB treat groups, and the difference has significance in statistics.Discussion:Diabetes is a kind of disease which severely harm mankind health, the diabetic alter merger cerebral pathological changes, nervous system defection and damage compare non- diabetic more severity.The diabetes cerebral disease is the main complications of the diabetic and one of the cause of deaths, with non- diabetes cerebral disease comparison,diabetes cerebral patient with the result that Mutilation rate, the case fatality rate all significantly increased, prognosis is worse, severely dangerous to mankind health.According to report way diabetes cerebral disease above 90% is ischemia cerebrovascular disease, only minority performance subarachnoid hemorrhage and cerebral hemorrhage.Therefore, the cerebrovascular disease of the diabetes is subjected more and more to domestic and international scholars.EGB, as an antioxidant drug, is concluded to have potent and promising therapeutic effect for stroke treatment.The injection of ginaton was the fourth era standard preparation of extracts from Ginkgo biloba, currently,and be applicated extensively in clinical treatment ischemic cerebrovascular disease. EGB contains many different flavone glycosides and terpenoides.It has the variety beneficial pharmacology activity and the depressant effect to variety enzyme, can direct the anti- oxidizeand clear the free radicals, extend blood vessel, improve the brain artery blood-supply . It can be specificitly antagonize platelet activating factor (PAF), ease brain dropsy, improve brain discharge, strengthen a nerve cell metabolism function.After focal cerebral ischemia, the neuron death in ischemia of cerebral issue has two kinds of dissimilarity of way: neorobiosis and cell apoptosis , both can be keep concomitance.To the cell of the ischemia core area, because of long time severity ischemia and anoxia , with neuron neorobiosis is lord, is irreversible process.But ischemia semidarkness is located on surroundings of cerebral ischemia focus, its cell death of modality is the main regard neuron apoptosis as principle.Therefore in cerebral ischemia earlier period,it can be given the valid of brain protection, salvaged the nerve cell in cerebral ischemia semidarkness area, decreased and conversedthe cell apoptosis, which is the key of decrease the physical cerebral ischemia volume and treat acute cerebral ischemia.This research result manifestation, the cell of apoptosis be mostly located on in the periphery area of cerebral ischemia , in control group it is clear existence that a great deal of of the cell of apoptosis at the semidarkness area of cerebral ischemia , such as cerebral ischemia edge seamy side and sea horse...etc. is most outstanding.Though the number of comparison of cell apoptosis in two group at the time observe point, EGB treatment group in the periphery area of cerebral ischemia , TUNEL masculine gender cell number obviously more reduced than in ischemia control group which is thus clear that a great deal of the cell of, P<0.01, have statistics to learn meaning. Summarizably, Elucidation gingko product can decrease the occurrence amount of cell apoptosis in which cerebral infarction semidarkness in diabetes merger cerebral infarction focus rats. To cell apoptosis have obvious of repress a function, to impatient the cerebral ischemia have explicit of brain protection function.The cell apoptosis is subjected to the adjust and control of related variety gene of apoptosis, includes of proto-oncogene B cell lymphoid lump-2(Bcl-2),Bax etc. The Bcl-2 and Bax are the importance controling gene proteinum to ischemia brain injured , reflection the cell apoptosis but a contrary set of the function.Bc1-2 is the important proteinum of repressing neuron apoptosis, with nerve cell survival close related. The Bax proteinum function is oppose to Bcl-2's, basic creature function is promote cell apoptosis.The Bcl-2 function of repressing cell apoptosis must to be realizated though the interaction that to be formed Bcl-2-Bax heterodimers with Bax. A figure type to the comparison of Bcl-2/Bax's determine the cell destiny that is existence or apoptosis, the increasing proportionality can repress cell apoptosis.The research indicates that Bcl-2 proteinum almost have no expression in the ischemia center area of two groups rats , Bax proteinum present only a little amount of expression.But the Bcl-2 of EGB treatment group have a great deal of expression in the area of the edge of ischemia and sea horse inside.Expression amount is obviously increased compared with the ischemia control group. Contrarily, in the center region of cerebral ischemia,we can detect significantly the expression increase about bax proteinum.In the meantime the expression decrease of Bax proteinum in the EGB tereat group, the number of immunity masculine cell in the cerebral ischemia periphery was obviously more fewer than that of the ischemia group at 3 days, 7 days and 14 days(P<0.01).The elucidation is that extract from gingko leaf can promote the expression of Bcl-2 proteinum , restrain the expression of Bax proteinum and prevent or decrease the occurrence that cell apoptosis because of ischemia.EGB treatment group, Bcl-2 of expression content after ischemia first attain high peak at 3days. along with injure time of extension, Bcl-2 expression content with apoptosis cell amount of increment but opposite decrease.This kind trend of distribute depend on time about Bcl-2 expression descend .It indicate that Bcl-2 proteinum have very importance function in the sensitivity of ischemia neuron,that may be one of the mechanisms about the neuron ego protection.Correspondingly, we can observe that Bax proteinum attain high peak just at 7 days after ischemia in this research.it can be conclude that when the elucidation nerve cell be subjected to injure, firstly start gene Bcl-2 expression with repress neuronic cell apoptosis, along with ischemia time of increment, the factor of promoting cell apoptosis start increase and make the peak of Bax proteinum expression appears the hysteresis, subsequently appear the neuronic cell apoptosis high peak of emergence, hint which the 7 days after MCAO may be the high peak of the cell apoptosis period.This experiment result manifestation, compared with the control group, at three time point of cerebral ischemia 3 days,7 days,14 days, Elucidation EGB possess of enhancing the founction Bcl-2 expression anddecreacing Bax expression, in order to elevated the ratio of Bcl-2/Bax protein, the difference has significance in statistics.In a word, the EGB can up-regulate Bcl-2 of expression in the periphery area of brain ischemia ,down-regulate that of Bcl-2, exaltation Bcl-2/Bax relative value, correspondly decrease neuronic cell apoptosis, match at each time point the amount of cell apoptosis in the EGB treatment group obviously lessen than that of ischemia control.In the meantime, the conclusion explanate the phenomena that neurologic impairment scores in the EGB treatment group lower than in the ischemia control group.By this token,the elucidation that EGB can repress cell apoptosis can be the protection function to the hurt of nerve function , promoting nerve function instaurationin the diabetes combine cerebral ischemia. Thus it provides the optly Medication time and course of treatment in the medical treatment for in the diabetes combine cerebral ischemia.Conclusions:1. The extracts from gingko leaf reduce the amount of neuronic cell apoptosis in the area of cerebral infarction semidarkness to the cell apoptosis have obvious repressive function.2. The extracts from gingko leaf can enhance the therapeutic potency possibly through inhibition of neuronal apoptosis; enhancing Bcl-2 expression,decreacing Bax expression and exaltation Bcl-2/Bax protein relative value might be one of the therapeutic mechanisms of EGB treated stroke in diabetic rats. |
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