Mechanism Of RIP3 On Methamphetamime-induced Dopaminergic Neuronal Necroptosis

Backgroud:Methamphetamine(METH),commonly known as "ice",is a new type of synthetic drug.It has become the second most widely used drug in the world due to its fast effect,long time of excitement,low price and simple synthesis technology.The abuse of METH has toxic damage effects on multiple organs,especially on the central nervous system.A large number of clinical data show that METH can directly damage brain neurons,leading to neuronal degeneration,necrosis,and a series of acute and chronic dysfunction.Studies on METH neurotoxicity have been associated with apoptosis and autophagy.However,the mechanism of METH-induced neuronal necroptosis has not been reported.Studies have shown that necroptosis is associated with the development of various diseases,including trauma,ischemia/reperfusion injury,and neurodegenerative changes.Therefore,we investigated whether METH can cause neuronal necroptosis,and we found that METH can cause the expression and activation of receptor-interacting protein kinase 3(RIP3),a key signaling molecule related to necroptosis.But,RIP3-mediated METH-induced neuronal necroptosis has not been reported.Objectives:Our study is to investigate the molecular mechanism of RIP3 on METH-induced dopaminergic neuronal necroptosis,further complementing METH-mediated cell death,and providing a theoretical basis for METH neurotoxic injury mechanism research and gene intervention.Methods:In this study,we first detected that whether METH can induce neuron necrosis in the specimen of METH abusers.We then constructed METH-treated model in vivo and in vitro,and use specific inhibitor and LV-shRIP3 to interfere with the expression and activation of RIP3.HE staining,immunohistochemical staining,Western Blot,TUNEL staining and immunofluorescence were used to detect the expression of RIP3 before and after intervention,and the changes of downstream signaling pathway molecules,and to explore the role of RIP3 in METH-induced neuronal necroptosis.Result:1.Neuronophagia appeared in the striatum of deceased exposed to METH,indicating that METH can promote neuronal necrosis,and expression of RIP3 protein was increased2.A similar phenomenon was observed in the striatum of the METH poisoning mice model,and the increment of RIP3 protein in the striatum of METH-treated mice was more obvious in necrotic areas labelled with TUNEL,indicating that RIP3 plays a key role in METH-induced cell necroptosis.3.After knocking down RIP3 expression,the number of death cells in the striatum got reserved,and the expression of pMLKL(S345)and pDRP1(S616)protein,NSE content in peripheral blood plasma and the mitochondrial fission decreased,indicating that RIP3 may mediate cell necroptosis via the downstream proteins MLKLand DRP1.4.METH can cause RIP and RIP3 interaction in primary dopaminergic neurons to phosphorylate RIP3.Activated RIP3 simultaneously activates downstream MLKL protein,thereby leading cell membrane rupture and causing cell necroptosis.5.Activated DRP1 translocated to mitochondria,causing mitochondrial fission,mitochondrial membrane potential decreasing,and intracellular ROS increasing,which mediates cell necroptosis.Conclusions:RIP3 plays an important role in METH-induced dopaminergic neuronal necroptosis,providing a potential molecular intervention target for neurotoxic therapy of METH.