Effect Of DNA Methylation Induced By AFB1 On CYP Expression In Broiler Liver And Intervention Of Curcumin

Aflatoxin B1(AFB1)is produced by the metabolism of Aspergillus flavus and Aspergillus parasiticus.It is one of the most toxic,carcinogenic and polluting mycotoxins,and is extremely harmful to human health and food safety.AFB1 is a pre-carcinogen.It is activated in the liver by the phase I metabolic enzyme cytochrome P450(CYP450)enzyme system to produce harmful AFB1-8,9-epoxide(AFB1-8,9-epoxide,AFBO).Biological macromolecules such as lipids,proteins and DNA cause damage,leading to cell apoptosis,necrosis and even carcinogenesis.DNA methylation is the earliest known epigenetic mechanism to be discovered.In the genomic DNA of animal cells,about 4% of cytosine(C)is in the form of 5-methylcytosine(5-m C),which is The most common form of methylation.The results of the study show that the general level of overall DNA hypomethylation and the hypermethylation of DNA in the promoter region of tumor suppressor genes are typical epigenetic changes in the course of cancer.Curcumin(Curcumin)is a polyphenol compound extracted from turmeric.It has a variety of pharmacological effects,including antioxidant,anti-inflammatory and anti-tumor effects.It also has a strong potential to protect liver cells.Studies have found that curcumin can effectively alleviate the toxic effects of AFB1,but the mechanism of curcumin’s alleviation of AFB1 poisoning in broilers is still not very clear.In particular,DNA methylation and AFB1-induced hepatotoxicity and the effects of curcumin are rarely reported.In this trial,64 1-day-old AA broilers were randomly divided into 4 groups,namely blank control group,AFB1 control group(1 mg/kg AFB1),curcumin + AFB1 intervention group(300mg/kg curcumin + 1 mg/kg AFB1)and curcumin control group(300 mg/kg curcumin),through the treatment of broiler serum physiological and biochemical indicators(ALT,AST,AKP,GGT),liver oxidative stress indicators(MDA,SOD,CAT,GSH,ROS),liver histopathology and the determination of AFB1-DNA adducts to evaluate the effects of curcumin intervention on AFB1 liver damage in broilers;Real-Time PCR was used to detect three phase I metabolic enzymes CYP1A1 in the liver of broilers in different treatment groups CYP1A2,CYP3A4 gene expression;Cocktail probe drug HPLC method was used to detect the enzyme activity of CYP1A2,CYP3A4;HPLC method was used to determine the overall liver DNA methylation level caused by AFB1,and RealTime PCR method was used to detect three types of DNA methyl transfer Gene expression levels of enzymes DNMT1,DNMT3 a,and DNMT3 b,and analyze the relationship between the overall level of DNA methylation,DNMTs,and CYP450 enzymes,and explore the role of epigenetic DNA methylation in liver damage caused by AFB1 The role and the effect of curcumin.The results of the study provide a theoretical scientific basis for the prevention and treatment of AFB1 hepatotoxicity and the further development of curcumin in veterinary clinics.The results are as follows:Combining the test results of serum indexes and histopathology,on the 28 th day of the test,the AFB1 model control group mice developed symptoms of liver injury,increased serum enzyme activity,decreased antioxidant enzyme activity,increased reactive oxygen generation,and degeneration of liver cells in broilers.Broiler production performance decreased;immunohistochemical tests showed that the AFB1-DNA adduct content of the model control group mice increased,and the small brown-black particles in the nucleus increased significantly.Curcumin can effectively improve the symptoms of liver injury caused by AFB1,indicating that curcumin can reduce the toxicity of AFB1 by inhibiting peroxidation and the production of AFB1-DNA adducts.Combined with the test results of CYP450 enzyme m RNA and enzyme activity detection,compared with the blank control group,the expression of CYP1A1 and CYP1A2 m RNA in the AFB1 control group was significantly up-regulated(P<0.05),the expression of CYP3A4 m RNA was extremely significantly up-regulated(P<0.01),and CYP1A2 The enzyme activity of CYP3A4 and CYP3A4 showed a similar trend to m RNA,and the addition of curcumin effectively reversed the above changes.It can be speculated that curcumin may reduce the expression of CYP1A1,CYP1A2 and CYP3A4 genes and protein levels,and reduce the conversion of AFB1 in the body.Hepatotoxicity of AFB1.The detection results of DNMTs m RNA and overall liver DNA methylation level showed that compared with the blank control group,the overall liver DNA methylation level and DNMT1,DNMT3 a,and DNMT3 b m RNA expression levels of broilers in the AFB1 model group were significantly reduced(P<0.01)The overall methylation level of liver DNA and DNMT1,DNMT3 a,DNMT3b m RNA expression in the curcumin+AFB1 group of broilers were significantly higher than the AFB1 model group,which proved that DNMT1,DNMT3 a and DNMT3 b work together to maintain the DNA methylation pattern,curcumin can Reduce the toxicity of AFB1 by increasing the overall methylation level of broiler liver DNA and the expression of DNMTs.Combined with the results of Pearson test and correlation analysis,the overall methylation level of broiler liver DNA is positively correlated with DNMT1,DNMT3 a and DNMT3 b levels,while the overall liver DNA methylation level and DNMTs gene expression of broiler chickens are both positively correlated with CYP1A1,CYP1A2 and CYP3A4.There is a negative correlation.To sum up,the results of this experiment show that feeding AFB1 will cause liver damage in broilers.The expression of CYP1A1,CYP1A2,CYP3A4 in the liver significantly increases,and the overall DNA methylation level and the expression of DNMTs are significantly reduced;administration of curcumin It can alleviate the liver damage of broilers caused by AFB1 and provide protection to the liver;curcumin reduces the expression of liver CYP450 enzymes(CYP1A1,CYP1A2,CYP3A4)and enzyme activity by enhancing the liver’s antioxidant capacity,and reduces AFBO adducts The production of AFB1-DNA,increase the overall DNA methylation level and the expression of DNMTs,protect the liver.The results of this study provide an important theoretical basis for enriching the mechanism of AFB1 induced hepatotoxicity and its prevention and treatment.