Protective Effect And Its Mechanism Of TIANTAIâ…¡ On AD Rats And Primary Cultured Neural Stem Cells And Neurons

Alzheimer disease(AD)is a progressive disease which the degeneration of the central nervous system is irreversible.It occurs in aging elders and its clinical manifestations include progressively memory declining,learning difficultly,dyscalculia,apraxia,agnosia,or even the AD patients can not take care of themselves and so on.the German doctor,Alois Alzheimer,first reported the clinical cases.the clinical reports such as Alzheimer diseases have been rapidly increasing Since 1907.With the global aging of the population,AD has became a fourth largestwhich threat to the health of the elders.Major neuropathological change of Alzheimer disease includes:lossof neurons;neurofibrillary tangles(NFT)and some senile plaques(SP),or neuritic plaques(NP)in the cerebral cortex and subcortical structure,which are responsible for higher brain functions;granulovacuolar degeneration(GD);neuropil threads(NT)and amyloid angiopathy(AAP)and so on.The inter-relationship among these pathological changes has been uncertain.The plaques are deposits found outside the neurons and are composed primarily of a small protein called amyloid-beta,or A-beta.The tangles are located inside neurons and their branching projections(axons and dendrites)and are made of filaments of a protein calledtau.Are the plaques and tangles responsible for the degeneration of brain neurons,or are they merely markers of where neuronal death has already occurred ? In the past decades,the weight of evidence has shifted toward the amyloid-cascade hypothesis,which posits that both A-betaand tau are intimately involved in causing Alzheimer's disease,with A-beta providing the initial insult.A-beta molecules in a test tube can assemble into fiberlike structures similar to those found in the plaques of Alzheimer's disease.The soluble assemblies as well as the fibers of A-beta are toxic to neurons cultured in Petri dishes,and the former can interfere with processes critical to learning and memory in mice.As we known that there is no effective treatment and drugs for AD now.It is the idealest method to prevent generateion and progress ofAD.Research datas show that the majority of AD cases are progressedfrom MCI(mild cognitive impairment)case.althoug it is lack of the definitive treatment and drugs on AD,Chinese traditional medicine is good at against the early stage of disease and Subhealth.Professor Wu has researched and practiced on the elderly brain diseases treated with Chinese traditional medicine for a long time He developed his own compound recipe TIANTAIâ…¡on Alzheimer's disease ,screening a lot of ancient and modern compound recipe on AD andcombing with the his clinical practices.It has been studying on anti-MCI and AD.On the basis of past researches about its pharmacodynamic effects I continue the study on AD rats and hope to discove the major effects and its possible mechanism.My research includes two major parts:The first experiment was studied on AD rats.The second one is study on primary stem cells and neurons.Sectionâ… .Experimental study of the protective effect and its mechanismof TIANTAI II on AD ratsFirst,Morris water maze was used for screening rats,which wereSD rats,SPF-class,male,240-270g weight,were rearing in barrier environment and seeding the conventional food and water.They were removed whose journey time was more than 30% to the average,the re-maining rats were used for experiments.The rats were anesthetized by intraperitoneal injection with 3% aqueous solution of sodium pentobarbital 1.5ml/kg weight.Then those ratswere pronely fixed in the brain three-dimensional positioning instruments.At first we conventionally open the skin at the top of the head,themarker point was beside 2.5mm and behind 3.6mm to the Bregma.Wedrilled two holes at the marker point with the special dental drill andinserted the microsyringes into the hippocampus 3.0mm deepth under the parietal bone.1μl of 5mM"aging"Aβ_25-35was slowly injected into the hippocampus in 5 minutes and the microsyringes were left for 10 minutes After that,the injured skins were carefully sutured and the ratswere treated by intramuscular injected 80 thousand unit of Penicillin for3days.From the 26th day after the operation,Morris water maze experiment was began,the experiment lasted for 5 days.the experimental resuIts of ability of study and memory were analyzed statistically.In addition to the normal control group of rats was subcutaneous injected 0.6ml/kg of 0.9% normal saline solution in the back and neck outside each rat,AD model group,sham-operated rats were subcutaneous injected 150mg/kg of 25% D-galactose each rat.At the end of the behavior experiment all rats were killed through the decapitation.The left brains were fixed with 4% paraformaldehyde for 24 hours,and were regularly stained by HE stain and we observed the morphology of organizational structureand neurons in the CA2,CA3 area of hippocampal We evaluate the AD model in accordance with the behavioral results and the histological results. The AD model rats were randomly divided into 5 groups,TIANTAâ… â…¡3 dose groups(10%,5%,2.5%),single drug(â„–1 drug)group andAD model group,while there was the establishment of normal controlgroup and the sham-operated group,10 rats in each group.TIANTAIâ…¡large-dose group rats were given oral 10%,and TIANTAIâ…¡medium-dose group rats were given oral 5% and TIANTAIâ…¡small-dose grouprats were given oral 2.5% of TIANTAIâ…¡,the volume was 10ml per 1kg weight.Single drug(â„–1 drug)group given oral the same volumeof 0.5%â„–1 drug.the rats of the sham-operated group and the normal control group were given the same volume of normal saline,the manner was same as the others The administration was once a day for30 days