Study On Anxiety-like Behavior And Mechanisim In Mice By Deletion Of Melatonin Receptor 1B (MTNR1B) In Astrocytes

PART I: ASTROCYTIC DELETION OF MELATONIN RECEPTOR2 LEADS TO ANXIETY-LIKE BEHAVIOR IN MICEPurpose:Melatonin has been shown to play anti-inflammatory and antioxidant roles through its receptors.Global knockout of MTNR1B gene in mice leads to symptoms such as anxiety and decreased attention.However,there have been no studies on mice with astrocyte-specific MTNR1B gene knockdown.Therefore,this study bred the MTNR1B cKO^GFAP mouse strain to explore changes in behavioral patterns and potential mechanisms in mice with astrocytic deletion of MTNR1B gene.Methods:Wild-type mice and MTNR1B cKO^GFAP mice of the same age were selected for cognitive function behavioral testing（including Morris water maze test and novel object recognition test）and emotion-related behavioral testing（including open field test and elevated plus maze test）.After completing the behavioral experiments,brain tissue was collected and subjected to immunofluorescence assays for Neu N,GAD67,and v GLUT1 expression levels in various brain regions（m PFC area,hippocampal CA1 area,CA3 area,and DG area）.The proportion of activated microglia was detected using immunofluorescence co-localization technology and flow cytometry.Golgi staining was used to measure dendritic spine density in wild-type and CKO mice.RNA was extracted from mouse brain tissue,and q RT-PCR was used to detect the expression levels of IL-1β,IL-6,and TNF-α.Results:（1）Differential expression analysis of GEO chip data showed that MTNR1B expression was significantly down-regulated in anxiety combined with depression and Alzheimer’s disease compared to the control group.（2）The area under the curve of ROC（AUC）of MTNR1B gene in patients with anxiety/depression was 0.722,indicating certain diagnostic value.（3）Analysis of the HPA database and immunofluorescence staining showed that MTNR1B gene was mainly expressed in the hippocampus in mouse brain tissue and mainly expressed in astrocytes.（4）Morris water maze results showed that specific knockdown of MTNR1B gene had no effect on spatial learning and memory ability in mice.（5）Novel object recognition test found no significant difference in recognition index between MTNR1B cKO^GFAP mice and wild-type mice.（6）Open field test revealed that compared to wild-type mice,MTNR1B cKO^GFAP mice spent less time exploring the central area of the open field,and the total distance explored was significantly reduced.（7）Elevated plus maze test found that compared to wild-type mice,MTNR1B cKO^GFAP mice spent less time exploring the open arms.（8）Immunofluorescence experiments showed that the number of neurons in the CA1 area,CA3 area,DG area,and prefrontal cortex area did not differ significantly between CKO mice and wild-type mice.（9）The fluorescence intensity of GAD67 in the hippocampal CA1area and CA3 area of MTNR1B cKO^GFAP mice was significantly lower than that in wild-type mice,but there was no significant difference in v GLUT1fluorescence intensity.（10）Dendritic spine density in the hippocampus of MTNR1B cKO^GFAP mice was significantly decreased.（11）Both immunofluorescence assays and flow cytometry detected a significantly higher proportion of activated microglia in the brains of CKO mice.（12）q RT-PCR experiments showed that the expression levels of IL-1β,IL-6,and TNF-αwere significantly upregulated in the brains of CKO mice.Conclusion:（1）MTNR1B cKO^GFAP mice were successfully bred.（2）Compared to wild-type mice,MTNR1B cKO^GFAP mice exhibited an imbalance of excitatory/inhibitory（E/I）neurons.（3）Compared to wild-type mice,microglia were overactivated in the brains of MTNR1B cKO^GFAP mice.（4）Compared to wild-type mice,synaptic connections were decreased in the brains of MTNR1B cKO^GFAP mice.（5）In comparison to wild-type mice,inflammation levels were upregulated in the brains of MTNR1B cKO^GFAP mice.PART II: MECHANISMS UNDERLYING THE ANXIETY-LIKE BEHAVIOR INDUCED BY ASTROCYTIC DELETION OF MELATONIN RECEPTOR2 IN MICEPurpose: The first part of the study demonstrated that MTNR1 B cKOGFAP mice exhibited anxiety-like behavior,E/I imbalance,increased inflammation,overactivated microglia,and synaptic loss.However,the mechanism by which the MTNR1 B gene regulates astrocyte function to affect mouse behavior is unclear.This section aims to explore this mechanism.Methods: RNA-seq was performed on hippocampal tissue from wildtype and MTNR1 B cKOGFAP mice to identify differentially expressed genes.KEGG and GO analyses were conducted,and single pathway GSEA analysis was performed on potential important pathways.IHA astrocytes were cultured in vitro and treated with the MTNR1 B antagonist 4-P-PDOT and LPS.Cell morphology was observed and analyzed.WB was used to detect the protein levels of i NOS,COX2,HO-1,Nrf2,CREB,AKT,and m TOR.q RT-PCR was used to detect the expression levels of IL-1β,IL-6,and TNF-α.Flow cytometry was used to detect the activation state of BV2 microglia cells.Detection kits were used to measure ROS,CAT,SOD,and GSH levels in cells.Results:（1）Single pathway GSEA analysis revealed that astrocytespecific knockdown of the MTNR1 B gene decreased nitric oxide synthesis,leading to decreased glutathione peroxidase levels and decreased reactivity of astrocytes to ROS.（2）When IHA cells were treated with LPS,cell size and length increased,and when cells were co-stimulated with LPS+4-P-PDOT,astrocytes further enlarged.（3）WB results showed that the MTNR1 B antagonist 4-P-PDOT increased the protein levels of i NOS and COX2 in LPS-induced IHA cells.（4）In vitro knockdown of the MTNR1 B gene also increased the levels of the inflammatory cytokines IL-6 and TNF-α in IHA cells.（5）Flow cytometry revealed that in vitro knockdown of the MTNR1 B gene increased the proportion of activated microglia（CD11bhighCD45+）induced by inflammation.（6）The MTNR1 B antagonist 4-P-PDOT significantly upregulated ROS accumulation in LPS-induced astrocytes,while GSH and CAT indices representing cellular antioxidant capacity were significantly decreased,and SOD levels were upregulated.（7）The MTNR1 B antagonist 4-P-PDOT further downregulated LPS-induced HO-1 levels.（8）WB results showed that neither the cytoplasmic nor nuclear Nrf2 protein levels in IHA cells were affected by LPS and 4-PPDOT treatment.（9）WB results showed that the MTNR1 B antagonist 4-PPDOT further downregulated the CREB signaling pathway levels induced by LPS in immortalized human astrocytes（IHA）.Conclusion:（1）An in vitro cell model with MTNR1 B knockdown was successfully established.（2）In vitro knockdown of the MTNR1 B gene increased inflammation levels in IHA cells.（3）In vitro knockdown of the MTNR1 B gene led to overactivation of BV2 microglia.（4）In vitro knockdown of the MTNR1 B gene caused an imbalance in the oxidative/antioxidant system.（5）The oxidative stress induced by in vitro knockdown of the MTNR1 B gene was mediated by the CREB/HO-1 signaling pathway.