Zhang, Wei MD*; Gao, Jun MD, PhD*†; Zhao, Tao MD*; Wu, Wenbin*; Bai, Yu MD†; Zou, Duowu MD, PhD*†; Li, Zhaoshen MD, PhD*†
Chronic pancreatitis (CP) is a disease with a succession of inflammatory infiltration and necrosis followed by fibrosis. The aims of treatment are to relieve symptoms, improve pancreas function, and reduce the risk of complications. Therapeutic strategies to treat CP are mostly symptomatic and supportive. Long-standing pain, a dominant feature of CP, is the core challenge for clinical physicians.1
Oral analgesic drugs, especially nonsteroidal anti-inflammatory drugs (NSAIDs), are widely prescribed for the treatment of pain in CP.1 Selective cyclooxygenase (COX) 2 inhibitor rofecoxib can inhibit chronic inflammatory changes and subsequent fibrosis in CP,2 which suggests it can act as a proactive choice rather than simply a painkiller. However, some studies indicated that there might be a moderately increased risk of vascular events even with short-term use.3 Nonselective COX inhibitors, although not specific to inflamed tissue, can suppress pain effectively. Cyclooxygenase 2 activity can be inhibited as well, suggesting NSAIDs might also play a protective role of pancreatic fibrosis. Besides the activity of COX-2, there are other matters to consider in the setting of CP. Nonsteroidal anti-inflammatory drugs induced the development of oxidative stress (OS)4 which was one of the most important mechanisms in fibrosis progression of CP. In addition, bacterial endotoxins, such as lipopolysaccharide (LPS), are known as a trigger factor in the initiation and progression of fibrosis in alcoholic CP.5 Once the intestinal barrier function is damaged by nonselective NSAIDs, translocation of bacteria, endotoxemia, and subsequent changes of local and systemic inflammatory response might aggravate the progress of CP.5,6
Considering nonselective COX inhibitors' pathogenic and antipathogenic roles in CP, their integrated effect on CP should be identified. Therefore, we analyzed the effect of naproxen on fibrosis and pain in trinitrobenzene sulfonic acid (TNBS)-induced CP rats. We found that high-dose naproxen (40 mg/kg per os [PO]) aggravated chronic inflammatory changes and subsequent fibrosis and decreased abdominal thermal withdrawal latencies in CP rats.
MATERIALS AND METHODS
Animals and Induction of CP
Male Sprague-Dawley rats (200-250 g) were used in all experiments. All procedures performed on animals were approved by the Institutional Animal Use and Care Committee of The Second Military Medical University, Shanghai, China. Animals were given free access to drinking water and standard food pellets until 12 hours before induction of CP, at which point food was withdrawn. Chronic pancreatitis was induced as previously described.7 Briefly, the common bile duct was closed temporarily near the liver with a small vascular clamp. A blunt 24-gauge needle was inserted into the duodenum and guided through the papilla into the duct and was secured with suture. A 2% solution (0.5 mL) of TNBS (Sigma-Aldrich, St Louis, Mo) in 10% ethanol in phosphate-buffered saline (0.1 mol/L), pH 7.4, was infused into the pancreatic duct over a period of 30 minutes. After 30 minutes, needle and suture line were removed, the hole in the duodenum was sutured, and the vascular clamp was removed restoring the bile flow, whereas in the control group, pancreases were just dragged lightly.
