The Experimental Research Of Paliperidone Protect Cortical Neurons Against Damages Induced By MK-801

Schizophrenia is a severe mental disorder with a life time prevalence of1%throughout the world population. This mental illness is characterized by psychotic or positive symptoms (hallucinations and delusions), and negative and cognitive symptoms (apathy, social withdrawal, decreased attention, decreased executive function, abnormal psychomotor speed of processing and impairment of verbal memory). Although there is a variety of theories, signaling pathways and other factors involve in the pathogenesis of schizophrenia, none of them can fully explain the underlying mechanisms. The aetiology and pathophysiology of schizophrenia still needs further exploration and study.MK-801(also named Dizocilpine), is a selective noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist. NMDA receptor is a subtype of glutamate receptors which are widely distributed in the cerebral cortex, hypothalamus, hippocampus, striatum, and spinal cord, and act as an excitatory neurotransmitter in the central nervous system. MK-801is widely used in the establishment of schizophrenia animal models of schizophrenia by blocking the noncompetitive NMDA receptor. The behavioral detections show that the sensorimotor function, learning and working memory, spatial orientation capacity are all subject to varying degrees of damage in the animal models of schizophrenia. The cortical neurons’ vacuolization and atrophy, the fracture of dendrites are also found in these models established by MK-801. In vitro studies reveal that MK-801can reduce cell viability, promote apoptosis-associated proteins expression and inhibit the outgrowth of neurites. Foreign scholars have successfully imitated schizophrenia neuronal damage with MK-801and established the in vitro model of schizophrenia.Non-classical antipsychotic drugs, also known as second-generation antipsychotics, are widely used in clinic. A great number of evidences have shown that second generation antipsychotics (SGAs) might offer neuroprotective effects. In clinical practice, SGAs are reported to improve the long-term outcome of schizophrenia, exhibit effective therapeutic efficacy for negative symptoms and cognitive deficits and have low incidence of extrapyramidal side effects. However, the underling molecular mechanisms have not been well understood, especially the intracellular signaling pathways related to their neuroprotective effects which can reduce the progressive morphological brain change in schizophrenia. However, the mechanisms of paliperidone exhibits neuron protection and anti-schizophrenia effects are still unclear, which needs further studies.In order to examine the mechanisms of antipsychotic as well as its neuron protective effects of paliperidone, we design a series of experiments as follows. In vitro experiments, cortical neurons are dissociated from embryonic mice at E15and cultured in DMEM/F12medium with10%fetal bovine serum. After incubation with DMEM/F12for five days, the neurons were stained with MAP-2to identify the purity of cortical neurons. MK-801with a series concentrations are selected to injure cortical neurons to measure its damage effects on primary cells in vitro. In order to investigate the protective effect, paliperidone of different concentrations are also added to the medium at the same time as MK-801exposure. MTT, LDH activity,[Ca2+]i and time course of [Ca2+]i assays are performed to identify the protection effects of paliperidone on cortical neurons treated with MK-801. The total length and density of neurites are also calculated to determine whether paliperidone can effectively promote neurite outgrowth. Besides, we examine the gene expression of Aktl and GSK3P, both of which are involved in the pathogenesis of schizophrenia.To explore whether the protective effects of paliperidone are mediated by Aktl/GSK3β signaling pathway, cortical neurons are pretreated with the Aktl inhibitor LY294002for1h before treated with MK-801and paliperidone.48h later, cells were double stained with Hoechst33342and PI, or subjected to MTT metabolism assay. In vivo experiments, mouse models of schizophrenia are established with MK-801. The animals are divided into three groups:normal control group, model group, and therapeutic group treated with paliperidone. The mouse behavior of different groups is analyzed to detect the therapeutic effects of paliperidone. We also measured the protein levels of p-Aktl and p-GSK3[3in mouse cortical tissues.The results of in vitro experiments show that the majority of cultured cortical neurons express MAP-2, which demonstrated the high purity of the cells. In contrast with normal control group, cortical neurons treated with a series concentration of MK-801exhibited apparently reduced viability, increased [Ca2+]i and LDH release, declined density and total length of neurites and decreased gene expressions of Aktl and GSK3β. Compared with MK-801, paliperidone inhibit MK-801induced neurotoxicity both in MTT metabolism assay and in LDH activity assay; time course studies reveal that paliperidone effectively attenuate the elevation of [Ca2+]i induced by exposure to MK-801. Moreover, paliperidone can significantly retard MK-801-mediated inhibition of neurite outgrowth and reverse MK-801-induced decreases of gene expression and phosphorylation of Aktl and GSK3β.Furthermore, these protective effects of paliperidone are blocked by pretreatment with a PI3K inhibitor LY294002. In vivo experiments, we successfully establish the animal model of schizophrenia with MK-801, the mice exhibit schizophrenia-like behaviors, such as hyper locomotion and stereotypy rotation. Compared with normal control group, both the number of hyper locomotion and stereotypy are significantly increased in model group. The decreasing protein levels of p-Aktl and p-GSK3(3are also observed in mouse cortical tissues. In the paliperidone therapeutic group, the number of hyper locomotion and stereotypy decreases compared to model group. Moreover, the protein levels of p-Aktl and p-GSK3β are also increased with paliperidone treatment.In conclusion, our results indicate that paliperidone can protect cortical neurons from damages caused by MK-801, and promote the outgrowth of neurites. These neuroprotection and antipsychotic effects may partly by up regulating Aktl/GSK3β signaling pathway.