Curcumin Alleviates Aflatoxin B1-induced Sub-acute Liver Injury Via Up-regulating Nrf2 And GST Expression

Aflatoxin B1（AFB1）is a kind of aflatoxin（AF）,classified as class 1 carcinogen due to its strong toxicity,great harm,and wide distribution.AFB1 is a pre-carcinogen,and it enters the body and metabolized by phase I drug metabolism enzymes to aflatoxin B1-8,9-epoxide（AFBO）.The liver is its main target organ,which can cause liver damage and even induce liver cancer.Liver-associated detoxification is the main way to eliminate the toxicity of metabolites,of which glutathione-S-transferase（GST）is the main detoxification enzyme found in the liver and aflatoxin B1-8,9-epoxide-glutathione（AFBO-GSH）is one of the main indicators to evaluate whether GST can detoxify AFB1.Curcumin is an active ingredient of turmeric,which has anti-inflammatory,anti-oxidative and immune functions.It activates the Nrf2-ARE pathway to fight against oxidative stress,but its mechanism of action is not yet clear,In particular,whether curcumin can interfere with hepatotoxicity of AFB1 by promoting the liver’s detoxification mechanism has not been reported.In this study,Arbor Acres（AA）broiler chickens with large domestic rearing capacity were selected as experimental animals.The effects of AFB1 on the expression of liver detoxification enzymes Nrf2and main phase II enzymes and the addition of curcumin were investigated in terms of mRNA,protein expression level and enzyme activity.Changes to AFB1 that caused the above effects.The detoxification ability of GST was evaluated by using high performance liquid chromatography to detect changes in the adduct AFBO-GSH content.This study will elucidate how subclinical liver injury in AFB1-induced broiler chickens and curcumin affect nuclear factor NF-E2-related factor（Nrf2）and its downstream target genes GSTA family and GSTM family subtypes（GSTA,GSTA3,GSTA4,and GSTM2）,and the epoxidase hydrolase-1（EPHX1）,relieving AFB1-induced subchronic liver injury in broilersThe results of the study are as follows:1.The effects of AFB1 and/or curcumin on the Nrf2 and II phase enzymes（GST and EPHX1）in the liver of broilers.The effects of of AFB1(5 mg·kg^-1)and curcumin in different doses(150 mg·kg^-1,300 mg·kg^-1 and450 mg·kg^-1)were studied by histopathological examination（HE staining）,RT-PCR and Western-Blot.The results showed that the expression of Nrf2 gene and protein in the AFB1 group was significantly lower than that in the blank control group（P<0.01）,and the expression of GSTA,GSTA3,GSTA4,GSTM2 and EPHX1 genes were significantly lower than relative to the blank control group（P<0.01）,and the expression of GSTA3 protein was significantly lower compared to the blank control group（P<0.01）.The pathological tissue section showed that the degeneration and necrosis of hepatocytes in AFB1 group were severe,indicating that AFB1 caused damage to broiler liver,and the AFB1 injury symptoms of the chicken liver tissue in the curcumin intervention group were significantly relieved,and the degree of remission was positively related to the dose of curcumin,indicating that curcumin has protective effect on the liver injury induced by AFB1.The curcumin control group had no significant difference compared with the blank control group（P>0.05）,indicating that the added curcumin concentration had no adverse effects on broilers.The medium and high dose curcumin could significantly increase the expression of GSTs and EPHX1 gene in the Nrf2 and phaseII（P<0.01）,and the high dose curcumin could significantly increase the expression of Nrf2,GSTA3 and GSTM2 protein（P<0.01）.It showed that curcumin preventedthe oxidative stress and liver injury induced by increasing Nrf2 and the expression of the downstream phase II enzymes pathway.After seventh days（thirty-fifth days）,the change trend of liver index in each group was similar to that of 28 days.The liver of AA broiler in AFB1 group still showed obvious liver injury state,and high dose curcumin still maintained a continuous protective effect on subchronic liver injury induced by AFB1 in AA broilers.The effects of AFB1 on Nrf2 and II phase enzyme（GST,EPHX1）in liver of broiler chicken had sequelae toxicity within 7 days of drug withdrawal,and the intervention of curcumin was sustained within 7 days of drug withdrawal.2.The effect of AFB1 and curcumin on GST activity and AFBO detoxification ability of broiler liver.After 28 days of continuous exposure,the activity of GST enzyme in the liver tissue of broilers decreased significantly compared with the blank control group（P<0.01）.The activity of GST enzyme in the curcumin intervention group was higher than that in the AFB1 group,but no significant difference was found.The liver microsomes were induced by BHA in the liver cytoplasm of mice,the cytoplasm of the control group,the cytoplasm of curcumin-fed broilers and the addition of the cytoplasm in the buffer solution,and the supernatants were analyzed by HPLC.The results showed that AFBO-GSH adducts were detected only in the supernatant of curcumin-fed group.After 7 days of withdrawal,the activity of GST and AFBO-GSH adduct were similar to those of 28 days.It is indicated that curcumin improves the activity of GST enzyme in liver tissues of broiler and promotes the binding of glutathione（GSH）to AFBO,thus it plays a role in detoxification,and its intervention is persistent within 7 days of withdrawal.The above results showed that feeding of 5 mg·kg^-1 AFB1 for 28 consecutive days could cause subchronic liver injury in AA broilers,and the injury was irreversible.The following phase II enzyme subtypes involved in detoxification were detected in broilers:GSTA,GSTA3,GSTA4,GSTM2 and EPHX1,and curcumin could improve the liver damage induced by the combination of upregulation of Nrf2 and the expression of its downstream genes.High dose intervention is the best one,which is one of the mechanisms of curcumin alleviating liver damage induced by AFB1 in AA broilers.It is suggested that the intervention dose of curcumin is 450 mg·kg^-1.