(P30-10-26) Insparin Protein Attenuates Fibrosis and Oxidative Stress in TGF-β1–Activated Hepatic Stellate Cells: Implications for MASLD and Liver Cancer

Objectives: Metabolic dysfunction–associated steatotic liver disease (MASLD) affects nearly one-third of U.S. adults and can progress to metabolic-associated steatohepatitis (MASH), an irreversible fibrotic condition that markedly increases the risk of cirrhosis and hepatocellular carcinoma (HCC). Fibrosis is the strongest predictor of liver-related mortality, yet effective antifibrotic preventive therapies remain limited.

Our previous in vivo studies showed that transgenic adipose-specific expression of Insparin, a 125 aa peptide derived from human adenovirus Ad36, reduced lipogenic gene expression, hepatic steatosis, and fibrosis development in mice on a 60% high-fat diet. To determine direct antifibrotic effects of Insparin, hepatic stellate cells (HSC) were stimulated with transforming growth factor-β1 (TGF-β1) into collagen-producing myofibroblast-like cells, increasing extracellular matrix (ECM) deposition, oxidative stress, and tissue scarring, and then exposed to E4orf1 transfection.

Methods: LX-2, an immortalized human HSC were cultured under standard conditions and stimulated with TGF-β1. Cells were assigned to four groups: (1) untreated control, (2) TGF-β1–stimulated fibrotic control, (3) Insparin treatment for 1h followed by 23h treatment with TGF-β1, (4) Insparin treatment for 5h following 19h of TGF-β1 treatment. Total RNA was isolated after 24h and expression of fibrosis, inflammation, and oxidative stress-related genes was quantified by RT-qPCR.

Results: Insparin treatment prevents the TGF-β1 attenuation of interstitial collagenases (MMP-1 and MMP-3) and overexpression of gelatinases (MMP-2). Further, Insparin significantly prevents fibrosis activation by reducing the expression of fibrotic genes (α-SMA, COL1A1, and COL4A1) and oxidative stress markers (NOX4).

Conclusions: Prevention of hepatic fibrosis is an important step in preventing HCC. Insparin reduces hepatic fibrosis and oxidative stress, which may limit steatosis-driven progression to MASH and HCC.

Funding Sources: No external funding