Epidermal growth factor prevents gut atrophy and maintains intestinal integrity in rats with acute pancreatitis

Abstract

Key Words: pancreatitis, epidermal growth factor, parenteral nutrition, total, intestinal mucosa, DNA, proteins

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 pathogenesis 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 one of the effective therapeutic strategies. Epidermal growth factor (EGF), which presents in large amount in the salivary and Brunner’s gland, and in a variety of secretions including saliva and milk, is a potent mitogen for small intestinal cells both in vivo and in vivo. Parenteral nutrition with administration 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 enzymes functionally[21,22].

The aim of this study is to evaluate the protective effects of EGF on intestinal barrier function in rats with acute pancreatitis under total parenteral nutrition (TPN).

MATERIALS AND METHODS

Materials

Forty-one male Sprague Dawley rats, each weighing approximately 210 g, were purchased from the Experimental Animals Center of Fourth Military Medical University. The rats were given water ad libitum and a standard rat food diet. They were subjected to alternate 12 h periods of darkness and light. After overnight fasting, the rats underwent placement of a central venous catheter through a right external jugular vein under sodium pentobarbital anesthesia (40 mg·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 35 g·L^-1 sodium taurocholate solution (1.0 mL·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 (0.1 mg·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].

Table 1 Composition of TPN solution (mL).

Composition	Volume
50% glucose	40
7% vamin*	16
20% intralipid*	11
Addamel*	0.3
Soluvit N*	0.3
Vitalipid N adult*	0.3
Heparin 60 U

*Products from Kabi Vitrum, Sweden.

Methods

On d 1 and d 5 after induced acute pancreatitis , every 8 animals in each group were anesthetized with 40 mg·kg^-1 sodium pentobarbital respectively. A midline abdominal incision was made and a 60 cm length of small intestine, 20 cm distal from the ligament of Treitz, was ligated at both ends. Then, 1.0 mL fluorescein isothiocyanate (FITC)-dextran 4000 (25 g·L^-1) solution was injected into the lumen of this ligated segment. A blood sample was withdrawn from the superior mesenteric vein 30 min later for the analysis of plasma FITC-dextran with a fluorescence spectrophoto meter at an excitation wave length of 480 nm, an emission wave length of 530 nm, and expressed as mg of FITC-dextran per L of plasma[29-31].

For histologic evaluation, 2 cm of proximal jejunal segement was fixed in 100 mL·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 segment. 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 and enzyme activity. The activities of sucrase and maltase were determined by the method of Dahlqvist[34]. Myeloperoxidase (MPO) activity was determined 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 5 d of TPN after induced acute pancreatitis, the mortality rate 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 (213 g ± 8 g in the EGF group vs 210 g ± 6 g in the control group) and there was no significant change on d 1 (214 g ± 9 g in the EGF group vs 211 g ± 6 g in the control group). But the final body mass gain was significantly greater in the EGF group than in the control group on d 5 (15 g ± 2 g in the EGF group vs 4 g ± 1 g 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 significantly as compared with the EGF group on d5 (P < 0.01, Table 2).

Table 2 Changes in mucosal wet mass, villus height and area (x^-± s, n = 8).

t/d	Group	m (wet mucosa)/mg·cm-1	h (villus)/μm2	a (villus)/μm
1	Control	35 ± 3	385 ± 32	40872 ± 5194
	EGF	35 ± 4	394 ± 37	41328 ± 4901
5	Control	30 ± 2	272 ± 21	18658 ± 2469
	EGF	44 ± 3b	409 ± 32b	43227 ± 5340b

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

Changes in intestinal permeability

Plasma FITC-dextran level in the control group increased significantly as compared with the EGF group on d 5 (P < 0.01, Table 3).

Table 3 Changes in plasma FITC-dextran (x^-± s, n = 8, mg·L^-1).

t/d	Group	FITC-dextran
1	Control	1.2 ± 0.5
	EGF	1.1 ± 0.4
5	Control	7.5 ± 0.7
	EGF	3.3 ± 0.7b

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

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 significantly as compared with that in the EGF group on d5 (P < 0.01, Table 4).

Table 4 Changes in activities of MPO, sucrase and maltase (x^-± s, n=8).

t/d	Group	z/m (MPO)/nkat·g-1	z/m (sucrase)/nkat·g-1	z/m (maltase)/nkat·g-1
1	Control	143.34 ± 111.67	0.54 ± 0.11	1.75 ± 0.32
	EGF	231.19 ± 8.34	0.49 ± 0.02	1.82 ± 0.21
5	Control	96.68 ± 13.35	0.19 ± 0.05	0.84 ± 0.16
	EGF	66.71 ± 13.20b	0.28 ± 0.08a	1.23 ± 0.24b

^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 clinical 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 progressive intestinal atrophy, which is characterized by reduction of mucosal mass, villus 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 d 5. This may be contributed to an anabolic effect of EGF[44]. On d 5, the significantly increased mucosal 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 DNA 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 metabolism of the infiltrating polymorphonuclear leukocytes (PMNs). It is believed that after specific membrane perturbation by stimuli, PMNs may exhibit a burst in 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 its infiltration. Evidence indicates that normal small intestine bears a low background of MPO activity, and the enzyme activity increased significantly in ischemic 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 d 5. 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 cell 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 mucosa, prevent increased intestinal permeability and maintain sucrase and maltase activities in acute pancreatitis rats under TPN.