The Role And Mechanism Of WNT/β-catenin Signaling Pathway In Regulating Psychiatric Disorder After Traumatic Brain Injury In Rats

Traumatic brain injury(TBI)is one of the most common nervous system(CNS)injury in clinical diagnosis and treatment,as well as identification and appraisal of forensic clinical medicine.It is difficult to treat and prevent memory disorder and mental damage after TBI.In general,secondary brain injury causes a series of neurological events and even death.Secondary brain injury is the material basis for psychiatric disease.After brain injury,autophagy and death related signaling pathways were activated and the internal environment in brain was destroyed significantly.Neuron,astrocyte and microglia is the most important cell in brain which participate regulation of brain function.In TBI brain tissue,reactive astrocyte could promote neuron synapses death and degradation.Meanwhile,microglia,as the immune cells in CNS,are activated and generated a large number of inflammatory factors post-TBI.The WNT/β-catenin signaling pathway have well-studied critical roles in the early developmental events and brain cell fate,such as proliferation and migration.However,the role of WNT/β-catenin signaling pathway in brain injury and even CNS injury and the mechanism of intercellular regulation are still unclear.In this paper,based on the WNT/βcatenin signaling pathway,we identified the WNT/β-catenin signaling pathway as the therapeutic target and the regulatory core pathway of intercellular interaction,and then,analyzed and discussed the mechanism of interaction post TBI.Objectives1.To establish an animal model of TBI and acute-neuroinflammation and to explore the expression difference of WNT/β-catenin signaling pathway.2.To investigate the protection mechanism of WNT/β-catenin signaling pathway postTBI.3.To explore the effect of WNT/β-catenin signaling pathway on anxiety-and depression-like behavior of animals suffered TBI.4.To investigate the role of WNT/β-catenin signaling pathway on morphology and function of neurons,astrocytes and microglia post-TBI.5.To investigate the effect of WNT/β-catenin signaling pathway on polarization of microglia and astrocytes post-TBI.6.To explore the effect of WNT/β-catenin signaling pathway on migration of astrocytes,and investigate the role of astrocytes migration on neuronal synaptic plasticity.7.To explore the regulation of WNT/β-catenin signaling pathway on neuron,astrocytes and microglia post-TBI.Methods1.TBI was performed using the controlled cortical impact(CCI)model and acuteneuroinflammation model was induced by intracerebroventricular(ICV)injection of LPS.2.The Wire Grip Test and Morris Water Maze were performed to detect the learning and memory function in sham rats and rats with change of expression of WNT/β-catenin signaling pathway.Meanwhile,Open Field Test,Light Dark box Test and Marble Burying Test were used to assess the anxiety-like behavior post-TBI and Sucrose Preference Test,Forced Swim Test,Social interaction Test were employed to investigate the depression-like and autism behavior.3.Immunofluorescence assay was used to determine the proliferation of astrocytes and microglia with regulation of WNT/β-catenin signaling pathway.4.Establishing primary cells injury model,LPS was used to induce injury on primary neuron culture,primary astrocyte culture and primary microglia culture.The expression of WNT/β-catenin signaling pathway was determined by Western blot.5.Astrocytes and microglia polarization markers were detected by RT-PCR and Western blot.6.The effect of WNT/β-catenin signaling pathway on proliferation of astrocytes was detected by EdU assay.7.Transwell co-culture were used to investigate migration of astrocytes and microglia and explore the function of migration on field of neuronal plasticity.8.The TUNEL assay was employed to detected the apoptosis regulation of WNT/βcatenin signaling pathway in primary astrocytes-neurons co-culture model.Results1.β-catenin level was significantly downregulated at 6 h in acute neuroinflammation model,and tendency of β-catenin expression was as same as that of TBI model.The WNT signaling was inhibited with activated microglia-condition medium(MCM)in primary neuron,and the off-peck time point is 8 hours and recovered at 48h.In astrocytes,the alteration of WNT was slight change from 1 hour to 8 hours,but down-regulation at 12 hours and lasted 48 hours2.The movement distance in salinomycin and DKK-1 group was decrease at 3 d,7 d and 14 d post-TBI significantly.Compare with sham group,the center distance of TBI,both in agonist and inhibitor group was downregulation at 3d and 7 d after TBI.In light-dark box test,light compartment times and number of light compartment entries significantly decreased if rats were suffered TBI.LiCl and Wnt3a could increase the light compartment time and entries compared with vehicle.In marble burying test,WNT agonist significantlydecreased the marble burying behavior as compared to vehicle group at 7d and 14d postTBI.3.LiCl and Wnt3a,as agonists,could reversed the β-catenin decrease at 1 d after TBI.In the meantime,the expression of inflammatory factors like iNOS and IL-1β was decrease in LiCl and Wnt3a treated groups compared with vehicle,salinomycin and DKK-1 group.4.Activation of WNT signaling will promote CD206 upregulation and formation M2 microglia as well as reduce apoptosis.In astrocytes,results showed that S100a10 and Tm4sf1 overexpressed in OGD/R and LiCl treatment groups and Arginase-1 and Ptx3 downregulated in LPS,activated-MCM and DKK-1 groups.5.The migration of microglia had no change in LiCl,DKK-1,LPS,LPS-MCM treatment group,but the number of astrocytes migration was increased significantly in LPS treated neuron group,especially in the astrocytes suffered LPS and DKK treatment6.The synapsis of primary neuron treated with M1-and DKK-microglia was inhibited and the number of synapsis increased compared with M2-and LiCl-microglia group.In astrocytes LiCl and OGD/R treatment could increase the length of synapsis grew longer than DKK-1 and LPS-MCM treatment group.7.The migration of microglia have no significant effect on the plasticity of neuron,and the migration of astrocytes could inhibit the synaptic elimination and plasticity of neuron.8.Activation of WNT/β-catenin signaling pathway could promote astrocytes to maintain resting state and polarization to A2 state,and reduce LPS-induced apoptosis.Conclusions1.The abnormal expression of WNT/β-catenin signaling pathway could be one of the most important pathophysiological mechanisms of memory and mental disorder post-TBI.2.The time course of WNT/β-catenin signaling pathway expression in neurons and astrocytes is different.The downregulation of WNT/β-catenin signaling pathway in astrocytes is a base of regulation.3.WNT/β-catenin signaling pathway could affect the polarization of glial cells(astrocytes and microglia).Activation of WNT/β-catenin signaling pathway could maintain the resting state of glial cells and promote the polarization of cells to anti-inflammatory.4.The migration of astrocytes is negative factor on the plasticity of neuron,and activation of WNT/β-catenin signaling pathway could inhibit the migration of astrocyte.5.Glial cells play an important role in maintaining neuronal plasticity,and regulation mode of WNT/β-catenin signaling pathway-astrocytes-neuron loop necessary for neuron plasticity.