Mechanistically, the associated signaling pathways include the accumulation of reactive oxygen species (ROS), endoplasmic reticulum (ER) stress, increased ceramide synthesis in hepatic cells, and so on. The pathogenesis of NAFLD is hypothesized to begin with abnormal accumulation of lipids in the liver due to a stress condition such as obesity and imbalanced nutrition uptake. Although lifestyle change would benefit improvement of hepatic steatosis, it is barely effective when NAFLD has progressed to NASH and further sever liver damage. In the last decade, NAFLD has been considered as a serious public healthy burden worldwide. 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 ultimately cryptogenic cirrhosis and hepatocellular carcinoma. 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. Palmitic acid induced predominant pyroptosis in HepG2 cells, and ER stress may be responsible for the induction of pyroptosis and subsequent cell death. 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). 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). Furthermore, we demonstrated that OA substantially alleviated either PA or TM induced ER stress and pyroptosis in HepG2 cells (P < 0.01).
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. The enhanced expressions of ER stress markers CHOP and BIP were found subcellular co-located to pyroptosis markers NLRP3 and ASC. Furthermore, we demonstrated that the PA stimulated ER stress was causally related to pyroptosis.
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). We 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). The blood chemistry were analyzed, and the liver histology and the expressions of related genes and proteins were determined in the liver tissues. 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 release of IL-1 beta and tumor necrosis factor alpha (TNF-α) was determined by ELISA. The morphology of pyroptosis was detected by acridine orange and ethidium bromide (AO/EB) staining. The expressions of pyroptosis and ER stress related genes or proteins were determined by real-time PCR, Western blot or immunofluorescence. Besides, the cells were treated with the chemical ER stressor tunicamycin (TM) with or without OA for 24 h as well. Human 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. 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.
In pyroptosis, gasdermin D (GSDMD) acts as a major executor, while NLRP3 related inflammasome is closely linked to caspase-1 activation.
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. 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 a novel programmed cell death.