Efficacy of a Novel Therapeutic, Based on Natural Ingredients and Probiotics, in a Murine Model of Multiple Food Intolerance and Maldigestion

Abstract

Patients with hypersensitive gut mucosa often suffer from food intolerances (FIs) associated with an inadequate gastrointestinal function that affects 15-20% of the population. Current treatments involve elimination diets, but require careful control, are difficult to maintain long-term, and diagnosis remains challenging. This study aims to evaluate the beneficial effects of a novel therapeutic of natural (NTN) origin containing food-grade polysaccharides, proteins, and grape seed extract to restore intestinal function in a murine model of fructose, carbohydrate, and fat intolerances. All experiments were conducted in four-week-old male CD1 mice. To induce FIs, mice were fed with either a high-carbohydrate diet (HCD), high-fat diet (HFD), or high-fructose diet (HFrD), respectively. After two weeks of treatment, several parameters and endpoints were evaluated such as food and water intake, body weight, histological score in several organs, gut permeability, intestinal epithelial integrity, and biochemical endpoints. Our results demonstrated that the therapeutic agent significantly restored gut barrier integrity and permeability compromised by every FIs induction. Restoration of intestinal function by NTN treatment has consequently improved tissue damage in several functional organs involved in the diagnostic of each intolerance such as the pancreas for HCD and liver for HFD and HFrD. Taken together, our results support NTN as a promising natural option in the non-pharmacological strategy for the recovery of intestinal dysregulation, supporting the well-being of the gastrointestinal tract.

Keywords: barrier integrity; food intolerances (FIs); grape seed extract; high-carbohydrate diet (HCD); high-fat diet (HFD); high-fructose diet (HFrD); maldigestion; pea protein; polysaccharides.

Figure 1

Timeline of FIs. The figure summarizes the timing of each experimental model. Five weeks for carbohydrate intolerance (the administration of NTN was conducted in the last two weeks); 14 weeks for lipid intolerance (the administration of NTN was carried out in the last two weeks); 15 weeks for fructose intolerance (the administration of NTN was carried out in the last two weeks).

Figure 2

Effects of NTN on body weight, food intake, histological damage of the pancreas and glucose-insulin levels in HCD intolerant mice. A slight increase in body weight and food intake was detected in HCD-mice compared to the control group (A,B); NTN administration reduced both parameters in HCD mice (A,B). Extensive neutrophil infiltration and tissue damage were observed in mice fed with HCD (E,G) compared to SD and SD + NTN animals (C,D,G). Administration of NTN was able to significantly counteract the extent of tissue damage and neutrophil infiltration in HCD mice (F,G). NTN administration reduced both glucose and insulin levels (H,I). Data are representative of at least three independent experiments. Values are means ± SEM. One-Way and Two-Way ANOVA test. * p < 0.05 vs. SD; ** p < 0.01 vs. SD; *** p < 0.001 vs. SD; # p < 0.05 vs. HCD; ## p < 0.01 vs. HCD.

Figure 3

Effect of NTN on adipose abdominal tissue and liver in HCD mice. HCD mice showed a significant tissue injury in adipose tissue (C,E); to the contrary, SD and SD + NTN mice showed no tissue damage (A,B,E). NTN restored physiological parameters, thus reducing neutrophil infiltration and adipocytes size (D,E). In addition, NTN was able to decrease liver weight (F). Data are representative of at least three independent experiments. Values are means ± SEM. One-Way ANOVA test. *** p < 0.001 vs. SD; # p < 0.05 vs. HCD; ### p < 0.001 vs. HCD.

Figure 4

Effects of NTN on intestinal tissue damage and permeability in HCD mice. Neutrophil infiltration and tissue damage was observed in mice fed with HCD (C,E) compared to SD and SD + NTN animals (A,B,E). Administration of NTN was able to significantly counteract the extent of intestinal tissue damage and neutrophil infiltration in HCD mice (D,E). FITC-dextran permeability assay of HCD mice jejunum exposed a marked increase of intestinal permeability; NTN exerted an important protective barrier effect (F). Data are representative of at least three independent experiments. Values are means ± SEM. One-Way ANOVA test. *** p < 0.001 vs. SD; ## p < 0.01 vs. HCD.

