Effect Of Zearalenone On Aflatoxin B1 Intestinal Injury In Pregnant Rats

Aflatoxin B₁（AFB₁）and zearalenone（ZEN）are two mycotoxins widely existing in feed raw materials and grain,oil and food.The mixed pollution of AFB₁and ZEN often occurs at home and abroad,which seriously threatens the health and safety of human and animals.As an important place for the digestion and absorption of nutrients,maintaining intestinal safety is very important for the health of mothers and young children during pregnancy.ZEN has estrogenic activity,and in special physiological stages such as mammalian pregnancy,it may increase the sensitivity of animals to toxins due to interfering with the body’s hormone metabolism and destroying tissue permeability.AFB₁ is harmful in many ways and also affects the digestive tract of animals.Whether ZEN can enhance the damage of AFB₁ to the intestinal tract of animals still needs to be studied.In this study,the contamination of AFB₁ and ZEN in 420 feed ingredients and feed products in the market was detected and counted,and the 48 pregnant rats were used as experimental animals and in the mean while the experimental animals were involved with control group,2 AFB₁ single group,2 AFB₁+1 ZEN group,in addition 2 AFB₁+10ZEN group.They were fed with basal diet,2 mg/kg AFB₁,2 mg/kg AFB₁+1 mg/kg ZEN,and then 2 mg/kg AFB₁+10 mg/kg ZEN,respectively.Sampling was carried out on the 14th day after the birth of the rat dams,and the morphological structure,mucosal permeability,digestive enzyme activity,antioxidant capacity,immune ability and changes of microbial flora of the intestinal tissue of the rat dams and offspring were analyzed and measured.ZEN affects the effect of AFB₁ on intestinal injury in rats.The main test results are as follows:1 Survey results of co-contamination of AFB₁ and ZEN in feed420 samples of feed and feed raw materials in the market in recent years were tested and counted.Among them,the co-contamination rate of AFB₁ and ZEN in 202feed ingredients was above 75%,and the co-contamination rate of AFB₁ and ZEN in218 compound feeds was above 50%.It indicates that there is common contamination of AFB₁ and ZEN in feed ingredients and feed products,which may cause common hazards.2 Effects on intestinal digestion and absorption function and offspring growth in ratsThe AFB₁ exposure caused that the average body weight in the pre-lactation and mid-term（0-14 d）was a significant decrease change（P<0.05）of the offspring.AFB₁exposure caused different degrees of damage to the small intestinal villi of the rat dams and offspring.In the 2 AFB₁+1 ZEN group,the crypt depths of the duodenum and jejunum of the rat dams were significantly increased（P<0.05）,and in the meanwhile the villi heights of the duodenum and jejunum of the offspring rats were showed a significantly higher than those of the AFB₁ single treatment group（P<0.05）.The activities of the three digestive enzymes in the jejunum of the rat dams and offspring decreased to varying degrees after AFB₁ exposure.Compared with the AFB₁ single treatment group,the activities ofα-amylase,trypsin and lipase in the 2 AFB₁+10 ZEN group of rat dams were significantly decreased（P<0.05）.In addition,the activities of trypsin and lipase in the 2 AFB₁+1 ZEN group also decreased significantly（P<0.05）.It shows that AFB₁ can cause pathological changes in the intestinal villi of dams and offspring,reduce the activity of intestinal digestive enzymes,cause abnormal intestinal digestion and absorption function,and cause poor growth of offspring.High-dose ZEN can aggravate the effect of AFB₁ on dams and offspring about effects of intestinal injury in rats.3 Effect on intestinal mechanical barrier function in ratsWhen it was matched to the AFB₁single treated group,the two treatment groups additionally supplemented with ZEN significantly increased the content levels of the three permeability indexes（P<0.05）.In the meanwhile,the m RNA expression of jejunum tight junction protein after exposure to AFB₁ which appeared a significant decline of rat dams（P<0.05）,and which had a downward trend of rat offspring.Moreover,compared with the AFB₁ alone treatment group,the expression of three tight junction proteins was down-regulated in the 2 mg/kg AFB₁+1 mg/kg ZEN group of rat dams.However,for the 2 mg/kg AFB₁+10 mg/kg ZEN group,the relative expression of the two protein genes ZO-1 and Occludin was significantly decreased（P<0.05）.It shows that AFB₁ can change the intestinal tissue structure of rat dams and offspring,increase the permeability