Mitochondrial alanyl-tRNA synthetase 2 mediates histone lactylation to promote ferroptosis in intestinal ischemia-reperfusion injury

Figure 1 Mitochondrial alanyl-tRNA synthetase 2 promotes intestinal ischemia-reperfusion injury and ferroptosis in mouse model. A: Protein level of mitochondrial alanyl-tRNA synthetase 2 (AARS2) in intestinal tissue. Protein levels of AARS2 in intestinal tissues normalized to β-actin. Bar graphs show densitometric quantification (mean ± SD); B: Hematoxylin and eosin staining of intestinal tissues; C: Serum level of Fatty acid-binding protein measured by ELISA; D: mRNA abundance of ZO-1 and occluding; E: Levels of Fe²⁺ and malondialdehyde; F: Protein levels of GPX4 and acyl-CoA synthetase long-chain family member 4 in intestinal tissues; G: Production of tumor necrosis factor-alpha and interleukin-6 measured by ELISA. ^aP < 0.01 vs sham, ^bP < 0.01 vs model. AARS2: Mitochondrial alanyl-tRNA synthetase 2.

Figure 2 Mitochondrial alanyl-tRNA synthetase 2 increases in vitro cell injury. A: Protein level of mitochondrial alanyl-tRNA synthetase 2 (AARS2) in cells. Protein levels of AARS2 in cells normalized to β-actin. Bar graphs show densitometric quantification (mean ± SD); B: Cell viability was detected by CCK-8; C: mRNA abundance of ZO-1 and occluding; D: Levels of Fe²⁺ and malondialdehyde; E: Protein levels of GPX4 and acyl-CoA synthetase long-chain family member 4 (ACSL4) in cells. Protein levels of GPX4 and ACSL4 in cells normalized to β-actin. Bar graphs show densitometric quantification (mean ± SD). ^aP < 0.01 vs cont, ^bP < 0.01 vs hypoxia-reperfusion. H/R: Hypoxia-reperfusion; AARS2: Mitochondrial alanyl-tRNA synthetase 2.

Figure 3 Mitochondrial alanyl-tRNA synthetase 2 epigenetically upregulates acyl-CoA synthetase long-chain family member 4 expression via histone lactylation. A and B: The (A) protein expression [Protein levels of acyl-CoA synthetase long-chain family member 4 (ACSL4) in Caco-2 cells normalized to β-actin. Bar graphs show densitometric quantification (mean ± SD)] and (B) RNA level of ACSL4 in Caco-2 cells that depleted of AARS2; C: The enrichment of H3K18 La in histone of ACSL4 gene; D: The luciferase activity of ACSL4 gene promoter. ^aP < 0.01 vs shNC.

Figure 4 Mitochondrial alanyl-tRNA synthetase 2 induces cell ferroptosis via regulating acyl-CoA synthetase long-chain family member 4. A: Protein level of acyl-CoA synthetase long-chain family member 4 (ACSL4) and GPX4 in cells. Protein levels of GPX4 and ACSL4 in cells normalized to β-actin. Bar graphs show densitometric quantification (mean ± SD); B: Cell viability was detected by CCK-8; C: mRNA abundance of ZO-1 and occluding; D: Levels of Fe²⁺; E: Malondialdehyde. ^aP < 0.01 vs hypoxia-reperfusion, ^bP < 0.01 vs hypoxia-reperfusion + shACSL4. H/R: Hypoxia-reperfusion; AARS2: Mitochondrial alanyl-tRNA synthetase 2.