Study on Protecting Effects of Parthenolide on Hepatic Injury in Rats with Seve- Re Acute Pancreatitis

Acta Medica Mediterranea, 2018, 34: 489

STUDY ON PROTECTING EFFECTS OF PARTHENOLIDE ON HEPATIC INJURY IN RATS WITH SEVE- RE ACUTE PANCREATITIS

GAOZHONG LI1,2, GUANGSHENG ZHAI3, HONG TAO4, JINGMEI CAO2, XIULIAN WANG2, ZHAOSHENG CHEN1, MIN LI2, KUNMING HUANG2, JIANQIANG GUO1* 1Department of Gastroenterology, the second hospital of Shandong University, No. 247 of Beiyuan Street, Jinan 250033, Shandong Province, China - 2Department of Gastroenterology, Zibo Central Hospital, No. 54 West of Gongqingtuan Road, Zhangdian District, Zibo 255036, Shandong Province, China - 3Department of Radiotheraphy, Zibo Central Hospital, No. 54 West of Gongqingtuan Road, Zhangdian District, Zibo 255036, Shandong Province, China - 4Division of Clinical Skill Training Center, Zibo Central Hospital, No. 54 West of Gongqingtuan Road, Zhangdian District, Zibo 255036, Shandong, China

ABSTRACT

Objective: Our study was designed to investigate the protective effects of parthenolide on hepatic injury in rats with severe acute pancreatitis (SAP). Methods: The SAP rat models were prepared and randomly devided into five groups，the model control group, parthenolide treated group with different doses of parthenolide, and the sham operated group. The levels of AMY, ALT, AST, IL-6 and TNF-α in serum, NF-κB expression and hepatic pathological changes in all groups were detected. Results: The levels of AMY, ALT, AST, IL-6 and TNF-α in serum and expression levels of NF-κB were lower in parthenolide treated groups than that in model control group. The model control group showed obviously pathological changes compared with sham group, while the administration of parthenolide dose-dependently inhibited the changes. Conclusion: Parthenolide demonstrated a well curative capability on rats with SAP.

Keywords: Severe acute pancreatitis; parthenolide; TNF-α; IL-6; NF-κB.

