Dong Li Chen, Wei Zhong Wang and Jun Yi Wang  Department of Gastrointestinal Surgery, Xijing Hospital, Fourth Military Medical University Xi＇an 710032, Shaanxi Province, China
Dr. Dong Li Chen, graduated from Fourth Military Medical University as a postgraduate in 1994, Doctor in charge of General Surgery, major in nutrition support and metabolism, having 15 papers published.
Presented at the International Symposium “Growth Factor and Nutrients in Intestinal Health and Disease”, Osaka, Japan, 31 October-3 November, 1998.
Correspondence to: Dr. Dong Li Chen, Department of Gastrointest inal Surgery, Xijing Hospital, Fourth Military Medical University Xi＇an 710032, Shaanxi Province, China
Telephone: 0086-29-3375265, Fax. 0086-29-3375261
Email. xjwcwk@fmmu.edu.cn.
Received: 2000-05-18 Accepted: 2000-06-23

Subject heading: pancreatitis; epidermal growth factor; parenteral nutrition, total; intestinal mucosa; DNA; proteins

Chen DL, Wang WZ, Wang JY. Epidermal growth factor prevents gut atro phy and maintains intestinal integrity in rats with acute pancreatitis. World J Gastroentero,2000;6(5):762-765

INTRODUCTION
There is abundant evidence that stressful insults such as acute pancreatitis may significantly alter the metabolism of the gut mucosa and therefore its barrier integrity, resulting in an increase in mucosal permeability and subsequent trans location of enteric bacteria and their endotoxins^[1-9]. The fact that most bacteria associated with acute pancreatic and peripancreatic infections are of enteric origin implies that the gut plays a major role in the pathogenesi s of pancreatic infection^[10-16].Thus various therapeutic modalities have been undertaken to maintain gut mucosal metabolism and function as well as to reduce the bacterial translocation during acute pancreatitis.
      In recent years, much attention has been focused on hormonal regulation as o ne of the effective therapeutic strategies. Epidermal growth factor (EGF), which presents in large amount in the salivary and Brunner＇s gland, and in a variet y of secretions including saliva and milk, is a potent mitogen for small intesti nal cells both in vivo and in vivo. Parenteral nutrition with administr ation of exogenous EGF has been shown to increase DNA and protein content in the small intestine^[17-20]. EGF can also regulate intestinal brush border e nzymes functionally^[21,22].
      The aim of this study is to evaluate the protective effects of EGF on intesti nal barrier function in rats with acute pancreatitis under total parenteral nutr ition (TPN).

MATERIALS AND METHODS
Materials
Forty-one male Sprague Dawley rats, each weighing approximately 210g, were purchased from the Experimental Animals Center of Fourth Military Medical University. The rats were given water ad libitum and a standard rat food die t. They were subjected to alternate 12h periods of darkness and light. Aft er overnight fasting, the rats underwent placement of a central venous catheter through a right external jugular vein under sodium pentobarbital anesthesia (40mg·kg^-1, intraperioneally). The central venous catheters were tunneled subscutaneously and attached to a spring coil/brass swivel mechanism, which allowed for free movement of the animals in cages. Acute pancreatitis was induced by intraductal infusion of 35g·L^-1 sodium taurocholate solution (1.0mL·kg^-1) after clamping the proximal end of the common bile duct and puncture through the duodenum into the biliary-pancreatic duct^[23-27]. On the day of cannulation (d0), rats were randomly divided into one of the two groups. The control group (n=21) was fed a conventional parenteral nutrition solution; the EGF group (n= 20) was fed besides the identical parenteral nutrition formula as in the control group, EGF (0.1mg·kg^-1) was injected subcutaneously twice daily.
      The TPN solutions were prepared in a laminar flow hood and were sterilized by membrane filtration. The composition of these solutions is shown in Table 1^[28].

Methods
On d 1 and d 5 after induced acute pancreatitis, every 8 animals in each group were anesthetized with 40mg·kg^-1 sodium pentobarbital respectively. A midline abdominal incision was made and a 60cm length of small intestine, 20cm distal from the ligament of Treitz, was ligated at both ends. Then, 1.0mL fluorescein isothiocyanate (FITC)-dextran 4000 (25g·L^-1) solution was injected into the lumen of this ligated segment. A blood sample was withdrawn from the superior mesenteric vein 30min later for the analysis of plasma FITC-dextran with a fluorescence spectrophoto meter at an excitation wave length of 480nm, an emission wave length of 530nm, and expressed as mg of FITC dextran per L of plasma^[29-31] .
      For histologic evaluation, 2cm of proximal jejunal segement was fixed in 100mL·L^-1(V·V^-1) formalin, embedded in paraffin, and stained with hematoxylin-eosin. Three paraffin sections were prepared from each fixed tissue sample, and each slide was analyzed. Villus height and area were measured in 10 well orientated villi, giving a total of 30 villis for each jejunal segme nt. Measurements were made in a blind fashion on coded slides, and mean values were obtained^[32,33].
      Samples of jejunal mucosa were scraped and used for the measurement of mass a nd enzyme activity. The activities of sucrase and maltase were determined by the method of Dahlqvist^[34]. Myeloperoxidase (MPO) activity was determin ed by the method of Bradley et al^[35]. The protein content in each sample was estimated according to the method of Read et al^[36].

