Protecting effects of dexamethasone on thymus of rats with severe acute pancreatitis.

Abstract

To study the protecting effects of dexamethasone on thymus of rats with severe acute pancreatitis (SAP).
The SAP rats were randomly assigned to the model group and dexamethasone-treated group, the other normal healthy rats were assigned to the sham operation group. The rat survival, thymus pathological changes, apoptotic index, as well as expression levels of NF-kappaB, P-selectin, Bax, Bcl-2, and Caspase-3 protein of all groups were observed, respectively, at 3 hours, 6 hours, and 12 hours. The contents of amylase and endotoxin in plasma as well as the contents of TNF-alpha, PLA2, and NO in serum were determined.
There was no marked difference between the model group and treated group in survival. The contents of different indexes in blood of treated group were lower than those of the model group to various degrees at different time points. The thymus pathological score was lower in treated group than in model group at 12 hours. The treated group in Caspase-3 protein expression of thymus significantly exceeded the model group at 12 hours. The apoptotic index was significantly higher in treated group than in model group.
Dexamethasone has protecting effects on thymus of SAP rats.

Full text

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Hindawi Publishing Corporation
Mediators of Inflammation
Volume 2007, Article ID 72361, 9 pages
doi:10.1155/2007/72361
ResearchArticle
Protecting Effects of Dexamethasone on Thymus of
Rats with Severe Acute Pancreatitis
Zhang Xiping,1Chen Li,2Lin Miao,3and Tian Hua2
1Department of General Surgery, Hangzhou First People’s Hospital, Hangzhou 310006, Zhejiang Province, China
2Department of General Surgery, Second Affiliated Hospital Medical College, Hangzhou 310003, Zhejiang Province, China
3Zhejiang University of Traditional Chinese Medical, Hangzhou 310053, Zhejiang Province, China
Correspondence should be addressed to Zhang Xiping, zxp99688@vip.163.com
Received 4 September 2007; Accepted 5 November 2007
Purpose.Tostudytheprotectingeffectsofdexamethasoneonthymusofratswithsevereacutepancreatitis(SAP).Methods.TheSAP
rats were randomly assigned to the model group and dexamethasone-treated group, the other normal healthy rats were assigned
to the sham operation group. The rat survival, thymus pathological changes, apoptotic index, as well as expression levels of NF-κB,
P-selectin, Bax, Bcl-2, and Caspase-3 protein of all groups were observed, respectively, at 3 hours, 6 hours, and 12 hours. The
contents of amylase and endotoxin in plasma as well as the contents of TNF-α, PLA2, and NO in serum were determined. Results.
There was no marked difference between the model group and treated group in survival. The contents of different indexes in blood
of treated group were lower than those of the model group to various degrees at different time points. The thymus pathological
score was lower in treated group than in model group at 12 hours.The treated group in Caspase-3 protein expression of thymus
significantly exceeded the model group at 12 hours. The apoptotic index was significantly higher in treated group than in model
group. Conclusion. Dexamethasone has protecting effects on thymus of SAP rats.
Copyright © 2007 Zhang Xiping et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
1.INTRODUCTION
Acute pancreatitis (AP) especially severe acute pancreatitis
(SAP) is a dangerous acute abdomen among diseases of di-
gestive system. In its early stage, a big amount of inflam-
matory mediators will be released and activated to cause
excessive immune response, resulting in cascade inflamma-
toryinjuryorinseverecasessystemicinflammatoryresponse
syndrome (SIRS) and multiple organ dysfunction syndrome
(MODS) [1–3]. As a basic physiological mechanism of body
to maintain normal morphous and function, apoptosis has
been found in multiple organs in systemic SAP complica-
tions [4–6]. At present, the sound therapeutic effects of large
dose of dexamethasone on SAP has been demonstrated but
further study still should be conducted for its therapeutic
mechanism. There are still few reports on thymus patho-
logical changes during SAP all over the world [7]. In this
experiment, the inflammatory mediators in blood, thymus
apoptosis, and protein expression of NF-κB, P-selectin, Bax,
Bcl-2, and Caspase-3 upon the onset of rat SAP have been
studiedtodiscusstheprotectingeffectsofdexamethasoneon
SAP complicated thymus injury and its mechanism. The tis-
sue microarray has also been applied to the pathohistological
examination of pancreatitis to improve the study efficiency,
which was first reported in this article around the world.
