Acute pancreatitis (AP) is characterized by the inappropriate activation, and inadequate clearance, of the protease trypsin in the pancreas, followed by a regional inflammatory response in the pancreas, often involving other organs.1 The pathogenesis of AP is complex and not yet clear, although much research has been carried out in recent years.2 Currently, the pathogenesis of AP is believed to be associated with the following factors: trypsin auto-digestion,3 production of oxygen free-radical species,4 microcirculation disorder,5 calcium overload,6 production of inflammatory mediators and cytokines,7 apoptosis, etc.8 When AP occurs, trypsin is abnormally activated and essentially digests pancreatic tissues; at the same time, inflammatory cells in the pancreas are recruited and activated, releasing a variety of mediators and cytokines which may cause a systemic inflammatory response syndrome (SIRS) and multiple organ dysfunction syndrome (MODS).9
If the release of enzymes can be reduced, the inflammatory reaction may be inhibited at the source. The level of pancreatin enzymes is related according to the death modes of pancreatic acinar cells.10 This study analyzed the reactions of isolated rat peritoneal macrophages to intracellular enzymes in the supernatants of pancreatic acinar cells that died by different modes. Its purpose was to clarify the intrinsic relationship between pancreatic acinar cell death, the release of cell contents and the inflammatory reaction of macrophages, which may provide new clues for taking measures to block this chain reaction and for treating AP in the future.
Rat pancreatic acinar cells, AR42J cells, were purchased from China Center for Type Culture Collection (CCTCC, Wuhan, China), and were cultivated in Ham's F12K medium (Sigma, USA) containing 10% fetal bovine serum (GIBCO BRL, USA) and incubated at 37°C, 5% CO2.
Collection and culture of peritoneal macrophages
Male Wistar rats ((250 ± 20) g) were provided by the Animal Research Center of the First Affiliated Hospital of Harbin Medical University (Harbin, China). The rats were treated in accordance with protocols approved by the local Animal Use and Care Committee. They were anesthetized by subcutaneous injection of 1% amobarbital (1 ml/100 g body weight). Abdominal incisions were conducted to reach the peritoneal tissues. Phosphate buffered saline (PBS; 30 ml) was injected into the abdominal cavity and the washing fluid was aspirated. The washing fluid was then centrifuged twice and cells prepared for cell suspension at 5×106 cell/ml in Roswell Park Memorial Institute 1640 medium (RPMI 1640) (Sigma, USA), containing 10% fetal bovine serum. The cell suspension was placed on a culture plate and incubated at 37°C, 5% CO2 for 2 hours. Phenol red-free RPMI 1640 medium was then used to rinse off non-adherent cells; the remaining cells were the purified peritoneal macrophages.
The experiment includes four groups: group A (the control group), group B (caerulein overstimulated group), group C (treated with caerulein and lipopolysaccharide), and group D (treated with caerulein and octreotide). 5×106/ml AR42J cells were added to culture plates and incubated for 24 hours. Caerulein (10 nmol/L, Sigma, USA) was then added to groups B, C, and D. Lipopolysaccharide (10 mg/L, Sigma) was added to group C. Octreotide (100 ng/ml, Novartis, Switzerland) was added to group D. The cells were cultivated for 24 hours. The culture supernatant was then discarded and rinsed with PBS. Fresh medium (2 ml) was added to cultures and incubated for another 6 hours. AR42J cells were collected to detect cell death using flow cytometery. One part of the supernatant was collected to detect amylase and lactate dehydrogenase (LDH) release. Another 1 ml of supernatant was inoculated into culture plates of macrophages and cultivated for 6 hours. Finally, macrophages were collected to detect NF-κB activation. The supernatant of the culture medium was collected to detect the TNF-α and IL-1β secretion. Each experiment was repeated five times.
Confocal laser microscopy and flow cytometry to detect apoptosis and oncosis
AR42J cells were collected to detect apoptosis and oncosis using the ApoAlert Annexin V-FITC Kit (BD Company, USA) according to manufacturer's recommendations. FITC-labeled annexin V was added to a final concentration of 2.5 mg/ml to cells. DNA was stained with propidium iodide (PI, 5 mg/ml). The slides were observed under a confocal laser microscope (Carl Zeiss, Germany), and the cells were counted with a flow cytometer (FACS Aria, BD Corporation, USA), FITC-conjugated annexin V and PI emissions were detected in the FL-1 and FL-2 channels, respectively.
