Ling-Gui-Zhu-Gan decoction improves hepatic steatosis through ALKBH1-mediated DNA N⁶-methyladenosine modification

Ling-gui-zhu-gan (LGZG) decoction, a traditional Chinese medicinal formulation with a long-established history, is frequently utilized in the treatment of fluid retention disorders, with a particular focus on metabolic dysfunction-associated steatotic liver disease (MASLD). Although its clinical efficacy has been substantiated, the precise mechanisms underlying its therapeutic effects on MASLD remain inadequately understood.

This study sought to elucidate the functional role and regulatory mechanisms of LGZG decoction in the treatment of MASLD.

A murine model of MASLD was induced through the administration of a high-fat diet (HFD), followed by intervention with LGZG decoction. The therapeutic efficacy of LGZG decoction in mice with MASLD was assessed through a comprehensive methodology, including triglyceride quantification, biochemical assays, hematoxylin-eosin staining, and oil red O staining. Additionally, dot blotting, methylated DNA immunoprecipitation sequencing, real-time quantitative polymerase chain reaction, western blotting, and immunofluorescence were employed to investigate the potential mechanisms of action of LGZG decoction in the context of MASLD. ALKBH1 knockdown or overexpression in AML12 cells was performed to verify the function of ALKBH1 on steatosis, and regulation of downstream targets.

The administration of LGZG decoction markedly ameliorated hepatic steatosis in mice with MASLD and enhanced the expression of AlkB homolog 1 (ALKBH1). ALKBH1 serves as a demethylase for DNA N⁶-methyladenosine (6 mA), facilitating the demethylation of DNA 6 mA and playing a pivotal role in the regulation of 6 mA modifications. The study demonstrated that LGZG decoction significantly diminished DNA 6 mA modification by upregulating ALKBH1 expression. Furthermore, LGZG treatment led to a reduction in 6 mA methylation on the promoters of pleckstrin and Sec7 domain containing 4 (PSD4) and ribosomal protein S23 (RPS23), which subsequently resulted in increased expression of PSD4 and RPS23 at both mRNA and protein levels. In addition, protein levels of ALKBH1 and PSD4 were significantly reduced in AML12 cells with free fatty acid treatment. Overexpression of ALKBH1 led to a significant decrease in TG levels, whereas ALKBH1 knockdown resulted in a significant increase in TG levels in AML12 cells. Overexpression of ALKBH1 also enhanced PSD4 expression, while ALKBH1 knockdown significantly suppressed PSD4 level.

The findings suggest that LGZG decoction represents an effective therapeutic strategy for MASLD, alleviating hepatic steatosis through the enhancement of ALKBH1-mediated 6 mA modification, reduction of 6 mA methylation on the PSD4 promoter, and upregulation of PSD4 expression.