Investigating the Mitochondria-Related Mechanisms of Ligustri Lucidi Fructus in Treating Diabetic Nephropathy: A Combined Approach of Network Pharmacology, Transcriptomics, and Experimental Validation

Abstract

Mitochondrial dysfunction and excessive oxidative stress within mitochondria are key pathological factors driving renal tubular injury in diabetic nephropathy (DN). While Ligustri Lucidi Fructus (LLF) is traditionally used in the treatment of DN, its precise mechanisms, particularly those involving mitochondria, remain poorly understood. This study identifies differentially expressed genes (DEGs) through differential expression analysis of the GSE142025 dataset, which includes data related to DN. Feature genes were selected from yielding overlapping genes by cross-referencing four distinct machine learning models. Genes with significant differential expression and consistent trends across both datasets were subjected to receiver operating characteristic curve analysis, and those with AUC > 0.7 in both datasets were defined as biomarkers. To investigate potential mechanisms of action, function enrichment, immune infiltration, network construction, and molecular docking analyses were performed. Finally, a DN mice model was constructed, further confirming the therapeutic effect of LLF on DN through assessments of blood glucose, urinary microalbumin levels, and histopathological examination. Meanwhile, the expression of these biomarkers was validated using RT-qPCR. The results indicated that four biomarkers were associated with mitochondria and related to the treatment of DN by LLF. These biomarkers were enriched in pathways related to ribosome function, valine, leucine, and isoleucine degradation, cytokine-cytokine receptor interactions, and peroxisomes. All biomarkers were negatively correlated with CD8+ T cells and activated mast cells while showing positive correlations with activated NK cells and naive B cells. Additionally, the binding energies of taxifolin, beta-sitosterol, and eriodictyol with the biomarkers were all below -5 kcal/mol. Animal experiments have confirmed that LLF significantly upregulates the expression of CAT and MAOA genes in kidney tissue. Identifying mitochondrial biomarkers for LLF treatment of DN offers novel insights into therapeutic strategies for the disease.