Abstract

BackgroundPyroptosis is a novel programmed cell death. It is identified as caspase-1 dependent and characterized by plasma-membrane rupture and release of proinflammatory intracellular contents inculuding IL-1 beta and IL-18. Pyroptosis is distinct from other forms of cell death, especially apoptosis that is characterized by nuclear and cytoplasmic condensation and is elicited via activation of a caspase cascade. In pyroptosis, gasdermin D (GSDMD) acts as a major executor, while NLRP3 related inflammasome is closely linked to caspase-1 activation. Given that pyroptosis has played a critical role in the progression of non-alcoholic steatohepatitis (NASH), here, we investigated whether the regulation of pyroptosis activation is responsible for the protective role of monounsaturated oleic acids in the context of hepatocellular lipotoxicity.MethodsHuman hepatoma cell line HepG2 cells were exposed to palmitic acid (PA) with or without oleic acids (OA) or/and endoplasmic reticulum (ER) stress inhibitor tauroursodeoxycholic acid (TUDCA) for 24 h. Besides, the cells were treated with the chemical ER stressor tunicamycin (TM) with or without OA for 24 h as well. The expressions of pyroptosis and ER stress related genes or proteins were determined by real-time PCR, Western blot or immunofluorescence. The morphology of pyroptosis was detected by acridine orange and ethidium bromide (AO/EB) staining. The release of IL-1 beta and tumor necrosis factor alpha (TNF-α) was determined by ELISA. Sprague–Dawley (SD) rats were fed with high fat diet (HFD) for 16 w, then, HFD was half replaced by olive oil to observe the protective effects of olive oil. The blood chemistry were analyzed, and the liver histology and the expressions of related genes and proteins were determined in the liver tissues.ResultsWe demonstrated that PA impaired the cell viability and disturbed the lipid metabolism of HepG2 cells (P < 0.01), but OA robustly rescued cells from cell death (P < 0.001). More importantly, we found that instead of cell apoptosis, PA induced significant pyroptosis, evidenced by remarkably increased mRNA and protein expressions of inflammasome marker NLRP3, Caspase-1 and IL-1beta, as well as cell membrane perforation driving protein GSDMD (P < 0.05). Furthermore, we demonstrated that the PA stimulated ER stress was causally related to pyroptosis. The enhanced expressions of ER stress markers CHOP and BIP were found subcellular co-located to pyroptosis markers NLRP3 and ASC. Additionally,TM was able to induce pyroptosis like PA did, and ER stress inhibitor TUDCA was able to inhibit both PA and TM induced ER stress as well as pyroptosis. Furthermore, we demonstrated that OA substantially alleviated either PA or TM induced ER stress and pyroptosis in HepG2 cells (P < 0.01). In vivo, only olive oil supplementation did not cause significant toxicity, while HFD for 32 w obviously induced liver steatosis and inflammation in SD rats (P < 0.05). Half replacement of HFD with olive oil (a mixed diet) has remarkably ameliorated liver abnormalities, and particularly inhibited the protein expressions of either ER stress and pyroptosis markers (P < 0.05).ConclusionPalmitic acid induced predominant pyroptosis in HepG2 cells, and ER stress may be responsible for the induction of pyroptosis and subsequent cell death. Monounsaturated oleic acids were able to ameliorate hepatocellular lipotoxicity both in vitro and in vivo, and OA mediated inhibition of ER stress and pyroptosis may be the underlying mechanisms.

Highlights

  • Non-alcoholic fatty liver disease (NAFLD) is characterized by steatosis at the first stage, and is able to progress to non-alcoholic steatohepatitis (NASH) or even cryptogenic cirrhosis and hepatocellular carcinoma [1, 2]

  • We found that long term high fat diet (HFD) significantly facilitated the protein expressions of both endoplasmic reticulum (ER) stress markers and pyroptosis markers including Glucose regulated protein 78 kD (GRP78),C/ EBP homologous protein (CHOP)(ER stress markers), NOD-like receptor family pyrin domain containing 3 (NLRP3), gasdermin D (GSDMD)/−N, Caspase-1, p20, and IL-1 beta, suggesting that ER stress and pyroptosis are important pathological process of NAFLD

  • For an integral pyroptosis, the release of mature IL-1 beta is processed by three major steps: the expression of pro-IL-1 beta by activating TLR2/4 pathway, the precursors are cut by cleaved-caspase-1, and matured IL-1 beta was released through GSDMD mediated-pore. These results suggested that simple ER stress, provoked by ER stressors only, was able to induce NLRP3 inflammasome, but unable to promote pro-IL-1 beta and pro-caspase-1 accumulation in HepG2 cells, probably due to lacking the activation of TLR4 pathway [35], which may be mechanistically different with palmitic acid (PA) induced ER stress and pyroptosis

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Summary

Introduction

Non-alcoholic fatty liver disease (NAFLD) is characterized by steatosis at the first stage, and is able to progress to non-alcoholic steatohepatitis (NASH) or even cryptogenic cirrhosis and hepatocellular carcinoma [1, 2]. It has been revealed that other types of programmed cell death, such as pyroptosis, may play an important role in NAFLD. Pyroptosis is a novel programmed cell death It is identified as caspase-1 dependent and characterized by plasma-membrane rupture and release of proinflammatory intracellular contents inculuding IL-1 beta and IL-18. Pyroptosis is distinct from other forms of cell death, especially apoptosis that is characterized by nuclear and cytoplasmic condensation and is elicited via activation of a caspase cascade. Given that pyroptosis has played a critical role in the progression of non-alcoholic steatohepatitis (NASH), here, we investigated whether the regulation of pyroptosis activation is responsible for the protective role of monounsaturated oleic acids in the context of hepatocellular lipotoxicity

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