Attenuation of ventilation-induced diaphragm dysfunction through toll-like receptor 4 and nuclear factor-κB in a murine endotoxemia model

Abstract

Mechanical ventilation (MV) is often used to maintain life in patients with sepsis and sepsis-related acute lung injury. However, controlled MV may cause diaphragm weakness due to muscle injury and atrophy, an effect termed ventilator-induced diaphragm dysfunction (VIDD). Toll-like receptor 4 (TLR4) and nuclear factor-κB (NF-κB) signaling pathways may elicit sepsis-related acute inflammatory responses and muscle protein degradation and mediate the pathogenic mechanisms of VIDD. However, the mechanisms regulating the interactions between VIDD and endotoxemia are unclear. We hypothesized that mechanical stretch with or without endotoxin treatment would augment diaphragmatic structural damage, the production of free radicals, muscle proteolysis, mitochondrial dysfunction, and autophagy of the diaphragm via the TLR4/NF-κB pathway. Male C57BL/6 mice, either wild-type or TLR4-deficient, aged between 6 and 8 weeks were exposed to MV (6 mL/kg or 10 mL/kg) with or without endotoxemia for 8 h. Nonventilated mice were used as controls. MV with endotoxemia aggravated VIDD, as demonstrated by the increases in the expression levels of TLR4, caspase-3, atrogin-1, muscle ring finger-1, and microtubule-associated protein light chain 3-II. In addition, increased NF-κB phosphorylation and oxidative loads, disorganized myofibrils, disrupted mitochondria, autophagy, and myonuclear apoptosis were also observed. Furthermore, MV with endotoxemia reduced P62 levels and diaphragm muscle fiber size (P < 0.05). Endotoxin-exacerbated VIDD was attenuated by pharmacologic inhibition with a NF-κB inhibitor or in TLR4-deficient mice (P < 0.05). Our data indicate that endotoxin-augmented MV-induced diaphragmatic injury occurs through the activation of the TLR4/NF-κB signaling pathway.

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

“…Although ROS and early cytokines are important to contribute to VIDD, and herein, we provide the evidence that using anti‐HMGB1 antibody can elevate the SOD activity and suppress the oxidants, inflammatory cytokines and VIDD comparable to the beneficial effects of ethyl pyruvate in this model (Figures ). In addition, we proved that MV induces diaphragmatic injury in a mouse model of endotoxaemia through activation of the TLR4/NF‐κB signalling pathway . This inspired us to investigate the role of HMGB1, a pivotal ligand of the TLR4 receptor, and develop an effective agent to improve VIDD during endotoxaemia.…”

“…After 1 hour of spontaneous respiration to allow for developing a septic response, the mice were subjected to MV for 8 hours. 9,31,36 Two doses of ethyl pyruvate (Sigma, St Louis, MO) were administered intraperitoneally. The first dose was administered 30 minutes before the mice were subjected to MV and the second dose was used after the mice were subjected to 4 hours of MV.…”

“…Total RNA (1 μg) was reverse transcribed using a GeneAmp polymerase chain reaction (PCR) system 9600 (PerkinElmer, Life Sciences, Inc, Boston, MA), as previously described. 31 The following primers were used for real-time polymerase chain reaction: HMGB1, forward primer 5′-TGGCAAAGGCTGACAAGGCTC-3′ and reverse primer 5′-GGATGCTCGCCTTTGATTTTGG-3′ and GAPDH as internal control, forward primer 5′-GGAGCGAGACCCCACTAACA-3′ and reverse primer 5′-ACATACTCAGCACCGGCCTC-3′ (Protech Technology Enterprise Co., Ltd., Taipei, Taiwan). 37,38 All quantitative PCR reactions using SYBR Master Mix were performed on a CFX96 Touch Real-Time PCR Detection system (Bio-Rad Laboratories, Inc, Hercules, CA).…”

“…Ethyl pyruvate, 100 mg/kg, was given intraperitoneally 30 Additional details, including measurement of diaphragm force-frequency relationships, immunoblot analysis, immunohistochemistry, mitochondrial isolation, measurement of diaphragmatic oxidative stress and antioxidant enzyme expression, TEM and TUNEL assay were performed as previously described. 9,31 3 | RE SULTS…”

“…In addition, we proved that MV induces diaphragmatic injury in a mouse model of endotoxaemia through activation of the TLR4/ NF-κB signalling pathway. 31 This inspired us to investigate the role of HMGB1, a pivotal ligand of the TLR4 receptor, and develop an effective agent to improve VIDD during endotoxaemia.…”