Oleic acid ameliorates palmitic acid induced hepatocellular lipotoxicity by inhibition of ER stress and pyroptosis

Abstract

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 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).

Conclusion: Palmitic 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.

Keywords: ER stress; Lipotoxicity; Oleic acid; Palmitic acid; Pyroptosis.

Fig. 1

Oleic acid protected HepG2 cells from palmitic acid induced Lipotoxicity. Viability of HepG2 cells was assessed using the CCK8 assay. a. and b. Alternatively, cells were treated with PA or OA alone for 12 h,24 h or 48 h. c. Cells were concomitantly incubated with PA and OA for 24 h. d. HepG2 were treated with 0.4 mM PA, 0.2 mM OA or combination of 0.4 mM PA plus 0.2 mM OA (PA/OA). The mRNA expression of key genes governing lipid metabolism were detected after 24 h treatment, and β-ACTIN was used as an internal control; e. Cells were stained with Oil Red O and lipid accumulation was visualized under a microscope at 200 × magnification after 24 h treatment. The data are presented as means ± SD for 3–5 biological replicates; *P < 0.05, **P < 0.01, *** P < 0.001vs. BSA;#P < 0.05,vs. PA; ns no significant differences between two connected groups

Fig. 2

Apoptosis is not the main form of cell death caused by palmitic acid induced Lipotoxicity. HepG2 were treated with 0.4 mM PA, 0.2 mM OA or combination 0.4 mM PA plus 0.2 mM OA (PA/OA). a and b. The mRNA expression of key genes governing apoptosis were detected after 24 h treatment, and β-ACTIN was used as an internal control. c. Representative western blots of Cleaved-caspase3/9 after 24 h treatment, and β-ACTIN was used as a protein-loading control. d. Apoptosis assay using FCM with AV/PI staining after 24 h treatment. The numbers at the lower or upper right indicate the percentage of sum of early and late apoptotic cells. The data are presented as means ± SD for 3–5 biological replicates; *P < 0.05, **P < 0.01, ***, P < 0.001vs. BSA; ns no significant differences between two connected groups

Fig. 3

Palmitic acid induced pyroptosis activation,and oleic acid protected HepG2 cell against pyroptosis. HepG2 were treated with 0.4 mM PA, 0.2 mM OA or their combination (PA/OA). a. The mRNA expressions of key genes governing pyroptosis were detected after 24 h treatment, and β-ACTIN was used as an internal control. b. HepG2 cells were stained with anti-GSDMD (red) antibody and DAPI, and then visualized under a microscope at 200× magnification after 24 h treatment. c. Cell supernatants were analyzed for IL-1β secretion by ELISA. d and e. Representative western blots of NLRP3, GSDMD/−N, pro-CAS-1, P20 and IL-1β after 24 h treatment, and β-ACTIN was used as a protein-loading control. f. The morphology of pyroptotic cells was visualized by AO/EB staining. All groups were visualized under a microscope at 50× and 200× magnification after 24 h treatment. The data are presented as means ± SD for 3–5 biological replicates; **P < 0.01, ***, P < 0.001vs. BSA;#P < 0.05,##P < 0.01,vs. PA; ns no significant differences between two connected groups

Fig. 4

Palmitic acid induced ER stress was associated with pyroptosis activation, and oleic acid protected HepG2 cells from ER stress. HepG2 were treated with 0.4 mM PA, 0.2 mM OA or their combination (PA/OA). a-b. The mRNA expressions (a) and protein expressions (b) of key genes governing ER stress were detected after 24 h treatment, and β-ACTIN was used as an internal control for qPCR and Western Blot, respectively. c. Representative western blots of GRP78 and CHOP after 24 h treatment, and β-ACTIN was used as a protein-loading control. d. HepG2 cells were exposed to PA or OA and subsequently labeled by immunofluorescence with anti-NLRP3 (red) and anti-CHOP (green) antibodies, respectively. e. All groups labeled for GRP78 (red) and ASC (green) as well. f. HepG2 cells were exposed to PA and was visualized under a microscope at 400× magnification after 24 h treatment. Inset shows zoomed of indicated region. The data are presented as means±SD for 3–5 biological replicates; *P < 0.05, **P < 0.01, vs. BSA; ^#P < 0.05, vs. PA; ns no significant differences between two connected groups

