Effect of gentiopicroside on experimental acute pancreatitis induced by retrograde injection of sodium taurocholate into the biliopancreatic duct in rats

Fitoterapia 102 (2015) 127–133

Contents lists available at ScienceDirect

Fitoterapia journal homepage: www.elsevier.com/locate/fitote

Effect of gentiopicroside on experimental acute pancreatitis induced by retrograde injection of sodium taurocholate into the biliopancreatic duct in rats Jian Lv, Wei-Liang Gu ⁎, Chang-Xun Chen School of Pharmacy, Shanghai University of Traditional Chinese Medicine, 1200 Cailun Road, Shanghai 201203, PR China

a r t i c l e

i n f o

Article history: Received 20 January 2015 Accepted in revised form 28 February 2015 Available online 7 March 2015 Chemical compounds studied in this article: Gentiopicroside (PubChem CID 88708) Sodium taurocholate (PubChem CID: 23666345) Keywords: Gentiopicroside Acute pancreatitis Sodium taurocholate Inflammation NF-κB p65

a b s t r a c t Gentiopicroside (otherwise known as Gentiopicrin), one of the main active ingredients from the traditional Chinese herb medicine Gentiana manshurica Kitag, presents the effect of attenuating acute pancreatitis in rats. The experimental acute pancreatitis was made by retrograde injection of sodium taurocholate into the biliopancreatic duct in rats. Gentiopicroside was given orally and it markedly reduced the pancreatitis-evoked increase of serum amylase and lipase activity, decreased the pancreas mass/body mass index, tissue water content, TNF-α and IL-1β concentrations, and attenuated the histopathological changes and NF-κB p65 protein expression in pancreatic tissue. The results indicate that the function of gentiopicroside on acute pancreatitis may be related to inhibiting the release of inflammatory mediators and NF-κB p65 protein expression. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Acute pancreatitis (AP) is one of the common acute abdominal ailments in clinical practice, characterized by abdominal pain and elevated activity of pancreatic enzymes in the blood, which carries considerable morbidity and mortality. AP can be divided into two groups in the clinic, about 80% to 85% develop an edematous pancreatitis, which is relatively easy to cure; the other 15% to 20% of patients develop a severe hemorrhagic necrotizing pancreatitis, some 10% to 20% of these die of multiorgan failure despite intensive careful treatment or surgery. Effective specific treatment strategies are limited due to a relatively poor understanding of its exact pathogenesis [1].

⁎ Corresponding author at: Department of Pharmacology, Shanghai University of Traditional Chinese Medicine, No. 1200 Cailun Road, Shanghai 201203, PR China. Tel./fax: +86 21 513 222 13. E-mail address: [email protected] (W.-L. Gu).

http://dx.doi.org/10.1016/j.fitote.2015.03.002 0367-326X/© 2015 Elsevier B.V. All rights reserved.

Gentiopicroside (PubChem CID 88708, Fig. 1) is one of the main active ingredients from the traditional Chinese herb medicine Gentiana manshurica Kitag, which has been widely used for treating jaundice, chronic liver disease, eczema, pain in the hypochondrium etc [2,3]. Gentiopicroside has a relatively rich source and exhibits obvious anti-inflammatory effects [2]. It can protect against chemically and immunologically induced hepatic injuries by inhibiting the production of tumor necrosis factor, promote bile secretion [3,4], produce significant analgesic effects against persistent inflammatory pain stimuli in mice [5], and inhibit concentration-dependently the spontaneous contractions of isolated guinea pig ileum by interfering with calcium influx into the smooth muscle cells [6]. Although the mechanism of AP has not been established, there is emerging evidence that up-regulation of inflammatory mediators, such as cytokines and chemokines, is central to the process [7]. And it is well known that in some cases, acute

128

J. Lv et al. / Fitoterapia 102 (2015) 127–133

The animals received humane care and had free access to a standard diet and drinking water. The animal experiment was approved by the Animal Care and Use Committee of Shanghai University of Traditional Chinese Medicine, and conformed to the Guide for Care and Use of Laboratory Animals, published by U.S. National Institutes of Health (NIH Publication No. 85-23, revised 1996). 2.3. Acute pancreatitis model in rats

