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The Role of Peroxynitrite in Hepatic Encephalopathy

Author: RongLin
Tutor: ShaoLiHua
School: Shandong University
Course: Occupational and Environmental Health
Keywords: hepatic encephalopathy nitrative stress Peroxynitrite 3-nitrotyrosine High Performance Liquid Chromatography
CLC: R747.9
Type: Master's thesis
Year: 2010
Downloads: 70
Quote: 1
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Abstract


ObjectiveThe hepatic encephalopathy (HE) is a serious harmful disease to human health, of which the pathogensis is not yet entirely clear. As we all know, in the blood of HE patients the level of the endotoxin, tumor necrosis factor increases, both of which are the promoter of nitric oxide synthase (NOS). The activation of NOS will promote the synthesis of nitric oxide (NO), and finally result in the significant increase of NO in vivo. Because of the decrease of the ability of the antioxidant defenses, superoxide anion (O2-) is also elevated at the same time. ONOO- is produced by the rapid integration of NO and O2-, and this reaction is almost irreversible. So the level of ONOO- may increase abnormally in serum of HE patients. The abnormally high level of ONOO- can directly affect the function of proteins by nitrating the specific residues of protein tyrosine (Tyr); and can lead to the dysfunction of nerve conduction by the oxidition of neurons membrane; and it also can cause liver, brain cell damage and mitochondrial energy synthesis disorders and so on. As a result, the role of ONOO" can not be excluded in HE pathogenesis. Therefore this study would investigate the role of ONOO- in the occurrence and development of HE by clinical research of the patients of liver injury and establishing the HE model of rats to reveal the pathogenic mechanism of ONOO- and provide the new ideas and scientific basis to the etiological factor and therapy of HE.Methods1. Clinical study of patients with liver injury1.1 The selection of the patients with liver injury Select 45 cases of viral hepatitis patients with HE and 30 cases of viral hepatitis patients with non-HE which were accurately diagnosed according to the ministry of health diagnostic criteria for viral hepatitis as well as 30 cases of health checkup persons in the same period. And they were divided into 3 groups according to the severity of disease:chronic hepatitis with HE group; chronic hepatitis with non-HE group and healthy people who did physical examination at the same period. Then patients in chronic hepatitis with HE group were further divided into 4 groups:phaseⅠ:pilot phase, phaseⅡ:pre-coma, phaseⅢ:sleeping on and phaseⅣ:coma period.1.2 The determination of several of biochemical indicators in serum Fasting blood, and serum were obtained after centrifugation. The contents of 3-nitrotyrosine (3-NT) in serum were detected by High-performance liquid chromatography (HPLC)-fluorescence detection method. The contents of malondialdehyde (MDA) were detected with the thiobarbituric acid method. And the vitality of superoxide dismutase (SOD) in serum was detected using xanthine oxides enzymaic method.2. Experimental study of HE rats2.1. The establishment of HE model of rats The model of HE in rats was established with thioacetamide (TAA). Male Wistar rats were divided into 4 groups randomly (15 in each group):blank control group (Ⅰ), HE model group (Ⅱ), ebselen treated group (Ⅲ) and solvent control group (Ⅳ). All the groups except groupⅠwere treated with TAA (300 mg/kg) by intraperitoneal injection (i.p.) at first day and treated with TAA (150 mg/kg) by i.p. in the following 2 to 4 days to manufacture the model of HE. At the same time rats in groupⅠwere treated with normal saline i.p. in the same dose. When rats in groupⅡappeared HE symptoms the four groups administered intragastrically (i.g.) with normal saline (Ⅰ,Ⅱ), ebselen (Ⅲ) and 0.5% Dimethyl sulfoxide (DMSO) (Ⅳ) respectively in the dose of 1 mL/100g.2.2. Determination of various biochemical indicators Tails of rats were cut to collect blood before treating with TAA. And after treated with TAA, rats in all groups were cut tails to collect blood when the HE symptoms occurred in rats of groupⅡ. After gavage treatment, when rats in groupⅡappearedⅢorⅣphase of HE symptoms, all the rats were decapitated and the blood were collected. And the contents of 3-NT in serum were detected by HPLC-fluorescence detection method. The activity of the ALT was detected by Reitman-Frankel. The contents of MDA were detected with the thiobarbituric acid method. And the vitality of SOD in serum was detected using xanthine oxides enzymaic method.2.3. The fabrication and stain of tissue section Liver tissues were obtained after rats in each group were sacrificed to make the paraffin section. And then the pathologic changes were observed by hematoxylin-eosin staining.Results 1. Analysis of clinical biochemical indicator1.1 Results of the detection of 3-NT, SOD and MDA in serum of HE patients Comparing the values of biochemical markers in patients’serum of chronic hepatitis with HE group with chronic hepatitis with non-HE group:The level of 3-NT in the serum of patients in chronic hepatitis with HE group was significantly higher than that in the serum of patients in chronic hepatitis with non-HE group(P<0.01); The activity of SOD in the serum of chronic hepatitis with HE group patients was notably lower than that in the serum of chronic hepatitis with non-HE group patients(P<0.01); The level of MDA in the serum of chronic hepatitis with HE group patients was remarkably higher than that in the serum of chronic hepatitis with non-HE group patients(P<0.01). Comparing the values of biochemical markers in patients’serum