Study On The Related Mechanism Of Curcumin Ameliorating Ochratoxin A–induced Intestinal Barrier Injury In Ducks

In recent years,the adverse effects caused by mycotoxins in amimals’ intestinal diseases have been increasing,which resulted in a significant reduction on the antioxidant status and immunity.Curcumin has been attributed with antioxidant,anti-inflammatory,antibacterial activities,and has shown highly protective effects against enteropathogenic bacteria and mycotoxins.OTA is one of the major intestinal pathogenic mycotoxins.The possible effect of curcumin on alleviation of enterotoxicity induced by OTA is unknown.The study was conducted to investigate the effects of dietary curcumin supplementation on tissue distribution of curcumin and its metabolites,intestinal antioxidation and key detoxification enzymes related genes expression in ducks.Furthermore,the possible effects of curcumin on the regulation of intestinal barrier function,mitochondrial function,protein synthesis,plasma metabolites,and gene expression of transporters in OTA-induced enteortoxicity were investigated.The study consisted of the following four experiments:In experiment 1: the study investigated the effects of increasing content of OTA on performance,antioxidant function and immunity in ducklings.A total of 1080 1-d-old male White Beijing ducklings with initial BW(43.5 ± 0.1 g)were randomly assigned to 6 treatment groups,each consisting of 6 replicates of 30 dirds.The basal diet(I)was a typical corn-soybean ration without antibiotics while the experimental diets(II to VI)substituted moldy corn(contained 12.7 mg/kg OTA)for fresh corn.The level of substitution in diets(II to VI)was 1%,2%,4%,8%,and 16%.The experiment lasted for 21 d.The results showed as follows: Compared with the control group,OTA at 0.5 mg/kg in the diet caused a significant decrease in the ADG and 21-d BW(P < 0.05).Ducks fed the diet containing 1.0 mg/kg of OTA had significantly decreased the plasma content of NO,and the activities of SOD and T-AOC(P < 0.05),and increased the lipid peroxide content of MDA(P < 0.05).With the OTA increasing,the plasma activity of DAO was significantly increased at 2.0 mg/kg,and further increased jejunal IL-1β and TNF-α levels,while decreased jejunal content of IL-10 and SIg A(P < 0.05).Histopathological examination showed that the villous blunting and epithelial denudation was observed when given 2.0 mg/kg of OTA.In experiment 2: the effects of dietary curcumin supplementation on tissue distribution of curcumin and its metabolites,antioxidation and detoxification related genes expression were examined in ducks.A total of 720 1-d-old male White Beijing ducklings with initial BW(58.6 ± 0.1 g)were randomly assigned to 4 dietary groups with 6 replicates per group and 30 ducks per replicate with the single factorial arrangement design.Ducks in control group were fed a basal diet without antibiotics and ducks in other groups were fed the dasal diet supplemented with 200,400,and 800 mg/kg curcumin respectively.The experiment lasted for 21 d.The results showed as follows: distribution of curcumin in the cecum,jejunum,liver,and plasma was 13.12 to 16.68 mg/g,75.50 to 575.40 μg/g,35.10 to 73.65 μg/g,and 7.02 to 7.88 μg/g,respectively.With the increasing of curcumin,the jejunal and hepatic content of curcumin,dihydrocurcumin,and tetrahydrocurcumin also increased,which showed a dose effect.Diet supplemented 400 mg/kg curcumin significantly increased jejunal activities of GSH-Px and T-AOC,and the transcript of HO-1 and Nrf2,reduced 8-OHd G and MDA content(P < 0.05).Adding 400 or 800 mg/kg curcurmin significantly increased the jejunum muscoal expression of CYP1A4,CYP2A6 and CYP2D17(P < 0.05),and decreased the expression of CYP1B1 and CYP2A6(P < 0.05).Furthermore,adding 400 or 800 mg/kg curcurmin significantly