Intermittent fasting ameliorates MAFLD by downregulating Lrg1: insights from bulk RNA sequencing and functional verification

Huafeng Chen¹Shilin Zhang¹Wenqiang Xie¹Jie Shen^1*Hua Liang^1,2*

• ¹Department of Endocrinology and Metabolism, The Eighth Affiliated Hospital, Southern Medical University (The First People's Hospital of Shunde, Foshan), Foshan, China
• ²Medical Research Center, The Eighth Affiliated Hospital, Southern Medical University (The First People's Hospital of Shunde, Foshan), Foshan, China

Background: The global prevalence of metabolic dysfunction-associated fatty liver disease (MAFLD) is increasing annually, significantly impairing patients' quality of life. Given the limitations of existing treatments, this study aims to investigate the effects of intermittent fasting (IF) on MAFLD and its underlying mechanisms. Methods: The liver tissues of four groups of mice were analyzed by bulk RNA sequencing: normal ad libitum diet (CD group), normal IF (iCD group), high-fat ad libitum diet (HFD group) and high-fat IF group (iHFD group). Differentially expressed genes (DEGs) were identified, followed by enrichment analyses including Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Set Enrichment Analysis (GSEA). Weighted gene co-expression network analysis (WGCNA) was used to identify related modules. The most highly correlated module genes were intersected with DEGs and analyzed by protein–protein interaction（PPI) network to identify key genes. The key genes were preliminarily verified by RT-qPCR. The function of the key gene was further verified by in vitro and in vivo experiments. Results: IF significantly improved metabolic abnormalities and hepatic lipid deposition in MAFLD mice. A total of 331 DEGs were identified between the HFD and CD group, 379 DEGs between the iHFD and HFD group, and 142 DEGs were found to be common to both comparisons. Enrichment analysis showed that DEGs were mainly enriched in pathways related to fatty acid metabolism and inflammatory responses. WGCNA identified red and blue modules are most strongly correlated with MAFLD traits. After intersecting with DEGs, 32 genes were obtained. Based on PPI network analysis, we identified five key genes. After knocking down one of the key genes, Lrg1, in vitro and in vivo, we confirmed that Lrg1 may promote the expression of lipogenic genes such as Srebf1, Scd1, and Fasn via the PI3K-AKT pathway, thereby accelerating MAFLD progression. Conclusions: Transcriptome analysis elucidated the potential mechanism by which intermittent fasting improves MAFLD, highlighting the important role of fatty acid metabolism and inflammatory responses. Several key genes regulating MAFLD through IF were identified. Knocking down the key gene Lrg1 inhibited the expression of lipogenic genes and effectively slowed MAFLD progression.