2.MATERIALS AND METHODS
2.1.Materials
Clean grade healthy male Sprague-Dawley (SD) rat in 250–
300g of body weight purchased from the Experimen-
tal Animal Center of Medical School, Zhejiang University
(Hangzhou, China). Sodium taurocholate and sodium pen-
tobarbital purchased from Sigma-Aldrich (Mo, USA). Dex-
amethasone injection purchased from Zhejiang Xinchang
(Shaoxing, China). The full automatic biochemical ana-
lyzer was used to determine the plasma amylase level (U/L).
Plasma endotoxin tachypleus amebocyte lysate kit was pur-
chased from Shanghai Yihua Medical Science and Technol-
ogy Corporation (Institute of Medical Analysis in Shanghai,
China),thecalculationunitforcontentisEU/mL.Theserum
nitrogen monoxidum (NO) kit was purchased from Nanjing
Jiancheng Bioengineering Research Institute (Shengzhen,
China), the calculation unit is μmol/L. The TNF-α ELISA
kit was purchased from Jingmei Bioengineering Corporation

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2Mediators of Inflammation
(Hangzhou,China),thecalculationunitforcontentispg/mL
(ng/L). The serum secretory phospholipase A2enzyme As-
say ELA kit (PLA2) was purchased from RdD system Ins and
the calculation unit is U/mL. The NF-κB, Bax, Bcl-2, and
P-Selectinantibody were purchased from Santa Cruz Com-
pany (Calif, USA). Caspase-3 antibody was purchased from
NeoMarkers Company (Calif, USA), DNA nick in situ end-
labeling (TUNEL) kit purchased from Takara Company. The
above determinations were all operated according to the in-
structions of the kits.
2.2.Methods
2.2.1.Animalgrouping
90 clean grade healthy male SD rats were prepared into
the SAP models via the improved Aho’s method [8] and
randomly divided into the model group (45 rats) and
dexamethasone-treated group (45 rats). Another 45 were se-
lected to be the sham operation group. In the next step, the
above groups were randomly divided into the 3 hours, 6
hours, and 12 hours groups with 15 rats in each group. The
dexamethasone-treated group was injected with dexametha-
sone injection via vena caudalis, 0.5mg/100g body weight
and single administration 15 minutes after successful prepa-
ration of SAP model. The sham operation group after receiv-
ing abdomen opening performed pancreas and duodenum
turning over and finally abdomen closing. The sham opera-
tion group and model group were injected with the saline of
the same volume via vena caudalis 15 minutes after the oper-
ation [9, 10].
2.2.2. Animalmodelpreparation
Fasting but water restraining were imposed on all rat groups
12 hours prior to the operation. The rats were anesthetized
by intraperitoneal injection of 2% sodium pentobarbital
(0.25mL/100g) after which the rats are laid and fixed, and
routine shaving, disinfection, and draping were performed.
Model group: after entering abdomen via median epigas-
trium incision, confirmed the bile-pancreatic duct and hep-
atichiluscommonhepaticduct,disclosedthepancreas,iden-
tified the duodenal papilla inside the duodenum duct wall,
and then used a No. 5 needle to drill a hole in the mesen-
terium avascular area. After inserting a segmental eqidural
catheter into the duodenum cavity via the hole, inserted into
the bile-pancreatic duct toward the direction of papilla in
a retrograde way, used the microvascular clamp to nip the
catheter head temporarily and meanwhile used another mi-
crovascular clamp to temporarily occlude the common hep-
atic duct at the confluence of hepatic duct. After connect-
ing the eqidural catheter end with the transfusion converter,
transfused 3.5% sodium taurocholate 0.1mL/100g by retro-
grade transfusion via the microinjection pump at the speed
of 0.2mL/min. Stayed for 4 minutes after injection and re-
moved the microvascular clamp and epidural catheter. After
checking for bile leakage, sutured the hole in the duodenum
lateral wall. Used the disinfected cotton ball to absorb up the
Table 1: Pathological score standard of thymus.
Pathological
score
Comparison
ofcortex
and medulla
thickness
Countof
epithelial cell
withnecrosis
in cortex
Countof
epithelial cell
withvacuole
degeneration
in medulla
1
1.5
2
2.5
3
3.5
Cortex is
thicker or
thinner
than or as
thick as
medulla.