Detection of pancreatic amylase and LDH release by chromatometry
AR42J cell culture supernatant was collected by centrifuge and analyzed strictly according to the operation manual of the pancreatic amylase test kit and LDH detection kit (Jiancheng Biotech, Nanjing, China).
Detection of NF-κB activation of macrophage by flow cytometry
Macrophages (5×106) were collected and Tris-HCl buffer (5 ml) was added immediately before cells were centrifuged for 5 minutes at 1200 r/min. Ten ml of 1% Triton X-100 (Sigma) abstergent solution was added and the cells were incubated at 4°C for 18 hours. A single-cell nuclear suspension was prepared using 50 μm nylon mesh filtration. The concentration of cell nuclei was adjusted to 1×106/ml. NF-κB p65 McAb (40 μl; Santa Cruz Biotechnology, Santa Cruz, USA) was added; the cells were then incubated for 20 minutes, and 1 μl FITC-labeled second antibody (Jackson Immuno Research, USA) was added. The cell nuclei were incubated for 20 minutes. Finally, 20 μl of PI was added and the cell nuclei were incubated for 30 minutes. The reaction system was analyzed by flow cytometery (FACS Aria, BD Corporation).
ELISA detection on TNF-α and IL-1β secretion of macrophage
Culture supernatant of macrophages was collected by centrifuge. The levels of TNF-α and IL-1β protein secreted by macrophages into the culture supernatant were determined by ELISA, which was performed according to operation manual of the ELISA Kit (Biosource, USA).
Data were represented as mean ± standard deviation (SD). The comparisons between two groups were conducted using analysis of variance (ANOVA) test. P <0.05 was considered statistically significant. Spearman rank correlation analysis was used to rate the relationship of two groups.
Assessment of apoptosis and oncosis of AR42J cells
As shown in Figure 1, oncotic cells and apoptotic cells were rarely found in the control group. In group B, more oncotic cells and apoptotic cells were observed. In group C, the number of oncotic cells increased, but the number of apoptotic cells decreased (compared with group B, P <0.05). In group D, the number of oncotic cells decreased significantly compared with group B, while the apoptotic cells increased (P <0.05).
The release of pancreatic amylase and LDH of pancreatic acinar cells
In group A, pancreatic amylase and LDH release were at a relatively low level. In group B, the release levels of the two enzymes were significantly higher than in group A. In group C, the levels were significantly higher than in group B (P <0.05). In group D, they were significantly lower than in group B (P <0.05) (Figure 2).
The results for NF-κB activation of macrophages
The results indicate that a lower level of NF-κB activation was observed in group A, while higher levels of NF-κB activation are seen in group B. In group C, the level of NF-κB activation was much higher than in group B (P <0.05). In group D, it was significantly lower than in group B (P <0.05) (Figure 3).
The detection results on TNF-α and IL-1β secretion of macrophages
The levels of TNF-α and IL-1β in group B were significantly increased compared to group A (P <0.05). The values were significantly higher in group C than in group B (P <0.05). The levels were significantly lower in group D than in group B (P <0.05) (Figure 4).
The increased release of the contents in pancreatic acinar cells, such as amylase (r=0.7935, P <0.05) and LDH (r=0.8102, P <0.05), changed directly with the occurrence of oncosis.
NF-κB activation are in line with the increased release of the cell contents, such as amylase (r=0.8325, P <0.05) and LDH (r=0.8643, P <0.05).
The changes in TNF-α secretion levels from macrophages correlates with the release of the cell contents, amylase (r=0.7936, P <0.05) and LDH (r=0.8064, P <0.05).
And IL-1β from macrophages to amylase (r=0.8435, P <0.05), to LDH (r=0.7847, P <0.05).
