Since severe acute pancreatitis (SAP) and obstructive jaundice (OJ) are common diseases, the study of their pathogenesis and treatment has been a hot spot in medical field for a long time [
In the present study, we have applied tissue microarray technology to examine the pathological changes in the spleen and thymus of SAP and OJ rats, and investigated the protective effect of
A total of 288 healthy male SD rats of clean grade, weighing between 270 and 330 g, were provided by the Laboratory Animal Research Center of the Zhejiang University of Traditional Chinese Medicine (China); Sodium taurocholate and sodium pentobarbital were purchased from Sigma Corporation, USA;
A total of 108 rats were used for SAP-associated experiments and randomly divided into sham-operated group, model control group, and treated group (
The rats were anesthetized with an intraperitoneal injection of 2.5% sodium pentobarbital (0.2 mL/100 g). Under aseptic conditions, the thigh skin was cut open to expose femoral vein and a transfusion passage was established through which continuous infusion was maintained using a microinfusion pump (1 mL/h/100 g). Subsequently, a median abdominal wall incision was made to expose duodenal papilla, and a number 5 syringe needle was used to prick a small hole in the mesenteric avascular area. The epidural catheter was first inserted into duodenal cavity via the hole, and then placed into the bile-pancreatic duct toward the direction of papilla. The catheter head was temporarily clamped using a microvascular clamp, and another microvascular clamp was used to occlude the common bile duct at the confluence of hepatic ducts to prevent a backflow of injected drugs into the liver. After connecting the epidural catheter end with the transfusion converter, 3.5% sodium taurocholate (0.1 mL/100 g) was transfused at a flow rate of 0.2 mL/min using a microinjection pump (produced by Zhejiang University, China). After completing the transfusion, microvascular forceps and epidural catheter were maintained for further 4 minutes and then removed. A check-up was then conducted to see whether bile leakage was present. After suturing the hole in the lateral wall of the duodenum, the abdominal cavity was closed conventionally. Sham-operated groups were performed just by moving the pancreas and duodenum after opening the abdominal cavity. Fifteen minutes after successful operation, a single dose of
After rats were anesthetized with an intraperitoneal injection of 2.5% sodium pentobarbital (0.2 mL/100 g), the abdominal cavity was opened to identify and dissociate common bile duct along the hepatoduodenal ligament. For rats in model control groups and the treated groups, the proximal end of common bile duct was double-ligated with surgical threads, common bile duct was cut off, and a layered suture of the abdominal wall was performed to close the abdominal cavity. For rats in sham-operated groups, common bile duct was only dissociated but not ligated, and a layered suture of the abdominal wall was also performed to close the abdominal cavity. An intraperitoneal injection of
(1) At various time points after operation, the mortality rate of rats was recorded. At the corresponding time points after operation, SAP or OJ rats were anesthetized with 2.5% sodium pentobarbital and killed to take blood samples and tissue specimens of spleen and thymus that were subsequently subjected to pathological examination and morphological observation under a light microscope. At 12 hours after operation in SAP experiment, the ultrastructural changes in the spleen and thymus were also observed under an electron microscope. The pathological severity score of spleen and thymus in SAP groups and spleen in OJ groups were analyzed, respectively.
(2) Determination of the contents of plasma endotoxin and serum
(3) Tissue microarray technology was used to prepare pathological sections of spleen and thymus, and then conducted immunohistochemical staining of Bax protein and TUNEL staining. The changes in the expression levels of Bax and the apoptosis index of lymphocyte in the spleen and thymus were observed. Firstly, tissue microarrays sections of spleen and thymus were prepared, the diameter was 1.5 mm. The Envision two-step method was used to detect the expression levels of Bax, a protein in the spleen and thymus. The evaluation standard were as follows: (1) the staining intensity was evaluated according to the extent of cell coloration: “
(4) The results were subjected to statistical analysis.
The compiled data were first input into an Excel sheet, and then read into SPSS15.0 for further analysis. Normal data were expressed as means (standard deviation) while nonnormal data were expressed as medians (interquartile range). Analysis of variance and pairwise comparisons were used for normal data, whereas nonnormal data were subjected to nonparametric test, among which Kruskal-Wallis H test was used for pairwise comparisons and Mann-Whitney U test for multiple comparisons. Yates' chi-square test (
One and five rats died in model control groups at 3 and 12 hours after operation, respectively; three died in treated group at 12 hours after operation; no rats died in the remaining groups. There was no marked difference in mortality rate between the time points at 3 and 6 hours after operation. At 12 hours after operation, only the mortality rate in model control group was significantly higher than that in sham-operated group (
At all time points after operation, the contents of plasma endotoxin in model control group were significantly higher than those in sham-operated group (
Comparison of endotoxin and PLA2 level of SAP groups (
Index | Time | Sham-operated group | Model control group | Treated group |
---|---|---|---|---|
Endotoxin(EU/mL) | 3 hours | 0.3 (0.5) | ||
6 hours | 0.3 (0.2) | |||
12 hours | 0.3 (0.1) | |||
3 hours | 31.5 (10.1) | |||
6 hours | 40.4 (12.1) | |||
12 hours | 46.0 (12.7) |
Note: Compare to sham-operated group,
At all time points after operation, the contents of serum
Model control group—6 hours in SAP experiment spleen (several apoptoic cells), TUNEL
Treated group—6 hours in SAP experiment spleen (several apoptoic cells) TUNEL,
Spleen congestion and mitochondrial vacuolation were seen.