Drugs and Experimental Design
Naproxen (Zhejiang Charioteer Pharmaceutical Co, Ltd, Taizhou, China) was suspended in 0.5% carboxymethyl cellulose (CMC; Sigma-Aldrich, St Louis, Mo) solution. Naproxen was administered PO (directly injected into mouth) or intraperitoneally.8 All the doses were administered in a constant volume of 0.5 mL/100 g of body weight. The doses of naproxen were selected according to Kumar et al.9
The animals were randomly divided into 8 groups of 8 animals each (Table 1). To observe the effect of CP and naproxen, dead rats were replaced with new ones to maintain 8 animals in each group. Chronic pancreatitis was induced in groups A1 to A6. Groups C1 and C2 underwent surgical procedure, but pancreases were just dragged lightly. Because pancreatic fibrosis develops 3 weeks after TNBS administration, monomorphonuclear and polymorphonuclear cell infiltrations decline gradually by the sixth week7; naproxen treatment was started 2 weeks after the induction of pancreatitis for 3 weeks. Groups A1 and A2 received naproxen per se 20 and 40 mg/kg (PO) once daily for 3 weeks, respectively. Groups A3 and A4 received naproxen per se 20 and 40 mg/kg (intraperitoneally [IP]) once daily for 3 weeks, respectively. Groups A5 and A6 (vehicle-treated CP group) received 0.5% CMC solution (groups A5, PO and A6, IP) daily for 3 weeks. Groups C1 and C2 and the vehicle-treated control group received 0.5% CMC solution (group, C1, PO and C2, IP) daily for 3 weeks. At the end of 3 weeks of treatment, the rats underwent behavioral tests, then they were killed, and blood or tissue samples were collected for analysis.
For histopathological examination, pancreatic tissue was fixed in 10% buffered formalin and embedded in paraffin; 4-μm sections were stained with hematoxylin and eosin (H&E). All specimens were scored by 2 pathologists who were unaware of the origin of the specimens. Evaluation of the pancreas was performed according to van Westerloo et al.10 Three pancreas sections were randomly selected and scored from each rat. Median scores were calculated. Areas of abnormal architecture were defined and quantified as 0, absent; 1, rare; 2, minimal (<10%); 3, moderate (10%-50%); or 4, severe (>50%). Within these areas, the presence of glandular atrophy and fibrosis were each scored as 0, absent; 1, minimal (<0%); 2, moderate (10%-50%); and 3, severe (>50%). The content of inflammatory cells (mainly neutrophils) and edema were scored on a 0-to-4 scale.
Assessment of Pancreatic Fibrosis
Morphometry of Van Gieson-Stained Sections
The amount of intrapancreatic collagen was quantified using image analysis of standard Van Gieson (VG)-stained pancreatic sections.11 Quantitative analysis of collagen was performed by morphometric analysis as previously described.10 Three digitized pictures of each pancreatic section were viewed through an Olympus BH2 microscope (Olympus Corporation, Tokyo, Japan). The total amount of collagen stained on each submitted section was calculated by Medical Image Quality Analyze System (MIQAS) (Qiuwei Biotechnological Co Ltd, Shanghai, China). Briefly, the pancreas was distinguished from the background according to a difference in light density, and a measurement of the total pancreatic tissue area was performed. In the second step, the amount of collagen (stained in red) was measured and was finally expressed as a percentage of the total pancreatic surface.
Intrapancreatic hydroxyproline was quantified using the detection kit according to Reddy and Enwemeka12 and the manufacturer's instructions (Jiancheng Bioengineering Institute, Nanjing, China). Hydroxyproline content is expressed as micrograms of hydroxyproline per gram pancreatic tissue.
Activated pancreatic stellate cells (PSCs) were demonstrated by immunostaining for α-smooth muscle actin (α-SMA). α-Smooth muscle actin immunostaining was performed on formalin-fixed, paraffin-embedded tissues as described previously.13 Stained slides were examined and scored under a photomicroscope by 2 blinded observers using MIQAS medical image quantitative analyzer system. Positive areas of α-SMA (+) cells in pancreatic sublobular and lobular atrophies were calculated.12 Positive cells located in or near blood vessel were ignored.
