Rapid restoration of colonic goblet cells induced by a hydrolyzed diet containing probiotics in experimental malnutrition

Purpose: To investigate the effects of the addition of probiotic bacteria to a hydrolyzed diet on the recovery of goblet cells during renutrition in an animal model of malnutrition. Methods: Twenty-six male Wistar rats (200-250g) were included in the study. Six were kept under normal conditions (sham group) while twenty received an aproteic diet for 15 days, and were randomized thereafter to receive a hydrolyzed diet containing (n=6; probiotic group) or not (n=6; hydrolyzed group) probiotics (106 cfu/g of Streptococcus thermophilus e Lactobacillus helveticus); or immediately killed (n=8; aproteic group). Histological slides containing cecal and sigmoid biopsies were used to counting the number of goblet cells and the goblet cells/colonocytes ratio. Results: Malnutrition diminished the population of goblet cells in all sites. Goblet cells/ colonocytes ratio of the probiotic group was significantly greater than hydrolyzed group at the ceccum (0.39 ± 0.03 vs. 0.34 ± 0.02; p=0.02). Only rats fed with probiotics showed complete restoration of the normal goblet cells/colonocytes ratio at the sigmoid (0.37 ± 0,02 vs. 0.22 ± 0,03; p<0,001). Conclusion: Streptococcus thermophilus and Lactobacillus helveticus added to a renutrition diet enhance the recovery of mucocal atrophy induced by malnutrition and especially induce a rapid restoration of goblet cells population in the malnourished colonic mucosa.


Introduction
One of the key functions of the intestine is to prevent lumen bacteria and toxins from reaching systemic circulation, organs or tissues 1 .The intestine is the main immunological organ: it contains 50% of all reticuloendothelial and other immune cells and it produces the greatest amount of secretory IgA 2 .The gut flora is one of the main constituents of this defense barrier and is considered the first line of defense of the gut.In fact, failure in this mechanism, results in an increased antigen and toxin transport across the gut mucosa 1 .Protein malnutrition disrupts the normal ecology of microflora 3 particularly strict anaerobes 4 and thus producing overgrowth of certain members of harmful flora.Nonspecific mechanisms that include intestinal flora, anatomical barriers (mucosa and epithelium), secretory substances such as lysozymes, IgA, and mucus are affected by malnutrition 5 .Malnutrition induced by dietary restriction produces a series of metabolic changes that lead to a reduction in body weight, depression of immunocompetence and altered of digestive system 1 .These changes have a profound impairment and severe alterations of morphology on variables such as brush border, enzymatic activity, mucosal mass, protein DNA contents and mucosal integrity [6][7][8][9] .The injured gastrointestinal mucosal barrier may lead to an increased passage of macromolecules across the intestine 7 .Refeeding rapidly restores the morphology and function of the intestine (repair of the gut atrophy and normalization of intestinal permeability) in rats.The extent of changes depends on the amount of food consumed and quality of dietary nitrogen 7,10 .Hydrolyzed diets, including those containing hydrolyzed proteins as nitrogen source, are frequently used to recover malnourished patients 11 .Tripeptides and dipeptides are more efficiently utilized than free amino acids, have greater nutritional value, are better absorbed, and increase the nitrogen retention than intact protein, contributing to enhance the gain of weight 11,12 .Some organisms as probiotic bacteria have potentially beneficial applications 13, 14 .Probiotic bacteria may improve not only the nutritional status and physiology, but also the intestinal microflora, production of IgA, and the immune response [14][15][16] .Probiotic bacteria can be used as innovative tools for treating dysfunctions of the gut mucosal barrier including acute gastroenteritis, food allergy and inflammatory bowel disease 14,15 .The mucus layer component of the intestinal secretion, which is rich in IgA, is produced by mucous-secreting goblet cells.We hypothesized that probiotics may enhance the number of goblet cells during malnutrition recovery.Thus, the aim of the present study was to investigate the effects of the addition of probiotic bacteria to a hydrolyzed diet on the recovery of goblet cells during renutrition in an animal model of malnutrition.

Methods
Twenty-six male Wistar rats (200-250g) were included in the study.The experiment followed the COBEA guidelines (Brazilian Committee on Experimental Animal Care) adopted by the Federal University of Mato Grosso.Animals were kept in a laboratory environment of light/ dark cycles for three days prior to the experiment.All animals had free access to water.

