<% vol = 15 number = 1 nextlink = 38 prevlink = 29 titolo = "AN EXPERIMENTAL STUDY OF THE ROLE OF CELLULAR APOPTOSIS AND ITS MECHANISM IN PULMONARY INJURY IN SCALDED RATS" volromano = "XV" data_pubblicazione = "March 2002" header titolo %>

Fusheng Shi1, Liu Xusheng2, Di Guiping, Yang Zhengguo1, Luo Xiangdong2, Yang Zongcheng2

1 Department of Burns, 322nd Hospital of PLA, Datong, 037006, People’s Republic of China
2 Institute of Burns Research, South-western Hospital, Third Military Medical University, Chongqing, People’s Republic of China

SUMMARY.Our aim in this study is to explore the role and mechanism of gene modulation of cell apoptosis in pulmonary injury in scalded rats. Apoptosis of pulmonary parenchymal cells in scalded rats was observed with the TUNEL method. The gene expressions of Fas/FasL, NF- k B/I k B, and BcL-2 and the expression of VE-calcium adhesion molecules in pulmonary tissue and post-burn pulmonary permeability were observed by the immunohistochemistry technique. It was found that apoptosis occurred in the pulmonary tissue cells of scalded Wistar rats. In particular, apoptosis was more obvious in alveolar epithelia and pulmonary vascular endothelia than in other cells. Pulmonary permeability in scalded rats was evidently increased when measured by 99Tm labelled albumin. Simultaneously, the expression of VE-calcium adhesion molecules of pulmonary vascular endothelia decreased, and this was well correlated with pulmonary cellular apoptosis. In the pulmonary tissue of scalded rats, the expression of Fas/FasL was markedly up-regulated and that of Bcl-2 was down-regulated. Also, the increased expression of Fas/FasL was correlated with the increase of pulmonary tissue cellular apoptosis. The expression of NF- k B/I k B was in accordance with that of Fas/FasL. We conclude that apoptosis occurred in the alveolar epithelia and pulmonary vascular endothelia of scalded rats, which might be related to the pathogenesis of pulmonary injury and increased pulmonary microvascular permeability and inter-endothelial conjunction injury in scalded rats. The activation of pulmonary tissue Fas/FasL, NF- k B/I k B, and Bcl-2 systems in scalded rats might participate in post-burn pulmonary injury and the signal transduction of pulmonary tissue cell apoptosis.


Post-burn multiple organ dysfunction is one of the major causes of death in severely burned victims and its pathogenesis is complicated. In recent years there has been an ever-increasing number of studies of the relationship between apoptosis and organ injury. However, the role of apoptosis is still unclear, as also the part it plays in the pathogenesis of post-burn organ injury. In this study we therefore studied pulmonary microvascular permeability, cell apoptosis, vascular endothelial injury, the expression of VE-cadherin, and the expression of the Fas/FasL, NF- k B/I k B, and Bcl-2 system in scalded rat pulmonary tissue, with a view to exploring the role of cell apoptosis in the pathogenesis of pulmonary injury in scalded rats and finding new ways of managing post-burn organ injury.

Materials and methods

  1. Materials. The apoptosis test kit came from the BM Co., Germany. The immunohistochemical staining SP test kit was from Beijing Zhongshan Biological Technique Co. The rabbit anti-human multiple cloned Fas antibody (C-20, SC-715) FasL antibody (N-20, SC-834), multiple cloned and antibodies NF- k B, I k B, Bcl-2 were all from American Santa Cruz Co. The single antibody for VE-cadherin was from Sigma Co. The Real Color Image Analyzer was made by the Leica Co., USA (model MD20). A laser confocal electronic scanner was used.
  2. Animals and grouping. Thirty healthy Wistar rats weighing 180-220 g were provided by the experimental animal centre of the Third Military Medical University. The rats were employed as control and were randomly divided into a normal control group (C) and 3 post-burn hour (PBH), 6 PBH, 12 PBH, and 24 PBH groups. The rats were housed for one week before the experiment.
  3. Determination of pulmonary microvascular permeability in scalded rats. The rats in S groups were catheterized via femoral vein and injected with 0.2 ml 99Tm-labelled stannite albumin at dose of 80-100 uCi (from China Academy of Atomic Energy). The rats were sacrificed by decapitation 30 min later and the internal organ tissue was harvested and weighed before counting its count per minute cpm for 30 sec in a gamma-radiometry. The amount of injected radioactive albumin was recorded in terms of its cpm value. The cpm value in every 100 mg of organ tissue was recorded and the ratio of the cpm of organ tissue to total injected amount was employed to reflect pulmonary microvascular permeability.
  4. The modified TUNEL method was employed for in situ detection of pulmonary cellular apoptosis.1 Apoptotic cells were counted with a laser confocal electronic scanner. The percentage of apoptotic cells under high-power field (x 400) was graded to 5 grades: 0: < 2%; 1: 3-9%; 2: 10-19%; 3: 20-39%; 4: > 40%.
  5. The immunohistochemical staining of Fas/FasL, NF- k B/I k B, and Bcl-2 VE-cadherin was processed with reference to the test kit. IHC staining was carried out with normal rabbit serum or 0.0l mol/l PBS as direct antibody. The image was analysed with a Real Color Image Analyzer 9MD20, Leica Co., Germany. The BMP images were taken under a microscope (x 200) with a grey degree of 50-250. The average grey-degree value of each slide was obtained with 10 images.