Figure 5

Effects of NTN administration on intestine epithelial integrity in HCD mice. A high percentage in the expression of ZO-1 (A,B,E) and Occludin (F,G,J) were found in intestinal tissues of SD mice, conversely HCD decreased such expressions (C,E,H,J). NTN has appreciably restored the levels of ZO-1 (D,E) and Occludin (I,J). Data are representative of at least three independent experiments. Values are means ± SEM. One-Way ANOVA test. *** p < 0.001 vs. SD; ## p < 0.01 vs. HCD.

Figure 6

Effects of NTN on liver tissue, fat mobilization and gut permeability in HFD mice. HFD-fed mice showed a significant increase in body weight compared to the sham group (A); NTN considerably decreased weight gain (A). No significant differences were detected in mice food intake (B). Significant hydropic degeneration and steatosis were observed in mice fed with HFD (E,G) compared to SD and SD + NTN animals (C,D,G). Administration of NTN was able to significantly counteract the extent of liver damage (F,G). In addition, NTN administered mice decrease NEFA and TG levels compared to HFD mice (H,I). FITC-dextran permeability of jejunum was very low in SD mice (J). Contrarily, after HFD, mice displayed an increased intestinal permeability that was reduced by NTN administration (J). Data are representative of at least three independent experiments. Values are means ± SEM. One-Way ANOVA test. *** p < 0.001 vs. SD; # p < 0.05 vs. HFD; ## p < 0.01 vs. HFD; ### p < 0.001 vs. HFD.

Figure 7

Effects of NTN administration on intestine epithelial integrity in HFD mice. High expressions of ZO-1 and Occludin have been found in intestinal tissues of the SD group and SD + NTN group ((A,B,E) and (F,G,J) respectively) compared to the HFD group ((C,E) and (H,J) respectively). The administration of NTN restored the expression of ZO-1 and Occludin proteins ((D,E) and (I,J) respectively). Data are representative of at least three independent experiments. Values are means ± SEM. One-Way ANOVA test. *** p < 0.001 vs. SD; ## p < 0.01 vs. HFD.

Figure 8

Effects of NTN on adipose damage in HFD mice. HFD led to a remarkable increase in neutrophil infiltration and adipocyte size (C,E) compared to SD and SD + NTN mice (A,B,E). Administration of NTN was able to significantly counteract the extent of adipose tissue due to HFD (D,E). Data are representative of at least three independent experiments. Values are means ± SEM. One-Way ANOVA test. *** p < 0.001 vs. SD; # p < 0.05 vs. HFD.

Figure 9

Effects of NTN on liver damage in HFrD. No considerable variations were detected in body weight and food intake in HFrD mice (A,B). Significant macrovesicular and microvesicular steatosis was observed in mice fed with HFrD (E,G) compared to SD and SD + NTN mice (C,D,G). The administration of NTN was able to significantly counteract the extent of liver damage in HFrD mice (F,G). Data are representative of at least three independent experiments. Values are means ± SEM. One-Way ANOVA test. *** p < 0.001 vs. SD; ### p < 0.001 vs. HFrD.

Figure 10

Effects of NTN on intestinal features in HFrD mice. Significant infiltration of inflammatory cells and tissue damage was observed in mice fed with HFrD (C,E) compared to SD and SD + NTN mice (A,B,E). Administration of NTN was able to significantly counteract the extent of intestinal tissue damage (D,E). A gut permeability assay exhibited an evident increase of intestinal permeability in HFrD jejunum compared to the SD group (F); NTN showed protective properties decreasing gut permeability (F). Data are representative of at least three independent experiments. Values are means ± SEM. One-Way ANOVA test. *** p < 0.001 vs. SD; ## p < 0.01 vs. HFrD.

Figure 11

Effect of NTN on intestinal epithelial integrity in HFrD mice. Intestinal tissues of SD and SD + NTN mice displayed high expressions of ZO-1 (A,B,E) and Occludin (F,G,J) proteins, contrariwise TJs expressions were reduced after HFrD (C,E,H,J). NTN two-week treatment significantly restored ZO-1 (D,E) and Occludin (I,J) expressions. Data are representative of at least three independent experiments. Values are means ± SEM. One-Way ANOVA test. *** p < 0.001 vs. SD; # p < 0.05 vs. HFrD; ### p < 0.001 vs. HFrD.