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α-Tocopherol Treatment Ameliorates Chronic Pancreatitis in an Experimental Rat Model Induced by Trinitrobenzene Sulfonic Acid
Original Paper Received: June 24, 2009 Accepted after revision: March 13, 2010 Published online: February 10, 2011
Pancreatology 2011;11:5–11 DOI: 10.1159/000309252
␣-Tocopherol Treatment Ameliorates Chronic Pancreatitis in an Experimental Rat Model Induced by Trinitrobenzene Sulfonic Acid X.C. Li a X.L. Lu b H.H. Chen a
Departments of a Geratology and b Gastroenterology, Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, China
Key Words ␣-Tocopherol ⴢ Chronic pancreatitis ⴢ Trinitrobenzene sulfonic acid
Abstract Objective: To investigate the effects of ␣-tocopherol on pancreatic fibrosis and survival in rats with experimental chronic pancreatitis induced by trinitrobenzene sulfonic acid (TNBS). Methods: Chronic pancreatitis was induced in male Sprague-Dawley rats by infusion of TNBS into the pancreatic duct. ␣-Tocopherol (300, 600 or 900 mg/kg) was orally administered to rats with experimental pancreatitis (treatment group) daily for 4 weeks. The relative pancreatic weight, pancreatic pseudocyst and death rate were observed. Paraffinembedded tissue samples were sliced, stained by hematoxylin-eosin and histopathologically examined. Results: ␣Tocopherol administration significantly ameliorated the pancreatic weight loss induced by TNBS in chronic pancreatitis rats compared to the control group. There were pancreatic pseudocysts in 69% of the ␣-tocopherol group, and in 100% of the control group. ␣-Tocopherol administration led to a significant increase of the survival rate. The histopathologic scores were higher in the control group than in the ␣-tocopherol group. Subgroup analysis of histopathologic
© 2011 S. Karger AG, Basel and IAP 1424–3903/11/0111–0005$38.00/0 Fax +41 61 306 12 34 E-Mail [email protected] www.karger.com
Accessible online at: www.karger.com/pan
scores revealed that a high dose of ␣-tocopherol results in less pancreatic injuries. Conclusion: ␣-Tocopherol treatment elevates survival rate, extenuates fibrosis and increases relative pancreatic weight in the chronic pancreatitis model. ␣Tocopherol therapy in chronic pancreatitis is now required to confirm these findings and establish the role of this treatment in the management of this disabling condition. Copyright © 2011 S. Karger AG, Basel and IAP
Introduction
Chronic pancreatitis is a progressive chronic inflammatory disease characterized by glandular atrophy, ductal changes and extensive fibrosis. Pancreatic fibrosis is a characteristic histopathological feature of chronic pancreatitis. Accumulating evidence from both in vivo and in vitro studies indicates a key role for activated pancreatic stellate cells (PSCs) in the fibrotic process. PSCs are now known to be activated by several factors that are upregulated during pancreatic injury, including growth factors, cytokines and oxidant stress. Significant lipid peroxidation occurs in chronic pancreatitis and is associated with active synthesis of collagen by PSCs. It has been demonstrated that oxidative stress is principally involved
Xiao-Chun Li, MD Department of Geratology, Second Affiliated Hospital School of Medicine, Zhejiang University, 88#, Jiefang Road Hangzhou 310009, Zhejiang Province (China) E-Mail pchen_helen @ 163.com
in the pathogenesis of chronic pancreatitis with fibrosis [1–4]. Although the mechanism responsible for pancreatic fibrosis was unknown for many years, targeting PSCs might be an effective therapeutic approach in chronic pancreatitis. Repeated administration of diethyldithiocarbamate, a superoxide dismutase inhibitor, causes inner and intralobular fibrosis with atrophy of acinar cells in the pancreas [5]. Recent studies report that the antioxidant capacity in patients with chronic pancreatitis is decreased [6]. Rickmann et al. [7] have reported the finding that tocotrienols induce activated PSC death, consisting of apoptosis and autophagy, by targeting the mitochondrial permeability transition pore. ␣-Tocopherol is one of the two naturally occurring subclasses of vitamin E compounds, and is also the most important lipid-soluble antioxidant. It has been reported that administration of ␣-tocopherol results in an approximately sevenfold elevation of mitochondrial content and that the rate of superoxide anion radical generation by submitochondrial particles is inversely related to their ␣-tocopherol content [8, 9]. ␣-Tocopherol can inhibit superoxide generation of neutrophils stimulated by protein kinase C [10]. Treatment with combined antioxidant preparation, which contains selenium, -carotene, L-methionine, vitamin C and ␣-tocopherol, can significantly reduce intractable abdominal pain in patients with confirmed chronic pancreatitis [5]. In addition to its antioxidant activity, ␣-tocopherol can also decrease the level of collagenase expression induced by aging [11]. So far, ␣-tocopherol has shown great promise for being useful in the treatment of chronic pancreatitis. As described above, we consider that the antioxidant treatment might be an option in treatment strategies in chronic pancreatitis. In the present study, we evaluated the efficiency of ␣-tocopherol in management of chronic pancreatitis induced by trinitrobenzene sulfonic acid (TNBS) to explore an important approach for the treatment of pancreatic fibrosis in the future. Methods The experiment was approved by the Institutional Animal Use and Care Committee of the Zhejiang University School of Medicine and performed in accordance with the National Institutes of Health Guidelines for the Care and Handling of Animals. Male Sprague-Dawley rats weighing 250–500 g were obtained from the Zhejiang University School of Medicine (Hangzhou, China). Before the experiment, the animals were fed standard rat chow and water. They were kept under controlled temperature and 12-hour light/dark cycles for at least 1 week.
