Relieving Transportation Stress Injury In Rat Small Intestinal Mucosal Epithelium By Regulating MiR23b-mediated Autophagy By L-Arg

During the summer transportation,livestock is stimulated by high temperature,vibration and other factors,which leading to intestinal ischemia injury,disruption of cell supply and nutrient limitation.The organism realizes energy recycling by autophagy.In this study,the transportation stress model and the heat stress model of rat and DEC-6 cell were selected to study the function and mechanism in stress,and two models were highly correlated.The purpose of this research is to investigate the stress response mechanism of intestinal epithelial cell and the protective effect of L-arginine.1.Mechanism of transportation stress injury on jejunum epithelium in ratsA rat transportation stress model of heat and shake combined stress was used.The results showed that under 35°C and O.lg relative centlrifugal force stress conditions,rats weight,rectal temperature,cortisol and HSP27/70 serum mRNA expression were significantly increased.Intestinal mucosal villus epithelial cell damaged,lamina propria exposed,nuclear shrinked,endoplasmic reticulum swelled,and the villi top occurred apoptosis.Gene microarray analysis assessed 93 differentially expressed genes associated with apoptosis,ER stress,and autophagy.67 are involved in endoplasmic reticulum unfolded protein response,and the 11 is involved in autophagy.P53,MAPK and mTOR related signaling pathways may be activated.Q-PCR results verified the result of gene microarray.The results of immunohistochemistry and Western blot showed that Casp3,Casp12,and LC3 were significantly increased in the small intestinal villi,and the content of mTOR and P-AKT decreased.It is suggested that ER stress is involved in the mechanism of stress injury,and the autophagy induced by ER stress may be the mechanism of cell survival in the injury response.2.Functional study of MiR23b in IEC6 cellsHeat stress model of IEC-6 cells was used,and the model was associated with rat transportation stress model.The results showed miR23b decreased after heat stress.Target genes of miR23b was predicted by software and verified by dual luciferase reporter gene assay.UBE20 is the target gene of miR23b.The result of deep sequencing showed that after knockdown miR23b,142 differentially expressed genes were mainly distributed in autophagy,ubiquitination,apoptosis and stress response in GOs and pathways.The analysis of these differentially expressed genes suggested that miR23b may regulate autophagy by regulating the level of cellular ubiquitination and participate in the regulation of cellular stress.3.Regulation of L-Arg on rmiR23b in stressHeat stress model of DEC-6 cells that stressed consecutive 1.5 hours in 44OC,the cells were obviously damaged and the number of autophagic bodies increased obviously,and the activity of autophagy was increased showed by GFP-LC3 tracing technique.5mM L-Arg could increase the expression level of miR23b in heat stress and inhibit the autophagy of IEC-6 cells induced by heat stress,regulated the expression of UBE20,UBE2R2,UBE2D1,UBR3,Smurf2,and decreased the expression of Casp3 and Casp9.The trend was as same as the over expression miR23b group in heat stress.The rats in group L-Arg were treated with L-Arg for 7 days.The results showed that compared with the stress group,the body weight of the rats increased after adding L-Arg,and the serum biochemical indexes were recovered.The morphological observation showed that the mucosal barrier injury of the jejunum was alleviated and the integrity of intestine was improved.MiR23b decreased after stress,while L-Arg could up regulate miR23b,and the trend of autophagy was consistent with in vitro tests.It is suggested that L-Arg had protective efifects on intestinal mucosal injury in rats.Conclusions:1L Transport stress induces the injury of rat jejunum mucosa cells,activates endoplasmic reticulum stress,and induces autophagy by regulating Akt-TSC-mTOR pathway.2.miR23b participate in the process of stress response.UBE20 is the target gene of miR23b,and miR23b may regulate autophagy via ubiquitin proteasome pathway.3,L-Arg regulates autophagy and ubiquitination by regulating miR23b,thereby alleviating epithelial cell damage caused by stress.