1–3
1–3
4–10
4–10
>10
>10
Flaky
necrosis
Flaky
necrosis
1–3
4–5
>5
4
4.5
>5
anesthesia on the abdominal cavity and close the abdomen
[8].
2.2.3. NF-κB,P-selectin,Bax,Bcl-2,and
Caspase-3proteinexpression
Applied tissue microarrays to prepare thymus microar-
ray sections; adopted streptavidin peroxidase (SP) method
for immunohistochemical staining; observed the NF-κB, P-
selectin, Bax, Bcl-2, and Caspase-3 protein expression of thy-
musunderlightmicroscope,respectively,andcarriedoutthe
comprehensive assessment according to the positive cell per-
centage: positive cell count < 10% means (−); positive cell
count 10–20% means (+); positive cell count 20–50% means
(++); positive cell count >50% means (+++) [9].
2.2.4. Apoptoticindex
Applied the tissue microarrays to prepare the thymus mi-
croarray sections; Adopted DNA nick in situ end-labeling
(TUNEL) technology for staining. Observed the thymus
apoptotic cells and calculated apoptotic index, respectively.
The TUNEL staining technique was applied to observe the
changes of thymus apoptotic cells and the apoptotic indexes
were calculated. Apoptotic index = apoptotic cell count/total
cell count × 100% [9].
2.2.5.Pathologicalscorestandardofthymus
There was no pathological score standard of thymus around
world. Therefore, we have made a quantitative scoring stan-
dard according to the thymus pathological changes of differ-
ent experimental groups, see Table 1.

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Zhang Xiping et al.3
2.3.Observationindexes
2.3.1.Survival
Examined the rat mortality at 3 hours, 6 hours, and 12
hours after operation and calculated the survival, observed
the gross changes of thymus.
2.3.2.Pathologicalchanges
After mercy killing, rats anesthetized by sodium pentobarbi-
tal in batches, collected the thymus and fixed them accord-
ing to the related requirements, observed the pathological
changes of thymus after HE staining, and performed thymus
pathological score according to the self made standards see
Table 1.
2.3.3. Differentindexesinblood
The contents of plasma amylase and endotoxin, serum NO,
TNF-α, and PLA2were determined via blood sampling from
heart.
2.3.4. Differentproteinsexpressionandapoptoticindex
To observe NF-κB, P-selectin, Bax, Bcl-2, and Caspase-3 pro-
tein expression and apoptotic index of thymus.
2.4.Statisticalmethods
The statistical analysis was conducted to the arranged ex-
perimental results by applying the SPSS11.5 software. The
Kruskal-Wallis test or variance analysis (only applied to
PLA2)wasapplied tothethreegroupcomparison.TheBonf-
feroni test was also applied to comparison. There are statisti-
cal significances when P < .05.
3.RESULTS
3.1.Survival
Model group: mortality, respectively, was 0% (0/15), 0%
(0/15), and 13.33% (2/15) at 3 hours, 6 hours, and 12 hours
while survival was 86.67% all along. The sham operation
group and dexamethasone-treated group survived at all time
points with 100% survival while there was no marked differ-
ence between the model group and dexamethasone-treated
group (P > .05) [8–10].
3.2.Comparisonofplasmaamylasecontent
The model group and dexamethasone-treated group sig-
nificantly exceeded the sham operation group at all time
points (P < .001). No marked difference between the
dexamethasone-treated groupand model group at 3 hours
and6hours(P >.05).Thedexamethasone-treatedgroupwas
significantly less than the model group at 12 hours (P < .01),
see Table 2.
3.3. Comparisonofplasmaendotoxincontent
The model group and dexamethasone-treated group sig-
nificantly exceeded the sham operation group at all time
points (P < .001). No marked difference between the
dexamethasone-treated group and model group at 3 hours
(P > .05). The dexamethasone-treated group was signifi-
cantly less than the model group at 6 hours and 12 hours
(P < .01), see Table 2.
3.4. ComparisonofserumNOcontent
The model group and dexamethasone-treated group signifi-
cantly exceeded the sham operation group at all time points
(P < .001). The dexamethasone-treated group was signifi-
cantly less than the model group at 3 hours and 12 hours
(P < .01), see Table 2.
