The Molecular Mechanism Of Combination Of Glycyrrhizic Acid And Probiotics For Alleviating The Hazards Of Weaned Piglet Growth And Gut Health Induced By Deoxynivalenol

Mycotoxins are toxic secondary metabolites produced by molds during growth process,which widely exist in ingredients or feeds.It is a great threat to the health of animals to cause huge economic losses,which seriously affect the development of animal husbandry and feed industry.Among various mycotoxins,deoxynivalenol（DON）,AFB1,and ZEN are more harmful to humans and animals,and the detection rate and over-standard rate of DON in feedstuffs or diets are very high in China.The primary target of DON is the intestinal tract when animals consume contaminated diets.DON can destroy the integrity of intestinal barrier function,affect the morphology of intestine and the protein structure of intestinal epithelial cells,thereby causing oxidative stress,inflammation and apoptosis.In recent years,numerous studies have shown that biodegradants such as probiotics and nutritional intervention can effectively reduce the toxicity of DON.Therefore,in view of the serious pollution of DON in feed ingredients in China and the toxic effects of DON,the present research aims to find a new type of feed additive that can effectively remove or degrade DON and improve immunity to alleviate the damage caused by DON to animals.Firstly,the alleviative effects for the toxicity of DON by three kinds of nutritional additives such as chlorogenic acid（CGA）,astilbin（AST）and glycyrrhizic acid（GA）were studied with porcine intestinal cells IPEC-J2,respectively.The nutritional additive with better alleviative effect is GA,and its acting mechanism is further elucidated through transcriptomics analysis.Then,the orthogonal method is used to optimize the best ratio of GA,Saccharomyces cerevisiae and Enterococcus faecalis to alleviate and degrade the toxicity of DON,and explore its immune protection mechanism on pig intestinal epithelial cells.After that,the damages of DON on piglet growth performance,nutrient digestion and absorption,immune function,intestinal and liver health,and the compatibility of GA and compound probiotics（GAP）mitigation mechanism for piglet induced by DON were studied through piglet feeding experiment.Finally,16S rRNA gene sequencing,transcriptomics and metabonomics and other multi-omics sequencing technologies are employed to analyze the effects of GAP and DON on piglet gut microbes,jejunum gene expression,serum metabolites and biochemical parameters,and the relationship among them,providing a theoretical reference for the use of this new feed additive in pig production to efficiently degrade mycotoxins,prevent piglet diarrhea,and provide protection for animal and human health.The main results of this study were as follows:（1）The effect of GA,CGA and AST on alleviating DON-induced IPEC-J2 cell damages:using porcine intestinal epithelial cells 1PEC-J2 as a model,the toxic effects of DON and the cell damage repair effects of CGA,AST and GA on DON were studied.The results showed that IPEC-J2 cells treated with 0.5 μg/mL DON for 6 h induced inflammation and apoptosis,and significantly reduced cell viability（P<0.05）,and significantly increased the release rate of lactate dehydrogenase（LDH）（P<0.05）,early and late apoptotic cell rate（P<0.01）,and significantly up-regulated the relative mRNA abundances of IL-6,IL-8,TNF-α,COX-2,NF-κB,Bax,Caspase 3,Occludin and ASCT2（P<0.05）,and significantly down-regulated the relative mRNA abundances of Bcl-2,ZO-1,PePT1,GLUT2 and SGLT1（P<0.05）.When 40 μg/mL CGA was pretreated for 1 h followed by DON incubation for 6 h or 20 μg/mL AST and 400 μg/mL GA were incubated with DON for 6 h,all could significantly increase cell viability to 98.32%,111.25%and 96.85%,respectively,and reduce LDH release rate and apoptosis rate（P<0.05）,reduce the expression of inflammation and apoptosis factor-related genes and proteins（P<0.05）,and increase the expressions of tight junction proteins and nutrient transport-related genes（P<0.05）.The results showed that the addition of CGA,AST and GA could reduce the cell toxicity induced by DON,increase the rate of viable cells,enhance the intestinal barrier function and improve the transportation and absorption of nutrients,which will lay theoretical foundation for the application of plant extracts such as CGA and GA serving as a feed additive to reduce the harm of mycotoxins in animal production.In addition,considering the economic cost,GA was selected as a nutritional additive for next research.