| As the toxic mechanisms of deoxynivalenol(DON),and the effectiveness and safety of montmorillonite(MMT)were not very clear still now,four experiments were conducted to determine the toxic effects of DON on growth performance,gut health,and the protective effects of MMT in piglets.Furthermore,DON,s effects on epithelial barrier integrity,and the underlying mechanisms were also investigated here.Another in vivo experiment was performed to evaluate the use of MMT as a protective agent in piglets.Exp.1 Effects of DON on growth performance and gut health and protective effects of MMT in pigletsA 2×2 factorial experiment was carried out.A total of twenty-four 4-week-old DLY piglets(initial weight:7.5 kg)were randomly allotted into four treatments(6 pens of one piglet per pen):control group(corn-soybean basal diet,C),MMT group(the rice bran and hull in basal diet was replaced by 0.5%MMT,MMT),DON group(pure DON with a final concentration was 2 mg/kg,DON),DON and MMT group(diet of DON group + 0.5%MMT,DON + MMT).The trial lasted for 28 days.The toxic effects of DON on growth performance,nutrients digestibilities,serum biochemistry,jejunum morphology and pathological changes,gut health-related genes expression,and the protective effects of MMT were investigated in this experiment.The results were as follows:1.DON significantly decreased piglets' ADG and ADFI in each stages(P<0.05).MMT had no significant effect on growth performance(P>0.05).There was no interaction between DON and MMT(P>0.05).2.DON did not affect nutrients digestibilities of piglets(P>0.05).MMT significantly increased piglets' digestibilities of organic matters,crude protein and energy(P ? 0.05).There was no interaction between DON and MMT(P>0.05).3.DON significantly decreased serum AKP activity and increased concentration of IgA of piglets(P ? 0.05).MMT had no significant effect on serum biochemistry and immune globulins concentrations(P>0.05).There was no interaction between DON and MMT(P>0.05).4.DON significantly decreased jejunum villus height and villus height/crypt depth ratio of piglets(P? 0.05),lead to villus breaking,fusion,lamina propria edema,and resulted in a reduction of goblet cell as well.Compared with DON group,jejunum villus height and villus height/crypt depth ratio were significantly increased for piglets in DON +MMT group(P ? 0.05).Montmorillonite alleviated the mucosa injury caused by DON.5.DON significantly downregulated mRNA expression of jejunum CLDN3,transfer TNF-a,GLUT2,ileum CLDN4,ZO1 and colon CLDN1(P ? 0.05),upregulated ileum SGLT-1 gene expression(P ? 0.05).The mRNA levels of jejunum MUC2 and ileum PepT-1 had a tendency to decrease(0.05<P ? 0.1).Compared with DON group,0.5%MMT significantly decreased SGLT-1 mRNA expression in ileum(P ? 0.05).These results indicated that DON decreased piglets' growth performance,affected serum biochemistry and IgA concentration,changed jejunum morphology,impaired jejunum mucosa and affected gut health-related genes expression in piglets.MMT improved nutrient digestibility and alleviated the mucosa injury by DON.Exp.2 Effects of DON on cell viability and epithelial barrier integrity of IPEC-J2 cellsIPEC-J2 cell was used as an in vitro model to study the effects of DON on cell viability and epithelial barrier integrity.Firstly,a 3×3 factorial design was conducted to determine the cell viability of IPEC-J2 cells.Differentiated cells were incubated with 1000,2000,4000 ng/ml DON for 24,48 and 72 h,respectively.The results showed that both DON concentration and incubate time significantly affected cell viability(P ? 0.05).Concentration and time had a significant interaction(P ? 0.05).Secondly,IPEC-J2 cells were incubated with 0,1000,and 4000 ng/ml DON for 1,24 and 48h to evaluate the effects of DON on tight junction proteins(TJs)CLDN1,CLDN3,CLDN4,OCLN and ZO1 mRNA expression and protein levels.The results showed that both DON concentration and incubate time significantly affected CLDN1,CLDN4,OCLN and ZO1 mRNA expression(P?0.05),DON concentration and incubate time had a significant interaction(P<0.05).DON concentration also significantly affected CLDN3 mRNA expression(P ? 0.05).The protein levels of CLDN3 and CLDN1,CLDN3,CLDN4,OCLN were significantly decreased when IPEC-J2 cells were treated with 1000 and 4000 ng/ml DON for 24h(P?0.05).Treatment of IPEC-J2 cells with both concentrations of DON for 48 h significantly decreased CLDN1,CLDN3,CLDN4 and OCLN protein levels(P ? 0.05).There was no significant effect of DON on ZO1 protein expression(P>0.05).At last,IPEC-J2 cells were incubated with 1000,2000,4000 ng/ml DON for 1,24,and 48h to evaluate the effect of DON on epithelial barrier integrity through the measurement of transepithelial electric resistance(TEER).The result showed that the TEER of IPEC-J2 cells was significantly decreased after DON treatments(P ? 