1. Introduction
The Western diet characterises by the high consumption of energy-dense ultra-processed foods, rich in saturated fats and simple carbohydrates. In this dietary pattern, more than 14% of the calorie intake comes from sugars, with sweet products and sweetened beverages as the main contributors to sugar consumption [
1,
2]. As a result, concerns have raised regarding the impact of these food products on the global epidemic of obesity [
3]. The World Health Organization recommends limiting added sugars to one-tenth of the daily calorie intake, namely 50 g per day for a 2500-calorie diet as well as European Food Safety Authority (EFSA) [
4]. Nevertheless, a typical 330-mL can of a regular soft drink contains up to 40 g of free sugars (>600 mmol L
−1). In addition, most foods marketed as healthy choices contains large portions of sugar (e.g., some breakfast cereals, yogurt, and other sugar-sweetened dairy products) and daily intake per capita in some developed countries can reach up to 145 g [
1].
As we reported in previous articles, rats fed a Western-style cafeteria diet (rich in sucrose and saturated fats) not only gain body weight and increased organ weights (liver, white adipose tissue, thymus, kidney, brown adipose tissue) but also manifest mild intestinal inflammation and enhanced permeability (intestinal dysfunction) [
5,
6,
7].
Proanthocyanidins are a group of flavonoids present in a variety of botanical sources such as cocoa, nuts, fruit and spices, which have demonstrated among others, antioxidant, anti-inflammatory and immunomodulatory effects in vivo [
8,
9], and could offer a safe adjunctive support to prevent the deleterious effects of unbalanced diet (“junk diet”), overweight and obesity [
10,
11,
12].
Oral administration of grape seed proanthocyanidins extract (GSPE) to rats has demonstrated beneficial effects against the increased intestinal permeability, inflammation and body weight developed because of a long-term cafeteria diet. We previously demonstrated that an intermittent GSPE treatment for 5 weeks during the administration of cafeteria diet normalized intestinal permeability and inflammation to levels comparable to the standard diet group up to 17 weeks in contrast to the non-GSPE cafeteria fed group, which maintained increased intestinal permeability and inflammation. The myeloperoxidase (MPO) activity, a marker of intestine’s inflammation, increased in the cafeteria diet group and decreased in preventive GSPE-receiving group [
5,
7].
The histochemical parameters of the gastrointestinal tract of rats have been studied previously by Sharma et al. [
13]. However, the effect of Cafeteria diet on intestinal morphology has been partially described and the effect of GSPE supplementation together with cafeteria diet in Wistar rats has not been studied.
In this study, we aimed to investigate the effect of cafeteria diet with or without a GSPE preventive treatment in the morphology and cell composition of small and large intestines. In addition, what would be the contribution of GSPE to putative morphological changes in the context of the typical Western diet? Does GSPE promotes alterations in the morphology of intestine? How any alteration correlates with functional/gene expression parameters in small and large intestine and in the whole body is addressed again.
4. Discussion
It is widely known that the Western diet induce changes in the gastrointestinal tract functionality. Published articles from authors and from our research group report alterations of intestinal permeability and inflammation in rats fed with a high fat/refined carbohydrate diet [
6,
7,
16,
17]. On this basis, first, our aim in the present study was to assess the structural responses of small and large intestine of Wistar rats to cafeteria diet rich in added sucrose (and sucrose-derived fructose) and to GSPE supplementation.
This study shows that chronic ingestion of cafeteria diet exerts an increase in the height of rat duodenal villi and total epithelium, and in the villus/crypt ratio, contributing to the overall absorption area or M surface area amplification. We suggest that this is a dietary adaptation to increase the caloric availability of food intake, as has been observed in other publications linking dietary macronutrients consumption to morphological changes in the intestine.
It is known that Wistar rats respond to protein content by modulating the dimensions of the villus in the small intestine [
18]. We have administered a cafeteria rich diet that enhances the intake of refined carbohydrates and fat in detriment to protein content. However, we also find this adaptive response in the increase in villus height, which depends on the continuous renewal of the intestinal epithelium through the multiplication and differentiation of stem cells at the base of the crypt, and the migration of mature cells along the villus [
18,
19].
