Exploring the antidepressant mechanisms of Guipi pill: A focus on gut-brain axis and immunity modulation

Figure 1 Schematic illustration of the study flow. GEO: Gene Expression Omnibus; MDD: Major depressive disorder; IBD: Inflammatory bowel disease; DEGs: Differentially expressed genes; eQTL: Expression quantitative trait loci; PBMC: Peripheral blood mononuclear cell; LD: Linkage disequilibrium; CUMS: Chronic unpredictable mild stress; OFT: Open field test; WMT: Water maze test; IVW: Inverse-variance weighted; MR: Mendelian randomization; MR-PRESSO: MR-pleiotropy residual sum and outlier; GZMA: Granzyme A.

Figure 2 Identification of differentially expressed genes in inflammatory bowel disease and depression patients by bioinformatics. A: Heatmap of the differentially expressed genes (DEGs) from inflammatory bowel disease (IBD) patients; B: Volcano plot of the DEGs from IBD patients; C: The heatmap of the DEGs from depression patients; D: Volcano plot of the DEGs from depression patients; E: Venny diagram analysis on the DEGs from IBD patients and depression patients. MDD: Major depressive disorder; IBD: Inflammatory bowel disease.

Figure 3 Animal behavioral test. A: Representative trajectory plots of rats in the open field test; B and C: Total distance and central distance traveled by rats in the open field test; D and E: Morris water maze experiment (spatial exploration experiment). ^aP < 0.05 vs control; ^bP < 0.05 vs chronic unpredictable mild stress model; ^cP < 0.01 vs chronic unpredictable mild stress model; ^dP < 0.01 vs control. CUMS: Chronic unpredictable mild stress; FH: Fluoxetine hydrochloride; GPP-L: Guipi pills-low dose; GPP-M: Guipi pills-medium dose; GPP-H: Guipi pills-high dose.

Figure 4 Effects of Guipi pills on the serum corticosterone, corticotropin-releasing hormone, and neuropeptide Y in chronic unpredictable mild stress-induced depression rats. ^aP < 0.01 vs control; ^bP < 0.05 vs chronic unpredictable mild stress model; ^cP < 0.01 vs chronic unpredictable mild stress model. CORT: Corticosterone; CRH: Corticotropin-releasing hormone; NPY: Neuropeptide Y; CUMS: Chronic unpredictable mild stress; FH: Fluoxetine hydrochloride; GPP-L: Guipi pills-low dose; GPP-M: Guipi pills-medium dose; GPP-H: Guipi pills-high dose.

Figure 5 Transcriptomics analysis of the colonic tissues from rats. A: The volcano plot of the differentially expressed genes (DEGs) from Guipi pills vs chronic unpredictable mild stress model group; B: The heatmap of the DEGs from Guipi pills vs chronic unpredictable mild stress model group; C: Kyoto Encyclopedia of Genes and Genomes enrichment analysis of the up-regulated DEGs; D: Kyoto Encyclopedia of Genes and Genomes enrichment analysis of the down-regulated DEGs. CUMS: Chronic unpredictable mild stress; GPP: Guipi pills; KEGG: Kyoto Encyclopedia of Genes and Genomes.

Figure 6 Venny analysis of the differentially expressed genes from Guipi pills vs chronic unpredictable mild stress and differentially expressed genes from inflammatory bowel disease patients and depression patients. DEGs: Differentially expressed genes; GPP: Guipi pills.

Figure 7 Forest plot of Mendelian randomization analysis between depression and GZMA, GZMK, and KCNJ2 genes. MR: Mendelian randomization; CI: Confidence interval; SNP: Single nucleotide polymorphism.

Figure 8 Mendelian randomization analysis results of GZMA and depression. A: Forest plot of Mendelian randomization (MR). The forest plot showed that most single nucleotide polymorphism effects were located to the left of the line of no effect, suggesting that up-regulation of GZMA reduced the risk of depression; B: Funnel plot of MR; C: Scatter plot of MR; D: Result of leave-one-out analysis. MR: Mendelian randomization; SNP: Single nucleotide polymorphism.

Figure 9 Mendelian randomization results for gene GZMA visualized in forest plot. CI: Confidence interval; OR: Odds ratio.

Figure 10  The molecular mechanism by which Guipi pills exerted antidepressant effects through the gut-brain axis. Following Guipi pills intervention, it specifically upregulated GZMA expression in CD8+ NC cells by modulating the intestinal immune microenvironment. GZMA activated the cyclic adenosine monophosphate (cAMP)/protein kinase A/cAMP response element-binding protein signaling pathway by inhibiting phosphodiesterase 4B, thereby promoting GPX4 transcriptional upregulation and enhancing ferroptosis resistance. Concurrently, cAMP response element-binding protein pathway activation further facilitated neuroplasticity. These effects collectively alleviated neuroinflammation, protected neurons, and ultimately ameliorated depressive symptoms. GPP: Guipi pills; cAMP: Cyclic adenosine monophosphate; PKA: Protein kinase A; CREB: Cyclic adenosine monophosphate response element binding protein; PDE4B: Phosphodiesterase 4B.