Protective effects of early treatment with propofol on endotoxin-induced acute lung injury in rats

J. Gao*,1, B. X. Zeng1, L. J. Zhou2 and S. Y. Yuan1

1 Department of Anesthesiology, Union Hospital and 2 Department of Child and Adolescent Health and Maternal Care, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, Hubei, China

*Corresponding author. Email: gaoju_003@163.com

Accepted for publication: September 24, 2003


    Abstract
 Top
 Abstract
 Introduction
 Methods and results
 Comment
 References
 
Background. To investigate the effects of propofol administration on acute lung injury in endotoxin-induced shock in rats.

Methods. Seventy-six male Wistar rats were randomly assigned to one of five groups: (i) saline control; (ii) endotoxin alone (receiving lipopolysaccharide (LPS) 8 mg kg–1 i.v.); (iii) pretreatment with propofol 1 h before LPS; (iv) simultaneous treatment with propofol and LPS; (v) post-treatment with propofol 1 h after LPS. During the 5 h after LPS injection, survival rates were recorded. Lung tissue was sampled to measure values of nitrite/nitrates (NO2–/NO3–) and tumour necrosis factor (TNF)-{alpha} in bronchoalveolar lavage (BAL) fluid, and wet-to-dry lung weight ratio, pulmonary permeability index, BAL protein and expression of inducible nitric oxide synthase (iNOS) and nitrotyrosine (NT).

Results. Compared with the endotoxaemic group, both the pre- and simultaneous treatment groups showed significantly improved 5 h survival rates, and attenuated endotoxin-induced increased BAL fluid NO2– /NO3– and TNF-{alpha}, iNOS mRNA and NT expression in lung tissue, and decreased pulmonary microvascular permeability. These beneficial effects were blunted in the post-treatment group.

Conclusions. These findings indicate that early administration of propofol may provide protective effects against endotoxin-induced acute lung injury.

Br J Anaesth 2004; 92: 277–9

Keywords: anaesthestics i.v., propofol; complications, acute lung injury; complications, endotoxaemia; pharmacology, nitric oxide; toxicity, peroxynitrite


    Introduction
 Top
 Abstract
 Introduction
 Methods and results
 Comment
 References
 
Acute lung injury (ALI) or acute respiratory distress syndrome (ARDS), a major cause of mortality in intensive care units, is characterized by hypoxaemia, pulmonary infiltrates, increased microvascular permeability and endothelial barrier disruption.1 Among many possible mediators for ALI, nitric oxide (NO), reactive oxygen species and pro-inflammatory cytokines have been implicated in the pathogenesis of sepsis-associated lung injury. Recent evidence indicates that large amounts of NO produced by inducible NO synthase (iNOS) react rapidly with the superoxide radical to form peroxynitrite (ONOO), which has been found in the early stages of endotoxin shock and is proposed to play an important role in the pathogenesis of sepsis-associated lung injury and ARDS.2 3 Propofol is known to be a peroxynitrite and free-radical scavenger.4 5 Treatment with propofol after endotoxin injection reduced the production of inflammatory cytokines and mortality rates in rats.6 Propofol may therefore be able to attenuate endotoxin-induced ALI. The current study was undertaken to clarify the effects of treatment with propofol on endotoxin-induced ALI.


    Methods and results
 Top
 Abstract
 Introduction
 Methods and results
 Comment
 References
 
The current study was approved by the Animal Care Committee of Tongji Medical College and was performed in accordance with National Institutes of Health guidelines for animal use.

After an i.p. injection of pentobarbital sodium 30 mg kg–1, 76 adult pathogen-free male Wistar rats were randomly assigned to one of five groups: (i) control group (n=8), receiving saline only; (ii) endotoxaemic group (n=17), receiving an i.v. bolus injection of lipopolysaccharide (LPS, O55:B5, Sigma Chemical, St Louis, MO, USA) 8 mg kg–1; (iii) pretreatment group; (iv) simultaneous treatment group and (v) post-treatment group (all n=17). Endotoxin was used as for the endotoxaemic group. Propofol 5 mg kg–1 i.v. bolus followed by infusion at 10 mg kg–1 h–1 up to 5 h after LPS injection was given 1 h before, simultaneously with or 1 h after the injection of LPS in the pretreatment, simultaneous treatment and post-treatment groups, respectively. During the 5 h after LPS injection, survival rates were recorded. The rats were killed 5 h after LPS injection. Lung injury was evaluated by measuring the following parameters: (i) concentrations of nitrites/nitrates (NO2–/NO3–) and tumour necrosis factor (TNF)-{alpha} concentrations in bronchoalveolar lavage fluid (BALF); (ii) protein concentration in BALF (BALFprot), wet-to-dry lung weight ratio (W/D), pulmonary permeability index (PPI) as indicators of high pulmonary vascular permeability; (iii) nitrotyrosine (NT) Western blot and iNOS mRNA expression in lung tissue.

Statistical analyses were performed by one-way ANOVA followed by Bonferroni post hoc test. Comparisons between survival rates were made with Kaplan–Meier analysis. A value of P<0.05 was considered significant.

Compared with the endotoxaemic group, both the pre- and simultaneous treatment groups showed significantly decreased BALF NO2–/NO3– and TNF-{alpha} and BALFprot concentrations, W:D and PPI values (P<0.05) (Table 1), and attenuated endotoxin-induced increased NT expression (Fig. 1) and iNOS mRNA (data not shown). These beneficial effects were blunted in the post-treatment group. In propofol-treated groups, especially the pretreatment group, there was an improvement in 5 h survival rates (P<0.05). Survival rates 5 h after LPS injection were 100%, 35.3%, 88.2%, 76.5% and 58.8% for the control, endotoxaemic, pretreatment, simultaneous treatment and post-treatment groups, respectively.


