Costunolide Prevents Free Fatty Acid-Induced Hepatocyte Steatosis by Activating the Autophagy Pathway

Abstract

Background: Costunolide can inhibit liver fibrosis and the progression of hepatocellular carcinoma. Therefore, this study intends to explore whether Costunolide can interfere with hepatocytes simple steatosis by inducing autophagy. Methods: Initially, Transformed Human Liver Epithelial-2 (THLE-2) cells were treated with different concentrations (0, 5, 10, 20, 30, 40, and 50 μM) of Costunolide. Moreover, a simple steatosis model of THLE-2 cells was induced by treating them with free fatty acids. Furthermore, Rapamycin was used to activate, and 3-methyladenine was used to inhibit the autophagy pathway in THLE-2 cells. Autophagy flux was measured using tandem confocal microscopy. Cell viability, lipid deposition, and triglyceride content were evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, oil red O staining, and the triglyceride content assay kit, respectively. Additionally, the protein expressions of autophagy-related genes, such as Beclin-1 and microtubule-associated protein 1 light chain 3-II and -I, as well as mitogen-activated protein kinase pathway were assessed using Western blot analysis. Results: Costunolide treatment increased the number of autophagosomes and autolysosomes, and significantly reduced deposition of lipid and triglyceride content in free fatty acids-induced fatty liver cells (p < 0.001). However, free fatty acids inhibited Beclin-1 expression and decreased microtubule-associated protein 1 light chain 3-II/I ratio, while Costunolide treatment reversed this effect and induced autophagy in THLE-2 cells (p < 0.05). Furthermore, free fatty acids activated mitogen-activated protein kinase 14 and extracellular-signal-regulated kinase phosphorylation, thereby activating the mitogen-activated protein kinase pathway, while Costunolide treatment inhibited the activation of this pathway (p < 0.001). Additionally, Costunolide and rapamycin exhibited similar effects on autophagy in free fatty acids-treated THLE-2 cells, and 3-methyladenine reversed the effects of Costunolide (p < 0.05). Conclusions: Costunolide interferes with simple steatosis of hepatocytes by inducing autophagy.