Role Of Gut Microbiota In Colon Damage Induced By Subacute DON Exposure

Deoxynivalenol(DON),a kind of trichothecenes mainly produced by Fusarium graminearum and Fusarium culmorum,is the most common mycotoxin with the highest contamination rate.The world mycotoxin survey report showed that China’s contamination rate of DON was 86% in 2020,ranking first among the contamination rates of all countries or regions in the world.DON can contaminate food such as grains and milk,and the total DON intake of residents in different countries or regions around the world ranged from 0.2~14.5 μg/kg bw/d.Acute DON exposure can cause gastrointestinal symptoms such as vomiting,diarrhea and anorexia in humans and animals,while chronic DON exposure cause toxic effects on target organs such as anorexia,growth retardation,immunotoxicity,and reproductive toxicity,posing a serious threat to the health of humans and animals.Hence,the high contamination rate of DON in food is a critical public health problem that requires our timely attention.The intestine,a vital digestive organ in humans and animals,is the first barrier against the invasion of pathogens and external toxic or harmful substances for the host.DON can exert many toxic effects on intestine,such as changes in intestinal structure,destruction of epithelial barrier,and damage to the immune response of intestinal mucosa.At present,small intestine was the target organ in most of in vivo studies on the effects of DON exposure on intestine,but similar research on large intestine(colon)is still relatively lacking.Among all parts of gastrointestinal tract,microbial diversity and abundance were richest in the colon,in which the bacterial concentration was as high as 1011~1012 CFU/m L.Gut microbiota plays a key part in maintaining the integrity of intestinal barrier function.Previous studies suggested that DON can interact with gut microbiota after entering the gut of humans and animals along with food or feed.DON may affect metabolic activity and host health by altering the composition and relative abundance of gut microbiota.Conversely,gut microbiota can metabolize or degrade DON by biological enzyme transformation or hydrolysis to reduce its toxicity and bioavailability.Thus,it can be concluded that gut microbiota may exert an important role in DON-induced intestinal damage.However,the current researches only focused on the changes in composition and relative abundance of gut microbiota induced by DON exposure,and the exact role of gut microbiota in DONinduced intestinal damage remains unclear.Therefore,it is necessary to explore the effect of DON exposure on the colon and to determine the role of the gut microbiota in it.In summary,this study intended to investigate the subacute toxicity of DON to initially explore the effect of DON exposure on colon,and took 100 times of human intake(10 μg/kg bw/d)as the intervention dose of DON(1 mg/kg bw/d)in mice by oral administration for 4 weeks based on the aforementioned DON exposure in humans(0.2-14.5 μg/kg bw/d)and the standards of subacute toxicity experiment to set intervention dose and period(covering 100 times of the actual exposure in human and 4 weeks),in order to initially explore the effects of subacute DON exposure on colon and gut microbiota,and reveal the exact role of gut microbiota in the effects of subacute DON exposure on colon by antibiotics treatment and fecal microbiota transplantation.This study aimed at providing a new theoretical basis for exploring the mechanism of DON-induced intestinal damage and opening up new research directions for the prevention or treatment of DON-induced intestinal damage.Part 1 Effects of subacute DON exposure on colon and gut microbiota in miceObjective(1)To investigate whether subacute DON exposure can lead to colon damage in mice.(2)To explore whether subacute DON exposure can cause changes in gut microbiota and fecal metabolites in mice.Methods(1)After being randomly divided into two groups by body weight,thirty 6-week-old SPF male C57BL/6j mice received deionized water(CTR group,n = 15)and DON(1 mg/ kg bw/d,DON group,n = 15)by oral gavage for 4 weeks(once a day),respectively.(2)Mice feces were collected for the sequencing and analysis of 16 S r DNA amplicon and metagenome as well as the detection and analysis of targeted metabolomics after gavage to observe the changes of gut microbiota and fecal metabolites.