continuously.all rats except the normal group were subcutaneous injected 150mg/kg of 25% D-galactose each rat for 50 days.The behavioral experiments were stared from the 26th day of the administration,including the training of study,locational navigation and3D-space exploration At the end of the behavior experiment all ratswere killed through the decapitation.The left brains were fixed with 4%paraformaldehyde for 24 hours,and were regularly stained by HE stainand we observed the morphology of organizational structure and neurons in the CA2,CA3 area of hippocampal We evaluate the AD model in accordance with the behavioral results and the histological results We studied the morphology and number value of the cholinergic neuron in telencephalon by immunohistochemistry,we studied the changes of thepositive cells of anti-ChAT antibody And we studied the neuronal apoptosis in brain tissue by TUNEL method The right brain was storedin liquid nitrogen for studing the expression of CHOP protein in the brains.by Western Blotting.Three brain samples were randomly selected each group,and we extracted the total RNA from each sample brain The RNA samples were tested,the value of the rato OD₂₆₀/OD₂₈₀ were over 1.86 and met the experimental requires.We synthesized and amplified cDNA and hybridizated with the RatRef-12 gene chip.And we scaned the genic chipand analysis the difference of the genic expression of 12 rat brainsCompared the AD model group with the others,the distance and the positioning time were shorter And the distance and time of locatioal navigation were shorter,the distance of the space exploration was shorter and the times crossing the security platform increased.IIDoes-large group and dose-medium group of TIANTAI had conspicuously no difference TIANTAI II can significantly improve the ability of learningand memory of AD rats. Experimental study of HE staining and anti-ChAT mmunohistochemistry on the brains shows that the number of the neurons in CA2,CA3area of hippocampal of TIANTAIâ…¡groups(large dose and medium dose)were more than one of AD rats.And the number of cholinergicneurons in forebrain was more than the multi-AD model group(P＜0.05)Expression of CHOP was less than AD model group.And apoptosis neurons was less than AD group.We scaned the genic chip and analysis the difference of the genic expression among ADmodel group,TIANTAIâ…¡group(large dose),single herb(â„–1)groupand sham-operated group We found a lot of different expression of the gene.Sectionâ…¡.Experimental study of the effect of TIANTAIâ…¡-containingcerebrospinal fluid.on primary cultured neural stem cells and neuronsTIANTAIâ…¡-containing cerebrospinal fluid was made for future cel1 biology experiments.18 Rabbits(Clean class,2.4-2.7 kg weight,malewere half.Female was half)were randomly divided into 3 groups There were the normal group,TIANTAIâ…¡group and single drug(â„–1)groups.Each group had 6 rabbits.Each rabbit of Tiantaiâ…¡Group wasgiven oral 10ml/kg of 20% TIANTAIâ…¡for 7days,twice a day.An hour after the end of administration,the rabbits were killed bybleeding to death We have got 800-1000μl of TIANTAIâ…¡-containing cerebrospinal fluid each rabbit with the microsyringe from fourth ventricle through the foramen magnum.The CSF samples of the same group were mixed and centrifugated 12000rpm for 10 minutes at 4℃.The supernatant from the CSF were sterilized with经孔径0.22μm filter and reserved the bacteriafree CSF containing TIANTAIâ…¡CSF at-70℃Cfor a long time.Neural stem cells(NSC)from fetal rats were primary cultured andtransformated Neurospheres formation:a single suspension of EB weregot by trypsinization for 5 minutes with 0.25% trypsin in 1 mmol/L EDTA(GIBCO/Invitrogen,USA),followed by quenching with 10% FCS.The cells were washed twice with basic neurosphere medium composed of DMEM:F12(1:1),0.6% glucose,2 mmol/L glutamine,3 M sodium bicarbonate,5 mmol/L HEPES buffer,25μg/ml insulin,100μg/ml transferring,20 nmol/L progesterone,60μmol/L putrescine,30 nmol/L selenium chloride [all from Sigma(USA)except glutamine(sigma)from GIBCO/Invitrogen(USA)] and plated onto T75 flasks(Greiner,Germany)at a density of 1×10(5)cells/ml in the neurosphere medium(basic neurosphere medium supplemented with 20 ng/ml fibroblast growth factor-2)(Invitrogen,USA)and 2μg/ml heparin sulfate(Sigma,USA).Cellswere grown for five to seven days and formed neurospheres.NSC were cultured with 10% fetal bovine serum(FBS)and could differentiate into astrocytes,oligodendrocyte cells,neurons,etc.And we found NSC differentiate into Cholinergic neurons incubated with the extract from feta1 chick leg muscles and TIANTAIâ…¡-containing CSF could promote theNSC into cholinergic neurons.The neurons from the fetal rat(E16-18)hippocampus were culturedwith the medium(includes Neurobasl A medium,2-Mercaptoethanol,EGF,bFGF,Glutamine,penicillin,stepmycin)at 37℃5%CO₂.Thecultured cells were identified as neurons and were used for the followthe experiment.When neurons grew to the logarithmic phase and were incubated with 10μM of Aβ_25-35for 24-hour and the AD cell modelwas made And Aβ_25-35caused neurons apoptosis and injured MTT method was used for analysis of neurons' growth capacity and AD model.neurons were grown well with TIANTAIâ…¡-containing cerebrospinal fluid.With acridine orange-ethidium bromide staining,we counted the value of neirons 1 under fluorescence microscope and calculated the apoptotic index.All results were statistical analyzed.To sum up,TIANTAI II has a better role in anti-AD of rats,themechanism involves anti-apoptosis,anti-endoplasmic reticulum stress,andpromote the differentiation and transformation of NSC And It can promote the NSC differentiate to cholinergic neurons in particular.Thereis whether or not occurrence of differentially expressed genes,we hopethe further studies on TIANTAIâ…¡.