| Alzheimer’s disease (AD) is a neurodegenerative disease accompanied by main clinical manifestations of progressive memory impairment, and which is a common and frequently encountered disease in the aged. Now, the world has faced to the population aging, the incidence of AD is increasing. Then, the effort of the pathology and treatment for the disease has become a focus in the international pharmaceutical industry.AD is a neurodegenerative disease with multi-pathogenetic participated, which is more complex involved in many factors. The neuron loss, glial cells and related substances abnormal increase, neurotransmitters and their receptors decrease, intracellular calcium overload, extracellular amyloid beta protein (Aβ) deposited for forming the senile plaques (SP), tau protein hyperphosphorylation for producing neurofibrillary tangle (NFT) in extracellular, the neuron apoptosis and neuro-inflammation are closely associated to the AD. It is pivotal to establish a suitable in vivo/in vitro model, and investigate its pathogenesis and treatment for AD.After a long period of study, the deposition of AP in the brain is considered as an important role in the pathogenesis of AD. A(3peptides injected by intracerebroventricl can induce animal memory impairment, and if combination with aluminum and recombinant human transforming growth factor-β1(RHTGF-β1), a multiple injured AD model established by three factors together can comprehensively simulate human AD pathological and physiological features and is closer to that of human AD multiple pathogenic characteristics. So, any strategies improved on neurological disorders with this animal model can be predicted a possibility for treatment of AD.Scutellaria Barbata Flavonoids (SBF), a novel flavonoid, is isolated from aerial parts of Scutellaria barbata, and scutellarein is the major ingredient. It has been reported that SBF possesses effects in antipyretic, antibacterial, antimutation, antioxiditive and other medical properties. SBF has obvious improvement on memory impairment and neuropathology disorders with ovariectomized rats, but for AD model of Aβ25-35combination with aluminum and RHTGF-β1reported. In this study, the rats were intracerebroventricularly injected Aβ25-35in combination with aluminium trichloride (AICI3) and RHTGF-(31to establish a brain injury model, and confirmed the effects and multi-pathway and multi-targets’ mechanism of SBF in memory deficits and neuropathological damages by measurement of learning and memory, neuropathology, glial cells and related to substrates, as well as the apoptotic factors of mitochondria pathway.PART ONE Improvement of Scutellaria Barbata flavonoids on memory deficits induced by compound Aβ25-35in ratsObjective:To establish the model of memory impaired and neurologicdamaged by intracerebroventricular injection of amyloid beta protein25-35(Aβ25-35) in combination with A1C13and RHTGF-β1in rats and detect the effects of Scutellaria Barbata flavonoids (SBF) on memory impaired and neurologicdamaged with this compound Aβ25-35rats.Methods:300-350g male SD rats were adaptive feeding for one week in clean animal lab before the operation. The rats received intracere-broventricular injection of RHTGF-β11μl (10ng) on the day1of operation, and then intracerebroventricularly injected Aβ25-35and A1C13from the day2of operation. Aβ25-354μg/d and1%AlC133μL/d were injected in every morning for consecutive14d and in every afternoon for consecutive5d, respectively. Sham control rats were conducted the same operation, but intracerebroventricularly injected the equal volume saline. On the day45of operation, all rats were performed the Morris water maze training for successful memory impairment model screening. The swimming scores of every operated rat and sham control rats on the day4of water maze training were used for calculating the successful model screening ratio (SR). When SR of any rat was more than0.2, the rat was confirmed to be a successful model rat. On the day49of operation, all successful model rats were randomly divided into4groups, model control (model control group) and3doses of SBF group rats. The SBF group rats were administered35,70and140mg/kg SBF for38d, and model control and sham control rats were given an equal volume of saline. On the day31administered of drug that is on the day79operation, all the rats were carried out the consecutive7d Morris water maze task for memory ability detection. The positioning navigation task on the day1and2of Morris water maze training was used to evaluate the rats’memory acquirement, the probe trial on the day3of Morris water maze was used to detect the memory retention, the reversal trial on the day of4,5and6of Morris water maze was used to evaluate the rats’ memory reproducibility and visible platform trial was used to test the rats’ swimming speed. On the second day of the last water maze task, that was the86d after operation, all the rats were decapitated, the microanatomy of brain was observed with naked eye and the morphologic structure/substructure of neuron were examined with light/electron microscope by hematoxylin-eosin (HE)/thiamine or uranyl acetate and lead nitrate-sodium citrate staining, respectively. Results:In the4d successful model rats’ screening by Morris water maze task, the time taken in finding the hidden platform (latency) progressively declined in all rats. If the SR value was greater