In the present study, the main source of carbohydrate intake was sucrose, which was taken daily at a rate of 46 g per 100 g of diet. A daily intake of a minimum of 100 mL of milk with sugar was also recorded, which means that sucrose was the main source of energy followed by fat. Sucrose is broken down into fructose and glucose, which are postulated to be the main agents causing this adaptive response [
20], increasing the surface area to optimize and enhance the absorption of these components [
21]. In this regard, Taylor et al. reported a 25–40% villus length increase in the duodenum and proximal jejunum in mice fed with high-fructose corn syrup compared to control ones [
22]. All this evidence together with our results suggest that not only protein content is able to affect and determine intestinal morphology, but also carbohydrates. In addition, fat intake and the overall caloric content of the diet have also been reported to influence intestinal morphology by increasing absorption parameters [
23].
While in the small intestine we could observe an increase in the intestinal absorption surface which is adapted to absorb the maximum amount of energy in the cafeteria group (junk diet); in the colon, where the maximum absorption of water but not nutrients occur, this adaptation is not found.
Regarding cell content adaptation in the intestine, it is seen that goblet cell content increases along the intestinal tract of Wistar rats and that cafeteria diet causes an increase in goblet cell content in the duodenum. Goblet cells are specialized in the synthesis and production of mucus that grows from the lower to the upper gastrointestinal tract [
19], as we observed in the present study.
With respect to the presence of mature and immature inflammatory forms in the duodenum analysed by expert histopathologists, we found a basal level of inflammation in standard diet consuming animals and a significant increase of inflammatory cells infiltration (rate of total inflammation accumulated) when rats consumed cafeteria diet.
Cafeteria diet also influence intestinal barrier function. Gene expression analysis of barrier related proteins showed a negative correlation between myosine light chain kinase (MLCK) and villus height in the duodenum. The biological meaning of this negative correlation between MLCK gene expression and villus height in this context is difficult to explain. Paracellular permeability is principally determined by the phosphorylation level of the regulatory light chain of myosin-2 (MLC-2), which is regulated by the enzyme MLCK. Regulation of MLC-2 phosphorylation by MLCK leads to contraction of the actin skeleton thereby increasing the permeability of the paracellular barrier in intestinal epithelial cells [
24]. In our work, the groups with higher villus height also showed higher intestinal permeability, which is a paradox. However, with intestinal permeability established after 17 weeks of junk diet, the effect of MLCK at the expression level is not as relevant as its activity.
On the other hand, we also found a positive correlation between final body weight gain and duodenal villus height and a positive correlation between villus height and food intake. Previous results from our group showed a positive correlation between body weight gain and food calorie intake in Wistar rats on a standard or cafeteria diet, and also demonstrated that there was a modulation of food intake by GSPE treatment [
6,
17].
Grape seed proanthocyanidin extract called GSPE is a natural, non-nutritive compound that is added to the diet. In the present study, we wanted to see if GSPE had any effect on modulating gut morphology. We believe that the fact that GSPE did not modify intestinal morphology at the concentrations tested in the corrective treatment of 100 and 500 mg/kg/day (low and high) or in the preventive supplementation is indicative of the absence of deleterious effects of the extract.
However, the SIT treatment with proanthocyanidins was able to reduce and normalize villus-to-crypt ratio, the villus and epithelium heights and M-ratio, indicating that this treatment is optimal for animals fed an unbalanced cafeteria diet. As well as grape seed-proanthocyanidin extract derived from
Vitis vinifera, other plant-derived compounds such as sapogenin extract of Jamaican bitter yam (
Dioscorea polygonoides) are able to modify the structure of intestine thus ameliorating deleterious effects of junk diets [
25].
GSPE extract also normalised the goblet cell content of the duodenum in the intermittent and corrective treatments to levels comparable to those of animals fed standard diet, only with significance in the CORR500 group. The preventive treatment was not able to prevent the increase in goblet cell numbers caused by the cafeteria intervention. Thus, the reduction in goblet cell numbers with GSPE treatments compared to the CAF group only occurred in the duodenum, but not in the ileum and colon.
Regarding diet-associated inflammation, chronic GSPE did not change the index of acute, chronic or total inflammation in the duodenum; but, as previously reported, GSPE treatments decrease MPO activity, an indicator of neutrophil infiltration, in the same animals [
17,
26].
In summary, the sucrose-rich cafeteria diet chronically administered to rodents caused morphological changes in the intestine by increasing the surface area of intestinal absorption, probably due to the glucose and fructose content following sucrase action, as an adaptative response. Treatment with 500 mg GSPE/kg.day intermittently every two weeks was the optimal dose-time of supplementation, as it prevented the increase in intestinal absorption surface area induced by chronic ingestion of the cafeteria diet.