Resolvin D1 protects against inflammation in
experimental acute pancreatitis and associated
lung injury
Yong Liu, Dan Zhou, Fei-Wu Long, Ke-Ling Chen, Hong-Wei Yang, Zhao-Yin Lv, Bin
Zhou, Zhi-Hai Peng, Xiao-Feng Sun, Yuan Li and Zong-Guang Zhou
Linköping University Post Print
N.B.: When citing this work, cite the original article.
Original Publication:
Yong Liu, Dan Zhou, Fei-Wu Long, Ke-Ling Chen, Hong-Wei Yang, Zhao-Yin Lv, Bin Zhou,
Zhi-Hai Peng, Xiao-Feng Sun, Yuan Li and Zong-Guang Zhou, Resolvin D1 protects against
inflammation in experimental acute pancreatitis and associated lung injury, 2016, American
Journal of Physiology - Gastrointestinal and Liver Physiology, (310), 5, G303-G309.
http://dx.doi.org/10.1152/ajpgi.00355.2014
Copyright: American Physiological Society
http://www.the-aps.org/
Postprint available at: Linköping University Electronic Press
1 Resolvin D1 protect against inflammation in experimental acute pancreatitis and associated
lung injury
Yong Liu1, 2, Dan Zhou3, Fei-Wu Long1, 2, Ke-Ling Chen1, Hong-Wei Yang1, 2, Zhao-Yin Lv1,
Bin Zhou1, Zhi-Hai Peng4,
Xiao-Feng Sun
1, 5,
Yuan Li1, Zong-Guang Zhou1, 21 Institute of Digestive Surgery and State Key Laboratory of Biotherapy, West China Hospital,
Sichuan University, Chengdu, China; 2 Department of Gastroenterological Surgery, West China
Hospital, Sichuan University, Chengdu, China; 3 Department of Pharmacy, West China Hospital,
Sichuan University, Chengdu, China; 4 Department of General Surgery, Shanghai First People's
Hospital, Shanghai Jiaotong University, Shanghai, China; 5 Department of Oncology, Department
of Clinical and Experiment Medicine, Linköping University, Linköping, Sweden
Running title: Resolvin D1 protect against acute pancreatitis
Correspondence and request for reprints to: Yuan Li, MD, PhD; Zongguang Zhou, MD, PhD,
Institute of Digestive Surgery and State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, No.1 Ke-Yuan-Si-Lu, Gao-Peng-Da-Dao, Chengdu, Sichuan, 610041, China.
2 Abstract
Acute pancreatitis is an inflammatory condition that may lead to multi-systemic organ failure with
considerable mortality. Recently, resolvin D1 (RvD1) as an endogenous anti-inflammatory lipid
mediator has been confirmed to protect against many inflammatory diseases. This study was designed to investigate the effects of RvD1in acute pancreatitis and associated lung injury. Acute
pancreatitis varied from mild to severe was induced by cerulein or cerulein combined with LPS,
respectively. Mice were pretreated with RvD1 at a dose of 300ng/mouse 30 min before the first
injection of cerulein. Severity of AP was assessed by biochemical markers and histology. Serum
cytokines and myeloperoxidase (MPO) levels in pancreas and lung were determined for assessing the extent of inflammatory response. NF-κB activation was determined by western blotting. The
injection of cerulein or ceulein combined with LPS resulted in local injury in the pancreas and
corresponding systemic inflammatory changes with pronounced severity in the ceulein and LPS
group. Pretreated RvD1 significantly reduced the degree of amylase, lipase, TNF-α and IL-6
serum levels, the MPO activities in the pancreas and the lungs, the pancreatic NF-κB activation, the severity of pancreatic injury and associated lung injury, especially in the severe acute
pancreatitis model. These results suggest that RvD1 is capable of improving injury of pancreas
and lung, and exerting anti-inflammatory effects may through the inhibition of NF-κB activation
in experimental acute pancreatitis, with more notable protective effect in severe acute pancreatitis.
