Ajugol Improves Liver Fibrosis by Modulating Bile Acid and Arachidonic Acid Metabolism in Mice

ABSTRACT Ajugol (AJ), the primary component extracted from the root of Rehmannia glutinosa , exhibits protective effects against metabolic dysregulation and chronic inflammation. This present work aims to study the therapeutic effect of AJ on liver fibrosis and to explore the underlying mechanism. Liver fibrosis was induced in C57BL/6J mice through the administration of 15% CCl 4 , followed by intragastric dosing of AJ at 20 and 40 mg/kg. We conducted a comprehensive evaluation involving histopathological assessment, serum biochemistry, RNA sequencing, and targeted metabolomics. In vitro models consisted of DCA‐stimulated AML12 and RAW264.7 cells. Compared to untreated fibrotic mice, AJ intervention decreased the levels of serum ALT and AST, as well as hepatic hydroxyproline, α‐SMA, and COL1A1. Transcriptomic analysis revealed that AJ significantly regulated key genes involved in primary bile acid (BA) biosynthesis and arachidonic acid (ARA) metabolism. AJ downregulated CYP7A1 expression and reduced the accumulation of toxic deoxycholic acid (DCA) in the mouse liver. Additionally, AJ decreased the expressions of COX‐2, PTGR1, PTGR2, and HPGDS, as well as the levels of downstream metabolites prostaglandins PGD2 and PGE2, thereby improving ARA metabolism via the COX pathway. The anti‐inflammatory effects of AJ were evidenced by a decrease in pro‐inflammatory cytokines IL‐1β, IL‐6, TNF‐α, and macrophage markers such as F4/80 and CD11b. In vitro, AJ alleviated DCA‐induced hepatocellular injury and inhibited pro‐inflammatory cytokine production in macrophages. DCA inhibited nuclear FXR activation, impairing hepatocyte function and activating inflammatory response. AJ effectively restored these functions. AJ improved hepatic inflammation and fibrosis, partly by alleviating the harmful effects of toxic BA accumulation. This highlights AJ as a promising novel therapeutic agent for liver fibrosis.