Mechanisms Of Deoxynivalenol-induced Inhibition On Pig Intestinal Epithelial Cell Proliferation And Stem Cell Expansion

Deoxynivalenol（DON）,which is produced by fusarium,is one of the most widely distributed mycotoxins in the world.Pig is the most sensitive farm animals to deoxynivalenol deposure which can disturb the renewal process of intestinal epithelium and then destroy their structure and function.Considering the fact that intestinal epithelial renewal is driven by stem cells located at the buttom of crypts,I hypothesized that low dose of deoxynivalenol may inhibit proliferation of intestinal epithelium and expansion of stem cells in a short time.A total of five experiments were conducted to vertify the hypothesis.In Experiment I,IPEC-J2 cells（an epithelial cell line derived from newborn piglet jejunum）were used to study the effects of DON dose and treatment time on proliferation,barrier function,and expression levels of proteins of interest.The results from 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyl tetrazolium bromide（MTT）and 5-Ethynyl-2′-deoxyuridine（EdU）incorporation assays showed that cell proliferation was sinificantly（P<0.05）decreased by 500 ng/mL DON treatment for 2 h.Effects of DON on trans-epithelial electrical resistance（TEER）and permeability of monolayer IPEC-J2 cells were tested using tranwell models.The results indicated that TEER was reduced（P<0.05）by 500 ng/mL DON treatement for at least 12 h.However,the trans-epithelial absorption of fluorescein sodium salt（FS）was not changed（P>0.05）by 500 ng/mL DON treatment for 2 h.In addition,protein levels of quescent intestinal stem cell（ISC）marker,B cell specific moloney murine leukemia virus insertion site 1（Bmi1）,proliferating cell nuclear antigen（PCNA）,β-catenin,c-Myc,and cyclin D1 were decreased（P<0.05）.Whereas,Axin2 expression level was increased（P<0.05）.The results suggest that proliferation of pig intestinal epithelial cells was inhibited by DON treatment with lower dose and fewer time when compared to barrier function.What’s more,Wnt/β-catenin signaling may involve in DON-induced cell proliferation inhibiton.Experiment II was conducted to study the effects of DON in vivo treatment for 2 h on small intestine morphology,barrier function,and expression of proteins of interest.A total of6 piglets were divided into two groups of similar body weight（BW）each.The Control and DON groups were orally administrated with 0 and 0.3 mg DON/kg BW,respectively.Two hours after oral intake of DON,jejunums were ligated and FS absorption was evaluated.The results showed that there was no difference（P>0.05）on plasma FS concentration between the Control and DON groups.HE staining indicated that DON treatment had no change on villi morphology,but hypertrophy was existed on crypt regions.The results of Western blotting showed that protein levels of mammalian target of rapamycin（mTOR）,ribosomal S6 protein kinase 1（S6K1）,phospho（p）-S6K1 and eukaryotic initiation factor 4E（eIF4E）in mTOR signgaling pathway,β-catenin,c-Myc and cyclin D1 in Wnt/β-catenin signaling pathway,tight junction proteins ZO-1 and Occludin,and Bmi1 were decreased（P<0.05）in small intestines of the DON group when compared with those of the Control group.Moreover,glycogen synthase kinase 3β（GSK3β）,a negative regulatory factor in Wnt/β-catenin signaling pathway,was increased（P<0.05）in the DON group.The results suggest that DON exposure in a short period can inhibit the activities of mTOR and Wnt/β-catenin signaling pathways and the protein level of Bmi1,which may relate to the hypertrophy of crypt regions.As indicated in the Experiment II,DON may damage pig ISCs in short time.Therefore,the objective of Experiment III was to establish pig ISCs culture methods and related evaluation techniques,which is foudation for further studies.（1）The structure of crypt-villi axis isolated from 1-d piglet was analysized under a microscope,and then preliminary methods for crypt isolation were developed.