These findings indicate that RvD1 may constitute a novel therapeutic strategy in the management of
severe acute pancreatitis.
3
Introduction
Acute pancreatitis is an inflammatory disorder of the exocrine pancreas. Although most patients
suffer a mild and limited disease, about one-fifth of cases develop acute respiratory distress
syndrome and multiple organ dysfunction, accompanied by high mortality (8). Specific and effective interventions for this disease are not available largely because of a lack of understanding
of the early cellular events in its pathophysiology. Nuclear factor-κB (NF-κB) is a ubiquitous
inducible transcription factor and composed of a group of structurally related transcriptional
proteins (15). In pancreas, the predominant form of NF-κB is p65/p50 heterodimer (12, 28).Under
control conditions, NF-κB is kept inactive in the cytoplasm through sequestration in complexes
with the inhibitor of κB (IκB) proteins, such as IκB-α and IκB-β. The sequestration prevents NF-κB migration into the nucleus, its’ binding to DNA, and transcriptional activation. Typically, in response to an inducing stimulus the IκBs are phosphorylated on specific Ser residues by IκB kinases (IKKs), which results in IκB ubiquitylation and proteasome-mediated degradation, allowing NF-κB nuclear translocation (11, 28). NF-κB is activated early in acinar cells during acute pancreatitis and increases expression of multiple pro-inflammatory genes, such as TNF-α
and IL-6 (9, 36). In most studies, pharmacologic inhibition of NF-κB resulted in an amelioration
of inflammatory response, necrosis, and other parameters of pancreatitis severity (7, 29, 31).
Moreover, a recent study showed that increased acinar cell NF-κB activity correlated with higher
cytokine expression and greater severity of acute pancreatitis using novel genetic mouse models
(13). Thus, specific and effective drugs which inhibiting NF-κB activation can be useful in therapy of acute pancreatitis. However, no innovative drugs are so far available in the clinical setting (23).
4
docosahexaenoic acid (DHA), which are enriched in some fish oils, are believed to exert
beneficial effects on a wide range of inflammatory disorders (30, 40), including acute pancreatitis
(1, 24). EPA and DHA originate the lipid mediators known as resolvins, which regulate critical
cellular events in the resolution of inflammation (32, 33). Resolvin D1 (7S, 8R, 17S-trihydroxy DHA, RvD1) is one of the resolvins and is derived from DHA (34). RvD1 can inhibit neutrophil
activation (16, 38), regulate cytokines (18, 39) and inhibit the activation of NF-κB pathway in
endotoxin (lipopolysaccharide, LPS) induced inflammatory response (4, 6, 18, 21, 42). It has also
been identified to reverses chronic pancreatitis-induced chronic pain (27).
Overall, these observations prompted us to hypothesize that RvD1 maybe have protective effects on acute pancreatitis though suppressing inflammatory response. To test this hypothesis,
we induced pancreatitis in mice, producing different degrees of severity by repeated injections of
cerulein with or without lipopolysaccharide (LPS). Local injuries of pancreas and lung were
assessed by established parameters, and systemic inflammation was determined through assaying
the serum TNF-α and IL-6 levels and myeloperoxidase (MPO) activity. The effects of RvD1 on inflammatory response in acute pancreatitis were studied in detail. Our findings provide a novel
5
MATERIAL AND METHODS
Animals and Reagents
Adult male C57BL/6 mice (20-25 g) were obtained from the Animal Centre of Sichuan University
(Chengdu, China), maintained on a 12 h light/12 h dark cycle at 22˚C, given water ad libitum, fed standard laboratory chow, and allowed to acclimatize for a minimum of 1 week. Mice were
randomly assigned to control or experimental groups. All experiments were conducted with the
approval of the Animal Research Committee at Sichuan University. Cerulein and
lipopolysaccharide (LPS) were purchased from Sigma Chemical (Sigma-Aldrich, St. Louis,
Missouri, USA). RvD1 was purchased from Cayman Chemical (Cayman, Michigan, USA). Antibodies against NF-κB p65 subunit and histone H3.1 were purchased from Cell Signaling
Technology (CST, Massachusetts, USA). Other items were purchased from standard suppliers or
as indicated in text.
