K-80003 Inhibits Liver Fibrosis Through Activation Of AMPKα By Inducing RXRα Interaction With CaMKKβ In Hepatic Stellate Cells

Liver fibrosis is a key step in the progression of chronic liver disease to cirrhosis.It is a compensatory repair response of the body.The main feature of liver fibrosis is the deposition of extracellular matrix,and the main source of extracellular matrix is activated hepatic stellate cells(HSCs).Thus,inhibiting the activation of HSCs is an attractive approach for developing new therapeutics for treating liver fibrosisasscoaietd diseases including NASH.The activation of HSCs is characterized by their transformation into myofibroblast-like cells and the depletion of lipid droplets that contain 70 to 80%of all vitamin A in the human body.The biological effects of vitamin A are mainly mediated by two classes of nuclear receptors,the retinoic acid receptor(RAR)and retinoid X receptor(RXR),which are highly expressed in HSCs.Emerging evidence indicates that vitamin A and derivatives are potent regulators of HSC activation,suggesting that targeting vitamin A receptors represents a promising approach for developing novel therapies for treating liver fibrosis and associated liver diseases.The aim of this study is to examine the effect of RXR and ligands on the activation of HSCs in vitro and in animals and the underlying molecular mechanism.In vitro assays were used to evaluate the anti-fibrotic effect of natural and synthetic RXRα modulators.The most potent anti-fibrotic compound identified,K-80003,was further evaluated in human and mouse primary hepatic stellate cells as well as in the carbon tetrachloride(CCl4)animal model for its inhibitory effect on liver fibrosis.CRISPR-Cas9 technology was used to construct RXRα-KO CFSC cells to study the role of RXRα in HSC activation and K-80003 effect.Various cellular and molecular approaches were taken to study the mechanism by which RXRa mediates the effect of K-80003 on inhibiting HSC activation and fibrotic responses.Our results identified K-80003,which binds RXRα by a unique mechanism,as a potent anti-fibrotic agent.K-80003 potently suppressed the fibrotic effect of TGF-β in vitro and inhibited liver fibrosis in the CCl4 animal model.K-80003 effectively inhibited the activation,proliferation and migration of HSCs and modulated mitochondrial function in a RXRα-dependent manner.The anti-fibrogenic effect of K80003 was largely attributed to its activation of AMPKα in a RXRα dependent manner.Mechanistic studies revealed that K-80003 inducced interaction between phosphorylated RXRα and CaMKKβ,thus promoting CaMKKβ interaction with AMPKα and subsequently AMPKα activation.Our results identify K-80003 as a potent inhibitor of HSC activation and liver fibrosis through its activation of AMPKα and unravel a new mechanism of K-80003 activation of AMPKα through the RXRα/CaMMK/AMPKα pathway for modulating liver fibrosis,which provides important mechanistic insight into AMPKα activation and new therapeutic strategies for developing RXRα-based anti-fibrosis agents.