(3)H&E,RT-q PCR,WB,IHC and cytometric bead array were used to observe or detect the related conditions of colon histopathology,colon physical barrier(occludin,ZO-1,claudin-3 and claudin-4),inflammatory cytokines(TNF-α,IL-1β,IL-6,MCP-1 and IL-10),oxidative stress(MDA,SOD,GPx and CAT)and apoptosis(percentage of TUNEL positive cells,Bax,cleaved caspase 3 and cleaved caspase 7)in mice.Results(1)Compared with that in the CTR group,food intake and body weight of mice in the DON group were significantly decreased in the third and second-fourth week(P < 0.05),respectively,and no significant difference was observed in the colon length and weight of mice in the DON group(P > 0.05).There were slight infiltration of inflammatory cells and loss of a small amount of goblet cells in colon tissues of the DON group relative to that of the CTR group(P < 0.05).No significant difference was observed in the levels of inflammatory cytokines(TNF-α,IL-1β,IL-6,MCP-1 and IL-10)in colon tissues between the two groups(P > 0.05).(2)The m RNA expressions of the tight junction proteins occludin,ZO-1,claudin-3 and claudin-4 in the colon tissue of the DON group were all decreased(P < 0.05)relative to that of the CTR group.Compared with that of the CTR group,the protein expressions of occludin,ZO-1 and claudin-3 were decreased(P < 0.05),but the protein expression of claudin-4 was increased in the DON group(P < 0.05).(3)OTU PCA of the gut microbiota in the mice of the CTR group and the DON group showed that the distance of samples within the group were closer,and that between the groups were dispersed(P < 0.05).Compared with that in the CTR group,the relative abundance of Barnesiella and Enterorhabdus in the DON group was reduced(P < 0.05),while the relative abundance of Clostridium Xl Va,Clostridium sensu stricto,Pantoea,Parabacteroides,Roseburia,Butyricimonas,Erysipelotrichaceae incertae sedis and Vampirovibrio was increased in the DON group(P < 0.05).There was no difference in the Shannon and Simpson index of α diversity between the two groups(P > 0.05),but both PCo A and Anosim of β diversity showed the significant difference of microbial diversity between the CTR group and the DON group(P < 0.05).(4)Compared with that of the CTR group,the abundance of 206 KOs(genes)of gut microbiota in the mice of the DON group was ascended(P < 0.05),while the abundance of 90 KOs was descended(P < 0.05).Twenty-six differential KEGG metabolic pathways were characterized by Lef Se analysis(Level 3,LDA > 2,P < 0.05).Among them,metabolic pathways related to anti-inflammatory and antioxidant effects(ko00510,ko00592,ko00950,ko00250 and ko00020)were less abundant in the DON group than in the CTR group,and metabolic pathways related to mitochondrial metabolism,apoptosis and the degradation and metabolism of DON(ko00660,ko01040,ko00253,ko01054 and ko00625)were more abundant in the DON group than in the CTR group.(5)Compared with that in the CTR group,5 fecal metabolites of the mice in the DON group were elevated(P < 0.05),namely 2,2-dimethyladipic acid,isohyodeoxycholic acid(b HDCA),hyodeoxycholic acid(HDCA),2-methylhexanoic acid and N-acetylserine;2 fecal metabolites of the mice in the DON group were dropped(P < 0.05),namely taurodeoxycholic acid(TDCA)and xylose.Bile acid(TDCA,b HDCA and HDCA)was the main category of differential metabolites.(6)MDA was remarkably increased while SOD,GPx and CAT were significantly decreased in the colon tissue of the DON group relative to that of the CTR group(P < 0.05).In addition,percentage of TUNEL positive cells,Bax and cleaved caspase 3 were all significantly elevated(P < 0.05),but cleaved caspase 7 was significantly reduced in colon tissues of the DON group relative to that of the CTR group(P < 0.01).(7)Spearman correlation analysis showed that not only there were significant correlations among differential bacterial genera,differential metabolic pathways(Level 3)and differential metabolites(P < 0.05),but also differential bacterial genera/differential metabolites were significantly correlated with oxidative stress and apoptosis in colon tissues(P < 0.05).Conclusion Subacute DON exposure can not only lead to pathological changes in the colon,destroy the integrity of the colonic mechanical barrier,and induce oxidative stress and apoptosis in colon tissues,but also cause changes in the gut microbiota and fecal metabolites.Gut microbiota may be involved in subacute DON exposure-induced colon damage in mice.Whether gut microbiota can play a role in subacute DON exposure-induced colon damage in mice needs to be further explored.Part 2 Effects of FMT from mice after subacute DON exposure on the colon in recipient miceObjective To investigate whether FMT from mice after subacute DON exposure could lead to colon damage in recipient mice.Methods(1)After being treated with antibiotics(Ab)cocktail for 27 days,twenty 6-week-old SPF male C57BL/6j mice were divided into two groups by body weight randomly,and then received FMT from mice in the CTR group(Ab-FCTR group,n = 10)and the DON group(Ab-FDON group,n = 10)severally for 3 days(once a day).After FMT,mice were fed normally for 4 weeks.