Antagonistic Roles Of Itaconate In Β2-Microglobulin-Induced Depression-and Anxiety-Like Behaviors And The Underlying Mechanisms

β2-microglobulin(B2M)is a newly discovered endogenous molecule that causes depression and anxiety.A large amount of evidence shows that the generation of B2M is significantly increased under the state of disease,suggesting that B2M is involved in comorbidity of depression and other diseases.Therefore,it is significant to explore ways to prevent B2M-induced depression for treating comorbidity of depression and other diseases.Itaconate,as a metabolite of tricarboxylic acid cycle,exerts a wide range of anti-inflammatory effect.Based on the neuroinflammation plays an important role in promoting the development of depression and anxiety,we will explore the antagonistic effect of Itaconate on B2M-induced depression and anxiety and its underlying mechanism.Kynurenine pathway(KP)is an important pathway of tryptophan metabolism in the brain.Under normal physiological conditions,quinolinic acid(QA),a downstream product of KP,is converted into NAD+ through quinolinate phosphoribosyl transferase(QPRT)to maintain homeostasis.In the stress state,the disorder of KP metabolism leads to the accumulation of QA and the decrease in NAD+production,which is an important factor for the occurrence of depression-and anxiety-like symptoms.Therefore,we will explore whether Itaconate prevents B2M-induced depression-and anxiety-like behaviors and investigate the underlying mechanisms from modulating the metabolic pathway of KP in the hippocampus of rats.Thus,we will research as follows:(1)To clarify the antidepressant-and antianxiety-like effects of Itaconate on B2M-exposed rats,and to specify the inhibitory roles of Itaconate on B2M-induced hippocampal neuroinflammation as well as synaptic plasticity disorders;(2)To reveal that Itaconate antagonizes B2M-induced metabolic imbalance of KP in the hippocampus of rats;(3)To confirm that QA accumulation,as the main characteristic of KP imbalance,elicits depression-and anxiety-like behaviors in rats.(4)To clarify that intercepting the conversion of QA to NAD+abolishes the antidepressant-and antianxiety-like effects of Itaconate in B2M-exposed rats.Part Ⅰ:Itaconate antagonizes B2M-induced depression-and anxiety-like behaviors and ameliorates neuroinflammation as well as synaptic plasticity dysfunction in hippocampusObjective:As an important neuroimmune protein,B2M has been proved to be an important endogenous depression-associated factor.The occurrence of neuroinflammation causes synaptic plasticity disorder,which plays an important role in the development of depression and anxiety.Itaconate is an anti-inflammatory metabolite.In the present study,we will explore the antagonistic effects of Itaconate on B2M-induced depression-and anxiety-like behaviors and whether this effect of Itaconate is involved in modulating neuroinflammation and synaptic plasticity.Methods:B2M(0.9 μg,1 μl,single dose,i.c.v.)and OI(4-Octyl Itaconate,a derivative of Itaconate,0.06 mg,0.18 mg,0.6 mg,3 μl,single dose,i.c.v.)were injected into the lateral ventricle of SD rats,respectively.Six days after administration,the depression-like behavior of rats was evaluated by source preference test(SPT),tail suspension test(TST)and forced swimming test(FST).The anxiety-like behaviors of rats were evaluated by novelty-suppressed feeding test(NSFT)and elevated plus-maze test(EPMT),as well as the motor function and anxiety-like behaviors of rats was evaluated by open field test(OFT).The activation of microglia in CA1,CA3 and DG region of hippocampus was observed by immunofluorescence,the levels of hippocampal inflammatory cytokines were detected by Meso Scale Discovery(MSD).The expressions of synaptic plasticity-related proteins SYN1,SYP,MAP2 and PSD95 in the region of CA1,CA3 and DG in hippocampus were detected by immunofluorescence,and the changes of synaptic ultrastructural in hippocampus were observed by transmission electron microscope.Results:1.Itaconate exerts antidepressant-like effect in B2M-exposed rats.OI significantly increased the source preference rate in SPT and reduced the immobility time in the TST in B2M-exposed rats.OI also shortened the immobility time of B2M-exposed rats in the FST,indicating the antidepressant effect of Itaconate in rats treated with B2M.2.Itaconate exerts the antianxiety-like effect in B2M-exposed