Research On The Metabolism And Mechanism Of Exogenous Hydrogen Sulfide On The BACE-1and PS1in APP Processing In Primary Culture Neurons

Part1: Effects of exogenous hydrogen sulfide on the viability andapoptosis of primary culture neurons.Objective: To observe the influence of exogenous Hydrogen Sulfide(H2S) on the viability and apoptosis of primary culture neurons. Methods:Cortical neurons were cultured by modified enzyme and mechanicaldigestion culture method. Sodium Hydrosulfide (NaHS) was used asexogenous H2S donor. The neurons were intervened by differentconcentration of NaHS (0,5,10,20,30,40,50,100and200μmol/L). Aftertreatment of neurons for24h, viability of neurons were measured by3-(4,5)-dimethylthiahiazo(z-y1)-3,5-diphenytetrazoliumromide(MTT)method,the apoptosis were measured by Hoechst33258staining. Results:The survival rate of cultured neurons were95.1±2.3％and the purity ofneurons were92.6±4.1％. In the concentration of5~20μmol/L NaHS，viability of neuron were not decreased. In the concentration of40~100μmol/L NaHS, viability of neuron were decreased significantly （P<0.05）,and decline even more in the concentration of200μmol/LNaHS.There were no obvious apoptosis in neurons after NaHS interventionat the concentration of5~10μmol/L.however,the apoptosis wassignificantly induced（P<0.05）by the NaHS in the range of50~100μmol/L.Conclusions: In the lower concentration of NaHS (5~20μmol/L),theviability and apoptosis of neuron are not affected, and lower viability andapoptosis are induced by NaHS above the30μmol/L concention. PART2: Effects of exogenous Hydrogen Sulfide on the Amyloidpeptide precursor, β-site amyloid precursor protein cleaving enzyme1and Presenilin1in primary culture neuronsObjective: To investigate the effects of exogenous Hydrogen Sulfide(H2S) on the Amyloid peptide precursor (APP),β-site APP enzyme1(BACE-1) and Presnilin1(PS1) in primary cultureneurons.Methods:Cortical neurons were cultured in vitro and SodiumHydrosulfide(NaHS) was used as exogenous H2S donor. The study weredivided into Control, NaHS5,10,20,30,40and50μmol/L group. Aftertreatment of neurons with different group for24h, the Aβ1-42level weretested by ELISA, the BACE-1and PS1mRNA expression were measured by QPCR. the expression of APP,BACE-1and PS1protein were measuredby Western blot. Results: Aβ1-42level decreased in the concentration of5~20μmol/L NaHS, and significantly decreased(P<0.05) in the range of10~20μmol/L.meanwhile, Aβ1-42level increased in the concentration of40~50μmol/L NaHS;accordingly, BACE-1and PS1mRNA decreased inthe concentration of NaHS5~30μmol/L, and significantly decreased in20~30μmol/L NaHS ((P<0.05),In the concentration of40~50μmol/LNaHS,the BACE-1and PS1mRNA increased. There were no changes inAPP protein expression after intervention by different concentration ofNaHS. But the BACE-1and PS1protein expression decreased significantly(P<0.05) in20μmol/L NaHS and10~20μmol/L NaHS. above theconcentration of30μmol/L,the BACE-1and PS1protein expressionincreased. Conclusions: exogenous H2S reduces the Aβ1-42,expression ofBACE-1and PS1mRNA and proteins in the lower concentration (within30μmol/L NaHS) and APP protein levels is not affected by differentconcentration of NaHS. PART3: The role of PI3K/Akt and MAPK/ERK signal pathway in theeffects of exogenous Hydrogen Sulfide on the β-site amyloid precursorprotein cleaving enzyme1and Presnilin1in primary culture neurons.Objective: To explore the role of PI3K/Akt and MAPK/ERKsignaling pathway in the effects of exogenous Hydrogen Sulfide(H2S) onthe β-site APP cleaving enzyme1(BACE-1) and Presnilin1(PS1) in primaryculture neurons. Methods: Cortical neurons were cultured in vitro andSodium Hydrosulfide (NaHS) was provided as exogenous H2S donor. Thestudy were divided into four groups：Control, NaHS20μmol/L, NaHS20μmol/L plus PI3K/Akt signal pathway inhibitor LY294002(20μmol/L)（NaHS+LY294002group）and NaHS20μmol/L plus MAPK/ERK signalpathway inhibitor PD98059(20μmol/L)（NaHS+PD98059group）.After24hours intervention, BACE-1and PS1protein level were measured byWestern blot. Results: BACE-1and PS1protein decreased significantly(P<0.05) after20μmol/L NaHS intervention. BACE-1and PS1proteinwere not changed in NaHS+LY294002group(P>0.05).however, BACE-1and PS1protein decreasd significantly in NaHS+PD98059group(P<0.05).meanwhile, comparing with NaHS+LY294002group,BACE-1and PS1protein expression in NaHS20μmol/L group andNaHS+PD98059group decreased significantly（P<0.05）.there were nodifference between NaHS20μmol/L group and NaHS+PD98059group.Conclusions: Exogenous H2S may exerts its reducing effects of BACE-1 and PS1protein expression through PI3K/Akt but not MAPK/ERKsignaling pathway.