Soluble Oligomers And Fibrillar Species Of Amyloidβ-Peptide Differentially Affect Cognitive Functions,Hippocampal Inflammatory Response And Effect By Piper Kadsura Ohwi

Background:Alzheimer’s disease is the most common degenerative disease of the central nervous system, its typical clinical manifestations are increasing memory loss, cognitive dysfunction, abnormal behavior and social barriers. Pathological changes of main are neurofibrillar tangles (NFT), senile plaque formation (SP) and neuron loss. The pathogenesis of AD is complex, so far it’s cause is unclear, since in the1980of the20th century, forming a lot of hypotheses, now the widely recognized hypothesis is beta-amyloid protein hypothesis, the main composition of SP is Aβ, Aβ produced by β, γ apocrine enzyme hydrolysis Aβ precursor protein(APP).Occurrence, development of AD and Aβ’s metabolic disorders due to abnormal deposition of basal forebrain are closely related. But the study found that in recent years, the Aβ oligomers compared with fibrillar Aβ with higher neurotoxicity, is the main toxic substances in the pathogenesis of AD. Currently The whole animal behavior of Aβ oligomer experiment results also controversial.comparative study of whole animal level between different forms of aggregation of Aβ on less.The current research on the pathogenesis of AD tips, chronic inflammation of central nervous system injury induced by Aβ activation of glial cells may be a pathological key led to AD. involved in the inflammatory response is mainly microglial cells (Mi). located in Toll like receptor(TLRs) of the surface of Mi and its auxiliary receptor CD14are involved in the activation process of Aβ Mi,but the conclusion more from the research of fibril Aβ, TLRs can identify oligomers of AP still inconclusive.In addition, on the process of AD’s research, the animal model is always the key. But now all kinds of models are insufficient, still without a kind of animal model can fully reproduce simulation of AD pathology, biochemistry, behavior and other aspects of the full feature. This experiment use osmotic pump will Aβ1-42polymeric and oligomeric continuous perfusion to the lateral ventricle of the rat AD model was established, so that a large number of A βslowly diffuse distribution in aged rat brain, solve the similar model A short-term aggregation in local injection point better, closer to the clinical pathological process of AD.Traditional Chinese medicine Piper Kadsura is the Piperaceae pepper plants of the genus wind vine stems, Chinese Pharmacopoeia records having through main and collateral channels, rheumatism and relieving pain efficacy, modern medical research has found its anti-inflammatory and neuroprotective effects, and has been applied in treatment and research for AD in recent years.The research and application of fibrillar Aβ1-42and Aβ1-42oligomers intracerebroventricular continuous perfusion of rat model of AD preparation, observation of two different forms of aggregation of A f3on the behavior of rats, hippocampus nerve cell ultrastructure and Toll like receptor4(TLR4) and the expression of inflammatory factors the effects of simultaneous application of therapeutic intervention, futokadsura stem extract, observe its effect on these indexes, in order to further reveal the pathogenesis of AD, explore a safe and effective treatment method.Objective:To investigate the fibrillar Aβ1-42and A β1-42oligomers on the behavior of rats, hippocampus nerve cell ultrastructure and TLR4nuclear factor-κ B (NF-κB). tumor necrosis factor alpha (TNF-α) expression changes of influence; application of Piper Kadsura stem prognostic index above.Methods:60healthy male SD rats, were randomly divided into10groups:normal control group (Control group), sham operation group (Sham group), fibrillar Aβ+Vehicle group1(FAβ group, intracerebroventricular infusion of polymeric Aβ+ cetonitrile and three acetic acid fluoride die), polymeric Aβ+Piper Kadsura Ohwi (PKO) group (FAp+PKO group, intracerebroventricular infusion of polymeric Aβ+acetonitrile and three trifluoroacetic acid, molding, intraperitoneal injection of PKO treatment), polymeric Aβ+Dimethyl sulfoxide (DMSO)group (FAβ+DMSO group, intracerebroventricular infusion of polymeric Aβ+acetonitrile and three trifluoroacetic acid, intraperitoneal injection of DMSO), Vehicle1groups (group Vehl, intracerebroventricular injection of not Aβ, but only perfusion, acetonitrile and three trifluoroacetic acid), Aβ oligomer+Vehicle2group(ApO group, intracerebroventricular perfusion A P oligomer+high density lipoprotein and HEPES model), Aβ oligomer+PKO (APO+PKO group, high density lipoprotein and HEPES molding, intraperitoneal injection of PKO treatment), Ap oligomer+DMSO group (APO+DMSO group, ventricle instillation of Ap oligomer+high density lipoprotein and HEPES, intraperitoneal injection of DMSO), Vehicle2groups (Veh2group, ventricle no injection of Aβ, only perfusion of high density lipoprotein and HEPES). Using micro-osmotic pump infusion fibrillar A β1-42or A β1-42oligomer dimer into the lateral ventricle method of making animal model. A day after modeling for FAP+PKO and APO+PKO group rats were intraperitoneally injected with concentration of10%containing2.5%DMSO PKO, according to the weight of administration (1ml/100g), FAP+DMSO and ApO+DMSO rats every day only received intraperitoneal injection of2.5%DMSO (1ml/100g), continuous administration for35days. Thirty-first days after the model of learning and memory function in Morris water maze test evaluation in rats, the experiment was divided into two parts of space exploration and constant-bearing