The Studies Of The Effect Of Monosaccharides On Mice Neuropsychological Behavior And The Role Of HMGB1 Protein In Depressive Disorder

PART 1: MONOSACCHARIDES INDUCE DEPRESSIVE/ANXIETY-LIKE BEHAVIORS AND IMPAIRED MEMORY AND THE PROBABLE PATHOGENESIS IN MICEBackground:It is reported that between 2005 and 2010,the average sugar consumption of all age and all gender groups exceeded 10% in developed countries.D-glucose(Glc)is the most widely distributed monosaccharide,and the mammalian brain mainly relies on Glc as its energy source.Studies have confirmed that major depressive disorder(MDD)are related to Glc metabolic disorders,the role of other monosaccharides that can pass through the blood-brain barrier is not yet clear,such as D-ribose(RIB),D-mannose(MAN),D-xylose(XYL),and L-arabinose(ARA).It has been reported that the average urine RIB level of Alzheimer's disease(AD)patients is significantly increased than that of healthy people,and excessive RIB can cause cognitive impairment,but there is still poor understanding of its physiological and pathological mechanisms.Additionally,AD patients are often accompanied by MDD.It remains to be further explored whether RIB metabolic abnormalities play an important role in it.Objective:1.Explore the effects of RIB,Glc,MAN,XYL,and ARA in the proliferation of cultured neuro-2a(N2a)cells.And the effects of low-and high-dose RIB and MAN on mice depressive-and anxiety-like behavior,as well as spatial learning and memory ability.2.Explore the underlying molecular pathogenic mechanisms of RIB and MAN causing abnormal neuropsychiatric behavior in mice.Methods:1.CCK8 assay was used to detect the effects of different concentrations of RIB,Glc,MAN,XYL,and ARA on the proliferation of cultured N2 a cells.2.Mice were intraperitoneally injected with low-(0.4 g/kg)or highdose RIB(4 g/kg),low-(0.48 g/kg)or high-dose MAN(4.8 g/kg),or 0.9%saline for four weeks.A series of behavioral tests were used to evaluate the effects of RIB and MAN on the depressive-and anxiety-like behavior,as well as spatial learning and memory ability.3.Ultra-performance liquid chromatography-mass spectrometry and ELISA assay were used to detect the concentrations of RIB and MAN in the hippocampus,prefrontal cortex,cerebral cortex,and hypothalamus after RIB and MAN injection.4.Histopathological results of the CA1,CA2,CA3,and dentate gyrus were analyzed using hematoxylin-eosin(HE)staining.5.Widely targeted metabolomics and RNA-Sequencing analysis was used to investigate the differential metabolites(DEMs)and differential genes(DEGs)in the hippocampus after RIB and MAN treatment.And R language and IPA software were performed to investigate the underlying molecular pathogenic mechanisms.Molecular analysis was used to detect the level of these signaling pathways.Results:1.Among the five monosaccharides,RIB and MAN significantly reduced cell viability(P < 0.05).2.In the 4 g/kg RIB group,the sucrose preference was significantly reduced,while the percentage of immobility time was significantly increased(P < 0.05).In the open-field test,compared with controls,the number of rearing was significantly decreased in the 0.4 and 4 g/kg RIB groups and in the 4.8 g/kg MAN group(P < 0.05),while the central distance in the 4 g/kg RIB group was significantly reduced(P < 0.05).In the Morris water maze,no significant difference was found in escape latency in different concentrations of RIB and MAN treated groups.However,in the probe trial,the 0.4 and 4g/kg RIB groups and the 4.8 g/kg MAN group all displayed a significantly decreased frequency of target platform crossings(P < 0.05)as well as lower percent time and distance traveled in the target quadrant(P <0.05).3.The levels of RIB were significantly increased in the cerebral cortex,hippocampus,and hypothalamus after treatment(P < 0.05),not in the prefrontal cortex(P > 0.05).Moreover,the levels of MAN were significantly increased in the cerebral cortex,hippocampus,and prefrontal cortex after treatment(P < 0.05),not in the hypothalamus(P > 0.05).4.HE staining revealed that the hippocampus of mice only in the 4 g/kg RIB group showed condensed and deeply-stained pyramidal cells(P < 0.05).5.Six DEMs and 190 DEGs were identified in the RIB-treated mice,of which 51 DEGs were highly correlated with 4 DEMs.Moreover,17 DEMs and 264 DEGs were identified in the MAN-treated mice,of which 109 DEGs were highly correlated with 15 DEMs.The potential pathogenic mechanisms of RIB and MAN are related to the insulin-POMC-MEK-TCF7L2 and MAPK-CREB-GRIN2A-Ca MKII signaling