Oleanolic acid-targeted LAMP1 mediates autophagy and lysosome to alleviate high-fat-induced hepatic lipid deposition

Abstract

Non-alcoholic fatty liver disease (NAFLD) threatens aquaculture worldwide and is closely associated with autophagic disturbance. Activity ingredients from Sedum sarmentosum Bunge (SSB) have been shown to alleviate high-fat diet (HFD)-induced NAFLD in tilapia. However, the precise active ingredients and their involvement in regulating autophagy remain unclear. This study aims to investigate the impact of the special active compounds in SSB responsible for ameliorating NAFLD and to elucidate their molecular mechanisms involving autophagy in hepatocytes. Ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was employed to characterize the active ingredients of SSB. The pharmacological effects of oleanolic acid (OA) were evaluated in HFD-induced tilapia through analysis of growth parameters, biochemical indicators, and hepatic pathology. In vitro, the effects of OA combined with rapamycin or chloroquine on the free fatty acids (FFA)-induced HepG2 cells were assessed by measuring lipid accumulation, autophagy, and lipophagy. OA target proteins were identified using an affinity-based probe combined with chemical proteomics. OA notably decreased the hepatosomatic indices, improved liver function, and suppressed lipid deposition in HFD-induced tilapia. Transmission electron microscopy revealed that OA mitigated hepatocellular structural damage and enhanced autophagosome formation. In FFA-induced HepG2 cells, OA treatment enhanced antophagy, autophagic flux and lipophagy, as evidenced by an increased LC3II/I ratio, elevated autolysosome formation, and enhanced colocalization of LC3 with Bodipy. Notably, rapamycin and chloroquine respectively promote and inhibited these effects. Chemical proteomics identified LAMP1 as a direct binding target of OA, which was further validated by the MicroScale thermophoresis (MST) and drug affinity responsive target stability (DARTS) assays. This study identifies OA as a critical active compound from SSB that ameliorates lipid deposition by regulating the autophagy-lysosome pathway via targeting LAMP1, providing a robust foundation for the development of functional feed additives transitioning from traditional herbal extracts to active ingredients in aquaculture.