Because no severity score standard for pathological changes of spleen were reported in literature, we established the standard according to the extent of pathological damage of spleen, which were shown in Table
Pathological severity score standard of spleen.
Score | Observation indexes |
---|---|
0 point | Normal |
1 point | Necrosis of follicle center |
2 points | blood sinus dilation or arteriolar sclerosis |
3 points | Necrosis of follicle center, blood sinus dilation and arteriolar sclerosis |
Comparision of pathological severity score of spleen and
Index | Time | Sham-operated group | Model control group | Treated group |
---|---|---|---|---|
Spleen | 3 hours | 0.0 (1.0) | 1.0 (1.0) | 0.0 (1.0) |
6 hours | 0.0 (0.0) | 1.0 (1.0) | 0.0 (1.0) | |
12 hours | 0.0 (0.0) | 1.0 (1.0) | 0.0 (1.0) | |
Thymus | 3 hours | 0.0 (0.0) | 0.0 (0.0) | 0.0 (0.0) |
6 hours | 0.0 (0.0) | 0.0 (0.0) | 0.0 (0.0) | |
12 hours | 0.0 (0.0) | 0.0 (0.0) | 0.0 (0.0) |
Note: Compare to sham-operated group,
At 12 hours after operation, the staining intensity of Bax protein of spleen in model control group was significantly higher than that in sham-operated group (
Comparison of pathological indexes of spleen of SAP groups (
Index | Time | Sham-operated group | Model control group | Treated group |
---|---|---|---|---|
Staining intensity of Bax | 3 hours | 0.0 (1.0) | 1.0 (2.0) | 1.0 (1.5) |
6 hours | 0.5 (1.0) | 1.0 (0.5) | 1.0 (1.5) | |
12 hours | 0.0 (1.0) | 1.0 (1.0) | ||
Product of the staining Intensity and positive rate of Bax protein | 3 hours | 0.0 (1.5) | 2.0 (2.0) | |
6 hours | 1.0 (3.0) | 2.0 (1.5) | 2.0 (3.0) | |
12 hours | 0.0 (2.0) | 2.0 (3.0) | ||
Apoptotic indexes | 3 hours | 0.0 (0.0) | 0.0 (0.04) | 0.0 (0.01) |
6 hours | 0.0 (0.0) | 0.0 (0.03) | 0.0 (0.0) | |
12 hours | 0.0 (0.0) | 0.0 (0.08) | 0.0 (0.0) |
Note: Compare to sham-operated group,
At 12 hours after operation, the product of the staining intensity and positive rate of Bax protein of spleen in model control group was significantly higher than that in sham-operated group (
The expression of NF-
At all time points after operation, no significant difference in the apoptosis index of spleen was noted between sham-operated group and model control group, between sham-operated group and treated group, as well as between model control group and treated group (
Sham-operated group—12 hours in SAP experiment thymus (normal), Electron microscope
Model control group—12 hours in SAP experiment thymus (apoptoic cells obviously increase in thymus), Electron microscope
Treated group—12 hours in SAP experiment thymus (normal thymic lymphocyte and reticulate epithelium) Electron microscope
The severity score of pathological changes of thymus was conducted according to the standard reported in literature [
The expression of Bax and NF-
It was noted that 2, 4, 4, and 7 rats died in model control groups on 7, 14, 21, and 28 days after operation, respectively; and 3, 2, and 4 rats died in treated groups on 14, 21, and 28 days after operation. The mortality rates on 7 days after operation showed no marked difference among three experimental groups. On 14 and 21 days after operation, the mortality rates in sham-operated groups were significantly lower than those in model control groups (
At all time points after operation, the contents of plasma endotoxin in sham-operated group were significantly lower than those in model control group and the treated group (
Comparison of endotoxin and PLA2 level of OJ groups (
Index | Time | Sham-operated group | Model control group | Treated group |
---|---|---|---|---|
Endotoxin(EU/mL) | 7 days | 0.2 (0.3) | ||
14 days | 0.2 (0.4) | |||
21 days | 0.2 (0.05) | |||
28 days | 0.3 (0.04) | |||
PLA2(U/mL) | 7 days | 208.1 (26.6) | 570.3 (239.6) | 514.4 |
14 days | 198.5 (46.3) | |||
21 days | 207.2 (38.6) | |||
28 days | 203.0 (31.0) |
Note: Compare to sham-operated group,
On 14, 21, and 28 days after operation, the contents of serum PLA2 in sham-operated group were significantly lower than those in model control group and treated group (
