Effects Of Chitosan-Zinc Complex On Intestinal Microbial Colonization And Barrier Function Of Weaned Piglets And The Underlying Mechanism

In pig production,dietary supplementation with high-dose inorganic zinc can eliminate the weaning stress of piglets and improve their growth.However,sometimes high-dose zinc exceeds the physiological needs of piglets.A large amount of zinc ions not absorbed by the intestine are excreted with feces,causing severe environmental pollution.Since July 1,2018,China has officially implemented the"zinc reduction"policy,so it is urgent to find a good alternative to high-dose zinc.Studies have shown that organozinc produced by complexing zinc ions to chitosan is positive to implement the zinc reduction strategy of weaned piglets,but the mechanism of the effect of chitosan-zinc complex on intestinal health of weaned piglets still needs to be further elucidated.Intestinal microorganisms play an important role in the healthy growth of piglets after weaning.They can affect the intestinal barrier function through direct（interact with intestinal cells）and indirect（produce metabolites）methods.Due to the huge difference in the number of microorganisms living in the small intestine and large intestine of monogastric mammals,the degradability of chitosan complexes in different intestinal segments is different,which implies that the effect of chitosan-zinc complex on the colonization of bacteria in small and large intestine of weaned piglets,as well as the intestinal barrier function,may be different.In addition,it is reported that nano-zinc particles are earlier to diffuse into the submucosa of the intestinal tract and interact with intestinal epithelial cells more efficiently.In this study,nano-scale（50-150 nm）chitosan-zinc complex（CP-Zn）synthesized in our laboratory previously is used to feed weaned piglets.This study is aimed to research the effect of CP-Zn on the growth performance,colonization of intestinal microorganisms and the barrier function of weaned piglets,and explore its potential mechanism.This study can provide a theoretical basis for the application of CP-Zn in weaned piglets.1.The effect of CP-Zn on the growth performance and small intestine antioxidant function of weaned pigletsThis chapter aims to study the effect of CP-Zn on the growth performance and small intestine antioxidant function of weaned piglets,and explore its potential mechanism.A total of 60 piglets（Duroc×Landrace×Large White,male）weaned at day 21 with an average body weight of 6.21±0.49 kg were randomly divided into five treatment groups,12 piglets in each treatment.The five dietary treatment groups are as follows:（1）basal diet（CON group）;（2）the basal diet supplemented with 100 mg/kg Zn from Zn SO₄（Zn SO₄group）;（3）the basal diet supplemented with 100 mg/kg Zn from CP-Zn（CP-Zn group,and it contains 766 mg/kg chitosan（CS））;（4）the basal diet supplemented with 100 mg/kg Zn from Zn SO₄+766 mg/kg CS（CS+Zn SO₄group）;（5）the basal diet supplemented with 766mg/kg CS（CS group）.On the 15^thday of the trial,six piglets from each group were randomly selected by gavage with ETEC K88(10¹⁰CFU for each piglet)and slaughtered48 hours later.Serum,stomach,chyme and mucosa of the duodenum and jejunum,and longissimus dorsi were collected for subsequent testing.The weight,feed intake and diarrhea rate（from day 1 to day 15）were recorded.In addition,IPEC-J2 stimulated by LPS was used to further explore the mechanism of CP-Zn in vitro.The results showed that compared with the piglets in CON group and the Zn SO₄group,the ADG and jejunal villus height in the CP-Zn group were significantly increased（P<0.05）,while the F:G and diarrhea rate were significantly decreased（P<0.05）.In terms of digestion and absorption,compared with the CON group and the Zn SO₄group,CP-Zn improved the activity of jejunum carbohydrate-related enzymes（amylase and sucrase）and the m RNA expression level of nutrient transport related genes（SGLT1,GLUT2 and EAAC1）（P<0.05）.In muscle,compared with CON group and Zn SO₄group,CP-Zn up-regulated the expression levels of m TOR pathway related proteins（IRS1,p-m TOR and p-p70S6K）（P<0.05）.Besides,compared with the CON group and the Zn SO₄group,CP-Zn increased the T-SOD and GSH-px enzyme activities of the jejunal mucosa（P<0.05）,and decreased the MDA level（P<0.05）.Furthermore,CP-Zn increased the expression levels of Nrf2 pathway related proteins（Nrf2,NQO1 and HO1）in jejunum（P<0.05）.The in vitro experiment showed that CP-Zn reduced the content of MDA and ROS in IPEC-J2 challenged by LPS,and up-regulated the expression levels of Nrf2 pathway related proteins（Nrf2,NQO1 and HO1）.The above results indicate that dietary supplementation of CP-Zn improves the growth