0.05).These results revealed that DON impaired the integrity of intestinal epithelial barrier through affecting the expression of TJs mRNA and protein.Exp.3 Pathways involved in DON's effects on intestinal epithelial tight junction proteins levelsIPEC-J2 cell was used as an in vitro model to explore the pathways involved in DON's effects on intestinal epithelial tight junction proteins levels.Firstly,IPEC-J2 cells were treated with a final concentration of 20 ?g/ml protein synthesis inhibitor CHX and 4000 ng/ml DON for 24 h,respectively,to compared the effects of CHX and DON on protein levels of CLDN1,CLDN35 CLDN4 and OCLN.The results showed that compared to control,both CHX and DON significantly decreased protein levels of CLDN1,CLDN3,CLDN4 and OCLN(P ? 0.05).The protein expressions of these four TJs were not different between CHX group and DON group(P>0.05).In order to investigate if PKC pathway was involved in the inhibition of DON's effect on TJs protein expression,differentiated IPEC-J2 cells were pre-treated with 20?mol/1 pan-PKC inhibitor bisindolylmaleimide I,hydrochloride(Bis)for 2 h,then incubated with 4000 ng/ml DON for 24 h.TJs protein levels were measured here.The results showed that,compared to control,4000 ng/ml DON significantly decreased the protein levels of CLDN1,CLDN3,CLDN4 and OCLN(P ? 0.05).There were no significant difference of CLDNs protein levels between Bis+DON group and control(P>0.05),but the expression of OCLN protein was downregulated compared to control(P ?0.05).Proteasome inhibitor MG-132 was used to investigate if protein degradation process participated in the toxicity of DON on TJs protein levels.IPEC-J2 cells were pre-treated with 10 ?mol/1 MG-132 for 2 h,then incubated with 4000 ng/ml DON for 24 h.TJs protein levels were measured here.The results showed that,compared to control,4000 ng/ml DON significantly decreased the protein levels of CLDN1,CLDN3,CLDN4 and OCLN(P?0.05),which was in consistence with the previous results.There were no significant differences of CLDN1 and CLDN3 protein expression between MG-132 +DON treatment and control(P>0.05).Compared to control,the protein level of CLDN4 in MG-132 + DON group was significant lower(P ? 0.05),but significantly higher than that in DON group(P ? 0.05).These results revealed that PKC and UPS pathways played roles in the regulation of DON on TJs protein expression through inhibiting protein synthesis and promoting protein degradation.Exp.4 Evaluation of graded levels of MMT as a protective agent in diets for pigletsA total of one hundred and twenty 28-d-old DLY piglets(initial weight:8.0kg)were randomly allotted into dietary treatments with graded supplementing-MMT levels(0,0.5%,1.0%,2.5%and 5.0%)with 6 replicate pens per treatment and 4 pigs per pen.All diets were fed for 28 d.Growth performance,serum biochemistry,serum antioxidant capacity,histomorphology,and mineral contents in serum and bones were measured here.The results showed that,as the addition levels of MMT increased,1.ADG and G:F of piglets changed in a quadratic manner,ADFI decreased in a linear and quadratic manner(P ? 0.05).Compared to control,the ADG,ADFI and G:F of piglets in 1.0%MMT group significantly increased(P? 0.05),5.0%MMT significantly decreased piglets' ADFI(P ? 0.05).2.The relative liver weight changed in a linear and quadratic manner(P ? 0.05),liver injury aggravated.MMT had no significant effect on jejunum morphology and relative organ weight of spleen and kidney(P>0.05).3.The activities of alanine aminotransferase(ALT)and aspartate aminotransferase(AST)increased in a linear and quadratic manner(P ? 0.05).4.The contents of serum Cu and Zn were linearly decreased(P<0.05),Mg concentration reduced in a linear and quadratic manner(P?0.05).Cu,Mg,Mn and Zn contents in the bone from the pigs of 2.5%and 5.0%MMT groups showed a significant reduction compared to control(P ? 0.05).MMT did not affect serum and bone contents of Ca,P,Na and K(P>0.05).5.The content of serum total antioxidant capacity(T-AOC)changed in a linear and quadratic manner,T-AOC concentrations in the piglets fed 2.5%and 5.0%MMT were lower than that in control group(P? 0.05).Serum superoxide dismutase(SOD)activity.changed in a quadratic manner(P ? 0.05),and activity of glutathione peroxidase(GSH-PX)decreased in a linear and quadratic manner(P ? 0.05).The piglets in 1.0%MMT group showed a higher SOD and GSH-PX activities compared with control(P:? 0.05).Compared to control,5.0%MMT significantly increased piglets' serum malondialdehyde(MDA)concentration(P:? 0.05).These results indicated that the growth and health of piglets were not impaired when dietary MMT level was? 1%,but when the additive level of MMT>2.5%,growth performance,liver function,serum and bone mineral concentrations and serum antioxidant capacity of piglets were negatively affected.Therefore,the safe additive level of MMT in diet was no more than 1%in this study.In summary,DON impaired piglet's growth performance,affected intestinal epithelial tight junction proteins expression through PKC and UPS pathways,and destroyed the integrity of epithelial barrier.Mycotoxin adsorbent MMT can partially alleviated jejunum mucosa injury,reflected a certain protective effect against the toxicity induced by DON.Low supplementation of MMT(? 1%)did not impair piglet' s growth and health.Therefore,the safe additive level of MMT in diet was no more than 1%in this study. |
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