# Si-Ni-San improves the deposition of lipid droplets in MAFLD through modulating the FXR-GPAT4 axis

## TL;DR

Si-Ni-San, a traditional Chinese medicine, reduces liver fat buildup in MAFLD by regulating FXR and GPAT4 proteins.

## Contribution

The study reveals a novel mechanism by which Si-Ni-San modulates the FXR-GPAT4 axis to treat MAFLD.

## Key findings

- Si-Ni-San reduces lipid droplet accumulation in MAFLD models by upregulating FXR and downregulating GPAT4.
- FXR transcriptionally inhibits GPAT4 expression, as confirmed by dual-luciferase reporter assays.
- Active components of Si-Ni-San bind to FXR and GPAT4, as shown through molecular docking and SPR analysis.

## Abstract

Metabolic-associated fatty liver disease (MAFLD) is a common metabolic disease with complex pathogenesis and lack of effective treatment. Si-Ni-San (SNS), a traditional Chinese medicine, has emerged as a promising candidate for MAFLD treatment. However, the protective mechanism remains unclear.

C57BL/6N mice were fed with high-fat diet (HFD) for 12 weeks to establish MAFLD mouse model. Concurrently, oleic acid-induced HepG2 cells were used in vitro as a cellular model for MAFLD. The effects of SNS and the positive drug obeticholic acid on hepatic lipid droplets deposition in MAFLD mice and cell models were evaluated. The expression levels of farnesoid X receptor (FXR) and glycerol 3-phosphate acyltransferase 4 (GPAT4) were detected by western blot. The siRNA and dual-luciferase reporter assay were used to detect the interaction between FXR and GPAT4. High-performance liquid chromatography (HPLC) was used to identify the active components in the SNS aqueous solution, and their binding affinities to targets were detected through molecular docking, molecular dynamics simulations, and surface plasmon resonance (SPR).

The active ingredients of SNS were identified by HPLC. SNS ameliorated hepatic lipid droplets deposition in both mouse and cellular models of MAFLD. SNS up-regulated the expression of FXR and down-regulated the expression of GPAT4 in hepatic tissues, thereby modulating proteins involved in hepatic lipolysis and lipophagy. FXR reduced lipid droplets accumulation by inhibiting GPAT4. The dual-luciferase reporter assay confirmed that FXR transcriptionally regulated and inhibited GPAT4 expression. Furthermore, molecular docking and molecular dynamics simulations predicted potential interactions between the active components of SNS and the FXR and GPAT4 proteins, with the binding affinity for FXR being subsequently confirmed through SPR analysis.

This study provided a new mechanistic exploration for FXR in improving MAFLD and broadened the research direction on the mechanisms by which SNS reduced hepatic lipid droplets deposition. It also offers a molecular dynamics basis for subsequent studies on how active components in SNS exert their effects through binding to FXR.