| Alzheimer's disease is one kind of disease characterized by recent memory handicap,senile plaque(SP),neurofibrillary tangle(NFT),and neuron loss in pathology and gradually neuron's functional deterioration clinically,and it seriously affect the patient's cognition function,memory function,language function and space cognition function,social activities ability,personal living ability and emotion personality etc.The cause and nosogenesis of disease are still not clear.Several theories now widely recognized are:neurotransmitter impairment theory;calcium homeostasis imbalance theory;free radicals injury theory;gene mutation theory;energy dysbolism theory;nerve cell apoptosis theory;excitatory transmitter toxicity;β-Amyloid deposition theory etc.LYS, the root of one plant named Millettia pulchra Kurz var laxior(Dunn)Z.Wei,is a commonly used Chinese medicinal herb in Guangxi province.It has the function of supplying QI and blood,increasing the human ability of immunity,anti-aging, anti-stress etc.It can also make the body strong and alleviate fatigue so as to be applied to treat poor memory in old people and children's hypophrenia clinically. It can also be used for physical weak person and postnatal weakness women. Our research is to extract polysaccharides from LYS and to observe the effects of LYS polysaccharides on anti-dementia and its mechanism.In these experiments,NG108-15 cells were used to establish cell model of Alzheimer's disease induced by Aβ25-35.Kunming mice were used to establish learning and memory disorder model induced by scopomamine,40%ethanol, D-galactose respectively.Senescence accelerated mouse(SAM)was used to establish the AD model as well.The subgroup and administration of Kunming mice are:(1)normal control group:normal sodium,30ml·kg-1;(2)model group: normal sodium,30ml·kg-1;(3)pisacetam control group:pisacetam,0.5g·kg-1;(4) low-dose of LYSP group:LYSP,45mg·kg-1;(5)mid-dose of LYSP group: 90mg·kg-1;(6)high-dose of LYSP group:LYSP,180mg·kg-1.After treatment, the effects of LYSP on correct frequency in Y-water maze test,Ach and AchE in mice serum or brain were detected.The subgroup and administration of SAM are:(1)SAMR1 group:normal sodium,30ml·kg-1;(2)SAMP8 untreated control group:normal sodium,30ml·kg-1;(3)huperzine A control group:huperzine A, 0.02mg·kg-1;(4)low-dose of LYSP group:LYSP,45mg·kg-1;(5)mid-dose of LYSP group:90mg·kg-1;(6)high-dose of LYSP group:LYSP,180mg·kg-1.After treatment,the effects of LYSP on escape latency in Morris water maze test, SOD,GSH-Px,MDA,NO,Ach and AchE in mice serum or brain,NE,DA, 5-HT,Glu,Gin,Asp,Asn,Gly,GABA,APP,Aβ,BACE,PS1,PS2 and ApoE in mice brain were observed.The main results are as follows:1.The extraction,identification and analysis of LYSP compositionLYSP didn't contain protein and nucleic acid.It showed high purity of polysaccharides when identified by means of Sephadex G-75 column or high performance gel permeation chromatography(HPGPC).It was composed of glucose and arabinose.2.Improvement of LYSP on the learning and memory abilities in learning and memory disorder modelScopomamine,40%alcohol,D-galactose and senescence accelerated mouse prone/8 were used to establish learning and memory disorder model respectively.Then Y-water maze and Morris water maze test were used to assess the change of learning and memory abilities of each group.The results showed that all kinds of dementia model were significant impaired in learning and memory abilities,and LYSP-treated group could significantly improve the learning and memory abilities.3.Protective effect of LYSP on NG108-15 dementia cells modelLYSP and the serum containing LYSP were used to culture NG108-15 cell model of Alzheimer induced by Aβ25-35.The cell morphology,proliferation, survival rate,neurite outgrowth and average length were observed.The results showed that LYSP or the serum containing LYSP could protect NG108-15 cells from Aβ25-35 neurotoxicity:the cells morphology was at good condition,and the cell viability,proliferation,survival rate,neurite outgrowth and average length were significantly elevated.4.Effects of LYSP on free radical metabolism in SAMP8Spectrophotometry was used to detect SOD and GSH-Px activities,MDA and NO contents in SAMP8 serum and brain.The results showed that SOD and GSH-Px activities were decreased significantly,and MDA and NO contents were increased significantly in model group or SAMP8 serum and brain. LYSP-treated group could enhence SOD and GSH-Px activities and decrease significantly MDA and NO contents in SAMP8 serum and brain.It suggested that LYSP could scavenge effectively free radical for anti-dementia goal.5.Effects of LYSP on cholinergic neurotransmitter in dememntia modelSpectrophotometry was used to detect Ach content and AchE activity in dementia animal model induced by D-galactose and SAMP8 model serum and brain.The results showed that AchE activity decreased significantly and Ach content had no significant difference in SAMP8 serum and brain.LYSP-treated group could decrease AchE activity.It suggested that LYSP could reduce the degradation of Ach by decreaseing AchE activity for anti-dementia goal. 6.Effects of LYSP on monoamine neurotransmitter in SAMP8 brainThe NE,DA and 5-HT contents in brain were detected with HPLC.The results showed that the NE,DA and 5-HT contents in SAMP8 brain were decreased significantly and LYSP-treated group could increase the NE,DA and 5-HT contents.It suggested that LYSP has obvious anti-dementia effects,which could regulate effectively the disorderly metabolized monoamine transmitter.7.Effects of LYSP on amino acids neurotransmitter in SAMP8 brainThe contents of amino acids neurotransmitter in brain were detected with HPLC.The results showed that the contents of GLU,GLN,ASP and GABA of SAMP8 group increased significantly in brain,and the contents of ASN and GLY were not significant increased.In LYSP-treated group,the contents of GLU and ASP decreased significantly,but the content of GLY was not obvious changed.The contents of GLN,ASN and GABA were decreased by treatment with LYSP.It suggested that LYSP could obviously decrease the contents of GLU and ASP,retrieve the metabolic disorder of amino acid neurotransmitter in SAMP8 brain,and make the balance of the excitatory and inhibitory amino acid neurotransmitter.8.Effect of LYSP on expressions of APP and AB in SAMP8 brainAPP and Aβaverage optical density and their immunoreactive neurons in brain tissue were determined by immunohistochemical analysis and computer image analysis.The results showed that LYSP-treated groups could decrease significantly APP and Aβaverage optical density and their immunoreactive neurons in brain tissue of SAMP8.It showed a dose-effect relationship in some degrees.It suggested that LYSP could reduce or inhibit the over-production of APP and Aβin the brain,so as to protect the neurons from the neurontoxicity ofβ-Amyloid. 9.Effect of LYSP on dementia-related gene expressions in SAMP8 brainThe mRNA contents of BACE,APP,PS1,PS2 and ApoE in brain tissue were assayed by reverse transcription polymerase chain reaction.The results showed that the LYSP-treated groups could reduce the mRNA contents of BACE,APP,PS1 and PS2,which showed a dose-effect relationship in some degrees.But it could not affect the content of ApoE.It suggested that LYSP could inhibit the expressions of BACE,APP,PS1,and PS2 in SAMP8 brain.In summary,the present results suggest that LYSP is effective in anti-dementia.As a possible mechanism,LYSP can scavenge free radicals, reduce the degradation of Ach,regulate effectively the disorderly metabolized monoamine and amino acid neurotransmitter,inhibit the over-production of APP and Aβ,and inhibit the expressions of dementia-related gene.
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