6
Pancreatology 2011;11:5–11
Induction of Chronic Pancreatitis Chronic pancreatitis was induced in male Sprague-Dawley rats by infusion of TNBS into the pancreatic duct by the method described by Puig-Divi et al. [12]. Animals were fasted overnight with free access to water. Anesthesia was generated by sodium nembutal (50 mg/kg body weight, i.p.). Heat loss was prevented during the operation by placing the animals on a warming tray together with a hot incandescent light, and an infrared warming lamp was used postoperatively until the animals woke up. A single subcutaneous dose of ceftriaxone 10 mg (Roche Products Pty Ltd., Dee Why, N.S.W., Australia) was given before the operation. The abdomen was shaved, prepared with povidone-iodine and then covered with a fenestrated sterile drape. A midline upper abdominal incision was made under sterile conditions. The entry point of the pancreatobiliary duct into the duodenum was identified and the duodenum was opened through a horizontal 0.5-cm antimesenteric incision. The ampulla of Vater was cannulated with polyethylene tubing (O.D. 0.62 mm; Dural Plastics, Auburn, N.S.W., Australia) and sutured so that the tip lay 3–5 mm within the pancreatobiliary duct. The pancreatobiliary duct was occluded with a vascular clamp at the hilum of the liver to prevent entry of the infusate into the liver. 2% TNBS (Sigma Chemical Co., USA) solution in phosphate-buffered saline (pH 8.0) with 10% ethanol was infused for 60 min to a total volume of 0.4 ml (Microinfusion Pump; Zhejiang University Medical Instruments Co., China). The hilar clamp was removed and, after a 5-min washout period, the cannula was also removed. The duodenum was closed with a single interrupted layer of absorbable sutures and the abdominal wound was closed in two layers. Bupivacaine (0.25%, 1.5 ml) was infiltrated into the abdominal wound for postoperative analgesia and normal saline (30 ml/kg body weight) was given via subcutaneous injection to maintain hydration. Postoperatively, rats were transferred to individual cages and were given O2 by mask until they were awake and moving. Rats were fasted for 72 h, but allowed free access to water after 48 h. Hydration was maintained by twice daily subcutaneous injections of sterile fluid (60 ml/kg/day; normal saline alternating with 4% dextrose containing 0.18% saline) until the animal began drinking voluntarily. Analgesia with subcutaneous buprenorphine 0.1 ml was given when necessary for postoperative analgesia. Administration of ␣-Tocopherol 72 h after the operation, the rats (n = 60) with experimental pancreatitis were randomly divided into two groups. ␣-Tocopherol (300, 600 or 900 mg/kg, Zhejiang Xianju Pharmaceutical Co., Ltd., China), dissolved in 5% Tween 80, was orally administered daily to the rats with experimental pancreatitis (treatment group, n = 45). Another group (n = 15) received an equal volume of 5% Tween 80 as a control. Standard rat chow and water were freely available from the 3rd day and weight gain was recorded weekly. Histopathology Animals were euthanized by inhalation of CO2 at the end of the 4th week. The pancreas was dissected as quickly as possible, the connective tissue ablated, and weighed. The portions were fixed in 10% neutral buffered formalin for histological examination. Paraffin-embedded tissue samples were sliced and stained by hematoxylin-eosin. A participating pathologist was blinded to the study protocol to evaluate all histological slides using a previously described scoring system [5] with modifications. Pericellu-
Li /Lu /Chen
lar and interlobular fibrosis, collagen content, and sublobular and lobular atrophy were evaluated semiquantitatively (table 1).
No pseudocyst Pseudocyst
30 25
Rats (n)
Statistical Analysis The data were analyzed with SPSS PC version 11.0. Continuous values were presented as means 8 SD. A 2 test was used to test the differences of nominal values between different groups. ANOVA and covariance analysis were used to evaluate the differences of continuous variants, and Kaplan-Meier survival analysis was chosen to test the differences of the death rate between the groups. p ! 0.05 was considered statistically significant.
35
20 15 10 5 0 Control
Treatment 300 mg/kg 600 mg/kg 900 mg/kg
Results
Body Weight and Relative Pancreatic Weight Both the treatment and control groups were weighed at the beginning and the end of the experiment. After 4 weeks, the weight of the control rats increased by 23.14% (from 337.45 8 18.52 g to 415.00 8 43.02 g), whereas the weight of the ␣-tocopherol treated rats increased by 42.30% (from 260.76 8 15.36 g to 370.52 8 49.73 g). As shown in table 2, the increase in mean body weight was significantly greater for the treatment group than for the control group (109.76 8 48.83 g vs. 77.55 8 33.76 g, p = 0.000). In the treatment group, there was no difference in change of body weight among rats treated with different dosages of ␣-tocopherol (p = 0.060). Pancreatic weight is a simple but reliable marker for assessing the degree of pancreatic regeneration after injury. Relative pancreatic weight is expressed by the ratio of pancreatic weight to body weight. Relative pancreatic weight increased significantly in the treatment group in comparison to the control group (2.84 8 0.46 vs. 1.91 8 0.31, p = 0.000). Pancreatic Pseudocysts and Survival Rate Pancreatic pseudocysts were found in all 11 rats in the control group. Conversely, 29 of the 42 rats treated (69%) were sick with pancreatic pseudocysts (p = 0.000). Pancreatic pseudocysts were observed in the 300 mg/kg (92.3%), 600 mg/kg (64.3%), and 900 mg/kg (53.3%) treatment subgroups, respectively (fig. 1, p = 0.000). The incidence of pancreatic pseudocyst was inversely related to the dosage of ␣-tocopherol used (p = 0.045). At the conclusion of the study, 4 of 15 rats had died, corresponding to a survival rate of 73.3% in the control group. In contrast, ␣-tocopherol administration led to a significant increase in survival, with a survival rate of 93.3% (p = 0.040). In treatment subgroups, the death rate was 13.3% (2/15), 6.7% (1/14) and 0% (0/15) for the 300, 600 and 900 mg/kg dosages, respectively (p = 0.417). The Kaplan-Meier log survival function of the control group and ␣-to␣-Tocopherol Treatment
Fig. 1. Effect of ␣-tocopherol on pancreatic pseudocyst formation
in chronic pancreatitis rats. ␣-Tocopherol treatment significantly decreased the pancreatic pseudocyst formation compared with the control (p = 0.000). The ␣-tocopherol treatment also has the tendency to be dose-dependent as shown in subgroup analysis (p = 0.000): larger doses result in less pancreatic pseudocyst formation.
