Study On The Mechanism Of Diet-induced Colonic Injury Based On The Synergistic Toxicity Of Enterotoxins

Inflammatory bowel disease（IBD）is a multifactor-induced refractory gastrointestinal disease.Due to complicated pathological mechanism,it is affected by many factors.Therefore,the pathological mechanisms and treatments of IBD are still not clear.At present,the pathogenic mechanism of inflammatory bowel disease mainly covers environmental,genetic,dietary and immune factors.Therapeutic programs are mostly based on anti-inflammatory,anti-bacterial,and immune-improving treatments.It is difficult to obtain satisfactory treatment results.Therefore,the research on pathogenic factors and inducing mechanism of IBD has become a hot research issue.Related researches can provide experimental reference for the prevention of IBD and the development of new drugs.With the development of economic society and improvement of living standards,the incidence of IBD in developing countries has increased,which suggest that dietary changes may have a certain impact on IBD incidence.The influence of different dietary cultures on the body is profound.It is often said that“indifferent from the mouth”means that poor dietary habits can lead to the development of diseases.Moreover,the processes of digestion,absorption,and excretion of food are directly related to the intestinal tract.How does these changes in dietary styles affect the health of the intestines?This issue deserves further study.In the previous study,we found that high-fat diet can induce obesity and liver damage in animals,but the colonic injury is not obvious.And high dosages of alcohol can induce inflammatory infiltration of the colon with mild colon injury.Moreover,high fat diet combined with low dosage of alcohol can induce dysfunction of gut microbiota,severe colonic inflammation and colon damage.These results suggested that under the mixed diets may exacerbate the colonic injury but the underlying mechanism needs further study.In recent years,study on the effects of gut microbiota and toxic metabolites on the body has received extensive attentions.Toxic metabolites,most of which can be excreted and inactive by the body’s immune function,but in the case of severe dysbacteriosis induced by unhealthy dietary habbits,it may lead to elevated such as lipopolysaccharide（LPS）and steroidal toxoids.The dysfunction of the intestinal flora,accumulation of endogenous toxins and LPS,continues to stimulate the immune system and epithelial cells of the colon,and eventually induces inflammation.Under long-term chronic inflammation,the body’s immune system may collapse.Then may lead to colonic inflammation and tissue damage.But its related mechanism is still not clear ObjectiveThe main purpose of this study is to compare the characteristics of different dietary patterns on colonic injury and explore the underlying mechanism of diet-induced colon injury.Methods1.Study on the effect and mechanism of three different diets on colonic injuryThree animal experiments were set normal group and experimental group respectively.The experimental groups with 3 different treatments,1:high-fat diet for24 weeks;2:Alcohol（4000 mg/kg）administration for 8 weeks;Alcohol（2000 mg/kg）+high-fat diet for 8 weeks.Then,detection indicators mainly include:（1）animal weight and dietary amount;（2）serum biochemistry:determination of blood lipids related to TC,TG,AST,ALT and DAO;（3）serum and colon inflammatory cytokines:TNF-α,IL-1β;（4）determination of skatole,indole and LPS in serum and colon;（5）pathological examination of the colon（HE）;（6）gut microbiota（16S rDNA,V4）;（7）the protein expression of TLR4,MyD88,IκBα,p50,p65,AhR,ARNT in colon;（8）the protein of p65 and p50 in colon by immunohistochemistry.2.Explore the toxic effects and synergistic effects of different intestinal toxins on NCM460 cellsAlcohol and oleic acid were used to simulate a model of alcohol+high fat diet stimulation in animal experiments in vitro.To investigate the effect of different concentrations of single toxin on normal or alcohol+oleic acid-induced NCM460 cells and determined cell viability,IC₅₀,cell toxicity,pro-apoptotic effects and further explore its molecular mechanisms.Detection indicators:（1）model screening;（2）effects of different toxins on normal and modeled NCM460 cells;（3）toxicity comparison;（4）cell apoptosis;（5）protein expression