（2）After comparing the structure and size of cryps form several ages of piglets,5-d piglets were chosen for following studies.（3）The advantages and disadvantages of 3 different methods for isolation crypt were summarized.Then a new improved method was put forward,with which 95%purity crypt can be obtained in short time.（4）After isolated from piglet smal intestine,the crypts were immediately used to explore in vitro three-dimensional（3D）culture systems.Finally,a basic culture system with Matrigel as extracellular matrix and advanced DMEM/F12 containing N2,B27,glutamine,N-acetyl-cysteine,epidermal growth factor（EGF）,Noggin,R-Spondin 1,nicotinamide,SB202190,LY2157299,fetal bovine serum（FBS）and Wnt3a conditional medium as culture media was built.The basic culture system is suitable for pig enteroid expansion in vitro.Moreover,the supplementation of Caudal type homeobox 2（Cdx2）conditional medium in middle-late culture period increased（P<0.05）enteroid forming efficiency and budding efficiency,when compared to the basic culture system.（5）The basic system supplemented with Jagged-1 can be used for culturing of single cells digested from small intestinal crypts（named as crypt cells）.（6）A populaton of CD44⁺CD24^lowCD166⁺cells were obtained using flow cytometry.When cultured in crypt cells culture system,the cell population could effectively expand into organoid,which suggest that the cell population had a large content of ISCs.（7）Techniques of lentivirus infection,MTT,and immunofluorescence staining were also set up.This is the first report of research on pig ISCs.Experiment IV was to clarify the effects of DON on expansion of pig ISCs.Firstly,5-d piglets were divided into the Control and DON groups which were orally administrated with solvent and 0.3mg DON/kg BW,respectively.After treatment for 2 h,the intestinal crypts from both groups were isolated cultured in the same methods.Secondly,crypt cells from crypts of normal piglets were cultured in a 3D system for 5 d,and then treated with DON for2 or 24 h.The results showed that enteroid forming efficiency and budding efficiency were significantly decreased（P<0.05）by DON treatment in vivo.Additionally,after treated with250 or 500 ng/mL DON for 24h,enteroid budding efficiency was highly significantly decreased（P<0.01）.The results of EdU incorporation asssy showed that the expansion of enteroid was inhibited after treated with 500 ng/mL DON for 2 h.Besides,fluorescence intensity of Bmi1 and protein level of leucine-rich-repeat-containing G-protein-coupled receptor 5（Lgr5）were both decreased（P<0.05）in 500 ng/mL DON group.The results suggest that DON treatment in short time,either in vivo or in vitro,can inhibit enteroid expansion.In Experiment V,an isobaric tag for relative and absolute quantitation（iTRAQ）-based proteomics was employed to further investigate the underlying mechanisms of shor-term treatment of DON on proliferation of intestinal epithelial cells and expansion of ISCs.A total of 6 small intestinal samples from the Control and DON groups in Experiment IIwere used.A total of 5175 proteins were identified,among which 94 proteins expressed differentlty between groups.Sixty-nine proteins were upregulated while 25 proteins were downregulated.Gene Ontology（GO）analysis combined with Kyoto Encyclopedia of Genes and Genomes（KEGG）pathway analysis revealed that those differently expressed proteins are enriched in phosphoinositide 3-kinase（PI3K）/Akt signaling pathway,Janus kinase/signal transducers and activators of transcription（JAK/STAT）signaling pathway,cell endocytosis,and ATP-binding cassette（ABC）transporters.In summary,although intestinal barrier is not changed by short-term exposure to DON,the proliferation of pig intestinal epithelial cells and expansion of ISCs are sensetively inhibited.The potential mechanisms may involve in ABC transporters,PI3K/Akt/mTOR signaling,JAK/STAT signaling and Wnt/β-catenin signaling.In general,this study provides a new insight into molecular mechanisms of DON-induced injury of intestinal epithelium and laid the foundation for the research of pig ISCs.