Study On The Function Of Uric Acid In The Redox Equilibrium Of Chicken

Many studies have shown that uric acid?UA?plays an important role in the antioxidant system,while high concentrations of UA can induce oxidative stress.In this study,animal model of broilers with different serum UA and chicken cardiac muscle cellular model damaged by hydrogen peroxide?H₂O₂?were established to investigate the effects of UA on antioxidant enzyme activities,antioxidants contents and Nrf2/ARE antioxidant pathway changes in vivo and in vitro.To the best of our knowledge,this is the first research which illustrates the "double-sided" effect,that both antioxidant and pro-oxidation role of UA on oxidative stress in broiler chickens.1.The difference of antioxidant system in normal physiological condition with high or low serum uric acidBroiler chickens grown under natural environment with different circulating UA concentrations?p<0.001?were firstly selected by detecting serum UA repeatedly to investigate the role of UA in the maintenance of redox balance.The results showed that Low UA?LUA?-chickens had higher?p<0.05?protein carbonyl level in cardiac muscle and kidney while there were no obvious difference?p>0.05?in the activities of SOD,CAT,GPX,and XOD and concentrations of GSH and AsA in cardiac muscle and kidney between High UA?HUA?and LUA chickens.Then,change of nuclear factor E2-related factor 2/antioxidant responsive element?Nrf2/ARE?signal pathway was investigated in HUA and LUA chickens.Although there were no significant statistical differences?p>0.05?in expression levels of Nrf2 and Nrf2/ARE downstream antioxidant-related genes between two group chickens,however,the extensively lower expression level of Nrf2 and antioxidant-related genes in cardiac muscle and kidney in LUA chickens indicating the broilers with low concentrations of serum UA were more likely to sustain oxidative damage than those with high concentrations serum UA.2.Effects of low protein diet supplemented with glycine on serum uric acid and antioxidant systemGlycine is a substrate in the biosynthesis of uric acid and other components like glutathione,heme,creatine,and nucleic acid.Therefore we added glycine in low-protein diets?12.54 MJ metabolizable energy and 17%crude protein,same metabolizable energy with the control group and low protein group?to investigate the role of increasing UA concentration in alleviating the oxidative stress caused by low-protein?LP?diet.The results showed that UA level was significantly decresed in serum and cardiac muscle in broilers feeding with LP diet.MDA and protein carbonyl contents and XOD activity increased significantly while GPX activity and GSH and AsA contents decreased significantly in cardiac muscle in LP group chickens.Similarly,MDA and protein carbonyl contents increased significantly while CAT,SOD,and GPX activities and AsA content decreased significantly in kidney in LP group chickens,indicating feeding with LP diet could induce oxidative stress in chickens.In contrast,MDA and protein carbonyl contents were decreased in cardiac muscle and kidney in broilers feeding with LP added with glycine diet?LP+Gly?when compared with LP group chickens.In addition,both transcription?p<0.05?and protein level?p<0.05?of Nrf2 decreased significantly,and the expression of Nrf2 signal pathway downstream genes like SOD2,GPX7,CAT and GCLC were down-regulated.Those results above indicated that LP diet could result in oxidative stress and the activation of Nrf2 pathway.The supplementation of glycine partially alleviated the oxidative damage by increased the synthesis of antioxidant substances such as uric acid and GSH.3.Effects of injecting urate oxidase on serum uric acid and antioxidant systemIn most species of mammals,urate oxidase can breaks down serum UA into allantoin to maintain the balance of UA level.In our previous study,we found that the serum UA content decreased significantly?p<0.05?on the third day after continuous injection with urate oxidase intraperitoneally in broiler chickens.On the basis of this result,serum and tissue samples were collected from chickens with low serum UA concentration and antioxidant parameters were measured.The results showed that UA concentration decreased significantly in cardiac muscle.In addition,SOD and GPx activities and GSH and AsA contents decreased significantly in cardiac muscle upon injection with urate oxidase for three days.As for Nrf2/ARE pathway,both transcription and protein expression level of Nrf2 and antioxidant related genes such as sodl,sod2,gpx7,gclc and gclm were down-regulated.Those results indicated that the rapid decrease of UA could induce oxidative damage in blood circulation system by reducing antioxidant enzyme activities and GSH generation,leading to the imbalance of redox status in cardiac muscle.4.Effects of feeding yeast extract on serum uric acid and antioxidant systemYeast extract contains biological molecular chemical compounds and metabolic product.Large doses of yeast extract can interfere with normal metabolism,activate purine metabolism pathway,and promote UA formation.To investigate the effects of abnormally high concentration UA on antioxidant balance in broilers,broiler chickens were feeding with diet supplement with 10%yeast extract powder for 4 weeks to establish the abnormally high UA animal model.The results showed that the serum UA level increased significantly at the third week upon feeding with diet added with yeast extract compared with control group chickens.In kidney,protein carbonyl content increased significantly,while GSH content and SOD activity decreased significantly.In addition,expression of antioxidant related genes such as SOD2,GPX7,and GSR were up-regulated significantly.In contrast,there was no obvious difference of oxidative parameters in cardiac muscle between broilers with high serum UA level and control group chickens.Those results indicated that the abnormal increased serum UA could induce oxidative damage in kidney,but not in cardiac muscle,in a tissue specific manner5.Effects of feeding different protein levels dietary on serum uric acid and antioxidant systemTo further investigate the effect of UA in antioxidant balance,animal model of chickens with different concentrations of UA was established.Chickens were feeding with diets adding different levels of crude protein,and serum UA concentration and oxidative parameters were measured.The results showed that the serum UA revealed a gradual increase in chickens feeding diets with different concentrations