The Effect Of Vitamin A And Its Derivatives In Oxidative Stress Model Of Duck Intestinal Epithelial Cells

Preliminary studies have found that plasma endogenous retinoic acid content is significantly reduced when laying ducks are at the peak stress of laying cages,and whether retinoic acid plays an important role in the repair of laying ducks’stress-induced damage.The purpose of this experiment was to investigate the effect of vitamin A and its derivative retinoic acid on H₂O₂-induced oxidative stress injury of duck intestinal epithelial cells（IEC）.Specifically,it includes two parts:the preliminary research basis（experiment 1）and experiment 2:Experiment one:Laying duck cages can solve the problem of environmental pollution in traditional breeding.However,ducks are waterfowl,and after being"caught on the shelf",they will experience caged stress and cause intestinal mucosal damage.The purpose of this experiment was to analyze the effects on the production performance of laying ducks and the comprehensive changes of plasma metabolites at different stages of basketing,so as to provide a reference for follow-up research.The feed intake and egg production of each group were recorded.On the first day of the experiment,the body weight of the laying ducks was measured every 5 days（days 5,10,and15）.Determination of plasma oxidative stress indicators.Changes of metabolites in plasma of traditional rearing（TB）and caged 5（CR5）,10（CR10）and 15（CR15）days were determined by liquid chromatography-mass spectrometry（LC-MS/MS）,and the differences were analyzed by KEGG Metabolite-related pathways.The results showed that compared with the other groups,the CR10 group had decreased feed intake,decreased body weight,and increased plasma total superoxide dismutase（T-SOD）levels.Compared with TB group,134,381 and 190 differential metabolites were identified in CR5,CR10 and CR15 groups.PCA results showed that the selected components were able to adequately discriminate the TB and CR10 groups.KEGG analysis showed that the difference of plasma metabolites between CR5 group and TB group was mainly related to ovarian steroid production,unsaturated fatty acid biosynthesis and phenylalanine metabolism.The differences in plasma metabolites between CR10 and TB groups were mainly related to PTS,aldosterone synthesis and secretion,vitamin digestion and absorption,and the biosynthesis of steroid hormones.In conclusion,by comparing the production performance,oxidative stress indicators,differential metabolite quantities and PCA of laying ducks,the effects of different cage stages on the production performance of laying ducks were discussed.From the test results,it can be inferred that the ducks are in a sensitive stage for the first 5 days after being basketed,and about 10 days after being basketed is a stress peak,and then the laying ducks continue to adapt to the cage environment.Plasma endogenous retinoic acid（RA）content was significantly reduced when laying ducks were at the peak stress of basketing,indicating that RA may play an important role in the repair of laying ducks stress damage.Experiment two:Plasma endogenous RA levels were significantly reduced and the intestinal barrier was damaged when laying ducks were at the peak of cage stress.RA is a derivative of VA and exerts its main physiological function.Because in vivo,when VA and RA are affected by oxidative stress,VA can be converted into RA,showing dynamic changes.In order to simulate this process,we designed such an experiment added various concentrations of VA/RA and H2O2-induced oxidative stress in primary duck IEC.We simulated this process by adding different concentrations of VA/RA to verify whether RA plays an important role in the repair of intestinal stress injury.The experiment was divided into 5 groups:control group1（CG1）,control group 2（CG2）,high concentration RA group（TG1）(VA:RA=1×10^-8:3×10^-8 mol/L),medium concentration RA group（TG2）(VA:RA=2×10^-8:2×10^-8 mol/L)and low concentration RA group（TG3）(VA:RA=3×10^-8:1×10^-8 mol/L).The control group 1 did not receive any treatment,and both the positive control group and the experimental group were treated with 50μmol/L H₂O₂ for4 h to induce oxidative stress.Differentially expressed genes were screened by RNA-Seq,KEGG pathway enrichment and GO analysis were performed,and verified by RT-q PCR.Cell viability was measured by CCK-8 assay,and cell transmembrane electrical resistance（TEER）was used to measure intestinal barrier integrity and tight junction permeability.RNA-seq analysis showed that the addition of VA/RA changed the expression of CAMs and PPAR signaling pathway related genes.Compared with CG1,50μM H₂O₂ treatment significantly reduced cell viability.Compared with CG2,the cell viability and TEER of TG1,TG2 and TG3 were significantly increased（P<0.05）.The relative expression of Nrf2 and HO-1 increased with increasing RA concentration（P<0.05）.The gene expression levels of ZO-1,claudin-1 and Occludin in TG1,TG2 and TG3 were higher than those in CG2（P<0.05）.High concentrations of RA(VA:RA=1×10^-8 M:3×10^-8 M)enhanced the expression of tight junction protein markers ZO-1,Occludin and claudin-1 at the m RNA level,enhanced cell viability,decreased intestinal permeability,and alleviated H₂O₂-induced intestinal epithelial barrier dysfunction.