Protective Effect Of Endocannabinoid2-AG Against LPS Induced Neurotoxicity In Primary Cultured Neurons From Rat Caudate Nucleus And Its Mechanism

Objective To establish a method for primary cultured neurons from caudate nucleusof rodent for the study on the characteristics of the morphology and identification ofneurons.To study the characteristics of ion channels by using whole cell patch-clamptechnique.To study the mechanism and its associated signal transduction pathway ofprotective effect of endocannabinoid2-AG against LPS induced neurotoxicity in primarycultured neurons from rat Caudate nucleus.Methods (1) Primary cultured neurons from caudate nucleus in neonatal wistar ratswithin24h after birth were processed by using enzyme digestion in the presence ofneurobasal A medium containing B27.(2)Neuron-specific enolase (NSE) immuno-histochemical staining was used to identify neurons. The dopaminergic neurons wasdetected by using tyrosine hydroxylase (TH) Immunohistochemical staining.(3)Theneurons were then used to record both the voltage-gated sodium currents and voltage-gatedpotassium currents using the whole cell patch-clamp technique.(4)By Hochest stain,tostudy2-AG inhibited the cellular apoptosis induced by LPS.(5)Using Caspase3activityassay kit,studied the effect of2-AG on LPS-induced elevated activities of caspase-3incultured neurons of caudate nucleus.(6) Effect of2-AG on LPS-induced expression ofErkⅠ/Ⅱ,phosph-ErkⅠ/Ⅱ,p38MAPK,phosph-p38MAPK,NF-κB,p-NF-κB,COX-2in cul-tured neurons of caudate nucleus was determined by western blot.Results (1) The caudate nucleus neurons isolated from newborn rats grow well underour experimental conditions.(2)NSE immunohistochemical staining and TH immuno-histochemistry results showed that more than90%of cultured cells were dopaminergicneurons.(3) Neurons from caudate nucleus in primary culture with high quality could beobtained, as indicated by the smooth membrane, elasticity and the activities of primary ironchannels (4) After12hours’ LPS treatment, the caudate nucleus neurons showed typicalcharacteristics of apoptosis, while adding the2AG, the karyopyknosis number of cells wassignificantly reduced.(5) After LPS treatment, the Caspase-3activity of caudate nucleusneurons was significantly enhanced,2-AG partially inhibited this activity,enhanced SR1(CB1receptor antagonist) may cancel the effects of2-AG..(6) Endocannabinoid2-AGinhibit expression of COX-2, phosph-ErkⅠ/Ⅱ, p-NF-κB phosph-p38MAPK, and thiseffect at least partially mediated by the CB1receptor.Conclusion Successfully established a model for primary cultured neurons from caudate nucleus of rodent,neurons of the caudate nucleus, the cell has smooth and elasticmembrane. Endocannabinoid2-AG Protects neurons against LPS-induced neuronaldamage. The protective effect of endocannabinoid2-AG against LPS inducedneurotoxicity in primary cultured neurons from rat Caudate nucleus may be achieved byinhibiting the expression of COX-2, and the CB1receptor mediated this effect. Theprotective effect may be related to the inhibition of the MAPK/NF-κB signal transductionpathway. The study provides a theoretical basis for an understanding of neurodegenerativediseases and new therapeutic approaches and methods.