of crude proteins without affecting the production performance.The MDA and protein carbonyl contents were gradually increased,while several antioxidant enzymatic activities were decreased in kidney and cardiac muscle with the decrease of serum UA level.In addition,the expression of antioxidant-related genes such as SOD2,GSS,GCLC,and GCLM were up-regulated in cardiac muscle,and Nrf2 gene and protein expression level were also changed in the same tendency with the gradual decreasing serum UA level in cardiac muscle.In line with the previous experiments,those results showed that feeding with diets with different crude protein level changed serum UA level in broiler chickens.Excessively low serum UA level could reduce the antioxidant enzymatic activities and antioxidants contents,up-regulate expression level of Nrf2/ARE pathway,leading oxidative damage in several tissues especially cardiac muscle and kidney.It also indicated that the long-term decrease of UA could cause the body to resist oxidative damage under the condition of natural growth.6.Effect of UA on antioxidant system of cardiac muscle cellsTo investigate the effect of high concentrations of UA on antioxidant system of cardiac muscle cells,identified chicken cardiac muscle cells cultivated in vitro were treated with different concentrations of UA.The results showed that MDA and protein carbonyl contents were increased and CAT and SOD enzymatic activities were decreased in cardiac muscle cells treated with 1200?M UA after 6h and 12h.In addition,both gene and protein expression levels of Nrf2?p<0.05?were significantly increased,and genes expression of SOD2,GSS SOD1,GCLC,GCLM were up-regulated 12h after treatment.The results indicated that high concentration of UA could induce oxidative damage in cultivated chicken cardiac muscle cells,leading to the decrease of antioxidant enzymatic activities and activation of Nrf2/ARE pathway which could resist oxidative damage.7.Protective effect of uric acid on cardiac muscle cells injured by hydrogen peroxideTo investigate Effect of different concentrations of UA in oxidative chicken cardiac muscle cells,cardiac muscle cells were treated with different concentrations of hydrogen peroxide?H₂O₂?.Cell viability was decreased and MDA and protein carbonyl contents were increased in cells treated with H₂O₂,indicating the successful establishment of cellular model.Then,H₂O₂ damaged cells were treated with different concentrations of UA.The results showed that MDA,protein carbonyl contents and accumulation of reactive oxygen species?ROS?were decreased and SOD activity was increased in cells treated with 300?M UA.Both mRNA and protein expression level of Nrf2 and several antioxidant related genes were up-regulated in H₂O₂-damaged cells treated with 300?M UA,indicating UA within normal physical range could alleviate oxidative damage induced by H₂O₂.However,MDA and protein carbonyl contents and accumulation of ROS were increased significantly while cell viability and SOD enzymatic activity were decreased significantly in oxidative damaged cardiac muscle treated with 1200?M UA.In addition,both mRNA and protein expression level of NrF2 and several antioxidant related genes were down-regulated in H₂O₂-damaged cells treated with 1200?M UA.Furthermore,we found that intracellular UA content was higher in H₂O₂-damaged cells,indicating that H₂O₂ treatment could increase cell membrane permeability which made it easier for UA to enter cells.Above all,normal physiological concentration of UA had no obvious detrimental effect on redox balance in chicken cardiac muscle cells while it protects H₂O₂-damaged cells from oxidative stress by activating Nrf2 pathway,urate with higher concentration than normal level triggers oxidative stress,induces ROS formation and aggravates oxidative stress in H₂O₂-damaged cells by inactivating Nrf2 pathway.8.Effects of the interaction with glucose and urate on antioxidant system of cardiac muscle cellsTo investigate Effect of different concentrations of UA in oxidative chicken cardiac muscle cells,cardiac muscle cells were treated with different concentrations of glucose.Cell viability was decreased and MDA and protein carbonyl contents were increased in cells treated with glucose,indicating the successful establishment of cellular model.Then,glucose-damaged cells were treated with different concentrations of UA.The results showed that MDA contents and accumulation of reactive oxygen species?ROS?were decreased and GSH content was increased in cells treated with 300?M UA.Several antioxidant related genes were up-regulated in glucose-damaged cells treated with 300?M UA,indicating UA within normal physical range could alleviate oxidative damage induced by glucose.However,MDA and protein carbonyl contents and accumulation of ROS were increased significantly while cell viability,SOD enzymatic activity and GSH content were decreased significantly in oxidative damaged cardiac muscle treated with 1200?M UA.In addition,both mRNA and protein expression level of Nrf2 and several antioxidant related genes were down-regulated in glucose-damaged cells treated with 1200?MUA.Furthermore,we found that intracellular UA content was higher in glucose-damaged cells,indicating that glucose treatment could increase cell membrane permeability which made it easier for UA to enter cells.Above all,Normal physiological concentrations of urate partially alleviate oxidative damage caused by glucose,urate with higher concentration than normal level triggers oxidative stress,induces ROS formation and aggravates oxidative stress in glucose-damaged cells by inactivating Nrf2 pathway.In summary,compared with kidney,liver,pancreas,cardiovascular tissue,the cardiac muscle is more sensitive to the changes of uric acid.Physiological concentration of serum uric acid has antioxidant properties,uric acid lowering will cause oxidative damage,excessive uric acid plays a prooxidant effect.Physiological concentration of uric acid can alleviate oxidative damage caused by hydrogen peroxide on cardiac muscle cells,high uric acid increased the hydrogen peroxide damage.High glucose induced the oxidative damage on cardiac muscle cells,physiological concentration of uric acid can alleviate oxidative damage caused by high glucose,high uric acid can aggravate the oxidative damage.Suggesting uric acid plays an important role in the antioxidant system.Therefore,it's of great importance to properly utilize the antioxidant and pro-oxidation characteristics of UA to maintain the body's redox balance.