Table 1. Histopathologic scoring system in chronic pancreatitis
rats Histology
Scores
Definitions
Pericellular fibrosis
0 1 2 3
absent fibrosis limited with 1–2 lobule(s) fibrosis in <50% of lobules fibrosis in >50% of lobules
Interlobular fibrosis
0 1 2 3
absent fibrosis between 2 and 3 lobules fibrosis in <50% of lobules fibrosis in >50% of lobules
Collagen content
0 1 2
absent thin fibrous band around cells or acini moderate thickened fibrous band around cells or acini marked homogeneous collagen around cells or acini
3 Sublobular atrophy
0 1 2 3
absent focal atrophy in 1–2 lobule(s) focal atrophy in <50% of lobules focal atrophy in >50% of lobules
Lobular atrophy
0 1 2 3
absent total atrophy in 1–2 lobule(s) total atrophy in <50% of lobules total atrophy in >50% of lobules
Pancreatology 2011;11:5–11
7
log survival function
log survival function
0
+
0 +
–0.1
+
–0.1
log survival
log survival
+
–0.2
–0.3
Group Control Treatment + Control-censored + Treatment-censored
Group Control 300 μg 600 μg 900 μg + Control-censored + 300 μg-censored + 600 μg-censored + 900 μg-censored
–0.2
–0.3
+
–0.4
+
–0.4 5.00
10.00
a
15.00
20.00
25.00
30.00
5.00
10.00
15.00
b
Days
Fig. 2. Kaplan-Meier log survival function of the control group and the ␣-tocopherol treatment group and its subgroups. a The Kaplan-Meier log survival function of the control group in comparison with the treatment group. The treatment group signifi-
20.00
25.00
30.00
Days
cantly decreased the death rate compared with the control group (p = 0.040), while the death rate between the treatment subgroups had no obvious difference as shown by log survival function in (b).
Table 2. Body weight changes and relative pancreatic weight of chronic pancreatitis rats in ␣-tocopherol-treated groups and control
Group
Subgroup
n
Body weight changes g, mean8SD
Pancreatic weight/body weight ratio g/kg, mean8SD
␣-Tocopherol-treated
300 mg/kg 600 mg/kg 900 mg/kg total control
13 14 15 42 11
88.46842.88 106.29851.09 131.47845.21 109.76848.83* 77.55833.76
2.4780.43* 2.8480.45* 3.1780.14* 2.8480.46* 1.9180.31
Control * p = 0.000, vs. control.
copherol treatment groups is shown in figure 2a and b. The control group had a marked death rate compared to the treatment group (fig. 2a; p = 0.040), while the death rate between the treatment subgroups had no obvious difference as shown by log survival function in figure 2b. This result suggested that ␣-tocopherol treatment might improve the survival rate of chronic pancreatitis. Histopathologic Analysis There was a marked morphological change in the pancreas induced by TNBS in the control group, such as segment glandular atrophy, mononuclear cell infiltration, 8
Pancreatology 2011;11:5–11
destruction of acini, and intralobular or interlobular and periductal fibrosis (fig. 3a). Scattered acinar units appeared to have acquired a ductal phenotype with loss of zymogen granules, the presence of bluish cytoplasm and a more centrally located nucleus. In treatment groups, when we compared the pathological change of the pancreas by hematoxylin-eosin staining, decreased evidence of pancreatic fibrosis and pancreatic atrophy could be found (fig. 3b, c, d) compared with the control group. The histopathologic scores of the pancreas were evaluated according to the criteria described in table 1. The mean histopathologic scores of the pancreas were significantly deLi /Lu /Chen
a
b
c
d
Fig. 3. Histopathological findings of ␣-tocopherol treatment in chronic pancreatitis rats. a Pancreatitis control: scattered acinar units appeared to have acquired a ductal phenotype with loss of zymogen granules, the presence of bluish cytoplasm and a more centrally located nucleus. Pancreas taken from groups treated with ␣-tocopherol 300 mg/kg (b), 600 mg/kg (c) and 900 mg/kg
(d). ␣-Tocopherol treatment significantly decreased the synthesis of collagens and the extent of pancreatic fibrosis and atrophy in comparison with the pancreatitis control group (a). These findings were more typically found in the ␣-tocopherol 900 mg/ kg group (hematoxylin-eosin staining, original magnification: !200).
creased in the groups treated with different amounts of ␣-tocopherol (fig. 4; p = 0.009). The treatment subgroup analysis of pathological scores implied that the increase in the dosage of ␣-tocopherol was associated with the decrease of the histopathologic score (fig. 4).
Chronic pancreatitis is characterized histologically by an irreversible and irregular scarring of exocrine parenchyma with ductal changes by inflammatory processes
[13]. Morphologically it is also defined as progressive pancreatic fibrosis and atrophy of the pancreatic exocrine cell mass [14]. It is important to establish an appropriate animal model in order to study the pathogenic processes. In the present study, the chronic pancreatitis model was induced in rats by infusion of TNBS into the pancreatic duct. At the end of the 4th week, pancreatic tissue samples in both the treatment and control groups showed a different extent of the histopathological features of chronic pancreatitis, such as pancreatic fibrosis and atrophy (fig. 3), suggesting that the formation of chronic pancreatitis in the experimental rat model was successful [15].
␣-Tocopherol Treatment
Pancreatology 2011;11:5–11
Discussion
9
Histopathologic scores
14 12 10 8
10
6
7
6.29
4
4.93
2 0 Control
300 mg/kg
600 mg/kg
900 mg/kg
Fig. 4. Pathological scores of ␣-tocopherol treatment in chronic pancreatitis rats. The mean scores of pancreatic histopathologic change were significantly decreased in the groups treated with ␣tocopherol (300, 600 and 900 mg/kg) compared with the control (p = 0.009). In subgroup analysis, higher doses of ␣-tocopherol resulted in lower pathological scores (300 vs. 600 mg/kg, p 1 0.05; 300 vs. 900 mg/kg, 600 vs. 900 mg/kg, p ! 0.05).