increased the jejunum muscoal expression of GST,MRP6 and ABCB1(P < 0.05).In experiment 3: the effects of dietary curcumin supplementation on OTA-induced oxidative stress,intestinal barrier,mitochondrial dyfunctions,protein synthesis,transporters,and plasma metabolites were examined in young ducks.A total of 540 1-d-old male White Beijing ducklings were randomly assigned into controls(fed only the basal diet),a group fed an OTA-contaminated diet(2.0 mg/kg feed),and a group fed the same OTA-contaminated feed plus 400 mg/kg of curcumin.Each treatment consisted of 6 replicates,each containing 30 ducklings and treatment lasted for 21 d.There was a significant decrease in ADG and increased feed: gain caused by OTA(P < 0.05);curcumin co-treatment prevented the decrease in BW and ADG compared with the OTA group(P < 0.05).Curcumin decreased the concentrations of DAO,IL-1β,TNF-α,and MDA and increased the activity of GSH-Px induced by OTA in the jejunal mucosa of ducks(P < 0.05).Histopathological and ultrastructural examination showed clear signs of enterotoxicity caused by OTA and were largely prevented by curcumin supplementation.OTA inhibited the m RNA expression of HO-1,Nrf2,RPS6K1,MTOR and protein expression of PI3 K,p-Akt,p-TOR,p-S6K1 in jejunal mucosa(P< 0.05),while curcumin supplemention effectively reversed decling expression of PI3 K,p-Akt,p-TOR and p-S6K1(P < 0.05).Additionally,curcumin increased jejunal mucosa OCLN and TJP1 m RNA and protein levels,and decreased those of ROCK1(P < 0.05).Notably,curcumin inhibited the increased expression of apoptosis-related genes,and downregulated TFAM,TFB1 M,and TFB2 M caused by OTA without any effects on POLRMT(P < 0.05).In experiment 4: the purpose of this study were to investigate the protective effects of dietary curcumin on ducks challenged with OTA through systemic and characteristic metabolites in the plasma and intestinal transporters.12 samples of each group of the trial 3 were selected.Metabolomics measurement showed that OTA exposure caused significantly elevated the plasma levels of lactate,uric acid,and myo-inositol,but reduced the plamsa levels of malic acid,citric acid,fumaric acid,glutamic acid,and glycerolin plasma.Concentrations of plasma glutamic acid,malic acid,and citric were increased and lactate,glycine,proline,and myo-inositol were decreased in CUR+OTA group than in the OTA group.Furthermore,the m RNA expression of jejunal glucose transporters(SGLT1)and amino acid and small peptide transporters(CAT1,EAAT4,and Pep T1)in ducks fed the diet with OTA were significantly downregulated(P < 0.05),and y+LAT2 was significantly upregulated compared to the controls(P < 0.05).The expression of SGLT1,CAT1,and EAAT4 in ducks fed curcumin in addition to OTA were significantly increased than with the toxic diet alone(P < 0.05).There were no significant effect in the expression of SLC1A4 and b0,+AT among treatments(P > 0.05).In conclusion,OTA inhibited growth performance,induced intestinal oxidative stress,inflammation,and lipid peroxidation.A diet containing 2.0 mg OTA/kg could induce enterotoxigensis in ducks.Adding 400 mg/kg curcumin enhanced antioxidant and phase-II detoxifying enzymes,and blocked metabolic activationof allogenic substances.Curcumin protected ducks from OTA-induced impairment of intestinal barrier function,mitochondrial integrity.In addition,curcumin mainly alleviated endogenous metabolic changes of the tricarboxylic acid cycle and glycolysis,and promoted nutrients absorption induced by OTA.The possible mechanism was through the enhanced antioxidant and detoxifying enzymes activities,clearance of ROS,the inhibition of mitochondrial apoptosis,and promoting protein synthesis by activation of Nrf2 and PI3K/Akt/TOR signaling pathways.Meanwhile,inhibited the Rho/ROCK related cytoskeleton-associated protein kinase pathway.