Effectes And Mechanisms Of Atorvastatin On The Expression Of Synapse-associated Proteins Cultured By Rat Cortical Neuron In Vitro

ObjectiveTo investigate whether atorvastatin can promote the quantities ofsynapse-associated proteins expression cultured by the milk rat corticalneurons in vitro and the signaling mechanisms responsible for this effect.MethodsThe cerebral cortex of within24hour-old Sprague-Dawley (SD) rats werecollected under sterile conditions to cultured cortical neuron in vitro, After beingcultured for4days for experiment, The experiment was divided into two parts,the first part investigates the effects of atorvastatin on SYP and PSD-95expression. cortical neurons were divided into control group, treatment group ofatorvastatin different concentrations, treatment group of atorvastatin at differenttime points. The equivalent cultured medium containing0.1%DMSO was addedto control group; after the neurons were cultured for4days, atorvastatin ofdifferent concentrations(0.1,1,5,10μmol/L) were added to the atorvastatintreatment group and were then observed48hours later; after the neuronswere cultured for4days, atorvastatin1μmol/L were added to the different timegroup for12h,24h,48h,96h. Inverted phase contrast microscope, tiny tubesrelated proteins-2(MAP2) and β-tubulin immunofluorescence stain wereemployed to observe the grow of dendrite and axon. Immunfluorescence andWestern blot were used to measure the expression of synaptophysin (SYP), postsynaptic density protein95(PSD95). The second part investigates themechanisms of atorvastatin on SYP and PSD-95expression. cortical neuronswere divided into control group, atorvastatin10μmol/L group, inhibitor group,atorvastatin+inhibitor group. control group: The equivalent cultured mediumcontaining0.1%DMSO was added to control group atorvastatin10μmol/Lgroup: after cultured for4days atorvastatin10μmol/L were added to corticalneurons then observed48hours later; inhibitor group: The PI3K inhibitorLY294002、LY303511、Akt inhibitor Triciribine、mTOR inhibitor Rapamycin fourinhibitors were dissolved in DMSO respectively, when using diluted to LY294002(30μmol/L), LY303511(20μmol/L), Triciribine (1μmol/L), Rapamycin (100nmol/L) separately added to neurons cultured for4days then observed48hours later. atorvastatin+inhibitor group. LY294002(30μmol/L), LY303511(20μmol/L), Triciribine (1μmol/L), Rapamycin (100nmol/L) four inhibitors wereadded30min prior to addition of atorvastatin10μmol/L then observed48hourslater. Through the semi-quantitative analysis of western blot detection whetheratorvastatin can act against inhibitors result in expressions on SYP and PSD-95,we speculated atorvastatin effects on SYP and PSD95expression whetherthrough activations of the PI3K/Akt/mTOR signaling pathways. Through thesemi-quantitative analysis of western blot to futher investigate p-PDK1, p-Akt,p-mTOR, p-p70s6k, p-4E-BP1protein expression and make clear the signaltransduction mechanism.ResultsThe results showed that morphology of neurons, atorvastatin5μmol/L canpromote neurite growth in cultured neurons, increased dendritic branching andinterconnected into a dense network; Immunofluorescence showed thatatorvastatin can significantly increase of dendrites and axon length, andsignificantly up-regulation SYP and PSD-95protein expression levels; Westernblot showed that different concentrations of atorvastatin treatment group could significantly increases of SYP and PSD-95protein expression, and in adose-dependent manner, atorvastatin1μmol/L was significantly （p<0.01）; Theresults also showed at different time of atorvastatin treatment group couldsignificantly increases of SYP and PSD-95protein expression, atorvastatin1μmol/L for12hours can increases of SYP and PSD-95protein expression levels（p<0.05）, SYP expression peaked when effected for96hours(p<0.01),PSD-95expression peaked when effected for48hours(p<0.01); atorvastatin10μmol/L can significantly increases of SYP and PSD-95proteinexpression(p<0.01). Application of PI3K inhibitor LY294002, Akt inhibitorTriciribine and mTOR inhibitor Rapamycin can significantly blockedatorvastatin-induced increases of SYP and PSD-95(p<0.01); It explainatorvastatin-induced increases of SYP and PSD-95protein expression levelmay be a certain relationship with the PI3K/Akt/mTOR signal transductionpathway. Further research proved that atorvastatin (10μ mol/L) could alsosignificantly increases of p-PDK1, p-Akt, p-mTOR, p-p70s6k and p-4E-BP1protein expression level (p<0.01). Application of PI3K inhibitor LY294002canblocked atorvastatin-induced increases of p-PDK1, p-Akt, p-mTOR, p-p70s6kand p-4E-BP1(p<0.05). In contrast the inactive analogue of LY294002,LY303511did not have an effect. Application of Akt inhibitor Triciribine couldsignificantly blocked atorvastatin-induced increases of p-Akt, p-mTOR (p<0.01).Application of mTOR inhibitor Rapamycin can blocked atorvastatin-inducedincreases of p-p70s6k (p<0.05), Application of mTOR inhibitor Rapamycin cansignificantly blocked atorvastatin-induced increases of p-4E-BP1(p<0.01).ConclusionsThese results suggest that activation of PI3K/Akt/mTOR signal transductionpathway is responsible for the atorvastatin-induced promote neurite growth andincreases SYP and PSD-95protein expression in cultured cortical neuron.