Abdominal thermal hyperalgesia was measured by the latency of withdrawal to thermal stimuli in the noxious range as previously described.14 The animals were placed in Plexiglas boxes on an elevated glass plate through which a high-intensity light beam was shone. After a 60-minute habituation period, a radiant heat stimulus was applied by concentrating a beam of light through a hole in the light box onto the abdominal area. The light beam and timer were immediately stopped when the animal withdrew, allowing the measurement of time between the start of the light beam and the withdrawal event. Forty-five minutes were allowed between each trial, and tests were repeated 3 times. A withdrawal event to radiant heat applied to the abdomen was defined as abdominal withdrawal (either abdominal musculature contraction or lifting of the abdomen through postural adjustment) accompanied by head turning toward the stimuli and licking of the abdominal area. The experimenter was blinded to the type of treatment that the animals had received.
Detection of Tumor Necrosis Factor α in Blood Serum
Blood samples were drawn via the left ventricle. The serum tumor necrosis factor α (TNF-α) concentration was determined by enzyme-linked immunosorbent assay following the manufacturer's instructions (R&D Systems China Co Ltd, Shanghai, China). The cytokine contents in the serum were expressed as picograms per milliliter.
Data were expressed as mean ± SD and were analyzed using SPSS PC version 13.0 (SPSS Inc, Chicago, Ill). Independent samples t test or 1-way analysis of variance (ANOVA) followed by Tukey honestly significant difference as a post hoc test was used to evaluate the differences of the groups. General linear model (repeated measures) was used to evaluate differences of the withdrawal latencies after thermal stimulation of the groups. Mann-Whitney U test was used to evaluate the differences of categorical values (histological evaluation and positive areas of VG staining, the expression of α-SMA). P < 0.05 was considered statistically significant.
Model of TNBS-Injected CP
Eighty percent of the animals in groups A5 and A6 survived the experiment period, 75% in groups A1 and A3, 62.5% in group A4, and only 50% in groups A2. The rats were also observed weekly for weight changes. Rat weights increased slightly after the injection of TNBS and stabilized after the second week of cannulation. High-dose (40 mg/kg) naproxen-treated animals had dark stools but maintained their dietary intake. In the first week of naproxen administration, significant weight reduction was observed in group A2 (24 ± 14.75 g), whereas in group A5, rat weights increased (33.13 ± 12.52 g), and the difference in weight changes between A2 and A5 was significant (P < 0.001; Fig. 1).
Effect of Naproxen Treatment on Systemic Inflammation
Only jaundiced CP rats showed a higher level of serum TNF-α compared with the control groups (Fig. 2A; P < 0.05; 169.61 ± 39.95 vs 47.62 ± 14.72 pg/mL). Naproxen treatment did not affect serum TNF-α level, irrespective of its doses or routes of administration (Fig. 2B).
Effect of Naproxen Treatment on Pancreatic Damage
All rats injected with TNBS (groups A1-A6) displayed histopathological features of CP at the time of killing, as reflected by glandular atrophy, fibrosis, edema, and inflammatory cell infiltrates (Table 2 and Figs. 3E, F; #P < 0.05 vs control groups). Rats with CP that were treated with high-dose naproxen (40 mg/kg PO) showed more severe pancreatic damage, especially fibrosis and inflammation (Table 2 and Figs. 3A, B, D; *P < 0.05 vs group A5 or A6). Low-dose (20 mg/kg) and high-dose naproxen treatments administered by intraperitoneal injections did not affect pancreatic damage in CP rats (Table 2).
Effect of Naproxen Treatment on Pancreatic Fibrosis
To evaluate the degree of fibrosis in the pancreas, VG-stained pancreas sections were analyzed using computer-assisted digital analysis, and pancreatic hydroxyproline content was quantified. Positive areas of VG-stained sections (group A5 vs C1 and group A6 vs C2) in the CP groups were higher compared with the control groups (Figs. 4A, D; 5.77 ± 1.65% vs 2.1 ± 0.39% and 5.72 ± 1.35% vs 2.3 ± 0.40%, respectively; P < 0.05).
Hydroxyproline content (group A5 vs C1 and group A6 vs C2) also increased after induction of CP (Fig. 5; 534.83 ± 234.34 vs 335.67 ± 60.55 μg/g and 503.85 ± 103.89 vs 320.36 ± 57.6 μg/g, respectively; P < 0.05).
