Identification of Ferroptosis -Related Genes in MAFLD/MASH and HQHF Validation

Sutong Liu,1– 3,* Lihui Zhang,1– 3,* Junjiao Xu,1 Qing Zhao,1 Dongfang Shang,1 Xiaoyan Liu,1 Qiang Zhang,1 Minghao Liu,1– 3 Wenxia Zhao1– 3 1The First Affiliated Hospital of Henan University of Chinese Medicine, Zhengzhou, 450000, People’s Republic of China; 2Collaborative Innovation Center of Prevention and Treatment of Major Diseases by Chinese and Western Medicine, Henan Province, Zhengzhou, 450000, People’s Republic of China; 3Collaborative Innovation Center of Research and Development on the Whole Industry Chain of Yu-Yao, Henan Province, Zhengzhou, 450000, People’s Republic of China*These authors contributed equally to this workCorrespondence: Minghao Liu, Email [email protected] Wenxia Zhao, Email [email protected]: Metabolic associated fatty liver disease (MAFLD) and its progressive form, Metabolic Dysfunction-Associated Steatohepatitis(MASH), are prevalent liver disorders with significant health implications. This study aims to identify differentially expressed genes (DEGs) associated with MAFLD/MASH and explore their potential link to ferroptosis, a form of regulated cell death.Methods: We conducted differential expression analysis using two datasets from the GEO database. Genes related to ferroptosis were identified from the Genecards and FerrDb databases, and the intersection genes were obtained by intersecting the DEGs with ferroptosis-related genes. Functional enrichment was performed using GO and KEGG pathways. The clinical diagnostic value of the intersection genes was evaluated through ROC analysis. Finally, the research findings were validated using a high-fat diet (HFD) mouse model, observing the improvement in gene expression with HQHF treatment.Results: We utilized two datasets, GSE89632 and GSE63067, from the GEO database, comprising 39 samples, including 24 healthy controls. Differential expression analysis revealed significant gene expression changes in NAFLD and NASH compared to healthy controls. These DEGs were visualized using volcano plots. Ferroptosis-related genes were identified from Genecards and FerrDb databases, resulting in 1937 unique genes. Venn analysis revealed intersections between DEGs and ferroptosis genes, highlighting potential regulatory roles. Functional enrichment analysis using GO and KEGG pathways indicated significant involvement in cellular components and signaling pathways, such as PPAR and HIF-1. The clinical diagnostic value of intersecting genes was assessed using ROC analysis, demonstrating promising diagnostic potential. In addition, a high-fat diet (HFD) mouse model was used to validate the research findings, showing that HQHF treatment can alleviate lipid deposition and inflammation in the liver. Transmission electron microscopy results indicated that HQHF prevented mitochondrial damage and significantly reduced the content of Fe2+ in the liver, alleviating iron deposition. We verified the reliable genes with diagnostic value through qPCR, and the results suggested that HQHF can lower the transcription levels of P4HA1, NR4A1, and EFEMP1, while increasing the transcription levels of ENo3, GRIA3, ME1, and FADS2, confirming our ROC results.Conclusion: This study provides insights into the molecular mechanisms underlying MAFLD/MASH and suggests ferroptosis-related genes as potential diagnostic biomarkers and therapeutic targets. Further research is warranted to explore these findings in clinical settings.Keywords: metabolic associated fatty liver disease, ferroptosis, HQHF