The molecular mechanism of Xiaoyaosan in treating major depressive disorder: Integrated analysis of DNA methylation and RNA sequencing of the arcuate nucleus in rats.

Ethnopharmacological relevance: Xiaoyaosan, a classic Chinese herbal formula, exhibits promising antidepressant effects. However, its specific antidepressant mechanisms remain incompletely understood. Previous studies have highlighted the significant role of DNA methylation in the pathogenesis of major depressive disorder (MDD). Yet, whether the effects of Xiaoyaosan are linked to DNA methylation and its regulation remains unclear.

Aim of the study: This study aims to explore and verify the molecular mechanism of Xiaoyaosan in treating MDD via integrated analysis of DNA methylation and RNA sequencing.

Materials and methods: In this study, a chronic unpredictable mild stress (CUMS) model was established to induce MDD in rats, which were subsequently orally treated with Xiaoyaosan, with fluoxetine as a positive control. Antidepressant effects were assessed by the open field test, sucrose preference test, and forced swimming test. Whole-genome bisulfite sequencing (WGBS) and bulk RNA sequencing were performed in the arcuate nucleus of hypothalamus to assess methylation changes and identify differentially expressed genes. Bioinformatics analyses were conducted to explore methylation alterations, RNA sequencing profiles, and their shared epigenetic as well as gene expression changes, to identify candidate genes. Finally, RT-PCR was used to validate the key differential genes.

Results: Xiaoyaosan effectively reversed depressive-like behaviors. Further, Xiaoyaosan treatment involved multiple epigenetic modifications. The results of differentially methylated genes showed that there were 1353 overlapped genes between M-vs-C-hypo gene and X-vs-M-hyper gene, 5326 overlapped genes between M-vs-C-hyper gene and X-vs-M-hypo gene. GO and KEGG enrichment analyses indicated these intersecting genes were involved in biological regulation, transcription factors, appetite and endocrine control systems, etc. The analysis of differentially expressed genes from RNA sequencing revealed that there were 25 overlapping genes between the M vs C hypomethylated group and the X vs M hypermethylated group, while 81 overlapping genes were identified between the M vs C hypermethylated group and the X vs M hypomethylated group. Those differential genes regulated by methylation enriched in processes related to brain and neuronal growth, neuropeptide and hormone activation, as well as biological processes and molecular functions associated with protein translation, synthesis, transport, and localization. The integrated analysis of DNA methylation and RNA sequencing screened 14 potentially differential genes, which were associated with appetite regulation, energy metabolism, and neuroreceptor ligands. PCR verification found that Lmx1b, Abcc5, Gpc3 and Cfb showed statistical differences.

Conclusions: The antidepressant mechanism of Xiaoyaosan involves the biological regulation in the arcuate nucleus of hypothalamus, including transcription factors, neurotransmitter regulation, neural development, appetite regulation peptides, and endocrine control systems. The methylation level and regulation at the gene locus of Lmx1b, Abcc5, Gpc3, and Cfb may play a key role in the treatment of Xiaoyaosan. These findings provide new insights into the therapeutic mechanisms of Xiaoyaosan.