View this table:
[in this window]
[in a new window]
 
Table 1 Lung injury parameters 5 h after injection of lipopolysaccharide. Data are mean (SD) (n=6 for each group). *P<0.05 vs control group; {dagger}P<0.05 vs endotoxaemic group; BALF, bronchoalveolar lavage fluid; TNF, tumour necrosis factor
 


View larger version (15K):
[in this window]
[in a new window]
 
Fig 1 Western blot analysis for nitrotyrosine. Lanes are as follows: l, endotoxaemic group; 2, pretreatment group; 3, simultaneous treatment group; 4, post-treatment group; 5, control group. The intensity of the bands is greater in the endotoxaemic group compared with the propofol treatment groups. A prominent band of nitrotyrosylated protein is demonstrated at approximately 60 kD.

 

    Comment
 Top
 Abstract
 Introduction
 Methods and results
 Comment
 References
 
There is evidence that the toxicity of NO is primarily mediated by the highly cytotoxic agent peroxynitrite. Large amounts of NO produced by iNOS react rapidly with the superoxide radical to form peroxynitrite, which has been proposed to play a significant role in cellular damage associated with overproduction of NO. The peroxynitrite reacts with protein tyrosine residues to form NT, a stable oxidation product. This reaction can alter cell signalling processes and plays an important role in the mechanism of apoptosis and subsequent barrier dysfuction.2 3 7

Studies in vitro have reported that propofol protects endothelial cells against the toxicity of peroxynitrite and reduces NT in bronchial cells exposed to NO itself.8 9 Our present study demonstrates that, in an endotoxin model, early treatment (including both pre- and simultaneous treatment) with propofol significantly attenuates endotoxin-induced increased expression of lung peroxy nitrite and iNOS mRNA, as well as NO and TNF-{alpha} production. Reduced NO production caused by down-regulation of mRNA expression for iNOS would contribute to a decrease in peroxynitrite generation in our current study. Values of W:D, PPI and BALFprot, which were used as indicators of pulmonary microvascular permeability, were decreased significantly in the pre- and simultaneous-treatment groups. Backman and colleagues10 showed that peroxynitrite infusion into a rabbit lung increased capillary permeability and lung weight, consistent with a pathogenic role for peroxynitrite in lung injury. Our findings suggest that the protective effects of early propofol treatment on endotoxin-induced endothelial barrier dysfuction and high microvascular permeability is caused, at least in part, by suppression of iNOS-NO-peroxynitrite dependent pathway. In contrast, the attenuation of endotoxin-induced ALI is reduced with propofol 1 h after LPS treatment. In addition, it has been reported that attenuation of TNF-{alpha} and NO production may improve the high mortality rate in septic shock. In this study, we present evidence that early treatment of LPS-exposed rats with propofol dramatically improved 5 h survival rates. These findings suggest that the improved mortality is associated with propofol’s suppression of TNF-{alpha} and NO production in animals with endotoxic shock.

Collectively, propofol can inhibit the production of pro-inflammatory cytokines, and appears to be an iNOS inhibitor and a peroxynitrite and free-radical scavenger. Through these mechanisms, propofol may attenuate endotoxin-induced ALI.


    References
 Top
 Abstract
 Introduction
 Methods and results
 Comment
 References
 
1 Fulkerson WJ, MacIntyre N, Stamler J, et al. Pathogenesis and treatment of adult respiratory distress syndrome. Arch Intern Med 1996; 15: 29–38

2 Sittipunt C, Steinberg KP, Ruzinski JT, et al. Nitric oxide and nitrotyrosine in the lungs of patients with acute respiratory distress syndrome. Am J Respir Crit Care Med 2001; 163: 503–10[Abstract/Free Full Text]

3 Gu Z, Ling Y, Cong B. Peroxynitrite mediated acute lung injury induced by lipopolysaccharides in rats. Zhonghua Yi Xue Za Zhi 2000; 80: 58–61[Medline]

4 Kaman S, Demiryurek AT. Propofol is a peroxynitrite scavenger. Anesth Analg 1997; 84: 1127–9[ISI][Medline]

5 Green TR, Bennett SR, Nelson VM. Specificity and properties of propofol as an antioxidant free radical scavenger. Toxicol Appl Pharmacol 1994; 129: 163–9[CrossRef][ISI][Medline]

6 Taniguchi T, Kanakura H, Yamamoto K. Effects of post-treatment with propofol on mortality and cytokine responses to endotoxin-induced shock in rats. Crit Care Med 2002; 30: 904–7[CrossRef][ISI][Medline]

7 Blaylock MG, Cuthbertson BH, Galley HF, et al. The effect of nitric oxide and peroxynitrite on apoptosis in human polymorphonuclear leukocytes. Free Radic Biol Med 1998; 25: 748–52

8 Mathy-Hartert M, Mouithys-Mickalad A, Kohnen S, et al. Effects of propofol on endothelial cells subjected to a peroxynitrite donor (SIN-1). Anaesthesia 2000; 55: 1066–71[CrossRef][ISI][Medline]

9 Huang CJ, Slovin PN, Nielsen RB, et al. Diprivan attenuates the cytotoxicity of nitric oxide in cultured human bronchial epithelial cells. Intensive Care Med 2002; 28: 1145–50[CrossRef][ISI][Medline]

10 Beckman DL, Mehta P. Effect of peroxynitrite on fluid accumulation in the isolated perfused rabbit lung. Med Sci Res 1999; 27: 147–8[ISI]