Ginsenoside Rb1 And Ginsenoside Rg3 Inhibit The Neurotoxicity Of β-amyloid Peptide By Up-regulating Neprilysin Gene Expression

Alzheimer's disease is a primary neurodegenerative disorder,the commonest cause of senile dementia.AD is characterized by a variety of pathological features,such as extracellular senile plaques,intracellular neurofibrillary tangles,extensively neuronal or synaptic loss.The mechanisms underlying the pathogenesis of AD remain unclear, and many hypothesis were proposed,among of them,amyloid cascade hypothesis was extensively approved.Amyloid-βpeptide is the main ingredient of senile plaques, contains 39-43 amino acids and is produced from amyloid precursor protein by sequential cleavage involvingβ-secretase andγ-secretase.Aβis a normal metabolite which,under physiological conditions,is constantly produced and quickly degraded. Amyloid cascade hypothesis posited that due to genetic defects such as mutations in APP,Presenilin 1,or Presenilin 2,Aβproduction is increased which lead to Aβaccumulation,accumulating Aβwill initially oligomerize,gradually form fibrils,and culminate in microscopically visible amyloid plaques,soluble and fibrillar Aβand associated plaque proteins are toxic to neurons,resulting in altered neuronal ionic homeostasis and oxidative injury,synaptic loss,the formation of neurofibrillary tangles,and eventual neuronal death and AD.A similar phenotype can also occur with reduction in the Aβcatabolic pathways.Therefore,in order to overcome AD,it is necessary to lower the Aβlevels in the brain,and the therapeutic strategies should aim at inhibiting Aβproduction,promoting Aβdegradation,inhibiting Aβaggregation, and cleating Aβdeposits.Formerly,much more attention has been paid to the abnormal Aβproduction.However,although overproduction of Aβis critical to the pathogenesis of some forms of familial AD,there is still little evidence to suggest that increased Aβproduction is important in Aβdeposition in aging and sporadic AD. Recently the role of Aβdegradation in Aβhomeostasis has been increasingly recognized,as several enzymes that degrade Aβhave been identified,such as neprilysin,endothelin-converting enzyme,insulin-degrading enzyme and angiotensin-converting enzyme.Among of these enzymes,neprilysin is believed to be the key enzyme in degrading Aβin brain.Therefore,in our experiment we chosed neprilysin as target enzyme to screen more than ten natural remedies which have been approved beneficial to neural system,aimed at obtaining potential chemicals enhancing neprilysin enzyme activity and then useful for chemotherapy of AD.To realize this aim,we established a cell strain highly secreted Aβ,and used it as AD model cell strain to study the therapeutic effects of the natural remedies which could enhance neprilysin enzyme activity.And,in this experiment,we also cloned the promoter sequence of neprilysin gene to study the underlying mechanism of compounds enhancing neprilysin enzyme activity.PART ONE SCREENING POTENTIAL NATRUAL REMEDIES ENHANCING NEPRILYSIN ENZYME ACTIVITYObjective:In attempt to obtain potential chemicals useful for chemotherapy of AD by screening natural remedies which could enhance neprilysin enzyme activity.Methods:(1) MTT was performed to identify the optimal drug concentration and acting time.(2) The effects of natural remedies on neprilysin enzyme activity were detected by Neprilysin peptidase assay.(3) Neprilysin peptidase assay was performed to furtherly identify the effect of ginsenoside Rb1 or ginsenoside Rg3 on Neprilysin enzyme activity,is dose dependent or time dependent?Results:(1) The results of MTT assay showed that 10mg/L tea polyphenols,25mg/L vitamine E,5μmol/L quercetin,20mg/L rutin,2mg/L emodin,5μmol/L resveratrol,5μmol/L curcumin,100μmol/L puerarin,5μmol/L huperzine A, 20μmol/L ginsenoside Rg1,50μmol/L ginsenoside Rb1,50μmol/L ginsenoside Rg3,50μmol/L ginsenoside Re,50μmol/L presenegenin,1mg/L timosaponin A3 has no significant or little effect on cell survival ability,and the optimal effect appeared from 48 to 72 hours.