Statistical analysis
Data were expressed as x-±s as indicated in each table. The significance of any difference between the two groups was determined with the Student＇s t test. Differences were considered statistically significant at P＜0.05.

RESULTS
Mortality rate 
During the 5d of TPN after induced acute pancreatitis, the mortality ra te was similar in the two groups. Specifically, 4.8% (1/21), 23.8% (4/21) in the control group and 5.0% (1/20), 20.0% (3/20) in EGF group on d 1 and d 5 respectively. This did not reach statistical significance.

Changes in body mass
The initial body mass in the two groups was similar (213g±8g in the EGF group vs 210g±6g in the control group) and there was no significant change on d1 (214g±9g in the EGF group vs 211g±6g in the control group). But the final body mass gain was signifi cantly greater in the EGF group than in the control group on d5 (15g± 2g in the EGF group vs 4g±1g in the control group, P＜0. 01).

Changes in mucosal wet mass, villus height and area
Mucosal wet mass, villus height and area in the control group decreased signific antly as compared with the EGF group on d5 (P＜0.01, Table 2) .

Changes in intestinal permeability
Plasma FITC-dextran level in the control group increased significantly as comp ared with the EGF group on d5(P＜0.01,Table 3).

Changes in activities of sucrase, maltase and MPO
Activities of sucrase and maltase in the control group decreased significantly as compared with those in the EGF group on d 5(P＜0.05, P＜0.01, respectively). However, MPO activity in the control group increased s ignificantly as compared with that in the EGF group on d5 (P＜0.01, Table 4).

Table 1 Composition of TPN solution (mL)

*Products from Kabi Vitrum, Sweden.
Table 2 Changes in mucosal wet mass, villus height and area (x-±s, n=8)

^bP＜0.01, vs control group at same time.
Table 3 Changes in plasma FITC-dextran (x-±s, n=8, mg·L^-1)

^bP＜0.01, vs control group at same time.
Table 4 Changes in activities of MPO, sucrase and maltase (x-±s, n=8)

^aP＜0.05, ^bP＜0.01 vs control group at same time.