Statistical analysis

The SPSS program package was employed in the data processing. The data were expressed as mean ± standard deviation (* = p < 0.05,** = p < 0.01).


1. Changes and distribution of cellular apoptosis of pulmonary parenchyma in scalded rats. These are shown in Table I. Alveolar epithelia and pulmonary vascular endothelia were the main positive staining cells by the TUNEL method. There were signs of apoptosis in some other mediator cells and pulmonary inflammatory cells. Cellular apoptosis appeared in pulmonary tissue at PBH 3 (apoptotic rate, 2-3%; mean value, 2%). The number of apoptotic cells increased with the passing of post-burn time (Table II). Cellular apoptosis was rarely found in normal pulmonary tissue (apoptotic rate, 0-1%).

<% createTable "Table I","Grading of pulmonary cellular apoptosis in scalded rats",";Group;Sample amount (n);Grading of apoptosis@;3 PBH;6;1 (0-1)*@;6 PBH;6;2 (1-2)*@;12 PBH;6;2 (2-3)**@;24 PBH;6;3 (2-3)**@;Control;6;0","PBH = post-burn hour",4,300,true %> <% createTable "Table II","Dynamic changes in pulmonary permeability at different post-burn times in scalded rats (¯ ± s, cpm/100 mg tissue)",";PBH;0;3;6;12;24@;Cpm;l.95 ± 0.32;4.90 ± 0.25;6.70 ± 0.90;9.07 ± 0.41;9.87 ± 1.45","'PBH = post-burn hour; Cpm: count per minute'",4,300,true %>

2. Pulmonary microvascular permeability and the expression of VE-cadherin (Table III). Permeability was expressed by cpm per 100 mg tissue; it increased with the passing of post-burn time and correlated positively with the grading of pulmonary cellular apoptosis (r = -0.89). The expression of VE-cadherin decreased and was negatively correlated with the grading of pulmonary cellular apoptosis (r = -0.87).

<% createTable "Table III","Expression of pulmonary VE-cadherin in scalded rats (¯ ± s)","; Control;6 PBH;12 PBH;24 PBH@;Area;520.1 ± 10.3;369.8 ± 12.1**;298.5 ± 11.6**;211.2 ± 16.l**@;IOD;258 3 ± 11.2;211.4 ± 9.3**;111.3 ± 10.8**;120.1 ± 10.1**","'Area = expression area (Ìm2); IOD = expression intensity (OD value)';** Post-burn values were compared with control values (p < 0.0l)",4,300,true %>

3. Changes in the expressions of pulmonary cellular Fas/FasL and Bcl-2 in scalded rats (Table IV). The expression of Fas antigen (Ag) in pulmonary tissue cells was up-regulated. The positive cells were mainly distributed in pulmonary epithelia and alveolar vascular endothelia. Compared with that in control group, the expression of Fas Ag in scalded groups was clearly higher (p < 0.0l), as also that of FasL Ag. However, the expression of Bcl-2 in scalded rats was evidently lower than that in control groups (Figs. 1-3).

