|
ObjectiveTo investigate whether Atorvastatin(Ato) can protect hippocampal neuronsfrom Aβ1-42induced neurotoxicity.MethodsThe brain of0~24h-old Sprague-Dawley (SD) rats were collected understerile conditions, animal anatomy microscope isolated hippocampi, cut intopieces and digested with0.125%trypsin. Primary hippocampal neurons werecultured in dulbecco modified eagle medium(DMEM)/F12mediumsupplemented with10%fetal bovine serum (FBS) and10%horse serum. After7d cultured, hippocampal neurons were divided into three parts, the first part ofhippocampal neurites were observed in normal cultured natural growthconditions. The normal cell culture do not join any drug every3d half of mediumwas changed once, was observed by inverted phase contrast microscope andrecorded3d,7d and14d growth of neuronal processes. The second part ofAβ1-42induced neurite development of hippocampal neurons cultured in vitroneurotoxicity. Experiment were divided into three groups: normal control group;Different concentrations of Aβ1-42(10、25、50、100nmol·L-1) and different time ofAβ1-42(24h、48h、96h) group. The third part of Ato can protect hippocampalneurons from Aβ1-42induced neurotoxicity. Experimental groups: normal controlgroup; Join the aggregation state of Aβ1-42treatment group of Aβ1-4250nmol·L-1for48h. Aβ1-42+Ato group: Ato5μmol·L-1were given1h before Aβ1-4250nmol·L-1added for48h. The Ato5μmol·L-1treatment group of hippocampalneurons to join Ato5μmol·L-1for48h. Application of microtubule associatedprotein-2(MAP2) immunofluorescence staining after measurement of total length of neurite branching (TDBL) and a protrusion number (PDN), to observethe neuronal development. β-Tubulin staining growth and hippocampal neuronsto observe the neuronal axons. Neuronal apoptosis was quantified by scoringthe percentage of cells with apoptotic nuclear morphology after Hoechst33258staining. Immunofluorescence staining and the cellular extracts were preparedfor Western blotting of active caspase3, synaptophysin (SYP), andpost-synaptic density protein95(PSD95).ResultsApplication of inverted phase contrast microscope observation indicatedthat, hippocampal neurons were cultured in3-4d, neurons exhibited typicalneuron shape, such as bipolar and multipolar neurons, mostly fusiform orconical shape, smooth surface, strong refraction, the visible part of neuronalgrowth significantly. Cultured of6-7d, hippocampal neuronal neurite lengthincreased thick growth, cells were large and bright, halo is obvious, because thecell growth between just connected into a sparse network, so used to counting,measuring neuronal dendrites and inhibition of neuronal growth factors arepossible. Cultured hippocampal neuron8-10d, neural cells began to gather andcontinues to increase, the halo is more obvious, cell growth between theprotrusions connected into a more dense neural network. With the prolongationof the culture time, the neurites arranged in a crisscross pattern, more difficult toidentify projections originate. Cultured hippocampal neuron14-15d,hippocampus neuron growth well, cell bodies and neurite processes ofsustainable, cell body no longer increases, gathered more apparent,protuberant continue. First with different concentrations of Aβ1-42(10、25、50、100nmol·L-1) for48h, inverted phase contrast microscopy andimmunofluorescence staining results showed, from the Aβ1-4225nmol·L-1projections are very obvious degenerative changes characteristic inhippocampal neurons, mainly for axonal varicosity change、the total dendriticlength and number of primary dendrites decreased, and showed a concentrationdependent, with Aβ1-4250nmol·L-1and Aβ1-42100nmol·L-1effect is significant,Aβ1-4210nmol·L-1effect is not obvious. so after the final concentration of Aβ1-42 50nmol·L-1were used. Secondly, based on the effects of differentconcentrations of Aβ1-42treatment group of projections, we selected the Aβ1-4250nmol·L-1optimum concentration of7d neurons were cultured for24h,48h,96h, results show, Aβ1-4250nmol·L-1for24h, can make TDBL in culturedneurons and PDN has decreased, after48h the TDBLand PDN further reducedthe most obvious. Thirdly, Ato5μmol·L-1were given1h before Aβ1-4250nmol·L-1added for48h, the performance of the TDBL and PND compared withAβ1-42groups were markedly prolonged, protruding varicose changes andneurite retraction.Western results further prove that, by adding different concentrations of Aβ1-42(10、25、50、100n mol·L-1) for48h in addition to Aβ1-4210nmol·L-1outsideall can reduce SYP, PSD95protein expression level. The level of Aβ1-4250nmol·L-1and Aβ1-42100nmol·L-1effect is the most obvious; at the same time,different time of Aβ1-42(24h,48h,96h) also reduced the expression level ofSYP, PSD95protein,48h more obvious. Ato5μmol·L-1were given1h beforeAβ1-4250nmol·L-1added for48h, prevented the Aβ-induced decrease inSYP and PSD95, and reduced Aβ-induced apoptotic morphology and activecaspase3protein level.Conclusions1、Aβ1-42can cause injury of hippocampal neurons cultured in vitro.2、Ato can protect hippocampal neurons against Aβ neurotoxicity andthe damage may be related to activate caspase3.
|