Weight loss in patients with chronic pancreatitis is a common condition which is a result of the decreased absorption of essential substances in the ingested food caused by the deteriorated function of the exocrine pancreas. In our study, the amount of weight loss was significantly lower in the ␣-tocopherol-treated rats with chronic pancreatitis than in the nontreated rats at the end of the experiment. Moreover, the relative pancreatic weight in treated rats was greater than that in the control group. This might be indicative of effective restoration of pancreas function, implying that ␣- tocopherol has the effect of improving pancreatic function in chronic pancreatitis. The decrease in incidence of pancreatic pseudocyst and death resulting from chronic pancreatitis in treated rats might also be an important piece of evidence. In our study, although histopathological features of chronic pancreatitis were observed in both treated and control rats, the histopathologic changes of chronic pancreatitis were alleviated by administrating ␣-tocopherol. Furthermore, the histopathologic scores were reversely related to the dosage of ␣-tocopherol. These findings suggest that ␣-tocopherol might have a direct effect on the pancreas in ameliorating chronic pancreatic injuries. Pancreatic fibrosis is a characteristic histopathological feature of chronic pancreatitis, and activated PSCs play a key role in the fibrogenesis. In vivo, PSCs rapidly proliferate following injury and have been proposed to be the major source of pancreatic extracelluar matrix compo10
Pancreatology 2011;11:5–11
nent during pancreatic fibrogenesis. The accumulating evidence clearly places the PSC at the center of pancreatic fibrosis [16, 17]. It has been known that PSCs can be activated by oxidant stress. Oxidant stress is the molecular and cellular damage resulting from excessive reactive oxygen species (ROS) production or from reduced endogenous antioxidants. It has been demonstrated that increased production of ROS in the course of pancreatitis activates nuclear factor B (NF-B), which subsequently results in transcription of various inflammatory cytokines such as TNF-␣ and ILs in acinar and PSC, leading to tissue damage, PSC proliferation and fibrosis [18]. On the other hand, it has been shown that antioxidant treatment prevents NF-B activation in PSCs, acinar cells and neutrophils in acute pancreatitis [19]. More important findings are that oxidative stress is primary rather than secondary to infiltration of inflammatory cells in the pancreas and the pancreas is more vulnerable to prolonged oxidative stress compared with the liver and kidney [5, 10, 20]. Based on these observations, the drugs which could inhibit or prevent oxidative stress might be developed and used for the treatment of chronic pancreatitis with fibrosis. Taurine, 2-aminoethanesulfonic acid, has been shown to increase the effect of glutathione peroxidase and is protective against lipid peroxidation and reperfusion injury. Recent data showed that taurine treatment improved morphological pancreatic injury as well as markers of oxidative stress (GPx, superoxide dismutase, MDA) of chronic pancreatitis in an experimental rat model. Experimental in vitro augmentation of submitochondrial particles with varying amounts of ␣-tocopherol caused up to a 50% decrease in the rate of superoxide anion radical generation. We inferred that one of the cytoprotective effects shown by ␣-tocopherol in the current model of chronic pancreatitis is possible through an antioxidative effect. This study showed that infusion of TNBS into the pancreatic duct caused chronic pancreatitis in rats similar to histological changes observed in humans with chronic pancreatitis. The data from this short study showed that in experimental chronic pancreatitis, ␣-tocopherol treatment significantly decreased the extent of pancreatic tissue damage (fig. 1, 3, 4), prevented weight loss (table 2) and increased the survival rate (fig. 2). Improved morphological pancreatic injury suggested that ␣-tocopherol might have a direct effect on the pancreas in ameliorating chronic pancreatic injuries. This work suggested that ␣tocopherol, an antioxidant, could prevent and ameliorate the extent of pancreatic fibrosis and might be an option in treatment strategies in chronic pancreatitis. However, Li /Lu /Chen
further detailed experiments and clinical trials will be required to test the potential clinical application. As the mechanisms involved in the progression of pancreatic fibrosis become elucidated, it may be possible in the future to develop novel therapeutic strategies. Antioxidants might become an important approach for the treatment of chronic pancreatitis. Our study suggests that ␣-tocopherol treatment increases the survival rate, extenuates pancreatic fibrosis and increases relative pan-
creatic weight in a chronic pancreatitis model. ␣-Tocopherol may become a useful therapeutic strategy in the future. Acknowledgement This work was supported by grants from the Open Funds of the Zhejiang Provincial Key Lab of Geriatrics and Youth Foundation of Zhejiang University.
References 1 Apte MV, Wilson JS: Mechanisms of pancreatic fibrosis. Dig Dis 2004;22:273–279. 2 Mas MR, Isik AT, Yamanel L, Inal V, Tasci I, Deveci S, Mas N, Comert B, Akay C: Antioxidant treatment with taurine ameliorates chronic pancreatitis in an experimental rat model. Pancreas 2006; 33:77–81. 3 Yoo BM, Oh TY, Kim YB, Yeo M, Lee JS, Surh YJ, Ahn BO, Kim WH, Sohn S, Kim JH, Hahm KB: Novel antioxidant ameliorates the fibrosis and inflammation of ceruleininduced chronic pancreatitis in a mouse model. Pancreatology 2005; 5:165–176. 4 Talukdar R, Saikia N, Singal DK, Tandon R: Chronic pancreatitis: evolving paradigms. Pancreatology 2006; 6:440–449. 5 Matsumura N, Ochi K, Ichimura M, Mizushima T, Harada H, Harada M: Study on free radicals and pancreatic fibrosis – pancreatic fibrosis induced by repeated injections of superoxide dismutase inhibitor. Pancreas 2001;22:53–57. 6 Altavilla D, Famulari C, Passaniti M, Galeano M, Macri A, Seminara P, Minutoli L, Marini H, Calo M, Venuti FS, Esposito M, Squadrito F: Attenuated cerulein-induced pancreatitis in nuclear factor-kappaB-deficient mice. Lab Invest 2003;83:1723–1732. 7 Rickmann M, Vaquero EC, Malagelada JR, Molero X: Tocotrienols induce apoptosis and autophagy in rat pancreatic stellate cells through the mitochondrial death pathway. Gastroenterology 2007; 132:2518–2532.
