Intestinal conditioner pronutrients are molecules of plant origin that improve the physiology of the digestive epithelium and have a positive effect on productive parameters.

OBJECTIVE

An in vitro trial aimed to reveal which are the molecular mechanisms of intestinal conditioner pronutrients and how they have a positive effect on the activity and regeneration of enterocytes.

ANALYSIS TECHNIQUE

To assess the effect of pronutrients on the mRNA-protein translation rate, a technique called SUnSET (Surface Sensing of Translation) was used.

The SUnSET technique is a non-radioactive method to monitor protein synthesis in cell populations, based on the use of puromycin. Puromycin is a tyrosyl-tRNA analog that can be incorporated into newly synthesized proteins through a peptide bond, so the detection of puromycin by an anti-puromycin antibody (a technique called ELISA Western Blot) can be correlated with the rate of translation mRNA-protein. On the other hand, cycloheximide is an inhibitor of protein synthesis in eukaryotic cells.

Image 1: Protein and protein synthesis with puromycin

PROCEDURE

• In vitro assays were performed in 12-well plates, where 1.2 × 10⁵ cells/ml were added in a final volume of 2 ml per well.
• After 4 days of culture, confluence was observed and the medium was replaced with different supplements, following the scheme described below:
  • TREATMENT A (Negative control): No treatment.
  • TREATMENT B: Cycloheximide and puromycin were added.
  • TREATMENT C: Only puromycin was added.
  • TREATMENT D: Puromycin and intestinal conditioner pronutrients (dilution 1: 1000) were added.
  • TREATMENT E: Puromycin and intestinal conditioner pronutrients (dilution 1: 10000) were added.
• The SunSET technique was used to evaluate the synthesis of new proteins (RNA translation rate) in the cultures.

Image 2: ELISA Western Blot results. Treatments classified with letters (different order of the treatments between repetitions)

RESULTS AND INTERPRETATION (Image 2)

A darker signal in the ELISA Western Blot technique correlates with a higher amount of puromycin, and, therefore, a higher rate of RNA-protein translation.

• Treatment A did not show any protein synthesis as it does not contain puromycin that can work as a marker.
• Treatment B showed that protein synthesis was inhibited by the presence of cycloheximide.
• Treatment C showed basic protein synthesis thanks to the marker activity of puromycin, without any other supplement.
• Treatment D showed protein synthesis in enterocytes treated with pronutrients at a 1:1000 dilution, while treatment E was at a 1:10000 dilution. These two groups showed an increase in the protein synthesis thanks to the use of intestinal conditioner pronutrients. The effect was most clearly seen in treatment D.

CONCLUSIONS

Intestinal conditioner pronutrients, molecules of plantorigin, have shown an inductive effect on the rate of mRNA-protein translation in their target cell, the enterocytes. The effect was greater with a higher dose of the supplement (treatment D, 1:1000 dilution).

A higher rate of mRNA-protein translation is correlated with an increase in the production of tissue-specific proteins, which implies a better function of the target organ and a higher productive yield.

Intestinal conditioner pronutrients are manufactured by Biovet S.A. and are marketed under the name Alquernat Nebsui.