Fig. 1. Structural formulas of gentiopicroside.

pancreatitis is induced by biliary, pancreatic duct obstruction or bile reflux. The bile reflux into the pancreas may lead to acute pancreatic inflammation accompanied by severe pain. Our earlier studies had shown that gentiopicroside could promote bile secretion and inhibit inflammation [2,3], we speculated that gentiopicroside should have some beneficial effects on AP. Therefore, a pancreatitis model of rats caused by retrograde injection of sodium taurocholate into the biliopancreatic duct was designed to investigate the effect of gentiopicroside. 2. Materials and methods 2.1. Drugs and reagents Gentiopicroside (purity: greater than 98%) was provided by the Department of Phytochemistry, Shanghai University of Traditional Chinese Medicine. Sodium taurocholate was purchased from Shanghai Kai Yang Biotechnology Co., Ltd. The amylase and lipase kits were purchased from Nanjing Jiancheng Bioengineering Co., Ltd, China. Tumor necrosis factor (TNF)-α and interleukin (IL)-1β radioimmunoassay kits were from Puer Weiye Beijing Bioengineering Co., Ltd, China. Nuclear factor kappa-B (NF-kB) p65 immunohistochemical kit was from Zhongmei Bioengineering Co., Ltd.

Acute pancreatitis was induced by using the improved Aho's method [8]. Male Sprague–Dawley rats weighing 220–250 g were fasted for 12 h before the experiments, but had free access to water. The animals were anesthetized by intraperitoneal injection of 40 mg/kg sodium pentobarbital, laid and fixed on table, routinely shaved, disinfected and draped. Laparotomy was performed through a midline incision. The pancreas was disclosed, the duodenal papilla inside the duodenum duct wall was identified, and then a No. 5 needle was used to drill a hole in the avascular area of mesentery. A catheter was inserted into the duodenal cavity via the hole, toward the direction of the papilla in a retrograde way. A microvascular clamp was used to nip the duct end temporarily and meanwhile another one was used to temporarily occlude the common hepatic duct at the confluence of the hepatic duct. After connecting the tube end with the transfusion converter, 3% sodium taurocholate (0.1 mL/100 g) was given by retrograde transfusion using microinjection pump (Shanghai Anjie Electronic Equipment Co., Ltd, China) at a speed of 0.2 mL/min. 4 to 5 min after injection, the microvascular clamp and the catheter were removed, and the hole in the lateral wall of duodenum was sutured. Thereafter, all devices were removed and the laparotomy was closed in two layers. In the sham operation group, only exploratory laparotomy was performed, without retrograde transfusion of 3% sodium taurocholate. In this study, gentiopicroside was given to the treated groups by gavage an hour before and three hours after the induction of acute pancreatitis in rats at 100 mg/kg, and 400 mg/kg respectively. The given doses of gentiopicroside were achieved from our previous studies, which demonstrated obvious effects on anti-inflammation and bile secretion promotion [2,4]. The sham operation group and the model control group were given double-distilled water of equivalent volume at the corresponding time points before and after the operation. 2.4. Biochemical measurements Blood was collected from the abdominal aorta 12 h after the induction of acute pancreatitis, and centrifuged at 2325 g and 4 °C, for 10 min. The supernatant was collected for the measurement of serum amylase and lipase activity according to the supplier's specifications. Amylase and lipase activity was expressed as U/L.

2.2. Animals

2.5. Pancreas mass index measurements

Male Sprague–Dawley rats weighing 220–250 g were provided by Shanghai Slac Laboratory Animal Co. Ltd. All animals were maintained in a 12 h light:12 h dark cycle room with the temperature at 23 ± 1 °C, and the humidity of 40 ± 5%.

After the exsanguination of the rats, the pancreas was quickly taken out, separated from the fat and lymph nodes, then weighed. The pancreas mass index was expressed as the ratio of pancreas mass/body mass.

J. Lv et al. / Fitoterapia 102 (2015) 127–133

2.6. Water contents of the pancreas tissue measurements To assess tissue edema, a portion of the pancreatic tissue was taken after weighing of the whole pancreas, weighed and dried in a 80 °C oven for 24 h until the weight was constant, and the water content [(wet weight − dry weight) / wet weight] of the pancreas was obtained.

