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Lipopolysaccharide inhibits lipophagy in HepG2 cells via activating mTOR pathway

HOU Zheng-Ping, LI Yan-Ping, ZHAO Lei, CHEN Ya-Xi, RUAN Xiong-Zhong^*

Key Laboratory of Metabolism on Lipid and Glucose, Center for Lipid Research, Chongqing Medical University, Chongqing 400016, China

Abstract

This study aimed to investigate the effect of lipopolysaccharide (LPS) on lipophagy in hepatocytes and the underlying mechanism. Human hepatoma cell line HepG2 was cultured in vitro, treated with 0.1 mmol/L palmitic acid (PA), and then divided into control group (0 μg/mL LPS), LPS group (10 μg/mL LPS), LPS+DMSO group and LPS+RAPA (rapamycin, 10 μmol/L) group. Lipid accumulation in hepatocytes was observed by oil red O staining. The autophagic flux of the cells was assessed using confocal laser scanning microscope after being transfected with autophagy double-labeled adenovirus (mRFP-GFP-LC3). The level of intracellular lipophagy was visualized by the colocalization of lipid droplets (BODIPY 493/503 staining) and lysosomes (lysosome marker, lysosomal associated membrane protein 1, LAMP1). The expression levels of mammalian target of rapamycin (mTOR), phosphorylated mTOR (p-mTOR), ribosome protein subunit 6 kinase 1 (S6K1), p-S6K1, LC3II/I and P62 protein were examined by Western blot. The results showed that the number of red lipid droplets stained with oil red O was significantly increased in LPS group compared with that in control group (P < 0.001). Moreover, in LPS group, the number of autophagosomes was increased, while the number of autophagolysosomes and the colocalization rate of LAMP1 and BODIPY were significantly decreased (P < 0.05). Meanwhile, the ratios of p-mTOR/mTOR and p-S6K1/S6K1, the ratio of LC3II/LC3I and the protein expression of P62 were significantly increased (P < 0.05) in LPS group. Furthermore, compared with LPS+DMSO group, RAPA treatment obviously reduced the number of lipid droplets and autophagosomes, and raised the number of autophagolysosomes and the colocalization rate of LAMP1 and BODIPY (P < 0.05). In conclusion, the results demonstrate that LPS inhibits lipophagy in HepG2 cells via activating mTOR signaling pathway, thereby aggravating intracellular lipid accumulation.

Key words: lipopolysaccharide; hepatocytes; lipophagy; lipid accumulation

Received: 2020-10-19　　Accepted: 2021-03-11

Corresponding author: 阮雄中　　E-mail: xiongzruan@foxmail.com

DOI: 10.13294/j.aps.2021.0022

Citing This Article：

HOU Zheng-Ping, LI Yan-Ping, ZHAO Lei, CHEN Ya-Xi, RUAN Xiong-Zhong. Lipopolysaccharide inhibits lipophagy in HepG2 cells via activating mTOR pathway. Acta Physiol Sin 2021; 73 (5): 813-820 (in Chinese with English abstract).