Induction of experimental pancreatitis
For cerulein pancreatitis, C57BL/6 mice were treated by 7 hourly intraperitoneal injections (IP) of cerulein (50μg/kg/h). More severe acute pancreatitis model was induced by administration of
cerulein in combination of LPS (20), mice were injected intraperitoneally with cerulein in the
same way as those in the cerulein acute pancreatitis model except that LPS was added (10 mg/kg)
with the last injection of cerulein. Controls received comparable injections of normal saline (NS).
RvD1 (dose of 300ng/mouse based on preliminary data) was administered to the mice by IP 30
min before the first injection of cerulein. Mice were killed 8 h and 24 h after the first injection of cerulein. For RvD1 therapeutic treatment group, RvD1 (dose of 300ng/mouse ) was administered
6
injection of cerulein.
Histological examination
Fresh specimens of murine pancreas and lung were fixed in 4% paraformaldehyde in
phosphate-buffered saline (PBS, pH 7.4).Tissues were embedded in paraffin, and 5 mm sections were processed for hematoxylin-eosin (H&E) staining by standard procedures. Then multiple
randomly chosen microscopic fields from at least three mice in each group were examined by two
pathologists in blind manner. For pancreatic injury, the scoring was on a scale of 0-3 (0 being
normal and 3 being severe) according to four items: presence of vacuolization, interstitial edema,
interstitial inflammation, the number of acinar cell necroses, as previously described (17). For lung injury, the scoring was on a scale of 0-4 (0= minimal damage, 1= mild damage, 2= moderate
damage, 3= severe damage, 4= maximal damage) according to four items: alveolar congestion,
hemorrhage, infiltration or aggregation of neutrophils in air space or the vessel wall, and thickness
of the alveolar wall/hyaline membrane formation, as previously described (14).
Measurement of amylase and lipase
Serum amylase and lipase were determined by means of a commercially available kit (R&D
System, MN, USA), and expressed as units per liter (U/L).
Measurement of cytokines
The pro-inflammatory cytokines TNF-α and IL-6 in serum were measured using Luminex assay kit according to the manufacturer’s instructions (R&D Systems, MN, USA). Assays were performed in duplicate using the Luminex 100 System (Austin, Texas, USA) Digital images of the bead array were captured following laser excitation and were processed on a computer
7
software (MiraiBio, Alameda, CA, USA).
Measurement of MPO activity
The extent of neutrophil infiltration was measured in both pancreatic and lung tissue by
quantifying myeloperoxidase (MPO) activity as previously described (35) . The enzyme activity was determined using MPO detection kit according to the manufacturer’s instructions (Nanjing
Jiancheng Bioengineering Institute, Nanjing, China). The activity was expressed as units per
milligram of wet tissue and calculated as % of control as previously described (20) .
Western blot analysis
Pancreatic tissue samples collected at 8h after first injection were homogenized, nuclear protein was extracted separately using the Nuclear Protein Extraction Kit (Viagene Biotech, Ningbo,
China) according to the manufacturer's instructions. The concentrations of protein were determined using the BCA method (Pierce, Rockford, USA). Each 20 μg aliquot of protein was loaded in a 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel, and then
transferred onto polyvinylidene difluoride membranes (Millipore, Massachusetts, USA). After complete protein transfer, the membranes were blocked with 5% milk powder solution for 1 h at room temperature, and incubated at 4˚C overnight with rabbit monoclonal anti- NF-κB p65 subunit diluted at a 1:1000 dilution in 5% milk powder solution. For internal reference, a rabbit
monoclonal anti- histone H3.1 antibody (1:1000 dilution) was used. After washing the membranes,
goat polyclonal anti-rabbit immunoglobulin G secondary antibody (CST, Massachusetts, USA)
conjugated to horseradish peroxidase was applied in a 1:5000 dilution and incubated for 1 h at room temperature. Finally, antibody binding was visualized using the enhanced
8 Statistical analysis
Data are expressed as mean ± SEM. All data were analyzed by one-way ANOVA with a posttest
analysis (Newman-Keuls). In all cases, a p value of < 0.05 was selected as criterion for statistical
9
RESULTS
Effects of RvD1 on the severity of experimental acute pancreatitis
We evaluated the severity of experiment pancreatitis through histological score based on the
extent of tissue edema, vacuolization, inflammation, and necrosis. The pancreas histological picture was normal in both control and RvD1 alone treated mice. Cerulein treated mice displayed
histological signs of acute pancreatitis characterized by interstitial edema, vacuolization, and
infiltration of neutrophil and mononuclear cells with little parenchyma necrosis and hemorrhage.
