Maes Karen, Stamiris Angela, Thomas Debby, Cielen Nele, Smuder Ashley, Powers Scott K, Leite Felipe S, Hermans Greet, Decramer Marc, Hussain Sabah N, Gayan-Ramirez Ghislaine
1Respiratory Muscle Research Unit, Laboratory of Pneumology and Respiratory Division, Katholieke Universiteit Leuven, Leuven, Belgium. 2Department of Critical Care Medicine, McGill University Health Centre and Meakins-Christie Laboratories, McGill University, Montréal, QC, Canada. 3Department of Applied Physiology and Kinesiology, University of Florida, Gainesville, FL. 4Department of Kinesiology and Physical Education, McGill University, Montreal, QC, Canada. 5Medical Intensive Care Unit, General Internal Medicine, University Hospitals Leuven, Leuven, Belgium.
Crit Care Med. 2014 Dec;42(12):e772-82. doi: 10.1097/CCM.0000000000000685.
Diaphragm dysfunction develops during severe sepsis as a consequence of hemodynamic, metabolic, and intrinsic abnormalities. Similarly, 12 hours of controlled mechanical ventilation also promotes diaphragm dysfunction. Importantly, patients with sepsis are often treated with mechanical ventilation for several days. It is unknown if controlled mechanical ventilation exacerbates sepsis-induced diaphragm dysfunction, and this forms the basis for these experiments. We investigate the effects of 12-hour controlled mechanical ventilation on contractile function, fiber dimension, cytokine production, proteolysis, autophagy, and oxidative stress in the diaphragm of septic rats.
Randomized controlled experiment.
Animal research laboratory.
Adult male Wistar rats.
Treatment with a single intraperitoneal injection of either saline or Escherichia coli lipopolysaccharide (5 mg/kg). After 12 hours, the saline-treated animals (controlled mechanical ventilation) and half of the septic animals (lipopolysaccharide + controlled mechanical ventilation) were submitted to 12 hours of controlled mechanical ventilation while the remaining septic animals (lipopolysaccharide) were breathing spontaneously for 12 hours. They were compared to a control group. All animals were studied 24 hours after saline or lipopolysaccharide administration.
Twenty-four hours after saline or lipopolysaccharide administration, diaphragm contractility was measured in vitro. We also measured diaphragm muscle fiber dimensions from stained cross sections, and inflammatory cytokines were determined by proteome array. Activities of calpain, caspase-3, and proteasome, expression of 20S-proteasome α subunits, E2 conjugases, E3 ligases, and autophagy were measured with immunoblotting and quantitative polymerase chain reaction. Lipopolysaccharide and/or controlled mechanical ventilation independently decreased diaphragm contractility and fiber dimensions and increased diaphragm interleukin-6 production, protein ubiquitination, expression of Atrogin-1 and Murf-1, calpain and caspase-3 activities, autophagy, and protein oxidation. Compared with lipopolysaccharide alone, lipopolysaccharide + controlled mechanical ventilation worsened diaphragm contractile dysfunction, augmented diaphragm interleukin-6 levels, autophagy, and protein oxidation, but exerted no exacerbating effects on diaphragm fiber dimensions, calpain, caspase-3, or proteasome activation.
Twelve hours of controlled mechanical ventilation potentiates sepsis-induced diaphragm dysfunction, possibly due to increased proinflammatory cytokine production and autophagy and worsening of oxidative stress.
在严重脓毒症期间，由于血流动力学、代谢及内在异常，膈肌功能障碍会逐渐发展。同样，12小时的控制性机械通气也会促使膈肌功能障碍。重要的是，脓毒症患者常接受数天的机械通气治疗。目前尚不清楚控制性机械通气是否会加剧脓毒症诱导的膈肌功能障碍，这构成了这些实验的基础。我们研究12小时控制性机械通气对脓毒症大鼠膈肌收缩功能、纤维尺寸、细胞因子产生、蛋白水解、自噬及氧化应激的影响。
随机对照实验。
动物研究实验室。
成年雄性Wistar大鼠。
单次腹腔注射生理盐水或大肠杆菌脂多糖（5毫克/千克）进行治疗。12小时后，生理盐水处理的动物（控制性机械通气）及半数脓毒症动物（脂多糖+控制性机械通气）接受12小时的控制性机械通气，而其余脓毒症动物（脂多糖）自主呼吸12小时。将它们与对照组进行比较。所有动物在给予生理盐水或脂多糖24小时后进行研究。
给予生理盐水或脂多糖24小时后，体外测量膈肌收缩力。我们还从染色的横切面测量膈肌肌纤维尺寸，并通过蛋白质组芯片测定炎性细胞因子。用免疫印迹法和定量聚合酶链反应测量钙蛋白酶、半胱天冬酶-3和蛋白酶体的活性、20S蛋白酶体α亚基、E2共轭酶、E3连接酶的表达及自噬情况。脂多糖和/或控制性机械通气分别降低膈肌收缩力和纤维尺寸，并增加膈肌白细胞介素-6的产生、蛋白质泛素化、Atrogin-1和Murf-1的表达、钙蛋白酶和半胱天冬酶-3的活性、自噬及蛋白质氧化。与单独使用脂多糖相比，脂多糖+控制性机械通气使膈肌收缩功能障碍恶化，增加膈肌白细胞介素-6水平、自噬及蛋白质氧化，但对膈肌纤维尺寸、钙蛋白酶、半胱天冬酶-3或蛋白酶体激活无加剧作用。
12小时的控制性机械通气会增强脓毒症诱导的膈肌功能障碍，可能是由于促炎细胞因子产生增加、自噬及氧化应激恶化所致。