Significant in-depth research on apoptosis has been conducted by researchers since the concept of apoptosis was introduced; researchers have found that two different morphological changes, cell pyknosis and cell swelling, often exist in the same lesion. Majno named these patterns apoptosis and oncosis and considered them to be two different modes of cell death.11 Apoptosis is programmed cell death, in which neither leaking of the cell contents nor an inflammatory response occurs. 12 Oncosis, on the other hand, has a variety of causes and is characterized by cell membrane damage, cell contents leaking and an inflammatory response.13 Oncosis is more clinically important with regard to pancreatic acinar cells, because these cells contain a variety of digestive enzymes; therefore leaking of these enzymes may lead directly to the damage of adjacent cells and to pancreas autodigestion.14 More importantly, leakage of cell contents may recruit and activate inflammatory cells, such as macrophages, inducing them to secrete excessive inflammatory factors into the blood, causing damage of distant organs, or even leading to SIRS, MODS, just as described in "inflammatory cytokines theory".15
AP is a common, critical, often fatal illness. It develops rapidly, and can easily be complicated by SIRS and MODS. Therefore, early intervention treatment is important. In this study, we observed the release of pancreatic digestive enzymes from pancreatic acinar cells dying by different modes. We also observed different levels of macrophage stimulation by these enzymes and different levels of inflammatory response as a result. If a relationship exists between these reactions, drugs that adjust the modes of cell death can be beneficial for the treatment of AP.
Most researchers use rat models to study AP, but the interaction between the various cells is extremely complicated in vivo, making it difficult to observe separately the relationship between acinar cells and macrophages. Therefore, it is necessary to isolate the cells from the body. In our previous study, we successfully detected apoptosis and oncosis of rat pancreas acinar cells,1 but the isolation of pancreatic acinar cells presents some difficulty. To improve efficiency and experimental repeatability, this study used a cell model to study AP. We chose AR42J pancreatic acinar-like cells derived from rat pancreatic cancer cells, which possess many characteristics of acinar cells, such as the secretion of amylase.16 Under overstimulation with caerulein, a cholecystokinin analogue, excessive secretion of digestive enzymes, cytoplasm vacuolization and cell death can be induced in these cells, which is similar to the pathological changes of AP.17,18 In our experiment, after AR42J cells were treated with caerulein, we found that the secretion of amylase and the amount of apoptosis and oncosis increased significantly, supporting the validity of this model. On this basis, we used the endotoxin lipopolysaccharide to mimic aggravated AP19 and octreotide to mimic the alleviation of AP;20 we observed apoptosis, oncosis and the release of cell contents under different conditions. Results indicated that oncosis increased while apoptosis decreased in the lipopolysaccharide aggravation group, while apoptosis increased and oncosis decreased in the octreotide alleviation group. The increased release of the contents in pancreatic acinar cells, such as amylase and LDH, changed directly with the occurrence of oncosis.
Studies indicated that excessive activation of inflammatory cells and secretion of inflammatory mediators play a key role in the development of AP.21 In order to observe the effects of different types of cell contents on inflammatory cells, we used the supernatant of the culture medium of AR42J cells to treat the isolated rat peritoneal macrophages, and then analyzed NF-κB activation and the release of TNF-α and IL-1β from the macrophages.
NF-κB is a common transcription factor.22 Normally, NF-κB, in combination with its inhibitor IκB, exists in the cytoplasm, but enters the nucleus after activation and combines with target genes to induce the transcription of many other factors. Rakonczay considered that excessive activation of NF-κB can cause overproduction of inflammatory factors, which may lead to inflammatory injury. Our results indicated that activation of NF-κB was significantly higher in group C while it was significantly lower in group D, compared with group B. These changes are in line with the increased release of the cell contents, such as amylase and LDH, suggesting that the activation of NF-κB is related to the release of cell contents and to cell death modes.
To further observe the inflammatory response of macrophages, we detected changes in TNF-α and IL-1β secretion. Inflammatory factors such as TNF-α and IL-1β are related to the activation of NF-κB.23 TNF-α and IL-1β are mainly secreted by mononuclear cells and macrophages; they are peptides with broad biological activities and play important roles in disease prevention and repair processes. However, excessive secretion of these cytokines can increase inflammation and cause other damage. TNF-α is an important factor for AP-caused SIRS and MODS, which may be major indicators of the severity of AP. IL-1β is an important inflammatory enhancement cytokine, which also plays an important role in the development of AP. Our results indicated that changes in TNF-α and IL-1β secretion levels from macrophages correlates with the release of the cell contents, again showing the intrinsic relationship between macrophage inflammatory response, the release of cell contents and the cell death modes of pancreatic acinar cells. The results suggest that rapid development of AP and inflammatory responses may be prevented through the adjustment of the modes of cell death. This may eventually provide new ideas for AP therapies.
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