High-dose naproxen administered orally (group A2) increased the positive areas of VG-stained sections (Figs. 4B, D; 8.89 ± 0.78% vs 5.77 ± 1.65%; P < 0.05). Hydroxyproline content was also higher in group A2 compared with group A5 (Fig. 5; 821.67 ± 274.96 vs 534.83 ± 234.34 μg/g; P < 0.05).
The routes of administration affected pancreatic collagen content. The positive area of VG-stained sections in group A2 was higher than in groups A3 and A4 (Figs. 4C, D; 8.89 ± 0.78 vs 4.51 ± 1.07 and 6.43 ± 1.94, respectively; P < 0.05). Hydroxyproline content was also higher in group A2 compared with groups A3 and A4 (Fig. 5; 821.67 ± 274.96 vs 557.04 ± 166.99 and 559.38 ± 178.38 μg/g, respectively; P < 0.05).
Immunohistochemistry for α-SMA
α-Smooth muscle actin (+) periacinary-activated PSCs were scored in the 8 groups. No α-SMA (+) expression was observed in normal parenchyma of the pancreas except around the vascular structures in sham-operation groups.
The number of α-SMA (+) PSCs in group A2 were higher than group A5 (Fig. 6; 42 ± 15% vs 21.8 ± 11.55, P < 0.05). No difference was found between groups A4 and A6.
All rats were tested at the fifth week after operation. The observer performing the behavioral testing was blind to the animals' condition. The rats injected with TNBS (group A5 vs C1 and group A6 vs C2) demonstrated increased sensitization to thermal stimuli on the abdominal area, that is, decreased withdrawal latencies (Fig. 7; 6.52 ± 1.49 vs 10.67 ± 5.36 seconds and 6.67 ± 1.61 vs 10.98 ± 4.25 seconds, respectively; P < 0.05). High-dose naproxen treatment (group A2 vs A5 and group A4 vs A6) decreased thermal withdrawal latencies in CP rats (Fig. 7; 5.02 ± 1.54 vs 6.52 ± 1.49 seconds and 5.17 ± 1.31 vs 6.67 ± 1.61 seconds, respectively; P < 0.05). No change in the withdrawal latencies was observed in CP rats treated with low-dose naproxen (Fig. 7; P > 0.05).
In this study, we demonstrated that (1) high-dose naproxen (40 mg/kg) administered orally aggravated pancreatic damage and fibrosis in TNBS-induced CP rats; and (2) instead of relieving pain, high-dose naproxen treatment reduced the withdrawal latencies to heat stimuli in CP rats.
One of the most important mechanisms in fibrosis of CP is OS. Pancreas has a low antioxidant capacity; TNBS injection into the pancreatic duct can induce OS.7 In the course of pancreatitis, production of reactive oxygen species can activate nuclear factor-κB15 and stellate cells16 in the pancreas, which aggravates pancreatic damage and fibrosis. It has also been demonstrated that several antioxidant drugs not only can improve pancreatic tissue OS and fibrosis13,17 but also can alleviate pain in CP.18 Convincing evidence was found that naproxen could induce OS in the vasculature and liver. Nonsteroidal anti-inflammatory drugs, including naproxen, could increase nicotinamide adenine dinucleotide phosphate oxidases and superoxide content in the aorta and heart.4 Moreover, naproxen also induced the OS in the liver microsomes and the isolated hepatocytes of rats that could impair biliary excretion.19 Thus, OS or impaired biliary excretion induced by high-dose naproxen might aggravate pancreatic damage and fibrosis.
