GGA Induce The Expression Of HSP70 In Hippocampus Of AD Rats And The Study On The Anti-apoptotic Mechanisms Of HSP70 In AD

PART ONE:GGA INDUCE THE EXPRESSING CHANGE OF HEAT SHOCK PROTEIN 70 IN HIPPOCAMPUS OF THE ALZHEIMER DISEASE RATS AND IT'S INFLUENCE ON THE ETHOLOGY AND HISTOLOGY OF THE RATSBackground and purpose Alzheimer disease(AD)is a central nervous system degenerative disease which character is progressive decline of memory and cognition's disturbance.The etiopathogenisis of this disease has not been clarified still.Nowadays,some studies have showed that there are some expressing changes of heat shock protein (HSP)in AD brain and these changes are related to the survival of neuron. HSP is a highly conservative polypeptide protein family with important physiological functions,which is induced by stress in cell.HSP can maintain normal structures and functions of protein as molecular chaperone and protect cell from oxidation and apoptosis and enhance cell's endurance in stress.It has been proved that Geranylgeranylacetone (GGA)can induced HSP70 expression in stomach intestine,liver,heart, arnphiblestrodes and central nervous system.GGA has been applied in cerebral vascular disease model in external and studies has showed GGA can induced HSP70 expression in brain of rats and produce a neuroprotective effect.Nowadays,there has no research in external and internal on whether GGA could be applied in AD.In our study,we built AD model and observed the HSP70 expression and the changes of ethology and histology in AD model rats,as well as in the rats which were administrated with GGA.We studied that GGA induced HSP70 expression in hippocampus of AD rats and it's neuroprotective effect and discussed the mechanisms,in order to provide theory and new method for clinical treatment of AD.Methods 144 healthy male Sprague-Dawley rats were randomly divided into control group,AD model group and GGA group(48 rats in each group).Each group were divided into four subgroups according to the time after operation(1^st,7^th,14^thand 21^stday),12 rats in each subgroup.The rats in AD model group and GGA group were injected with Aβ_1-42into hippocampus and the rats in control group were injected with corresponding dissolvant into hippocampus.After operation,the rats in GGA group were given GGA with a dose of 800mg/kg/d through intragastric administration for 21 days and the rats in the other groups were given Sodium Chloride.Y maze test was used to study the learning and memory ability of the rats before injection and at the 7^th,14^thand 21^st day after injection into hippocampus.The rats were executed as soon as the Y maze test finished at corresponding time point after operation.The brains were removed for frozen sections and the neuronal morphologic changes in hippocampal CA1 area and Aβprecipitation in brain were observed by HE stain and Congo red stain respectively.The expression of HSP70 in hippocampus of rats was detected by RT-PCR and Western-blot technique.Results 1.Compared the groups with the learning and memory ability, the difference among the three groups was no significant(P＞0.05)before injection.The learning and memory ability of the rats in model group significantly decreased at the 14^thday after injection than those in control group(P＜0.05),especially at 21^stday(P＜0.01).But the learning and memory ability of the rats improved significantly in GGA group at the 14^thand 21^stday(P＜0.05).2.The cells in hippocampal CA1 area of the rats in control group aligned regularly and the cellular structure was intact.But these cells in model group aligned irregularly and the cellular structure was indiscriminate and the neuron decreased.These dysfunctions were remarkable at 21^stday after operation.Compared with model group,the alignment of cells and the cellular structure in GGA group was improved visibly and the hippocampal neuron increased.3.Through Congo red stain for the sections of the brains of the rats in model group,we found that there were some positive staining arounded by some microglias concentrated between hippocampus CA1 area and dentate gyrus.These positive staining might be Aβprecipitation.No similar Aβprecipitation be found in brains of rats in control and GGA group.4.There were no difference in HSP70 expression in hippocampus among the three group at 1^stday after operation(P＞0.05).Compared with control group,the HSP70 expressions in hippocampus tissues of the rats in model group decreased gradually at the 7^th,14^th,and 21^stday after injection Aβ_1-42and these decreases were significant(P＜0.05).In GGA group,the HSP70 expressions in hippocampus increased than model group at corresponding time point(P＜0.05),especially at the 14^thand 21^st (P＜0.01)Conclusions 1.The toxic effect of Aβ_1-42make the expression of HSP70 in hippocampus of AD rats low and the low expression of HSP70 may impair the neuroprotection of HSP70.This mechanisms maybe participate the pathological proceeding of AD.2.The increasing expression of HSP70 in hippocampus may inhibit toxic effect of Aβ_1-42and