of chronic hepatitis with non-HE group with normal control group:the level of 3-NT in the serum of chronic hepatitis with non-HE group was markedly higher than that in the serum of normal control group(P<0.01). The activity of SOD in the serum of chronic hepatitis with non-HE group was obviously lower than that in the serum of normal control group(P<0.01); And the content of MDA in the serum of chronic hepatitis with non-HE group was remarkably higher than that in the serum of normal control group(P<0.01).1.2 Results of the detection of 3-NT, SOD and MDA in serum of patients at different phases of HE patients All the biochemical markers in serum ofⅠ-Ⅱphases andⅢ-Ⅳphases of viral hepatitis patients with HE were significantly different (P<0.01). With the HE symptoms worsened the levels of 3-NT in patients’ serum increased gradually(P<0.05), the activity of SOD was decreased(P<0.05), and the content of MDA increased gradually (P<0.05).1.3 The correlation analysis of 3-NT with SOD and MDA in serum of HE patients There was significant correlation between 3-NT and SOD, MDA in serum of HE patients, and in which the content of 3-NT and the activity of SOD showed negative correlation.2. The general state and the dissection of rats After two days’injection of TAA, the rats presented the HE symptom ofⅠorⅡperiod. After reducing the quantity of TAA according to the condition of each rat, rats could aggravate to the symptom ofⅢorⅣperiod and die at last. After gavaging rats in groupⅢthe development of HE symptoms delayed and the survival time extended. While in groupⅣthe development of HE symptoms almost the same with that of groupⅡ. 3. The analysis of biochemical indexes in serum of rats3.1 HE serum biochemical indicators of self controlled When the symptom of HE appeared, the level of 3-NT in serum of rats suffering from HE was significantly higher (P<0.01) than that before the appearance of HE symptom, the activity of ALT elevated significantly(P<0.01), while the activity of SOD decreased markedly (P<0.05), and the content of MDA in the serum increased notably(P<0.01).3.2 Comparison of biochemical indexes between HE rats and normal rats After the symptoms of HE appeared, the biochemical indexes in serum of groupsⅡ,ⅢandⅣcompared with groupⅠ, it was found that the level of 3-NT in serum of groupsⅡ,ⅢandⅣrats was objectively higher than that was in groupⅠ(P<0.01). The activity of ALT in serum of groupsⅡ,ⅢandⅣrats was obviously higher than that was in groupⅠ(P<0.01). The activity of SOD in serum of groupsⅡ,ⅢandⅣrats was lower than that was in groupⅠ(P<0.05). And the content of MDA in serum of groupsⅡ,ⅢandⅣrats was significantly higher then that was in groupⅠ(P<0.01). The impact to the experiment results from stimulating factors such as the diet and i.p. was ruled out.3.3 Comparison of biochemical indexes in serum of rats in each group after gavage treatment Results of analysis of variance pairwise comparison indicate that after gavaging treatment, the level of 3-NT, ALT and MDA in serum of groupⅡandⅣrats was significantly higher than in groupⅠandⅢrats(P<0.01); while the difference of the level of 3-NT, ALT and MDA between groupⅡandⅣ,ⅠandⅢwas not remarkable(P>0.05); the activity of SOD in serum of groupⅡandⅣrats was significantly lower than in groupⅠandⅢrats(P<0.01); while the difference of the activity of SOD between groupⅡandⅣ,ⅠandⅢwas not remarkable (P>0.05)3.4 Comparison of the biochemical indexes in serum of rats in each group at different stages of the experiment The changes of the biochemical index in serum of rats in groupⅠat different stages of the experiment were not significantly(P>0.05). The comparison of the biochemical indexes in serum of rats in groupⅡandⅣat different stages of the experiment showed that:the contents of 3-NT and MDA increased notably(P<0.01) after rats being treated with TAA. And the activity of ALT was enhanced significantly(P<0.01), while the activity of SOD decreased markedly (P<0.05). Comparing the biochemical indicators in serum after the gavage with before: the levels of 3-NT and MDA increased notably(P<0.01), the activity of ALT was enhanced significantly(P<0.01), and the activity of SOD decreased markedly (P<0.05). The comparison of the biochemical indexes in serum of rats in groupⅢat different stages of the experiment showed that:the contents of 3-NT and MDA increased notably (P<0.01) after rats being treated with TAA. And the activity of ALT was enhanced significantly (P<0.01), while the activity of SOD decreased markedly (P<0.05). Compared the biochemical indicators in serum after the gavage with before:results showed that the levels of 3-NT and MDA decreased notably (P<0.01), the activity of ALT was decreased significantly (P<0.01), and the activity of SOD enhanced markedly (P<0.05).4. The result of histology observation Varying degrees of necrosis, collapse and disappearances were observed in rat liver cells in groupⅡandⅣ. And serious degeneration was existed in liver cells if not necrotic. There were many inflammatory cells in necrotic liver. The higher the HE degree was, the more obvious pathological changes of liver were. As compared groupⅢwith groupⅠ, there were no significant changes with hepatic lobule structure, while the volume of liver cells increased.ConclusionsThis study discussed the possible mechanism of ONOO- in HE from histology and biochemistry points at both clinical and animal experiments, and revealed that the protein nitration caused by abnormal increase of ONOO- might be one of the pathogenesis of HE. It suggested that drugs which inhibiting protein nitration such as ebselen might be benefited to the control of the development of HE.

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