| The residues of tyrosine are vulnerable to be oxidized. Tyrosine oxidized products(TOP) including dityrosine(DT) and 3-nitrotyrosine(3-NT) are the markers of oxidative damage in vivo. Whether dietary TOP affect the accumulation of DT and 3-NT in body are still unclear. This topic performed the acute toxicity, 28 days-gavage and long-term feeding experiment to study the impacts of TOP on the redox status of myocardial, blood pressure and myocardial collagen metabolism. We also explored the pathway of myocardial energy metabolism and collagen metabolism. Preparation of TOP was referred to the method of Kurahashi. The main research contents are as follows:1. Acute toxicity experiment: 20 rats, male and female each half, with 110 g-120 g. Each rat was given 5000 mg/kg·bw of TOP. The mortality and poisoning symptoms were recorded.2. 28 days-gavage experiment: 80 rats, male and female each half, weighting 110 g-120 g, Samples(physiological saline, tyrosine 50 mg/kg·bw, TOP 250 mg/kg·bw, TOP 1250 mg/kg·bw) were given to rats through oral administration for 28 days, body weight was recorded each week, oxidative redox and biochemical indexes were determined after 28 days.3. Animals feeding experiment: Normal feeding(Control), Tyr 0.44 %, TOP1 0.22 %, TOP2 0.44 %, blood pressure was detected the at the end of 23 weeks, the indicators of oxidative stress of myocardial were detected respectively at the end of 6 weeks, 12 weeks, and 24 weeks. Activity of myocardial enzyme, vascular active substances, myocardial collagen metabolism and free fatty acids were determined. Myocardial mitochondria was observed. Myocardial redox related genes were detected using Real-time fluorescent quantitative PCR. The indicators of collagen degradation, lipid metabolism and expression of p38 MAPK pathway were also examined.The results are as follows:1. During the acute toxicity experiment, rats have no symptoms of poisoning performance nor death.And the toxicity dose of TOP on both female and male rats were more than 5000 mg/kg·bw.According to the acute toxicity grading standard, TOP belongs to non-toxic level.2. After 28 days-gavage, 250 mg/kg·bw group of the male rat had significant difference with the Control and the Tyr group(P < 0.05), while 1250 mg/kg·bw group of the female rat have significant difference with the Control and the Tyr group(P < 0.05). Meanwhile, 250 mg/kg group of the female rat have significant difference with the Control and the Tyr group in the second week(P < 0.05), and 1250 mg/kg·bw group of the male rat have significant difference with the Control and the Tyr group in the first week(P < 0.05). Biochemical tests in males including glutamic oxalacetic transaminase and glutamic-pyruvic transaminas showed significant differences in 250 mg/kg·bw group(P<0.05). Urea nitrogen, creatine kinase and creatine kinase isoenzyme all showed significant differences in the 1250 mg/kg·bw group(P<0.05). Biochemical tests in females of the 1250 mg/kg·bw group had significant differences compared with the Control(P<0.05). ROS, MDA, DT, AOPP of blood and myocardial increased significantly in the group of 1250 mg/kg·bw(P<0.05). T-AOC, CAT, SOD, GSH-Px decreased significantly(P<0.05). Dose TOP cause the obvious clinical toxic symptoms in 28 days-gavage experiment was less than 1250 mg/kg·bw. The main target organs were the liver, kidney and heart.3. After 6 week feeding, T-AOC, CAT, SOD, GSH-Px of TOP2 group decreased significantly compared with the Control and Tyr group in rats myocardial(P<0.05). At the end of 24 th week, T-AOC, CAT, SOD, GSH-Px, GSH/GSSG of TOP2 group all decreased significantly(P<0.05). ROS, DT, MDA, PC, AOPP of TOP2 group increased significantly compared with the Control and Tyr group(P<0.05) from the 6th week to the 24 th week. All the changes had dose-related effects. Gene expression of Nrf2, Trx-1, NF-κB, and AP-1 were significantly up-regulated(P<0.05) in rat myocardial after 24 weeks feeding.4. After 23 weeks feeding, compared with the Control and Tyr group, systoclic, diastolic and mean blood pressure of rats in TOP2 group were all elevated significantly(P<0.05). Blood flow and blood flow rate dropped significantly(P<0.05). After 24 weeks feeding, value of Ang II of rats in TOP groups elevated significantly while NO decreased significantly(P<0.05).5. After 24 weeks feeding, serum CK and CK-MB activities and myocardialⅠCTP、PⅢNP contents of rats in TOP groups all increased significantly compared to the control and Tyr groups(P<0.05), and all the changes had dose-related effects. Myocardial collagen degradation gene and inflammatory factors(TIMP-1, TIMP-2, PDGFB, IL-1, IL-6, TNF-α) were significantly up-regulated(P<0.05), while MMP-2, MMP-9 were significantly down- regulated(P<0.05). Myocardial electron microscopic showed abnormalities in heart tissue, such as myocardial fiber fracture and dissolution, mitochondria swelling and vacuole formation. Moreover, RT-PCR shows that, gene expression of PGC-1α, SIRT1, ERRα, PPAR-α were significantly down-regulated in myocardial(P<0.05), by the mean time, FFA increased significantly and T-ATPase activity declined significantly(P<0.05)all the changes had dose-related effects.6. The expression of Trx-1, TNF-α, PDGFβ in rat myocardial were positively correlated with levels of ROS, MDA, DT and AOPP respectively, however, the expression of SIRT1, PPAR-α in rat myocardial were negative correlated with levels of ROS, MDA, DT and AOPP respectively(P<0.05).TOP may lead to accumulation of oxidized protein products both in rat blood and myocardial, which induce the imbalance of redox state and oxidative damage to myocardial both in short-time gavage and long-time feeding. The oxidative damage has both dose and time related effects. Long-term feeding of TOP leads to higher blood pressure, abnormal collagen synthesis and degradation and induce myocardial energy metabolism abnormality, which may cause function failure in heart. |
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