Icariin ameliorates minimal change disease by regulating the mitochondrial dysfunction pathway: An integrated strategy of network pharmacology, bioinformatics, and experimental validation

Hao Wu^1,2Rong Wu³Dian Zhong¹Enlai Dai^1*Li Chen⁴Guozhong Xue²Xuping Li²Hanyu Wang²

• ¹Gansu University of Chinese Medicine, Lanzhou, China
• ²Affiliated Hospital of Gansu University of Chinese Medicine, Lanzhou, China
• ³Lanzhou University First Hospital, Lanzhou, China
• ⁴The Second People's Hospital of Baiyin Municipality, Baiyin, China

Background: Minimal change disease (MCD) involves mitochondrial dysfunction, and icariin (ICA) has therapeutic potential; however, the exact mechanism by which ICA regulates mitochondrial dysfunction remains to be elucidated. This study investigated ICA targets and mitochondrial dysfunction-related genes (MDRGs) involved in MCD pathogenesis.Methods: First, the differentially expressed genes (DEGs) between MCD and controls were identified using differential expression analysis. Differential MCD-ICA target genes were obtained by intersecting the DEGs and MDRGs with ICA target genes. The four algorithms of Cytoscape were subsequently used to screen differential MCD-ICA target genes for candidates, which were refined through expression validation, machine learning, and ROC analysis to pinpoint the key genes. Next, a nomogram model of MCD was constructed. Moreover, gene set enrichment analysis (GSEA), immune infiltration, molecular regulatory network analysis, and molecular docking were performed using the key genes. Finally, reverse transcription quantitative polymerase chain reaction (RT-qPCR) was used to validate the expression of the key genes in rat samples. In parallel, mitochondrial morphology was examined using transmission electron microscopy, and the ATP content in renal tissue was measured using colorimetric detection.Two key genes (ANPEP and XDH) were identified, both down-regulated in MCD. Furthermore, these findings were confirmed using RT-qPCR, with ICA intervention reversing their expression. In addition, the key gene-based nomogram had a good prediction effect. Molecular docking confirmed strong binding between ICA and each of the key genes. GSEA revealed that the top 3 most prominent pathways shared between the 2 key genes included neutrophil degranulation and the innate immune system, with differential immune cell infiltration noted between the MCD patients and controls (e.g., resting dendritic cells and eosinophils). A total of 12 transcription factors coregulated the genes XDH and ANPEP. Transmission electron microscopy and colorimetry confirmed that ICA intervention alleviated mitochondrial dysfunction.ANPEP and XDH were identified to be associated with ICA therapy and MDRGs in MCD patients. Furthermore, the potential ameliorating effect of ICA on MCD could be achieved by alleviating mitochondrial dysfunction. This work provides a potential theoretical basis for MCD treatment.