performance of weaned piglets and improve the antioxidant capacity in small intestine by up-regulating the Nrf2 signaling pathway.2.The effect of CP-Zn on the the small intestine barrier function and serum metabolites of weaned pigletsThis chapter aims to study the effect of CP-Zn on the the small intestine barrier function and serum metabolites of weaned piglets.The animal experiment design is the same as the first chapter.The jejunum and its mucosa of piglets in the Zn SO₄group,CP-Zn group and CS+Zn SO₄group（with the same concentration of Zn）were sampled for detecting immunohistochemistry and related molecular indexes.In addition,the piglet serum metabonomics analysis was performed based on the LC-MS platform.The results showed that in terms of the intestinal barrier,compared with the Zn SO₄group,CP-Zn significantly increased the protein expression levels of Occludin and Claudin-1 in the jejunal mucosa（P<0.05）,and reduced the levels of serum endotoxin and diamine oxidase（DAO）（P<0.05）.In terms of apoptosis,compared with the Zn SO₄group,CP-Zn reduced the number of jejunal epithelial TUNEL⁺cells and the expression levels of apoptosis-related protein（Bax and Cleaved caspase3）（P<0.05）.Compared to Zn SO₄group,CP-Zn decreased the levels of MDA,low-density lipoprotein（LDL-C）,urea nitrogen（BUN）,aspartate aminotransferase（AST）in serum（P<0.05）,and increased albumin level（P<0.05）.Moreover,the results of serum metabolomics showed that there were 64 different metabolites between the CP-Zn group and the Zn SO₄group.Overall,PCA analysis showed that the metabolites of the two treatment groups showed different clusters.In detail,compared with the Zn SO₄group,the levels of some essential amino acids and small peptides（L-methionine,L-lysine and L-tryptophan;γ-glutamyl tyrosine,phenylalanyl tryptophan,glutylleucine,hydroxyprolyryl tyrosine and aspartyl leucine）were up-regulated in the serum of piglets in the CP-Zn group（P<0.05）;while some long-chain fatty acids（linoleic acid,docosahexaenoic acid,16-hydroxyhexadecanoic acid,palmitic acid,docosahexaenoic acid and arachidonic acid）were down-regulated in the serum of piglets in the CP-Zn group（P<0.05）.The above results indicate that dietary supplementation of CP-Zn can improve the apoptosis and barrier function of the small intestine of weaned piglets,reduce the level of serum endotoxin level,and further changes the composition of serum metabolites.3.The effect of CP-Zn on jejunum inflammatory response and mucosal microbial composition of weaned pigletsThis chapter aims to study the effect of CP-Zn on the jejunal inflammation and mucosal microbial composition of weaned piglets,and explore its potential mechanism.The animal experiment design is the same as that in Chapter 1.The relevant samples of piglets in the Zn SO₄group,CP-Zn group and CS+Zn SO₄group（with the same concentration of Zn）were collected.The methods of ELISA,RT-q PCR and western blot were used to detect jejunum mucosal inflammation related indicators.The 16S r RNA（V3-V4）sequencing technology was used to analyze the microbial composition of jejunal mucosa chyme;gas chromatography was used to detect the content of short-chain fatty acids（SCFAs）in jejunal chyme.The results showed that compared with the Zn SO₄group,CP-Zn significantly decreased the levels of TNF-α,IL-1βand MPO（P<0.05）,decreased the m RNA and protein expression levels of TLR4 and TLR5 and the protein expression levels of My D88 and NF-κB pathway related proteins（p-IκB and p-p65）（P<0.05）in the jejunal mucosa（P<0.05）.In terms of microbial composition,PCo A analysis（based on the Bray-curtis algorithm at the OUT level）showed that the microbial composition of the jejunum mucosa in the CP-Zn group and the Zn SO₄group presented different clusters.Compared with the Zn SO₄group,the Shannon index was decreased,while the Simpson index was increased in the CP-Zn group（P<0.05）.Furthermore,compared with the Zn SO₄group,CP-Zn increased the relative abundance of Firmicutes and Lactobacillus in the jejunal mucosa（P<0.05）,and decreased the relative abundance of Proteobacteria,Streptococcus,Actinobacillus,Turicibacter,Veillonella,Romboutsia and Escherichia-Shigella（P<0.05）.Spearman analysis showed that some bacteria（Proteobacteria,Streptococcus,Actinobacillus,Turicibacter,Veillonella,Romboutsia,Escherichia-Shigella）were positively correlated with inflammation-related parameters（TLR4,TLR5,TNF-α,IL-1βand MPO）（P<0.05）.In addition,CP-Zn did not change the content of SCFAs in piglet jejunum chyme（P>0.05）.The above results indicate that dietary supplementation of CP-Zn reduced the abundance of opportunistic pathogenic bacteria in the jejunum mucosa of weaned piglets,further decrease the expression levels of