Recombinant human thioredoxin suppresses lipopolysaccharide-induced bronchoalveolar neutrophil infiltration in rat
Introduction
Acute respiratory distress syndrome (ARDS) is defined as the acute onset of severe lung injuries, bilateral infiltrate on chest X-ray and severe hypoxia with poor prognosis; the mortality rate is 40–60% (Doyle et al., 1995, Ware and Matthay, 2000). The syndrome is caused by several direct lung injury including pneumonia and aspiration of gastric contents, and by indirect lung injury such as sepsis. Regulating the extravasation and infiltration of neutrophils is regarded as one of the initial critical steps for treating patients with ARDS/acute lung injury (ALI). Treatment of initial disorders, respiratory control by oxygen and mechanical ventilation, circulatory assist and nutritional support are indispensable. In addition, glucocorticoid and sivelestat, a recently developed neutrophil elastase inhibitor (Kawabata et al., 2000, Nogami et al., 2000, Zeiher et al., 2002), are used. However, the prognosis is very poor and the more frequent cause of death is multiple organ failure rather than simple respiratory failure (Ware and Matthay, 2000). Therefore, novel therapeutic drugs are required for improving the prognosis.
Human thioredoxin (hTRX) is a ubiquitously expressed, multifunctional protein that has a redox-active disulfide/dithiol within the conserved -Cys-Gly-Pro-Cys- sequence (Holmgren, 1985, Nakamura et al., 1997, Tagaya et al., 1989). TRX protects cells against oxidative stress by scavenging reactive oxygen species. In addition, TRX suppresses inflammation by regulating the neutrophil activation and extravasation and exerts the anti-inflammatory effect (Nakamura et al., 2001). Administration of recombinant human thioredoxin (rhTRX) attenuates several diseases associated with neutrophil infiltration including cytokine- or anticancer drug-induced lung injuries (Hoshino et al., 2003), transient focal cerebral ischemia (Hattori et al., 2004), and autoimmune myocarditis (Liu et al., 2004). However, it has never been investigated whether rhTRX administered after the onset of injuries can suppress the infiltration of neutrophils.
In this study, we investigated the therapeutic effect of rhTRX against ALI in rats injected intravenously with lipopolysaccharide (LPS), which is an animal model of sepsis-induced ARDS/ALI. The results suggest that the tissue accumulation of intravenously administered rhTRX is marginal and the plasma concentrations of rhTRX play important roles to suppress the LPS-induced extravasation of neutrophils from the circulation into the bronchoalveolar space.
Section snippets
Animals
Specific pathogen-free (SPF) male Wistar rats weighing 220–240 g and 6-week-old female nude mice were obtained from Nihon SLC (Hamamatsu, Japan). The animal study protocol was approved by the Animal Research Committee, Graduate School of Medicine, Kyoto University.
Reagents
rhTRX was provided from Ajinomoto Inc. (Tokyo, Japan). LPS from Escherichia coli 0111:B4 (L2630) was purchased from Sigma (St. Louis, MO). LPS in normal saline at 5 mg/ml was stored at − 30 °C until use. Irinotecan hydrochloride
Pharmacokinetics of intravenously administered rhTRX
Previous studies referred to in the introduction demonstrated that intraperitoneal administration of 2 mg/kg rhTRX rescues animals in several oxidative stress-associated disease models. In order to develop and apply rhTRX to clinical use, we tested in the current study the intravenous injection of the recombinant protein. First, we tried bolus injection of rhTRX, however, we could not rescue the rats administered with LPS (data not shown). Therefore, we next measured the plasma concentration of
Discussion
A previous study demonstrated that not only overexpression of hTRX in transgenic mice but also intraperitoneal administration of rhTRX ameliorates ALI induced by inflammatory cytokines or bleomycin (Hoshino et al., 2003). In the current preclinical study, we used an LPS-induced ARDS model in the rat and examined the effect of intravenously administered rhTRX. According to our previous study, rhTRX in plasma at levels higher than 1000 ng/ml is required for preventing neutrophils from tissue
Conclusion
We have demonstrated in this study that continuous intravenous administration of rhTRX suppresses LPS-induced bronchoalveolar neutrophil infiltration by an anti-chemotactic effect. Because rhTRX is considered as a unique and promising agent regulating inflammation and oxidative stress, we are to use rhTRX for treating ARDS patients with poor prognosis as a clinical trail and investigate its clinical efficacy. In this context, a translational research project is in progress in our institute,
Acknowledgements
We thank Dr. Wenrui Liu for technical assistance and Ms. Aya Ono for secretarial help. This work was supported by funds from the Ministry of Education, Culture, Sports, Science and Technology, Japan and Redox Bioscience, Inc. (Kyoto, Japan).
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