The treatment of cerulein in combination with LPS caused more severe pathological changes in
the pancreatic tissue, with an obvious edema, inflammation, vacuolization, and many local acinar cells necrosis (Fig.1). In contrast, treatment with RvD1 significantly reduced the morphological
changes seen in both models of acute pancreatitis, and decreased the severity of experiment acute
pancreatitis (Fig.2D). Some parameters used to quantify the severity of acute pancreatitis were
also measured, such as amylase and lipase. Low levels of serum amylase and lipase activity were
evidenced in control and RvD1alone treated mice. Injection of cerulein with or without LPS enhanced serum amylase and lipase activity compared with control mice. Treatment with RvD1
showed a marked reduction in the activity of these pancreatitis markers (Fig. 2A and B).
Effects of RvD1 on inflammatory cytokines in experimental acute pancreatitis
To assess pancreatic inflammatory response, we investigate the pro-inflammatory cytokines
TNF-α and IL-6, two of the main mediators of the acute-phase response whose levels are useful for predicting the severity of acute pancreatitis (19, 22). Low TNF-α and IL-6 levels were
10
revealed a moderate increase only at early stage after cerulein treatment compared with control
animals, while the serum obtained from cerulein plus LPS injected mice showed a marked
increase in both cytokines levels. In contrast, treatment with RvD1 significantly reduced the
cerulein or cerulein plus LPS induced increase of TNF-α and IL-6 levels, especially in cerulein plus LPS injected mice (Fig. 3).
Effects of RvD1 on pancreatic MPO activity in experimental acute pancreatitis
MPO was assessed as a quantitative marker of neutrophil infiltration in pancreatic inflammatory
disease. Low levels of MPO were detected in the pancreas of control and RvD1alone treated mice. In contrast, the pancreas obtained from cerulein with or without LPS injected mice showed a
marked increase in MPO activity. Treatment with RvD1 inhibited cerulein with or without LPS
induced increase in MPO pancreatic levels, and the inhibition role of RvD1 on MPO activity
seems more effective in cerulein plus LPS induced mice (Fig. 2C).
Effects of RvD1 on pancreatic NF-κB activation in experimental acute pancreatitis
Pancreatic tissue samples were collected at 8 h after first cerulein administration, and the levels of
NF-κB p65 subunit in the nucleus were measured by western blot (Fig. 4A). Cerulein and cerulein
plus LPS both induced increased levels of NF-κB p65 subunit in the nucleus, with a more
pronounced increase of NF-κB p65 subunit in cerulein plus LPS induced mice. In contrast,
treatment with RvD1 inhibited cerulein with or without LPS induced NF-κB p65 subunit increase in the nucleus (Fig. 4B).