（2）Using high-throughput transcriptome sequencing technology to reveal the protective mechanism of GA on DON-induced oxidative stress,inflammation and apoptosis of IPEC-J2 cells:Cells were treated according to the control group（CON）,DON,GA and GA+DON group（DON indicates 0.5 μg/mL DON incubation for 6 h,GA indicates 400 μg/mL GA incubation for 6 h,GA+DON indicates 400 μg/mL GA and 0.5 μg/mL DON incubation for 6 h）.After the above treatments,the cells and supernatants of different treatments were collected,and the DON,IL-8,Caspase 3 and NF-κB contents were detected by ELISA method,and the corresponding RNA was extracted for transcriptome sequencing analysis.The results showed that DON could induce oxidative stress by significantly increasing the content of malondialdehyde（MDA）and reducing the activities of catalase（CAT）and superoxide dismutase（SOD）（P<0.01）,and it significantly increased the expressions of IL-8,Caspase 3 and NF-κB in the cell supernatant（P<0.01）,whereas GA addition significantly increased the activity of CAT and SOD and decreased MDA,IL-8,Caspase 3 and NF-κB contents（P<0.01）.RNA-seq technology was used to conduct a comprehensive analysis of the gene expression patterns of each group,and the differentially expressed genes（DEGs）were screened.The GO,KEGG enrichment analysis and PPI analysis of DEGs revealed that GA could inhibit the production of inflammatory factors and chemokines by activating TNF,Toll-like receptors and NF-κB signaling pathways,and significantly decrease oxidative stress and reduces cell apoptosis,and had a certain immunomodulatory effect,which indicated that GA could reduce intestinal oxidative stress and inflammation induced by DON.（3）The effect of combination of GA,Saccharomyces cerevisiae and Enterococcus faecalis on alleviating IPEC-J2 cell damage induced by DON:MTT method was used to study the whole bacterial liquid,cells and supernatant of Saccharomyces cerevisiae and Enterococcus faecalis for alleviating DON cytotoxicity.The orthogonal method was used to optimize the optimal ratio of GA,Saccharomyces cerevisiae and Enterococcus faecalis for cell viability,and its effect on DON degradation and the expressions of cytokines and other proteins were also determined.The results showed that the optimal counts of Saccharomyces cerevisiae and Enterococcus faecalis could significantly promote IPEC-J2 cell viability（P<0.05）,and the optimization of GA,Saccharomyces cerevisiae and Enterococcus faecalis（GAP）had the best effect on cell viability.The best ratio was 400μg/mL GA,1×106 CFU/mL Saccharomyces cerevisiae and 1×106 CFU/mL Enterococcus faecalis,which not only significantly alleviated the toxic effect of DON,but also achieved the highest degradation rate of DON（34.69%）.In addition,GAP also significantly reduced the relative mRNA abundances of IL-8,Caspase 3 and NF-κB and the protein expressions of Bax,TNF-α and COX-2 proteins（P<0.05）,and significantly increased ZO-1,Claudin-1 and PePT1 protein expressions（P<0.05）.It was inferred that the combination of GA,Saccharomyces cerevisiae and Enterococcus faecalis had the synergistic effect of enhancing cell viability and DON degradation,which could protect IPEC-J2 cells from DON-induced damage by reducing DON cytotoxicity,reducing cell apoptosis and inflammation,improving intestinal barrier function,and regulating nutrient absorption and transport.（4）The effect of GAP on alleviating piglet growth and intestinal damages induced by DON:In this experiment,160 42-day-old pigelts（Landrace×Large white）were selected and randomly divided into four groups,which were fed with the normal diet（CON）,moldy wheat diet containing 1040 μg/kg DON（DON）,normal diet supplemented with GA and compound probiotics（GAP）,and moldy wheat diet supplemented with GA and compound probiotics（GPD）,four repetitions in each group,10 piglets in each repetition（half castrated male and half female）,the experimental period was 28 d.The results showed that compared with the CON group,the DON group significantly reduced daily gain（ADG）,daily feed intake（ADFI）,fecal amylase activity and serum SOD activity（P<0.05）,and significantly increased ADFI/ADG,diarrhea rate,elimination rate and serum alanine aminotransferase（ALT）,aspartate aminotransferase（AST）,LDH,MDA,IL-8,Caspase 3 and NF-κB levels（P<0.05）.Furthermore,compared with the DON group,the GAP group and GPD group significantly increased ADG,ADFI,fecal amylase activity,crude protein and crude fat digestibility and serum SOD activity（P<0.05）,significantly reduced ADFI/ADG,diarrhea rate,elimination rate and the expressions of AST,ALT,LDH,MDA,IL-8,Caspase 3 and NF-κB in serum（P<0.05）.The morphology of piglet jejunum and liver showed that DON severely damaged the structure and morphology of piglet jejunum and liver.Compared with the DON group,the GAP group and GPD group significantly increased the villus height and villus height/crypt depth（V/C）in jejunum（P<0.05）,significantly reduced the crypt depth（P<0.05）,and reduced inflammation of the jejunum and liver.In addition,GAP addition also significantly reduced the residual amount of DON in feces,serum,jejunum contents and liver.In the jejunum of piglet,compared with the control group,the DON group significantly up-regulated the expressions of Caspase 3,IL-8,COX-2,GULT2 genes,and COX-2,TNF-α and ASCT2 proteins（P<0.05）,while down-regulated the expressions of Bcl-2 and IL-10,ZO-1,Occludin and Claudin-1 genes（P<0.05）and the expressions of ZO-1,Claudin-1 and PePT1 proteins（P<0.05）.After adding GAP,it significantly down-regulated the expressions of inflammation and apoptosis genes and proteins,and significantly increase the expressions of tight junction proteins and nutrient transport factor genes and proteins.In the liver of piglet,compared with the control group,the DON group significantly increased the expressions of IL-8,COX-2 and Claudin-1 genes and decreased the expression of Bcl-2,ZO-1,Occludin,GLUT2 and SGLT1（P<0.05）;while the addition of GAP significantly reduced the expressions of Caspase 3,IL-8,COX-2 and TNF-α（P<0.05）,and significantly increased the expressions of Bcl-2,ZO-1,Occludin,Claudin-1,GLUT2,ASCT2 and PePTl and the protein expressions of ZO-1,Claudin-1 and ASCT2（P<0.05）.These results indicated that DON destroyed the intestinal barrier and nutrient transport by inducing inflammation and apoptosis in the jejunum and liver,thereby affecting the growth performance of piglets;however,adding GAP could significantly alleviate the jejunum and liver damage induced by DON,and promote the healthy growth of piglets.