| Deoxynivalenol（DON）is a mycotoxin with the highest contamination rate and overstandard rate in agricultural products and feeds,which seriously threatens human health and animal husbandry.Pigs are the most sensitive stock to DON,and their gut and immune system are the main targets of DON,Melatonin（MEL）is a neurohormone mainly secreted by the pineal gland.It has significant antioxidant and anti-inflammatory functions,and is involved in the regulation of mammalian circadian rhythms,anti-oxidative stress,regulation of glucose,lipid metabolism balance and other Physiological activities.This study investigated the protective effect and specific mechanism of melatonin（MEL）on DON-induced toxicity of porcine intestinal epithelial cell line（IPEC-J2）,in order to alleviate the cytotoxicity of DON.Research offers new directions and perspectives.The study begins with the examination of the effects of DON exposure on IPEC-J2 cell activity,reactive oxygen species,and apoptosis,as well as the preliminary investigation of the cytotoxic effect of DON on IPEC-J2;Cells were pre-exposed to MEL,and then exposed to DON,to detect cell viability,apoptosis and oxidative stress and other indicators to explore the alleviating effect of MEL on DON-induced IPEC-J2 cytotoxicity.In addition,RNA-seq technology was used to explore the related genes and signaling pathways that MEL alleviated DON-induced cytotoxicity.The role and mechanism of tight junction and autophagy signaling pathways in MEL alleviating DONinduced cytotoxicity were explored by studying the activity of signaling pathways.The main test results of this study are as follows:1.In order to explore the harm of DON to intestinal health of piglets,different concentrations（0,0.5,1,1.5,2,and 2.5 μg/mL）of DON were used to treat cell viability for cell culture time（24,48 and 72 h）,and it was found that IPEC-J2 cell viability was decreased in a ratio-and time-dependent interaction effects with DON,IPEC-J2 cell viability decreased to 55.1%after 24 h of culture and DON concentration of 1 μg/mL.The expression changes of apoptosis,inflammation and tight junction related genes were detected by qRT-PCR.The results revealed that compared with the control group,the expressions of caspase-8,caspase9 and Bax genes were markedly increased after 1 μg/mL DON treatment（P<0.01）,and the mRNA expression level of IL-6,IL-1βTNF-α,COX-2 inflammation-related genes was significantly raised（P<0.01）,the tight junction-related genes Claudin-1 and Claudin-3 were significantly dropped（P<0.01）.In addition,the effect of DON on mitochondrial function was detected,and the intracellular reactive oxygen species（ROS）content was detected by fluorescent probe DCFH-DA.The results showed that the fluorescence intensity of ROS in the DON treatment group was dramatically enhanced（P<0.01）,the mitochondrial membrane potential of IPEC-J2 cells decreased under the induction of DON.This indicated that the DONinduced IPEC-J2 cell injury model was successfully constructed in this study.2.Based on the damage of DON to porcine intestinal cells and the anti-inflammatory and antioxidant properties of MEL,this study plumbed the protective effect of MEL on the cytotoxicity of DON on porcine PEC-J2 cells and its specific mechanism.The activity of IPEC-J2 cells pretreated with different concentrations of MEL for 24 h and then treated with DON（1 μg/mL）for 24 h was detected,and it was uncovered that MEL（1μM）significantly alleviated the DON-induced decrease of IPEC-J2 cells.Cell apoptosis was detected by flow cytometry,and the results showed that MEL pre-treatment reduced DON-induced apoptosis rate（P<0.05）.The expression levels of apoptosis-related proteins Bcl-2 and Bax were further detected.,MEL pre-treatment increased the ratio of Bcl-2/Bax（P<0.05）.The secretion of inflammatory factors was detected by ELISA.The results showed that MEL pre-treatment could significantly reduce the expressions of inflammatory factors IL-6,IL-1β and TNF-αcompared with DON treatment group（P<0.05）.Using ROS and TMRE kits to detect the effect of MEL on DON-induced mitochondrial damage in IPEC-J2 cells,it was found that MEL alleviated the DON-induced increase of ROS and the decrease of mitochondrial membrane potential（P<0.01）.The above results illustrate that MEL has a mitigating effect on DON-induced toxicity.3.To further plumb the specific mechanism by which MEL alleviates DON-induced cytotoxicity.In this study,transcriptome sequencing was performed on the experimental groups（control group,DON treatment group and DON+MEL group）.Transcriptome sequencing analysis found that a total of 9838 differentially expressed genes were identified between the DON treatment group and the control group（P<0.05）,including 4627 upregulated genes and that of 5244 down-regulated genes.A total of 2134 genes were identified that were differentially expressed between the DON+MEL group and DON treatment group（P<0.05）,among which the expression of 1249 genes was up-regulated and that of 885 genes was down-regulated.Combined analysis between experimental groups（DON treatment vs control group;DON+MEL group vs DON treatment group）the 1476 differentially expressed genes were found.These differential genes were mainly enriched peptide metabolic process（GO:0006518）,translation（GO:0006412）,peptide biosynthetic process,ribosomes（GO:0043043）,ribosomal（GO:0044391）,ribosome（GO:0005840）functional items and signaling pathways such as ribosomes,oxidative phosphorylation,and autophagy.qRT-PCR revealed that the expression trends of differentially expressed genes CLDN1,MAP2K1,SOD2,IL-18,ZFP36L2,FAM172A,CHUK and KLF5 were consistent with RNA-seq sequencing results.The mechanism analysis of the screened key signaling pathways such as autophagy and tight junction showed that MEL pre-treatment could alleviate DON-induced degradation of ZO-1,Claudin-1 and Occludin.Meanwhile,MEL down-regulated the expression of DONinduced autophagy proteins LC3-II/LC3-I,Atg5,and Beclin 1,and increased the phosphorylation of mTOR and AKT.In conclusion,MEL attenuated part of DON-induced cellular damage through the autophagy and tight junctions signaling pathway. |
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