Background: 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. 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. Methods: 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. 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. Results: 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). 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

The original article possessed a minor typo in first author, Xin Zeng’s name which has since been corrected.

Necroptosis and pyroptosis are lytic and inflammatory types of programmed cell death that require the membrane destruction predominantly driven by the mixed lineage kinase domain-like (MLKL) and gasdermin D (GSDMD) proteins. However, the crosstalk between them remains largely unknown. Here, our research discloses that endoplasmic reticulumn transmembrane protein inositol-requiring enzyme-1α (IRE-1α) is a potential modulator of both necroptosis and pyroptosis, paricularly in liver pathology. Interestingly, enhanced expression of IRE-1α triggers hepatic pyroptosis, while defective IRE-1α level activates hepatic necroptosis, and both processes are closed related to the activity of GSDMD. To elucidate unknown crosstalk, by using pharmacological and genetic methods, we first demonstrated that IRE-1α suppresses necroptosis by promoting the expression of GSDMD and cleaves caspase-8 and by inhibiting the expression of receptor-interacting serine/threonine-protein kinase 1 (RIPK1), RIPK3 and MLKL. Unexpectedly, excess IRE-1α initiates pyroptosis by increasing GSDMD and NLRP3 levels. Our work clearly provides insight into the modulation of IRE-1α to dominate necroptosis and pyroptosis and suggests that IRE-1α may be a promising therapeutic target for drug discovery in both types of tissue injuries.

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Necroptosis and pyroptosis are lytic and inflammatory types of programmed cell death that require the membrane destruction predominantly driven by the mixed lineage kinase domain-like (MLKL) and gasdermin D (GSDMD), respectively. However, the crosstalk between them remains largely unknown. Here, we disclose that inositol-requiring enzyme-1α (IRE-1α) is a potential modulator of both necroptosis and pyroptosis, particularly in liver pathology. In vivo, we found that pharmacological suppression of IRE-1α resulted in serious acute liver failure, which may be attributable to the downregulation of GSDMD and caspase-8 while remarkable upregulation of necroptosis markers receptor-interacting serine/threonine-protein kinase 1 (RIPK1), RIPK3 and MLKL. However, by using thapsigargin (THP) to moderately restore the IRE-1α level, liver failure was distinctly alleviated. Conversely, ER stressor tunicamycin (TM) promoted IRE-1α activity, which initiated liver pyroptosis by increasing GSDMD and NLRP3. But maintaining the IRE-1α balance by moderate inhibition of IRE-1α effectively improved mouse survival. In vitro, we demonstrated that inhibition of IRE-1α led to distinct necroptosis accompanied by the reduction of GSDMD in LO-2 cells and mouse primary hepatocytes. Nevertheless, by using lipopolysaccharide (LPS) to specifically inspire the GSDMD level, necroptosis was obvious ameliorated. In addition, overexpression of IRE-1α in LO-2 cells obviously increased pyroptotic markers, such as GSDMD and NLRP3, but downregulated the necroptosis markers p-MLKL and p-RIPK3. In conclusion, enhanced expression of IRE-1α triggers hepatic pyroptosis, while IRE-1α deficiency activates hepatic necroptosis, and both processes are closed related to the activity of GSDMD in mice. So, IRE-1α may be a promising therapeutic target in tissue injuries.

Obesity is a recognized epidemic worldwide, and the accumulation of excess free saturated fatty acids (SFAs) in cells induces cellular lipotoxic damage and increases the risk of a wide spectrum of metabolic diseases including type 2 diabetes (T2D) and nonalcoholic fatty liver disease (NAFLD). Monounsaturated fatty acids (MUFAs) and polyunsaturated fatty acids (PUFAs) have been reported to combat SFA-induced cellular damage. However, the comparative studies of the two types of unsaturated fatty acids (UFAs) are still limited. We investigated the effects of different MUFAs and PUFAs in the human hepatocyte line L-02 cells in vitro, and in high-fat-diet (HFD)-induced obese C57BL/6 mice in vivo. The results of the in vitro study showed that SFAs induced significant cellular lipotoxic damage, but the combination of MUFAs/PUFAs with SFAs significantly improved the impaired cell viability. Particularly, oleic acid (OA) was superior to eicosapentaenoic acid (EPA), Docosahexaenoic acid (DHA), and arachidonic acid (AA) in terms of its anti-apoptotic effect and inhibition of endoplasmic reticulum (ER) stress. In vivo, both olive-oil-enriched (HFD + OO) and fish-oil-enriched high-fat diets (HFD + FO) reduced hepatic steatosis and improved insulin sensitivity in obese mice. However, FO induced an abnormal increase in serum aspartate aminotransferase (AST) and an increase in the oxidative stress indicator Malondialdehyde (MDA). Liver-targeted lipidomic analysis showed that liver lipid metabolites under the two types of UFA dietary interventions differed from the HFD group, modulating the abundance of some lipid metabolites such as triglycerides (TGs) and glycerophospholipids. Furthermore, the FO diet significantly increased the abundance of the associated FA 20:5 long-chain lipid metabolites, whereas the OO diet regulated the unsaturation of all fatty acids in general and increased the abundance of FA 18:1 in the overall lipid metabolites, especially TGs, which may primarily contribute to the FO, and OO drove protection in NAFLD.