Histomorphometric study of role of lactoferrin in atrophy of the intestinal mucosa of rats

M. Caruso, A. Demonte, V. A. Neves

Department of Food and Nutrition, School of Pharmaceutical Sciences, S?o Paulo State University-UNESP, Araraquara, Brazil.
UNIARA-University Centre of Araraquara, Araraquara, Brazil.

DOI: 10.4236/health.2012.412198                

Abstract

The purpose of this work was to study the effects of oral administration of lactoferrin (Lf) in rats subjected to atrophy of the small intestine induced by a diet based on soy protein concentrate as the main protein source. We used 24 male Wistar rats aged 40 days, kept in individual cages under appropriate conditions of temperature, light and humidity. The animals were divided into four groups (n = 6); 1) group SL received soy-based food and, once a day, a supplement of 200mg/kg of Lf administered by gavage; 2) group Si received the soy feed without supplement of Lf; 3) group CL received a diet based on casein plus Lf; 4) group Ci received the casein diet without supplement of Lf. At the end of fifteen days, a 10 mm segment of the initial portion of the small intestine was sectioned and subjected to morphometry of the intestinal crypts and villi and assessment of the number and size of myofibroblasts. Comparison between groups showed that the length of the villi was similar in groups Ci and CL and higher in CL than in SL; SL than in Si, in Ci than in SL, and in Ci than in Si to Ci. The crypt depth was similar in SL and CL, SL and Ci and Ci and CL and was higher in Si than in Ci and in Si than in SL. The number of myofibroblasts was higher in SL than in CL, in SL than in Si, in CL than in Ci, and in SL than in and Ci; between Ci and Si there was no difference. The area of myofibroblasts was similar between the groups SL and CL and Si and Ci and higher in SL than in Si, and in Cl than in and Ci, and in SL than in Ci. All statistical analysis assumed significance when p < 0.05. From these results, we conclude that lactoferrin increases the number and size of the pericrypt myofibro-blasts and stimulates rapidly the regeneration of atrophied villi.

Keywords

Intestinal Villi; Lactoferrin; Myofibroblast; Atrophy

1. INTRODUCTION

The basic functional unit of absorption in the intestine is the “crypt-villus”. Enterocytes proliferate in the crypt and migrate toward the top of the villi where they are discarded at the end of their useful life. The reproduction rate of these cells in the crypt and the rate migration may be altered in various physiological or pathological states and also by the action of various substances [1]. Nutritional factors may be directly responsible for altering the intestinal mucosa [2]. The gut-nutrient relationship is extremely complex and involved, where even the mere presence or absence of nutrients in the intestinal lumen may induce changes or adaptations, such as atrophy or hypertrophy of the mucosa. There are reports that soybean meal can cause an increase in villous atrophy and some authors assume those immunologically active proteins, such as glycinin and b-conglycinin, are responsible for hypersensitivity reactions, and thus cause villous atrophy [3]. Feeding pigs on a diet with soybean meal as the sole source of protein, or on “corn/soy” diets for pigs, resulted in a lower villus height in the middle portion of the small intestine, as well as deeper in all areas of the small intestine [4], compared to diet with casein as the main protein observed by Caruso and Demonte [5] in rats subjected to dietary soy protein concentrate as the sole protein source.

Lactoferrin (Lf) is a glycoprotein belonging to the transferring group well recognized for its antimicrobial action and also for promoting cell growth and differentiation [6]. The molecular mechanisms by which lactoferrin acts at the cellular level are largely unknown, but specific receptors for lactoferrin have been described in various cell types and tissues of mammals, such as monocytes, lymphocytes, platelets, liver, epithelial cells of the mammary glands and intestines [7]. Some studies have shown the involvement of this protein in stimulating the growth of epithelial cells such as keratinocytes [8], enterocytes in Caco-2 culture [9], epithelium of the intestinal lymphoid tissue [10] and endometrial stromal cells [11]. Similar actions have also been demonstrated for cells of mesodermal origin, such as osteoblasts [12], lymphoblastic T cells [13] and also in angiogenesis [14]. Myofibroblasts (Mf), cells of mesodermal origin, which are differentiated fibroblasts that have been to express smooth muscle cell phenotype, are often located beneath the skin, as well as at the edge of the crypts and in the lamina propria of intestinal villi. They are the source of various cell growth factors, such as FGF (Fibroblast Growth Factor) and HGF (Hepatocyte Growth Factor), and also of basement membrane proteins [15].

