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