<% createTable "Table IV","Changes in expressions of pulmonary Fas, FasL, and Bcl-2 in scalded rats (¯ ± s)","; Number§1,2§Fas§1,2§FasL§1,2§BCl-2@;  Control;Scald;Control;Scald;Control;Scald@;Area;6;264.2 ± 12.3;837.2 ± 26.1;212.2 ± 2.1;617.3 ± 45.5**; 313.2 ± 5.1;117.3 ± 5.5**@;IOD;6;102.3 ± 11.2;212.1 ± 35. 1*;100.1 ± 3.1;163.1 ± 13.5**;210.1 ± 8.3;131.1± 3.5**","",4,300,true %>
<% immagine "Fig. 1","gr0000012.jpg","The change in expression of Fas antigen (Ag) in pulmonary tissue cells was up-regulated in scalded rats compared with control.",230 %> <% immagine "Fig. 2","gr0000013.jpg","The change in expression of FasL antigen (Ag) in pulmonary tissue cells was up-regulated in scalded rats compared with control.",230 %>
<% immagine "Fig. 3","gr0000014.jpg","The change in expression of BCl-2 antigen (Ag) in pulmonary tissue cells was down-regulated in scalded rats compared with control.",230 %>

4. Changes in the expressions of pulmonary tissue cellular, NF- k B/I k B in scalded rats (Table V). The expression of pulmonary tissue cellular NF- k B was manifestly up-regulated in scalded rats. The positive cells were predominantly coated in pulmonary epithelia and alveolar vascular endothelia. The expression in scalded rats was significantly higher than that in control groups (p < 0.0l), as also that of I k B (Figs. 4,5).

<% createTable "Table V","Changes in expression of pulmonary NF- k B and I k B in scalded rats (¯ ± s)","; Number§1,2§NF- k B§1,2§I k B@;  Control;Scald;Control;Scald@;Area;6;214.2 ± 12.3;837.2 ± 26.1**;212.2 ± 2.1;617.3 ± 45.5**@;IOD;6;102.3 ± 11.2;212.1 ± 35.1**;100.1 ± 3.1;163.1 ± 13.5**","",4,300,true %>
<% immagine "Fig. 4","gr0000015.jpg","The change in expression of pulmonary tissue cellular NF- Í B was manifestly up-regulated in scalded rats.",230 %> <% immagine "Fig. 5","gr0000016.jpg","Changes in expression of pulmonary tissue cellular I Í B in scalded rats. The expression of pulmonary tissue cellular I Í B was manifestly down-regulated in scalded rats.",230 %>


1. - Pulmonary tissue cellular apoptosis plays roles in early pulmonary injury in scalded rats. This was indicated by the decrease in the expression of endothelial VE-cadherin along with the increase of pulmonary apoptotic cells, the widening of inter-endothelial conjunction, and the increase of pulmonary microvascular permeability in scalded rats. Apoptosis, also known as programmed cell death, is a highlight of recent studies. Apoptosis and death are two basic patterns of cell death. There are essential differences in morphology, cell death process, and induced inflammation between the two patterns. According to previous theories, histocytes suffered from necrosis induced by ischaemia, hypoxia, and secondary injury during severe trauma, shock, and acute injury. There have been several studies indicating that cell apoptosis not only plays important roles in cellular selection and differentiation and in the scavenging of senile cells in the physiological state but also participates in the pathogenesis of many diseases. The roles of histocyte apoptosis in the pathogenesis of post-burn internal organ injury have received increasing attention.2,3

This study indicates the occurrence of apoptosis of pulmonary vascular endothelia and alveolar epithelia in scalded rats. In addition to the increase of apoptotic cells, pulmonary vascular permeability increased, as also the expression of endothelial VE-cadherin, which suggested that the apoptosis of pulmonary alveolar epithelia and vascular endothelia might participate in the pathogenesis of pulmonary injury in scalded rats. The increase of microvascular permeability was usually due to injury to the inter-endothelial conjunction and to vascular endothelial injury. Conversely, the increase in vascular permeability has also been regarded as the result of endothelial contraction due to an increment in intra-endothelial calcium concentration. However, recent studies suggested that endothelial apoptosis was one of the main factors leading to the increase of vascular permeability. The factors that could induce cell apoptosis have been recognized to be: 1. bacterial infection; 2. cytokines; 3. stress reaction; and 4. ischaemia and reperfusion. Sepsis induced by bacterial infection is one of the commonest causes of the multiple organ dysfunction syndrome (MODS). LPS is a key activator of the systemic inflammatory response syndrome. Various studies have indicated that LPS and sepsis could induce apoptosis in many cells.4-7 The endothelium is an active participator in the inflammatory reaction. It possesses many important biological functions and its apoptosis may not only induce the increase in vascular permeability but also cause alterations of other vascular functions.