rats.OI significantly increased the central distance of B2M-exposed rats in OFT.In the NFST,OI shortened the feeding latency of B2M-exposed rats,while there was no significant difference of the food intake in 10 min among the rats.Moreover,OI treatment significantly increased the number of entries into and the time spent in the open arm in B2M-treated rats,while the number of total arm entries was no change in the EMPT.These data indicated the antianxiety-like effect of Itaconate in rats treated with B2M.3.Itaconate antagonizes the hippocampal neuroinflammation in B2M-exposed rats.The expressions of IBA1 and CD68,as well as the number of IBA1+cells co-localized with CD68 were increased in B2M-treated rats,indicating that B2M induces excessive activation of microglia in hippocampus.While treatment with OI significantly decreased the expressions of IBA1 and CD68,as well as the number of IBA1+ cells co-localized with CD68 in hippocampal of B2M-exposed rats.Furthermore,the productions of pro-inflammatory cytokines TFN-γ,TNF-α,IL-1β,IL-6 and KC/GRO were increased,while the levels of anti-inflammatory cytokines IL-4,IL-5,IL-10 and IL-13 were decreased,in the hippocampus of B2M-exposed rats.While OI treatment significantly decreased the levels of pro-inflammatory cytokines and increased the levels of anti-inflammatory cytokines in the hippocampus of B2M-treated rats.These results indicated that Itaconate ameliorates neuroinflammation in the hippocampus of B2M-exposed rats.4.Itaconate ameliorates hippocampal synaptic plasticity disorder in B2M-exposed rats.The expressions of synaptic plasticity-related proteins SYN1,SYP,MAP2 and PSD95 in CA1,CA3 and DG region of hippocampus were significantly decreased in B2M-exposed rats,while OI treatment increased the expressions of SYN1,SYP,MAP2 and PSD95 in the hippocampus of B2M-exposed rats.Moreover,the synapse density,synaptic surface density,synaptic interface curvature,the length of synaptic active zone,and the thickness of postsynaptic density(PSD)were significantly decreased,as well as the synaptic cleft width was increased,in the section of CA1,CA3 and DG in the hippocampus of B2M-exposed rats.While OI significantly increased the synapse density,synaptic surface density,synaptic interface curvature,the length of synaptic active zone,and the thickness of PSD,as well as decreased the synaptic cleft width,in the section of CA1,CA3 and DG in the hippocampus of B2M-exposed rats.These data indicated that Itaconate ameliorates the disorder of hippocampal synaptic plasticity in the B2M-exposed rats.Conclusion:Itaconate antagonizes B2M-induced depression-and anxiety-like behaviors and ameliorates B2M-induced neuroinflammation as well as synaptic plasticity dysfunction in the hippocampus of ratsPart Ⅱ:Itaconate redresses the imbalance of Kynurenine pathway metabolism in the hippocampus of B2M-exposed ratsObjective:Metabolic disorder of kynurenine pathway(KP)is an important factor in inducing neuroinflammatory response and plays an important role in the occurrence of depression.In order to further reveal the mechanism underlying the antidepressant-like role of Itaconate,we will further explore the corrective effect of Itaconate on the metabolic disorder of KP in the hippocampus of B2M-exposed rats.Methods:The enzymes of KP,such as IDO,KMO,KYNU,HAAO and QPRT,in hippocampus were detected by Western Blot,and the metabolites of KP,such as KYN,3-HK,3-HAA,QA,and NAD+,in hippocampus were detected by LC-MS/MSResults:1.Itaconate regulates the expressions of key enzymes of KP in the hippocampus of B2M-exposed rats.The expressions of IDO1,KMO,KYNU,and HAAO were increased,as well as the expression of QPRT was decreased,in the hippocampus of B2M-exposed rats,which indicated that B2M induces metabolic disorder of KP.While OI treatment decreased the expressions of IDO,KMO,KYNU,and HAAO,and increased the expression of QPRT,which indicated that Itaconate regulates the metabolism of KP in the hippocampus of B2M-exposed rats.2.Itaconate regulates the KP metabolites in the hippocampus of B2M-exposed rats.The intermediates of KP,such as KYN,3-HK,3-HAA,and QA,were increased,while the production of NAD+was decreased,in the hippocampus of B2M-treated rats,indicating the imbalance of KP metabolism induced by B2M in the hippocampus.While OI treatment decreased the levels of KYN,3HK,3HAA,and QA and increased the level of NAD+in