Tehranolide Attenuates Lipid Accumulation in Steatotic HepG2 Cells via cAMP/AMPK/SIRT1-Mediated Autophagy Activation

Metabolic dysfunction-associated steatotic liver disease (MASLD) is a prevalent metabolic condition marked by abnormal lipid buildup within hepatocytes, leading to inflammation and liver injury. Hepatic lipid accumulation can be driven by multiple factors, including high glucose, free fatty acids, and lipotoxic stress. Autophagy, which may be influenced by metabolic regulators such as cyclic adenosine monophosphate (cAMP), AMP-activated protein kinase (AMPK), and sirtuin 1 (SIRT1), is suggested to contribute to the maintenance of hepatic lipid homeostasis. Tehranolide, a sesquiterpene lactone derived from Artemisia diffusa and structurally related to artemisinin, is believed to have hepatoprotective effects similar to artemisinin. This work is the first to assess the impact of tehranolide on lipid accumulation with emphasis on autophagy/AMPK/SIRT1 signaling in steatotic human hepatoma-derived cells (HepG2).

This investigation was undertaken to evaluate the potential of tehranolide to reduce lipid accumulation in a high-glucose-induced steatotic hepatocyte model, potentially involving autophagy-related signaling pathways such as cAMP, AMPK, and SIRT1.

A high-glucose-induced steatotic model was established in HepG2 cells. After determining the effective concentration of tehranolide by means of the MTT assay, lipid-loaded cells received treatment with tehranolide. The content of intracellular triglycerides (TGs) was determined via Oil Red O staining and commercial kits. The expression of lipid metabolism-related genes [fatty acid synthase (FASN), sterol regulatory element-binding protein 1c (SREBP-1c), and SIRT1] and autophagy markers [light chain 3 (LC3), beclin-1] was analyzed by quantitative real-time polymerase chain reaction (qRT-PCR), while protein levels of LC3-I, LC3-II, AMPK, and phosphorylated AMP-activated protein kinase (p-AMPK) were evaluated by Western blotting. Intracellular cAMP levels, lactate dehydrogenase (LDH) release, and inflammatory cytokines were also quantified using commercial kits.

Tehranolide significantly decreased intracellular TG levels, downregulated lipogenic genes (FASN, SREBP-1c), and upregulated the lipolytic gene SIRT1. It increased the expression of autophagy-related markers (beclin-1 and LC3-II). Furthermore, tehranolide increased intracellular cAMP and AMPK phosphorylation, while inhibition of SIRT1 or blockade of autophagy attenuated these effects. In addition, tehranolide reduced glucose-induced cytotoxicity and suppressed pro-inflammatory cytokine production in HepG2 cells.

Tehranolide attenuates lipid accumulation and inflammatory responses in high-glucose-induced steatotic HepG2 cells, potentially involving autophagy-related processes, which may be linked to cAMP, AMPK, and SIRT1. These findings suggest that tehranolide may represent a potential modulator of hepatocellular lipid metabolism in glucose-induced steatosis, warranting further validation in more comprehensive in vitro and in vivo models.