SLC25A1-targeted gene therapy attenuates liver fibrosis through NEDD4-mediated ubiquitination and degradation of PPARγ.

Abstract

Restoring lipid droplet (LD) content has been reported to reverse hepatic stellate cell (HSC) activation during liver fibrosis. Although the mitochondrial citrate carrier SLC25A1 is known to drive metabolic reprogramming in cancer and steatohepatitis, its specific role in regulating LDs remains unclear. This study aimed to elucidate the role of SLC25A1 in controlling LD homeostasis during HSC activation and to determine whether therapeutic inhibition of SLC25A1 could ameliorate liver fibrosis by restoring peroxisome proliferator-activated receptor-γ (PPARγ)-dependent lipid storage. Knockdown of Slc25a1 was achieved using either an adeno‑associated viral vector expressing Slc25a1 short hairpin RNA or hyaluronic acid-modified, HSC membrane-biomimetic nanovesicles containing Slc25a1 small interfering RNA (HA@JMNVs/siSlc25a1). Both approaches effectively attenuated HSC activation in three distinct mouse models, induced by carbon tetrachloride, methionine- and choline-deficient diet, and bile duct ligation, respectively. Mechanistically, SLC25A1 deficiency reduced cytosolic acetyl-coenzyme A levels in activated the human immortalized hepatic stellate cell line LX-2, resulting in reduced overall acetylation of neural precursor cell expressed, developmentally downregulated 4 (NEDD4). This reduction subsequently suppressed the NEDD4-mediated ubiquitination and degradation of PPARγ at lysine 197 (K197). The metabolic-post-translational signaling cascade increased perilipin-2 (PLIN2) transcription, inhibited lipophagy, and ultimately restored LD accumulation, thereby reversing HSC activation. Collectively, these findings establish the SLC25A1-regulated metabolic axis as a promising therapeutic target and offer a robust preclinical proof-of-concept for targeted gene therapies against liver fibrosis.