| Aflatoxin B1(AFB1)has been evaluated by the International Agency for Research on Cancer as one of the most toxic chemical carcinogens known,and the liver is its main target organ.In addition,the gastrointestinal tract is the direct organ exposed to mycotoxins.AFB1 exposure can damage its mucosal barrier function and destroy the structure of gut microbiota.The balance of gut microbiota is very important for animal digestion,absorption and immune regulation.Therefore,maintaining gut microbiota homeostasis may be a potential pathway to improve AFB1 toxicity.Total flavonoids of Rhizoma Drynariae(TFRD)are natural flavonoids extracted from dry rhizomes of ferns,which has protective effect on AFB1-induced liver injury in broilers.Therefore,the purpose of this study was to investigate the mechanism of AFB1-induced liver injury and the improvement of TFRD.In this study,90 one-day-old Arbor Acres(AA)broilers were randomly divided into 3 groups: control group(CON),AFB1 group,AFB1 and TFRD co-treatment group(A+T).AFB1(100 μg/kg body weight,dissolved in 4% ethanol)was injected into the crop of AFB1 group and A+T group for 7 days to establish AFB1 exposure model.The CON group was injected with the same dose of 4% ethanol into the crop and fed the basal diet.At the same time,broilers in the A+T group were supplemented with 125 mg/kg TFRD in the basal diet.To explore the toxic mechanism of AFB1 on liver injury in broilers,and the protective effect of TFRD on AFB1-induced liver injury by regulating gut-liver axis interaction.The main research contents and results are as follows:(1).To investigate the effect of TFRD on intestinal microbial homeostasis and intestinal barrier in AFB1-exposed broilers.The 16 S r RNA sequencing analysis of duodenal contents of broilers in each group showed that TFRD could improve the gut microbiota disorder caused by AFB1 exposure,and the microorganisms related to bile acid metabolism such as Faecalibacterium,Subdoligranulum,Lactobacillus and[Ruminococcus] were significantly different between groups(p < 0.05).At the same time,TFRD supplementation effectively improved the morphological and structural damage of the duodenal intestine,and improved the intestinal barrier by increasing the number of crypt goblet cells and the mRNA expression of and mucin MUC2(p <0.05),as well as the mRNA and protein expression levels of tight junction proteins Occludin and Claudin-1(p < 0.05).(2).To explore the role of bile acid metabolism after AFB1 exposure.In this study,plasma samples of broiler chickens were collected,and used extensive targeted metabolomics techniques to study the differences in metabolites between plasma samples under different treatment conditions.A total of 773 metabolites were identified in the plasma of the CON group,AFB1 group,and A+T group by UPLC-MS/MS detection platform,self-built database and multivariate statistical analysis.The identified metabolites were further analyzed.A total of 34 metabolites with significant changes were screened in the CON group and AFB1 group,of which15 were upregulated and 19 were downregulated.A total of 57 metabolites with significant changes were screened in the AFB1 group and A+T group,of which 17 were upregulated and 40 were downregulated.KEGG enrichment analysis of differential metabolites showed that differential metabolites in the CON group and AFB1 group were mainly enriched in serotonergic synapse,arachidonic acid metabolism,sphingolipid signaling pathway,fatty acid biosynthesis and other pathways.The differential metabolites in the AFB1 group and A+T group were mainly enriched in primary bile acid biosynthesis,linoleic acid metabolism,bile secretion and sulfur metabolism.Then,the common metabolites with significant differences between the CON and AFB1 groups,the AFB1 and A + T groups were used as potential biomarkers and analyzed by Wayne,and 9 common differential metabolites were found.Including creatine,3-N-methyl-L-histidine,L-carnitine,tryptophan caffeine,L-tyrosine methyl ester 4-sulfate,taurolithocholic acid,Ala-Tyr,and Carnitine C8:0.Among them,secondary bile acids taurolithocholic acid showed an increasing trend after AFB1 exposure and decreased significantly after TFRD supplementation(p < 0.05).In addition,the relative content of tryptophan betaine also increased significantly after AFB1 exposure(p < 0.001),and decreased significantly after TFRD supplementation(p < 0.001).It is suggested that both may play a detoxification role in AFB1-exposed plasma metabolites in broilers.Furthermore,Pearson correlation analysis showed that differential plasma metabolites were closely related to gut microbiota.For example,taurolithocholic acid was significantly positively correlated with [Ruminococcus] and Lactobacillus(p < 0.05),and significantly negatively correlated with Subdoligranulum(p < 0.05),further demonstrating that gut microbiota disorders affect bile acid metabolism.(3).To explore the molecular mechanism of TFRD alleviating AFB1-induced liver injury.Based on the above results,the levels of antioxidant enzyme activity and the mRNA expression levels of key regulators of lipid metabolism and key genes of ferroptosis were detected.Firstly,TFRD effectively alleviated AFB1-induced liver pathological damage,reduced alanine aminotransferase(ALT)(p < 0.001)and aspartate aminotransferase(AST)(p < 0.01)activities,increased superoxide dismutase(SOD)(p < 0.01),glutathione peroxidase 1(GPX1)(p < 0.01)and catalase(CAT)(p < 0.05)activities,reduced malondialdehyde(MDA)levels(p < 0.01),and improved antioxidant capacity.In addition,liver oil red staining showed severe lipid accumulation in AFB1 group(p < 0.001),and serum total cholesterol(TC)(p < 0.01)and triglyceride(TG)(p < 0.001)were increased,suggesting abnormal lipid metabolism,while TFRD supplementation effectively reversed the above changes.The genes expression of key regulators of lipid metabolism,PPARα,CPT-1A,SREBP1 and ACC,increased under AFB1 exposure(p < 0.01),further indicating that AFB1 induced abnormal liver lipid metabolism.Since AFB1 exposure led to oxidative stress and abnormal lipid metabolism,we speculated that ferroptosis occurs in the liver.Real-time quantitative PCR detection showed that AFB1 exposure led to a significant increase in the mRNA expression of ferroptosis-inducing gene ACSL4(p <0.01),and the mRNA expression of ferroptosis-inhibiting genes GPX4(p < 0.05)and FTH1(p < 0.001)was significantly downregulated,indicating that AFB1 exposure may promote ferroptosis in the liver,and TFRD supplementation can effectively inhibit the occurrence of ferroptosis in the liver.Subsequent Pearson correlation analysis showed that plasma metabolites were closely related to liver lipid metabolism genes and ferroptosis related regulatory factors,suggesting that AFB1 was absorbed into the blood circulatory system through the gastrointestinal tract,and then had toxic effects on the liver.In summary,TFRD plays a protective role in AFB1-induced liver injury by improving gut microbiota homeostasis and gut barrier function,correcting lipid and bile acid metabolism,inhibiting liver oxidative stress and ferroptosis.These findings provide new perspective that AFB1 induces liver injury,and provide a theoretical basis for the development of Chinese herbal feed additives to improve AFB1 hepatotoxicity. |
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