Data Sheet 1_Metformin exacerbates diabetic amyotrophy via oxidative stress and gut microbiota alterations.xlsx

ObjectiveThis study aims to explore the targets and mechanisms by which metformin (MET) exacerbates diabetic amyotrophy (DA) using a streptozotocin (STZ)-induced diabetic SD rat model.
MethodThe targets of MET were acquired from 10 databases, including HIT and TCMSP. The DA targets were obtained from the GeneCards database. The associated targets were imported into the Venn analysis platform to draw Venn diagrams, and their intersections were visualized. A target protein–protein interaction (PPI) network was constructed using the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) and Database for Annotation, Visualization, and Integrated Discovery (DAVID) databases and Cytoscape software, and the core targets were identified. GO enrichment and KEGG pathway analyses were performed on the targets, and the construction of the “MET exacerbated DA target-pathway” network was carried out. Molecular docking was performed using Schrödinger Maestro 13.5 software to determine the binding free energy and binding mode of MET with the target protein. The expression of NOS1 and 3-nitrotyrosine proteins in gastrocnemius muscle tissue was detected by WB. The expression of 3-nitrotyrosine protein in gastrocnemius muscle tissue was detected using an enzyme-linked immunosorbent assay (ELISA). The localization of NOS1 in the gastrocnemius muscle tissue was detected using immunohistochemistry (IHC). 16S rDNA gene sequencing was performed to detect structural changes in the fecal microbiota.
ResultsNetwork toxicology analysis revealed 19 intersecting proteins. GO functional enrichment and KEGG pathway analyses demonstrated that the obtained targets were involved in molecular functions, biological processes, and cellular components and were involved in pathways such as drug metabolism—other enzymes, peroxisomes, and nucleotide metabolism. Based on the crystal structures of potential target proteins, complex structural models of target–MET were constructed through molecular docking (extra precision [XP] mode of flexible docking), followed by MM-GBSA analysis. The IHC results indicated that MET significantly promoted the redistribution of NOS1 from the membrane region to the cytoplasm. WB results showed that MET significantly (p < 0.001) promoted the expression of 3-nitrotyrosine in the diabetic SD rat model but had no significant effect on the expression of NOS1. ELISA results demonstrated that MET significantly (p < 0.001) promoted the expression of 3-nitrotyrosine in the diabetic SD rat model. The 16S rDNA gene sequencing results showed that MET led to a significant (p < 0.05) increase in the abundance of Desulfovibrio in the intestinal tract of rats.
ConclusionMET exacerbates diabetic amyotrophy by promoting the increased relative abundance of Desulfovibrio, inducing muscle oxidative stress, and causing NOS1 dysregulation.