| In many age-related diseases,prevalence of neurodegenerative disease increased year by year.Alzheimer's disease(AD)also called senile dementia is a neurodegenerative disease accompanied by main clinical manifestations of progressive memory impairment.In recent years'study of AD,the deposition of(3-amyloid protein(A?)is considered as the main structural material of senile plaques(SP)in the brain of AD,and its toxic effect plays an important role in the mechanism of AD pathology.A? peptides injected by intraventricular can induce animal memory impairment,and if combination with aluminum chloride and TGF ?-1,a multiple injury AD model established by three drugs together can comprehensively simulate human AD multiple pathological and physiological features.Programmed cell death/Apoptosis which has close connection with central nervous system aging and AD occurrence is an active physiological death of gene regulation in order to maintain tissue homeostasis.Apoptosis is one of the reasons for the neuron loss of neurodegenerative disease.Neuronal apoptosis is activated by internal and external factors through cell's own genetic program,and accelerate neuronal apoptosis will induce AD.The main pathway of apoptosis is roughly divided into mitochondrial signaling pathway,endoplasmic reticulum road and death receptor pathway according to its initiate and apoptosis transduction pathway.Mitochondria is now generally considered to be the center of the control of apoptosis and the main place to produce oxygen free radicals,and mitochondrial dysfunction or mitochondria mediated neuronal apoptosis plays an important role in the occurrence and development process of degenerative disease.A(3 is mainly through activation of the mitochondrial apoptotic pathway induce neuronal apoptosis and cause neuronal loss,which is vital in the pathogenesis and pathological process of AD.Scutellaria barbata flavonoids(SBF),a group of flavonoid extracted and separated from Scutellaria Barbata D(Don),has a protective effect on the nerve due to its multi-hydroxy structure with a strong reduction.Modem pharmacological found that SBF has a variety of pharmacological effects on antioxidation,antimutagenic,antitumor,memory impairment and neuropathological changes.In this study,the rats were intracerebroventricularly injected A?25-35 in combination with AICl3 and RHTGF-?1 to establish the brain injury model and detected the effects and its mechanism of SBF on mitochondrial apoptotic pathway abnormalities in rat brain.The aim of study is to provide an experimental foundation of SBF for against dementia.Objective:To investigate the effection of SBF on mitochondrial apoptotic pathway abnormalities in rat brain induced by complex A?25-35.Methods:Healthy 300-350g male rats,12h light/dark illumination,conventional pellets feed,free drinking water,adaptive feeding a week,12 hours of fasting before surgery.Intraperitoneal injection of 10%chloral hydrate(300mg/kg)for surgery anesthesia,and intraventricular injection of RHTGF-?1 10ng on the first day of operation,and then intraventricularly injected A?25-35 and AlCl3 from the second day of operation.A?25-35 4?g/d and 1%AlCl3 3?l/d were consecutive injected in every morning for 14d and in every afternoon for 5d,respectively.The sham rats were conducted the same operation,but intraventricularly injected the equal volume saline.On the 45th day of operation,the rats were performed the Morris water maze training for successful memory impairment model screening.On the 49th day of operation,the successful model rats were randomly divided into 4 groups,A(3 group and 3 doses of SBF group rats.The drug group rats were administered into 35,70 and 140 mg/kg·d-1 SBF and A(3 group and sham operated group rats were given an equal volume of saline.After continuous administration 36d,that on the 86d of operation all the rats were sacrificed by decapitation for indicators detection.Western blotting method was for B-cell lymphoma/leukemia-x/1 gene,Bcl-2 antagonist/killer in cortical mitochondria and Cysteinyl aspartate specific protease-3 protein expression in cortical cytoplasm determination.RT-PCR method detected Cytochrome C,Apoptotic protease activating factor-1,Cysteinyl aspartate specific protease-9 mRNA expression in cortical cytoplasm.Results:1.Determination of the expression of Bak protein in cortical mitochondria.The protein relative expression of Bak on A?25-35 damaged rats in cerebral cortex detected by western blotting.The results indicated that compared with the sham group,the Bak protein expression of cerebral cortex mitochondria in A? group was significantly increased(P<0.01).However,the increased expression can be obviously reversed by SBF at does of 35,70 and 140 mg/kg,as compared with model group(P<0.01).2.Determination of the expression of Bcl-xL protein in cortical mitochondria.The protein relative expression of Bcl-xL on A?25-35 damaged rats in cerebral cortex mitochondria detected by western blotting.The results indicated that compared with the sham group,the Bcl-xL protein expression of cerebral cortex mitochondria in A? group was significantly decreased(P<0.01).However,the decreased expression can be obviously reversed by SBF at does of 35,70 and 140 mg/kg,as compared with model group(P<0.01).3.Determination of the expression of Caspase-3 protein in cortical cytoplasm.The protein relative expression of Caspase-3 on A?25-35 damaged rats in cortical cytoplasm detected by western blotting.The results indicated that compared with the sham group,the Caspase-3 protein expression of cortical cytoplasm in A? group was significantly increased(P<0.01).However,the increased expression can be obviously reversed by SBF at does of 35,70 and 140 mg/kg,as compared with model group(P<0.01).4.Determination of the expression of Cyt-C mRNA in cortical cytoplasm.The Cyt-C mRNA relative expression of A?25-35 damaged rats in cortical cytoplasm detected by RT-PCR.The results indicated that compared with the sham group,the Cyt-C mRNA expression of cortical cytoplasm in A?group was significantly increased(P<0.01).However,the increased expression can be obviously reversed by SBF at does of 35,70 and 140 mg/kg,as compared with model group(P<0.01).5.Determination of the expression of Apaf-1 mRNA in cortical cytoplasm.The Apaf-1 mRNA relative expression of A?25-35 damaged rats in cortical cytoplasm detected by RT-PCR.The results indicated that compared with the sham group,the Apaf-1 mRNA expression of cortical cytoplasm in A(3group was significantly increased(P<0.01).However,the increased expression can be obviously reversed by SBF at does of 35,70 and 140 mg/kg,as compared with model group(P<0.01).6.Determination of the expression of Caspase-9 mRNA in cortical cytoplasm.The Caspase-9 mRNA relative expression of A?25-35 damaged rats in cortical cytoplasm detected by RT-PCR.The results indicated that compared with the sham group,the Caspase-9 mRNA expression of cortical cytoplasm in A(3group was significantly increased(P<0.01).However,the increased expression can be obviously reversed by SBF at does of 35,70 and 140 mg/kg,as compared with model group(P<0.01).Conclusion:1.SBF can significantly reduce the expression of apoptotic genes Bak in cortical mitochondria of rat brain induced by complex A?25-35 and improve the expression of anti-apoptotic genes Bcl-xL.2.SBF can obviously reduce the expression of Cyt-C,Apaf-1 and Caspase-9 mRNA and Caspase-3 protein in the cortex cytosol of rat brain induced by complex A?25-35.3.SBF has firmly improvements on rats impaired memory induced by A?25-35 combined with AlCl3 and RHTGF-(31,and its effective mechanism may reduce the expression of apoptotic genes Bak protein in cortical mitochondria,improve the expression of anti-apoptotic genes Bcl-xL protein in cortical mitochondria,reduce the expression of Caspase-3 protein in the cortex cytosol,reduce the expression of Cyt-C,Apaf-1 and Caspase-9 mRNA in the cortex cytosol.Therefore,SBF can inhibite neuronal apoptosis and play a protective effect on the nerve.The effect of SBF may help improve memory disorders and treatment of AD. |
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