129

was scored as follows: 1, b25%; 2, 25–50%; 3, 50–75%; and 4, N75%. The final score was determined by adding the intensity to extent of positivity scores, yielding a range from 1 to 7 Scores; 1–2 were defined as negative expression (−), 3 as weak staining pattern (+), 4 as moderate staining (++), and N5 as strong staining (+++). 2.10. Statistical analysis

2.7. TNF-α and IL-1β concentration measurements Another portion of pancreatic tissue sample was homogenized in cold NaCl solution (0.9%) in a glass homogenizer, then centrifuged, and the supernatant was used for TNF-α and IL-1β assays. TNF-α and IL-1β were measured using radioimmunoassay kits according to the instructions (Beimian Dongya Biotechnique Institute, Beijing, China), and expressed as pg/mg prot. 2.8. Histopathological evaluation For histological evaluation, the pancreas was taken out 12 h after the induction of pancreatitis, fixed in 10% formaldehyde, and embedded in paraffin. Full-length (4 μm) sections were taken and stained with hematoxylin and eosin for histological evaluation. The degree of tissue damage was examined in a standard fashion by a single pathologist, using an established scoring system [9]. Edema, inflammation, hemorrhage and necrosis of the pancreas were each graded from 0 to 4. Edema: 0 = absent, 1 = focally increased between lobules, 2 = diffusely increased between lobules, 3 = tense acini and widely separated lobules, 4 = gross lobular separation; inflammation: 0 = absent, 1 = around ductal margins, 2 = in parenchyma (b50% of lobules), 3 = in parenchyma (51%– 75% of lobules), 4 = massive collections and abscesses; hemorrhage: 0 = absent, 1 = blood in parenchyma (b25%), 2 = blood in parenchyma (25%–50%), 3 = blood in parenchyma (50%–75%), 4 = blood in 100% of lobules; necrosis: 0 = absent, 1 = periductal parenchymal destruction, 2 = focal parenchymal necrosis (b 20%), 3 = diffuse loss of lobules (20%– 50%), 4 = severe loss of lobules (N50%). 2.9. Immunohistochemical assay of NF-κB p65 expression in pancreas The pancreas was fixed in 10% formaldehyde, embedded in paraffin, and routinely sliced. The sections were treated according to the supplier's specifications. Briefly, they were baked at 56 °C for 2 h and dewaxed in a routine fashion. Peroxidase was blocked for 15 min with 0.3% H2O2, and then incubated with a primary antibody. Then, the sections were incubated with a secondary antibody over night at 4 °C. At last, freshly prepared diaminobenzidine (DAB) solution was added for coloration and counter-stained with hematoxylin. Positive signals were detected as nucleus staining presenting a yellow color. Routine dehydration and transparence were carried out, followed by neutral gum mounting. The NF-κB p65 protein expression of the pancreas was observed under a light microscope, and the comprehensive assessment was carried out according to the standard described by Meng [10]. The intensity of positivity was scored as follows: 0, negative; 1, light-yellow; 2, brown-yellow; and 3, brown-black. The extent of positivity

The results of the parametric tests were expressed as mean ± standard deviation (mean ± SD). Statistical analysis was performed by one-way analysis of variance (ANOVA) for multiple comparisons, followed by Dunnett's test to evaluate the difference between two groups. Nonparametric values were expressed as ranked data. The significance of differences in the histopathological scores and immunohistochemical scores was assessed by the Ridit test. Values of P b 0.05 were considered statistically significant, and P b 0.01 was considered very significant for all analyses. 3. Results 3.1. Effect of gentiopicroside on activity of serum amylase and lipase in rats As shown in Fig. 2, the activity of serum amylase and lipase was markedly higher 12 h after AP induction in the model group than in the sham operation group(P b 0.01). After the rats were treated with gentiopicroside at high- and low-dose, the activity of serum amylase and lipase significantly reduced (P b 0.01). 3.2. Effect of gentiopicroside on mass index and water content of pancreas in rats The pancreas mass index was markedly greater in the model group than in the sham operation group (P b 0.01). After the rats were treated with high- and low-dose of gentiopicroside, the pancreas mass index was significantly reduced (both P b 0.05, Fig. 3A). The water content of pancreas was significantly higher in the model group than in the sham operation group (P b 0.01). Treatment with a high-dose of gentiopicroside significantly reduced the pancreas water content (P b 0.05, Fig. 3B). 3.3. Effect of gentiopicroside on levels of TNF-α and IL-1β in pancreatic tissue in rats As shown in Fig. 4, the level of TNF-α in pancreatic tissue in the model group was markedly greater than in the sham operation group (P b 0.05). This was markedly lower in the high-dose gentiopicroside treatment group (P b 0.05). The level of IL-1β in the pancreatic tissue in model group was obviously higher than in the sham operation group (P b 0.01). This was lower in both gentiopicroside treatment groups (P b 0.01). 3.4. Effect of gentiopicroside on histopathological changes in rats Histopathological findings were well correlated with the biochemical signs of acute pancreatitis. It was found that in the

