The Mechanism Research Of Protective Effect Of Endocannabinoid2-AG Against Homocysteine-induced Neurotoxicity In Primary Cultured Rat Caudate Nucleus Neurons

Objective: Our research mainly study the mechanism and associated signal pathway ofprotective effect of endocannabinoid2-Arachidonoylglycerol(2-AG), treatment with2-AGthrough exogenous way and treatment with monoacylglycerol lipase(MAGL) inhibitorsURB602to increase the concentration of2-AG, against homocysteine(Hcy) inducedcytotoxicity in primary cultured Caudate nucleus(CN) neurons.Methods:(1) Add endocannabinoid2-AG or URB602to primary cultured CNneurons(cells were used in experiments in7days in culture), after12hours using westernblot determined the expression change of COX-2、NF-κB、p-NF-κB、IκB-α、ERK l/2、p-ERKl/2、p38MAPK、p-p38MAPK、caspase-3、cleaved caspase-3、Bcl-2、p-Bcl-2inducedby Hcy.(2) Hoechst staining method to detect apoptosis, aiming at surveying the protectiveeffect of2-AG and URB602.(3) The CN neurons were used to record the voltage-gatedsodium currents by using the whole cell patch-clamp technique when administration2-AGand Hcy.Results:(1) Both2-AG and URB602not only inhibit excessive expression of COX-2,p-NF-κB, p-ERK1/2but also suppress the reduced expression of IκB-α、p-Bcl-2, this effectmediated by the cannabinoid type-1(CB1) receptor.(2) Under Hcy treatment12hours, theCN neurons showed typical characteristics of apoptosis, such as the nucleus pycnosis ordebris shaped thick dense, while adding the2-AG or URB602, the karyopyknosis numberof CN neurons was significantly reduced. And this effect was blocked by CB1receptorantagonists Rimonabant (SR141716, SR1).(3) Our results revealed that in primarycultured CN neurons2-AG effectively restrained Hcy-induced increase of sodium currentin a CB1receptor-dependent way. Hcy induced sodium channel activation curve shift tothe left obviously, reduced the activation threshold and increased cell excitability, thephenomenon disappeared after dealing with2-AG, and the existence of the SR1canpartially blocked the effect of2-AG. Hcy and2-AG had no effect on the inactivation ofsodium current.Conclusion:2-AG and MAGL inhibitors URB602both protect primary cultured CNneurons from Hcy-induced neurotoxicity. And neuroprotective roles of the2-AG may bethrough the suppression of expression of COX-2and ERK1/2/NF-κB signal transductionpathway in a CB1receptor-dependent way. This research will help understand themolecular mechanism of the neuroprotective role of endocannabinoids2-AG against Hcy’s neurotoxic effects and implicate new strategy and direction for further revealing thepathogenesis of neural degeneration diseases such as Alzheimer’s disease.