Studies On Effects Of Ginsenoside Re On Alzheimer's Disease Based On One-Compound-Multiple-Targets Strategy

Alzheimer's disease(AD)is the most common cause of cognitive impairment in older patients and is expected to increase greatly in prevalence along with aging of populations in the next future. It is characterized by the development of senile plaques and neurofibfillary tangles,which are associated with neuronal loss.AD,characterized by progressive memory loss,decline in language skills and other cognitive impairments,has been a major threat to aging population.Although the etiology of AD is not very clear,multiple factors,such as amyloid-β(Aβ)and tau protein aggregation,excessive metal ions (e.g.,Cu²⁺,Zn²⁺,Fe³⁺),oxidative stress and reduced acetylcholine(Ach)level,have been considered to play important roles in the pathogenesis of AD.This provides diverse targets for screening AD-modifying drugs.Indeed,numerous synthetic or natural molecules have been screened to decrease Aβproduction(e.g.,β-secretase inhibitor),to prevent Aβor tau aggregation,to chelate transition metals,to scavenge reactive oxygen species(ROS)or to inhibit acetylcholinesterase(AchE)or monoamine oxidase(MAO).However,the success of the one-drug-one-target strategy is limited,which has stimulated the search for more efficient combined weapons to combat AD.According to the signal transduction of estrogen receptorβsubtype,we established the drug screening model based on cell and reporter gene.The functional response was the marker among drug screening.The molecules that can change the activity of transcription factor and affect to the cell control were screened based on the cell level.In this thesis,a recombinant vector pTAL/ERE-SEAP was constructed by inserting a synthetic sequence which composed of five ERE bounded elements in front of promoter of pTAL/SEAP vector,pTAL/ERE-SEAP was then transfected into HEK293 cells and stably transfected monoclonal cell line with recombinant vector was obtained and was evaluated,the result indicate that a sensitive and efficient screening model that can screen the small molecular compounds with estrogen receptorβsubtype-like activity was established 342 samples were screened and screening times went beyond 1000 to find the biology simulacrums.Ginsenoside Re indicates weak estrogen receptorβsubtype-like activity. According to the signal transduction of NF-κB,we also established the drug screening model based on cell and reporter gene.A recombinant vector pTAL/NF-κB-SEAP was constructed by inserting a synthetic sequence which composed of seven NF-κB bounded elements in front of promoter of pTAL/SEAP vector,pTAL/NF-κB-SEAP was then transfected into HEK293 cells and stably transfected monoclonal cell line with recombinant vector was obtained and was evaluated. Ginsenoside Re indicates inhibitory effect of NF-κB signal transduction induced by lipopolysaccharide(LPS).In present experiment,ginsenoside Re could protect PC12 cells against the injuries caused by exposing of PC 12 cells to 5mmol·L^-1glutamate for 15min followed by incubation with serum-free medium for 24h,which resembled the excitotoxin in vivo system.Furthermore,the ginsenoside Re could decrease the[Ca²⁺]i of PC12 cells in Mg²⁺-free Hanks and D-Hanks solution but there was no effect on basal[Ca²⁺]i in Hanks solution.The studies also indicated that ginsenoside Re can inhibit the increases of[Ca²⁺]i induced by KCl and glutamate.In conclusion,ginsenoside Re may protect PC12 cells against glutamate excitotoxic damage through suppressing the excessive entry of Ca²⁺ and the release of the calcium stores.To study the protective effects of ginsenoside Re on the neurons damaged by glutamate,the rat primary cultured neurons were damaged by glutamate,the membrane lipids fluidity was studied with fluorescence polarization(P)and mean microviscosity(η)with DPH as a probe;the protective effects of ginsenoside Re on this model were evaluated by lactate dehydrogenase(LDH)efflux assay and colormetric MTT assay.Results indicated that the fluorescence polarization and mean microviscosity of ginsenoside Re were lower than glutamate model group in a dose-depended manner.Treatment of ginsenoside Re resulted in the decrease of LDH release from primary cultured neurons damaged by glutamate and increase in the absorbance(A)at 540nm tested by colorimetric MTT assay.No proliferation effects on normal primary cultured neurons were observed.The results suggested that ginsenoside Re had a protective effect on rat primary cultured neurons damaged by glutamate,it might be relative to the improvement of membrane lipids fluidity.In present experiment,the exposure of SHSY5Y ceils to 10μmol·L^-1Aβ_1-40for 24h produced an obvious decrease in cell viability measured by MTT and increase in the extra cellular LDH release measured by LDH kit.Pretreated with different concentration of ginsenoside Re,the cell damage was greatly decreased.In order to investigated the effect of ginsenoside Re on mitochonddal swelling and H⁺-ATPase activity,the adult male SD rats weight 250-300 g were subjected to either sham surgery and middle cerebral occlusion(MCAO)of brain ischemia-reperfusion.It was observed that ginsenoside Re(5,10 or 20 mg/kg,P.O.for 7 days,once a day)significantly decreased mitochondrial swelling and prevented reduction of H⁺-ATPase activity.In conclusion,the study demonstrated a significant effect of ginsenoside Re on targets relative to AD.Ginsenoside Re has estrogen receptorβsubtype-like activity,anti-inflammation, suppressing the excessive entry of Ca²⁺and the release of the calcium stores,protects cells against injury induced by Aβ,it also may improve membrane lipids fluidity of neurons,decreased mitochondrial swelling and prevented reduction of H⁺-ATPase activity.