130

J. Lv et al. / Fitoterapia 102 (2015) 127–133 Sham operation

Sham operation

Model

Model+gentiopicroside(400mg/kg)

##

**

70000

**

60000 50000 40000 30000

Serum activity of lipase (U/L)

Serum activity of amylase (U/L)

80000

1000

Model+gentiopicroside(100mg/kg)

90000

Model

##

Model+gentiopicroside(100mg/kg)

900

Model+gentiopicroside(400mg/kg)

800 700 600 500

**

400

**

300 200 100 0

20000

A

B

Fig. 2. Effect of gentiopicroside on serum activity of (A) amylase and (B) lipase in rats. Compared with the sham operation group: ##, P b 0.01; compared with the model group: **, P b 0.01.

sham operation group the entire normal glandular architecture was preserved, while in the model group, various pathologic manifestations such as severe edema, substantial inflammatory infiltration, a large area of hemorrhage and necrosis, and lobule outline damage were observed in the pancreatic tissue of the rats (P b 0.05 compared with that in sham operation group). However, these histopathological changes were alleviated in both gentiopicroside treatment groups. The more favorable result was seen in the high-dose group (P b 0.05), where just local neutrophil infiltration was occasional (Fig. 5; Table 1). 3.5. Effect of gentiopicroside on NF-κB p65 expression in pancreas of rats In the sham operation group, a little expression of NF-κB p65 in the pancreas was found. However, the expression of NFκB p65 in the pancreas was significantly increased in the model group compared to that in the sham operation group (P b 0.05). In the gentiopicroside treatment groups, the expression of NFκB p65 in the pancreas was reduced, especially in the high-dose group, which was significantly less than that in the model group (P b 0.05, Fig. 6; Table 2). Sham operation

Sham operation

Model

Model

77

Model+gentiopicroside(400mg/kg)

##

*

0.5 0.4 0.3 0.2 0.1

*

Model+gentiopicroside(100mg/kg)

##

Model+gentiopicroside(400mg/kg)

pancreas water content

Pancreas mass index

Pancreas tissue mass index, in accompaniment with the serum activity of amylase and lipase is an important marker of acute pancreatitis. In our study, the experimental acute pancreatitis was induced by retrograde infusion of sodium taurocholate into the pancreaticobiliary duct. This is the most commonly used model related to AP. The result of our study showed that the pancreas tissue mass index and the activity of serum amylase and lipase were obviously increased, and the morphological abnormalities were significant. Treatment with gentiopicroside demonstrated a beneficial effect on experimentally induced pancreatitis. AP is usually accompanied by well-established inflammatory reactions. These involve a number of inflammatory mediators, which contribute to the initiation and progression of both local pancreatic destruction and systemic manifestations. Besides local pathological injuries, severe acute pancreatitis can lead to systemic inflammatory response syndrome, or even the complication of multiple organ injury, and further multiple organ dysfunction syndromes, resulting in quite high mortality.

Model+gentiopicroside(100mg/kg)

0.7 0.6

4. Discussion

76

*

75 74 73 72 71 70

0

A

B

Fig. 3. Effect of gentiopicroside on pancreas (A) mass index and (B) water content in rats. Compared with the sham operation group: ##, P b 0.01; compared with the model group: *, P b 0.05.