DISCUSSION
The effects of EGF on intestinal integrity were investigated in an experimental acute pancreatitis model in rats. TPN was used in both groups to mimic the clini cal setting in as much as acute pancreatitis patients are often nourished by TPN . Acute pancreatitis can lead to ischemic damage of intestinal mucosa. Administration of TPN even to healthy experimental animals is associated with progress ive intestinal atrophy, which is characterized by reduction of mucosal mass, vil lus height and area, mucosal wall thickness, etc^[37-41], so that combination of acute pancreatitis and TPN might lead to more damage to the gut mucosa than TPN or acute pancreatitis alone. EGF was selected because it has been suggested to be a potent mitogen for small intestinal cells both in vitro and in vivo^[42-44]. A previous study by our group also demonstrated that EGF could increase DNA and protein content in the small intestine^[45].
      In this study, the body mass gain in the EGF group was significantly greater than in the control group on d5. This may be contributed to an anabolic effect of EGF^[44]. On d5, the significantly increased mucosa l mass, villus height and area in jejunum were also found in the EGF group as compared with the control group. This is because that EGF can enhance intestinal glutam ine influx and supply more energy for mucosal regeneration so as to attenuate in testinal atrophy. And it is also related to the increased mucosal protein and DN A content in small intestine^[46,47].The results suggest that the administration of exogenous EGF may prevent intestinal atrophy in rats with acute pancreatitis under TPN.
      For the assessment of barrier function of intestinal mucosa, a permeability test can be a suitable method. Pantzar et al^[31] suggested that nondegr adable dextrans could be used as permeability markers and reflected the proteoly sis-independent passage of proteins through the small intestinal epithelia. Because there may be a paracellular route through the tight junctions for the markers with M_r below 30000 instead of a transcellular route as sugg ested for the larger molecules. In the present study, permeability of the small intestine to FITC-dextran 4000 (mean M_r, 4000), through the tight junctions of the intestinal epithelia, increased significantly in the control group as compared with the EGF group on d 5. The results indicate that EGF may prevent an increase in permeability of the small intestine to FITC-dextran 4000 in rats with acute pancreatitis under TPN.
      Tissue damage can be caused either directly or indirectly by the oxidative met abolism of the infiltrating polymorphonuclear leukocytes (PMNs). It is believed that after specific membrane perturbation by stimuli, PMNs may exhibit a burst i n oxygen consumption and start to generate active oxygen metabolites, which may lead to oxidative stress in tissues. So, the accumulation of PMNs in affected or gans is considered to be one of the causative factors of multiple organ failure (MOF). MPO is an essential enzyme for PMNs function and a useful indicator of it s infiltration. Evidence indicates that normal small intestine bears a low backg round of MPO activity, and the enzyme activity increased significantly in ischem ic small intestine followed by PMNs infiltration^[48,49]. MPO activity also increased in the lung of rats with acute pancreatitis^[50,51].It is still unclear whether PMNs infiltration may be involved in the damage of small intestine in acute pancreatitis. In the present study, MPO activity in the control group increased significantly as compared with the EGF group on d5. It indicates that EGF may reduce PMNs accumulation in intestinal mucosa, thus minimizing oxidative stress in rats with acute pancreatitis under TPN.
      Sucrase and maltase, which lie in villus brush border, are two kinds of impor tant disaccharidases. They are often used as the markers of the normal cel l proliferation and digestive function in small intestine^[29,30,34]. In this study, activities of sucrase and maltase in the control group decreased significantly compared with the EGF group on d 5. Maintenance of sucrase and maltase activities indicates that EGF may alleviate damage of jejunum in rats with acute pancreatitis under TPN.
      In summary, the present study demonstrated that treatment with EGF can lead to body weight gain, reduce gut atrophy and PMNs accumulation in intestinal mucos a, prevent increased intestinal permeability and maintain sucrase and maltase ac tivities in acute pancreatitis rats under TPN.