(2)Mice feces were collected for the sequencing and analysis of 16 S r DNA amplicon before and after antibiotics treatment to observe the effects of antibiotics in depleting gut microbiota.Mice feces of the Ab-FCTR group and Ab-FDON group were collected for the sequencing and analysis of 16 S r DNA amplicon and metagenome as well as the detection and analysis of targeted metabolomics after FMT(normal feeding for 4 weeks),in order to observe the colonization of gut microbiota from donor mice in recipient mice and changes in gut microbiota and fecal metabolites in recipient mice.(3)H&E,RT-q PCR,WB,IHC and cytometric bead array were used to observe or detect the related conditions of colon histopathology,colon physical barrier(occludin,ZO-1,claudin-3 and claudin-4),inflammatory cytokines(TNF-α,IL-1β,IL-6,MCP-1 and IL-10),oxidative stress(MDA,SOD,GPx and CAT)and apoptosis(percentage of TUNEL positive cells,Bax,cleaved caspase 3 and cleaved caspase 7)in mice.Results(1)After antibiotics treatment,the number of OTU was reduced by 82.3%,and Shannon and Simpson index of α diversity were remarkably decreased(P < 0.001).Both PCo A and Anosim analysis of β diversity showed that there was a statistically significant difference in the species diversity of gut microbiota in mice before and after antibiotic treatment(P < 0.01).(2)After FMT,the top 10 dominant phyla of gut microbiota in mice of the recipient group(Ab-FCTR group and Ab-FDON group)were the same as that of the donor group(CTR group and DON group).Among the top 20 dominant genera in the recipient group,18 genera were consistent with the donor group.The Shannon and Simpson index of α diversity and the PCo A and Anosim analysis of β diversity in the recipient group were similar to that in the donor group.(3)The relative abundance of Parabacteroides,Butyricimonas and Clostridium XVIII was increased(P < 0.05),while the relative abundance of Helicobacter and Anaeroplasma was decreased in the Ab-FDON group(P < 0.05),compared with that in the Ab-FCTR group.Among them,the changes of Parabacteroides and Butyricimonas in recipient group were consistent with that in the donor group.There were 38 increased KO and 59 decreased KO the Ab-FDON group relative to that in the Ab-FCTR group.A differential metabolic pathway ko00270 was revealed by Lef Se analysis(Level 3,LDA > 2,P < 0.05),whose abundance in the Ab-FDON group was higher than that in the AbFCTR group.(4)Compared with that in the Ab-FCTR group,6 fecal metabolites of the mice in the AbFDON group were elevated(P < 0.05),which were norcholic acid(Nor CA),hydroxyphenyllactic acid,3-Oxocholic acid(3-DHCA),HDCA,deoxycholic acid(DCA),and deoxyglycolic acid(GDCA);3 fecal metabolites of the mice in the AbFDON group were dropped(P < 0.05),namely methylmalonic acid,maltotriose and maltose/lactose.Bile acid(DCA,HDCA,GDCA,Nor CA and 3-DHCA)was the main category of differential metabolites,and the change in HDCA between recipient groups was consistent with that between donor groups.(5)Spearman correlation analysis demonstrated that differential bacterial genera were significantly correlated with differential metabolites(P < 0.05),but there was no significant correlation between differential metabolic pathways and differential bacterial genera/differential metabolites(P > 0.05).(6)Food intake,body weight as well as colon length and weight of mice were of no significant changes in the Ab-FDON group relative to that in the Ab-FCTR group(P > 0.05).There were no significant changes in pathology and the expressions of inflammatory cytokines TNF-α,IL-1β,IL-6,MCP-1 and IL-10 of mice colon between Ab-FCTR group and Ab-FDON group(P > 0.05).(7)Compared with that in the Ab-FCTR group,the m RNA expressions of tight junction proteins occludin and claudin-3 were reduced(P < 0.01)and the m RNA expression of ZO-1 was in a decreasing trend(P = 0.054)in the colon tissue of the Ab-FDON group.However,the differences in the m RNA expression of claudin-4 and the protein expressions of occludin,ZO-1,claudin-3 and claudin-4 were of no significance between the two groups(P > 0.05).(8)There were no significant differences in MDA,SOD,GPx and CAT in colon tissues between Ab-FCTR group and Ab-FDON group.Compared with that of the Ab-FDON group,percentage of TUNEL positive cells,Bax and cleaved caspase 3 and cleaved caspase 7 were also not significantly changed in colon tissues of the Ab-FDON group(P > 0.05).Conclusion Although FMT from mice after subacute DON exposure did not fully recapitulate the pathological phenotype and molecular-level changes related to colon damage of donor mice in recipient mice,it reduced the expression of tight junction proteins at the transcriptional level in colon tissues and led to the changes of gut microbiota and its metabolites in recipient mice.Gut microbiota was involved in the subacute DON exposure-induced colon damage in mice.