Nobiletin Attenuates Inflammation and Modulates Lipid Metabolism in an In Vitro Model of Intestinal Failure-Associated Liver Disease

<b>Background</b>: Intestinal failure-associated liver disease (IFALD) is a serious complication in patients receiving parenteral nutrition, often exacerbated by inflammation, lipid overload, and oxidative stress. Nobiletin (NOB), a polymethoxylated flavone, is known for its anti-inflammatory and lipid-regulating properties. <b>Methods</b>: We employed an in vitro model using THLE-2 human hepatocytes and primary human cholangiocytes exposed to Intralipid (INT) and lipopolysaccharide (LPS) to simulate IFALD conditions. NOB was tested at non-toxic concentrations (10 and 25 µM) to assess its protective effects. MTT viability assays, multiplex bead-based immunoassays (MAGPIX), RT-qPCR, and Western blotting were used to evaluate changes in inflammation markers, gene expression, and protein signaling. Moreover, ALT and AST activities were used to assess hepatocellular injury. <b>Results</b>: NOB maintained high cell viability in THLE-2 hepatocytes and cholangiocytes, confirming its low cytotoxicity. NOB normalized ALT and AST activities in both tested cell lines, but the effect reached statistical significance only for ALT in cholangiocytes. Under IFALD-like conditions (LPS+INT), NOB significantly preserved metabolic activity in both cell types. In THLE-2 and cholangiocytes, NOB markedly reduced the phosphorylation of pro-inflammatory proteins JNK, NF-κB, and STAT3, indicating a broad inhibition of inflammatory signaling. Moreover, in THLE-2 cells, NOB upregulated lipid metabolism-related genes (<i>PRKAA2</i>, <i>CYP7A1</i>, and <i>ABCA1</i>) and decreased oxidative stress, thereby enhancing the nuclear translocation of Nrf2 and increasing SOD1 level, which supports the activation of antioxidant defenses. <b>Conclusions</b>: NOB exhibits hepatoprotective properties under IFALD-like conditions in vitro, likely through modulation of inflammation-related signaling and lipid metabolism pathways.