Effect Of Uric Acid On The Intestinal Oxidative Stress In Chickens

This study aimed to research the effects and mechanism of intestinal uric acid on the oxidative stress in intestine to verify whether intestinal uric acid plays an oxidation role in intestinal tract,and to explore the synthesis mechanism and active defense capacity of uric acid in gut.This research has furtherly provided new theoretical basis for the composition of antioxidant system and defense capacity in intestine,and preliminary explored the biological function of uric acid in intestine,and opened the new field of uric acid related studies in gut.Trial 1 The effects of precursors on uric acid synthesis in intestinal epithelial cells were studied.The chyme and duodenum,jejunum,ileum,liver,kidney,gallbladder,pancrea were collected from 42-days of age broilers.And the expression levels of uric acid synthesis enzymes in these organs and distributions of uric acid,allantoin,uric acid transporters in gut were measured.The results showed that the gene expression levels of key enzymes in de novo purine synthesis pathway in small intestine were very low.And the gene expression levels of key enzyme in purine nucleotide salvage pathway were existed and high in small intestine.Particularly,the expression levels of APRT,ADA,GDA and XDH mRNA were higher.The uric acid content in chyme and uric acid transporter mRNA levels of front intestine were significantly higher than those in the back intestine,and the allantoin content was also similarly.Used the hypoxanthine or inosine at concentrations of 0,100μM,1 mM and 10 mM to treat for 24 h or 36 h,the cells and culture medium were collected.And the uric acid,uric acid synthesis enzymes and transporter expression levels were also detected.Results showed that compared with control group,the treatment of hypoxanthine and inosine can significantly increase the amount of uric acid inside and outside the intestinal epithelial cells.The duodenums cultured in vitro by everted gut sac method were treated with 0,100μM and 1mM inosine for 2 h,and the culture medium inside and outsied the gut sac were collected to detect the uric acid content.The results showed that inosine significantly increased the uric acid content secreted from duodenum.The above results indicated that the uric acid may be synthesised by purine nucleotide salvage-decomposion pathway in small intestine.And the intestine may be an important organ to produce uric acid.Trial 2 The establishment of acute oxidative stress model of intestinal epithelial cells was studied.The H₂O₂ was selected to mold,and the test conditions were improved.The intestinal epithelial cells were cultured,and treated by the gradient concentrations of H₂O₂ for2 h to detect the cells vitality,antioxidant sysytem,LDH enzyme activity to select the appropriate treatment concentration.The selected treatment concentrations of H₂O₂ were used to treat the cells for gradient times to test the cells viability to select the treatment time.The oxidative stress model was validated by the MDA content,one of oxidative stress markers.The results showed that when the H₂O₂ concentration was 2 mM or above,the cells viability and the activity of antioxidant enzymes decreased significantly,and the LDH enzyme in medium increased significantly.When the intestinal epithelial cells were treated with 2 mM H₂O₂ for 2 h,the MDA content of cells was significantly decreased.The above results showed that the intestinal epithelial cells treated with 2 mM H₂O₂ for 2 h can successfully construct a stable acute oxidative stress model.Trial 3 The effects of uric acid on the oxidative stress of intestinal epithelial cells were researched.The 7 groups of Control,H₂O₂,150μM UA+H₂O₂,300μM UA+H₂O₂,600μM UA+H₂O₂,1200μM UA+H₂O₂,3600μM UA+H₂O₂ were set.The uric acid was used to pretreat the intestinal epithelial cells for 6 h,then the oxidative stress condition of 2 mM H₂O₂treating for 2 h was used to treate the cells.The cells vitality,oxidative stress markers,antioxidant system,Nrf2-ARE pathway expression level were measured.Results showed that compared with H₂O₂ group,the pretreatment of uric acid significantly improved the cell viability,and the MDA and protein carbonyl content of cells remarkably decreased with the enhancing of the antioxidant system.Compared with the control group,H₂O₂ significantly increased the Nrf2 protein level inside the nucleus of cells.However,after the uric acid treating,the Nrf2 protein level inside the nucleus significantly increased compared to H₂O₂group.At the same time,part of target genes expression levels elevated remarketably.The above results showed that uric acid can strongly enhance the antioxidant capacity of intestinal epithelial cells through the Nrf2-ARE pathway to alleviate the oxidative stress.Trial 4 The effects of acute oxidative stress on the synthesis of uric acid in intestine of chicken were studied.The 2 mL Fe²⁺solution at 0,0.32,0.8,2 and 5 mg/mL were respectively administered into the digestive tract of 1-day of age male chicks.After 4 h,the small intestine of chicks was collected.And the oxidative stress markers,antioxidant system,Nrf2-ARE pathway and gene expression of uric acid synthesis enzymes were detected.For duodenum,the results showed that when the Fe²⁺concentration was equal to 2 mg/m L or greater,the MDA and protein carbonyl content increased,and the antioxidant enzymes cativity and the expression of Nrf2-ARE pathway decreased.At the same time,the V/C of small intestine segments declined significantly.The genes expression levels of key enzymes significantly decreased at the Fe²⁺concentration equal to or higher than 2 mg/m L.The intestinal epithelial cells were treated with 0,250μM,500μM,1 mM,2 mM H₂O₂ for 2 h,and the expressions of the UA synthesis enzymes and transporters were detected.The results showed that the mRNA expression levels of XDH and SLC2A9 increased significantly at low concentration,while the 2 mM H₂O₂ significantly decreased the gene expression levels of uric acid synthesis enzymes and transporters.The results indicated that oxidative stress significantly decreased the ability of intestinal uric acid synthesis and secretion.Trial 5 The effects of low levels of oxidation stimulation on the uric acid synthesis in intestinal epithelial cells were researched.The H₂O₂ at concentrations of 0,50μM,100μM,200μM,400μM was used to treat the intestinal epithelial cells for 36 h.After finishing the treatment,the cells and supernatant were collected to detect the uric acid content.And the gene expression levels of uric acid synthesis enzymes and uric acid transporters in intestinal epithelial cells were measured.The results showed that H₂O₂ significantly increased the uric acid content inside and outside cells and the XDH mRNA of cells.The expression level of ABCC4 mRNA showed the significantly rise with the increase of H₂O₂ concentration.The duodenums cultured in vitro by the mean of everted gut sac were treated with the H₂O₂ at the concentrations of 0,50μM and 200μM.The culture medium inside and outside the gut sac was collected to detect the uric acid content.The results showed that the uric acid secreted from duodenum increased significantly with the increase of H₂O₂ concentration.The above results demonstrated that low level of oxidative stimulation promoted the intestinal uric acid synthesis and secretion.