Ducosahexaenoic Acid Provides Protection To Cerebral Cortical Neurons From Impairment Induced By Amyloid Beta Protein 25-35 In Rats

Objective Alzheimer's disease(AD) is a serious degenerative disease of the central nervous system,both amyloid beta protein(Aβ) and the senile plaques(SPs) formed by aggregated Aβare the specific pathological changes in Alzheimer's disease.Aggregated Aβhas a wide range of toxicity,which is considered to be the key pathogenic substance of AD.A large number of studies in vitro and in vivo have shown that Aβhave toxicity on many kinds of cells, especially on the hippocampus and cortical neurons.Therefore,prevention and antagonism of the neurotoxicity induced by Aβhave already become the central target for the prevention and treatment of AD.Studies have shown that the direct toxic effect of Aβis associated with the disruption of neuronal Ca²⁺ homeostasis,which made the neuronal intracellular calcium concentration significantly increased.Docosahexaenoic acid(DHA) is a polyunsaturated fatty acid(PUFA) primarily found in marine fish and algae.DHA is the major n-3 PUFA constituent of neuronal membrane in grey matter of cerebral cortex.DHA has been shown in several epidemiological and in vivo studies to have protective effects against AD and cognitive alterations.Researches have also shown that DHA could attenuate the neurotoxicity of Aβeffectively,DHA plays a role such as neuroprotective effect,anti-apoptotic effect,and so on.Our experiment before have showed that the reduction of DHA could increase the inflammatory response and promote the activation of caspase,which aggravate the cell apoptosis.This experiment is designed to observe the protection of docosahexaenoic acid on primary culture cortical neurons of rat exposed to amyloid beta protein 25-35,to study whether DHA provide protection to calcium overloading of neurons induced by Aβ,to study the neuroprotective mechanism or path of DHA.Methods1.Primary culture of Wistar rat cortical neurons:First cerebral cortex of the newborn Wistar rat(1 day) were made into cell suspension,then the cell suspension was planted into a 96 plate or a Petri dish which has been coated with L-polylysine,then placed the 96 plate or Petri dish which has been planted with cell suspension into the 37℃incubator including 5 percent of carbon dioxide in volumn percentage for cuture.Routinely,cerebral cortical neurons cultured for 7 day in vitro were devided into 5 experimental groups.2.Experimental groups:(1) Control group:without any treatment factor; (2) Abeta group:Aggregated Aβ_25-35(25μM);(3) Low DHA-Abeta group:DHA(20μM) +Aggregated Aβ_25-35(25μM);(4) Middle DHA-Abeta group:DHA(50μM) +Aggregated Aβ_25-35(25μM);(5) High DHA-Abeta group:DHA(100μM) +Aggregated Aβ_25-35(25μM).Cerebral cortical neurons cultured for 7 day in vitro were devided into 5 experimental groups,then add DHA of different dose(the final concentration of DHA was 20,50,100μmol / L) into Low DHA-Abeta group,Middle DHA-Abeta group and High DHA-Abeta group for cuture last for 24 hours,then add aggregated Aβ_25-35(final concentration of Aβ_25-35 was 25μmol / L) according to the request of grouping for chronic cuture last for 24 hours or for acute administration.3.Detection Method:(1) CCK-8(Cell Counting Kit-8) staining was used to detecte the survival rate of cortical neurons.(2) LSCM(laser-scanning confocal imaging system) was used to detecte the changes of intracellular free calcium concentration represented by relative fluorescence intensity(F/F_b) in neurons labeled with fluorescent dye Fluo-3/AM(5μM).4.Statistical analysis All the statistics were represengted by mean±standard deviation ((?)±s),then dealed with statistical software SPSS13.0,the significance between different treatment groups using one-way ANOVA(analysis of variance) test,groups whose analysis of variance homogeneous were compared with LSD(least significant difference) test,groups whose analysis of variance heterogeneous were compared with Games-Howell test,the standard of testing wasα= 0.05,P＜0.05 indicating significant difference.Results1.Attenuation of DHA on the neuronal morphology change induced by AβCerebral cortical neurons cultured for 8 day in vitro which have been given DHA of different dose for 24 hours,then treated with aggregated Aβfor 24 hours according to the request of grouping.Compared with Control group,neurons of Abeta group showed significant neurotoxicity,we observed that the majority of neurons cell body swelled into round,significant disappearance of neurite,and neurons growth from adherent to suspension,there was a large number of dead neurons.There was significant difference between Abeta group and DHA-Abeta groups,in DHA-Abeta groups the neurotoxicity effect almost disappeared.Compared with Abeta group,few neurons of low DHA-Abeta and high DHA-Abeta group displayed retraction of neurite of different degree,and rarefaction of the neurofibril network,neurons of the middle DHA-Abeta group showed stronger cell growth,longer and more neurite,more complex neurofibril network structure.The results showed that DHA has antagonism on Aβinduced neurotoxicity(neuronal morphology and structure change),the middle DHA-Abeta showed the strongest effect.2.Attenuation of DHA on decreasing of the neurons survival rate induced by Aβ_25-35Neurons survival rate of Abeta group decreased significantly,there was significant difference between Abeta group and control group(n=8,P＜0.05);DHA could attenuate this effect,there was significant difference(n=8,P＜0.05);Significant difference also existed between groups of DHA-Abeta of different dose,and the middle DHA-Abeta is the most effectfull(n=8,P＜0.05).3.Attenuation of DHA on increasing of neurons intracellular free calcium concentration induced by Aβ_25-35Neurons intracellular free calcium concentration increased apparently and persistently when neurons exposed to Aβ_25-35,there was significant difference between Abeta group and control group(n=15,P＜0.05);DHA could attenuate this effect,there was significant difference(n=15, P＜0.05),Significant difference also existed between groups of DHA-Abeta of different dose, and the middle DHA-Abeta is the most effectfull(n=15,P＜0.05).Conclusions1.Aβ_25-35 can reduce neurons survival rate in vitro,and increase neurons intracellular free calcium concentration apparently and persistently in vitro.2.Calcium overloading induced by amyloid beta peptide 25-35,may be the central neurotoxicity of amyloid beta peptide 25-35.3.DHA can partly decrease calcium overloading induced by amyloid beta peptide 25-35,it maybe the important mechanism of DHA attenuating the neurotoxicity induced by amyloid beta peptide 25-35.