Our experiment had the objective of determining the possible influence of oral administration of lactoferrin on the sub-epithelial pericrypt sheath myofibroblasts and the repopulation of the intestinal mucosa of rats, atrophied by the use of soy as the only source of protein.

2. MATERIALS AND METHODS

2.1. Animals

A total of 24 male albino Wistar rats, aged 40 days, average weight of 123.8 ± 1.2 grams, were allowed to adapt for three days, with free access to commercial feed (Labina^®, Purina, Brazil) and water under the following conditions: forced ventilation, controlled humidity and temperature (50% - 60% RH and 22˚C ± 1˚C), and a 12:12 h light and dark cycle. After adaptation, the animals were first weighed and separated into four groups of six animals, identified and transferred to individual cages. The four groups (n = 6) received different types of feed, with or without supplements of lactoferrin and were identified with the following codes: Ci (casein alone), CL (casein plus lactoferrin), Si (soybean alone); and SL (soybean plus lactoferrin). The weighing was repeated on the fifth, tenth and fifteenth day of the experiment.

2.2. Diets

Diets were prepared in compliance with the American Institute of Nutrition (AIN-93) recommendations [16]. Each animal received 10 g of their daily diet (AIN-93) and water ad libitum. All procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals [17]. Groups CL and SL received 200 mg/kg lactoferrin (Bioferrin 1000^®, Apolactoferrin, Glanbia Nutritionals, Carlsbad, USA) dissolved in 1.0 mL of distilled water (200 mg/mL) by gavage, once daily. The dose and method of administration were based on other experiments [18-21].

2.3. Experimental Procedure

For fifteen days, group SL ate a feed with soya as the only protein source and received the lactoferrin by gavage once a day, while group Si received the soy diet without the lactoferrin. A third group (CL) received a diet with casein as only protein source and lactoferrin by gavage, while the last group (Ci) received the casein diet without lactoferrin.

2.4. Surgical Procedure

At the end of fifteen days the animals were anesthetized with ketamine (50 mg/kg)/xylazine (5 mg/kg). Each animal was subjected to trichotomy of the ventral region and antisepsis with 1% povidone-iodine solution. By laparotomy, a 10 mm long segment was sectioned from the initial portion of the small intestine, 4 cm from the xiphoid process, 15 mm from the gastro-duodenal junction and termino-terminal anastomosis was performed with the edges of the bowel, for its use in another experiment. The sectioned segments of intestines were examined by conventional histological techniques for histomorphometry of the villi and intestinal crypts and immunohistochemistry to assess of the number and size of the myofibroblasts. The experimental procedure was approved by the Ethics Committee for Animal Use in Research (CEUA), Faculty of Pharmaceutical Sciences UNESP (res. 06/2011).

2.5. Treatment of Collected Material

After being fixed in 10% formaldehyde (in pH 7.5 buffer), the segments were subjected to the standard technique of dehydration in increasing battery of ethyl alcohol, xylene and embedded in paraffin wax. Thin sections (4 µm thick) were cut in the conventional microtome, and laid on both untreated and silanized microscope slides. For the observation and measurement of intestinal villi and crypts, slide were stained with Hematoxylin and Eosin (HE). The Avidin-Biotin Peroxidase (ABP) immunohistochemical technique devised by Hsu et al. [22] was used to reveal myofibroblasts. This indicates, in brown tones, the presence of these cells. The silanized slides were used for good adhesion of the histological sections. After dewaxing and rehydration of the sections, the slides were immersed in citrate buffer (pH 6.0) exposed to microwave oven for 15 minutes, then transferred to phosphate buffer with hydrogen peroxide + 3% methanol for 20 minutes, to block endogenous peroxidase. Slides were incubated in primary antibody anti- α-smooth muscle actin (Monoclonal Mouse Smooth Muscle Actin Clone 1A4 Code M0851, Dako Cytomation, USA) for 12 hours at 4˚C. After washing in phosphate buffer, biotinylated antibody was applied to the slides for 30 minutes at room temperature. After another bath in phosphate buffer, we applied the complex streptavidin/peroxidase for 30 minutes, at room temperature. The slides were again washed in phosphate buffer, treated with diaminobenzene to reveal the immune reaction and counterstaining with hematoxylin.