5

#

Sham operation

Model

Model

Model+gentiopicroside(100mg/kg)

4

Model+gentiopicroside(400mg/kg)

3

2

*

1

131

Sham operation

0

A

Concentration of IL-1β (ng/mg prot)

Concentration of TNF-α (ng/mg prot)

J. Lv et al. / Fitoterapia 102 (2015) 127–133

0.25

##

Model+gentiopicroside(100mg/kg) Model+gentiopicroside(400mg/kg)

0.2 0.15

** **

0.1 0.05 0

B

Fig. 4. Effect of gentiopicroside on (A) TNF-α and (B) IL-1β levels in pancreas in rats. Compared with the sham operation group: #, P b 0.05, ##, P b 0.01; compared with the model group: *, P b 0.05, **, P b 0.01.

Various pro-inflammatory cytokines, such as TNF-α and IL1β are currently believed to play an important role in promoting local tissue destruction and remote organ failure in the course of AP [11,12]. The severity of pancreatitis has been shown to correlate with TNF-α and IL-1β levels [13]. Specific treatments that target the reduction of TNF-α and IL-1β levels may reduce the severity of AP. IL-1β is an important factor in host defense and is also an important mediator of various disorders including septic shock, adult respiratory distress syndrome, and multiple organ failure. IL-1β has shown to be a significant cytokine for the development of AP. It plays a crucial role in the release of other inflammatory mediators such as phospholipase A2 (PLA2). IL-1β can

also injure tissue by activating inflammatory mediators such as prostaglandin, leukotriene and platelet activating factor. IL-1β has been shown to trigger firm adhesion of monocytes for vascular migration to the site of infection. The IL-1β promoter region contains binding sites for a number of important transcriptional factors including NF-κB [14]. TNF-α is implicated as an important mediator of the inflammatory response in AP. It activates proline-rich tyrosine kinase 2 to cause cytoskeletal disorganization and NF-κB to cause inflammatory response, and it triggers cell death signaling through divergent mechanisms mediated by protein kinase C. It is able to promote the release of inflammatory mediators such as IL-1β, IL-6, and TXA2 [15,16] to induce activation of the

Fig. 5. Effect of gentiopicroside on histopathological changes in rats. (H&E stain, magnification 200×). (A) Sham operation group; (B) Model group; (C) Model plus gentiopicroside 100 mg/kg group; (D) Model plus gentiopicroside 400 mg/kg group.

132

J. Lv et al. / Fitoterapia 102 (2015) 127–133

Table 1 Effect of gentiopicroside on pathological score in pancreas tissue.

Table 2 Effect of gentiopicroside on NF-κB p65 expression in pancreas tissue.

Group

Dose (mg/kg)

Pathological score 0

1

2

3

4

Sham operation Model# Model plus gentiopicroside Model plus gentiopicroside⁎

– – 100 400

8 0 0 2

0 0 0 6

0 1 4 0

0 5 3 0

0 2 1 0

n = 8. Compared with the sham operation group: #P b 0.05; compared with the model group: ⁎P b 0.05.

neutrophil, release a large number of oxygen free radicals and other pro-inflammatory cytokines [17], and cause granulocyte infiltration, organ damage and destruction of cell membrane stability, leading to pancreatic tissue necrosis. Our study showed that the model rats were accompanied with increased TNF-α and IL-1β in the pancreatic tissue; treatment with gentiopicroside reduced these cytokine levels. These results indicate that the function of gentiopicroside in antagonizing AP correlates well with its inhibition of TNF-α and IL1β levels. Studies in recent years have shown that NF-κB presents high expression state in acute pancreatitis and plays an important role in the onset and turnover of acute pancreatitis together with other inflammatory cytokines [18–20]. The inhibition of NF-κB has been shown to ameliorate the inflammatory effects of pancreatitis [21,22]. NF-κB, a protein with multi-attribute transcription capabilities, occurs normally in the cell as a heterodimer or a homodimer formed by polypeptides P50 and P65. The NF-κB p65 is one of the important composing members of the NF-κB/Rel

Groups

Dose (mg/kg)

Sham operation Model# Model plus gentiopicroside Model plus gentiopicroside⁎

– – 100 400

Rank of NF-κB p65 expression −

+

++

+++

7 0 0 2

1 0 3 5

0 3 5 1

0 5 0 0

n = 8. Compared with the sham operation group: #P b 0.05; compared with the model group: ⁎P b 0.05.