DOI: 10.19193/0393-6384_2018_2_79

Received November 30, 2017; Accepted January 20, 2018

Introduction If the SIRS is severe, then proinflammatory mediators can cause early multiple (respiratory, car- Severe acute pancreatitis (SAP) is one of the diovascular, renal, and hepatic) organ failure. SAP most common acute abdomens in clinical practice(1), is often associated with high morbidity and mortali- which can cause systemic inflammatory response ty due to the development of pancreatic and extra- syndromes (SIRS) such as effusion of blood vessel, pancreatic necrosis, their subsequent infection and shock and multiple organ functional disturbances or multisystem organ failure (MOF)(5-7). Inflammatory even multiple organ dysfunction syndrome(2-4). SAP mediators is critical for progression of SAP and has generally undergoes two phases. During the first 1 been receiving increasing attention in recent years. or 2 week, a pro-inflammatory response occurs, SAP is a potentially lethal inflammatory condition which results in systemic inflammatory response of the pancreas that involves both peripancreatic syndrome (SIRS), a sterile response in which sepsis tissue and remote organs. Local inflammatory reac- or infection rarely occurs. tion in pancreas leads to systemic SIRS and MOF, 490 Gaozhong Li, Guangsheng Zhai et Al which is believed to be the capital cause of mortali- 60%, and with free access to food and water. All ty.8,9 Inflammatory components include interleukin procedures were in accordance with the Guidelines (IL)-6, tumor necrosis factor (TNF)-a, IL-8, IL-10, of the Animal Experiments of Yuhuangding and IL-1b are considered to be required for SAP Hospital of Yantai, Affiliated Hospital of Qingdao development(10). A recent study has shown that pro- Medical University. inflammatory stimuli such as IL-6 and TNF-a play a central role in the initiation and progression of Model of SAP SAP.11 Despite overall reduced mortality in the last The rats were fasted overnight with free access decade, SAP is a devastating disease that is associ- to water 12 h before experiments. Surgical anesthe- ated with mortality ranging from less than 10% to sia was performed by intraperitoneal injection of as high as 85%(12-16). 10% chloral hydrate. SAP was induced by retro- Parthenolide is a sesquiterpene lactone that grade infusion of 1% sodium taurocholate (0.1 was purified from the shoots of feverfew mL/100 g body weight) into the common pancreatic (Tanacetum parthenium), a traditional herbal medi- duct through epidural catheter and duodenal papil- cine that has been used for the treatment of arthritis, la.21 Five minutes later artery clamp was removed, fever and migraine in China(17). The nucleophilic tube was drawn, the abdominal wall was sutured to nature of its methylene-γ-lactone ring and epoxide establish the animal model of SAP. Duodenum and group enables rapid interactions with biological the pancreas of rats in control group was only sites. These interactions have been found to be injected saline instead of sodium taurocholate then related to its ability to induce oxidative stress, and gently put back to the abdomen. it shows multiple anti-cancer and pro-apoptotic characteristics. Previous studies have demonstrated Animal groups that parthenolide has strong anti-inflammatory Fifty rats were randomly divided into the effects and is a potent nuclear factor kappa-B (NF- sham-operated group, the model control group and κB) inhibitor by specifically inhibiting the IκB parthenolide treated group. In sham-operated group, kinase complex(8-20). Therefore, the present study only exploratory laparotomy was performed, name- was focused on the preventive effect of partheno- ly after entering abdominal cavity, checking pan- lide in rats model with SAP. creas and duodenum and then closing abdomen(22-23). Both sham-operated group and model control group Materials and methods were injected equivalent volume saline at the corresponding time points during SAP operation. After Chemicals SAP operation, rats in all groups were allowed free Parthenolide was purchased from Beijing access to water. The rats in parthenolide treated Institute of Biological Products (Beijing, China) group were injected parthenolide intraperitoneally and it’s purity was determined to be ≥ 98% by by low (100 μg/kg), middle (200 μg/kg) and high HPLC measurement. Sodium taurocholate was pur- (300 μg/kg) dose. Rats in sham-operated group and chased from USA Sigma Company. TNF-a and IL-6 model control group were intraperitoneally infused enzyme-linked immunosorbentassay (ELISA) kits with tween 80 (0.05%). were obtained from R&D Systems, Inc. (Minneapolis, MN, USA). Monoclonal antibodies ELISA assay against NF-κB p65 subunit and β-actin were pur- After the completion of treatments, the ani- chased from Santa Cruz Biotechnology, Inc. (Santa mals were sacrificed under deep anesthesia with Cruz, CA, USA). chloral hydrate (300 mg/kg, i.p.) and bloodletting via inferior vena cava. Blood samples were collect- Animals ed and centrifuged (4°C) at 3000 r/min for 15 min. Fifty healthy male Sprayer (SD) rats at 250- The upper serum was separated immediately and 280 g of body weight (7-8 weeks old) were pur- was stored at -20°C for future analysis. ELISA kits chased from the Experimental Animal Center of (BioSource International, Camarillo, CA, USA) Suzhou StairMaster technology Co. Ltd. (SPF was performed to determine serum levels of TNF-α grade, Certificate No. SCXK20140007). All rats and IL-6 according to the manufacturer’s instruc- were kept in a regulated environment with a con- tions. Changes of α-amylase (AMY), alanine trolled temperature of 22-24℃ and humidity of 50- aminotransferase (ALT) and aspartate aminotrans- Study on protecting effects of Parthenolide on hepatic injury in rats with severe acute pancreatitis 491 ferase (AST) in serum were determined by ELISA Twelve hours after modeling, the content of kits, respectively. AMY, AST and ALT in serum of model control group and parthenolide treated groups were signifi- Histopathological evaluation cantly higher than that in sham-operated group (P < The hepatic specimens was collected and fixed 0.05). The parthenolide treated groups were obvious- in 10% formalin for 24 h, embedded in paraffin, ly lower than the model control group (P < 0.05), and stained with hematoxylin-eosin (HE) and which appeared to be dose-dependent (Figure 1). examined by light microscopy. The content of IL-6 and TNF-α in serum Western Blot analysis As shown in Figure 2, 12 h after induction of Hepatic tissues were lysed by the nuclear SAP, the serum IL-6 and TNF-α level in model extract kit following the manufacturer’s instruc- control group was obviously higher than that in tions. SDS-PAGE was performed using 50 or 100 sham-operated group, whereas parthenolide treat- g/l acrylamide gels. Proteins were electrotransfered ment attenuated the increase of serum IL-6 (Figure to polyvinylidene fluoride membranes and probed 2A) and TNF-α (Figure 2B), which appeared to be with primary antibody (anti- NF-κB-p65, 1:500; β- dose-dependent. actin, 1:500). The membranes were incubated with corresponding horseradish peroxidase-linked secondary antibody. They were developed by enhanced chemiluminescence (Amersham International, Buckingham, UK), and exposure to X-ray film.