In the experiment performed by Abello et al.,8 porcine aorta endothelium was cultured in vitro and stimulated with LPS. The hot-shock protein reaction was then induced, producing apoptosis but not necrosis. Zhang et al.4 found endothelial apoptosis after in situ hybridization in rats after injection of LPS. Apoptosis of the endothelium may occur in cases of hot shock and oxidized stress reaction. These considerations suggest that post-burn cellular apoptosis participates in the injury of multiple organs.

In this study, the expression of VE-cadherin of pulmonary vascular endothelia was attenuated, together with the apoptosis of pulmonary histocytes. Ultrastructural examination indicated pulmonary endothelial swelling or cytolysis, and broadening and separating of the perinuclear gap, karyopyknosis, margination of heterochromatin, and widening of inter-endothelial conjunction in scalded rats. Between the endothelia are the tight junction, adhesion junction, gap junction, and syndesmosis, of which the adhesion junction or zonular adherent is the most important inter-endothelial junction.8 The adhesion junction is a kind of cytoplasmic junction mediated by cadherin; it is also a junction between cytoplasm and the net structure composed of intracellular cytoplasmic protein and actin filament, and its structural change can cause alteration of the cytoskeleton structure and lead to endothelial deformity. All these procedures could modulate the exudation of macromolecular matter and blood cells. Cadherin or B412 is a special adhesion molecule of endothelia and also essential matter of the intercellular junction. Endothelial injury could not only cause exudation of large amounts of fluid to interstitial space, enlarge the gaps between capillary and tissue, and aggravate tissue cellular hypoxia but also lead to massive accumulation of inflammatory cells in local tissue and directly lead to injury of tissue cells.

2. - Gene modulation of cell apoptosis in pulmonary parenchymal injury in scalded rats. The Fas/FasL system is one of the important signal passages in cell apoptosis. Previous studies have indicated that Fas MRNA and protein could be expressed on type II alveolar epithelia and immunological epithelia, and that this was correlated with apoptosis. This suggested that Fas-dependent apoptosis participated in alveolar renewal and restoration and in the modulation of the systemic inflammation reaction. This study indicates that Fas antigen was only weakly expressed in the pulmonary tissue of normal rats. On the other hand, the expression of Fas antigen was manifestly up-regulated in the pulmonary tissue cells of scalded rats (p < 0.01). There was almost no expression of FasL in normal pulmonary tissue, while the expression was clearly enhanced in the pulmonary tissue cells of scalded rats (p < 0.05). The increment of Fas and FasL expressions was paralleled by the increment of pulmonary tissue cell apoptosis, which suggested that activation of the Fas/FasL system might participate in the pathogenesis of pulmonary tissue cellular injury in scalded rats. It is reasonable to assume that the Fas/FasL system participated in pulmonary tissue cell apoptosis by means of following mechanism: 1. the expression of Fas antigen on target cells was up-regulated;9 the FasL expression of lymphocytes infiltrated in the lungs was up-regulated, inducing the combination of Fas and FasL antigens on target cells to form compounds and thus induce cell apoptosis. However, the physiological cell death induced by FasL was characterized by short distance, high concentration, and a short half-life period.100 It was thus difficult to explain the apoptosis of tissue cells that were far away from lymphocytes. Recent studies have indicated that FasL MRNA could be expressed in epithelia11 and that this might also be related to the immunological privilege and immunological homeostasis of the host.12,13 The present study indicates that FasL could be expressed in partial alveolar epithelia. Consequently, we believe that apart from the above mechanism, the participation of the Fas/FasL system in pulmonary cell apoptosis in scalded rats is also related to the cell apoptosis induced by the auto-expression of FasL in pulmonary epithelia. However, the Fas/FasL system is not a unique pattern of cell apoptosis in this pathological period. It has been shown in previous studies that the TNF-·, Bcl-2/Bax system (another pair of cell apoptosis related genes) and 53P could induce apoptosis and participate in the process of target cell injury in systemic uncontrolled inflammatory response, which suggests a sophisticated mechanism of pulmonary tissue cell apoptosis in scalded rats.14-16