3.5. ComparisonofserumTNF-αcontent
The model group and dexamethasone-treated group sig-
nificantly exceeded the sham operation group at all time
points (P < .001). No marked difference between the
dexamethasone-treated group and model group at 3 hours
(P > .05). The dexamethasone-treated group was signifi-
cantly less than the model group at 6 hours and 12 hours
(P < .05), see Table 2.
3.6. ComparisonofserumPLA2content
The model group and dexamethasone-treated group signifi-
cantly exceeded the sham operation group at all time points
(P < .001). The dexamethasone-treated group was signifi-
cantly less than the model group at all time points (P < .001),
see Table 3.
3.7.Pathologicalchangesofthymusunder
lightmicroscopeofallgroups
3.7.1. Shamoperationgroup
Inshamoperationgroup,histologicalfindingsofthymusat3
hours,6hours,and12hoursareconsistent,thymusstructure
is normal, boundary between cortex and medulla is clear,
cortex/medulla thickness ratio is around 2 ∼1 : 1, lobule
visible, envelope is intact, epithelial cell is with “starry sky”
changes scattered in cortex, condensed deep purple-blue cell
nucleus; many slightly stained epithelial reticular cells in star
shapeandwithmultipleprotuberances,withbignucleusand
rich kytoplasm in medulla; medulla structure is more loose
than cortex, few epithelial cell nucleus slightly stained, and
some epithelial cell “vacuole like”.
3.7.2. Modelgroup
In model group, thymus cortex gradually thinned at 3 hours,
6 hours, and 12 hours, “starry sky” like epithelial cells with
fragmented nucleus, fewer lymphocytes, slightly stained ep-
ithelial cell nucleus in medulla, and more chromatin-losing
cells “vacuole like” than in normal group.

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4Mediators of Inflammation
Table 2: Comparison of contents of different indexes in blood (M(QR)).
Groups index
Sham operation
6h
2117
(324)
0.015
(0.007)
15.0
(7.5)
4.90
(2.60)
Model
6h
8149
(1540)
0.055
(0.025)
62.5
(27.5)
77.54
(42.16)
Dexamethasone-treated group
3h6h
6739
(2310)(2258)
0.030
(0.014)(0.012)
50.0
(20)(12.5)
38.40
(26.60) (26.40)
3h
2038
(346)
0.015
(0.007)
10.0
(12.5)
3.30
(3.60)
12h
1725
(434)
0.016
(0.005)
15.0
(10.0)
3.70
(2.30)
3h
7423
(2275)
0.035
(0.017)
72.5
(17.5)
46.13
(37.95)
12h
9195
(1298)
0.059
(0.020)
70.0
(13.75)
67.30
(32.13)
12h
7791
(1863)
0.042
(0.018)
45.0
(17.5)
38.70
(28.5)
Amylase (U/L)
7839
Endotoxin (EU/mL)
0.040
NO (μmol/L)
60.0
TNF-α (ng/L)
58.30
Table 3: Comparison of PLA2in serum (X ±S).
3h
Sham operation
group
Model group
103.69 ±20.82 119.85 ±17.74 121.29 ±17.01
Dexamethasone-
treated group
Groups
6h 12h
18.70 ±4.4016.70 ±3.8318.52 ±11.31
53.96 ±15.4067.75 ± 27.95 65.27 ±26.21
Table 4: Comparison of pathological score of the thymus (M(QR)).
Groups
Sham operation group
Model group
Dexamethasone-treated group
3h 6h12h
0.0 (1.0)
2.0 (0.0)
2.0 (1.5)
0.0 (2.0)
2.5 (2.0)
2.5 (1.0)
0.0 (1.0)
3.0 (1.5)
2.5 (0.5)
3.7.3. Dexamethasone-treatedgroup
In treated group, cortex slightly thinned, more “starry sky”
like cortex epithelial cells than in normal group, much less
lymphocytes, and many “vacuole like” epithelial cells in
medulla.
3.8. Comparisonofthymuspathological
scoresofallgroups
The scores were higher in model group and treated group
than in sham operation groupat different time points (P <
.001). The score was lower in treated group than in model
group at 12 hours (P < .05), see Table 4.
3.9. Changesofdifferentproteinsexpression
The expression of NF-κB, P-selectin, Bax, and Bcl-2 in thy-
mus was negative in all groups at different time points, see
Figure 1.
Figure 1: Treated group: 12 hours; NF-κB × 200.