2.6. Analysis of Slides

Observations and measurements were taken with an Olympus BX 50 optical microscope (Olympus America Inc., USA), at magnifications 100× and 400×, in three different fields. The images were captured by a Sony SSC-DC54 digital camera (Sony Electronics Inc., Japan) and sent to a Pentium IV computer equipped with an E1940 LG Flatron monitor (LG Electronics, USA). All measurements were performed with the program ImageJ 1.44p (Natl. Inst. of Health, USA). The Length of the Villus (LV) was measured from its basal region, which coincides with the top of the crypts. A line was drawn between two points on the base and another line perpendicular to it, toward the point at the apex of the villus. The length of the second line, in µm, provided by the image analyzer, was taken as the length of the villus (Figure 1). The depth of the crypt (DC) was measured from its base to the crypt-villus transition. These structures were measured on both sides (right and left) of the cut, to reduce, by averaging, any changes in measurement due to a possible tilt of the material during embedding of the segment in wax. For each animal, 160 villi and 80 crypts were measured giving a total of 960 villi and 480 crypts for each diet group. The number of myofibroblasts was determined by direct counting. Areas of myofibroblasts were extracted by ImageJ 1.44p, from the images by individually marking them by means of the Freehand Select tool, as shown in Figure 2.

2.7. Statistical Analysis

Two-way Analysis of Variance (ANOVA) for repeated measures was used, with a significance level (alpha) p ≤ 0.05, to compare the values obtained in the measurement of all villi and crypts on both sides of the section, instead of simply taking the value. The weights of animals in

Figure 1. Measurement (µm) of the length of a villus with the straight line tool of ImageJ. (Magnificatiion: 100×).

Figure 2. Determination of the total area (µm²) of the myofibroblast pericrypt sheath, with the freehand selections tool, in ImageJ. Marked myofibroblast (red) and unmarked myofibroblasts (black arrow). (Magnification: 400×).

different groups were compared with the Student t-test, also at a significance level (alpha) p ≤ 0.05. Linear Regression and Correlation analysis were used to compare the values obtained for the variables studied. The software used was PAST 1.99, Excel (Microsoft Co.) and BioEstat 5.0.

3. RESULTS

Animal weight (g): the Student t-test showed that the means on the first day were similar in all groups (p > 0.05). On the fifth day, there was a very significant difference between groups Ci and Si favoring the casein group (p < 0.001). Between groups CL and SL, the weight of casein group was again higher (p = 0.001). Comparing groups SL and Si showed a significantly higher weight for the lactoferrin group (p < 0.05). Between groups Ci and SL, the comparison was favorable to the casein group (p = 0.001). There was no significant difference between the groups CL and Ci. On the tenth day, comparisons between groups CL and SL, Ci and SL and CL and Ci showed no significant difference. Between groups Ci and Si the casein group showed more growth (p < 0.001). The groups SL and Si showed a highly significant difference, favorable to the lactoferrin group (p < 0.001). On the fifteenth day, there was a very significant difference between the groups Ci and Si, the casein group having grown more (p < 0.001), while groups SL and Si showed a highly significant difference favor of the lactoferrin group (p < 0.001). No differences were observed between groups Ci and CL; CL and SL or Ci and SL The average weights of the groups are compared in Table 1.

3.1. Length (Height) of Villi (LV)

The comparisons between groups are shown in Table 2. Comparisons between the groups Ci and Si, between

Table 1. Intergroup¹ comparisons of mean body weight (g).

Table 2. Intergroup¹ comparisons of mean Villus Length (LV) in µm.

CL and SL and between Ci and SL all showed that the length of the villi was significantly higher in animals fed casein than in those fed soybean (p < 0.001). In the comparison between groups Si and SL, LV was significantly higher in animals that received lactoferrin supplementation (p < 0.001). There was no significant difference in LV between the groups Ci and CL.

3.2. Depth of Crypts (DC)

Table 3 compares the increased Depths of the Crypts (DC), in micrometers (µm), between pairs of groups. The comparison between Ci and Si indicates that the depths of the crypts are significantly different between the two protein sources, being greater for animals fed soy. In the

Conflicts of Interest

The authors declare no conflicts of interest.

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