Statistical analysis Data were presented as means ± standard deviation (SD). Statistical comparisons between Figure 1. Comparison of AMY, ALT, AST in serum. two groups were analyzed by t test. Values p < A: The content of AMY in serum; B: The content of ALT in 0.05 was considered statistically significant. serum; C: The content of AST in serum. *p < 0.05 compared with Sham operated group; #p < 0.05 compared with Model Results control group. Serum AMY, ALT and AST content Pathological findings of liver tissue Sham group: Twelve hours after modeling, complete structure of hepatic lobules, occasional inflammatory cell infiltration in portal area were observed. Most liver cells presented normal mor- phology (Figure 3A). In the model control group, there was obviously damaged hepatic lobule structure, further increased range and area of cell necrosis, residual metamorphic liver cells only at periph- ery of partial hepatic lobules; relatively large area of inflammatory cell infiltration within lobules or portal area, obvious congestion in sinus hepaticus (Figure 3B). The changes of parthenolide treated group were milder than that of the model control group, which appeared to be dose dependent. The group treated with low or middle dose of partheno- Figure 1. Comparison of AMY, ALT, AST in serum. A: The content of AMY in serum; B: The content of lide showed obviously pathological changes than ALT in serum; C: The content of AST in serum. *p < high dose parthenolide treated group (Figure 3C 0.05 compared with Sham operated group; #p < 0.05 and 3D). The group treated with high dose of compared with Model control group. parthenolide showed slight swelling of liver cells, mild dilation and hyperemia change of sinus hepati- 492 Gaozhong Li, Guangsheng Zhai et Al cus. Punctate necrosis or slight focal necrosis of Western blot revealed that the expression of liver cells, no obvious lamellar necrosis, inflamma- NF-κB was significantly (P < 0.05) increased in the tory cell infiltration in portal area were also nucleus of liver in model control group compared observed (Figure 3E). to the sham-operated group. The administration of parthenolide dose-dependently inhibited the elevation of NF-κB in SAP rats (Figure 4).