3.10. Comparisonofthymusapoptosis
countsofallgroups
Lymphocytes were apoptotic cells of thymus. In sham op-
eration group, apoptotic cells were found, respectively, in 2
and 1 rat at 6 hours and 12 hours, and the apoptotic index
was between 10 per 10000 and 24 per 10000 as shown in Fig-
ures 2 and 3. In model group, apoptotic cells were found in
2 rats at 12 hours, and the apoptotic index was between 6
per 10000 and 8 per 10000 as shown in Figure 4. In treated
group, apoptotic cells were found, respectively, in 6, 9, and
8 rats at 3 hours, 6 hours, and 12 hours, and the apoptotic
index was between 2 per 10000 and 76 per 10000 as shown
in Figures 5 and 6. The counts were higher in treated group
than in sham operation group and model group at different
time points (P < .05). There was no marked difference be-
tween model group and sham operation group (P > .05), see
Table 5.
3.11.ComparisionofCaspase-3proteinofthymusof
allgroups
The dexamethasone-treated group significantly exceeded the
sham operation group at different time points (P < .01).
The model group significantly exceeded the sham operation
group at 3 hours and 6 hours (P < .05). The dexamethasone-
treated group significantly exceeded the model group at 12
hours (P < .05), see Tables 6 and 7, and Figures 7–10.

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Zhang Xiping et al.5
Figure 2: Sham operation group: 6 hours; Tunel × 200.
Figure 3: Sham operation group: 12 hours; Tunel × 200.
3.12. Correlationanalysis
The amylase of the model group was positively correlated
with PLA2at 3 hours (P < .05). The amylase of the model
group was positively correlated with NO at 6 hours (P < .05).
The NO of the dexamethasone-treated group was positively
correlated with PLA2(P < .05) and apoptotic indexes were
negatively correlated with PLA2 at 3 hours (P < .05). The
TNF-αcontentofthedexamethasone-treatedgroupwaspos-
itively correlated with PLA2at 6 hours (P < .05). Apoptotic
indexes of the dexamethasone-treated group were negatively
correlated with PLA2at 12 hours (P < .05).
4. DISCUSSIONS
When SAP occurs, pancreatins such as trypsin, pancrelipase,
and amylopsin will be activated and excessively released [11],
resulting in necrosis of pancreas and tissues around it. The
absorbed necrotic tissue and bulk toxic substances will cause
severe systemic inflammatory reaction. The inflammatory
mediators are TNF-α, PLA2, NO, endotoxin, and so on. As
one of the final common mediators in cascade reaction of in-
flammatory mediators [12], NO can be regarded as a study
index for the changes of SAP state. TNF-α which is an im-
portant cytokine participates in SAP inflammatory cascade
reaction [13, 14]. In SAP cases, endotoxin may reach tissues
such as lung and liver due to intestinal mucosa damage [15].
The abnormal release and activation of PLA2are closely re-
Figure 4: Model group: 12 hours; Tunel × 200.
Figure 5: Treated group: 12 hours; Tunel × 200.
Table 5: Comparison of apoptosis index of the thymus (M(QR)).
Group
Sham operation group
Model group
Dexamethasone-treated
group
3h
0.00 (0.00)
0.00 (0.00)
6h
0.00 (0.00)
0.00 (0.00)
12h
0.00 (0.00)
0.00 (0.00)
0.00 (0.06) 0.06 (0.12)0.02 (0.30)
lated with SAP severity, and the PLA2inhibitor can improve
the pathological changes of SAP [16, 17]. The activation of
NF-κB, which is a transcription factor participating in the
regulation of inflammatory molecule expression and regu-
latesinflammatorymediatorexpression,isthekeyinitialstep
of inflammatory reaction [18, 19]. P-Selectin is a member of
the family of cell adhesion molecule and is expressed in most
architectonic blood vessels of the normal human body. How-
ever, the content is very low and the expression can be signif-
icantly increased when in acute inflammation [20, 21]. It is
also an important indicator of inflammation [20, 22]. This
study found that NF-κB and P-selectin were negative in all
groups and showed that they were not related to the inflam-
matory reaction of thymus in SAP rats.
In this experiment, the plasma endotoxin content and
contents of NO, TNF-α, and PLA2in serum were all lower
in treated group than in model group, and were negatively
correlated with thymus pathological changes, demonstrating