Model control group—28 days in OJ experiment spleen (several apoptoic cells), TUNEL
Treated group—28 days in OJ experiment spleen (several apoptoic cells), TUNEL
Model control group—21 d in OJ experiment (++) spleen, Bax
The severity score standard for pathological changes of spleen was cited in Table
Comparision of pathological severity score of spleen in OJ group (
Index | Time | Sham-operated group | Model control group | Treated group |
---|---|---|---|---|
Pathological severity score of spleen | 7 days | 0.0 (0.0) | 0.0 (0.0) | 0.0 (0.0) |
14 days | 0.0 (1.0) | 0.0 (2.0) | 0.0 (0.0) | |
21 days | 0.0 (0.0) | 1.0 (2.0) | 0.0 (0.0) | |
28 days | 0.0 (0.0) | 0.0 (1.0) | 0.0 (0.0) |
Note: Compare to sham-operated group,
On 7, 14, and 21 days after operation, the staining intensity of Bax protein of spleen in model control group was significantly higher than that in sham-operated group (
Comparison of pathological indexes of spleen in OJ groups (
Index | Time | Sham-operated group | Model control group | Treated group |
---|---|---|---|---|
Staining intensity of Bax | 7 days | 0.0 (2.0) | ||
14 days | 2.0 (2.0) | 2.0 (0.0) | ||
21 days | 1.0 (2.0) | |||
28 days | 1.0 (2.0) | 1.0 (2.0) | 0.0 (2.0) | |
Product of staining intensity and positive rate of Bax | 7 days | 0.0 (2.0) | ||
14 days | 2.0 (4.0) | 4.0 (2.0) | ||
21 days | 2.0 (4.0) | |||
28 days | 1.0 (4.0) | 0.5 (1.5) | 0.0 (2.0) | |
Apoptotic indexes | 7 days | 0.0 (0.0) | 0.0 (0.0) | |
14 days | 0.0 (0.0) | 0.0 (0.0) | ||
21 days | 0.0 (0.0) | 0.0 (0.002) | 0.0 (0.0) | |
28 days | 0.0 (0.0) | |||
Staining intensity of NF- | 7 days | 0.0 (0.0) | 0.0 (1.0) | 0.0 (0.0) |
14 days | 0.0 (1.0) | 0.0 (1.0) | 0.0 (0.5) | |
21 days | 0.0 (0.0) | 0.0 (0.0) | 0.0 (0.0) | |
28 days | 0.0 (0.0) | 0.0 (0.0) | 0.0 (0.0) | |
Product of staining intensity and positive rate of NF- | 7 days | 0.0 (0.0) | 0.0 (1.0) | 0.0 (0.0) |
14 days | 0.0 (1.0) | 0.0 (2.0) | 0.0 (0.5) | |
21 days | 0.0 (0.0) | 0.0 (0.0) | 0.0 (0.0) | |
28 days | 0.0 (0.0) | 0.0 (0.0) | 0.0 (0.0) |
Note: Compare to sham-operated group,
Treated group—21 d in OJ experiment (+) spleen, Bax
On 7, 14, and 21 days after operation, the products of the staining intensity and the positive rate of Bax protein of spleen in model control group were significantly higher than those in sham-operated group (
At all time points after operation, no significant difference in the staining intensity of NF-
At all time points after operation, no significant difference in the product of the staining intensity and the positive rate of NF-
On 7, 14, and 28 days after operation, the apoptosis indexes of spleen in model control group were significantly higher than those in sham-operated group (
Sham-operated group—28 d in OJ experiment thymus (negative TUNEL
Model control group—28 d in OJ experiment thymus (many apoptoic cells), TUNEL
Treated group—28 d in OJ experiment thymus (several apoptoic cells), TUNEL
On 7, 21, and 28 days after operation, the staining intensity of Bax protein of thymus in model control group was significantly higher than that in sham-operated group (
Comparison of pathological indexes of thymus in OJ groups (
Index | Time | Sham-operated group | Model control group | Treated group |
---|---|---|---|---|
Staining intensity of Bax | 7 days | 0.0 (0.0) | 0.0 (1.0) | |
14 days | 0.0 (1.0) | 1.0 (1.0) | 0.0 (1.0) | |
21 days | 0.0 (0.0) | 0.0 (1.0) | ||
28 days | 0.0 (0.0) | 0.0 (1.0) | ||
Product of staining intensity and positive rate of Bax | 7 days | 0.0 (0.0) | 0.0 (1.0) | |
14 days | 0.0 (1.0) | 0.0 (1.0) | 0.0 (1.0) | |
21 days | 0.0 (0.0) | 0.0 (1.0) | ||
28 days | 0.0 (0.0) | 0.0 (1.0) | ||
Apoptotic indexes | 7 days | 0.0 (0.0) | 0.0 (0.0) | 0.0 (0.0) |
14 days | 0.0 (0.0) | 0.0 (0.01) | 0.0 (0.0) | |
21 days | 0.0 (0.0) | 0.0 (0.0) | ||
28 days | 0.0 (0.0) | 0.0 (0.0) | ||
Staining intensity of NF- | 7 days | 0.0 (0.0) | 0.0 (2.0) | 0.0 (1.0) |
14 days | 0.0 (2.0) | 1.0 (2.0) | 0.0 (1.5) | |
