SFAs facilitates ceramide's de novo synthesis via TLR4 and intensifies hepatocyte lipotoxicity.

Background: Non-alcoholic steatohepatitis (NASH), an advanced manifestation of non-alcoholic fatty liver disease (NAFLD), is characterized by hepatocyte injury, inflammation, and fibrosis. Saturated fatty acids (SFAs) have emerged as key contributors to hepatocyte lipotoxicity and disease progression. Toll-like receptor 4 (TLR4) acts as a sentinel for diverse ligands, including lipopolysaccharide (LPS) and endogenous molecules like palmitic acid (PA)-induced ceramide (CER) accumulation, promoting hepatocyte demise. However, the intricate mechanisms underlying TLR4's modulation of ceramide metabolism and their concerted effect on SFA-mediated hepatotoxicity remain elusive.

Methods: A NASH mouse model with liver-specific TLR4 knockdown was established through palm oil feeding and AAV2/8 tail vein injection. Histological and biochemical assessments were conducted to evaluate the mice's condition and liver damage extent. Liquid chromatography-mass spectrometry (LC-MS) was employed to quantify ceramide levels in liver tissues, offering insights into NASH mechanisms.

Results: The PO-fed model exhibited elevated serum ALT, AST, and liver TG levels, enhancing lipid accumulation and hepatocellular damage. TLR4 knock-down reduced liver mass and the liver-to-body weight ratio, signifying a decreased hepatic burden. Histopathological evaluations revealed substantial improvement in hepatic steatosis in TLR4-silenced PO-fed mice, with diminished lipid droplets and inflammatory infiltrates. LC-MS analysis showed a marked decrease in long-chain ceramides (C14, C16, C20) in TLR4-knockdown PO-fed mice. Furthermore, expression of MyD88, SPTLC1, SPTLC2, and inflammatory markers IL-1β, IL-6, TNF-α were significantly attenuated.

Conclusion: SFAs activate the TLR4 signaling pathway via MyD88, fostering ceramide de novo synthesis, which exacerbates hepatocyte lipotoxicity and accelerates NASH progression.