the intermediates of KP in the hippocampus of B2M-treated rats,which indicated that Itaconate restores the KP metabolites levels in the hippocampus of B2M-exposed rats.Conclusion:Itaconate corrects the KP metabolic imbalance in the hippocampal of B2M-exposed rats.Part Ⅲ:Quinolinic acid causes depression-and anxiety-like behaviors as well as hippocampal neuroinflammation and synaptic plasticity disorder in ratsObjective:The accumulation of quinolinic acid is an important signal of KP metabolic disorder.In order to further clarify that rectifying the metabolic disorder of KP and reducing the accumulation of QA are the crucial link of Itaconate to inhibit B2M-caused depression and anxiety,we will also confirm that QA induces hippocampal neuroinflammation and synaptic plasticity disorder as well as depression-and anxiety-like behaviors.Methods:QA(Quinolinic acid,50 nmol,150 nmol,500 nmol,1μ,single dose,i.c.v.)was injected into the lateral ventricle of SD rats.Six days after administration,the depression-and anxiety-like behaviors of rats were evaluated by SPT,TST,FST,NSFT,EPMT and OFT.The activation of microglia in CA1,CA3 and DG region of hippocampus was observed by immunofluorescence,the levels of hippocampal inflammatory cytokines were detected by MSD.The changes of synaptic plasticity were detected by immunofluorescence and transmission electron microscope.Results:1.QA induces depression-like behaviors in rats.Compared with the control group,QA significantly decreased the source preference rate in the SPT and increased the immobility time in the TST.QA also increased the immobility time in the FST.These results indicated that QA induces depression-like behaviors in rats.2.QA induces anxiety-like behaviors in rats.Compared with the control group,QA significantly decreased the central distance of rats in the OFT.In the NSFT,QA increased the time of feeding latency,while there was no significant difference of the food intake in 10min among the rats.Moreover,QA treatment significantly decreased the number of entries into and the time spent in the open arm,while the number of total arm entries was no change of rats in the EMPT.These data indicated that QA induces anxiety-like behaviors in rats.3.QA induces hippocampal neuroinflammation in rats.Compared with the control group,QA increased the expressions of IBA1 and CD68,as well as the number of IBA1+cells co-localized with CD68 in the hippocampus of rats,indicating that QA induces excessive activation of microglia in hippocampus.Furthermore,QA increased the secretion of pro-inflammatory cytokines TFN-γ,TNF-α,IL-1β,IL-6 and KC/GRO,while decreased the levels of anti-inflammatory cytokines IL-4,IL-5,IL-10 and IL-13,in the hippocampus of rats.These data indicated that QA induces hippocampal neuroinflammation.4.QA leads to synaptic plasticity dysfunction in rats.Compared with the control group,the expressions of synaptic plasticity-related proteins SYN1,SYP,MAP2 and PSD95 in the CA1,CA3 and DG region of hippocampus were significantly decreased in QA-exposed rats.Moreover,QA decreased the synapse density,synaptic surface density,synaptic interface curvature,the length of synaptic active zone,and the thickness of PSD,as well as increased the synaptic cleft width,in the hippocampus(CA1,CA3 and DG)of rats.These data indicated that QA induces hippocampal synaptic plasticity disorder.Conclusion:QA induces depression-and anxiety-like behaviors,and its underlying mechanism is closely related to hippocampal neuroinflammation and synaptic plasticity disorder.Part Ⅳ:Blocking the conversion of QA to NAD+ reverses the antagonistic effect of Itaconate on depression-and anxiety-like behaviors in B2M-exposed rats.Objective:As an important active molecule in KP metabolism,the increase of QA level represents the activation of KP pathway and the disorder of KP metabolism.While Itaconate restores the balance of KP metabolism via reducing the accumulation of QA and promoting the production of NAD+ in the hippocampus of B2M-exposed rats.Therefore,in order to further clarify that the antagonistic mechanism of Itaconate on B2M-induced depression-and anxiety-like behaviors in rats is to correct the metabolic imbalance of KP and promote the transformation of QA into NAD+,we used phthalic acid(PA),a specific inhibitor of quinolinic acid phosphoribose transferase(QPRT),to induce the metabolic imbalance of KP by promoting the