Effect of silibinin on CFLAR-JNK pathway in oleic acid-treated HepG2 cells

Abstract

Aims: Silibinin is a flavonolignan from milk thistle with many pharmacological activities including lipid-lowering and antioxidant. Caspase 8 and Fas-associated protein with death domain-like apoptosis regulator (CFLAR) is an important target gene in regulating non-alcoholic steatohepatitis (NASH). At present, the effect of silibinin on CFLAR-JNK pathway related to NASH was unknown. Here the effect of silibinin on CFLAR-JNK pathway and its downstream target genes involved in lipid metabolism, glucose uptake, oxidative stress and inflammatory response were studied in oleic acid (OA)-treated HepG2 cells.

Main methods: OA-treated HepG2 cells were employed as a in vitro model of steatosis, insulin resistance and oxidative stress. The model cells were then treated by silibinin (5, 20, 50, and 100 μM) for 24 h and detected for the related indicators as follows: (1) cellular triglycerides (TG), nitric oxide (NO) and glucose uptake; (2) the mRNA levels of the sterol regulatory element binding protein-1C (SREBP-1C), patatin-like phospholipase domain containing 3 (PNPLA3) and peroxisome proliferator activated receptor-α (PPARα); (3) the protein levels of PPARα, SREBP-1C, PNPLA3, CFLAR, phosphorylated c-Jun N-terminal kinase (pJNK), phosphatidylinositol 3-kinase (PI3K), phosphorylated serine-threonine protein kinase (pAKT), nuclear factor E2-related factor 2 (NRF2), cytochrome P450 2E1 (CYP2E1) and 4A (CYP4A).

Key findings: Compared to the control, OA-treatment led to a result as follows: (1) increased the intracellular levels of TG and NO; (2) up-regulated the protein expression of SREBP-1C, PNPLA3, pJNK, CYP 2E1 and CYP 4A; (3) decreased the uptake of 2-NBDG; (4) down-regulated the protein expression of CFLAR, PPARα, PI3K, pAKT and NRF2. Compared to OA-treated HepG2 cells, silibinin treatment could improve the indicators as follows: (1) decreased the intracellular levels of TG and NO; (2) down-regulated the protein expression of SREBP-1C, PNPLA3, pJNK, CYP 2E1 and CYP 4A; (3) increased the uptake of 2-NBDG; (4) up-regulated the protein expression of CFLAR, PPARα, PI3K, pAKT and NRF2.

Significance: Silibinin can ameliorate some metabolic alterations and induce some molecular changes by activating the CFLAR-JNK pathway and thereby regulating its downstream target genes involved in lipid metabolism (PPARα, SREBP-1C and PNPLA3), glucose uptake (PI3K-AKT), oxidative stress (NRF2, CYP2E1, CYP4A) and inflammatory response(NO) in OA-treated HepG2 cells demonstrating its possible use in ameliorating various symptoms of NASH.

Keywords: CFLAR; HepG2 cells; NASH; NRF2; Silibinin.