Silent information regulator sirtuin 1 ameliorates acute liver failure via the p53/glutathione peroxidase 4/gasdermin D axis

Figure 1 Ferroptosis and pyroptosis occur in human acute liver failure. A: The levels of aspartate aminotransferase, alanine aminotransferase, tumour necrosis factor alpha, interleukin (IL)-1β, and IL-6 were changed in serum samples of healthy individuals and patients with acute liver failure (ALF) (n = 30 in each group); B: Iron staining (scale bar: 100 µm and 33.3 µm) of normal liver tissue and ALF liver tissue; C: Detection of iron in normal liver tissue and ALF; D: Western blot analyses of glutathione peroxidase 4, solute carrier family 7a member 11, p53, and acyl-CoA synthetase long-chain family member 4 proteins were performed in healthy individuals and patients with ALF. Data are presented as the mean ± SD of three independent experiments; E: Western blot analysis of gasdermin D protein expression (n = 3); F: Western blot analysis of silent information regulator sirtuin 1 and Ac-p53 protein expression in healthy individuals and patients with ALF (n = 3). ^aP < 0.05, ^bP < 0.01. ALF: Acute liver failure; AST: Aspartate aminotransferase; ALT: Alanine aminotransferase; IL: Interleukin; GPX4: Glutathione peroxidase 4; SLC7A11: Solute carrier family 7a member 11; ACSL4: Acyl-CoA synthetase long-chain family member 4; GSDMD: Gasdermin D; SIRT1: Silent information regulator sirtuin 1.

Figure 2 Inhibition of the p53/glutathione peroxidase 4/gasdermin D signaling pathway attenuates acute liver failure in vivo. A: Macroscopic examinations of mouse livers and microscopic examinations of liver sections stained with hematoxylin and eosin (100 ×); B: Administration of pifithrin-α and liproxstatin-1 and gasdermin D (GSDMD) knockout improved the median survival in mice treated with D-galactosamine/lipopolysaccharide; C: Detection of interleukin (IL)-1β, tumour necrosis factor alpha, IL-6, IL-2, and C-C motif ligand 2 in mouse serum with multi-factor kit. Data are presented as the mean ± SD of five independent experiments; D: Western blot analyses of p53, glutathione peroxidase 4 (GPX4), solute carrier family 7a member 11, Acyl-CoA synthetase long-chain family member 4 (ACSL4), and GSDMD proteins (n = 3 in each group). Data are presented as the mean ± SD of three independent experiments; E: Western blot analyses of GSDMD protein expression (n = 3 in each group). Data are presented as the mean ± SD of three independent experiments; F: IIron detection with an iron kit. Data are presented as the mean ± SD of five independent experiments; G: Quantitative real-time polymerase chain reaction analyses of p53, GPX4, SLC7A11, ACSL4, and GSDMD mRNA expression (n = 3 in each group). Data are presented as the mean ± SD of three independent experiments. ^aP < 0.05, ^bP < 0.01. ALF: Acute liver failure; AST: Aspartate aminotransferase; ALT: Alanine aminotransferase; IL: Interleukin; GPX4: Glutathione peroxidase 4; SLC7A11: Solute carrier family 7a member 11; ACSL4: Acyl-CoA synthetase long-chain family member 4; GSDMD: Gasdermin D; SIRT1: Silent information regulator sirtuin 1; LPS: Lipopolysaccharide; D-GalN: D-galactosamine.

Figure 3 Inhibition of the p53/glutathione peroxidase 4/gasdermin D signaling pathway exacerbates acute liver injury in vitro. A and B: The levels of alanine aminotransferase and aspartate aminotransferase were changed in HL7702 cells (n = 3); C-E: The levels of iron, reactive oxygen species, and glutathione were changed in HL7702 cells (n = 3); F: Quantitative real-time polymerase chain reaction analyses of p53, glutathione peroxidase 4 (GPX4), solute carrier family 7a member 11 (SLC7A11), Acyl-CoA synthetase long-chain family member 4 (ACSL4), and gasdermin D (GSDMD) mRNA expression (n = 3 in each group); G: Western blot analyses of p53, GPX4, and GSDMD protein expression (n = 3 in each group); H: Western blot analyses of SLC7A11 and ACSL4 protein expression (n = 3 in each group). ^aP < 0.05, ^bP < 0.01. ALF: Acute liver failure; AST: Aspartate aminotransferase; ALT: Alanine aminotransferase; IL: Interleukin; GPX4: Glutathione peroxidase 4; SLC7A11: Solute carrier family 7a member 11; ACSL4: Acyl-CoA synthetase long-chain family member 4; GSDMD: Gasdermin D; SIRT1: Silent information regulator sirtuin 1; LPS: Lipopolysaccharide; D-GalN: D-galactosamine.

Figure 4 Activation of the p53/glutathione peroxidase 4/gasdermin D signaling pathway exacerbates alanine aminotransferase. A: Macroscopic examinations of mouse livers and microscopic examinations of liver sections stained with hematoxylin and eosin (100 ×); B: Administration of nutlin-3α and RAS-selective lethal 3 shortened the survival time of mice treated with lipopolysaccharide/D-galactosamine; C: Detection of interleukin (IL)-1β, tumour necrosis factor alpha, IL-6, IL-2, and C-C motif ligand 2 in mouse serum with multi-factor kit; D: Western blot analyses of p53, glutathione peroxidase 4, solute carrier family 7a member 11, acyl-CoA synthetase long-chain family member 4, and gasdermin D (GSDMD) protein expression (n = 3 in each group); E: Western blot analyses of GSDMD protein expression (n = 3 in each group); F: Iron detection with an iron kit. ^aP < 0.05, ^bP < 0.01. ALF: Acute liver failure; AST: Aspartate aminotransferase; ALT: Alanine aminotransferase; IL: Interleukin; GPX4: Glutathione peroxidase 4; SLC7A11: Solute carrier family 7a member 11; ACSL4: Acyl-CoA synthetase long-chain family member 4; GSDMD: Gasdermin D; SIRT1: Silent information regulator sirtuin 1; LPS: Lipopolysaccharide; D-GalN: D-galactosamine; RSL3: RAS-selective lethal 3.