(2) We found that tea polyphenols,ginsenoside Rb1 and ginsenoside Rg3 could significantly enhance Neprilysin enzyme activity,117±2.8%,131±4.0%,121±1.6%respectively,compared with control cells(untreated SK-N-SH cells).(3) 20μmol/L ginsenoside Rb1 could effectively enhance Neprilysin enzyme activity,(120±7.9)%compared with control cells,when the concerntration reached to 100μmol/L,the Neprilysin enzyme activity rised to(131±2.3) %,and the optimal effect of ginsenoside Rb1 appeared at 72h.(4) 25μmol/L ginsenoside Rg3 could effectively enhance Neprilysin enzyme activity,(117±4.5)%compared with control cells,when the concerntration reached to 100μmol/L,the Neprilysin enzyme activity rised to(140±8.5) %,and the optimal effect of ginsenoside Rg3 appeared at 72h.Conclusion:Identified the optimal drug concentration and acting time.Tea polyphenols,ginsenoside Rb1 and ginsenoside Rg3 could significantly enhance neprilysin enzyme activity.The effects of both ginsenoside Rb1 and ginsenoside Rg3 are dose-dependent and time-dependent.PART TWO CONSTRUCTION AND IDENTIFICATION OF SK-N-SH CELLS EXPRESSING SWEDISH MUTANT APP695 AND STUDYING OF THE NEURON-PROTECTIVE EFFECTS OF GINSENOSIDE RB1 AND RG3Objectives:Construct expression vector pcDNA3.1-sweAPP695,construct AD model cells highly expressing Swedish mutant APP695 and detect the therapeutic effects of ginsenoside Rb1 and ginsenoside Rg3 on it.Methods:(1) Constructed eukaryotic expression vector pcDNA3.1-sweAPP695 and identified by restriction enzyme digestion and DNA sequencing.(2) Transfected plasmid pcDNA3.1-sweAPP695 into SK-N-SH cells with Lipofectamine^TM 2000,used G418 to screen positive cells.3-4 weeks later, cell number of each clone reached about 50,then picked out one clone and digested with trypsinase and inoculated into 50ml flask and continued culturing,identified with Western Blot and ELISA.(3) Western Blot was performed to confirm the expression of sweAPP695 gene in stably transfected cells using antibody anti-APP.(4) Enzyme Linked Immunosobent Assay was performed to detect extracellular Aβ₄₀ and Aβ₄₂ of stably transfected cells.(5) After identification,positive cells were named as sweAPP-SK cells and used as AD model cells.Observed the morphological changes of sweAPP-SK cells,detected the cell proliferation ability by MTT assay and draw cell growth curve.(6) Marked the cells with reactive oxygen species probe H₂DCFD and detected intracellular reactive oxygen species by Flow Cytometry,then analyzed the changes of reactive oxygen species levels induced by sweAPP695 gene overexpression.(7) Marked the cells with Ca²⁺ probe Fluo-3 AM ester and detected intracellular [Ca²⁺]by Flow Cytometry,then analyzed the changes of[Ca²⁺]induced by sweAPP695 gene overexpression.(8) Gathered cells and analyzed the cell apoptosis by Flow Cytometry.(9) Treated cells with 50μmol/L ginsenoside Rb1 and 50μmol/L ginsenoside Rg3 for 72 hours respectively,gathered the cell culture medium,and measured the concentration of extrallelar Aβ₄₀ and Aβ₄₂ by ELISA,then analyzed the effects of ginsenoside Rb1 and ginsenoside Rg3 on Aβdegradation metabolism.(10) Treated cells with 50μmol/L ginsenoside Rb1 and 50μmol/L ginsenoside Rg3 for 72 hours respectively,markered the cells with reactive oxygen species probe,and detected intracellular reactive oxygen species by Flow Cytometry,then analyzed the effects of ginsenoside Rb1 and ginsenoside Rg3 on production of intracellular reactive oxygen species.(11) Treated with 50μmol/L ginsenoside Rb1 and 50μmol/L ginsenoside Rg3 for 72 hours respectively,markered the cells with Ca²⁺ probe,and detected the intracellular[Ca²⁺]by Flow Cytometry,then analyzed the effects of ginsenoside Rb1 and ginsenoside Rg3 on Ca²⁺ homeostasis maintainance.