Screening and functional validation of key genes in Helicobacter Pylori-induced macrophage M1 polarization: role in migraine-associated functional dyspepsia

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Abstract
Background: Migraine, a prevalent and debilitating neurovascular disorder, frequently co-occurs with functional dyspepsia. Emerging evidence suggests that Helicobacter pylori (H. pylori) infection may contribute to both conditions via neuroimmune pathways, though the molecular basis for this association has yet to be fully elucidated. Methods: H. pylori -related datasets from GEO were analyzed to identify differentially expressed genes (DEGs), followed by functional enrichment analyses including GO, KEGG, GSEA, and GSVA. Immune infiltration patterns were assessed, and CGRP-related hub genes were identified using machine learning approaches. The relationship between these hub genes and immune cell infiltration—particularly M1 macrophage polarization—was explored, and the interaction between CALCA and PNOC was analyzed by correlation analysis and protein-protein interaction networks. Experimental validation was performed using RT-qPCR in H. pylori -infected THP-1 macrophages and SH-SY5Y neuronal cells, along with loss- and gain-of-function experiments to investigate regulatory relationships. Results: Transcriptomic analysis of H. pylori -infected patients identified 683 differentially expressed genes enriched in immune and inflammatory pathways. Immune profiling further revealed prominent M1 macrophage polarization with increased γδT cells and B lymphocytes. Among the DEGs, four CGRP-related hub genes (PNOC, ICAM1, MMP9, and NFE2L1) were identified and found upregulated in H. pylori -infected macrophages. Correlation analyses showed PNOC, ICAM1, and MMP9 positively associated with M1 macrophages and their canonical markers, with knockdown of each gene attenuating M1 marker expression. In contrast, NFE2L1 showed no significant correlation, yet its knockdown increased M1 marker expression. Among the hub genes, only PNOC correlated positively with CALCA; both were upregulated in H. pylori -infected neuronal cells. Notably, PNOC knockdown in macrophages reduced CALCA expression in co-cultured neurons, while overexpression had the opposite effect, suggesting macrophage-derived PNOC may regulate neuronal CGRP expression. Conclusion: H. pylori infection may promote migraine and functional dyspepsia through gastric inflammation and neuroimmune signaling. This process involves four key genes—PNOC, ICAM1, MMP9, and NFE2L1—that regulate M1 macrophage polarization and pro-inflammatory responses. Among them, PNOC appears to link gastric inflammation to neurological symptoms by modulating neuronal CGRP. Together, these findings point to gut-brain axis involvement in this comorbidity and may inform future therapies targeting H. pylori eradication and CGRP-related pathways.

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