than0.2according to the latency of each model control and sham control rats taken for finding the hidden platform at the day4training in Morris water maze task. The rat was confirmed as successful model rat. The successful rate of memory impairment model in the present study was94.7%. In the positioning navigation experiment, model control rats always took longer latency to find the hidden platform on the day I and2test in the water maze task, as compared with the sham control (P<0.01). However, SBF can significantly shorten the time to reach the platform as compared with model control rats (P<0.01). In the probe trial, the model control rats took decreased time, distance and crossing number in target quadrant (the first quadrant) within60s, as compared with sham control rats (P<0.01). While the three dose of SBF at35,70and140mg/kg have differently attenuated the above decreases in swimming time, swimming distance and crossing number of model control rats in target quadrant(P<0.05, P<0.01). In the reversal trial, the model control rats always spent longer time to find the hidden platform on the day4,5and6of Morris water maze test (P<0.01). Interestingly, SBF35,70and140mg/kg can significantly shorten latency onto the platform of model control rats (,P<0.05, P<0.01). In the visible platform test, that was the7th Morris water maze training, the time of all rats in each group spent to reach the visible platform was no significantly difference. The result indicated that swimming speed of each group rats were approximate in water maze, which may eliminate disturbance of swimming score as their swimming speed discrepancy. On the86d of operation, all the rats were sacrificed by decapitation, the conspicuous histological changes of model control rats were viewed by naked vision, including lessened weight of brain, yellow surface of cerebral cortex, and cortical thinned or collapsed in some rats. The neuron of hippocampus and cerebral cortex was found a markedly pathological change by HE and thionine staining under light microscope observation, such as neuron loss, cell swelling and Nissl bodies decrease. Some neuron of cerebral cortex in model control rats occurred typical colliquative necrosis, including cell membrane broken, nucleus dissolved and a great inflammatory cell infiltrated in necrosis region. Electron microscopy found that the hippocampus neuron pyknosis, nuclear membrane pit, nucleolus margination, chromatin condensation, ribosome separation in cytoplasm, a large lipofuscin deposition, mitochondria swelling, endoplasmic reticulum expansion, astrocyte foot processes edema, myelin sheath lamellar damage, axon and myelinated nerve loss, a large excitatory neurotransmitter at presynaptic membrane. However, different dose of SBF by administration for38d differently reversed the above neuronal pathological changes induced by compound Aβ25-35which the neuron and Nissl body count increase, the typical colliquative necrosis and subcellular structure damages absents. Conclusion:Intracerebroventricular injection of Aβ25-35combined with AIC13and RHTGF-β1can result in an impairment of memory and damages of neuro-structure. Then, SBF was found that has markedly ameliorations on memory impaired and neuron damaged using this rats’model.PART TWO Inhibition of Scutellaria Barbata flavonoids on brain’s Aβ and NFT abnormal generation induced by compound Aβ25-35in ratsObjective:To determine the abnormal generation of Aβ and NFT in brain induced by compound Aβ25-35using the compound Aβ25-35successful model rats. And elucidate the improvement and effective mechanism of SBF on memory impaired and neuronal damaged through the inhibition of Aβ and NFT abnormal generation and regulation of three secretases α-, β-and γ-level. Methods:The all rats were decapitated60min after the last drug administered, and then the brain was gently separated on ice. The Congo red and silver nitrate staining was used for Aβ and NFT determination, respectively, and quantitative real-time polymerase chain reaction (qPCR) method was used for determination of a, β and γ secretases mRNA expression levels. Results:Compared with sham control, the cells number of Aβ congo red stained and NFT silver nitrate stained in model control were significantly increased (P<0.01). NFT cells in cerebral cortex presented the neurofibril thicken and disordered all over the cytoplasm and even extended the dendrite, and finally to form a deep staining and tailing state. However,35,70and140mg/kg SBF were differently inhibited Aβ and NFT formation in the brain by daily and orally administration for38d (P<0.05, P<0.01). The determination of three secretases α-, β-and γ-expression found that the mRNA expressions of β-and γ-secestase significantly increased (P<0.01) and decreased (P<0.05) in model control, respectively, as compared with sham control. However, the two doses of SBF70and140mg/kg can strongly reversed the increase in P-secretase mRNA expression (P<0.01), and the two doses of SBF35and140mg/kg further lowered y-secretase mRNA expression (P<0.05, P<0.01). Conclusions:Intracerebroventricular injection of Aβ25-35combined with AlC13and TGF-β1can dramatically cause Aβ and NFT abnormal generation, raise β-secretase expression and lower γ-secretase expression in rats’brain. SBF can inhibit Aβ and NFT overproduction and positively regulate β-and γ-secretases expressions, which suggested that