| The aim of this study is to investigate the effects of Cu/Zn-Mt on the intestinal microflora and barrier function of weanling pig and elucidate its underlying mechanisms.Cu/Zn-Mt was prepared by loading Cu/Zn with montmorillonite.The in vitro trail was designed to study the effects of Cu/Zn-Mt on bacterial activity,intestinal epithelial cell function and inflammatory response.The effects of Cu/Zn-Mt on the growth performance,diarrhea,intestine micrbiota and barrier function of weanling pig were also studied.Moreover,advant technologies(16S rRNA sequencing,transcriptomics,metabonomics)were used to elucidate Cu/Zn-Mt’s underlying mechanisms,which may provide scientific basis for further studies on an effective solution to the diarrhea of weanling pig.The results were showed as follows:1 Preparation,characterization,antibacterial and cytotoxicity studies of Cu/Zn-MtA series of modified montmorillonites(Cu-Mt,Zn-Mt,Cu/Zn-Mt with Zn/Cu ratio of 0.98,1.97,3.87)were prepared,and characterized using X-ray diffraction,fourier transformed infrared spectroscopy and scanning electron microscopy.The specific surface areas,antibacterial activity and cytotoxicity of the modified montmorillonites were investigated.In the modified montmorillonites,hydrated copper ions and zinc ions were exchanged in the interlayer space of montmorillonites and the particles were irregular shapes.The results showed that Cu/Zn-Mt enhanced antibacterial activity compared with Zn-Mt and Cu-Mt possibly due to the synergistic effect between Zn2+ and Cu2+.Among the Cu/Zn-Mt with different Zn/Cu raitos,Cu/Zn-Mt with a Zn/Cu ratio of 0.98 or 1.97 showed higher antibacterial activity against gram-negative bacteria(Escherichia coli),gram-positive bacteria(Staphylococcus aureus).Moreover,the antibacterial activity of Cu/Zn-Mt was associated with the enhancement of the specific surface area when particle size decreased.Cytotoxicity studies on IPEC-J2 cell showed a slight cytotoxicity of Cu/Zn-Mt.The present study further explored the antibacterial mechanism of Cu/Zn-Mt on E.Coli using transcriptomics,scanning electron microscopy,transmission electron microscope,DNA electrophoresis,etc.The results showed Cu/Zn-Mt could inhibit the growth of E.Coli through by absorption and Cu/Zn effect;Cu/Zn-Mt treatment could increase active oxygen,membrane permeability and DNA degradation,and inhibited biofilm formation;153 differentially expressed genes could be obtained by Cu/Zn-Mt treatment,including 133 up-regulated genes and 20 down-regulated genes;GO function annotaiton classfication indicated that the differentially expressed genes were divided into different function categories,in which many function categories were mainly involved,such as metal ions binding and transmembrane transport,biosynthesis and metabolic processes of protein,enzyme,nucleic acid;The KEGG enrichment analysis showed that 41 genes were annotated to 7 metabolic pathways.Nucleic acid polymerase,sulfur metabolism and amino acid metabolism have been mainly significantly enriched.Therefore,the results indicate that Cu/Zn-Mt,which was prepared with Cu and Zn supported in montmorillonite,showewd strong antibacterial activity and a slight cytotoxicity.Cu/Zn-Mt could kill bacteria through absorption and Cu/Zn effect;Cu/Zn-Mt could interfer the biosynthesis and metabolic processes of enzymes,proteins and nucleic acids by affecting metal ions binding and transmembrane transport,which could eventually lead to the death of bacteria.2 Effect of Cu/Zn-Mt on E.Coli induced epithelial barrier disruption in IPEC-J2 cellsThe present study investigated the beneficial effect of Cu/Zn-Mt on barrier function in an in vitro transwell model with porcine intestinal epithelial cell line IPEC-J2 cells,and subjected cells with E.Coli stimulation.This study was divided into two parts:Part 1,explore the effect of Cu/Zn-Mt pretreatment on E.Coli induced epithelial barrier disruption in IPEC-J2 cells;Part 2,explore the effects of Cu/Zn-Mt on epithelial barrier function after E.Coli challenge.In Part 1,the results showed that compared with the control group,E.Coli treatment significantly increased(P<0.05)the number of E.Coli in the upper and lower chambers of transwell.reduced(P<0.05)transepithelial electrical resistance(TER)and increased(P<0.05)fluorescein isothiocyanate-dextran 4kDa(FD4)permeability,disrupted tight junction protein expression(Occludin,ZO-1,Claudin-1,Claudin-2)distribution.These effects were prevented by Cu/Zn-Mt pretreatment.In Part2,the result showed that Cu/Zn-Mt could alleviate the epithelial barrier function induced by E.Coli and inhibited the disruption of tight junction protein expression and distribution(P<0.05).Also Cu/Zn-Mt could decrease((P<0.05)the mRNA expression of pro-inflammatory factor(CXCL1,CXCL2,CXCL6,IL-6,IL-8,TNF-α).Taken together,these results suggested that Cu/Zn-Mt could protect the epithelial barrier function through two ways:1.Inhibite the proliferation of E.Coli,which can reduce the intestinal epithelial barrier injury caused by E.Coli infection.2.Directly affect the intestinal epithelial cells and repair the damage of intestinal epithelial barrier.3 Effect of Cu/Zn-Mt on growth performance,intestine microbiota and intestinal barrier function of weaned piglets.The study was conducted to evaluate the effects of Cu/Zn-Mt on growth performance,intestine microbiota and intestinal barrier function of weaned piglets.A total of 72 pig(Duroc x Landracex Yorkshire;average initial weight of 6.36 kg weaned at 21 ±1 d age)were allotted to two treatments for two weeks.Each treatment had six pens of six pig.The two