(12) Treated cells with 20μmol/L extracellular Aβ_25-35 for 48 hours to induce cell apoptosis,and treated cells with ginsenoside Rb1 and ginsenoside Rg3 respectively,gathered cells and analyzed cell apoptosis by Flow Cytometry, then evaluated the protective effects of ginsenoside Rb1 and ginsenoside Rg3 on cell apoptosis.(13) Treated cells with 20μmol/L extracellular Aβ_25-35 for 48 hours to induce cell apoptosis,and treated cells with ginsenoside Rb1 and ginsenoside Rg3 respectively,extrated cell protein and detected the apoptosis associated protein Bcl-2 and Bax by Western Blot,then analyzed the effects of ginsenoside Rb1 and ginsenoside Rg3 on cell apoptosis.Results:(1) Restriction endonuclease digestion result showed that the direction and length of inserted sweAPP695 fragment is right.DNA sequence is correct.(2) The result of Western Blot showed that SK-N-SH cells transfected with pcDNA3.1-sweAPP695 highly expressed sweAPP695 protein.(3) The result of ELISA showed that both Aβ₄₀ and Aβ₄₂ in culture media were significantly increased after transfection,7.26 and 3.27 fold respectively.(4) After identifying by Western Blot and ELISA,the positive cells were named as sweAPP-SK cells,and used as AD model cells.(5) sweAPP-SK cells were larger than SK-N-SH,and proliferated slowly.(6) The results of Flow Cytometry showed that sweAPP-SK cells contained higher concentration of reactive oxygen species than SK-N-SH cells,2.48 fold.(7) The results of Flow Cytometry showed that[Ca²⁺]of sweAPP-SK cells was higher than that of SK-N-SH cells,2.76 fold.(8) While apoptosis analysis result showed that sweAPP695 gene over- expression did not induce significant cell apoptosis.(9) ELISA results showed that ginsenoside Rb1 could significantly decrease Aβ₄₀ in culture medium of sweAPP-SK cells,74±1.61%;while had no significant effect on Aβ₄₂,99±3.31%.Ginsenoside Rg3 could significantly decrease both Aβ₄₀ and Aβ₄₂ in culture medium of sweAPP-SK cells, 83±5.31%and 76±6.74%respectively.(10) The results of Flow Cytometry showed that the concentration of intracellular reactive oxygen species of sweAPP-SK cells decreased after being treated with ginsenoside Rb1 or ginsenoside Rg3,from 437.67 to 282.70 and from 437.67 to 271.19,respectively.(11) The results of Flow Cytometry showed that[Ca²⁺]of sweAPP-SK cells decreased after being treated with ginsenoside Rb1 or ginsenoside Rg3, from 109.85 to 64.35 and from 109.85 to 71.85,respectively.(12) Flow Cytometry results showed that cells treated with 20μmol/L extracellular Aβ_25-35 for 48 hours appeared apoptosis,the apoptosis rate was 13.4%,and when treated cells with ginsenoside Rb1 or ginsenoside Rg3 before adding Aβ_25-35,cell apoptosis rate decreased,0.40%and 0.36% respectively.(13) The results of Western Blot showed that the Bax protein of cells decreased after being treated with ginsenoside Rb1 or ginsenoside Rg3,while the Bcl-2 protein was not significantly affected.Conclusion:Successfully constructed expression vector pcDNA3.1-sweAPP695;and obtained cell strain highly expressed sweAPP695 and highly secreted Aβ₄₀ and Aβ₄₂,named as sweAPP-SK cell strain.Both the concentration of intracellular reactive oxygen species and[Ca²⁺]of sweAPP-SK cells are higher than those of SK-N-SH cells.Ginsenoside Rb1 could significantly decrease the concentration of extracellular Aβ₄₀,and ginsenoside Rg3 could significantly decrease the concentration of extracellular Aβ₄₀ and Aβ₄₂.Both ginsenoside Rb1 and ginsenoside Rg3 could significantly decrease intracellular reactive oxygen species so that to protect cells from oxidative damage.Both ginsenoside Rb1 and ginsenoside Rg3 could significantly decrease intracellular[Ca²⁺]so that to prevent cells from damage induced by[Ca²⁺]increase.Both ginsenoside Rb1 and ginsenoside Rg3 could inhibit bax gene expression thereby inhibiting cell apoptosis.PART THREE STUDYING OF THE UNDERLYING MECHANISM OF GINSENOSIDE RB1 AND GINSENOSIDE RG3 ENHANCING NEPRILYSIN ENZYME ACTIVITYObjective:Explore the underlying mechanisms of ginsenoside Rb1 and ginsenoside Rg3 enhancing Neprilysin enzyme activity.Methods:(1) Treated cells with ginsenoside Rb1 and ginsenoside Rg3 respectively, extracted cell protein,and performed Western Blot to detect the effects of ginsenoside Rb1 and ginsenoside Rg3 on neprilysin gene expression.