（5）The high-throughput sequencing technology was used to study the effects of DON and GAP on piglet intestinal and fecal microflora:16S rRNA sequencing technology was used to determine and analyze the microbiota in feces and jejunum contents of piglets.The results showed that adding GAP could significantly improve the diversity of microbes in feces and jejunum contents of piglets;DON significantly changed the flora structure of the feces and jejunum contents of piglets,significantly reduced the number of Firmicutes,and significantly increased the number of Proteobacteria and Actinobacteria in jejunum content,while significantly reduced the number of Firmicutes,and increased the number of Bacteroidetes and Actinobacteria in feces.At the genus level of jejunum content and feces,DON significantly reduced the number of Lactobacillus and increased the number of Clostridium_sensu_stricto₁;however,GAP addition could maintain the balance of intestinal flora and promote piglet growth by significantly increasing the beneficial bacteria such as Lactobacillus and reducing the harmful bacteria such as Clostridium_sensu_stricto₁.It was found that GAP intervention was beneficial to the difference in the metabolic function of intestinal flora through predicting the function of the flora in piglet feces and jejunum content.In addition,it was inferred that Lactobacillus and Streptococcus play an important role in the detoxification process of DON by analyzing the correlation between intestinal microbes and residual toxins and other factors,which provide a theoretical basis for improving intestinal health and growth performance of piglets by using this kind feed additives in animal production.（6）High-throughput transcriptome sequencing technology was used to analyze the effects of GAP on the gene expression profile in piglet jejunum induced by DON:The differentially expressed genes were screened by transcriptome sequencing analysis of the jejunum tissues in CON,DON and GPD groups.The results showed that 283,339,and 317 differentially expressed genes were screened in CON vs.DON,CON vs.GPD,DON vs.GPD,respectively,in which 86 differentially expressed genes were co-expressed in CON vs.DON and CON vs.GPD.Among them,31 differential genes were up-regulated in the CON vs.DON but down-regulated in DON vs.GPD;51 differential genes were down-regulated in the CON vs.DON,but up-regulated in the DON vs.GPD.These differential genes were mainly involved in cellular processes,metabolic processes,stimulus responses,cells,organelles,binding and catalytic activity.KEGG enrichment analysis showed that the addition of GAP promoted the digestion and absorption of protein,fat,vitamin and other nutrients in the intestine.In addition,it was found that the down-regulated genes induced by DON such as TNS1,KCNIP4,SCG3 were significantly positively related to Streptococcus,Lactobacillus and Rothia;and the down-regulated genes by GAP addition such as BARX2,PHGDH,ACTN2 were significantly negatively correlated with Escherichia-Shigella by analyzing the correlation between the co-expressed differential genes and the microbial flora in jejunum content,which further revealed the molecular mechanism of GAP for alleviating DON toxicity by regulating intestinal microbiota.（7）The molecular mechanism of GAP on alleviating the damage induced by DON was revealed based on multi-omics analysis:LC-MS metabolomics was used to study the changes of serum metabolites of piglets in CON,DON,GAP and GPD group,respectively.After multivariate statistical analysis of PCA and OPLS-DA,metabolic differences were identified and screened,and KEGG enrichment analysis was performed.The correlation between serum metabolites and jejunum genes and intestinal microbiota was analyzed by multi-omics.The results showed that 201,351,423,and 271 differential metabolites were screened in the CON vs.DON,CON vs.GAP,CON vs.GPD,DON vs.GPD,respectively.There were 69 common differential metabolites in CON vs.DON and DON vs.GPD.It was found that GAP could alleviate the mechanism of DON-induced metabolic disorders and damage in piglets by regulating lipid metabolism pathways such as glycerophospholipid metabolism,linoleic acid metabolism,α-linolenic acid metabolism,and fat digestion and absorption by KEGG enrichment analysis.In addition,the correlation among the total of 69 differential metabolites with ADG,ADFI,serum biochemical parameters,DON residues,co-expressed differential genes and microbiota were deeply analyzed,which showed that DON caused disturbance of intestinal microbiota,lipid and energy metabolism in piglets,and GAP could maintain the balance of intestinal microbiota and improve metabolic disorders,thereby alleviating the toxicity of DON and promoting healthy growth of piglets.