Fig. 5

ER Stress induced NLRP3 inflammasome -mediated pyroptosis activation. The ER stress in HepG2 cells were elicited by chemical ER stressor tunicamycin (TM) for 24 h. a. Cell viability of cells was assessed using the CCK8 assay. b. and c. The mRNA expression of key genes governing ER stress and pyroptosis were detected after 24 h treatment of 0.8 uM TM, and β-ACTIN was used as an internal control. d. Representative western blots of ER stress and pyroptosis makers, and β-ACTIN was used as a protein-loading control. e. HepG2 cells were exposed to TM and plus with TUDCA or OA followed by labeling with anti-GSDMD or anti-NLRP3 antibody and were visualized under a microscope at 400× magnification after 24 h treatment. f. Cells were exposed to TM, or plus with TUDCA, 4-PBA or OA, and cell viability was assessed using the CCK8 assay. g. The mRNA expression of key genes governing ER stress and pyroptosis were detected after 24 h treatment, and β-ACTIN was used as an internal control. h. and i. Representative western blots of GRP78, CHOP, NLRP3 and GSDMD-N after 24 h treatment, and β-ACTIN was used as a protein-loading control. The data are presented as means ± SD for 3–5 biological replicates; *P < 0.05, **P < 0.01, ***P < 0.001vs. BSA;#P < 0.05, ##P < 0.01,vs. TM; ns no significant differences between two connected groups

Fig. 6

Oleic acid abrogated pyroptosis in HepG2 cells through inhibiting ER stress. The ER stress in cells were inhibited by chemical chaperones 4-phenylbutyric acid (4-PBA) or tauroursodeoxycholic acid (TUDCA) for 24 h against ER stress. a. and b. Cell viability of HepG2 cells was assessed using CCK8 assay. c. The mRNA expression of key genes governing pyroptosis were detected after 24 h treatment, and β-ACTIN was used as an internal control. d. Representative western blots of NLRP3 and GSDMD-N after 24 h treatment, and β-ACTIN was used as a protein-loading control. e. HepG2 cells were exposed to PA or plus OA, TUDCA or 4-PBA followed by labeling with anti-GSDMD (red) antibody and DAPI staining, and were visualized under a microscope at 100× magnification after 24 h treatment. f. and g. Cell supernatants were analyzed for IL-1β and TNF-α secretion by HepG2 cells by ELISA. The data are presented as means ± SD for 3–5 biological replicates; *P < 0.05, **P < 0.01, and ***P < 0.001vs. BSA;#P < 0.05, ##P < 0.01vs.PA;ns no significant differences between two connected groups

Fig. 7

High-fat diet-induced ER stress-mediated pyroptosis and olive oil ameliorated this effect in a rat NAFLD model. a. H&E staining of liver sections from chow, HFD, HFD/OA and OA groups(N = 5). b. and c. Changes in liver weights and volume at the end of 32 w treatment. d. Changes in body weights of experimental animals from different groups. e-j. Serum levels of AST, AST, TG, TC,LDL-C and HDL-C. k. Scores of steatosis based on the representative H&E staining of liver sections from different groups. Grades of steatosis: 0 (no steatosis), 1 (< 33% steatosis), 2 (33–66% steatosis) or 3 (> 66% steatosis) (n = 5). l. and m. Representative western blots of ER stress and pyroptosis makers, and β-ACTIN was used as a protein-loading control. The data are presented as means ± SD for 3–5 biological replicates; *P < 0.05, **P < 0.01, and ***P < 0.001vs. Chow; #P < 0.05, ##P < 0.01, vs. HFD;ns no significant differences between two connected groups