Ethyl pyruvate attenuates ventilation-induced diaphragm dysfunction through high-mobility group box-1 in a murine endotoxaemia model

Abstract

Mechanical ventilation (MV) can save the lives of patients with sepsis. However, MV in both animal and human studies has resulted in ventilator-induced diaphragm dysfunction (VIDD). Sepsis may promote skeletal muscle atrophy in critically ill patients. Elevated high-mobility group box-1 (HMGB1) levels are associated with patients requiring long-term MV. Ethyl pyruvate (EP) has been demonstrated to lengthen survival in patients with severe sepsis. We hypothesized that the administration of HMGB1 inhibitor EP or anti-HMGB1 antibody could attenuate sepsis-exacerbated VIDD by repressing HMGB1 signalling. Male C57BL/6 mice with or without endotoxaemia were exposed to MV (10 mL/kg) for 8 hours after administrating either 100 mg/kg of EP or 100 mg/kg of anti-HMGB1 antibody. Mice exposed to MV with endotoxaemia experienced augmented VIDD, as indicated by elevated proteolytic, apoptotic and autophagic parameters. Additionally, disarrayed myofibrils and disrupted mitochondrial ultrastructures, as well as increased HMGB1 mRNA and protein expression, and plasminogen activator inhibitor-1 protein, oxidative stress, autophagosomes and myonuclear apoptosis were also observed. However, MV suppressed mitochondrial cytochrome C and diaphragm contractility in mice with endotoxaemia (P < 0.05). These deleterious effects were alleviated by pharmacologic inhibition with EP or anti-HMGB1 antibody (P < 0.05). Our data suggest that EP attenuates endotoxin-enhanced VIDD by inhibiting HMGB1 signalling pathway.

Keywords: endotoxaemia; ethyl pyruvate; high-mobility group box-1; mitochondria; ventilator-induced diaphragm dysfunction.

Figure 1

Electron microscopy and muscle force‐frequency activity of the diaphragm. Representative micrographs of the longitudinal sections of diaphragm (×20 000: upper panel; ×40 000: lower panel) were from the same diaphragms of non‐ventilated control mice and mice ventilated at a tidal volume (V_T) of 10 mL/kg (V_T 10) for 8 h with or without LPS administration (n = 3 per group). (A and B) Non‐ventilated control wild‐type mice with or without LPS treatment: normal sarcomeres with distinct A bands, I bands and Z bands; (C) 10 mL/kg wild‐type mice without LPS treatment (normal saline): increase of diaphragmatic disarray; (D) 10 mL/kg wild‐type mice with LPS treatment: disruption of sarcomeric structure with loss of mitochondrial swelling, streaming of Z bands and collection of lipid droplets; (E) 10 mL/kg wild‐type mice pretreated with ethyl pyruvate: reduction of diaphragmatic disruption. (F) Diaphragm muscle‐specific force production was measured as described in Materials and Methods2. Mitochondrial swelling with concurrent loss of cristae and autophagosomes containing heterogeneous cargo are identified by arrows. Ethyl pyruvate, 100 mg/kg, was given intraperitoneally 30 min before mechanical ventilation and 4 h after mechanical ventilation. *P < 0.05 vs the non‐ventilated control mice with LPS treatment; ^† P < 0.05 vs all other groups. Scale bar represents 500 nm. EP, ethyl pyruvate; Hz, hertz; LPS, lipopolysaccharide; N, Newton

Figure 2

Ethyl pyruvate abrogated endotoxin‐augmented mechanical ventilation‐induced oxidative stress, inflammatory cytokines, calpain, atrogin‐1 and MuRF‐1 expression in the diaphragm. (A) protein carbonyl groups (diaphragm), (B) SOD (diaphragm), (C) BAL fluid active PAI‐1 and (D) BAL fluid HMGB1 were from the non‐ventilated control mice and mice ventilated at a tidal volume of 10 mL/kg for 8 h with or without LPS administration (n = 5 per group). Western blots were performed using antibodies that recognize calpain (E), atrogin‐1 (F), MuRF‐1 (G) and GAPDH expression from the diaphragms of non‐ventilated control mice and mice ventilated at a tidal volume of 10 mL/kg for 8 h with or without LPS administration (n = 5 per group). Arbitrary units were expressed as relative calpain, atrogin‐1 and MuRF‐1 activation (n = 5 per group). Ethyl pyruvate, 100 mg/kg, was given intraperitoneally 30 min before mechanical ventilation and 4 h after mechanical ventilation. *P < 0.05 vs the non‐ventilated control mice with LPS treatment; ^† P < 0.05 vs all other groups. BAL, bronchoalveolar lavage; GAPDH, glyceraldehydes‐phosphate dehydrogenase; HMGB1, high‐mobility group box‐1; MuRF‐1, muscle ring finger‐1; PAI‐1, plasminogen activator inhibitor‐1; SOD, sodium dismutase

