Gut health immunomodulatory and anti-inflammatory functions of gut enzyme digested high protein micro-nutrient dietary supplement-Enprocal

Abstract

Background: Enprocal is a high-protein micro-nutrient rich formulated supplementary food designed to meet the nutritional needs of the frail elderly and be delivered to them in every day foods. We studied the potential of Enprocal to improve gut and immune health using simple and robust bioassays for gut cell proliferation, intestinal integrity/permeability, immunomodulatory, anti-inflammatory and anti-oxidative activities. Effects of Enprocal were compared with whey protein concentrate 80 (WPC), heat treated skim milk powder, and other commercially available milk derived products.

Results: Enprocal (undigested) and digested (Enprocal D) selectively enhanced cell proliferation in normal human intestinal epithelial cells (FHs74-Int) and showed no cytotoxicity. In a dose dependent manner Enprocal induced cell death in Caco-2 cells (human colon adencarcinoma epithelial cells). Digested Enprocal (Enprocal D: gut enzyme cocktail treated) maintained the intestinal integrity in transepithelial resistance (TEER) assay, increased the permeability of horseradish peroxidase (HRP) and did not induce oxidative stress to the gut epithelial cells. Enprocal D upregulated the surface expression of co-stimulatory (CD40, CD86, CD80), MHC I and MHC II molecules on PMA differentiated THP-1 macrophages in coculture transwell model, and inhibited the monocyte/lymphocyte (THP-1/Jurkat E6-1 cells)-epithelial cell adhesion. In cytokine secretion analyses, Enprocal D down-regulated the secretion of proinflammatory cytokines (IL-1beta and TNF-alpha) and up-regulated IFN-gamma, IL-2 and IL-10.

Conclusion: Our results indicate that Enprocal creates neither oxidative injury nor cytotoxicity, stimulates normal gut cell proliferation, up regulates immune cell activation markers and may aid in the production of antibodies. Furthermore, through downregulation of proinflammatory cytokines, Enprocal appears to be beneficial in reducing the effects of chronic gut inflammatory diseases such as inflammatory bowel disease (IBD). Stimulation of normal human fetal intestinal cell proliferation without cell cytotoxicity indicates it may also be given as infant food particularly for premature babies.

Figure 1

SDS-PAGE analysis of Enprocal and whey protein concentrate (WPC) digested with gut enzyme cocktail (pH7.2) for 4 hours.

Figure 2

Cell viability (A), Cell cytotoxicity (B) of Caco-2 and normal human FHs74-Int cells treated with undigested and digested Enprocal (Enprocal D) 100–8000 μg/ml of each for 48 hours. Cell viability was determined by trypan blue exclusion assay. Cell cytotoxicity was determined by LDH release assay. (C) Cell viability (trypan blue exclusion assay) of Caco-2 monolayers treated with Enprocal D (500–2000 μg/ml) and with other digested milk product controls. All treatments were performed in triplicate and assay was repeated three times independently with similar results. The mean for representative experiment was calculated and presented as a mean ± SD values. ** Indicates a highly significant P < 0.001 value from the control with media only. *Indicates a significant P < 0.05 value from the control with media only.

Figure 3

Cell cytotoxicity determined by LDH release assay (A) and cell death (apoptosis/necrosis) (B) following treatment with 500–2000 μg/ml of Enprocal D and other digested milk product controls. Cells were treated for 48 hours with milk products and stained by TUNEL analysis for apoptotic cells, Annexin-V-fluos analysis and counterstained with propidium iodide to reveal necrotic cells. Cell death is shown here in terms of apoptotic (A/I) and necrotic indices (N/I). All treatments were performed in triplicate and assay was repeated three times independently with similar results. The mean for representative experiment was calculated and presented as a mean ± SD values. ** Indicates a highly significant P < 0.001 value from the control with media only. *Indicates a significant P < 0.05 value from the control with media only.

