Rhein from a traditional herbal formula attenuates hepatic fibrosis via ALDH1A3-mediated retinoic acid metabolism

Abstract

Hepatic fibrosis is a prevalent outcome of chronic liver diseases. Although activation of hepatic stellate cell (HSC) is a primary driver of fibrogenesis, therapeutic strategies targeting retinoic acid (RA) metabolism in HSCs remain insufficiently investigated. This study aimed to elucidate the material basis of Huanggen formula (FHG), identify its principal bioactive constituents, and clarify the underlying antifibrotic mechanisms. The antifibrotic efficacy of FHG was assessed in a CCl4-induced mouse model. Widely-targeted and untargeted metabolomics were conducted to characterize FHG constituents and serum-absorbed components, followed by high-content screening to evaluate anti-HSC activation activity. Rhein was further examined in TGF-β1-stimulated LX-2 cells and CCl4-induced mice. Label-free proteomics, multiple reaction monitoring quantification, DARTS, CETSA, and molecular docking were performed to identify the molecular targets of rhein. ALDH1A3 inhibition, knockdown, and overexpression were employed to verify its functional role in LX-2 cells. FHG markedly alleviated metabolic disturbances and histopathological injury in fibrotic mice. Metabolomic analysis identified anthraquinones and flavonoids as the principal active classes, with rhein exhibiting potent anti-HSC activation effects. Rhein suppressed LX-2 activation, extracellular matrix (ECM) accumulation, and migration, and mitigated CCl4-induced hepatic fibrosis. Proteomic analysis indicated that rhein regulated ECM remodeling, mitochondrial function, and RA metabolism, accompanied by significant upregulation of DHRS4 and ALDH1A3. Mechanistically, rhein directly targeted to ALDH1A3, enhanced its expression, and increased intracellular ATRA level. The inhibition of ALDH1A3 partially abrogated the effects of rhein. Knockdown and overexpression experiments verify that ALDH1A3 is a critical negative regulator of HSC activation. Rhein is a key antifibrotic constituent of FHG and suppresses hepatic fibrosis by targeting ALDH1A3 and restoring RA metabolism. The ALDH1A3-ATRA axis represents a central mechanism and a promising therapeutic target. Herb and serum metabolomics defined FHG’s chemical basis, highlighting anthraquinones. High-content screening identified rhein as the principal antifibrotic compound. Rhein suppressed HSC activation by through modulation of RA metabolism via ALDH1A3. Rhein upregulated ALDH1A3 and elevated ATRA level to inhibit HSC activation. The ALDH1A3-ATRA axis is critical for regulation of HSC activation. Herb and serum metabolomics defined FHG’s chemical basis, highlighting anthraquinones. High-content screening identified rhein as the principal antifibrotic compound. Rhein suppressed HSC activation by through modulation of RA metabolism via ALDH1A3. Rhein upregulated ALDH1A3 and elevated ATRA level to inhibit HSC activation. The ALDH1A3-ATRA axis is critical for regulation of HSC activation.