An in vitro mechanistic study of cholinergic-associated mechanisms relevant to MASLD in HepG2 cells.

BACKGROUND/AIM: Polyunsaturated fatty acids (PUFAs) and the hepatic cholinergic axis are critical regulators of lipid metabolism and inflammatory signaling; however, their time-dependent interplay in hepatocytes remains incompletely understood. Clarifying how n-6 and n-3 PUFAs interact with cholinergic pathways may provide mechanistic insight into metabolic dysfunction-associated steatotic liver disease (MASLD).

MATERIALS AND

METHODS

HepG2 cells were used to examine the combined effects of butyrylcholinesterase (BChE) overexpression and PUFA exposure. Cells were treated with linoleic acid (LA; n-6) or α-linolenic acid (α-LA; n-3) for 24 or 48 h. Gene expression analyses were performed to assess pathways involved in de novo lipogenesis, β-oxidation, cholinergic signaling, and inflammatory responses. Cholinesterase activities were measured enzymatically, secreted cytokines were profiled, and molecular docking simulations were performed to evaluate potential α-LA interactions with the α7 nicotinic acetylcholine receptor (α7 nAChR) and M3 muscarinic acetylcholine receptor (M3 mAChR).

RESULTS

An early antilipogenic profile and changes suggestive of increased fatty-acid oxidation (↓SREBP-1c, ↓ACC1, and ↑CPT1A) were induced by α-LA at 24 h and were partially attenuated by 48 h. LA exhibited a biphasic pattern characterized by modest early suppression followed by a pronounced proinflammatory profile at 48 h (↑TNF, ↑IL6, and ↑COX-2). BChE overexpression was associated with increased expression of lipogenic and triglyceride-related genes, whereas cotreatment with PUFAs resulted in time- and class-specific modulation. Cholinergic markers exhibited divergent expression patterns: BCHE + α-LA increased CHAT and CHRNA7 expression at 48 h, whereas ACHE mRNA expression increased under both BCHE + LA and BCHE + α-LA conditions. Enzymatic analyses demonstrated increased total cholinesterase activity in the BChE and α-LA groups, while molecular docking supported the potential accommodation of α-LA within the binding pockets of α7 nAChR and M3 mAChR.

CONCLUSION

PUFA class and exposure duration jointly shape hepatocellular metabolic and inflammatory states, with cholinergic signaling acting as a modulatory node relevant to MASLD.