family with a relative molecular weight of 65,000. When resting, it is combined with its inhibitor IκB laying dormant in the cytoplasm where it cannot enter the cell nucleus [23,24]. A series of enzymes can be activated through a signal transduction pathway after the stimulation of TNF-α, IL-1, LPS to activate NF-κB followed by IκB kinase [25]. When IκB falls off the NF-κB complex, the activated NF-κB will move into the cell nucleus, bind to the κB sequence of the promoter or enhancer area of target gene and cause the transcription of many factors including cytokines, chemotactic factor, macrophage chemotactic peptide, cellular adhesion molecule, growth factor, immune receptor and acute phase reactive protein [26]. These manifold factors participate in tissue injury. In the present study, the NF-κB p65 expression, was accompanied by TNF-α and IL-1β levels that were markedly elevated after induction of pancreatitis, while gentiopicroside significantly reduced these expressions and improved the histopathological changes in the pancreas. In addition, the activity of amylase and lipase in serum and the pancreatic tissue water content were all decreased. It indicates that the

Fig. 6. Effects of gentiopicroside on NF-κB p65 expression in pancreas in rats (magnification 400×). (A) Sham operation group; (B) Model group; (C) Model plus gentiopicroside 100 mg/kg group; (D) Model plus gentiopicroside 400 mg/kg group.

J. Lv et al. / Fitoterapia 102 (2015) 127–133

administration of gentiopicroside seems to be effective in attenuating experimental pancreatic injury, which is likely to be related to its inhibition of the release of inflammatory mediators and the expression of NF-κB. 5. Conclusion Gentiopicroside has a relatively rich source; its chemical structure is stable and suitable for a variety of structural transformations as well. It indicates that gentiopicroside might be a potential therapeutic agent or precursor for treating acute pancreatitis. Further studies are needed to explore in more detail the role of gentiopicroside in anti-acute pancreatitis and the related mechanisms especially implicated with the molecular aspects. Disclosure of interest The authors declare that they have no conflict of interest concerning this article. Acknowledgment This study was supported by a grant from the National Natural Science Foundation of China (No. 81202944). We thank Prof. Chun Qing Song for providing gentiopicroside for this study. References [1] Chiang DT, Anozie A, Fleming WR, Kiroff GK. Comparative study on acute pancreatitis management. ANZ J Surg 2004;74:218–21. [2] Chen CX, Liu ZW, Sun ZR, Song CQ, Hu ZB. Studies on anti-inflammatory effect of gentiopicroside. Chin Tradit Herb Drugs 2003;34:814–6. [3] Liu ZW, Chen CX, Jin RM, Shi GQ, Song CQ, Hu ZB. Studies on liver protection and promoting bile secretion of gentioproside. Chin Tradit Herb Drugs 2002;33:47–50. [4] Kondo Y, Takano F, Hojo H. Suppression of chemically and immunologically induced hepatic injuries by gentiopicroside in mice. Planta Med 1994;60:414–6. [5] Chen L, Liu JC, Zhang XN, Guo YY, Xu ZH, Cao W. Down-regulation of NR2B receptors partially contributes to analgesic effects of Gentiopicroside in persistent inflammatory pain. Neuropharmacology 2008;54:1175–81. [6] Rojas A, Bah M, Rojas JI, Gutiérrez DM. Smooth muscle relaxing activity of gentiopicroside isolated from Gentiana spathacea. Planta Med 2000;66: 765–7. [7] Vaquero E, Gukovsky I, Zaninovic V, Gukovskaya AS, Pandol SJ. Localized pancreatic NF-kappaB activation and inflammatory response in taurocholate-induced pancreatitis. Am J Physiol Gastrointest Liver Physiol 2001;280:1197–208.