Refeeding after malnutrition
Eight animals malnourished (aproteic group (AG)) and all six animals of the sham group were killed on the 13 th day and necropsied for data collection.After 12 days of malnutrition induction the other 12 rats of the aproteic groups were randomized to two groups to be reefed with either a hydrolyzed diet (20g/day) (composition per 100g: 19.96g protein [80% dipeptides and 20% aminoacids], 64.47g carbohydrate [88% maltodextrin and 12% starch], 15.57g lipid [50% mid-chain triglycerids, 30% milk lipids and 20% corn oil]; hydrolyzed group (HG), n=6) or the same diet (16g/day) plus reconstituted milk (4.0g/day) containing 10 6 cfu/g of Streptococcus thermophilus e Lactobacillus helveticus (Bionan, Nestlé, Brazil) (final composition per 100g: 18.60g protein, 62.15g carbohydrate, 19.25g lipid, probiotic group (PG), n=6) for 15 days.Blood samples were collected on the 16 th day and then the animals were killed.The two diets were isoenergetic and isonitrogenous.The amount of food consumed was registered daily and the weight was obtained each four days.During necropsy the entire large bowel were dissected, freed from the mesentery, and weighted after the contents were gently removed.Two cm long full thickness biopsies from cecum (one cm distal to the ileocecal valve) and sigmoid (two cm above the peritoneal reflexion) were collected and sent for histological analysis in 10% formalin.Slides containing three or four histological sections of 4 µm-thick cut sagitally to the mucosa were stained with hematoxylin and eosin, and examined by a experienced blind examiner with an optical microscope (100 X magnification).The number of both goblet cells and colonocytes were obtained in the five best-oriented crypt.The ratio goblet cells/colonocytes was calculated in both cecum and sigmoid specimens.

Statistical analysis
One-way ANOVA or Kruskal-Wallis test followed by Tukey's test was used to compare groups.A 5% level was established as being statistically significant.Analyses were done using the 11.0 SPSS statistical package.

Results
Renutrition with either hydrolyzed or hydrolyzed plus probiotics promoted significant increase in the large bowel weights (p<0.01) when compared with aproteic animals.The length of the large bowel did not differ among the groups (Table 1).

Ceccum
The findings at the ceccum can be seen in Figures 1  and 2. Malnutrition severely diminished the population of goblet cells (14,2 ± 2 vs. 20±1 cells/crypt; p=0,01).After renutrition both diets assured a complete and similar restoration of the goblet cells number at the crypt (control group= 24±4 cells/crypt and probiotic group = 23±6 cells/ crypt; p= 0,98).The ratio goblet cells/colonocytes was significantly compromised with malnutrition.There was a decrease of approximately 31% in the ratio after the installation of malnutrition (0.39 ± 0.01 vs. 0.27 ± 0.02; p< 0.001).Both types of renutrition successfully restored the ratio from malnutrition status.However the goblet cells/ colonocytes ratio of the probiotic group was significantly greater than hydrolyzed group (0.39 ± 0.03 vs. 0.34 ± 0.02; p=0.02) and similar to non-malnourished animals (p=0.98).

Discussion
The findings of this present study have clearly confirmed that malnutrition diminishes the number of globlet cells at both the cecal and sigmoid sites.The data well demonstrated that only the administration of probiotic bacteria was able to restore the goblet cells/ colonocytes ratio in both large bowel sites.This suggests that probiotics may influence the production of goblet cells by the crypt and thus promote a more rapid colonic mucosal trophism.Nutrition plays a key role in maintaining the balance of the intestinal microflora, and malnutrition disturbs the ecological barrier, and induces histological damage 1 .The production of mucin is decreased in the malnourished gut mucosa 5 .This reduction in mucin concentration is related to the decreased number of goblet cells, is selective, and did not reflect all surface glycoproteins 5 .Some recent reports have consistently attested that probiotics may enhance and accelerate intestinal mucosal trophism 1,15,16 .Our findings showed that probiotics produce a more rapid restoration of goblet cells at the large bowel crypts.This effect produced by probiotics was seen earlier by Cano et al 15 .The innate immune system plays a crucial role in maintaining the integrity of the intestine and protecting the host against a vast number of potential microbial pathogens from resident and transient gut microflora 18,19 .The innate immune system at the gut mucosa not only provides the first line of defense against pathogenic microorganisms but also provides the biological signals that instruct the adaptive immune system to elicit a response 20 .Probiotics may stimulate the gut innate immune system 18,19,21 .The mechanisms by which probiotic bacteria affect the immune system are unknown yet, but many of them are attributed to an increase in the innate or in the acquired immune response 21 .Consistent with this argument, probiotic bacteria can stimulate the production of IgA 22,23 .The overall results suggest that probiotics may enhance mucosal trophism and especially increase the number of goblet cells at the crypt site.As goblet cells are the main mucus producing cells, and mucus is rich in IgA 24 , we can speculate that probiotics may enhance the local immune system response during renutrition.Although the findings an experimental study should be transposed to the clinical setting with caution it could be concluded that Streptococcus thermophilus and Lactobacillus helveticus added to a renutrition diet enhance the recovery of mucocal atrophy induced by malnutrition and especially induce a rapid restoration of goblet cells population in the malnourished colonic mucosa.Immediate clinical application of this would be the improvement of the gut immune response to pathogenic microorganisms.