11 Effects of RvD1 on severity of acute pancreatitis associated lung injury
We also evaluated the severity of acute pancreatitis associated lung injury based on histological
alternation including alveolar congestion, hemorrhage, neutrophils infiltration in air space and
thickness of the alveolar wall. The lung histological picture was normal in control and RvD1alone treated mice. In mice treated by cerulein with or without LPS, lung damage was characterized by
alveolar congestion, obvious hemorrhage, infiltration of neutrophil in air space and increased
thickness of the alveolar wall (Fig. 5). Treatment with RvD1 significantly reduced the histological
alterations of lung injury (Fig. 6B). MPO was also assessed as a quantitative marker of neutrophil
infiltration in lung inflammatory response. Low levels of MPO were detected in the lung tissues of control and RvD1alone treated mice. In contrast, the lung tissues obtained from cerulein with or
without LPS injected mice showed an increase in MPO activity, with a more pronounced increase
of MPO activity in cerulein plus LPS induced mice. In lung tissues of additional RvD1 treated
mice, there was significantly less MPO activity as compared with cerulein or cerulein plus LPS
induced mice, respectively (Fig. 6A). Overall, these finding indicated that pretreatment of RvD1 reduced the severity of acute pancreatitis associated lung injury.
Therapeutic treatment of RvD1 reduced the severity of experimental acute pancreatitis
We further evaluated the severity of experimental pancreatitis with RvD1 therapeutic treatment. Application of RvD1 4h after induction of acute pancreatitis, either by cerulein or cerulein plus
LPS, could significantly alleviate pancreatic inflammation, which was evaluated by pancreatic morphological changes (Fig. 7), pancreatic injury scoring, pancreatic MPO activity and serum
12 DISCUSSION
Acute pancreatitis is a potentially fatal disease characterized by wide clinical variation, ranging
from a mild self limiting to severe disease complicated by sepsis and multi-organ failure, leading
to high morbidity and mortality rates (8, 41). Currently, despite the development of new diagnostic tools and treatment options, there are several problems in the therapy of severe acute
pancreatitis (26, 44). In this study, we induced acute pancreatitis in two different mouse models
characterized by different degrees of severity. In mice, acute pancreatitis induced by cerulein
alone represents relatively mild type, focal necrosis possibly could be detected but customarily
little parenchyma necrosis occurs. Acute pancreatitis is a kind of special inflammatory disease that in could arouse systemic inflammatory responses (SIRS), which cause ‘injury in distant organ
more severe than that in pancreas’, this is typically observed clinically. In pancreatitis induced
SIRS, lung is the most susceptible organ and significant changes are primarily detect in lungs. Our
results demonstrate that cerulein alone induced a relatively mild model of acute pancreatitis with
moderate increased leukocyte infiltration in lung tissues. In contrast, combination of cerulein and LPS injection induced more severe model with deteriorated pancreatic inflammation, evident local
acinar necrosis, as well as drastic systemic inflammatory responses, accompanied by more severe
lung injury. Despite the difference in the two models of acute pancreatitis, we discovered the
significant therapeutic role of RvD1 in both models no matter the presence or absence of LPS,
though it is a little more protective in the severe form of the disease. Thus, RvD1could be a
representative agent of a novel class of drugs to be proposed for an innovative treatment of severe acute pancreatitis.
