Discovery and functional validation of a gut microbiota-metabolite-miRNA axis in diabetic encephalopathy

Abstract

Background

Diabetic encephalopathy (DE), a severe neurological complication of diabetes, is characterized by cognitive decline and neuronal damage. While gut microbiota dysbiosis has been implicated in diabetes pathogenesis, its specific role and molecular mechanisms in DE remain unclear.

Methods

A multi-omics approach integrating 16S rRNA sequencing and untargeted metabolomics was performed on fecal samples from 29 DE patients and 31 diabetic controls (DM). An in vitro DE model was established using high glucose (HG)-treated HT22 cells, which were further incubated with sterile fecal microbiota supernatant (FMS) from DE patients. Neuronal viability, apoptosis, oxidative stress markers (SOD, MDA, ROS), and miR-493-3p expression were assessed. The miR-493-3p/RAF1 interaction was validated using dual-luciferase reporter assays and Western blot.

Results

No significant differences in overall microbial diversity were identified in DE and DM cohorts. However, DE patients exhibited distinct gut microbiota composition, with elevated Verrucomicrobiotaand Bacteroidota, and reduced Proteobacteriaand Firmicutes. Metabolomic analysis revealed 160 differentially abundant metabolites enriched in amino acid and lipid metabolism pathways. In vitro, DE-derived FMS dose-dependently exacerbated HG-induced neuronal oxidative damage and apoptosis, concomitant with miR-493-3p upregulation. Inhibition of miR-493-3p attenuated these damaging effects and restored RAF1 expression. RAF1 was confirmed as a direct target of miR-493-3p, and its downregulation was critical in mediating FMS-induced neuronal injury.

Conclusion

This study identified a novel gut-brain axis pathway in DE, whereby gut microbiota dysbiosis and metabolic alterations promote neuronal damage via the miR-493-3p/RAF1 signaling axis. These findings provide new insights into DE pathogenesis and suggest potential therapeutic targets for this debilitating complication.