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8 Packer L, Weber SU, Rimbach G: Molecular aspects of alpha-tocotrienol antioxidant action and cell signalling. J Nutr 2001; 131: 369S–373S. 9 Lass A, Sohal RS: Effect of coenzyme Q(10) and alpha-tocopherol content of mitochondria on the production of superoxide anion radicals. FASEB J 2000;14:87–94. 10 Kanno T, Utsumi T, Takehara Y, Ide A, Akiyama J, Yoshioka T, Horton AA, Utsumi K: Inhibition of neutrophil-superoxide generation by alpha-tocopherol and coenzyme Q. Free Radic Res 1996;24:281–289. 11 Ricciarelli R, Maroni P, Ozer N, Zingg JM, Azzi A: Age-dependent increase of collagenase expression can be reduced by alpha-tocopherol via protein kinase C inhibition. Free Radic Biol Med 1999; 27:729–737. 12 Puig-Divi V, Molero X, Salas A, Guarner F, Guarner L, Malagelada JR: Induction of chronic pancreatic disease by trinitrobenzene sulfonic acid infusion into rat pancreatic ducts. Pancreas 1996; 13:417–424. 13 Kloppel G, Maillet B: Pathology of acute and chronic pancreatitis. Pancreas 1993; 8: 659– 670. 14 Keith RG: Definition and classification of chronic pancreatitis. World J Surg 2003; 27: 1172–1174.
15 Stevens T, Conwell DL, Zuccaro G: Pathogenesis of chronic pancreatitis: an evidencebased review of past theories and recent developments. Am J Gastroenterol 2004; 99: 2256–2270. 16 Neuschwander-Tetri BA, Bridle KR, Wells LD, Marcu M, Ramm GA: Repetitive acute pancreatic injury in the mouse induces procollagen alpha1(I) expression colocalized to pancreatic stellate cells. Lab Invest 2000; 80: 143–150. 17 Apte MV, Haber PS, Darby SJ, Rodgers SC, McCaughan GW, Korsten MA, Pirola RC, Wilson JS: Pancreatic stellate cells are activated by proinflammatory cytokines: implications for pancreatic fibrogenesis. Gut 1999; 44:534–541. 18 Kim H, Seo JY, Roh KH, Lim JW, Kim KH: Suppression of nf-kappaB activation and cytokine production by N-acetylcysteine in pancreatic acinar cells. Free Radic Biol Med 2000;29:674–683. 19 Blaisdell RJ, Giri SN: Mechanism of antifibrotic effect of taurine and niacin in the multidose bleomycin-hamster model of lung fibrosis: inhibition of lysyl oxidase and collagenase. J Biochem Toxicol 1995;10:203– 210. 20 Peeters-Joris C, Vandevoorde AM, Baudhuin P: Subcellular localization of superoxide dismutase in rat liver. Biochem J 1975; 150:31–39.
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11
Pancreatology 2011;11:5–11 DOI: 10.1159/000309252
␣-Tocopherol Treatment Ameliorates Chronic Pancreatitis in an Experimental Rat Model Induced by Trinitrobenzene Sulfonic Acid X.C. Li a X.L. Lu b H.H. Chen a
Departments of a Geratology and b Gastroenterology, Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, China
Key Words ␣-Tocopherol ⴢ Chronic pancreatitis ⴢ Trinitrobenzene sulfonic acid
Abstract Objective: To investigate the effects of ␣-tocopherol on pancreatic fibrosis and survival in rats with experimental chronic pancreatitis induced by trinitrobenzene sulfonic acid (TNBS). Methods: Chronic pancreatitis was induced in male Sprague-Dawley rats by infusion of TNBS into the pancreatic duct. ␣-Tocopherol (300, 600 or 900 mg/kg) was orally administered to rats with experimental pancreatitis (treatment group) daily for 4 weeks. The relative pancreatic weight, pancreatic pseudocyst and death rate were observed. Paraffinembedded tissue samples were sliced, stained by hematoxylin-eosin and histopathologically examined. Results: ␣Tocopherol administration significantly ameliorated the pancreatic weight loss induced by TNBS in chronic pancreatitis rats compared to the control group. There were pancreatic pseudocysts in 69% of the ␣-tocopherol group, and in 100% of the control group. ␣-Tocopherol administration led to a significant increase of the survival rate. The histopathologic scores were higher in the control group than in the ␣-tocopherol group. Subgroup analysis of histopathologic
© 2011 S. Karger AG, Basel and IAP 1424–3903/11/0111–0005$38.00/0 Fax +41 61 306 12 34 E-Mail [email protected] www.karger.com
Accessible online at: www.karger.com/pan
scores revealed that a high dose of ␣-tocopherol results in less pancreatic injuries. Conclusion: ␣-Tocopherol treatment elevates survival rate, extenuates fibrosis and increases relative pancreatic weight in the chronic pancreatitis model. ␣Tocopherol therapy in chronic pancreatitis is now required to confirm these findings and establish the role of this treatment in the management of this disabling condition. Copyright © 2011 S. Karger AG, Basel and IAP
Introduction
Chronic pancreatitis is a progressive chronic inflammatory disease characterized by glandular atrophy, ductal changes and extensive fibrosis. Pancreatic fibrosis is a characteristic histopathological feature of chronic pancreatitis. Accumulating evidence from both in vivo and in vitro studies indicates a key role for activated pancreatic stellate cells (PSCs) in the fibrotic process. PSCs are now known to be activated by several factors that are upregulated during pancreatic injury, including growth factors, cytokines and oxidant stress. Significant lipid peroxidation occurs in chronic pancreatitis and is associated with active synthesis of collagen by PSCs. It has been demonstrated that oxidative stress is principally involved
Xiao-Chun Li, MD Department of Geratology, Second Affiliated Hospital School of Medicine, Zhejiang University, 88#, Jiefang Road Hangzhou 310009, Zhejiang Province (China) E-Mail pchen_helen @ 163.com
in the pathogenesis of chronic pancreatitis with fibrosis [1–4]. Although the mechanism responsible for pancreatic fibrosis was unknown for many years, targeting PSCs might be an effective therapeutic approach in chronic pancreatitis. Repeated administration of diethyldithiocarbamate, a superoxide dismutase inhibitor, causes inner and intralobular fibrosis with atrophy of acinar cells in the pancreas [5]. Recent studies report that the antioxidant capacity in patients with chronic pancreatitis is decreased [6]. Rickmann et al. [7] have reported the finding that tocotrienols induce activated PSC death, consisting of apoptosis and autophagy, by targeting the mitochondrial permeability transition pore. ␣-Tocopherol is one of the two naturally occurring subclasses of vitamin E compounds, and is also the most important lipid-soluble antioxidant. It has been reported that administration of ␣-tocopherol results in an approximately sevenfold elevation of mitochondrial content and that the rate of superoxide anion radical generation by submitochondrial particles is inversely related to their ␣-tocopherol content [8, 9]. ␣-Tocopherol can inhibit superoxide generation of neutrophils stimulated by protein kinase C [10]. Treatment with combined antioxidant preparation, which contains selenium, -carotene, L-methionine, vitamin C and ␣-tocopherol, can significantly reduce intractable abdominal pain in patients with confirmed chronic pancreatitis [5]. In addition to its antioxidant activity, ␣-tocopherol can also decrease the level of collagenase expression induced by aging [11]. So far, ␣-tocopherol has shown great promise for being useful in the treatment of chronic pancreatitis. As described above, we consider that the antioxidant treatment might be an option in treatment strategies in chronic pancreatitis. In the present study, we evaluated the efficiency of ␣-tocopherol in management of chronic pancreatitis induced by trinitrobenzene sulfonic acid (TNBS) to explore an important approach for the treatment of pancreatic fibrosis in the future. Methods The experiment was approved by the Institutional Animal Use and Care Committee of the Zhejiang University School of Medicine and performed in accordance with the National Institutes of Health Guidelines for the Care and Handling of Animals. Male Sprague-Dawley rats weighing 250–500 g were obtained from the Zhejiang University School of Medicine (Hangzhou, China). Before the experiment, the animals were fed standard rat chow and water. They were kept under controlled temperature and 12-hour light/dark cycles for at least 1 week.