(2) Treated cells with ginsenoside Rb1 and ginsenoside Rg3 respectively, extracted cell RNA,and performed revese transeription-PCR to detect the effect of ginsenoside Rb1 on neprilysin gene expression at mRNA leve, performed real-time PCR to detect the effect of ginsenoside Rg3 on neprilysin gene expression at mRNA level.(3) To detect if the effect of ginsenoside Rb1 and ginsenoside Rg3 on neprilysin gene expression took place on initiation of transcription stage,we cloned 2.9Kb promoter fragment upstream of neprilysin gene and inserted it into pGL3-basic,a promoter-less luciferase reporter vector,to form 2.9Kb promoter-luciferase reporter plasmid(pGL3-nep2.9Kb),and identified it with enzyme digestion and DNA sequencing.Cotransfected pGL3-nep2.9Kb and pRL-TK with lipofectamin^TM 2000,tested the promoter activity of 2.9Kb-promoter fragment of neprilysin gene by Dual Luciferase Reporter gene assay.(4) Reconstructed the pGL3-nep 2.9Kb to pGL3-nep2.4Kb by SmaI digestion, cotransfected pGL3-nep2.4Kb and pRL-TK with lipofectamin^TM 2000, then tested the promoter activity of 2.4Kb-promoter fragment of neprilysin gene by Dual Luciferase Reporter gene assay. (5) The effects of ginsenoside Rb1 and ginsenoside Rg3 on promoter activity of 2.4Kb-promoter was also detected by Dual Luciferase Reporter gene assay.Results:(1) The results of Western Blot showed that cells treated with ginsenoside Rb1 and ginsenoside Rg3 respectively expressed more Neprilysin protein and their effects were dose-dependent,which indicated that both ginsenoside Rb1 and ginsenoside Rg3 enhanced Neprilysin enzyme activity by promoting neprilysin gene expression.(2) The reverse transcription-PCR result showed that ginsenoside Rb1 could promote neprilysin gene expression,and the real time-PCR result showed that 25μmol/L ginsenoside Rg3 could significantly enhance neprilysin gene expression,1.8 fold at 72h,while 50μmol/L Rg3 could significantly enhance neprilysin gene expression 2.9 fold at 48 h.(3) The result of the restriction enzyme digestion showed that 2.9Kb promoter fragment was correctly inserted into pGL3-basic in direction,and DNA sequencing result showed that the DNA sequence of 2.9Kb promoter fragment was right.But the result of Dual Luciferase Reporter gene assay showed that 2.9Kb fragment did not have promoter activity.(4) Reconstructed 2.9Kb promoter fragment to 2.4Kb fragment by SmaI digestion,DNA sequencing result indicated that DNA sequence of 2.4Kb was correct.The results of Dual Luciferase Reporter gene assay showed that the 2.4Kb-fragment presented promoter activity that was 10 fold stronger than pGL3-basic did.(5) The results of Dual Luciferase Reporter gene assay showed that ginsenoside Rb1 could enhance the promoter activity of 2.4Kb-fragment 1.46 fold,and ginsenoside Rg3 could enhance the promoter activity of 2.4Kb-fragment 1.4 fold.Conclusions:Ginsenoside Rb1 and ginsenoside Rg3 could enhance Neprilysin enzyme activity by promoting neprilysin gene expression.Cloned 2.9Kb promoter fragment spanned from -2288-+708bp of neprilysin gene,but it did not have promoter activity.2.4Kb promoter fragment spanned from -2288- +144bp of neprilysin gene.2.4Kb-promoter fragment proved to have promoter activity in neuroblastoma cells(SK-N-SH cells).Both ginsenoside Rb1 and ginsenoside Rg3 could enhance the promoter activity of 2.4Kb-promoter.Further work will focus on finding out the cis-acting element and trans-acting factors involed in this process.