Bupleuri Radix- Aurantii Fructus Immaturus pair attenuates metabolic dysfunction-associated fatty liver disease in association with modulation of PI3K/Akt-GSK3β/FoxO1 pathway: Evidence from multi-model validation including human iPSC-derived liver organoids
BAP improved glycolipid metabolism, insulin resistance, and hepatic steatosis in MAFLD mice. • Human iPSC-derived liver organoids were used to model MAFLD and evaluate BAP. • Network pharmacology and multi-model validation revealed the mechanism of BAP. Ethnopharmacological relevance Bupleuri Radix (Chaihu) and Aurantii Fructus Immaturus (Zhishi) is a classic herb pair from Si-Ni-San. In traditional Chinese medicine theory, they regulate liver-spleen qi movement through a “one ascending, one descending” dynamic, and are used for syndromes of liver depression and spleen deficiency with phlegm-stasis intermingling. This syndrome pattern closely mirrors the pathogenesis of metabolic dysfunction-associated fatty liver disease (MAFLD). However, the mechanisms underlying the anti-MAFLD effect of this herb pair remain unclear. Aim of the study To determine the effects and molecular mechanisms of the Bupleuri Radix- Aurantii Fructus Immaturus pair (BAP) in alleviating MAFLD. Materials and methods The quality of the BAP preparation used in this study was partially assessed by high-performance liquid chromatography (HPLC). Potential mechanisms were predicted via network pharmacology and functional enrichment analysis, and experimentally explored using high-fat high-sucrose diet mice, FFA-induced AML12 hepatocytes and human induced pluripotent stem cell (iPSC)-derived liver organoids. The PI3K/Akt-GSK3β/FoxO1 signaling pathway was further examined by LY294002 rescue assay. Results BAP ameliorated MAFLD-related hepatic phenotypes both in vivo and in vitro. Four representative quality control markers, namely synephrine, naringin, saikosaponin A, and saikosaponin D, were identified using validated reference standard methods. Network pharmacological analysis predicted that AKT1, GSK3B, and FOXO1 might be central targets, and the PI3K-Akt signalling pathway was identified as a potential pathway associated with the anti-MAFLD effects of BAP. GO and KEGG enrichment analyses revealed that the overlapping targets were significantly enriched in insulin signalling, lipid metabolism, and FoxO signalling pathways. In vivo, BAP treatment significantly reduced body weight gain, improved serum lipid profiles and liver function, alleviated insulin resistance, and attenuated hepatic steatosis and inflammation in HFSW-fed mice. Energy metabolism monitoring indicated that BAP markedly enhanced oxygen consumption and carbon dioxide production throughout the 24-hour light-dark cycle. In human iPSC-derived liver organoids, BAP reduced FFA-induced hepatocyte injury, lipid accumulation, and oxidative stress, and partially normalized the aberrant expression of genes involved in lipogenesis (SREBF1, FASN, SCD1), fatty acid oxidation (PPARA, CPT1A), and gluconeogenesis (G6PC). Western blot analysis demonstrated that BAP significantly upregulated PI3K expression and the phosphorylation levels of Akt (Ser473), GSK-3β (Ser9), and FoxO1 (Ser256), while downregulating p-GS (Ser641) in mouse livers, liver organoids, and AML12 cells. Moreover, co-treatment with the PI3K inhibitor LY294002 partially counteracted the beneficial effects of BAP on these proteins and counteracted the improvements in glycolipid metabolism, suggesting that the anti-MAFLD effects of BAP are associated with modulation of the PI3K/Akt pathway. Conclusions With BAP as the research subject, this study conducts cross-validation across a multi-model system including whole-animal models, hepatocytes and human liver organoids, and suggests that BAP ameliorates glucolipid metabolic disorders and slows MAFLD progression, effects that are closely associated with modulation of the PI3K/Akt-GSK3β/FoxO1 signaling pathway. These findings provide novel compatibility insights and experimental evidence for traditional Chinese medicine-based intervention of MAFLD. Download: Download high-res image (291KB) Download: Download full-size image
危险品化学品经营许可证（带存储）