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

DE patients showed distinct gut microbiota composition compared to diabetic controls despite no significant differences in overall microbial diversity.

• Study included 29 DE patients and 31 diabetic controls (DM)
• No significant differences in overall microbial diversity were identified between DE and DM cohorts
• DE patients exhibited elevated Verrucomicrobiota and Bacteroidota
• DE patients exhibited reduced Proteobacteria and Firmicutes
• Profiling was performed using 16S rRNA sequencing on fecal samples

Metabolomic analysis revealed 160 differentially abundant metabolites between DE patients and diabetic controls, enriched in amino acid and lipid metabolism pathways.

• Untargeted metabolomics was performed on fecal samples from 29 DE patients and 31 DM controls
• 160 differentially abundant metabolites were identified
• Metabolites were enriched in amino acid and lipid metabolism pathways
• Analysis was integrated with 16S rRNA sequencing in a multi-omics approach

DE-derived fecal microbiota supernatant (FMS) dose-dependently exacerbated high glucose-induced neuronal oxidative damage and apoptosis in vitro.

• An in vitro DE model was established using high glucose (HG)-treated HT22 cells
• HT22 cells were incubated with sterile fecal microbiota supernatant (FMS) from DE patients
• FMS exacerbated HG-induced neuronal damage in a dose-dependent manner
• Outcomes assessed included neuronal viability, apoptosis, and oxidative stress markers (SOD, MDA, ROS)

DE-derived FMS upregulated miR-493-3p expression in HG-treated HT22 neuronal cells.

• miR-493-3p expression was assessed in HG-treated HT22 cells incubated with DE patient-derived FMS
• FMS treatment resulted in concomitant miR-493-3p upregulation alongside exacerbated neuronal damage
• The upregulation occurred in a context of increased oxidative stress and apoptosis

Inhibition of miR-493-3p attenuated FMS-induced neuronal injury and restored RAF1 expression.

• Inhibition of miR-493-3p attenuated the damaging effects of FMS on HG-treated HT22 cells
• miR-493-3p inhibition restored RAF1 expression
• RAF1 downregulation was identified as critical in mediating FMS-induced neuronal injury
• These functional effects were assessed in the in vitro HG/FMS model

RAF1 was confirmed as a direct molecular target of miR-493-3p using dual-luciferase reporter assays and Western blot.

• The miR-493-3p/RAF1 interaction was validated using dual-luciferase reporter assays
• Western blot was also used to confirm the interaction
• RAF1 downregulation was described as 'critical in mediating FMS-induced neuronal injury'
• This established a direct mechanistic link between miR-493-3p and RAF1 in the context of DE neuronal damage