ATR-FTIR Assessment of the Antioxidant and Anti-Inflammatory Effects of (-)-Epicatechin in MASLD

Lipid peroxidation is a contributor and a result of metabolic dysfunction-associated steatotic liver disease (MASLD), related to oxidative injury and inflammatory/fibrogenic signaling, which eventually results in disease progression. Early (conjugated dienes, CDs) and propagation (lipid hydroperoxides, LOOHs) products, along with attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy assessment, could possibly be used to quantify the extent of this process, while TNF-α and TGF-β₁ reflect downstream inflammatory and profibrotic responses. (-)-Epicatechin (EC), a dietary polyphenol, shows antioxidant activity and could decrease the extent of lipid peroxidation and inflammation/fibrogenesis, thereby limiting MASLD progression. The aim of this study was to determine whether EC attenuates lipid peroxidation and pro-inflammatory/profibrotic signaling in an in vitro model of steatosis and whether ATR-FTIR could be used for such assessment in the context of steatosis and lipid peroxidation. Steatosis was induced in HepG2 cells with sodium oleate. EC (10-50 μM) was applied either as pretreatment before oleate exposure or as posttreatment during already established steatosis. Lipid peroxidation was quantified according to CDs, LOOHs, and the LOOH/CD ratio. ATR-FTIR band ratios and intensities related to lipid peroxidation (CO and C-H bands) were evaluated alongside biochemical results. TNF-α and TGF-β₁ concentrations were measured as they are potential indicators of inflammation and fibrogenesis in MASLD, respectively. Sodium oleate increased CDs, LOOHs, and the LOOH/CD ratio, consistent with enhanced lipid peroxidation, and elevated TNF-α and TGF-β₁. EC pretreatment reduced CDs and LOOHs and lowered the LOOH/CD ratio and the TNF-α and TGF-β₁ levels relative to sodium oleate control groups in a concentration-dependent manner. Although the posttreatment effects were less pronounced, EC posttreatment still decreased all these parameters in a concentration-dependent manner in steatotic HepG2 cells. ATR-FTIR spectra were consistent with the biochemical results where the ATR-FTIR carbonyl (CO) band intensity showed a positive correlation with the LOOH/CD ratio, TNF-α, and TGF-β₁ in the EC Pretreatment and Posttreatment models. Therefore, these results suggest that ATR-FTIR readings reflect quantitative changes in lipid peroxidation and associated inflammatory and fibrotic processes in steatosis.