Discussion

Along with the increasing morbidity and mortality of SAP, improving the clinical therapeutic effects on SAP and finding cheap alternates with precise therapeutic effects and fewer side effects have been the hot spots of clinical research(24-26). Parthenolide, sesquiterpene lactone existing in the leaves of feverfew, is considered to be the main component with biological activity in the extracts of this perennial plant. Infusions of feverfew con- taining parthenolide have been applied in patients to prevent migraine and rheumatic pain(27-28). Figure 3. Pathological changes of liver under light Previous studies have demonstrated that partheno- microscope. A: Sham operated group; B: Model control lide has strong anti-inflammatory effects and is a group; C: group treated with low dose of parthenolide; potent nuclear factor kappa-B (NF-κB) inhibitor by D: group treated with middle dose of parthenolide; E: specifically inhibiting the IκB kinase complex. The group treated with high dose of parthenolide; (HE present study showed that parthenolide can relieve ×200). the severity of hepatic injury in SAP procedure. Effect of parthenolide on nuclear NF-кB Alanine aminotransferase (ALT) is found expression mainly in liver cells, while aspartate aminotransferase (AST) is found mainly in cardiac muscle, followed by the liver. They are nonspecific intracel- lular functional enzymes and normally can be found in the serum at very low levels. The membrane per- meability of hepatic cells increases when they are damaged. As a result, ALT and AST in cytoplasm are released into the blood, resulting in an increase in the activities and contents of serum ALT and AST. Therefore, serum ALT and AST levels are sensitive parameters for the evaluation of hepatic cell damage. In the present study, the treatment of parthenolide obviously attenuated the content of ALT and AST in serum, which demonstrated the protective effect of parthenolide on hepatic injury in rats with SAP. Figure 4. Effect of parthenolide on nuclear NF-кB A recent study has indicated that pro-inflam- expression. The expression of NF-кB was increased in matory stimuli such as IL-6 and TNF-α play a cen- the nucleus of liver in model control group, while the tral role in the initiation and progression of SAP.1 administration of parthenolide attenuated the elevation The level of IL-6 in serum is a discriminator of of NF-кB in SAP rats. SAP: severe acute pancreatitis; Sham: Sham operated group; Model: Model control SAP, and when accompanied by multi-organ failure group; L-P: group treated with low dose of parthenolide; could be used as an early marker of a possible fatal M-P: group treated with middle dose of parthenolide; H- outcome(29). TNF-α can induce plenty of other P: group treated with high dose of parthenolide. *p < inflammatory cytokines and activate various 0.05 compared with Sham operated group; #p < 0.05 immune cells, thus inducing the pro-inflammatory compared with Model control group. Study on protecting effects of Parthenolide on hepatic injury in rats with severe acute pancreatitis 493 response(30). TNF-α and IL-6 can increase plasma tiple organ injury of rat with severe acute pancreatitis. extravasation, induce leukocyte adherence, and then World J Gastroenterol. 2007; 13(34): 4566-4573. (31) 4) Rau BM, Bothe A, Kron M, Beger HG. Role of early result in SIRS and MODS . Preventing the activity multisystem organ failure as major risk factor for pan- of TNF-α and IL-6 can markedly attenuate the sys- creatic infections and death in severe acute pancreatitis. temic amplification of SAP, and it is responsible for Clin Gastroenterol Hepatol. 2006; 4(8): 1053-1061. the increased morbidity and mortality associated doi: 10.1016/j.cgh.2006.05.030. with SAP. In this experiment, it was found that the 5) Werner J, Feuerbach S, Uhl W, Büchler MW. Management of acute pancreatitis: from surgery to content of serum IL-6 and TNF-α was significantly interventional intensive care. Gut. 2005; 54(3): 426- increased in SAP model group, while the adminis- 436. doi: 10.1136/gut.2003.035907. tration of parthenolide markedly down-regulated 6) van Baal MC, van Santvoort HC, Bollen TL, Bakker the levels of both IL-6 and TNF-α in serum, which OJ, Besselink MG, Gooszen HG. Systematic review of percutaneous