6
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Induction of Chronic Pancreatitis Chronic pancreatitis was induced in male Sprague-Dawley rats by infusion of TNBS into the pancreatic duct by the method described by Puig-Divi et al. [12]. Animals were fasted overnight with free access to water. Anesthesia was generated by sodium nembutal (50 mg/kg body weight, i.p.). Heat loss was prevented during the operation by placing the animals on a warming tray together with a hot incandescent light, and an infrared warming lamp was used postoperatively until the animals woke up. A single subcutaneous dose of ceftriaxone 10 mg (Roche Products Pty Ltd., Dee Why, N.S.W., Australia) was given before the operation. The abdomen was shaved, prepared with povidone-iodine and then covered with a fenestrated sterile drape. A midline upper abdominal incision was made under sterile conditions. The entry point of the pancreatobiliary duct into the duodenum was identified and the duodenum was opened through a horizontal 0.5-cm antimesenteric incision. The ampulla of Vater was cannulated with polyethylene tubing (O.D. 0.62 mm; Dural Plastics, Auburn, N.S.W., Australia) and sutured so that the tip lay 3–5 mm within the pancreatobiliary duct. The pancreatobiliary duct was occluded with a vascular clamp at the hilum of the liver to prevent entry of the infusate into the liver. 2% TNBS (Sigma Chemical Co., USA) solution in phosphate-buffered saline (pH 8.0) with 10% ethanol was infused for 60 min to a total volume of 0.4 ml (Microinfusion Pump; Zhejiang University Medical Instruments Co., China). The hilar clamp was removed and, after a 5-min washout period, the cannula was also removed. The duodenum was closed with a single interrupted layer of absorbable sutures and the abdominal wound was closed in two layers. Bupivacaine (0.25%, 1.5 ml) was infiltrated into the abdominal wound for postoperative analgesia and normal saline (30 ml/kg body weight) was given via subcutaneous injection to maintain hydration. Postoperatively, rats were transferred to individual cages and were given O2 by mask until they were awake and moving. Rats were fasted for 72 h, but allowed free access to water after 48 h. Hydration was maintained by twice daily subcutaneous injections of sterile fluid (60 ml/kg/day; normal saline alternating with 4% dextrose containing 0.18% saline) until the animal began drinking voluntarily. Analgesia with subcutaneous buprenorphine 0.1 ml was given when necessary for postoperative analgesia. Administration of ␣-Tocopherol 72 h after the operation, the rats (n = 60) with experimental pancreatitis were randomly divided into two groups. ␣-Tocopherol (300, 600 or 900 mg/kg, Zhejiang Xianju Pharmaceutical Co., Ltd., China), dissolved in 5% Tween 80, was orally administered daily to the rats with experimental pancreatitis (treatment group, n = 45). Another group (n = 15) received an equal volume of 5% Tween 80 as a control. Standard rat chow and water were freely available from the 3rd day and weight gain was recorded weekly. Histopathology Animals were euthanized by inhalation of CO2 at the end of the 4th week. The pancreas was dissected as quickly as possible, the connective tissue ablated, and weighed. The portions were fixed in 10% neutral buffered formalin for histological examination. Paraffin-embedded tissue samples were sliced and stained by hematoxylin-eosin. A participating pathologist was blinded to the study protocol to evaluate all histological slides using a previously described scoring system [5] with modifications. Pericellu-
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lar and interlobular fibrosis, collagen content, and sublobular and lobular atrophy were evaluated semiquantitatively (table 1).
No pseudocyst Pseudocyst
30 25
Rats (n)
Statistical Analysis The data were analyzed with SPSS PC version 11.0. Continuous values were presented as means 8 SD. A 2 test was used to test the differences of nominal values between different groups. ANOVA and covariance analysis were used to evaluate the differences of continuous variants, and Kaplan-Meier survival analysis was chosen to test the differences of the death rate between the groups. p ! 0.05 was considered statistically significant.
