| Objectives: To investigate the association between gut microbiota dysbiosis and reduced secretion of glucagon-like peptide-1(GLP-1)in patients with major depressive disorder(MDD)and to explore the specific mechanisms by which GLP-1 exerts its antidepressant effects through neuroprotection,aiming to provide experimental and theoretical evidence for the "microbiota-gut-brain" axis hypothesis of MDD.Methods:Part 1: A case-control study was conducted.A total of 80 newly diagnosed and untreated MDD patients were divided into two groups.One group of 40 patients provided blood samples,while the other group of 40 patients provided stool samples.Age-and gender-matched control samples were collected for both groups.ELISA was used to measure GLP-1 concentrations in plasma and feces,and brain-derived neurotrophic factor(BDNF)concentrations in plasma.q RT-PCR was employed to assess the relative abundance of seven target bacterial genera in the gut.Clinical data were analyzed to investigate the differences in plasma and fecal GLP-1 levels and their relationship with gut microbiota and plasma BDNF in MDD patients.Part 2: A corticosterone-induced cell model of MDD was established using HT-22 hippocampal neurons in mice.After GLP-1intervention,cell morphology was observed under fluorescence microscopy.Cell proliferation was assessed using the CCK-8 assay,and apoptosis and necrosis were detected using flow cytometry and Hoechst/Propidium Iodide double staining.Lactate dehydrogenase and glucose concentrations in cell culture supernatants were measured by colorimetry.Neurotransmitter levels(serotonin,norepinephrine,dopamine,and BDNF)were quantified by ELISA,and key proteins in the c AMP-CREB-BDNF signaling pathway were analyzed using Western blot.Part 3: An animal model of MDD was established in SD rats using chronic unpredictable mild stress(CUMS)for four weeks,followed by two weeks of drug treatment.Behavioral changes were compared between normal control,MDD,fluoxetine-treated(MDD+Flu),and fluoxetine combined with lactobacillus treatment(MDD+Flu+Lac.)groups.16 S r DNA sequencing was used to analyze the gut microbiota distribution in each group of rats.Hematoxylin and eosin staining was performed to observe morphological changes in colon mucosa and hippocampal tissues.Nissl staining was used to assess Nissl bodies in hippocampal neurons.Electron microscopy was employed to examine subcellular structures of hippocampal neurons.Immunofluorescence experiments were conducted to assess the expression of apoptosis-related proteins in hippocampal neurons.ELISA was used to measure neurotransmitter levels in rat serum and brain tissue.Western blot was employed to assess protein levels of GLP-1,apoptosis-related proteins,and key proteins in the c AMP-CREB-BDNF signaling pathway in rat colon and hippocampal tissues.Results:Part 1: 1.Plasma and fecal GLP-1 levels were significantly lower in the MDD group compared to the control group(Plasma: 0.81±0.22 vs.1.05±0.26,P<0.01;Fecal:2.23±0.46 vs.2.47±0.37,P<0.05);2.The MDD group exhibited gut microbiota dysbiosis,with higher relative abundance of Prevotella,Roseburia,and Escherichia coli compared to the control group(P<0.05),but lower relative abundance of Lactobacillus,Bacteroides,and Bifidobacterium(P<0.05);3.Fecal GLP-1 levels were positively correlated with the relative abundance of Lactobacillus in both MDD and control groups(MDD group:r=0.405,P<0.01;Control group: r=0.693,P<0.001);4.Plasma BDNF levels were lower in the MDD group compared to the control group(P<0.05),and this decrease correlated positively with GLP-1 levels(MDD group: r=0.554,P=0.01;Control group: r=0.633,P<0.01).Part 2: 1.Corticosterone(CORT)damage to HT-22 cells at 200μM for 48 hours effectively established an MDD cell model;2.GLP-1 at 50 n M reversed the cell damage induced by CORT,resulting in a significant decrease in cell death and apoptosis rate from18.567±0.662 to 10.677±0.606(P<0.0001),increased levels of serotonin(5-HT),norepinephrine(NE),dopamine(DA)in cell culture supernatant compared to the Model group(P<0.05 for all),and a significant decrease in glucose concentration(P<0.05);3.Key proteins in