Mechanism Of NPAS2 On Depression-like Behavior Induced By Neuroinflammation

Background:Depression is a major clinical feature of significant and persistent mood depression,which can lead to serious consequences.In recent years,the incidence of depression has been increasing year by year,but the causes of depression are still poorly understood.Therefore,it is urgent to search for intervention targets related to depression.The phenomenon of biological rhythm is closely related to our daily life,regulating all aspects of physiological activities of the human body.The NPAS2 protein as a key component of the biological clock,is involved in a wide range of transcriptional regulation,mediating biological responses to light,and has been implicated in psychiatric disorders.It is not clear whether the circadian rhythm molecule NPAS2 changes after depression,therefore,this study focuses on the role of rhythm gene NPAS2 in depression.Objective:(1)To determine the expression changes of the rhythm molecule NPAS2 in the central nervous system by establishing depression-like behavior induced by neuroinflammation.(2)To explore the role of NPAS2 gene knockout on depression-like behavior and the molecular mechanism.Methods:(1)C57BL/6J male mice(6-8 weeks)were successfully established inflammatory depression model by LPS,and verified the successful depression model by Forced Swimming Test and Tail Suspension Test.With the success of the depression model,the normal and depression group expression of NPAS2 protein in hippocampus,prefrontal cortex and amygdala was detected by Western blotting.The expression of NPAS2~+/Neu N~+and NPAS2~+/GFAP~+were determined by immunofluorescence staining,and the expression of NPAS2 in primary cultured astrocytes and neurons after LPS induced inflammation were verified by Western blotting.(2)NPAS2 knockout mice were constructed and induced inflammatory depression model by LPS,next to verify NPAS2 gene role in depression through behavioral tests.The expressions of IL-1α,IL-1β,IL-4,IL-6,TNF-α,IL-10 and TGF-βin the hippocampus of WT、WT-LPS、NPAS2 KO、NPAS2 KO-LPS groups were detected by Elisa and Western blotting.The changes of astrocyte marker(GFAP)and microglia marker(Iba-1)in hippocampus of mice in each group were verified by immunofluorescence staining.Finally,the expression of BDNF/Trk B signaling pathway molecules,synaptic related molecules PSD95 and SNAP25 were detected by Western blotting.Results:(1)The expression of NPAS2 protein in the hippocampus of depressed mice were increase,but there were no significant changes in the prefrontal cortex and amygdala.(2)The results of immunofluorescence staining showed that NPAS2 could be expressed in both astrocytes and neurons.After LPS stimulation,the expression of NPAS2increased in astrocytes,but there was no significant change in neurons,and the results were consistent in vitro experiments.(3)Compared with the WT-LPS group,NPAS2 KO mice were resistant to depression-like behaviour induced by LPS.In addition,the Elisa results showed that pro-inflammatory factors IL-1β,IL-6 and TNF-αin NPAS2 KO mice did not up regulate after LPS induction,and anti-inflammatory factors IL-10 and TGF-βlevels were increase.While IL-1αand IL-4 had no significant changes.And the results were consistent by Western blotting.(4)The changes of GFAP and Iba-1 in WT、WT-LPS、NPAS2 KO、NPAS2 KO-LPS group were detected by immunofluorescence staining.The results showed that LPS significantly increased the number of GFAP~+astrocytes in hippocampus,but did not change the number of astrocytes in NPAS2 KO mice.However,compared with WT group mice,LPS treatment did not change the expression of IBa-1~+microglia in the hippocampus of NPAS2 KO mice.(5)To test the expression of BDNF,p-Trk B,Trk B,PSD95 and SNAP25 proteins in the hippocampus in each group by Western blotting.The results showed that LPS could significantly reduce the expression of the above proteins,but the NPAS2 knockout mice could significant recovery the BDNF level.And the expression of the receptor p-Trk B was also increased.Compared with WT-LPS group,PSD95 and SNAP25 protein levels in NPAS2 KO-LPS group were increasedConclusions:The increase of NPAS2 protein in the hippocampus is involved in depression-like behavior in mice;NPAS2 knockout mice play an antidepressant role by increasing the level of synaptic plasticity proteins PSD95 and SNAP25 through the BDNF/Trk B signaling pathway.