Tuberc Respir Dis.  2006 Apr;60(4):437-450. 10.4046/trd.2006.60.4.437.

Pretreatment of Diltiazem Ameliorates Endotoxin-Induced Acute Lung Injury by Suppression of Neutrophilic Oxidative Stress

• Jang YS
• Lee YM
• Ahn WS
• Lee SC
• Kim KC
• Hyun DS

• KMID: 1719369
• DOI: http://doi.org/10.4046/trd.2006.60.4.437

Abstract

BACKGROUND: Acute respiratory distress syndrome (ARDS) is characterized by severe inflammatory pulmonary edema of unknown pathogenesis. To investigate the pathogenesis of ARDS associated with neutrophilic oxidative stress, the role of phospholipase A2 (PLA2) was evaluated by the inhibition of calcium channel.
METHODS
In Sprague-Dawley rats, acute lung injury (ALI) was induced by the instillation of E.coli endotoxin (ETX) into the trachea. At the same time, diltiazem was given 60 min prior to tracheal instillation of ETX. Parameters of ALI such as lung and neutrophil PLA2, lung myeloperoxidase (MPO), BAL neutrophils, protein, surfactant were measured. Production of free radicals from neutrophils was measured also. Morphological studies with light microscope and electron microscope were carried out and electron microscopic cytochemistry for detection of free radicals was performed also.
RESULTS
Diltiazem had decreased the ALI parameters effectively in ETX given rats and decreased the production of free radicals from neutrophils and lung tissues. Morphological studies denoted the protective effects of diltiazem.
CONCLUSION
Diltiazem, a calcium channel blocker, was effective in amelioration of ALI by the suppression of neutrophilic oxidative stress mediated by PLA2 activation.

Keyword

ARDS; PLA2; Free Radicals; Neutrophils

MeSH Terms

Acute Lung Injury*
Animals
Calcium Channels
Diltiazem*
Free Radicals
Histocytochemistry
Lung
Neutrophils*
Oxidative Stress*
Peroxidase
Phospholipases A2
Pulmonary Edema
Rats
Rats, Sprague-Dawley
Respiratory Distress Syndrome, Adult
Trachea
Calcium Channels
Diltiazem
Free Radicals
Peroxidase
Phospholipases A2

Figure

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  Figure 1 Changes in PLA2 activity in the lung. Comparing with control rats, ETX-treated rats show higher (P<0.001) PLA2 activity in the lung, however the PLA2 activity was down to the control level by the effect of diltiazem (P<0.001). Number of experiments are in the parentheses. *P<0.001, Control vs ETX #P<0.001, ETX vs ETX+diltiazem ETX; endotoxin-treated ETX+diltiazem; Diltiazem was treated prior to endotoxin

• 

  Figure 2 Comparison of PLA2 activity in the isolated human neutrophils. Endotoxin had increased (P<0.01) the production of oxidants from neutrophils compared with that of non-stimulated neutrophils. In contrast, pretreatment of diltiazem had decreased (P<0.01) the production of oxidants from endotoxin- charged neutrophils. Number of experiments are in the parentheses. *P<0.01, Control vs ETX #P<0.01, ETX vs ETX+diltiazem ETX; endotoxin-treated ETX+diltiazem; Diltiazem was treated prior to endotoxin

• 

  Figure 3 Changes in MPO activity in endotoxin and diltiazem treated rats. Endotoxin had increased (P<0.001) the pulmonary MPO activity which was reversed (P<0.001) by pretreatment of diltiazem before endotoxin insult in rats. Number of experiments are in the parentheses. *P<0.001, Control vs ETX #P<0.001, ETX vs ETX+diltiazem ETX; endotoxin-treated ETX+diltiazem; Diltiazem was treated prior to endotoxin