Figure 4

Effect of Enprocal D and other digested milk product controls on cell proliferation of normal human intestinal cells (FHs74-Int) determined by MTT assay. All treatments were performed in triplicate and assay was repeated three times independently with similar results. Each bar presented in the histogram was mean ± SD values of all experiments in triplicates. **P < 0.001 is the highly significant value from the control with media only or enzyme cocktail. *P < 0.05 is the significance value from the control with media only or enzyme cocktail.

Figure 5

Effect of Enprocal D and other digested milk product controls on transepithelial electrical resistance (TEER) (A), tight junction protein-ZO-2 expression (immunofluorescence in confocal microscopy) (B). Caco-2 monolayers (21 day old, differentiated cells) were incubated with 500 μg/ml of test samples and control (no treatment) for 48 hours as indicated. All treatments were performed in triplicate and assay was repeated three times independently with similar results. The effect on epithelial barrier integrity was measured (TEER results in ohms-cm²) and mean ± SD to relative TEER values were calculated with respect to media only controls and plotted. Significant differences (*P, < 0.05) and highly significant differences (**P, < 0.001) between the relative TEER levels as compared to control cells are indicated by asterisks.

Figure 6

Antioxidant enzymes activities for copper zinc superoxide dismutase (CuZnSOD) and manganese superoxide dismutase (MnSOD) (A), glutathione peroxidase (GPx), and catalase (CAT) (B) were determined after treating Caco-2 cells with 500 μg/ml of Enprocal D and compared with other digested milk product controls. All data was compared with the levels of positive control (H₂O₂/Menadione). All treatments were performed in triplicate and assay was repeated three times independently with similar results. Data are expressed as mean ± SD. **P < 0.001 is the highly significant value from the control with media only. *P < 0.05 is the significance value from the control with media only.

Figure 7

Cell surface activation marker expression on the human THP-1 (PMA differentiated) macrophages for MHC-I and MHC-II molecules (A) and co-stimulatory (CD80, CD86 and CD40) molecules (B) is modulated by Enprocal D and compared with other digested milk product controls in vitro. Responses were measured by the upregulation of the surface markers CD80, CD86 and CD40. A negative control of cells without treatment is shown. Positive control of LPS and IFN-γ is shown. All treatments were performed in triplicate and assay was repeated three times independently with similar results. The mean for each experiment was calculated and presented as a mean ± SD values. ** Indicates a highly significant P < 0.001 value from the control with media only. *Indicates a significant P < 0.05 value from the control with media only.

Figure 8

Inhibition of lymphocyte-epithelial (A) and monocyte-epithelial (B) cell adhesion by Enprocal D and other digested milk product controls. For adhesion assays, Caco-2 cell monolayers, grown in 96-well plates, were treated for 18 h with Enprocal D and other digested milk product controls. CMFDA-labeled Jurkat (A) or THP-1 (B) cells were then added. After incubation for 60 min at 37°C, nonadherent immune cells were removed by washing with HBSS and the monolayer-associated Jurkat cells or THP-1 was counted. Values are showed as the percent inhibition. All treatments were performed in triplicate and assay was repeated three times independently with similar results. The mean for representative experiment was calculated and presented as a mean ± SD values. ** Indicates a highly significant P < 0.001 value from the control with media only. *Indicates a significant P < 0.05 value from the control with media only.

Figure 9

Schematic drawing of a model on hypothesis of mechanism(s) of Enprocal's function in gut and immune health. Enprocal D working model depicting the activation of immune and strong gut cell integrity. 1 & 2) Oral administration and its digestion of Enprocal D maintains the human gut cell integrity and aids in gut cell proliferation with no cytotoxicity and no oxidative injury. 3) Modulation of Th1 and Th2 cytokine responses by Enprocal D appear to aid the production of antibodies and gut immune functions. 4) By enhanced activation of immune responses, Enprocal thus, may prevent pathogen invasion. With the strong gut cell integrity and downregulation of proinflammatory cytokines such as TNFα and IL-1β, Enprocal D may help in the management of chronic gut inflammatory diseases such as inflammatory bowel disease (IBD) and leaky gut syndrome.