ameliorate neural impairment and increase neural survival,so that improve the learning and memory ability of AD rats. PART TWO:GGA INDUCE THE EXPRESSION OF HEAT SHOCK PROTEIN 70 IN HIPPOCAMPUS OF THE ALZHEIMER DISEASE RATS AND IT'S INFLUENCE ON THE NEURONIC APOPTOSIS AND THE EXPRESSING CHANGES OF CYTOCHROMEC,CASPASE-9,FADD AND CASPASE-8 IN HIPPOCAMPUSNeuronic loss in hippocampus and cortex is one of the major pathological characters of AD.Studies to AD have showed that there are close relationship between the neuronic loss and cognitive impairment of AD.The major neuronic loss result from apoptosis induced by Aβ.The activation of caspase pathway is a main cause in triggering and executing neuronic apoptosis,which includes mitochondrial pathway and death receptor pathway in cell membrance.Some studies in vitro have showed HSP70 plays a key role in restriction of apoptosis.But the results of studies to the mechanisms,especially about the death receptor pathway,were indefinite,even contradictory.There has still no research on HSP70 inhibiting apoptosis in AD in vivo.In our study,we established AD models and observed the neuron apoptosis and the expression of HSP70 in hippocampus of the rats in AD group and GGA group.At the same time,We detected the expressing changes of apoptotic correlation factors including cytochrome C,caspase-9,FADD and caspase-8 in hippocampus of the rats in each group.According to the experiment,we discussed the mechanisms about HSP70 induced by GGA inhibiting apoptosis in the brain of AD,in order to provide theory and new method for clinical treatment of AD.Methods 36 healthy male Sprague-Dawley rats tested by Y-maze were randomly divided into control group,AD model group and GGA group(12 rats in each group).The rats in AD model group and GGA group were injected with Aβ_1-42into hippocampus and the rats in control group were injected with corresponding dissolvant into hippocampus. After operation,the rats in GGA group were given GGA with a dose of 800mg/kg/d through intragastric administration for 21 days and the rats in the other groups were given Sodium Chloride.The rats were executed at 21^stday after operation.The brains of 6 rats in each group were removed for frozen sections and used a dual procedure involving immunofluorescence for Hsp70 localization and TUNEL assay for cell apoptosis to correlate the pattern of Hsp70 expression and cell apoptosis, and the results were observed through confocal laser scaning microscope (CLSM).The brains of another 6 rats in each group were removed for detecting the expression of cytochrome C,caspase-9,FADD and caspase-8 in hippocampus by Western-blot technique.Results 1.There were few TUNEL-positive cells which represented apoptotic cells labeled by FITC-dUTP presenting green fluorescence in the nucleus and few HSP70-positive cells labeled by TRITC presenting red fluorescence in the cytoplasm in the areas from hippoamppal CA1area to dentate gyrus of the rats in control group.In hippocampus of the rats in model group,the TUNEL-positive cells increased prominently(P＜0.01)and HSP70-positive cells decreased significantly (P＜0.05)compared with the control group.However,in GGA group, the TUNEL-positive cells decreased notably(P＜0.01)and HSP70-positive cells increased prominently(P＜0.01)compared with the model group.Most cells which cytoplasm appeared HSP70-positive didn't labeled by FITC-dUTP in the nucleus,on the contrary,in the TUNEL-positive cells,the HSP70 expression in cytoplasm were slight or lacking.2.The expression of caspase-9 in hippocampus of the rats in model group was increased compared with the control group and the difference was significant(P＜0.05).The expression of caspase-9 in hippocampus of the rats in GGA group was decreased prominently(P＜0.05)compared with the model group.3.Compared with the control group,the expression of FADD in hippocampus of the rats in model group was increased significantly (P＜0.05),as well as GGA group.And the difference between model group and GGA group was not significant(P＞0.05). 4.The expression of caspase-8 in hippocampus of the rats in model group was increased compared with the control group and the difference was significant(P＜0.05).The expression of caspase-8 in hippocampus of the rats in GGA group was decreased notably(P＜0.05)compared with the model group and there were no difference between GGA group and control group(P＞0.05).5.The expression of cytochromeC in hippocampus of the rats in model group was increased significantly(P＜0.05)compared with the control group.The expression of cytochromeC in hippocampus of the rats in GGA group was decreased prominently(P＜0.05)compared with the model group.Conclusions 1.Aβ_1-42can precipitate neuron apoptosis by activating apoptotic pathway including mitochondrial and death receptor pathway. This mechanisms is important to the nerval impairment in AD.2.The neuroprotective effect of increasing expression of HSP70 in hippocampus of AD induced by GGA may be related to it's increasing antiapoptotic mechanisms and relieving neuron damage.