Study On The Effect And Mechanism Of 2,6-Dichloro-1,4-Benzoquinone On Intestinal Flora And Inflammatory Response In Mice

Objective:Disinfection byproducts（disinfection byproduct,DBPs）have been produced in the process of the disinfection of drinking water,the natural organic matter combines with disinfectant causing the generation of DBPs which cause toxic effects toward human body via drinking water.Recently,an emerging DBP,2,6-dichloro-1,4-benzoquinone（2,6-dichloro-1,4-benzoquinone,DCBQ）has been detected in drinking water and swimming pool,it also has higher concentration and higher toxicity than other regular DBPs.However,there are still limited researches related to the in vivo toxicity of DCBQ.Therefore,it is of great significance to study the in vivo toxicity of DCBQ and its poterntial mechanisim.To investigate the intestinal injury and effects on immune function of DCBQ on mice,detecting the alterations to immunity,gut microbiota,metabolome and the expression of the m RNA related to immune response,revael the toxic effects and its mechanism of DCBQ exposure,and provide references for studing the toxic effects of other DBPs.Methods:Male and female ICR mice（18～22 g）were purchased from the Laboratory Animal Center of Jilin University,China.After acclimatization,40 mice were randomly assigned into four groups（n=10,half male and half female）.Mice were treated daily with 0（sesame oil;vehicle control）,31.25,62.5 and 125 mg/kg body weight of DCBQ solution by intragastric administration for 4 weeks.The doses selected for DCBQ treatment were based on the median lethal dose(LD₅₀)of DCBQ on mice with acute toxicity test.The body weights were measured every three days and the conditions were recorded everyday.After treatment,all mice were anaesthetized and sacrificed to collect the tissues（small intestine,liver,kidney,heart,spleen,lung,testicle,epididymis,uterus,ovary and brain）,blood and feces.The blood samples were then centrifuged for the serum.All the tissue samples were weighted and the organ indexes were calculated.The intestinal histological changes were observed by hematoxylin and eosin staining.The levels of IL-12,IL-4,IL-6,IFN-γ,TGF-β,ROR-γt in serum were determined by using enzyme-linked immunoabsorbent assay method.The evaluation of T cells subsets was performed by flow cytometry.The expression of macrophages,M1 macrophage and M2 macrophage were measured by immunohistochemical staining.The expression of m RNA of NF-κB,IL-1β,IL-17,IL-10,IL-13,CCL11,IL-12,CCR5 were detected by quantitative real-time polymerase chain reaction.The changes of gut microbiome were detected by 16S r RNA gene amplification and sequencing technology.The changes of gut metabolome were detected by high performance liquid chromatography-mass spectrometry（LC-MS）.Results:1.The generate toxicity of DCBQ in mice:compared with the control group,the body weight in 125 mg/kg DCBQ group on exposure day 9,18,21 and 24 has significantly decreased（P<0.01）,the relative organ weight of brain has significantly increased in 62.50 mg/kg DCBQ group,and relative organ weight of liver has decreased in 62.50 mg/kg and 125 mg/kg DCBQ group（P<0.01）.2.Effects of DCBQ on intestinal histological changes in mice:compared with the control group,lots of inflammatory cells such as lymphocytes and eosinophils were observed to infiltrate in the intestines in all DCBQ expoursure groups,intestinal mucosa was destroyed,villous epithelial cells were not neatly arranged in 125 mg/kg DCBQ group.3.Effects of DCBQ on inmmune response in mice（1）Compared with the control group,the serum levels of IL-4 and IL-6 were significantly increased in 62.5mg/kg and 125 mg/kg DCBQ groups respectively（P<0.01）,the serum levels of IFN-γand ROR-γt were significantly decreased in all DCBQ groups（P<0.01）.（2）Compared with the control group,Th1 cells and Th17 cells were significantly decreased in all DCBQ groups（P<0.01）,while Th2 cells at 31.25 and 62.5mg/kg and T regulation（Treg）cells at all the three doses DCBQ groups were significantly increased（P<0.01）,and the Th1/Th2 ratio and Th17/Treg ratio were significantly decreased in all DCBQ treatment groups（P<0.01）.（3）Compared with the control group,the number of total macrophage,M1macrophage and M2 macrophage with the immunohistochemically staining have increased at both 62.5 and 125 mg/kg groups（P<0.01）,the ratio of M2/M1 was significantly improved at 62.5 mg/kg DCBQ group（P<0.01）.（4）Compared with the control group,the m RNA levels of NF-κB,IL-1β,IL-17,IL-10,CCL11 were significantly increased in the intestine tissue at both 62.5 and125 mg/kg groups（P<0.01）,the m RNA levels of IL-13 were significantly increased in all DCBQ treatment groups（P<0.01）,whereas the m RNA levels of IL-12 and CCR5were slightly decreased in DCBQ exposure groups（P<0.01）.4.Effects of DCBQ on the changes of the gut microbiota in mice（1）Compared with the control group,the gut microbiome’s composition and abundance were significantly changed in DCBQ exposure group（P<0.05）.（2）Compared with the control group,the abundance of Proteobacteria,Alloprevotella and Prevotellaceae showed significant differences in DCBQ exposure group（P<0.05）.（3）Compared with the control group,the abundance of Proteobacteria,Alloprevotella,Prevotellaceae and other differential bacteriaes were correlated with immune factors（P<0.01）.5.Effects of DCBQ on the metabolomic alterations in mice（1）Compared with the control group,42 differential metabolites were found in DCBQ exposure group（P<0.05）,the analysis of the metabolic pathways of the differential metabolites suggested that the mice underwent drastic changes in 28metabolic pathways with DCBQ treatmen,including unsaturated fatty acid synthesis,purine metabolism,amino acid synthesis and metabolism,lipid metabolism（P<0.05）.（2）Compared with the control group,the abundance of Proteobacteria,Alloprevotella,Prevotellaceae and other differential bacteriaes were correlated with metabolomic alterations at 62.5 mg/kg DCBQ exposure group（P<0.01）.Conclusions:1.DCBQ exposure can reduce the body weight and change the organ indexes of mice.2.DCBQ exposure can cause deterioration and intestinal inflammation.These suggest that DCBQ can induce intestinal immune response,leading to intestine injury.3.DCBQ exposure can affect immune function and disturbe the balance between Th1-induced immunity and Th2-induced immunity.4.DCBQ exposure can alter the composition of gut microbiome in mice,and it showed correlations with the altered immunological indicators.These suggerst that DCBQ can disturbe the immune response by altering the composition of gut microbiome.5.DCBQ exposure can change the fecal metabolomics profile in mice,and it showed correlations with the altered gut microbiome.These suggerst that DCBQ can disturbe the immune response by changing the fecal metabolites.