13
severe acute pancreatitis caused a marked reduction in the level/activity of the markers of
pancreatitis severity. In this study, RvD1 treatment decreased serum lipase and amylase activity in
experiment pancreatitis. The results of the present study also show that cerulein and LPS caused a
significant enhancement in the serum levels of TNF-α and IL-6. These inflammatory cytokines are two of the principal mediators of the acute-phase response, and they have been suggested as
markers for predicting the severity of acute pancreatitis (19, 22). In view of the well established
anti-inflammatory properties of RvD1 (2, 34), in this study, the administration of RvD1
significantly inhibited the production of TNF-α and IL-6. There are also evidences demonstrated
that blockades of these inflammatory cytokines attenuate the disease process in experimental pancreatitis (3, 25). Furthermore, TNF-α and IL-6 are basic regulators of all neutrophil functions
and MPO is a well known marker of neutrophil infiltration in inflammatory disease (10, 37). In the
present study, cerulein and LPS stimulation caused enhanced MPO levels in both pancreatic and
lung tissue, while RvD1 treated mice showed a significant decrease of the enzymatic activity, thus
suggesting a reduced recruitment of neutrophils inside the pancreatic and lung tissue. Both biochemical and molecular data very well correlated with the histological results. Indeed, in
pancreas samples obtained from cerulein and LPS injected mice, we observed a marked edema, an
increased neutrophil infiltration, local necrosis and a high degree of vacuolization those were
abated by treatment with RvD1. Moreover, cerulein and LPS induced lung injury characterized by
alveolar congestion, obvious hemorrhage, infiltration of neutrophil in air space and increased
thickness of the alveolar wall those were also attenuated by treatment with RvD1.The effects of RvD1 were confirmed in cerulein alone induced mild acute pancreatitis. Therefore, our findings
14
The signaling pathway responsible for the role of RvD1 in regulating inflammatory response
during the course of acute pancreatitis has been of interest. One important signaling molecule,
NF-κB, was identified as an important regulator of the expression of many inflammatory
mediators in the pancreas (5). There is an emerging body of evidence which suggests that NF-κB plays an important role in the early stage of acute pancreatitis, and that inhibiting this transcription
factor reduces the disease severity (7, 29, 31). Most researchers agree that blocking NF-κB
activation is beneficial in acute experimental pancreatitis (13, 28). Here, we show that NF-κB
activation gradually increased after induction of pancreatitis, especially in cerulein and LPS
induced severe acute pancreatitis, and positively correlated with an increase in serum pro-inflammatory cytokines, serum amylase and lipase, as well as the influx of inflammatory cells
into the pancreas. It is remarkable that recent researches have shown RvD1 is involved in the
regulation of NF-kB activation in the context of inflammation. Wang et al. demonstrated that
RvD1 markedly inhibited the activation of NF-κB and mitogen-activated protein kinases (MAPKs)
in a mouse model of LPS-induced acute lung injury (42). Consistent with their findings, Liao et al. reported that RvD1 attenuate lung inflammation of LPS-induced acute lung injury by suppressing
NF-κB activation through a mechanism partly dependent on peroxisome proliferator-activated receptor gamma (PPARγ) activation (21). Chen et al. reported that RvD1 inhibited endotoxin-induced NF-κB activation and suppressed inflammation in LPS-induced kidney injury
(4).
In line with these observations, our data showed that RvD1 significantly inhibited both the cerulein and cerulein in combination with LPS induced NF-κB activation in experiment
15
as neutrophil infiltration in both pancreas and lung were reduced, ameliorating the acute
pancreatitis and associated lung injury.
Furthermore, Wang et al. also showed that RvD1 has a therapeutic effect 8 hours after LPS
administration (43). Indeed, our further data confirmed that therapeutic treatment of RvD1 4 h after induction of acute pancreatitis also significantly reduced the severity of experimental acute
pancreatitis. Mice treated with therapeutic RvD1 showed significant decrease of digestive enzyme
activity and pancreatic MPO activity, as well as the histological results. In pancreas samples
obtained from cerulein and LPS injected mice, we observed a marked edema, an increased
neutrophil infiltration, local necrosis and a high degree of vacuolization those were abated by therapeutic treatment with RvD1 4 h after induction of acute pancreatitis. This data suggested that
RvD1 has a therapeutic effect in cerulein induced experimental acute pancreatits even through the
pancreatic inflmmation has been originated.
In conclusion, our data demonstrates that RvD1 is capable of improving injury of pancreas and
lung, and exerting anti-inflammatory effects may through the inhibition of NF-κB activation in experimental acute pancreatitis in mice, with even more notable protective effect in severe acute
pancreatitis. Therefore, our present findings provide the potential for the development of an
16 Grants
This study was supported by grants from the National Natural Science Fund of China (
NSFC key
project 30830100 and projects 81170439, 81470886, 81500486
) and The Project Sponsored by the Scientific Research Foundation for the Returned Overseas Chinese Scholars,State Education Ministry（no.20101174-4-2）.
Conflict of interest: No conflicts of interest, financial or otherwise, are declared by the authors.