REFERENCES
1    Wang XS, Gao YB, Zhang ZL. Influence of destroyed pancreatitic cover on mortality of pancreatitis rats.
      Shijie Huaren Xiaohua Zazhi,2000; 8:587-588
2    Gong ZH, Yuan YZ, Lou KX, Tu SP, Zhai ZK, Xu JY. Effects and mechanisms of somatostatin analogues on apoptosis of 
      pancreatic acinar cells in acute pancreatitis in mice.Shijie Huaren Xiaohua Zazhi,1999;7:964-966
3    Liu Q, Djuricin G, Rossi H, Bewsey K, Nathan C, Gattuso P, Weinstein RA, Prinz RA. The effect of lexipafant on bacterial
      translocation in acute necrotizing pancreatitis in rats.Am Surg,1999;65:611-616
4    Kotani J, Usami M, Nomura H, Iso A, Kasahara H, Kuroda Y, Oyanagi H, Saitoh Y. Enteral nutrition prevents bacterial 
      translocation but does not improve survival during acute pancreatitis.Arch Surg,1999;134:287-292
5    Zhou XZ, Mao QS, Chen YQ, Shen HX. Relationship between pancreatitis pathology and oxygen free radicals in rats. 
      Shijie Huaren Xiaohua Zazhi,2000;8:108 -109
6    Luiten EJ, Hop WC, Endtz HP, Bruining HA. Prognostic importance of gram negative intestinal colonization preceding 
      pancreatic infection in severe acute pancreatitis. Results of a controlled clinical trial of selective decontamination.
      Intensive Care Med,1998;24:438-445
7    Wang XD, Andersson R, Soltesz V, Leveau P, Ihse I. Gut origin sepsis, macroph age function, and oxygen extraction 
      associated with acute pancreatitis in the rat.World J Surg,1996;20:299-308
8    Wang XP, Yuan YZ, Xu JY. Effect of glutamine on acute pancreatitis in rats.Xin Xiaohuabingxue Zazhi,1993;1:204-205
9    Liu H, Ji YL. Analysis of 434 cases of acute pancreatitis.Xin Xiaohuabingxue Zazhi,1996;4:527-528
10  Ruan CP, Wang YH, Wang LG. Bacterial translocation from the gastrointestinal tract in rats with intestinal ischemia.
      Xin Xiaohuabingxue Za zhi, 1996;4:304-305
11  Wang ZF, Pan CE, Liu SG. Role of inflammatory mediators in acute pancr eatitis.Huaren Xiaohua Zazhi,1998;6:170-171
12  Gu JC, Qin ZY, Wang Y. Changes of TXA-2 and PGI-2 in acute necrotic pancreatitis combined with lung injury in 
      experimental rats. Shijie Huaren Xiaohua Zazhi,1999;7:275
13  Chen JZ, Dai ZB. Changes of intestinal microcirculation in acute necrotic pancreatitis with bacterial translocation.
      Shijie Huaren Xiaohua Zazhi, 1999;7:641
14  Li ZL. Diagnosis and treatment of multiple organ disorder and failure induced by severe infections.
      Shijie Huaren Xiaohua Zazhi,1999;7:1074-1076
15  Sahin M, Yol S, Ciftci E, Baykan M, Ozer S, Akoz M, Yilmaz O, Kuru C.Does large bowel enema reduce septic 
      complications in acute pancreatitis. Am J Surg,1998;176:331-334
16  Dong HL.Intestinal permeability test and its clinical significance. Shijie Huaren Xiaohua Zazhi,2000;8:562-563
17  Bulus N, Barnard JA. Heparin binding epidermal growth factor-like growth factor is a transforming growth factor beta 
      regulated gene in intestinal epithelial cells.Biochem Biophys Res Commun,1999;264:808-812
18  Svanberg E, Svaninger G, Soussi B, Lundholm K. Mouse extensor digitorum longus muscle preparation as a tool in 
      nutrition research: a quantitative comp arison to in vivo and cell culture experiments.Nutrition,1999;15:200-207
19  Petersen H, Haldosen LA. EGF modulates expression of STAT5 in mammary epithelial cells.Exp Cell Res,
      1998;243:347-358
20  Usui S.Progress of enteral nutrition.NipponGeka Gakkai Zasshi,1998;99:154-158
21  Darimont C, Gradoux N, de Pover A. Epidermal growth factor regulates fatty acid uptake and metabolism in Caco2 cells.
      Am J Physiol,1999;276(3 Pt1):G606-G612
22  Shin CE, Falcone RA Jr, Duane KR, Erwin CR, Warner BW. The distribution of endogenous epidermal growth factor 
      after small bowel resection suggests inc reased intestinal utilization during adaptation.J Pediatr Surg,1999;34:22-26
23  Plusczyk T, Rathgeb D, Westermann S, Feifel G. Somatostatin attenuates microcirculatory impairment in acute sodium
      taurocholate induced pancreatitis.Dig Dis Sci,1998;43:575-585
24  Bloechle C, Kusterer K, Kuehn RM, Schneider C, Knoefel WT, Izbicki JR.Inhibition of bradykinin B2 receptor preserves
      microcirculation in experimental pancreatitis in rats.Am J Physiol,1998;274(1 Pt 1):G42-G51
25  Aho HJ, Koskensalo SML, Nevalainen TJ. Experimental pancreatitis in the rats: sodium taurocholate-induced acute
      pancreatitis.Scand J Gastroenterol,1980;15:411-416
26  Xu CF, Jiang WP, Cai YL, Zhang YK, Liu SZ. Protective effect of verapamil on experimental acute pancreatitis.
      Xin Xiaohuabingxue Zazhi,1997 ;5:295-296
27  Lu XD, Liu GS, Chen YR. Experimental and clinical research of tumor necrosis factor alpha on acute pancreatitis.
      Xin Xiaohuabingxue Zazhi,1997;5:534
