Oleic acid ameliorates palmitic acid induced pyroptosis by attenuating endoplasmic reticulum stress in HepG2 cells
Authors: Zeng Xin , Liu Xiaohong , Luo Ruixi , Chen Younan 
Published: June 2021

Background

In the liver, chronic exposure to high FFAs in the context of hyperlipidemia or ectopic deposition of fat will result in nonalcoholic fatty liver disease (NAFLD) ultimately. In the present study, the mechanisms of saturated fatty acids induced cell death have been investigated. Particular attentions have been pay on caspase‐1 mediated pyroptosis and endoplasmic reticulum stress (ERS). In addition, the protective effects of a monounsaturated fatty acid, oleic acid (OA) against hepatic lipotoxicity was explored.

Methods

Human hepatoma cell line HepG2 was challenged by palmitic acid (PA) to induce lipotoxicity, and OA was supplemented concomitantly with PA to investigate its protective effects. Cell viability was determined by CCK8 assay and apoptosis was measured by flow cytometry. The expression of ER stress and pyroptosis related proteins was measured by Western blot or real‐time PCR. The morphology of pyroptosis was presented by PI staining and AO/EB Dyeingy. The expression level of gasdermin D (GSDMD), an executor of pyroptosis, was determined by Western blot and immunofluorescence assay.

Results

1) PA deteriorated cell viability of HepG2 cells in dose and time dependent manner. PA of 0.4 mM has remarkably decreased the cell viability (60%) compared to controls after 24 h incubation. Stimulation with OA of 0.2 mM was nontoxic to cell viability, but the combination of OA and PA robustly restored the cell viability to normal level. 2) Inconsistent with the results in cell viability, PA challenge for 24 h has not significantly increased cell apoptosis in HepG2. However, the expression of inflammasome and pyroptosis markers, including caspase‐1/p20, IL‐1 beta, IL‐18, NLRP3 and GSDMD was significantly up‐regulated, indicating that pyroptosis was activated obviously. In addition, the occurrence of pyroptosis was convinced by the increased AO/EB double positive cells as well as increased GSDMD expression after PA stimulation, suggesting a kind of gasdermin‐mediated programmed necrosis, which is a typical feature of pyroptosis. Interestingly, OA substantially suppressed the PA induced activation of pyroptosis, evidenced by significantly down‐regulated expression of above markers. 3) Meanwhile, the expression of ER stress related markers was found notably up‐regulated after 24 h PA exposure, including CHOP, phosphorylated PERK, and GRP78 on protein levels, and UPR genes, such as XBP1, CHOP, and GRP78 on mRNA levels. In consistence, OA attenuated PA triggered ER stress in HepG2 cells.

Conclusion

Compared to apoptosis, caspase‐1 mediated pyroptosis might be an essential form of cell death driven by excessive uptake of saturated fatty acid. Monounsaturated oleic acid was able to robustly protect HepG2 cells against lipotoxicity. The down‐regulation of GSDMD expression and consequently amelioration of pyroptosis may involve in the mechanisms of OA mediated protective action, and the OA mediated alleviation of PA triggered ER stress might be a possible upstream mechanism of its effects on pyroptosis.

Support or Funding Information

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.