treatments were:(1)Control group:basal diet;(2)Cu/Zn-Mt group:basal diet+ 2 g/kg Cu/Zn-Mt.The results showed that as compared with control group,Cu/Zn-Mt supplementation increased(P<0.05)average daily gain,gain:feed ratio,the ratio of jejunal villus height and crypt depth,microvilli height,tight junction protein expression(occludin,claudin-1 and ZO-1)in jejunum mucosa and the content of Cu and Zn in serum and ileum mucosa;In Cu/Zn-Mt group,the villi of the jejunum grew thicker,arranged regularly and evenly.The Cu/Zn-Mt addition decreased(P<0.05)fecal scores,the number of Escherichia coli and Streptococcus suis in jejunum and colon,the paracellular permeability of FD4 in jejunum;No difference was observed(P>0.05)on the expression of antibiotic resistance gene or Cu/Zn resistance genes in feces between control group and Cu/Zn-Mt group.The results indicated that Cu/Zn-Mt supplementation could improve growth performance and intestinal function in weaned piglets.Besides,this dose wouldn’t induce the increase of drug-resistant and Cu/Zn resistant bacteria.4 Effect of Cu/Zn-Mt on intestinal microbiota in weaned piglets:16sRNA sequencing and metabolomics analysis16sRNA sequencing and metabolomics were used to study the effects of Cu/Zn-Mt on intestinal microbiota in weaned pig.The 16sRNA sequencing results showed that Cu/Zn-Mt supplementation significantly influenced the composition of intestinal microbiota,reduced(P<0.05)the relative number of potentially pathogenic bacteria in the colon and increased the ability of stress tolerance.At the phylum level,the relative abundance of Firmicutes was significantly increased(P<0.05),while the relative abundance of Bacteroidetes and Proteobacteria were reduced(P<0.05).At the genus level,Cu/Zn-Mt supplementation significantly increased(P<0.05)the relative abundance of Coprococcus,Lactobacillus,Eubacterium,Akkermansia,f_Lachnospiraceae,Bifidobact.erium,Ros’eburia,Faecalibacterium and Enterococcus.The relative abundance of Clostridium,f_Erysipelotrichaceae,Turicibacter,Streptococcus,Prevotella,.f_Enterobacteriaceae was reduced(P<0.05).KEGG metabolic pathway analysis showed that Cu/Zn-Mt supplementation significantly increased(P<0.05)the metabolic pathways such as metal ion absorption metabolism,amino acid metabolism,anti-infection ability,and reduced(P<0.05)the enrichment of related inflammatory signaling pathways,lipopolysaccharide biosynthesis,apoptosis,bacterial invasion of intestinal epithelium and others.Metabolomics data showed that compared with control group,the Cu/Zn-Mt supplementation significantly changed(P<0.05)colonic metabolites(19 species),which was mainly involved butyrate metabolism,amino acid metabolism,glutamic acid,arginine,proline,histidine,etc.),and fat metabolism(bile acid synthesis,the synthesis of carnitine,fatty acid metabolism,arachidonic acid metabolism,linolenic acid and linoleic acid metabolism,mitochondrial-oxidation)short chain saturated fatty acids,purine metabolism,urea cycle,sugar metabolism(pyruvate metabolism),vitamin metabolism(nicotinate and nicotinamide metabolism),seroidogenesis.The results indicated that Cu/Zn-Mt supplementation could influence the composition and metabolic function of intestinal microbiota.5 Effect of Cu/Zn-Mt on E.Coli LPS-induced intestinal injury in weaned pigletsThis study was aimed at investigating whether dietary Cu/Zn-Mt could alleviate E.Coli LPS-induced intestinal injury in weaned pig,and explored the molecular signaling mechanism.Eighteen 21-d-old piglets were randomly divided into three groups(control.LPS and LPS + Cu/ZnMt).After 14 d of feeding,piglets in the LPS group and LPS+Cu/Zn-Mt group received i.p.administration of LPS,whereas piglets in the control group received saline.At 4 h post-injection,jejunum samples were collected for analysis.The results indicated that,compared with the control group,the piglets challenged with LPS significantly decreased(P<0.05)intestinal TER and tight junction protein,and increased(P<0.05)FD4 permeability,the mRNA expression of pro-inflammatory cytokine,which indicated that LPS induced intestinal barrier injury in pig.Compared with the LPS group,Cu/Zn-Mt supplementation significantly increased(P<0.05)intestinal TER and tight junction protein,and decreased the FD4 permeability,the mRNA expression of pro-inflammatory cytokine in pig.Transcriptome data showed that compared with LPS group,differentially expressed genes in Cu/Zn-Mt group are mainly expressed in extracellular exosome and nucleus.Cu/Zn-Mt supplementation could reduce intestinal inflammation through affecting many biological processes(inflammation,immune response,etc).Toll-like receptors signaling pathways,MAPK signaling pathways,cell factor receptor interactions,chemokine signaling pathways,the activity of transcription factors,NF-KB signaling pathways and other associated metabolic pathways were also involved.The results suggested that the addition of Cu/Zn-Mt could alleviate LPS-induced intestinal injury by alleviating intestinal inflammation in weaned piglets.In conclusion,above results demonstrated that Cu/Zn-Mt showed strong antibacterial activity,as well as repaired damaged epithelial cells and alleviat the inflammatory response reaction induced by E.Coli in vitro.The addition of Cu/Zn-Mt could improve growth performance,reduce diarrhea,and relieve weaning stress in weaned piglets.Its underlying mechanism might be associated with improved intestinal microbiota and alleviatied intestinal inflammation,which further protecting the intestinal epithelial barrier function of weaned piglets. |
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