of intestinal mucosa,and in the meanwhile the expression of three important the genes of tight junction protein in the jejunum were also down-regulated change,which leads to further impairment of intestinal barrier function.In addition,High-dose ZEN can also aggravate the effects of AFB₁ on the toxicity of the intestinal mechanical barrier in dams and offspring.4 Effect on intestinal stress injury in ratsAfter exposure to AFB₁,the jejunal MDA level of the rat dams was significantly increased（P<0.05）.In the meanwhile,the two treatment groups in which the rat dams were treated with ZEN significantly increased the jejunal MDA activity（P<0.05）and decreased the jejunal GSH-PX content（P<0.05）when they were compared with the AFB₁ single treatment group.Moreover,the activities of the two indicators T-AOC and SOD tended to decrease.Among them,2 AFB₁+10 ZEN group also significantly increased the MDA content compared with the 2 AFB₁+1 ZEN group（P<0.05）.The expression of inflammatory factors in the jejunum of rat dams and offspring increased after exposure to AFB₁.The m RNA expression of the two indicators of IFN-γand IL-6in the jejunum of the rat dams 2 AFB₁+10 ZEN group was significantly up-regulated（P<0.05）.The expression of apoptotic genes in the jejunum of rat dams and offspring increased after toxin exposure,while the expression of apoptotic genes in offspring rats showed a downward trend.Matched to the AFB₁single treated group,additional feeding of ZEN to dams could up-regulate the expression of apoptotic genes,and the 2AFB₁+10 ZEN group was more significantly up-regulated than the 2 AFB₁+1 ZEN group.This indicates that AFB₁ can cause oxidative stress injury in the intestinal tract of rats,and induce inflammation and apoptosis in the jejunal epithelial tissue of rat dams and offspring.High-dose ZEN can aggravate the effect of AFB₁ on the toxicity of intestinal stress injury in rat dams and offspring.5 Effect on intestinal biological barrier function in ratsPCoA principal axis analysis showed that there were significant differences in the species composition of intestinal microflora in rat dams（P<0.05）.The flora level analysis showed that the first two phyla with the highest relative abundance in the four treatment groups were Bacteroidota and Firmicutes,while Proteobacteria and Actinobacteriota,which accounted for less,had significant differences between the groups（P<0.05）.The genus-level analysis also found that the relative abundances of harmful bacteria g＿norank＿f＿Muribaculaceae and also includes Romboutsia increased significantly（P<0.05）after AFB₁ exposure,and in the meanwhile the relative abundance of beneficial bacteria Bacteroides showed a significant fall（P<0.05）.LEf Se species difference discriminant analysis showed that f＿Bacteroidaceae and g＿Bacteroides were significantly enriched in the control group,f＿Muribaculaceae and g＿norank＿f＿Muribaculaceae in the 2 AFB₁+1 ZEN group,and enriched f＿Peptostreptococcaceae and g＿Romboutsia in the 2 AFB₁+10 ZEN group.This indicated that AFB₁ could significantly change the composition of the rectal microflora in rats,induce the proliferation of intestinal pathogens,and impair the function of the intestinal biological barrier.High-dose ZEN can aggravate the risk of AFB₁ harming the gut microbes of rat dams.To sum up,the following conclusions are obtained in this study:（1）There is co-contamination of AFB₁ and ZEN in feed raw materials and feed products,and the co-contamination rate reaches 75.24%,which may cause the problem of common harm.（2）AFB₁ causes severe damage to the intestinal morphology and structure of rat dams and offspring,reduces the activity of intestinal digestive enzymes,causes abnormal intestinal digestion and absorption function,and causes malnutrition and growth retardation of offspring.High-dose ZEN can aggravate the effect of AFB₁ on intestinal injury in rat dams and offspring.（3）AFB₁ can increase the intestinal mucosal permeability of rat dams and offspring,affect the integrity of the tight junction structure of the jejunum,and cause intestinal oxidative stress and toxicity.High-dose ZEN can increase the intestinal sensitivity of pregnant rats,and aggravate the damage of intestinal barrier.（4）AFB₁ significantly changed the composition of the rectal microflora in dams,increases the relative abundance of pathogenic bacteria in the gut microbiota,and disrupts intestinal microbial homeostasis,High-dose ZEN can aggravate AFB₁ to increase the abundance of potential pathogenic bacteria in the gut in degree of risk.