Lactucin ameliorates FFA-induced steatosis in HepG2 cells by modulating mitochondrial homeostasis through the SIRT1/PGC-1α signaling axis

Abstract

Nonalcoholic fatty liver disease is a complex disease involving abnormal liver metabolism. Its strong association with metabolic dysfunction has led to a change in nomenclature to metabolism dysfunction-associated fatty liver disease (MAFLD). MAFLD pathogenesis involves abnormal accumulation of hepatic lipids that lead to the production of excess free fatty acids (FFAs), which in turn cause an imbalance in hepatic mitochondrial function. Lactucin, a natural compound extracted from Cichorium glandulosum Boiss. et Huet, regulates liver metabolism and protects the liver. However, the potential mechanisms underlying the lactucin-mediated effects in MAFLD require further investigation. In the present study, HepG2 cells were treated with FFAs to establish an in vitro model of MAFLD. Parameters related to lipid accumulation and mitochondrial function, including triglycerides (TG), oil red O-stained lipid droplets, reactive oxygen species (ROS), mitochondrial membrane potential (JC-1), adenine triphosphate (ATP), and complex III were analysed. Morphology of the mitochondria were evaluated by transmission electron microscopy. Furthermore, key proteins in the sirtuin 1 (SIRT1)/peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) signalling axis and mitochondrial quality control were analysed. The SIRT1 inhibitor EX-527 was used to verify the key role of the SIRT1 signalling pathway. Western blotting showed that lactucin upregulated the expression of SIRT-1, PGC-1α, Nrf1, Tfam, Mfn2, and Opa1, and promoted mitochondrial biosynthesis and kinetics. The results suggest that lactucin restores mitochondrial dynamic homeostasis by upregulating the SIRT1/PGC-1α signalling axis, thereby reducing FFA-induced lipid accumulation in HepG2 cells.

Keywords: HepG2 cells; Lactucin; Mitochondrial biosynthesis; Mitochondrial dynamics.

Graphical abstract

Fig. 1

Effects of lactucin on cell viability and intracellular lipid accumulation in HepG2 cells. (A) HepG2 cells were treated with 0 (1), 5 (2), 10 (3), or 20 (4) μmol/L lactucin for 48 h, and cell morphology was observed. (B) CCK-8 assay was used to measure the cell survival rate and IC50 (C). (D) The TG enzyme method was used to determine the changes in triglyceride content in HepG2 cells after FFA intervention and treatment with different concentrations of lactucin for 48 h. (E) RT‒PCR was used to detect the mRNA levels of SREBP-1 in cells. (F–G) Effect on intracellular lipid accumulation in HepG2 cells as observed by oil red O staining. (5) Control, (6) FFA (0.4 mM, OA:PA = 2:1), (7) FFA+5 μmol/L lactucin, (8) FFA+10 μmol/L lactucin, (9) FFA+20 μmol/L lactucin.

Fig. 2

Effect of lactucin on mitochondrial structure and ATP content in FFA-induced HepG2 cells. (A–B) Observation of mitochondrial ultrastructure by transmission electron microscopy. The red arrow indicates the same mitochondria at different magnifications. (1) Control, (2) FFA, and (3) FFA+20 μmol/L lactucin. (C) Intracellular ATP levels were measured using a kit.

Fig. 3

Effects of Lactucin on FFA-induced changes in the mitochondrial membrane potential and ROS levels in HepG2 cells. (A) Detection of intracellular ROS levels using the fluorescent probe DCFH-DA. (1) Control, (2) Rosup, (3) FFA, and (4) FFA+20 μmol/L lactucin. (B) The relative fluorescence intensity corresponding to each group of ROS experiments. (C) Changes in the intracellular mitochondrial membrane potential. (5) Control, (6) CCCP, (7) FFA, and (8) FFA+20 μmol/L lactucin. (D) The relative fluorescence intensity corresponding to each group of JC-1-treated cells.

Fig. 4

Effect of lactucin on mitochondrial biosynthesis genes in FFA-induced HepG2 cells. (A–D) Expression of SIRT1, PGC-1α, Nrf1, and Tfam mRNA. (E–L) Protein expression of SIRT1, PGC-1α, Nrf1, and Tfam in different experimental groups and the relative intensity of each band for GAPDH quantification.

Fig. 5

Effect of lactucin on mitochondrial dynamics genes in FFA-induced HepG2 cells. (A–C) Expression of Mfn2 mRNA and protein. (D–F) Expression of Opa1 mRNA and protein. (G–I) Drp1 mRNA and protein expression.

Fig. 6

The SIRT1 inhibitor EX527 reverses the effects of lactucin on lipid accumulation and mitochondrial function. (A) (1,5) Control, (2,6) FFA, (3,7) FFA+20 μmol/L lactucin, and (4,8) EX527 + FFA+20 μmol/L lactucin. HepG2 cells in different groups were treated for 48 h, and cell morphology was observed. (B, D) Oil red O staining was used to observe the changes in lipid accumulation in HepG2 cells of different groups. (C, E-F) Effects of EX527 on mitochondrial membrane potential and mitochondria, as observed under specific fluorescence. (9,14) Control, (10) CCCP (11,15) FFA (12,16) FFA+20 μmol/L lactucin, (13,17) EX527 + FFA+20 μmol/L lactucin. (G) Changes in triglyceride content in HepG2 cells after EX527 intervention as determined by TG enzyme assay. (H) Respiratory chain complex III activity was assayed using a CoQ-Cytochrome C reductase activity assay kit.

Fig. 7

The SIRT1 inhibitor EX527 reverses the effects of lactucin on mitochondrial quality control-associated proteins. (A–L) The effect of EX527 on intracellular SIRT1, PGC-1α, Nrf1, Tfam, Mfn2, and Opa1 protein levels was observed. The relative intensity of each protein band was quantified by GAPDH.

Fig. 8

Lactucin regulates mitochondrial biosynthesis and dynamics through the SIRT1/PGC-1α pathway.