of TLR4,MyD88,IκBα,p50,p65,AhR,ARNT;（6）the protein expression of p65 and p50 detected by immunofluorescence.3.The effects of different mixed enterotoxins on colon injury were verified in normal animalsForty C57 mice were randomly divided into control group（NC）,mixed high-dose toxins group（MH）,low-dose toxins group（ML）and LPS control group（LPS）respectively.Each group of toxins were injected intraperitoneally for 16 weeks.Indicators:（1）body weight;（2）serum biochemistry:TC,TG,AST,ALT and DAO;（3）serum inflammatory cytokines:TNF-α,IL-1β,IL-6 and IL-10;（4）HMGB1 in serum and colon;（5）pathological examination of the colon（HE）;（6）macrophage type was detected by immunofluorescence and determine the ratio of M1/M2.Results1.Study on pharmacodynamics and mechanism of three different diet patterns on colon injuryHigh-fat diet can increase body weight,serum LPS,and colon IL-1βsignificantly（P<0.05）.Body weight and TG in serum decreased significantly（P<0.01）after 8weeks of alcohol administration.AST,ALT,DAO in serum and TNF-αand IL-1βin colon increased significantly（P<0.01）and activated the NF-κB and AhR-mediated toxic metabolism pathway.Moreover,HFD increased the expression of p65 and p50 in colon.Under the stimulation of a mixed diet of alcohol and high fat diet,the body weight and gastrointestinal barrier stability decreased.At the same time,liver injury indexes,TNF-α,IL-1βconcentration in serum and colon increased significantly（P<0.01）.Besides,NF-κB and AhR-mediated metabolic signaling pathways were activated and increased expression of p65 and p50 in colon.These results suggest that alcohol can induce colon injury but mixed with high fat diet would induce severe colon mucosa and tissue damage.2.To explore the toxic effects and synergistic effects of different intestinal toxins on cells.The toxicity was found to be LPS>skatole>indole.Compared with three different enterotoxins,the toxicity of indole was significantly increased along with time,which suggested that indole may have more pronounced toxic effects.Except for skatole,we found that indole,LPS,and mixed groups increased the proportion of early apoptotic cells significantly.Besides,skatole can increase the late apoptosis of cells significantly.At the same time,skatole and mixed toxins can significantly activate the AhR pathway and increase the expression of p50 in the nucleus.These results showed that different intestinal toxins have synergistic damage stimulatory effects,which may be activated by the co-stimulation of NF-κB and AhR-mediated signaling pathway.3.The effects of different mixed enterotoxins on colon injury were verified in normal animalsThe high-dose toxin（MH）and low-dose toxin（ML）groups increased AST and ALT levels（P<0.01）in serum.This result suggesting that mixed toxins can induce liver injury.Toxins could destroy the stability of the intestine and increase the levels of TNF-α,IL-1β,IL-6,and IL-10 in serum（P<0.01）.The effect of MH was found to be the most toxicity between 4 groups.Compared with LPS and MH groups,the MH can increase inflammation significantly,especially for IL-1βin serum（P<0.01）.Histopathological examination revealed that the MH group had more severe colonic tissue damage and inflammatory infiltration than in the ML and LPS groups.The results of HMGB1 concentration in serum and colon showed the same trend as inflammatory factors,indicating that the body’s inflammation level was in an overall activated state.On this basis,the expression of M1 increased more than M2 type,which indicated that the imbalance of inflammation was caused by M1 type firstly.Besides,there is a clear dose-effect relationship between three toxin groups.ConclusionThe mixed diet induces disturbance of the gut microbiota,destroys the stability of the gastrointestinal tract,increased the accumulation of intestinal toxins in the colon,induced damage to the colonic epithelial cells,apoptosis of colonic cells and activated NF-κB and AhR signaling pathway in colon.Besides,these signaling eventually causes an imbalance in the body’s inflammation then,inflammatory infiltration and tissue damage in the colon.More importantly,the discovery of synergistic toxic effects between different toxins will help uncover the relationship between enterotoxins and IBD.This study provides new ideas and directions for the follow-up of more complex toxin researches in colon diseases.