As a proinflammatory enzyme, inducible COX-2 is considered the key target to treat inflammatory diseases. Cyclooxygenase 2 inhibitors have also been demonstrated to play an anti-inflammatory role in acute or chronic inflammation. Cyclooxygenase 2 is expressed in human tissues of CP20 and in activated PSCs; it regulates the responses of PSCs to multiple cytokines.21 In addition, celecoxib plays antifibrogenic effects in both bile duct ligation and thioacetamide rats through a proapoptotic effect on HSCs.22 In view of this, COX inhibitor may exert a beneficial role in CP. In the WBN/Kob rat, chronic inflammatory changes and subsequent fibrosis can be inhibited by specific COX-2 inhibitor rofecoxib; apart from this, migration of macrophages is COX-2 dependent.2 Nonetheless, COX-2 also has anti-inflammatory properties. In carrageenin-induced pleurisy model, COX-2 protein expression had 2 peaks. Cyclooxygenase 2 may play a proinflammatory role during the early phase but may aid resolution during the later phase by generating an alternative set of anti-inflammatory prostaglandins.23 In this study, we started naproxen treatment at the third week after TNBS injection, at which time point the severe acute necrotizing pancreatitis had resolved; the inhibited COX activity may be harmful to inflammation resolution. Therefore, low-dose naproxen did not display the therapeutical effect, and high-dose naproxen treatment aggravated pancreatic damage and fibrosis (Fig. 3) and reduced the thermal pain threshold instead (Fig. 7).
In addition, one of the most important adverse effects of NSAIDs is gastrointestinal mucosal damage. Oral administration of high-dose naproxen (40 mg/kg) can destroy the intestinal barrier, such as producing hemorrhagic damage,24 increasing gastrointestinal permeability.25 Once the intestinal barrier was destroyed, bacteria translocation and endotoxemia may occur subsequently. Serum endotoxin concentrations correlated with the severity of clinical acute pancreatitis.26 In CP, PSCs that express LPS receptors TLR4 and CD14 are activated by endotoxin both in vivo and in vitro.4 Convincing evidence is provided that endotoxin is a trigger factor for alcohol-related CP.4 Repeated administrations of LPS in alcohol fed rats could lead to acinar atrophy and significant fibrosis in the pancreas. In our study, CP rats treated with high-dose naproxen (40 mg/kg PO) had severe melena and anemia especially in the first 2 weeks of administration, implying the possibility of appearance of damaged intestinal barrier. Gastrointestinal injury by high-dose naproxen treatment is the result of topical effects instead of a systemic effect according to the results of serum TNF-α level (Fig. 2). Nevertheless, we failed to find differences in plasma diamine oxidase activity and level of endotoxin between groups A2 and A5 (data are not shown). Our chosen test time was the third week of naproxen treatment (the end point of the whole study) at which time point the intestinal damage may disappear or alleviate in a process of mucosal adaptation.
We are aware the current study has a number of limitations. The exact mechanisms of naproxen's pathogenic role in fibrosis progression of CP are not completely understood. In this study, we chose the TNBS-induced CP model. Infusion of TNBS into the pancreatic duct could provoke damage and scarring of the small intrapancreatic ducts whose pathological changes were consistent with small duct occlusion disease mimicking the human counterpart.7 It is not known whether other CP animal models treated with naproxen will manifest the same effects. In addition, pancreatic fibrosis in this model lasts for 3 weeks; the progression of pancreas inflammation and fibrosis after high-dose naproxen treatment continuously needs to be explored. Moreover, the long-term consequences of high-dose use of NSAIDs in CP patients needs to be observed.
In conclusion, we found that high-dose naproxen treatment (40 mg/kg PO) would aggravate pancreatic damage and fibrosis in TNBS-induced CP models, which suggests the potential risk of long-term use of NSAIDs as analgesic in clinical practice with CP.
The authors thank Professor Jenyu Wei (University of California at Los Angeles) for his instruction of manuscript revision. The authors are also thankful to Dr Lihua Ju (University of Fudan, Shanghai, China) and Dr Xiaohua Man (Changhai Hospital, Second Military Medical University, Shanghai, China) for histological and immunohistochemical analyses.
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