SBF improved the rats’memory deficits and neuronal damages induced by compound Aβ25-35partly derive from suppressing the Ap and NFT disorders. PART THREE Inhibition of Scutellaria Barbata flavonoids on brain’s neurogliocyte proliferation and activation induced by compound Aβ25-35in ratsObjective:To confirm the brain’s neurogliocyte proliferation and activation induced by compound Aβ25-35using the successful model rats. And, through examination of neurogliocyte number and related to activated proteins to define the improvement of SBF on memory impairment and neuropathological disorder induced by compound AP25-35on account of inhibition of neurogliocyte proliferation and activation. Methods:All the rats were decapitated60min after the last SBF administered, and then the brain was gently separated on ice. SABC immunohistochemical method were used to detect astrocyte (AS), neuronal nitric oxide synthase (nNOS) and induced nitric oxide synthase (iNOS) protein expressions, the silver carbonate was used for microglia (MG) number measurement, quantitative real-time polymerase chain reaction (qPCR) method was used for endothelial nitric oxide synthase (eNOS) mRNA expression, western blotting method was for determination of glial fibrillary acidic protein(GFAP), leukocyte common antigen (CD45), heat shock protein(HSP) protein expressions, RT-PCR method was used for detecting ApoE mRNA expression and ELISA methods were for measurement of interleukin-1beta (IL-1β), tumor necrosis factor-alpha (TNF-a), IL-6, IL-8) and IL-10levels, respectively. Results:The number of AS and MG, the protein expressions of GFAP/CD45/HSP70and iNOS, the mRNA expressions of ApoE and eNOS, and the levels of TNF/IL-6/IL-8were all significantly increased (P<0.01), and the protein expression of nNOS and the levels of IL-1β and IL-10were dramatically lowered (P<0.01) in model control rats’brain, respectively, as compared with sham control. Interestingly,35,70and140mg/kg SBF can differently reverse the above detrimental changes induced by compound Aβ25-35in rats’ brain. Conclusions:The compound Aβ25-35can cause neurogliocyte proliferation and activation and accompanied by the abnormal expressions of GFAP/CD45HSP70/iNOS/NOS and aberrant levels of IL-1β, TNF-α, IL-6, IL-8and IL-10. SBF can significantly reverse the above detrimental changes, which suggested that the improvement of SBF on memory deficits and neurologicdamages derived from suppressing the neurogliocyte proliferation and activation induced by compound Aβ25-35.PART FOUR Positive regulation of Scutellaria Barbata flavonoids on brain’s mitochrondria apoptosis pathway disorder induced by compound Aβ25-35in ratsObjective:To find the abnormal alterations of the brain’s apoptotic factors B cell lymphoma-2(BCL-2), BCL-2associated X protein (BAX) and cysteinyl aspartate specific proteinase-3(Caspase-3) in mitochrondria apoptosis pathway induced by compound Aβ25-35. And through examination of the above apoptotic factors to ascertain the improvement of SBF on memory impairments and neuropathological disorder induced by compound Aβ25-35in view of positive regulating the expressions of apoptotic factors of mitochondria pathway. Methods: All the rats were decapitated60min after the last drug administered, and then the brain was gently separated on ice. The western blotting method was used to detect the protein expressions of BCL-2and BAX and qPCR method was selected for determining the mRNA expression of Caspase-3. Results:The compound Aβ25-35can cause the deleterious changes of Bcl-2, Bax and Caspase in cerebral cortex. Compared with sham control, the protein expressions of BCL-2markedly lowered (P<0.05, P<0.01), and protein expressions of BAX and mRNA expression of Caspase-3were significantly increased (P<0.01, P<0.05), respectively. However, SBF35,70and140mg/kg can differently reverse the apoptotic factors disorders induced by Aβ25-35. The protein expression of BCL-2significantly increased (P<0.05), and the expressions BAX protein and Caspase-3mRNA dramatically decreased (P><0.05, P<0.01), respectively in three doses of SBF group, as compared with model control. Conclusions:The compound Aβ25-35can result in abnormal expression of BCL-2, BAX and Caspase-3of mitochondria apoptosis pathway, and three doses of SBF can differently reverse the above disturbances induced by compound Aβ25-35, which indicated that the amelioration of SBF on memory impairments and neuronal damages partly originated from the up-modulation on mitochondria apoptosis pathway disorder.CONCLUSIONSThe rats’model established by intracerebroventricular injection of Aβ25-35in combination with AlC13and RHTGF-β1appear memory impairment and symbolic neuronal disorders, and it is found that SBF can conspicuously improve the rats’memory acquirement, memory retention, memory reproducibility disorders and neuronal disturbance induced by compound A(325-35, using this rats’model, which the effective mechanism of SBF are primary from inhibiting Aβ and NFT abnormal generation, neurogliocyte proliferation and activation, and up regulating mitochondria apoptotic pathway. Then, the present studies suggest that the multi-regulation of SBF on serial molecule is the foundation on anti-AD, and clarify multi-pathway and multi-targets molecular mechanism of SBF in improvement of memory deficits and neuronal damages. |
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