Authorship: Yong Liu, Fei-Wu Long, Ke-Ling Chen, Hong-Wei Yang, Zhao-Yin Lv and Bin
Zhou performed experiments; Yong Liu and Dan Zhou analyzed data and contributed to the
writing; Zhi-Hai Peng and Xiao-Feng Sun edited and revised the manuscript; Yuan Liand
Zong-Guang Zhou conceived and designed the study and approved the final version of the
17 Figure legends
Figure 1.
Effects of RvD1 on histological changes in experimental acute pancreatitis. The histological examination was done at 8h and 24h after the first injection of cerulein.Representative micrographs of H&E-stained pancreatic sections at the indicated times are shown. Bar indicates 50 μm.
Acute pancreatitis was induced by cerulein (Cer) with the
presence or absence of LPS. Controls were injected with normal saline. RvD1
(300ng/mouse) was administered 30 min before the first injection of cerulein.
Figure 2. Effects of RvD1 on the severity of experimental acute pancreatitis. Serum amylase (A)
and lipase (B) activity were measured at 8h and 24h after the first injection of cerulein. MPO activity (C) in pancreatic tissue was measured at 8h and 24h after the first injection of cerulein,
data are expressed as % of control in each group. Histological changes of pancreatic injury at 8 h
and 24h was scored as shown (D). Results are expressed as means with the SEM of at least three
separate experiments with statistical significance at *p<0.05.
Figure 3. Effects of RvD1 on inflammatory cytokines in experimental acute pancreatitis. The
inflammatory cytokines TNF-α (A) and IL-6 (B) in the serum were determined at 8h and 24h after
the first injection of cerulein using Luminex assay. Results are expressed as means with the SEM
of at least three separate experiments with statistical significance at *p<0.05.
Figure 4. Effects of RvD1 on NF-κB activation in experimental acute pancreatitis. The activation
of NF-κB was determinate by detecting the levels of NF-κB p-65 subunit in the nucleus using
western blot analysis (A). Quantification of NF-κB p-65 subunit expression (B) in pancreatic tissue at 8 h and 24h after the first injection of cerulein was shown. Data are expressed as
18
three separate experiments with statistical significance at *p<0.05.
Figure 5. Effects of RvD1 on histological changes in acute pancreatitis associated lung injury.
The histological examination was done at 8 h and 24h after the first injection of cerulein.
Representative micrographs of H&E-stained lung sections at the indicated times are shown. Bar indicates 50 μm. Acute pancreatitis was induced by cerulein (Cer) in the presence or absence of
LPS. Controls were injected with normal saline. RvD1 (300ng/mouse) was administered 30 min
before the first injection of cerulein.
Figure 6. Effects of RvD1 on severity of acute pancreatitis associated lung injury. MPO activity
(A) in lung tissue was measured at 8h and 24h after the first injection of cerulein, data are expressed as % of control in each group. Histological changes of lung injury at 8 h and 24h was
scored as shown (B). Results are expressed as means with the SEM of at least three separate
experiments with statistical significance at *p<0.05.
Figure 7. Therapeutic effects of RvD1 on histological changes in experimental acute pancreatitis.
The histological examination was done at 24h after the first injection of cerulein. Representative micrographs of H&E-stained pancreatic sections are shown. Bar indicates 50 μm. Acute
pancreatitis was induced by cerulein (Cer) with the presence or absence of LPS. Controls were
injected with normal saline. RvD1 (300ng/mouse) was administered 4h after the last injection of
cerulein.
Figure 8. Therapeutic effects of RvD1 on the severity of experimental acute pancreatitis. Acute
pancreatitis was induced by cerulein (Cer) with the presence or absence of LPS. Controls were injected with normal saline. RvD1 (300ng/mouse) was administered 4h after the last injection of
19
of cerulein. MPO activity (C) in pancreatic tissue was measured at 24h after the first injection of
cerulein, data are expressed as % of control in each group. Histological changes of pancreatic
injury 24h was scored as shown (D). Results are expressed as means with the SEM of at least
20
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