Previous studies have indicated that cell apoptosis participates in pulmonary tissue reconstruction after injury,17 which suggests that apoptosis plays different roles in different periods of pulmonary tissue cells in scalded rats. Further studies on apoptosis of pulmonary tissue cells would help to explore the molecular mechanism of the gene modulation of the apoptosis of pulmonary tissue cells in scalded rats and further clarify the pathogenesis of post-burn MOF at cellular level. Nuclear Factor-KB (NF-KB) is a general name for proteins that could specifically bind KB sites located in the sites of initiators of multiple genes and promote transduction.18

It has been widely recognized that there are KB sites in the initiator foci of many genes of cytokines and adhering molecules related closely to inflammation and immune reaction. In addition, it has been shown in in vivo and in vitro experiments that the activation of NF-KB is correlated with over-expression of the above factors. The characteristics of post-burn pulmonary injury are neutrophil infiltration in pulmonary tissue induced by numerous pathogenic factors and over-expression of cytokines such as TNF, IL-1, IL-6, and IL-8 and of adhering molecules such as ICAM-1 and VCAM-1, accompanied by the production of multiple inflammatory mediators and spread injury of pulmonary tissue. Many internal organs other than the lungs are involved and ARDS and MODS develop. The correlation between post-burn pulmonary injury and NF-KB is involved owing to the important role of NF-KB in regulating the gene expression of multiple inflammatory factors. Generally speaking, intracellular NF-KB is in an inactive state without the ability to regulate transduction when there is no stimulation of the cells. At the same time, intracellular NF-KB combines with the inhibiting protein IKB and is trapped within the cytoplasm.

When the cells were stimulated by an extracellular signal, phosphorylation and degradation occurred. As a result, liberated NF-KB transferred quickly from the cytoplasm to the nucleus and thus enhanced transduction of some related genes. The present study suggests that there are enhanced expressions of NF-KB and IKB in post-burn pulmonary tissue cell apoptosis, which might participate in the modulation of cell apoptosis. This could provide new ideas at molecular level for the management of post-burn pulmonary tissue cell injury.

RESUME. Le but des Auteurs était d’explorer le rôle et le mécanisme de la modulation de l’apoptose des cellules dans les lésions pulmonaires des rats exposés à l’ébouillantement. L’apoptose des cellules parenchymales pulmonaires dans les rats ébouillantés a été observée utilisant la méthode TUNEL. La technique immunohistochimique a été utilisée pour observer les expressions des gênes de Fas/FasL, NF- k B/I k B, et BcL-2, comme aussi l’expression des molécules d’adhésion du calcium-VE dans les tissus pulmonaires après la brûlure. Les Auteurs ont trouvé que l’apoptose se vérifiait dans les cellules des tissus pulmonaires des rats Wistar exposés à l’ébouillantement. En particulier, l’apoptose était plus évidente dans l’épithélium alvéolaire et l’endothélium vasculaire pulmonaire par rapport aux autres cellules. La perméabilité pulmonaire dans les rats exposés à l’ébouillantement augmentait en manière évidente avec le mesurage moyennant l’albumine étiquetée avec 99Tm. En même temps l’expression de la molécule de l’adhésion du calcium-VE de l’endothélium vasculaire pulmonaire diminuait, étant bien corrélée avec l’apoptose cellulaire pulmonaire. Dans les tissus pulmonaires des rats exposés à l’ébouillantement, l’expression de Fas/FasL était notamment augmentée, tandis que celle de Bcl-2 diminuait. En outre, l’expression augmentée de Fas/FasL était corrélée avec l’augmentation de l’apoptose cellulaire des tissus pulmonaires. L’expression de NF- k B/I k B était en accord avec celle de Fas/FasL. Les Auteurs concluent que l’apoptose se vérifiait dans l’épithélium alvéolaire et l’endothèle vasculaire pulmonaire des rats exposés à l’ébouillantement, ce qui pourrait être lié à la pathogenèse de la lésion pulmonaire et de la perméabilité microvasculaire pulmonaire augmentée et à la lésion de conjonction inter-endothéliale dans les rats exposés à l’ébouillantement. L’activation du tissu pulmonaire Fas/(FasL, NF- k B/I k B et BcL-2 et des systèmes Bcl-2 dans les rats exposés à l’ébouillantement pourrait participer à la lésion pulmonaire après la brûlure et à la transduction du signal de l’apoptose des cellules tissulaires pulmonaires.


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<% riquadro "This paper was received on 9 October 2001.

Address correspondence to: Dr. Fusheng Shi, Departement of Burns, 322nd Hospital of PLA, Datong, 037006, People's Republic of China. " %>

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