navigation. Space exploration experiment recording rat escape latent period, constant-bearing navigation test recording rats in the Morris water maze mobile video, calculating the times of crossing platform, time ratio and distance ratiothrough the quadrant in the platform. Electron microscopic observation on the ultrastructure of hippocampus neural cells, real-timepolymerasechainreactio,(RT-PCR) used to detect the expression of TLR4mRNA in the hippocampus, immunoblotting Western blot (WB) to detect the expression of NF-κBP65, TLR4 protein, enzyme labeled immunosorbent assay (ELISA) for the detection of the expression of TNF-α.Result:(1) the water maze experiment results:Control group, Sham group, Veh1group and Veh2group were compared between the escape latency, the times of crossing the platform, the time ratio and distance ratio had no significant difference (P>0.05); FAβ group compared with FAβ+DMSO group, A β O group compared with Aβ O+DMSO group in escape latency, the times of crossing the platform time and distance ratio, the ratio of no significant difference (P>0.05); the number of FAβ group, FAβ+DMSO and AβO group, AβO+DMSO group compared with Control group, Sham group, Vehl group and Veh2group had significantly longer escape latency, significantly reduces times of across the platform, the time ratio and distance ratio was significantly decreased (all P <0.05); AβO and FAβ group had significantly longer escape latency, significantly decreased times of a cross the platform, the time ratio and distance ratio was significantly decreased (all P <0.05); the number of FAβ+PKO and AβO+PKO group compared with Control group, Sham group, Vehl group and the Veh2group had significantly longer escape latency, significantly reduces times of across the platform, the time ratio and distance ratio was significantly decreased (all P <0.05); but FAβ group, FAβ+DMSO compared with AβO group, AβO+DMSO escape latency significantly shortened significantly, cross platform Increase, time ratio and distance ratio also increased significantly (P <0.05).(2)to observe the ultrastructure of hippocampus neural cells of CA1region under electron microscope: Control group, Sham group. Vehl group and Veh2group in CA1area of hippocampus nerve cell ultrastructure had no obvious abnormalities; A βO group and the AβO+DMSO and FA β group and FAβ+DMSO were seen neural cell apoptosis and the damage of different degree, FAβ group+PKO group are in the most serious injury; Ap()+PKO group showed a small amount of AβOptosis of nerve cells, nerve cell damage of AβO group and the AβO+DMSO and FA Pgroup and FAβ+DMSO are lighter. (3)electron microscopy semithin sections in CA1area of hippocampus neural cells apoptosis were observed under light microscope:Control group, Sham group, Vehl group and Veh2groupCA1of rat hippocampus were few of apoptotic neurons apoptosis of neurons, but there was no significant difference between groups (P>0.05); FAβ group, FAβ+DMSO group, ApO group and ApO+DMSO group in hippocampal CA1region of rats large number of apoptotic neurons were seen, and the number of apoptotic neurons compared with Control group, Sham group, Vehl group and Veh2group were significantly increased (P <0.05), but the FAβ group compared with FAβ+DMSO group, AβO group compared with the Aβ O+DMSO group apoptosis cells had no significant difference (P>0.05); hippocampal CA1region of AβO rats apoptosis neurons compared with FAβ group increased significantly (P<0.05);FAβ+PKO group and ApO+PKO group compared with Control group, Sham group,Vehl group, Veh2group the number of apoptotic neurons was increased (P <0.05), but compared with FAβ group, FA P+DMSO group, AβO and AβO+DMSO group decreased significantly (P <0.05).(4) RT-PCR, WB, ELISA results:Between Control group, Sham group, Vehl group and Veh2group compared with TLR4, NF-kB, TNF-a expression had no significant difference (P>0.05); FAβ group and FAβ+DMSO group, AβO group and ApO+DMSO group compared with TLR4, NF-κB, TNF-a the expression of no significant difference (P>0.05); FAβ group, FAβ+DMSO group, AβO group and ApO+DMSO group compared with Control group, Sham group, Vehl group and Veh2group the expression of TLR4,NF-κB,TNF-α increased significantly (all P <0.05); AβO group in the expression of TLR4,NF-κB,TNF-α compared with FAβ group was increased (P<0.05); FAβ+PKO group and ApO+PKO group in the expression of TLR4, NF-kB, TNF-a compared with Control group, Sham group, Vehl group, Veh2group were increased (P <0.05). but compared with FAβ group, FAβ+DMSO group, ApO and AβO+DMSO groups expression in TLR4,NF-κB, TNF-a decreased obviously (P <0.05). (5) the results of partial correlation analysis:TLR4, NF-κB, TNF-α with the times of crossing platform to present the negative correlation (P=0.000), TLR4, NF-κB, TNF-α were positive correlation (P=0.000).Conclusion:(1) infusion Aβ1-42into one side of the lateral ventricle constant can be successfully established the animal model of AD;(2) fibrillar Aβ1-42and A β1-42oligomers can induce the apoptosis of nerve cells, injury and TLR4, NF-κB, TNF-α over-expression, resulting in impairment of learning and memory in rats.(3) Aβ1-42oligomers can be recognized by the TLR4, but also compaired with fibrillar Aβ1-42, the induced TLR4, NF-κB, the expression of TNF-α is higher, apoptosis, damage and rat learning and memory dysfunction is more serious.(4) the Piper Kadsura intervention can improve learning and memory dysfunction induced by A in rats, its mechanism may be associated with reduced inflammatory factors TLR4, NF-κB, the expression of TNF-α, inhibiting nerve cell apoptosis.