Effects Of Curcumin On P-glycoprotein And CYP450s In The Small Intestine Of AFB1-fed AA Broiler Chickens

Aflatoxin is a highly toxic secondary product of mold,the content and activity of AflatoxinB1(AFB1)in moldy feeds is the highest.Oral intake of feed containing AFB1 can cause acute,sub-chronic or chronic poisoning reactions in livestock and poultry,liver injury,even liver cancer.AFB1 is extremely harmful to the health of livestock and the poultry of the livestock industry.Therefore,blocking the entry of AFB1 into the body through the digestive tract is one of the important methods to prevent its toxicity to livestock and poultry.Curcumin is a fat-soluble phenolic pigment extracted from traditional Chinese medicine turmeric.It has anti-inflammatory,anti-oxidative and anti-cancer effects,and has a strong intervention effect on the toxicological effects of AFB1.P-glycoprotein(P-glycoprotein)is an ATP-dependent membrane transporter that is distributed in many tissues of the body.As a drug transporter,it can excrete the intracellular substrate to reduce the intracellular substrate concentration,thereby reducing the efficacy or reducing the toxicity.At present,the metabolism of AFB1 is mainly concentrated in the liver of its target organs,and the intestinal tract is also rich in drug transporters and phase-I metabolizing enzymes.Whether P-glycoprotein and phase-I metabolizing enzymes are involved in AFB1 in the intestinal absorption is rarely reported.Based on the sub-chronic intoxication model of AA broilers,this experiment explored the role of P-glycoprotein and phase-I metabolizing enzymes in the absorption and metabolism of AFB1 and the interaction between P-glycoprotein and phase-I metabolizing enzymes under curcumin intervention.In this experiment,1-day-old AA broilers were used as experimental animals to adapt to feeding for 3 days and randomly divided into 6 groups: 1)Blank control group(basal broiler chicken feed without AFB1 and curcumin);2)Curcumin control group(feed containing 450 mg/kg curcumin);3)Curcumin low-dose group(feed containing 150 mg/kg curcumin + 5 mg/kg AFB1);4)Curcumin middle-dose group(feed containing 300 mg/kg curcumin + 5 mg/ kg AFB1 diet);5)High-dose curcumin group(feed containing 450 mg/kg curcumin + 5 mg/kg AFB1);6)AFB1 poisoning model group(fed with 5 mg/kg AFB1 feed).After 28 days of feeding,the experimental animals were sacrificed,and the small intestine was rapidly isolated.After careful removal of the pancreas,mesentery,and fat,visual observation,pathological examination,and antioxidant(SOD,MDA)assays were performed.The alleviation effect of curcumin against AFB1-induced injury was studied.The gene expression of cytochrome P450 enzymes(CYP3A4,2A6,1A1,1A2)in duodenum,jejunum and ileum of broiler chickens was detected by Real-time quantitative PCR.The effect of curcumin on the absorption and metabolism of AFB1 in broiler intestine was analyzed and to enrich the research data of enteric drug transporters and CYP450 enzymes,to explore the first pass effect of AFB1 in the intestine,and to provide corresponding scientific experimental basis for the metabolic mechanism of AFB1.The results were as follows:Combined with histopathology and antioxidant test,the results showed that,on the 28 th day of this experiment,the addition of 5 mg/kg AFB1 to the diet can cause duodenal villi crushing and congestion in broilers,leading to a decrease in growth performance;significantly increased MDA content in the duodenum.(P<0.01),SOD activity was significantly reduced(P<0.01),antioxidant capacity was decreased,and intestinal injury was caused.By adding different doses of curcumin,the degree of duodenal injury decreased in a dose-dependent manner.The MDA content in the high-dose curcumin group was significantly decreased(P<0.01),and the SOD activity was significantly increased(P<0.01).On the 35 th day of the experiment,the duodenal villi congested in AFB1 model group and it had significantly increased MDA content(P<0.01)and SOD activity(P<0.05).With the different doses of curcumin added in groups,intestinal damage reducedThe results of P-glycoprotein gene expression and ATPase activity showed that on the 28 th day,P-glycoprotein mRNA expression and related ATPase activity in the small intestine of AFB1 model group were significantly reduced(P<0.01),indicating that AFB1 can inhibit the activity of P-glycoprotein in small intestine;longitudinal comparison revealed that the expression of P-gp mRNA in the duodenum and related ATPase activity was lower than that in the jejunum and ileum,indicating that the effect of AFB1 on P-gp is mainly in the duodenum.The curcumin additive showed a dose-dependent increase in P-gp mRNA expression.High-dose curcumin could cause AFB1 to excrete out of cells and reduce body injury by promoting the expression of P-gp.Longitudinal comparison found that the result in the curcumin added group in the duodenum were higher than those in the jejunum and ileum.On the 35 th day of the experiment,there was no significant difference in P-gp mRNA expression and related ATPase activity between the groups.The results of cytochrome P450 gene expression test showed that at the 28 th day,the expression levels of CYP1A1,CYP1A2,CYP2A6,and CYP3A4 mRNA in the AFB1 model group were significantly higher than those in the control group(P<0.05).There are four cytochrome enzymes in the tract,which can activate AFB1 biologically to AFBO,thereby producing cytotoxicity;vertical comparison found that the expression level of cytochrome isoenzyme in the duodenum was higher than that in the jejunum and ileum,The results showed that curcumin could inhibit the biological activation of AFB1 by inhibiting the expression of cytochrome enzyme,and alleviate its damage to the intestinal tract.On the 35 th day of the experiment,there was no significant difference in mRNA expression levels of the four P450 enzymes between the groups.The results of this experiment showed that adding 5 mg/kg of AFB1 to the diet can produce toxicity leading to intestinal damage in broilers by inhibiting the expression of P-gp and increasing the expression of CYP1A1,CYP1A2,CYP2A6 and CYP3A4;curcumin can decrease the absorption of AFB1 by increasing the expression of P-gp in the duodenum;curcumin can inhibit the CYP450 enzymes(CYP1A1,CYP1A2,CYP2A6 and CYP3A4)to reduce the activation of AFB1 in the small intestine.