pathways,respectively.6.The expression levels of Pomc,Tcf7l2,p-MEK1,and p-MEK2 were upregulated in the 4 g/kg RIB group(P < 0.05),while not in the 0.4 g/kg RIB group(P > 0.05).And the expression levels of Grin2 a,Camk2b,pp38 MAPK,and p-Ca MKII? were significantly increased accompanied by a decrease of p-CREB in the 4.8 g/kg MAN group(P < 0.05),not in the 0.48g/kg MAN group.Conclusions:RIB and MAN can pass through the blood–brain barrier and enter the brain tissue by intraperitoneal injection.High-dose RIB can cause mice depressive-and anxiety-like behaviors and spatial memory impairment,which may be related to the insulin-POMC-MEK-TCF7L2 pathway in the hippocampus.In contrast,high-dose MAN can only cause mice anxiety-like behavior and spatial memory impairment,which may be related to the MAPK-CREB-GRIN2A-Ca MKII pathway in the hippocampus.PART 2: EFFECTS AND POSSIBLE MECHANISMS OF HMGB1 IN DEPRESSIVE DISORDERBackground:Major depressive disorder(MDD)is a serious mental disease with an estimated global life-time prevalence of 21%.Numerous risk alleles for MDD have been identified,such as glucose metabolism disorder,but onset is closely associated with environmental factors,such as stressful life events.Sustained stress and the subsequent release of pro-inflammatory cytokines lead to chronic neuroinflammation and dysregulated autophagy,which in turn are associated with MDD.Brain-resident macrophages(microglia)are essential mediators of neuroinflammation in response to stress,and thus microglial activation is considered a major pathogenic process contributing to MDD.High mobility group box 1(HMGB1)is a danger-associated molecular pattern molecule released in response to physical and psychological stressors.Previous studies have identified that HMGB1 is highly correlated with MDD,however,it remains unclear how elevated brain HMGB1 influences m PFC function and depressive-like behavior.Objective:1.Explore the role of HMGB1 in depression and whether stressinduced activation of the HMGB1/nuclear factor-kappa B(NF-?B)p65/signal transducer and activator of transcription 3(STAT3)axis in the m PFC contributes to MDD pathogenesis by driving local microglial activation and autophagy.2.Explore the level of HMGB1 in MDD patient serum and the correlation between HMGB1 level and clinical characteristic.Methods:1.R language and IPA bioinformatics methods were used to analyze the whole transcriptome data in the dlPFC of post-mortem male MDD patients from a public database.2.MDD model mice were constructed by chronic social defeat stress(CSDS)and investigated the effects of chronic stress in microglial reactivity,autophagy as well as the HMGB1/NF-?B p65/STAT3 axis and proinflammatory cytokine in the m PFC.3.Using the targeted inhibitors of HMGB1 and NF-?B,HMGB1 recombinant protein,AAV overexpression and knockdown virus,and m RFPGFP-LC3 virus in vivo and in vitro,to exam the effects of HMGB1/NF-?B p65/STAT3 and ensuing neuroinflammation and autophagy in depressivelike behavior.4.The enzyme-linked immunosorbent assay was used to detect the level of HMGB1 in MDD patient serum and analyzed the possible factors that affect the level of HMGB1 by multiple regression analysis.Results:1.MDD is associated with the differential expression of genes indicative of microglial activation and autophagy,elevated proinflammatory cytokines,and activation of the HMGB1/NF-?B p65/STAT3 axis in the dlPFC.2.CSDS in mice enhanced depressive behaviors as well as microglial reactivity and autophagy in the m PFC.These responses were associated with robust activation of the HMGB1/NF-?B p65/STAT3 axis and proinflammatory cytokine upregulation in the m PFC.3.Pharmacological inhibition of HMGB1 or NF-?B prevented lipopolysaccharide-induced microglial activation and autophagy,while recombinant(r)HMGB1 protein reversed these effects.Depressive-like behaviors,neuroinflammation,autophagy,and HMGB1/NF-?B p65/STAT3 activation induced by CSDS were mimicked by exogenous administration of r HMGB1 or virally mediated overexpression of HMGB1 in the m PFC,and blocked by downregulation of NF-?B.Conversely,HMGB1 knockdown produced a resilient phenotype and eliminated the activation effects of CSDS-induced depression.4.Serum HMGB1 level is elevated in MDD patients and positively correlated with symptom severity.Conclusions:HMGB1 in the mPFC plays a key role in stress-induced MDD through activation of HMGB1/NF-?B p65/STAT3 axis and ensuing neuroinflammation and autophagy.It also can be used as a diagnostic biomarker and therapeutic target for MDD.