21 days | 0.0 (1.0) | 0.0 (1.0) | 0.0 (0.0) | |
28 days | 0.0 (1.0) | 0.0 (1.5) | 0.0 (2.0) | |
Product of staining intensity and positive rate of NF- | 7 days | 0.0 (0.0) | 0.0 (2.0) | 0.0 (2.0) |
14 days | 0.0 (4.0) | 2.0 (4.0) | 0.0 (2.0) | |
21 days | 0.0 (2.0) | 0.0 (2.0) | 0.0 (0.0) | |
28 days | 0.0 (1.0) | 0.0 (2.5) | 0.0 (4.0) |
Note: Compare to sham-operated group,
Model control group—7 d in OJ experiment thymus (+++), Bax
Model control group—21 d in OJ experiment thymus (++), Bax
On 7, 21, and 28 days after operation, the products of the staining intensity and the positive rate of Bax protein of thymus in model control group were significantly higher than those in sham-operated group (
At all time points after operation, no significant difference in the staining intensity of NF-
At all time points after operation, no significant difference in the product of the staining intensity and the positive rate of NF-
On 21 and 28 days after operation, the apoptosis indexes of thymus in model control group were significantly higher than those in sham-operated group (
At present, the immune organ injury in SAP and OJ has attracted more and more attention. An indispensable step to treat SAP and OJ is to help restore the function of the body's immune organs using drugs. SAP and OJ, as are diseases commonly seen in the departments of general surgery and digestive medicine, are sensitive, to a certain extent, to auxiliary treatment with Chinese traditional drugs.
When SAP develops, gut-derived endotoxins enter into the systemic circulation and cause intestinal endotoxemia which can increase the permeability of intestinal mucosa and facilitate the invasion of intestinal bacteria and endotoxins into the body. Thus, a vicious cycle is formed [
We think that the lower systemic immune function in OJ patients creates conditions for the occurrence of endotoxemia which can in turn induce the damage to the body's immune function. We speculate that immune function injury results from the damage to immune organs. Comparing the pathological changes of spleen and thymus, we found that varying degrees of pathological damage were present in the spleen and thymus of SAP rats and the spleen of OJ rats in model control group but pathological damage in the thymus of OJ rats was slight. After treatment with
The results of this study showed that the contents of plasma endotoxin in SAP and OJ rats in model control group were significantly higher than those in sham-operated group, indicating that SAP and OJ can increase the contents of plasma endotoxin in rats. We believe that SAP and OJ can lead to an increase in the contents of endotoxin not only through causing intestinal mucosal barrier dysfunction, invasion of intestinal flora into blood, and the dissolving bacterial cell wall [
Phospholipase
Apoptosis is a self-protective strategy employed by the body for removal of the destroyed cells through initiating programmed gene expression under certain pathophysiological conditions [
NF-
To sum up,
We claimed that this paper was original and would not have any financial interest in a company or its competitor, and that all authors meet standard for authorship. We abided the ethics in this animal experimental study. The ethics committee approval of our hospital was secured for the animal study reported, and all rats have not been abused and executive mercy killing was considered when the observing time in this study was over.
This work was supported by technological foundation project of Traditional Chinese Medicine Science of Zhejiang province (no. 2003C130; no. 2004C142), foundation project for medical science and technology of Zhejiang province (no. 2003B134), grave foundation project for technological and development of Hangzhou (no. 2003123B19), intensive foundation project for technology of Hangzhou (no. 2004Z006), foundation project for medical science and technology of Hangzhou (no. 2003A004), and foundation project for technology of Hangzhou (no. 2005224).