Figure 5 Silent information regulator sirtuin 1 interdicts the glutathione peroxidase 4/gasdermin D signaling pathway by intercepting p53 deacetylation. A: Macroscopic examinations of mouse livers and microscopic examinations of liver sections stained with hematoxylin and eosin (100 ×); B: Survival time of mice after resveratrol and EX527 treatments; C: Multi-factor kit for the Detection of interleukin (IL)-1β, tumour necrosis factor alpha, IL-6, IL-2, and C-C motif ligand 2 in mouse serum with multi-factor kit; D and E: Western blot analyses of silent information regulator sirtuin 1, acetylated p53, p53, glutathione peroxidase 4, solute carrier family 7a member 11, and Acyl-CoA synthetase long-chain family member 4 protein expression (n = 3 in each group). ^aP < 0.05, ^bP < 0.01. ALF: Acute liver failure; AST: Aspartate aminotransferase; ALT: Alanine aminotransferase; IL: Interleukin; GPX4: Glutathione peroxidase 4; SLC7A11: Solute carrier family 7a member 11; ACSL4: Acyl-CoA synthetase long-chain family member 4; GSDMD: Gasdermin D; SIRT1: Silent information regulator sirtuin 1; LPS: Lipopolysaccharide; D-GalN: D-galactosamine; RSL3: RAS-selective lethal 3; RSV: Resveratrol.

Figure 6 Silent information regulator sirtuin 1 interdicts the glutathione peroxidase 4/gasdermin D signaling pathway by intercepting p53 deacetylation. A: Western blot analyse of gasdermin D protein expression (n = 3 in each group); B: Iron detection with an iron kit; C: Silent information regulator sirtuin 1 (SIRT1) expression in liver tissue measured by immunofluorescence (100 ×); D: Percentage of SIRT1 immunofluorescence area; E: Percentage of p53 immunofluorescence area; F: p53 expression in liver tissue measured by immunofluorescence (100 ×). Data are presented as the mean ± SD of five independent experiments. ^aP < 0.05, ^bP < 0.01. ALF: Acute liver failure; AST: Aspartate aminotransferase; ALT: Alanine aminotransferase; IL: Interleukin; GPX4: Glutathione peroxidase 4; SLC7A11: Solute carrier family 7a member 11; ACSL4: Acyl-CoA synthetase long-chain family member 4; GSDMD: Gasdermin D; SIRT1: Silent information regulator sirtuin 1; LPS: Lipopolysaccharide; D-GalN: D-galactosamine; RSL3: RAS-selective lethal 3; RSV: Resveratrol.

Figure 7 Silent information regulator sirtuin 1 attenuates hepatocyte ferroptosis and pyroptosis via a p53/glutathione peroxidase 4/gasdermin D dependent mechanism in vitro. A and B: The levels of aspartate aminotransferase and alanine aminotransferase were changed in serum samples of HL7702 cells (n = 3); C and D: The levels of iron and reactive oxygen species were changed in HL7702 cells (n = 3); E: Western blot analyses of silent information regulator sirtuin 1, p53, and acetylated p53 protein expression (n = 3 in each group); F: Western blot analyses of gasdermin D and glutathione peroxidase 4 protein expression (n = 3 in each group); G: Western blot analyses of solute carrier family 7a member 11 and Acyl-CoA synthetase long-chain family member 4 protein expression (n = 3 in each group). ^aP < 0.05, ^bP < 0.01. ALF: Acute liver failure; AST: Aspartate aminotransferase; ALT: Alanine aminotransferase; IL: Interleukin; GPX4: Glutathione peroxidase 4; SLC7A11: Solute carrier family 7a member 11; ACSL4: Acyl-CoA synthetase long-chain family member 4; GSDMD: Gasdermin D; SIRT1: Silent information regulator sirtuin 1; LPS: Lipopolysaccharide; D-GalN: D-galactosamine; RSL3: RAS-selective lethal 3; ROS: Reactive oxygen species.

Figure 8 Silent information regulator sirtuin 1 activation ameliorates lipopolysaccharide/D-galactosamine-induced acute liver failure by inhibiting p53/glutathione peroxidase 4/gasdermin D to reduce ferroptosis and pyroptosis. Silent information regulator sirtuin 1 activation inhibits p53 deacetylation, thereby inhibiting the glutathione peroxidase 4 (GPX4)/gasdermin D (GSDMD) signaling pathway and reducing inflammatory response and iron deposition. Blocking the p53/GPX4/GSDMD signaling pathway attenuates ferroptosis and pyroptosis in acute liver failure. IL: Interleukin; GPX4: Glutathione peroxidase 4; SLC7A11: Solute carrier family 7a member 11; GSDMD: Gasdermin D; SIRT1: Silent information regulator sirtuin 1; ROS: Reactive oxygen species; GSH: Glutathione; TNF-α: Tumour necrosis factor alpha.