accumulation of QA in the brain,and to explore whether blocking the conversion of QA to NAD+ reverses the antagonistic effect of Itaconate on B2M-induced depression-and anxiety-like behaviors of rats.Methods:Rats were injected with B2M(0.9 μg,1 μl,single dose,i.c.v.)and OI(4-Octyl Itaconate,0.18 mg,3 μl,single dose,i.c.v.)in the absence or presence of PA(500 μM,1 μl,single dose,i.c.v.),respectively.Six days after administration,the metabolites of KP(QA and NAD+)in the hippocampus were detected by LC-MS/MS.The changes of synaptic plasticity were detected by immunofluorescence and transmission electron microscope.The activation of microglia in the region of CA1,CA3 and DG in hippocampus was observed by immunofluorescence,the levels of hippocampal inflammatory cytokines were detected by MSD.The depression-and anxiety-like behaviors of rats were evaluated by SPT,TST,FST,NSFT,EPMT and OFT.Results:1.PA blocks Itaconate-increased NAD+ levels in the hippocampus of B2M-exposed rats.PA abolished the improvement of OI on the production of hippocampal NAD+ in B2M-treated rats,indicating that PA reverses Itaconate-restored KP balance in the hippocampus of B2M-exposed rats.2.Inhibiting the conversion of QA to NAD+reverses the protection of Itaconate against B2M-induced hippocampal neuroinflammation in rats.PA abolished OI-decreased the expressions of IBA1 and CD68,as well as the number of IBA1+ cells co-localized with CD68 in B2M-treated rats.Furthermore,PA increased the levels of pro-inflammatory cytokines TFN-γ,TNF-α,IL-1β,IL-6 and KC/GRO,while decreased the levels of anti-inflammatory cytokines IL-4,IL-5,IL-10 and IL-13,in the hippocampus of B2M-exposed rats cotreated with OI,indicated that abolishing Itaconate-restored the metabolic balance of KP in the hippocampus of B2M-exposed rats reverses the protection of Itaconate against B2M-induced hippocampal neuroinflammation.3.Inhibiting the conversion of QA to NAD+reverses the protection of Itaconate against B2M-induced hippocampal synaptic plasticity disorder in rats.PA downregulated the expressions of synaptic plasticity-related proteins SYN1,SYP,MAP2 and PSD95,decreased the synapse density,synaptic surface density,synaptic interface curvature,the length of synaptic active zone,and the thickness of PSD,as well as increased the synaptic cleft width,in the hippocampus(CA1,CA3 and DG)of B2M-exposed rats cotreated with OI,indicated that abolishing Itaconate-restored the metabolic balance of KP in the hippocampus of B2M-exposed rats reverses the protection of Itaconate against B2M-induced hippocampal synaptic plasticity disorder.4.Inhibiting the conversion of QA to NAD+reverses the antidepressant-like role of Itaconate in B2M-exposed ratsPA significantly decreased the source preference rate in the SPT and increased the immobility times in the TST and the FST in the rats cotreated with B2M and OI,which indicated that abolishing Itaconate-restored the metabolic balance of KP in the hippocampus of B2M-exposed rats reverses the antagonistic effect of Itaconate against B2M-induced depression-like behaviors.5.Inhibiting the conversion of QA to NAD+reverses antianxiety-like role of Itaconate in B2M-exposed ratsFurthermore,PA significantly decreased the central distance of rats cotreated with B2M and OI in the OFT.In the NSFT,PA also increased the time of feeding latency in the rats cotreated with B2M and OI,while there was no significant difference of the food intake in 10 min among the rats.Moreover,in the EMPT,PA significantly decreased the number of entries into and the time spent in the open arm in the rats cotreated with B2M and OI,while the number of total arm entries was no change among the rats.These results indicated that abolishing Itaconate-restored the metabolic balance of KP in the hippocampus of B2M-exposed rats reverses the antagonistic effect of Itaconate on B2M-induced anxiety-like behaviors in rats.Conclusion:Blocking the conversion of QA to NAD+ reverses the protection of Itaconate against B2M-induced hippocampal neuroinflammation and hippocampal synaptic plasticity disorder,as well as depression-and anxiety-like behaviors in rats.ConclusionItaconate exerts antidepressant and antianxiety-like effects on B2M-exposed rats through alleviating the hippocampal neuroinflammation and improving the hippocampal synaptic plasticity by restoring the KP balance in the hippocampus of B2M-exposed rats.