catheter drainage as primary treatment for also demonstrated the protective effect of partheno- necrotizing pancreatitis. Br J Surg. 2011; 98(1): 18-27. lide on hepatic injury in rats with SAP. doi: 10.1002/bjs.7304. Some studies have found that Nuclear factor 7) Zerem E, Imamović G, Sušić A, Haračić B. Step-up kappa-light-chain enhancer of activated B cells approach to infected necrotising pancreatitis: a 20-year (NF-κB) is a key regulator of cytokine induction, experience of percutaneous drainage in a single centre. Dig Liver Dis. 2011; 43(6): 478-483. doi: which is usually elevated in the pancreas during 10.1016/j.dld.2011.02.020. SAP. Inhibition of NF-κB activation can down-reg- 8) Shrivastava P, Bhatia M. Essential role of monocytes ulate in the release of inflammatory cytokines(32). and macrophages in the progression of acute pancreati- NF-κB consists of five major proteins: RelA (p65), tis. World J Gastroenterol. 2010;16(32):3995-4002. 9) Dambrauskas Z, Giese N, Gulbinas A, Giese T, RelB, c-Rel, NF-κB1, and NF-κjB2. Activated NF- Berberat PO, Pundzius J, et al. Different profiles of κB is a transcription factor associated with a wide cytokine expression during mild and severe acute pan- range of cellular responses, including inflammation, creatitis. World J Gastroenterol. 2010; 16(15): 1845- immune regulation, survival, and proliferation(33). 1853. The majority of NF-κB activities have connection 10) Bhatia M, Brady M, Shokuhi S, Christmas S, Neoptolemos JP, Slavin J. Inflammatory mediators in (34) (35) with TNF-α, foreign antigens , oxidative stress , acute pancreatitis. J Pathol. 2000; 190(2): 117-125. doi: and exposure to radiation. Phosphorylation of IκB 10.1002/(SICI)1096-9896(200002)190:2<117::AID- by IκB kinase (IKK) on the cytosolic domain and PATH494>3.0.CO;2-K. subsequent ubiquitination and degradation of IκB 11) Ramudo L, Manso MA, Sevillano S, De DI. Kinetic study of TNF-a production and its regulatory mecha- leads to activation of NF-κB. Interestingly, nisms in acinar cells during acute pancreatitisinduced parthenolide is found to inhibit IKK-mediated by bile-pancreatic duct obstruction. J Pathol. phosphorylation of IκB by blocking the activity of 2005;206(1):9-16. IKK, thus leading to suppression of RelA/p65 12) Buter A, Imrie CW, Carter CR, Evans S, McKay CJ. activity in acute myeloid leukemia cells(36). Dynamic nature of early organ dysfunction determines outcome in acute pancreatitis. Br J Surg. 2002; 89(3): In conclusion, parthenolide can reduce the 298-302. doi: 10.1046/j.0007-1323.2001.02025.x. content of AMY, ALT, AST in serum to improve the 13) Johnson CD, Kingsnorth AN, Imrie CW, McMahon survival of rats with SAP. What’s more, it can alle- MJ, Neoptolemos JP, McKay C, et al. Double blind, viate the pathological changes of liver. We believe randomised, placebo controlled study of a platelet acti- vating factor antagonist, lexipafant, in the treatment that parthenolide can play certain role in future and prevention of organ failure in predicted severe SAP treatment. acute pancreatitis. Gut. 2001; 48(1): 62-69. 14) Horvath K, Freeny P, Escallon J, Heagerty P, Comstock B, Glickerman DJ, et al. Safety and efficacy of video- References assisted retroperitoneal debridement for infected pancreatic collections: a multicenter, prospective, single- arm phase 2 study. Arch Surg. 2010; 145(9): 817-825. 1) Kaplan A, Akkücük S, Ugur M, et al. Safety and effica- doi: 10.1001/archsurg.2010.178. cy of tirofiban hydrochloride (TH) in cerulein-induced 15) Beger HG, Rau BM. Severe acute pancreatitis: Clinical acute pancreatitis[J]. Acta Medica Mediterranea, 2015, course and management. World J Gastroenterol. 2007; 31(6): 1253. 13(38): 5043-5051. 2) Zhang Q, Ni Q, Cai D, Zhang Y, Zhang N, Hou L. 16) Bruennler T, Langgartner J, Lang S, Wrede CE, Klebl Mechanisms of multiple organ damages in acute necro- F, Zierhut S, et al. Outcome of patients with acute, tizing pancreatitis. Chin Med J (Engl). 2001; 114(7): necrotizing pancreatitis requiring drainagedoes 738-742. drainage size matter? World J Gastroenterol. 3) Zhang XP, Ye Q, Jiang XG, Ma ML, Zhu FB, Zhang 2008;14(5):725-730. RP, et al. Preparation method of an ideal model of mul- 17) Knight DW. Feverfew: chemistry and biological activi- 494 Gaozhong Li, Guangsheng Zhai et Al

