The Study Of The Metabolismof Oligopeptide(RVPSL) In An Lmitative Gastrointestinal Tract Environment

The study supported by Project of National Key Technology Research and Development Program for the 12 th Five-year Plan——‘Biological preparation technology research of food-derived bioactive peptides’, the project number is 2012BAD33B03, mainly engaged in digestion and metabolism research of peptides from egg white. This study took the activity peptitde RVPSL which from egg white as raw material, established gastrointestinal digestion and metabolism model and cell co-culture model by in vitro test. Application of molecular biology related technology to evaluate the cell model, and analysed the metabolites of cell co-culture model through ultra high performance liquid chromatography(HPLC). Finally, we got the optimal digestion model of gastrointestinal digestion to evaluate the metabolism of oligopeptide.First of all, this study established the gastrointestinal digestion and metabolism model. By using the method of simulated in vitro gastrointestinal tract, researched the absorption and metabolism of RVPSL which from egg white. We measured the quantity of digestion and metabolism of RVPSL by high performance liquid chromatograph, which can lay the foundation for further study of oligopeptide digestion and metabolism study. The result of simulated gastric environment shows that, when the metabolism of RVPSL reached 61.40% ± 3.02% of the initial content and the metabolic time is 0~30min in the gastric environment, we can get the highest metabolic rate. At the same time, the result of simulated intestinal environment shows that, when the metabolism of RVPSL reached 30.44%±5.82% of the initial content and the metabolic time is 60~90min in the intestinal metabolic, we can get the highest metabolic rate.In order to further study absorption and metabolism of egg white oligopeptides RVPSL in the small intestine, this study established Caco-2/HT29 co-culture cell model preliminarily. With the purpose of optimized the inoculation proportion of co-culture cell model, this study tested the transepithelial electrical resistance, and observed the cellular morphology with laser confocal microscope after stained the cell monolayer with FITC-Phalloidin. And then, we stained the mucous with alcian blue dyeing on the co-culture cell\model. Finally we analysed the representative gene expressions of different inoculation proportion of co-culture cell model with the methods of total RNA extraction, Real-time PCR and so on. Through the above research, we systematic evaluated different inoculation proportion of Caco-2/HT29 co-culture cell model from various aspects, and determined the ratio of 9:1(Caco-2:HT29) was the optimal proportion of co-culture cell model. This result will lay the foundation for subsequent absorption metabolism studies of egg white oligopeptide.We studied the metabolism of RVPSL using the optimized co-culture cell model which established in previous studies. The MTS test showed RVPSL had no reproductive toxicity on Caco-2 and HT29 cells. RVPSL were added on the AP side of the transwell insert, and than we detected the decomposition rate and metabolic rate of RVPSL in different time node using ultra high performance liquid chromatograph. The results showed that, in Caco-2/HT29 co-culture cell model, the decomposition rate of RVPSL was highest at 0~30 min(AP side), and the metabolic rate was highest at 30~60 min(BL side).Basing on the established Caco-2/HT29 co-culture cell model, we studied the influence of RVPSL to cytokine IL-6 and IL-8. After 2h of added different concentrations of RVPSL, we detected the concentration of cytokine IL-6 and IL-8. The results showed that, it can promote the production of IL-6 when the concentrations of RVPSL were higher than 3m M. However various concentrations of RVPSL almost had no effect on IL-8. In the results of detected the concentration of cytokine IL-6 and IL-8 after 12 h of added different concentrations of RVPSL, we had known that, it can promote the production of IL-8 when the concentrations of RVPSL were higher than 5m M. However various concentrations of RVPSL almost had obvious inhibitory effect on the IL-8.