Blueberry attenuates oxidative stress-associated signals and neutral lipid accumulation in 2D and 3D hepatic cell models

ABSTRACT Metabolic dysfunction-associated steatotic liver disease is associated with hepatic lipid accumulation, oxidative stress, inflammation, and fibrosis. This study examined phenolic profiles during customized INFOGEST digestion of whole blueberries and evaluated the effects of a minimally processed clarified blueberry homogenate (HCB) in 2D and 3D hepatic models. HCB was obtained by homogenizing fresh blueberries and removing the insoluble fibrous fraction. Total phenolic content, antioxidant activity, and selected polyphenols were analyzed by spectrophotometry and HPLC. Intestinal total phenolic content was approximately 94% of the gastric value. Simulated digestion released blueberry polyphenols and antioxidant activity. Catechin, rutin were detected across the digestive phases. A non-cytotoxic concentration of HCB, standardized to a total phenolic content of 15 μg/mL GAE, was selected for cellular experiments. In HepG2 cells under basal conditions, HCB reduced the basal oxidative stress-associated signal by 72%, decreased the p-ERK1/2/total ERK1/2 ratio and NF-κB protein levels, and increased SIRT1, PGC-1α and ATGL. HCB also modulated genes involved in redox regulation and iron-related processes. In HCB-treated HepG2 cells, SRS imaging showed a lower mean lipid droplet area, while neutral lipid-positive area and triglyceride-sensitive fluorescence were 70.6% and 67.5% lower, respectively.In HepaRG cells, HCB reduced OA/PA-induced neutral lipid accumulation by 61%. In TGF-β1-challenged HepG2/LX-2 spheroids, HCB attenuated neutral lipid accumulation and reduced α-SMA and COL1A1-positive areas by 74% and 58%, respectively, compared with TGF-β1-treated spheroids. In conclusion, these findings characterize the cellular responses to a minimally processed HCB across complementary 2D and 3D hepatic systems and provide a basis for further mechanistic, preclinical and clinical investigation.