• 

  Figure 4 Comparison of the degree of neutrophilic migration into the alveolar lumen in rats. Comparing with control rats, ETX-treated rats showed increased (P<0.001) migration of neutrophils into alveolar lumen denoted in the increased number of neutrophils in BAL fluid. In contrast, pretreatment of diltiazem before endotoxin charge had decreased (P<0.001) the migration of neutrophils into alveolar lumen denoted in the decreased number of neutrophils in BAL fluid compared with that of endotoxin-treated rats. Number of experiments are in the parentheses. *P<0.001, Control vs ETX #P<0.001, ETX vs ETX+diltiazem ETX; endotoxin-treated ETX+diltiazem; Diltiazem was treated prior to endotoxin

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  Figure 5 The effect of diltiazem on the generation of oxidants from isolated neutrophils. By stimulation of neutrophils by PMA, there was significant increased production of oxidants (P<0.001), which was down to significant lower level (P<0.001) by diltiazem. Number of experiments are in the parentheses. *P<0.001, Control vs ETX #P<0.001, ETX vs ETX+diltiazem ETX; endotoxin-treated ETX+diltiazem; Diltiazem was treated prior to endotoxin

• 

  Figure 6 Effect of diltiazem on the lung leak caused by endotoxin. Endotoxin had increased (P<0.01) lung leak denoted by the protein content (mg/ two lungs) in BAL compared with that of control rats. Diltiazem decreased (P<0.001) the lung leak denoted by the decreased content of BAL protein compared with that of endotoxin-treated rats. Number of experiments are in the parentheses. **P<0.01, Control vs ETX #P<0.001, ETX vs ETX+diltiazem ETX; endotoxin-treated ETX+diltiazem; Diltiazem was treated prior to endotoxin

• 

  Figure 7 The changes in the amount (µmol/ two lungs) of pulmonary surfactant in BAL. Endotoxin had increased (P<0.001) the secretion of phospholipid compared with that of control rats. Pretreatment of diltiazem decreased the secretion of phospholipid (P<0.01) in endotoxin-charged rats. Number of experiments are in the parentheses. *P<0.001, Control vs ETX ##P<0.01, ETX vs ETX+diltiazem ETX; endotoxin-treated ETX+diltiazem; Diltiazem was treated prior to endotoxin

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  Figure 8 Photographs of BAL-cytospinned film. 5h after in saline insufflated rats the compositions of cellular components in BAL were almost monocytes (a). In contrast, the cellular components were almost neutrophils in ETX-treated rats (b). In diltiazem and ETX-treated rats, the cellular components of BAL fluid were monocytes and neutrophils and they were intermingled (c). Bar indicates 50µm.

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  Figure 9 Histological changes in the lungs of rats given ETX and diltiazem with ETX. In control rats, alveolar septa and lumen are well preserved (a). 5h after ETX challenge, perivascular edema (asterik) and migration of inflammatory cells (arrow head) were observed. Atelectasis was found also(A, b) (b). Hyaline membrane was also found in alveolar lumen(h, 9-b). However, in rats given diltiazem and ETX, alveolar septa and lumen were relatively well preserved and pervascular edema was minimal (arrow head, c.) Bar indicates 50 µm.

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  Figure 10 Ultrastructural changes of the lung in endotoxin and endotoxin with diltiazem treated rats. Well preserved alveolar type II cells and type I cell were shown (a). After ETX challenge, there were interstitial edema (asterik), thinning of basement membrane (arrow head) and endothelial edema (arrow) were noted. Particularly, hypertrophy and vacuolization of lamellar bodies were prominent in alveolar type II cell (b). Bar indicates 2µm. In contrast, pretreatment of diltiazem reduced interstitial edema. And lamellar structure of the alveoar type II cells were relatively well preserved also (c). Adhesion of neutrophils were noted(arrow head in ETX-treated rats). but surrounding structures were relatively intact(10-c). Bar indicates 3µm. T2 : alveolar type II pneumocyte

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  Figure 11 Detection of hydrogen peroxide with CeCl3 cytochemical electron microscopy. In control and diltiazem with endotoxin treated rats, few cerrous perhydroxide deposits were found (a,c), however, dense deposits of cerrous perhydroxide granules were found along the alveolar lining (arrow head) of ETX-treated rats. Bar indicates 3µm.

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