35
20 15 10 5 0 Control
Treatment 300 mg/kg 600 mg/kg 900 mg/kg
Results
Body Weight and Relative Pancreatic Weight Both the treatment and control groups were weighed at the beginning and the end of the experiment. After 4 weeks, the weight of the control rats increased by 23.14% (from 337.45 8 18.52 g to 415.00 8 43.02 g), whereas the weight of the ␣-tocopherol treated rats increased by 42.30% (from 260.76 8 15.36 g to 370.52 8 49.73 g). As shown in table 2, the increase in mean body weight was significantly greater for the treatment group than for the control group (109.76 8 48.83 g vs. 77.55 8 33.76 g, p = 0.000). In the treatment group, there was no difference in change of body weight among rats treated with different dosages of ␣-tocopherol (p = 0.060). Pancreatic weight is a simple but reliable marker for assessing the degree of pancreatic regeneration after injury. Relative pancreatic weight is expressed by the ratio of pancreatic weight to body weight. Relative pancreatic weight increased significantly in the treatment group in comparison to the control group (2.84 8 0.46 vs. 1.91 8 0.31, p = 0.000). Pancreatic Pseudocysts and Survival Rate Pancreatic pseudocysts were found in all 11 rats in the control group. Conversely, 29 of the 42 rats treated (69%) were sick with pancreatic pseudocysts (p = 0.000). Pancreatic pseudocysts were observed in the 300 mg/kg (92.3%), 600 mg/kg (64.3%), and 900 mg/kg (53.3%) treatment subgroups, respectively (fig. 1, p = 0.000). The incidence of pancreatic pseudocyst was inversely related to the dosage of ␣-tocopherol used (p = 0.045). At the conclusion of the study, 4 of 15 rats had died, corresponding to a survival rate of 73.3% in the control group. In contrast, ␣-tocopherol administration led to a significant increase in survival, with a survival rate of 93.3% (p = 0.040). In treatment subgroups, the death rate was 13.3% (2/15), 6.7% (1/14) and 0% (0/15) for the 300, 600 and 900 mg/kg dosages, respectively (p = 0.417). The Kaplan-Meier log survival function of the control group and ␣-to␣-Tocopherol Treatment
Fig. 1. Effect of ␣-tocopherol on pancreatic pseudocyst formation
in chronic pancreatitis rats. ␣-Tocopherol treatment significantly decreased the pancreatic pseudocyst formation compared with the control (p = 0.000). The ␣-tocopherol treatment also has the tendency to be dose-dependent as shown in subgroup analysis (p = 0.000): larger doses result in less pancreatic pseudocyst formation.
Table 1. Histopathologic scoring system in chronic pancreatitis
rats Histology
Scores
Definitions
Pericellular fibrosis
0 1 2 3
absent fibrosis limited with 1–2 lobule(s) fibrosis in <50% of lobules fibrosis in >50% of lobules
Interlobular fibrosis
0 1 2 3
absent fibrosis between 2 and 3 lobules fibrosis in <50% of lobules fibrosis in >50% of lobules
Collagen content
0 1 2
absent thin fibrous band around cells or acini moderate thickened fibrous band around cells or acini marked homogeneous collagen around cells or acini
3 Sublobular atrophy
0 1 2 3
absent focal atrophy in 1–2 lobule(s) focal atrophy in <50% of lobules focal atrophy in >50% of lobules
Lobular atrophy
0 1 2 3
absent total atrophy in 1–2 lobule(s) total atrophy in <50% of lobules total atrophy in >50% of lobules
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7
log survival function
log survival function
0
+
0 +
–0.1
+
–0.1
log survival
log survival
+
–0.2
–0.3
Group Control Treatment + Control-censored + Treatment-censored
Group Control 300 μg 600 μg 900 μg + Control-censored + 300 μg-censored + 600 μg-censored + 900 μg-censored
–0.2
–0.3
+
–0.4
+
–0.4 5.00
10.00
a
15.00
20.00
25.00
30.00
5.00
10.00
15.00
b
Days
Fig. 2. Kaplan-Meier log survival function of the control group and the ␣-tocopherol treatment group and its subgroups. a The Kaplan-Meier log survival function of the control group in comparison with the treatment group. The treatment group signifi-
20.00
25.00
30.00
Days
cantly decreased the death rate compared with the control group (p = 0.040), while the death rate between the treatment subgroups had no obvious difference as shown by log survival function in (b).
Table 2. Body weight changes and relative pancreatic weight of chronic pancreatitis rats in ␣-tocopherol-treated groups and control
Group
Subgroup
n
Body weight changes g, mean8SD
Pancreatic weight/body weight ratio g/kg, mean8SD
␣-Tocopherol-treated
300 mg/kg 600 mg/kg 900 mg/kg total control
13 14 15 42 11
88.46842.88 106.29851.09 131.47845.21 109.76848.83* 77.55833.76
2.4780.43* 2.8480.45* 3.1780.14* 2.8480.46* 1.9180.31
Control * p = 0.000, vs. control.
copherol treatment groups is shown in figure 2a and b. The control group had a marked death rate compared to the treatment group (fig. 2a; p = 0.040), while the death rate between the treatment subgroups had no obvious difference as shown by log survival function in figure 2b. This result suggested that ␣-tocopherol treatment might improve the survival rate of chronic pancreatitis. Histopathologic Analysis There was a marked morphological change in the pancreas induced by TNBS in the control group, such as segment glandular atrophy, mononuclear cell infiltration, 8
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destruction of acini, and intralobular or interlobular and periductal fibrosis (fig. 3a). Scattered acinar units appeared to have acquired a ductal phenotype with loss of zymogen granules, the presence of bluish cytoplasm and a more centrally located nucleus. In treatment groups, when we compared the pathological change of the pancreas by hematoxylin-eosin staining, decreased evidence of pancreatic fibrosis and pancreatic atrophy could be found (fig. 3b, c, d) compared with the control group. The histopathologic scores of the pancreas were evaluated according to the criteria described in table 1. The mean histopathologic scores of the pancreas were significantly deLi /Lu /Chen
a
b
c
d
Fig. 3. Histopathological findings of ␣-tocopherol treatment in chronic pancreatitis rats. a Pancreatitis control: scattered acinar units appeared to have acquired a ductal phenotype with loss of zymogen granules, the presence of bluish cytoplasm and a more centrally located nucleus. Pancreas taken from groups treated with ␣-tocopherol 300 mg/kg (b), 600 mg/kg (c) and 900 mg/kg
(d). ␣-Tocopherol treatment significantly decreased the synthesis of collagens and the extent of pancreatic fibrosis and atrophy in comparison with the pancreatitis control group (a). These findings were more typically found in the ␣-tocopherol 900 mg/ kg group (hematoxylin-eosin staining, original magnification: !200).
creased in the groups treated with different amounts of ␣-tocopherol (fig. 4; p = 0.009). The treatment subgroup analysis of pathological scores implied that the increase in the dosage of ␣-tocopherol was associated with the decrease of the histopathologic score (fig. 4).