TLR4/5-NF-κB signaling pathway-related proteins and inflammatory cytokines in the jejunum.4.The effect of CP-Zn on the colonic microbial composition and its metabolites of weaned pigletsThis chapter aims to study the effect of CP-Zn on the colonic microbial composition,metabolites and immune response of weaned piglets.The animal experiment design is the same as the first chapter.The colonic chyme and mucosa,and mucosal chyme of colon were sampled for the determination of inflammation-related parameters,SCFAs,16S r RNA sequencing（V3-V4）and absolute quantification of bacteria.The results showed that the butyric acid content of piglet colonic chyme in the chitosan matrix treatment groups（CS group,CP-Zn group and CS+Zn SO₄group）was significantly higher than（P<0.05）that in the non-chitosan matrix treatment groups（CON group and Zn SO₄group）.In addition,compared with the CON group,CP-Zn treatment reduced the levels of MDA and pro-inflammatory factors TNF-αand IL-1β（P<0.05）;increased the level of anti-inflammatory factor IL-10（P<0.05）;decreased the expression levels of NF-κB pathway-related proteins（p-IκB and p-p65）in the colonic mucosa（P<0.05）.In terms of the microbial composition,PCo A analysis（based on the Bray-curtis algorithm at the OUT level）showed that the colonic mucosal microbial composition showed different clusters between chitosan matrix treatment groups（CS group and CP-Zn group）and non-chitosan matrix treatment groups（CON group and Zn SO₄group）.Compared with the CON group,the Shnnon index of the colonic mucosal bacteria in the CP-Zn group was decreased（P<0.05）,while the Simpson index was increased（P<0.05）.Furthermore,the relative abundance of Prevotella of the colonic mucosal in the CS group and the CP-Zn group was higher than that in the CON group and the Zn SO₄group（P<0.05）and the relative abundance of Escherichia-Shigella of the colonic mucosal in the CP-Zn group was lower than that in the CON group and the Zn SO₄group（P<0.05）.In addition,the results of absolute quantification showed that the copy number of Clostridium cluster IV in the colon chyme of piglets in the CS group and CP-Zn group was higher than that in the CON group and Zn SO₄group（P<0.05）.The above results indicate that dietary supplementation of CP-Zn changed the bacterial composition and butyric acid content of weaned piglets,and also reduced the inflammatory response of colonic mucosa.5.The effect of CP-Zn on the colonic barrier function and epithelial cell proliferation of weaned pigletsThis chapter aims to study the effect of CP-Zn on the colonic barrier function and the proliferation of epithelial cells in weaned piglets,and to explore its potential mechanism.The animal experiment design is the same as the first chapter.The colon of piglets was sampled and fixed in 4%paraformaldehyde for immunohistochemistry and immunofluorescence detection.The colon mucous of piglets was collected for western blot,RT-q PCR and other related molecular indicators detection.The concentration of zinc in serum and organs was measured by ICP-OES.The results showed that compared with the CON group and the Zn SO₄group,the CP-Zn and CS+Zn SO₄treatment groups significantly increased the m RNA expression levels of mucin（MUC1 and MUC2）and protein expression levels of barrier function（ZO-1 and Claudin-1）in the colonic mucosa of the piglets（P<0.05）.Compared with the CON group,the phosphorylation level of AMPK protein was significantly up-regulated in the colonic mucosa of the CP-Zn group,CS+Zn SO₄group and CS group（P<0.05）.The results of immunohistochemistry showed that CP-Zn and CS+Zn SO₄treatment groups significantly increased the number of Ki67⁺cells in the colonic epithelium of piglets compared to the CON group and Zn SO4 group（P<0.05）.Furthermore,the m RNA expression levels of WTN3 and Lgr5,and protein expression level ofβ-catenin in the colonic mucosa of piglets in the CP-Zn and CS+Zn SO₄groups were significantly higher than those in the CON group and Zn SO4 group（P<0.05）.In addition,compared with piglets in the CON group,CP-Zn and CS+Zn SO₄treatment groups significantly reduced the m RNA expression levels of colonic endoplasmic reticulum stress-related genes（GPR78,ATF4 and CHOP）（P<0.05）.In addition,the m RNA expression levels of ZIP7 and GPR39 in the colonic mucosa of piglets in the CP-Zn and CS+Zn SO4 treatment groups were higher than those in the CON and CS groups（P<0.05）.The above results indicate that dietary supplementation of CP-Zn can alleviate the endoplasmic reticulum stress of the colonic epithelium and activate the WNT/β-catenin signaling pathway in the colon,thereby increasing the renewal speed of the colonic epithelium and improving the colonic barrier function of weaned piglets.