Effect Of Transport Stress On Oxidative Damage And Autophagy Of Jejunum In Pigs

The integrity of the structure and function of intestinal mucosal epithelial cells plays an important role in animal growth and development,immune defense,nutrient metabolism,etc.As the main organ of stress response,intestinal tract is easily affected by various unfavorable factors,leading to oxidative stress and intestinal damage,destroying the integrity of intestinal mucosal epithelial cells and providing possibility for the occurrence of related diseases.Autophagy is a lysosomal-dependent protein degradation pathway characterized by cytoplasmic vacuolation,which plays an important role in maintaining cell structure and function integrity and regulating oxidative stress to alleviate cell damage.Transportation is an important link in pig breeding and slaughtering,transport stress can easily lead to pig death,disease occurrence and slaughter efficiency,which seriously affect the economic benefits of pig breeding.Therefore,we explore the effects of transport stress on the structure and function of pig intestines,and analyze its mechanism to play an important role in taking effective measures to alleviate transport stress and improve slaughter efficiency.This study focused on the effects of transport stress on intestinal damage and autophagy in pigs,RNA-Seq technology was used to screen differential genes and signaling pathways in intestinal damage,the regulation of differentially expressed genes on oxidative damage and autophagy of intestinal mucosal epithelial cells was analyzed at the cellular level.This provides a theoretical basis for elucidating the mechanism and regulation mechanism of transport stress.The main results of the study:1.Effects of transport stress on oxidative damage and autophagy of jejunum in pigsWe randomly divided 16 fattening pigs（（Dabai×Changbai）×Duroc）weighing 100±5 kg into two groups.The control group was directly transported to the slaughterhouse and rested for 24 hours.The experimental group was transported for 5 hours and slaughtered immediately.Blood and jejunum tissues were collected,serum MDA,LDH,SOD and LPS levels were detected.HE staining was used to analyze the morphology of jejunum,and the expression levels of NOX1,HO-1,tight junction protein,autophagy-related protein,PINK1 and Parkin were analyzed by QPCR and Western blot.The results showed that transport can cause jejunum villi damage,shedding,shortened length,deep crypts;elevated levels of MDA,LDH and endotoxin LPS in serum,decreased SOD levels;the expression of HO-1 and tight junction proteins（Claudin-1,Occludin and ZO-1）in jejunum was down-regulated,and the expression of NOX1 mRNA was up-regulated;Moreover,transport stress can induce autophagy levels by increasing the expression of autophagy-associated proteins（LC3,ATG5）,while increasing the protein expression levels of PINK1 and Parkin,and activating mitochondrial autophagy.It is indicated that transport stress can cause oxidative damage in the intestinal tract of pigs and activate the level of autophagy.2.Effect of transport stress on gene transcription level of jejunal tissue in pigsRNA was extracted from jejunal tissues,differentially expressed genes were screened by RNA-Seq technology,and GO,KEGG and protein interactions were analyzed,the accuracy of sequencing results was verified by QPCR.Compared with the control group,there were 1036 transcript differential genes in the transport stress group（FC>1.5,FDR<0.05）,of which 606 genes were up-regulated and 430 genes were down-regulated;The GO function annotation is mainly related to G-protein coupled receptor signaling,defense response,cytokine action,lipid catalysis process,membrane composition,extracellular region,etc.;KEGG pathway enrichment mainly with MAPK signaling pathway,PI3K-Akt signaling pathway,cytokine-cytokine receptor interaction,apoptosis,calcium signaling pathway,etc.;There is a good interaction between differentially expressed gene proteins associated with oxidative stress and ROS production,and PCR results are highly correlated with RNA-Seq.3.Effect of NOX1 on oxidative damage of pig intestinal epithelial cells（IPEC-1）Transport stress leads to an increase in NOX1 levels in pig jejunal tissues,while NOX1 is a key enzyme in ROS production.The role of NOX1 in oxidative damage and autophagy of IPEC-1 cells remains to be elucidated.In this study,the optimal concentration of NOX1 inhibitor（ML171）was screened,IPEC-1 cells were treated with this concentration inhibitor and 300μM H₂O₂,collect the cells and supernatant culture medium,detect the content of oxidative damage marker molecules（MDA,SOD,LDH）and the changes of cell ROS level,mitochondrial membrane potential level and mitochondria quantity;The RNA and protein in the cells were extracted,and the expression of tight junction protein gene,autophagy-related protein and SIRT1 and PGC-1αgenes and proteins were detected by QPCR and Western blot.Compared with H₂O₂treatment group,NOX1 inhibitor can decrease the content of MDA,LDH and ROS in IPEC-1 cells,up-regulate SOD activity;Inhibition of NOX1 can restore mitochondrial membrane potential levels and mitochondrial numbers,increase the expression of tight junction proteins Claudin-1 and ZO-1,inhibit the expression of autophagy-related proteins,and increase the expression of SIRT1 and PGC-1α.It is shown that inhibition of NOX1 expression can alleviate oxidative stress caused by H₂O₂,reverse H₂O₂-induced autophagy,and play an important role in maintaining intestinal mucosal barrier integrity.Based on the above results,the following conclusions are drawn:1.Transportation can cause decreased levels of antioxidants,accumulation of endotoxin,causing significant oxidative damage in the intestine,and activation of autophagy and PINK1/Parkin-mediated mitochondrial autophagy in pigs.2.RNA-Seq technology screened a total of 1036 intestinal oxidative damage transcript differential genes,of which 606 genes were up-regulated,430 genes were down-regulated,and differentially expressed genes associated with PI3K-Akt,oxidative stress,ROS production,apoptosis,and participates in many signaling pathway processes.3.Inhibition of NOX1 expression reduced the levels of ROS and autophagy in IPEC-1 cells,increased the expression of tight junction molecules in the intestinal mucosa and increased the activity of SIRT1/PGC-1αpathway,and alleviated the oxidative damage of IPEC-1 cells induced by H₂O₂.