Figure 3

Ethyl pyruvate and anti‐HMGB1 antibody inhibited endotoxin‐aggravated mechanical ventilation‐enhanced HMGB1 mRNA activation and HMGB1 protein expression. (A) Real‐time PCR performed for HMGB1 mRNA expression was from the diaphragms of non‐ventilated control mice and mice ventilated at a tidal volume of 10 mL/kg for 8 h with or without LPS administration (n = 5 per group). Arbitrary units were expressed as the ratio of HMGB1 mRNA to GAPDH (n = 5 per group). (B) Representative micrographs (x400) with HMGB1 staining of paraffin diaphragm sections and quantification were from the diaphragms of non‐ventilated control mice and mice ventilated at a tidal volume of 10 mL/kg for 8 h with or without LPS administration (n = 5 per group). Ethyl pyruvate, 100 mg/kg, was given intraperitoneally 30 min before mechanical ventilation and 4 h after mechanical ventilation. Anti‐HMGB1 antibody, 100 mg/kg, was administered intravenously 30 min before the start of ventilation. A dark‐brown diaminobenzidine signal identified by arrows indicates positive staining for HMGB1 in the diaphragm, whereas shades of bluish tan signify non‐reactive cells. *P < 0.05 vs the non‐ventilated control mice with LPS treatment; ^† P < 0.05 vs all other groups. Scale bars represent 20 μm. Anti‐H, anti‐HMGB1 antibody; PCR, polymerase chain reaction

Figure 4

Anti‐HMGB1 antibody suppressed endotoxin‐exacerbated mechanical ventilation‐mediated oxidative stress and inflammatory cytokines production. (A) protein carbonyl groups (diaphragm), (B) SOD (diaphragm), (C) BAL fluid active PAI‐1 and (D) BAL fluid HMGB1 were from the non‐ventilated control mice and mice ventilated at a tidal volume of 10 mL/kg for 8 h with or without LPS administration (n = 5 per group). Western blots were performed using antibodies that recognize calpain (E), atrogin‐1 (F), MuRF‐1 (G) and GAPDH expression from the diaphragms of non‐ventilated control mice and mice ventilated at a tidal volume of 10 mL/kg for 8 h with or without LPS administration (n = 5 per group). Arbitrary units were expressed as relative calpain, atrogin‐1 and MuRF‐1 activation (n = 5 per group). Anti‐HMGB1 antibody, 100 mg/kg, was administered intravenously 30 min before the start of ventilation. *P < 0.05 vs the non‐ventilated control mice with LPS treatment; ^† P < 0.05 vs all other groups

Figure 5

Inhibition of endotoxin‐augmented mechanical ventilation‐induced diaphragmatic injury by ethyl pyruvate and anti‐HMGB1 antibody. (A) Representative micrographs of the longitudinal sections of diaphragm (×40 000) were from the diaphragms of non‐ventilated control mice and mice ventilated at a tidal volume of 10 mL/kg for 8 h with or without LPS administration (n = 3 per group). Mitochondrial swelling with coexisting vacuole formation, loss of cristae and autophagosomes containing heterogeneous cargo is identified by arrows. (B) Diaphragm muscle‐specific force production was measured as described in Methods. (C and D) Western blots were performed using antibodies that recognize mitochondrial cytochrome C, LC3‐II and GAPDH expression from the diaphragms of non‐ventilated control mice and mice ventilated at a tidal volume of 10 mL/kg for 8 h with or without LPS administration (n = 5 per group). Arbitrary units were expressed as relative mitochondrial cytochrome C and LC3‐II activation (n = 5 per group). Ethyl pyruvate, 100 mg/kg, was given intraperitoneally 30 min before mechanical ventilation and 4 h after mechanical ventilation. Anti‐HMGB1 antibody, 100 mg/kg, was administered intravenously 30 min before the start of ventilation. *P < 0.05 vs the non‐ventilated control mice with LPS treatment; ^† P < 0.05 vs all other groups. Scale bars represent 500 nm. LC3‐II, light chain 3‐II; Mito‐Cyt C, mitochondrial cytochrome C

Figure 6

Ethyl pyruvate and anti‐HMGB1 antibody ameliorated endotoxin‐enhanced mechanical ventilation‐mediated caspase‐3 expression and apoptosis in the diaphragm. (A) Western blots were conducted using antibodies that recognize caspase‐3 and GAPDH expression from the diaphragms of non‐ventilated control mice and mice ventilated at a tidal volume of 10 mL/kg for 8 h with or without LPS administration (n = 5 per group). Arbitrary units were expressed as the ratio of cleaved caspase‐3 to GAPDH (n = 5 per group). (B and C) Representative micrographs (×400) with TUNEL staining of paraffin diaphragm sections and quantification were from the diaphragms of non‐ventilated control mice and mice ventilated at a tidal volume 10 mL/kg for 8 h with or without LPS administration (n = 5 per group). Ethyl pyruvate, 100 mg/kg, was given intraperitoneally 30 min before mechanical ventilation and 4 h after mechanical ventilation. Anti‐HMGB1 antibody, 100 mg/kg, was administered intravenously 30 min before the start of ventilation. Apoptotic cells are identified by arrows. A bright green signal indicates positive staining of apoptotic cells, and shades of dull green signify non‐reactive cells. *P < 0.05 vs the non‐ventilated control mice with room air; ^† P < 0.05 vs all other groups. Scale bars represent 20 μm. TUNEL, terminal deoxynucleotidyl transferase‐mediated dUTP‐biotin nick end‐labelling

Figure 7

Schematic figure illustrating the signalling pathway activation with mechanical ventilation and endotoxaemia. Endotoxin‐mediated augmentation of mechanical stretch‐induced inflammatory cytokine production and diaphragm injury was alleviated after the administration of ethyl pyruvate and anti‐HMGB1 antibody.31 HMGB1, high‐mobility group box‐1; LC3‐II, light chain 3‐II; LPS, lipopolysaccharide; MuRF‐1, muscle ring finger‐1; NF‐κB, nuclear factor κappa B; PAI‐1, plasminogen activator inhibitor‐1; ROS, reactive oxygen species; TLR4, toll‐like receptor 4; VIDD, ventilator‐induced diaphragm dysfunction. *Reference 31