The Mechanism Of Oxidative Stress And Fibrosis Change In Rat Liver And Kidney Induced By Tyrosine Oxidized Products

Amino acid residues in proteins are easily to be modified by various physical and chemical factors and are easily to be oxidized by the products of lipid peroxidation and glycation during food processing and storage,resulting in protein structure and function changesa and affecting food quanlity and nutrition.Tyrosine(Tyr)residues are susceptible to oxidation and nitration.The structure of tyrosine oxidized products(TOP)is stable and not easy to be enzymatic hydrolysis,therefore,TOP are widely detected in meat and dairy products.Studies have found that accumulation of TOP in biological system may be mistakenly incorporated into the protein synthesis pathway,leading to protein dysfunction and cell toxicity,which is related to tissue damage and a variety of chronic metabolic diseases.Interestingly,previous studies in our laboratory have found that dietary casein containing TOP can lead to elevated free radicals in tissues accompanied by accumulation of Dityrosine(Dityr)and advanced oxidation protein products(AOPPs),which revealed the potential biological damage of dietary TOP.However,the reason of Dityr and AOPPs deposition in tissues induced by TOP administration,the digestion and absorption process of dietary TOP and its impact on biological system are not clear and need further study.In this study,we investigated the absorption of dietary TOP by both in vivo and in vitro experiments and explored the effects of TOP on oxidative stress and fibrosis changes in liver and kidney.TOP was produced in our lab using H2O2-Cu oxidation system.HPLC-MS results showed that the main component of TOP is Tyr and dityrosine(Dityr)which accounts for about 22% of total TOP.Then the Dityr in TOP was separated and purified by HPLC.The collected Dityr samples were analyzed by NMR and were proved to be purity.The Ussingchamber model was used to simulate the absorption of TOP in vitro.The results showed that Dityr,the main component of TOP could be absorbed through the intestine of rat.Then we perforemed rat gavage experiment to evaluate TOP absorption in vivo,the results show that Dityr can be absorbed into through portal vein into the liver and can not be metabolized in the liver,resulting in increased level of Dityr in peripheral blood.28 days oral toxicity test were performed in both female and male rats to assess the safety of TOP.Female and male rats were administrated with different doses of TOP(50,250,1250 mg/kg bw/day)for 28 days.The results showed that low dose(50 mg/kg bw/day)TOP had no obvious influence on the growth properties of rats.But with the increased intragastric dose of TOP(250,1250 mg/kg bw/day)and the prolonged time of gastric infusion(over 2 weeks),the appetite and growth rate of rats were declined significantly.The impact effect was dose-related and time-related.The results also showed that intragastric administration of TOP increased the levels of serum aspartate aminotransferase(AST),alanine aminotransferase(ALT),creatinine(Cr)and blood urea nitrogen(BUN)while decreased total protein level.In addition,the liver and kidney index was elevated after TOP administration.The above results showed that TOP induced rat liver and kidney injury.Intragastric administration of TOP could also lead to the decreased antioxidant enzyme activity and the accumulation of oxidized protein products in the serum,liver and kidney,which reflects the oxidative stress in rats.Rats were fed with TOP at different doses of 2,4,8 g/kg diet for 6 w,12 w and 24 w respectively.The results showed that all doses of TOP at different feeding time could cause the decrease of feed intake and body growth rate,and induce oxidative stress in rat blood,liver and kidney.The Nrf2-ARE pathway in rat liver and kidney was activated by TOP to upregulate its downstream antioxidant enzyme genes to resist the body damage,but the antioxidant enzyme activity was significantly decreased.TOP also led to the occurrence of inflammation in liver and kidney.After TOP administration,serum biochemical indicators related to liver and kidney function were increased significantly and a large number of protein oxidation and lipid oxidation products were accumulated in rat liver and kidney.All the above changes were both dose-related and time-related.During all the experimental period,rats fed with Tyr showed no significant difference from those of the control group.After 24 weeks feeding with TOP at different doses of 2,4,8 g/kg diet,rat liver and kidney were analyzed by immunohistochemistry(IHC)and the results showed that different doses of TOP caused dose-related Dityr deposition in rat liver cell cords,glomerulus,renal capsule and renal interstitial.HE and Masson staining results demonstrated that TOP induced rat liver and kidney fibrosis changes.Different doses of TOP cells caused dose-related increased extracellular matrix(ECM)content,including procollagen type I carboxy terminal peptide(ICTP),type III procollagen amino terminal peptide(PIIINP),laminin(LN)and hyaluronic acid(HA),in rat liver and kidney.TOP induced fibrosis changes in liver and kidney was regulated by MAPKs/TGF-?1/Smads signaling pathway which was triggered by oxidative stress and inflammation.Mice were performed intragastric administration using pure Dityr(1 mg/kg body weight/day)and TOP(1 mg/kg body weight/day)for 10 w.At the end of the experiment,mouse liver and kidney showed oxidative stress and the accumulation of oxidized protein products.Both Dityr and TOP administration induced significantly changes of indexes related to liver and kidney function.The results of HE and Masson staining showed that Dityr and TOP induced fibrosis changes in mice liver and kidney,and the extracellular matrix(ECM)increased in the liver and kidney.Similar to the TOP feeding experiment results in rats,the Dityr and TOP induced liver and kidney fibrosis in mice were caused by oxidative stress and inflammatory via activating MAPKs/TGF-?1/Smads signal pathway.There was no difference between Dityr and TOP gavage group.The similar changes in Dityr and TOP treated mice showed that TOP induced oxidative stress and liver fibrosis were mainly caused by Dityr.The above results show that Dityr,the main product of TOP,can be absorbed directly into blood.Long term intake of TOP can cause oxidative stress and inflammation,and activate Nrf2-ARE pathway.TOP can also lead to liver and kidney damage and induce fibrosis of liver and kidney by triggering MAPKs/TGF-?1/Smads signaling pathway.The liver and kidney damage increases with the prolongation of TOP feeding time and the increase of TOP doses.In addition,the damage of Dityr on liver and kidney and its mechanism are similar to that of TOP,indicating that Dityr plays an important role in TOP induced liver and kidney injury.