133

[8] Zhang XP, Zhang L, He JX, Zhang RP, Cheng QH, Zhou YF, et al. Experimental study of therapeutic efficacy of Baicalin in rats with severe acute pancreatitis. World J Gastroenterol 2007;13:717–24. [9] Schmidt J, Rattner DW, Lewandrowski K, Compton CC, Mandavilli U, Knoefel WT, et al. A better model of acute pancreatitis for evaluating therapy. Ann Surg 1992;215:44–56. [10] Meng Y, Ma QY, Kou XP, Xu J. Effect of resveratrol on activation of nuclear factor kappa-B and inflammatory factors in rat model of acute pancreatitis. World J Gastroenterol 2005;11:525–8. [11] Laveda R, Martinez J, Munoz C, Penalva JC, Saez J, Belda G, et al. Different profile of cytokine synthesis according to the severity of acute pancreatitis. World J Gastroenterol 2005;11:5309–13. [12] Granger J, Remick D. Acute pancreatitis: models, markers, and mediators. Shock 2005;24(Suppl. 1):45–51. [13] Frossard JL, Hadengue A, Spahr L, Morel P, Pastor CM. Natural history of long-term lung injury in mouse experimental pancreatitis. Crit Care Med 2002;30:1541–6. [14] Domagala F, Martin G, Bogdanowicz P, Ficheux H, Pujol JP. Inhibition of interleukin-1beta-induced activation of MEK/ERK pathway and DNA binding of NF-kappaB and AP-1: potential mechanism for Diacerein effects in osteoarthritis. Biorheology 2006;43:577–87. [15] Satoh A, Gukovskaya AS, Edderkaoui M, Daghighian MS, Reeve Jr JR, Shimosegawa T, et al. Tumor necrosis factor-alpha mediates pancreatitis responses in acinar cells via protein kinase C and proline-rich tyrosine kinase 2. Gastroenterology 2005;129:639–51. [16] Xue DB, Zhang WH, Yun XG, Song C, Zheng B, Shi XY, et al. Regulating effects of arsenic trioxide on cell death pathways and inflammatory reactions of pancreatic acinar cells in rats. Chin Med J (Engl) 2007;120: 690–5. [17] Gulcubuk A, Altunatmaz K, Sonmez K, Haktanir-Yatkin D, Uzun H, Gurel A, et al. Effects of curcumin on tumour necrosis factor-alpha and interleukin6 in the late phase of experimental acute pancreatitis. J Vet Med A Physiol Pathol Clin Med 2006;53:49–54. [18] Algül H, Tando Y, Schneider G, Weidenbach H, Adler G, Schmid RM. Acute experimental pancreatitis and NF-kappaB/Rel activation. Pancreatology 2002;2:503–9. [19] Satoh A, Masamune A, Kimura K, Kaneko K, Sakai Y, Yamagiwa T, et al. Nuclear factor kappa B expression in peripheral blood mononuclear cells of patients with acute pancreatitis. Pancreas 2003;26:350–6. [20] O'Reilly DA, Roberts JR, Cartmell MT, Demaine AG, Kingsnorth AN. Heat shock factor-1 and nuclear factor-kappaB are systemically activated in human acute pancreatitis. JOP 2006;7:174–84. [21] Ethridge RT, Hashimoto K, Chung DH, Ehlers RA, Rajaraman S, Evers BM. Selective inhibition of NF-kappaB attenuates the severity of caeruleininduced acute pancreatitis. J Am Coll Surg 2002;195:497–505. [22] Satoh A, Shimosegawa T, Fujita M, Kimura K, Masamune A, Koizumi M, et al. Inhibition of nuclear factor-kappaB activation improves the survival of rats with taurocholate pancreatitis. Gut 1999;44:253–8. [23] Pande V, Ramos MJ. NF-kappaB in human disease: current inhibitors and prospects for de novo structure based design of inhibitors. Curr Med Chem 2005;12:357–74. [24] Yamamoto Y, Gaynor RB. IkappaB kinases: key regulators of the NFkappaB pathway. Trends Biochem Sci 2004;29:72–9. [25] Shishodia S, Aggarwal BB. Nuclear factor-kappaB activation: a question of life or death. J Biochem Mol Biol 2002;35:28–40. [26] Choi EK, Jang HC, Kim JH, Kim HJ, Kang HC, Paek YW, et al. Enhancement of cytokine-mediated NF-kappaB activation by phosphatidylinositol 3kinase inhibitors in monocytic cells. Int Immunopharmacol 2006;6: 908–15.