Chronic pancreatitis is characterized histologically by an irreversible and irregular scarring of exocrine parenchyma with ductal changes by inflammatory processes
[13]. Morphologically it is also defined as progressive pancreatic fibrosis and atrophy of the pancreatic exocrine cell mass [14]. It is important to establish an appropriate animal model in order to study the pathogenic processes. In the present study, the chronic pancreatitis model was induced in rats by infusion of TNBS into the pancreatic duct. At the end of the 4th week, pancreatic tissue samples in both the treatment and control groups showed a different extent of the histopathological features of chronic pancreatitis, such as pancreatic fibrosis and atrophy (fig. 3), suggesting that the formation of chronic pancreatitis in the experimental rat model was successful [15].
␣-Tocopherol Treatment
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Discussion
9
Histopathologic scores
14 12 10 8
10
6
7
6.29
4
4.93
2 0 Control
300 mg/kg
600 mg/kg
900 mg/kg
Fig. 4. Pathological scores of ␣-tocopherol treatment in chronic pancreatitis rats. The mean scores of pancreatic histopathologic change were significantly decreased in the groups treated with ␣tocopherol (300, 600 and 900 mg/kg) compared with the control (p = 0.009). In subgroup analysis, higher doses of ␣-tocopherol resulted in lower pathological scores (300 vs. 600 mg/kg, p 1 0.05; 300 vs. 900 mg/kg, 600 vs. 900 mg/kg, p ! 0.05).
Weight loss in patients with chronic pancreatitis is a common condition which is a result of the decreased absorption of essential substances in the ingested food caused by the deteriorated function of the exocrine pancreas. In our study, the amount of weight loss was significantly lower in the ␣-tocopherol-treated rats with chronic pancreatitis than in the nontreated rats at the end of the experiment. Moreover, the relative pancreatic weight in treated rats was greater than that in the control group. This might be indicative of effective restoration of pancreas function, implying that ␣- tocopherol has the effect of improving pancreatic function in chronic pancreatitis. The decrease in incidence of pancreatic pseudocyst and death resulting from chronic pancreatitis in treated rats might also be an important piece of evidence. In our study, although histopathological features of chronic pancreatitis were observed in both treated and control rats, the histopathologic changes of chronic pancreatitis were alleviated by administrating ␣-tocopherol. Furthermore, the histopathologic scores were reversely related to the dosage of ␣-tocopherol. These findings suggest that ␣-tocopherol might have a direct effect on the pancreas in ameliorating chronic pancreatic injuries. Pancreatic fibrosis is a characteristic histopathological feature of chronic pancreatitis, and activated PSCs play a key role in the fibrogenesis. In vivo, PSCs rapidly proliferate following injury and have been proposed to be the major source of pancreatic extracelluar matrix compo10
Pancreatology 2011;11:5–11
nent during pancreatic fibrogenesis. The accumulating evidence clearly places the PSC at the center of pancreatic fibrosis [16, 17]. It has been known that PSCs can be activated by oxidant stress. Oxidant stress is the molecular and cellular damage resulting from excessive reactive oxygen species (ROS) production or from reduced endogenous antioxidants. It has been demonstrated that increased production of ROS in the course of pancreatitis activates nuclear factor B (NF-B), which subsequently results in transcription of various inflammatory cytokines such as TNF-␣ and ILs in acinar and PSC, leading to tissue damage, PSC proliferation and fibrosis [18]. On the other hand, it has been shown that antioxidant treatment prevents NF-B activation in PSCs, acinar cells and neutrophils in acute pancreatitis [19]. More important findings are that oxidative stress is primary rather than secondary to infiltration of inflammatory cells in the pancreas and the pancreas is more vulnerable to prolonged oxidative stress compared with the liver and kidney [5, 10, 20]. Based on these observations, the drugs which could inhibit or prevent oxidative stress might be developed and used for the treatment of chronic pancreatitis with fibrosis. Taurine, 2-aminoethanesulfonic acid, has been shown to increase the effect of glutathione peroxidase and is protective against lipid peroxidation and reperfusion injury. Recent data showed that taurine treatment improved morphological pancreatic injury as well as markers of oxidative stress (GPx, superoxide dismutase, MDA) of chronic pancreatitis in an experimental rat model. Experimental in vitro augmentation of submitochondrial particles with varying amounts of ␣-tocopherol caused up to a 50% decrease in the rate of superoxide anion radical generation. We inferred that one of the cytoprotective effects shown by ␣-tocopherol in the current model of chronic pancreatitis is possible through an antioxidative effect. This study showed that infusion of TNBS into the pancreatic duct caused chronic pancreatitis in rats similar to histological changes observed in humans with chronic pancreatitis. The data from this short study showed that in experimental chronic pancreatitis, ␣-tocopherol treatment significantly decreased the extent of pancreatic tissue damage (fig. 1, 3, 4), prevented weight loss (table 2) and increased the survival rate (fig. 2). Improved morphological pancreatic injury suggested that ␣-tocopherol might have a direct effect on the pancreas in ameliorating chronic pancreatic injuries. This work suggested that ␣tocopherol, an antioxidant, could prevent and ameliorate the extent of pancreatic fibrosis and might be an option in treatment strategies in chronic pancreatitis. However, Li /Lu /Chen
further detailed experiments and clinical trials will be required to test the potential clinical application. As the mechanisms involved in the progression of pancreatic fibrosis become elucidated, it may be possible in the future to develop novel therapeutic strategies. Antioxidants might become an important approach for the treatment of chronic pancreatitis. Our study suggests that ␣-tocopherol treatment increases the survival rate, extenuates pancreatic fibrosis and increases relative pan-
creatic weight in a chronic pancreatitis model. ␣-Tocopherol may become a useful therapeutic strategy in the future. Acknowledgement This work was supported by grants from the Open Funds of the Zhejiang Provincial Key Lab of Geriatrics and Youth Foundation of Zhejiang University.
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