| Mycotoxins are toxic secondary metabolites produced by filamentous fungi.Among them,aflatoxin B1(AFB1),zearalenone(ZEA)and deoxynivalenol(DON)are the three most common mycotoxins with the highest contamination rate in agricultural products and feeds at present,which influence human and animal health.Swine jejunal epithelial cells(IPEC-J2)were used to study the accumulative cytotoxicity of AFB1,ZEA and DON.Compound probiotics,mycotoxin-degradation enzymes and glycyrrhizic acid were used to alleviate cytotoxicity of mycotoxins.The main results were as follows:(1)Response surface design of AFB1,ZEA and DON for studying accumulative cytotoxicity of mycotoxins.AFB1,ZEA and DON were selected as the three factors of Box-Behnke design.The different mycotoxin concentrations of 10,20,30 μg/L for AFB1,150,300,450μg/L for ZEA,500,1000,1500 μg/L for DON were used as the three coding levels of Box-Behnke design.The viability of IPEC-J2 cells influenced by AFB1,ZEA and DON was detected by MTT assay.The results showed that the lowest cell viability(the high-level cytotoxicity)was 32.32%when AFB1,ZEA and DON concentrations were 30,150 and 1500 μg/L,while the highest cell viability(the low-level cytotoxicity)was 53.01%when AFB1,ZEA and DON concentrations were 10,150 and 600 μg/L.(2)Synchronous degradation of AFB1,ZEA and DON by compound probiotics and mycotoxin-degradation enzyme.Bacillus subtilis K4,Saccharomyces cerevisae and Clostridium butyricum were selected and combined with mycotoxin-degradation enzymes in this study.The optimal compound probiotics was obtained by Latin square design.The result showed that the synchronous degradation rates of AFB1,ZEA and DON were 40.55%,56.05%,47.22%in the high-cytotoxicity group(P<0.05),and 43.05%,38.26%,80.49%in the low cytotoxicity group(P<0.05),when visible counts of Bacillus subtilis K4,Saccharomyces cerevisiae and Clostridium butyricum were 1×105 CFU/mL,respectively.The effect of combination between compound probiotics and mycotoxin-degradation enzymes on AFB1,ZEA and DON degradation rates showed that total mycotoxin-degradation rates were 190.08%,175.10%and 184.44%by single compound probiotics,single mycotoxin-degradation enzymes,and both combination at a ratio of 30:1,which was higher than other groups(P<0.05)under high-level cytotoxicity condition;total mycotoxin-degradation rate was 219.23%(P<0.05)by single mycotoxin-degradation enzymes,followed by 178.07%,181.84%and 170.33%by single compound probiotics,and both combination at a ratio of 30:1 and 3:2,which was higher than other groups(P<0.05)under low-level cytotoxicity condition(3)Relieving mycotoxin-induced IPEC-J2 cell injury by glycyrrhizic acid,compound probiotics and mycotoxin-degradation enzymes1)Effects of glycyrrhizic acid(GA)co-culturing with AFB1,ZEA and DON on the viability of IPEC-J2 cells.The results showed that the cell viability was increased by 49.40%,72.98%,78.45%in the high-level cytotoxicity group,and 30.76%,44.22%,60.09%in the low-level cytotoxicity group when GA contents were 200,400,600 μg/mL,compared with the mycotoxin group(P<0.05).2)Effects of compound probiotics,mycotoxin-degradation enzyme,glycyrrhizic acid co-culturing with AFB1,ZEA and DON on the viability of IPEC-J2 cells.The results showed that compared with the mycotoxin group,adding probiotics,glycyrrhizic acid and their combination at the same time,the cell viability was increased by 31.48%,69.42%,66.52%(P<0.05)in the high-level cytotoxicity group,and 53.10%,106.82%,100.21%(P<0.05)in the low-level cytotoxicity group.In adition,mycotoxin-degradation enzyme significantly reduced cell viability,so it was ignored in the subsequent experiments.3)Effects of mycotoxin-biodegradation products on the viability of IPEC-J2 cells.The results showed that compared with the control group,mycotoxin-biodegradation products could make cell viability reach 82%,indicating that the mycotoxin-biodegradation products had less cytotoxicity.4)Effects of compound probiotics and glycyrrhizic acid on apoptosis of IPEC-J2 cells induced by AFB1,ZEA and DON.The experiment was divided into control group(CON),low-level cytotoxicity group(ADZ),compound probiotics+glycyrrhizic acid group(CGA),and ADZ+CGA group.Cell apoptosis was detected by flow cytometry after co-culture for 24 h.The results showed that compared with ADZ group,the total apoptosis rate in ADZ+CGA group was decreased from 6.12%to 4.10%(P<0.05),the number of dead cells was decreased from 3.61%to 1.59%(P<0.05),the number of living cells was increased from 90.33%to 94.14%(P<0.05).5)The effect of CGA on the mRNA abundance and protein expression of related genes in ADZ-induced IPEC-J2 cells.The mRNA abundances of apoptotic genes suchas Bax,Bcl-2,Casepase-3,inflammatory related factors such as IL-8,TNF-α,NF-κB,tight junction proteins such as Occludin,Claudin-1,ZO-1,and nutrient transporter proteins such as ASCT2,GLUT2,PepT1,SGLT1 were determined by qPCR.The relative protein expression levels of TNF-α,COX-2,Claudin-1,ZO-1,SLC1A5 and SLC15A1 were detected by Western Blotting.The experimental design was the same as above.The results showed that compared with ADZ group,the mRNA abundances of caspase-3,TNF-α,NF-κB and ASCT2 in ADZ+CGA group were down-regulated(P<0.05),and mRNA abundances of Bcl-2,Bcl-2/Bax,ZO-1,Claudin-1 and PepT1 were up-regulated(P<0.05).Western Boltting results showed that ADZ+CGA could decrease TNF-α and Claudin-1 protein expressions(P<0.05),and increase COX-2 and SLC1A5 protein expressions(P<0.05),compared with ADZ group.SLC15A1 protein expression had insignificant change(P>0.05).In conclusion,the combination of compound probiotics with glycyrrhizic acid can not only degrade AFB1,ZEA and DON,but also effectively alleviate mycotoxin-induced cell damages. |
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