ty. Nat Prod Rep. 1995; 12(3): 271-276. 31) Kim H. Cerulein pancreatitis:oxidative stress, inflam- 18) Nam YJ, Lee DH, Lee MS, Lee CS. Sesquiterpene lac- mation, and apoptosis. Gut Liver. 2008; 2(2): 74-80. tone parthenolide attenuates production of inflammato- doi: 10.5009/gnl.2008.2.2.74. ry mediators by suppressing the Toll-like receptor-4- 32) Kim H, Seo JY, Roh KH, Lim JW, Kim KH. mediated activation of the Akt, mTOR, and NF-κB Suppression of NF-kappaB activation and cytokine pathways. Naunyn Schmiedebergs Arch Pharmacol. production by N-acetylcysteine in pancreatic acinar 2015; 388(9): 921-930. doi: 10.1007/s00210-015-1132- cells. Free Radic Biol Med. 2000; 29(7): 674-683. 3. 33) Baud V, Karin M. Is NF-kappaB a good target for can- 19) Zhang X, Fan C, Xiao Y, Mao X. Anti-inflammatory cer therapy? Hopes and pitfalls. Nat Rev Drug Discov. and antiosteoclastogenic activities of parthenolide on 2009;8(1):33-40. doi: 10.1038/nrd2781. human periodontal ligament cells in vitro. Evid Based 34) Tapia PC. Sublethal mitochondrial stress with an atten- Complement Alternat Med. 2014; 2014: 546097. doi: dant stoichiometric augmentation of reactive oxygen 10.1155/2014/546097. species may precipitate many of the beneficial alter- 20) Wang M, Li Q. Parthenolide could become a promising ations in cellular physiology produced by caloric and stable drug with anti-inflammatory effects. Nat restriction, intermittent fasting, exercise and dietary Prod Res. 2015; 29(12): 1092-1101. doi: phytonutrients: “Mitohormesis” for health and vitality. 10.1080/14786419.2014.981541. Meidical Hypotheses. 2006; 66(4): 832-843. doi: 21) Muhs BE, Patel S, Yee H, Marcus S, Shamamian P. 10.1016/j.mehy.2005.09.009. Inhibition of matrix metalloproteinases reduces local 35. Payne CM, Weber C, Crowley-Skillicorn C, et al. and distant organ injury following experimental acute Deoxycholate induces mitochondrial oxidative stress pancreatitis. J Surg Res. 2003; 109(2): 110-117. and activates NF-κB through multiple mechanisms in 22) Zhang XP, Zhang L, He JX, Zhang RP, Cheng QH, HCT-116 colon epithelial cells. Carcinogenesis. 2007; Zhou YF, et al. Experimental study of therapeutic effi- 28(1): 215-222. doi: 10.1093/carcin/bgl139. cacy of Baicalin in rats with severe acute pancreatitis. 36) Dai Y, Guzman ML, Chen S, Wang L, Yeung SK, Pei World J Gastroenterol. 2007; 13(5): 717-724. XY, et al. The NF (nuclear factor)-κB inhibitor 23) Zhang XP, Zhang L, Yang P, Zhang RP, Cheng QH. parthenolide interacts with histone deacetylase Protective effects of baicalin and octreotide on multiple inhibitors to induce MKK7/JNK1-dependent apoptosis organ injury in severe acute pancreatitis. Dig Dis Sci. in human acute myeloid leukaemia cells. Br J 2008; 53(2): 581-591. doi: 10.1007/s10620-007-9868- Haematol. 2010; 151(1): 70-83. doi: 10.1111/j.1365- 3. 2141.2010.08319.x. 24) Lese M, Tamasan A, Stoicescu B, Brânduşe M, Puia I, Mare C, et al. Surgical treatment in severe acute pancreatitis. Last 15 years of experience in Emergency County Hospital of Baia Mare. Chirurgia (Bucur). 2005; 100(5): 445-450. 25) Gunduz E, Zengin Y, Ulger B V, et al. Demographic Properties And Clinical Outcomes Of Acute Pancreatitis In Pregnancy: Our Experience Of 33 Patients[J]. Acta Medica Mediterranea, 2014, 30(30): 1285-1290. 26) Cappell MS. Acute pancreatitis: etiology, clinical pre- sentation, diagnosis, and therapy. Med Clin North Am. 2008;92(4):889-923, ix-x. doi: 10.1016/j.mcna.2008.04.013. 27) Levin M. Herbal treatment of headache. Headache. 2012;52(Suppl 2):76-80. doi: 10.1111/j.1526- 4610.2012.02234.x. 28) Magni P, Ruscica M, Dozio E, Rizzi E, Beretta G, Maffei Facino R. Parthenolide Inhibits the LPS- induced Secretion of IL-6 and TNF-α and NF-κB Nuclear Translocation in BV-2 Microglia. Phytother Res. 2012; 26(9): 1405-1409. doi: 10.1002/ptr.3732. 29) Lee JH, An CS, Yun BS, Kang KS, Lee YA, Won SM, et al. Prevention effects of ND-07, a novel drug candi- date with a potent antioxidative action and anti-inflam- ______matory action, in animal models of severe acute pan- Corresponding author creatitis. Eur J Pharmacol. 2012;687(1-3): 28-38. doi: JIANQIANG GUO 10.1016/j.ejphar.2012.04.048. Department of Gastroenterology, the second hospital of 30) Surbatovic M, Radakovic S. Tumor necrosis factor-a Shandong University, Jinan 250033 levels early in severe acute pancreatitis:is there predic- Shandong Province tive value regarding severity and outcome. J Clin (China) Gastroenterol. 2013; 47(7): 637-643.