28  Zhang GX, Lai JH, Jia TW, Wang WZ, Wang JY. Effect of epidermal growth factor on glutamine metabolic enzymes in 
      small intestine and skeletal muscle of parenterally fed rats.Nutrition,1997;13:652-655
29  Chen DL, Sando K, Chen K, Wasa M, Takagi Y, Okada A. Protective effect s of selenium supplementation in minimizing 
      5 fluorouracil induced lipid peroxi dative damage of the small intestine.J Trace Elem Exp Med,1997;10:163-171
30  Chen DL, Wu GS, Wang WZ, Wang JY. Neutrophil infiltration is involved in 5 fluorouracil induced lipid peroxidative 
      damage of the small intestine.Chin Med Sci J,1997;12:181-183
31  Pantzar N, Westrm BR, Luts A, Lundin S. Regional small intestinal permeability in vitro to different sized dextrans and 
      proteins in the rat. Scand J Gastroenterol,1993;28:205-211
32  Haque SMM, Chen K, Usui N, Iiboshi Y, Okuyama H, Masunari A, Cui L, Nezu R, Takagi Y, Okada A. Alanyl glutamine 
      dipeptide supplemented parenteral nutrition improves intestinal metabolism and prevents increased permeability in 
      rats. Ann Surg,1996;223:334-341
33  Chen DL, Wang WZ, Wang JY. Epidermal growth factor protects intestinal function of rats with acute pancreatitis 
      during total parenteral nutrition. JPEN J Parenter Enteral Nutr,1999;23:S159
34  Dahlqvist A. Assay of intestinal disaccharidases.Anal Biochem,1968;22:99-107
35  Bradley PP, Priebat DA, Christensen RD, Rothstein G. Measurement of cutaneous inflammation: estimation of neutrophil 
      content with an enzyme marker. J Invest Dermatol,1982;78:206-209
36  Read SM, Northcote DH. Minimization of variation in the respose to different proteins of the coomassie blue G dye 
      binding assay for protein.Anna Biochem,1981;116:53-64
37  Li J, King BK, Janu PG, Renegar KB, Kudsk KA. Glycyl-L-glutamine-enr iched total parenteral nutrition maintains small 
      intestine gut associated lymph oid tissue and upper respiratory tract immunity.JPEN J Parenter Enteral Nutr,
      1998;22:31-36
38  Chance WT, Foley NT, Thomas I, Balasubramaniam A. Prevention of parent eral nutrition induced gut hypoplasia by 
      coinfusion of glucagon like peptide 2.Am J Physiol,1997;273(2 Pt 1):G559-G563
39  Janu P, Li J, Renegar KB, Kudsk KA. Recovery of gut associated lymphoid tissue and upper respiratory tract immunity 
      after parenteral nutrition.Ann Surg,1997;225:707-717
40  Qin HL, Cui HG, Zhang CH, Wu DW, Chu XP. Effects of glutamine on struc ture and function of gut in endotoxemic rats.
      China Natl J New Gastroenterol,1996;2:69-72
41  Raina N, Cameron RG, Jeejeebhoy KN. Gastrointestinal, hepatic, and metabolic effects of enteral and parenteral 
      nutrition in rats infused with tumor necrosis factor.JPEN J Parenter Enteral Nutr,1997;21:7-13
42  Goodlad RA, Raja KB, Peters TJ, Wright NA. Effects of urogastrone epi dermal growth factor on intestinal brush border 
      enzymes and mitotic activity. Gut,1991;32:994-998
43  Gong LB, Yang X, Zhang WW, Li SL, Sun SY. Study on the content of serum epidermal growth factor, gastric acid 
      secretion and serum gastrin in duodenitis.China Natl J New Gastroenterol,1996;2:228-229
44  Zhou J, Wu K, Fernandes CL, Cheng AL, Finch PW. Keratinocyte growth fa ctor down regulates expression of the 
      sucrase isomaltase gene in Caco 2 intestinal epithelial cells.J Biol Chem,1998;273:33367-33373
45  Wang JY, Zhang LH, Song WL. Epidermal growth factor regulates intestin al glutamine uptake during total parenteral 
      nutrition. Clin Nutr, 1996;15:21-23
46  O＇Loughlin E, Winter M, Shun A, Hardin JA, Gall DG. Structural and function adapation following jejunal resection in 
      rabbits: effect of epidermal growth factor.Gastroenterology,1994;107:87-89
47  Jacobs DO, Evans DA, Mealy K, O＇Dwyer ST, Smith RJ, Wilmore DW. Combined effects of glutamine and epidermal 
      growth factor on the rat intestine.Surgery,1988;104:358-364
48  Konaka A, Nishijima M, Tanaka A, Kunikata T, Kato S, Takeuchi K. Nitric oxide, superoxide radicals and mast cells in
      pathogenesis of indomethacin induced small intestinal lesions in rats.J Physiol Pharmacol,1999;50:25-38
49  Cetinkale O, Konukoglu D, Senel O, Kemerli GD, Yazar S. Modulating the functions of neutrophils and lipid peroxidation 
      by FK506 in a rat model of thermal injury.Burns,1999;25:105-112
50  Gloor B, Todd KE, Lane JS, Rigberg DA, Reber HA. Mechanism of increase d lung injury after acute pancreatitis in IL-10
      knockout mice.J Surg Res,1998;80:110-114
51  Sugita H, Yamaguchi Y, Ikei S, Ogawa M. Effects of propentofylline on tumor necrosis factor alpha and cytokine-induced
      neutrophil chemoattractant production in rats with cerulein-induced pancreatitis and ndotoxemia.Pancreas,
      97;14:267-275

Reviews	Add
more>>