the c AMP-CREB-BDNF signaling pathway in cell culture supernatant were significantly reduced in the model group compared to the control group,and GLP-1treatment increased the expression of these proteins(PKA: 0.302±0.002 vs.0.439±0.025,P<0.0001;p-CREB: 0.391±0.005 vs.0.527±0.032,P=0.001;p-Trkb: 0.312±0.027 vs.0.396±0.016,P=0.001).Part 3: 1.Behavioral experiments following four weeks of CUMS exposure showed depressive-like behavior in SD rats.Treatment with the antidepressant fluoxetine(MDD+Flu)and fluoxetine combined with lactobacillus(MDD+Flu+Lac.)for two weeks improved depressive-like behavior in rats,with the MDD+Flu+Lac.group showing more significant improvement;2.16 S r DNA sequencing results revealed distinct dominant gut bacteria in each group of rats(Control group: Actinobacteria;MDD group:Selenomonadales and Negativicutes;MDD+Flu group: Clostridia;MDD+Flu+Lac.group:Lactobacillus);3.Levels of DA,5-HT,NE,and GABA in rat serum were significantly lower in the MDD group compared to the Control group(P<0.01 for all),and the MDD+Flu+Lac.group showed significant increases in these neurotransmitter levels(P<0.01).There were no significant differences between the MDD+Flu group and the MDD group(P>0.05);4.Compared to the control group,the MDD group exhibited colonic mucosal damage,inflammatory changes,and reduced GLP-1 levels(P<0.0001).Rats in the MDD+Flu and MDD+Flu+Lac.groups showed improved mucosal morphology and reduced inflammatory cell infiltration,with higher GLP-1 levels(P<0.0001).The improvement was more significant in the MDD+Flu+Lac.group;5.Compared to the control group,the MDD group exhibited inflammatory changes,apoptotic characteristics in hippocampal neurons,and reduced Nissl bodies.After antidepressant treatment,pathological morphology improved,and Nissl body count increased(Control group:228.93±33.33,MDD group: 168.73±44.49,MDD+Flu group: 193.80±33.13,MDD+Flu+Lac.group: 201.87±24.75,P=0.003).In rat brain tissue,DA,5-HT,NE,and GABA levels were lower in the MDD group compared to the Control group(P<0.05).The MDD+Flu+Lac.group showed significant increases in these neurotransmitter levels,with statistically significant differences compared to the MDD group(P<0.05).There were no significant differences between the MDD+Flu group and the MDD group(P>0.05).Compared to the control group,the MDD group exhibited decreased levels of synaptic remodeling protein PSD-95,mature neuron marker protein MAP2,and cell proliferation marker Brd U(P<0.01).The MDD+Flu+Lac.group showed higher levels of PSD-95 and Brd U compared to the MDD group(P<0.05).There were no significant differences between the MDD+Flu group and the MDD group(P>0.05).Compared to the Control group,the MDD group showed increased levels of apoptotic proteins caspase-3 and BAX and decreased levels of anti-apoptotic protein BCL-2 in hippocampal tissue.The MDD+Flu and MDD+Flu+Lac.groups exhibited decreased levels of caspase-3 and BAX and increased levels of BCL-2,with statistically significant differences compared to the MDD group(P<0.05).There were no significant differences between the MDD+Flu group and the MDD group(P>0.05).Compared to the control group,the MDD group showed decreased levels of key proteins in the c AMP-CREB-BDNF signaling pathway,including p-PKA,p-ERK1/2,p-CREB,and BDNF(P<0.0001).After antidepressant treatment,these protein levels increased to varying degrees(P<0.05).The MDD+Flu+Lac.group showed a more significant increase in protein levels.Conclusion: Reduced GLP-1 secretion in the gut of MDD patients is associated with gut microbiota dysbiosis.Gut-derived GLP-1 may influence central BDNF production through the "microbiota-gut-brain" axis and contribute to the pathogenesis and development of MDD.GLP-1 can reverse corticosterone-induced damage to HT-22 cells,exerting a neuroprotective effect,and this mechanism may be associated with increased activity of the c AMP-CREB-BDNF signaling pathway.Adjunctive treatment with lactobacillus improves gut microbiota dysbiosis in MDD rats,increases hippocampal GLP-1 levels,and enhances the efficacy of antidepressant treatment. |
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