过氧亚硝酸阴离子在肝性脑病中作用的研究
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摘要
目的：
肝性脑病(hepatic encephalopathy, HE)是严重危害人类健康的疾病,有关其发病机制尚未完全明确。众所周知,HE病人血液中内毒素、肿瘤坏死因子升高,这两者均是一氧化氮合酶(nitric oxide synthase, NOS)的促进剂。NOS被激活,促进一氧化氮(nitric oxide, NO)的合成,使体内NO大量增加,而且由于抗氧化防御能力下降,体内超氧阴离子自由基(superoxide anion,O2-·)也常见升高。NO与O2-·会迅速反应生成过氧亚硝酸阴离子(peroxynitrite, ONOO-),该反应几乎不可逆,因而HE病人体内ONOO可能异常增高。由于异常增高的ONOO可特异性使蛋白质酪氨酸(tyrosine, Tyr)线基硝基化而直接影响到蛋白质的功能；可使神经元细胞膜氧化从而导致神经传导功能障碍；可能造成肝、脑细胞线粒体破坏及能量合成障碍等,所以,在HE发病过程中,ONOO的作用不能排除。因此本研究通过对临床肝损伤病人及大鼠HE模型血清氧化／硝基化指标的检测,探讨ONOO在HE的发生发展中的作用,揭示ONOO-的致病机制,为HE病因的研究及其治疗提供新的思路和科学依据。
研究方法：
1.对临床肝损伤病人的研究
1.1研究对象的选取与分组选取同一时期根据卫生部病毒性肝炎诊断标准明确诊断的病毒性肝炎伴HE患者45例和病毒性肝炎非HE患者30例以及同一时期的健康查体者30例,根据疾病严重程度将其分为3组：病毒性肝炎伴HE组,病毒性肝炎非HE组,健康对照组。HE的诊断：病毒性肝炎患者除肝功能严重损伤外,临床上出现一系列无其他原因可解释的神经精神症状,并按下列条件分期：Ⅰ期：前驱期,Ⅱ期：昏迷前期,Ⅲ期：昏睡期,Ⅳ期：昏迷期。
1.2各种生化指标的测定空腹采血,离心分离后取血清。用高效液相色谱-荧光检测法检测血清中3-硝基酪氨酸(3-NT)含量；硫代巴比妥酸法检测血清中丙二醛(MDA)含量；黄嘌呤氧化物酶法检测血清中超氧化物歧化酶(SOD)的活力。
2.对大鼠HE模型的研究
2.1.大鼠HE模型的建立及分组用硫代乙酰胺(Thioacetamide, TAA)建立大鼠HE模型。雄性Wistar大鼠随机分为4组(每组15只)：空白对照组(Ⅰ)、HE模型组(Ⅱ)、依布硒啉处理组(Ⅲ)、溶剂对照组(Ⅳ)。除Ⅰ组外,其它各组第1 d均腹腔注射TAA 300 mg/kg,第2-4 d,每日分别注射TAA 150 mg/kg,直至出现HE症状；Ⅰ组给予等量生理盐水腹腔注射。TAA处理组出现HE症状后,Ⅲ组每天经口灌胃依布硒啉1 mL/100g,Ⅰ组和Ⅱ组灌胃等量生理盐水,Ⅳ组灌胃等量0.5%二甲基亚砜(DMSO)。
2.2.各种生化指标的测定各组大鼠于TAA处理前断尾法取血；采用TAA处理后,TAA处理组大鼠出现Ⅱ期HE症状时,各组大鼠断尾取血；灌胃处理后,在Ⅱ组大鼠出现Ⅲ期或Ⅳ期HE症状后,各组大鼠均断头处死,取血。用高效液相色谱-荧光检测法检测血清中3-NT含量；赖氏法检测血清中ALT的活力；硫代巴比妥酸法检测血清中MDA含量；黄嘌呤氧化物酶法检测血清中SOD的活力。
2.3.组织病理切片的制作及染色各组大鼠断头处死后取肝组织,制作石蜡切片,进行苏木素-伊红染色观察组织病理改变。
结果：
1.临床生化指标分析
1.1 HE病人血清中3-NT、SOD和MDA的检测结果病毒性肝炎伴HE组病人血清生化指标与病毒性肝炎非HE组比较：病毒性肝炎伴HE组病人血清中3-NT的含量明显升高(P<0.01),SOD的活性显著降低(P<0.01),MDA的含量明显升高(P<0.01)。病毒性肝炎非HE组病人血清生化指标与健康对照组比较：病毒性肝炎非HE组病人血清中3-NT的含量显著升高(P<0.01),SOD活性明显降低(P<0.01),MDA的含量显著升高(P<0.01)。
1.2不同时期HE病人血清中3-NT、SOD和MDA的检测结果病毒性肝炎伴HE组病人Ⅰ-Ⅱ期与Ⅲ-Ⅳ期血清中各生化指标的差异均具有显著性(P<0.01)。随HE病情加重,病人血清中3-NT的含量升高(P<0.01),SOD的活性降低(P<0.01),MDA的含量升高(P<0.01)。
1.3 HE病人血清中3-NT和SOD、MDA的相关性分析HE病人血清中3-NT和SOD、3-NT和MDA之间存在明显相关性(P<0.05),其中3-NT含量和SOD活性呈负相关。
2大鼠一般状态及解剖观察腹腔注射TAA 2 d后,即可出现HEⅠ期或Ⅱ期症状。根据每只大鼠的具体情况减量注射TAA后,在3-7 d内,可继续发展至Ⅲ期或Ⅳ期症状直至死亡。Ⅲ组大鼠在出现HE症状后灌胃依布硒啉,发现HE的发展速度减缓、症状减轻,存活时间延长。Ⅳ组大鼠在出现HE症状后灌胃DMSO,其病情进展基本同Ⅱ组。
3大鼠血清生化指标分析
3.1 HE大鼠血清生化指标的自身前后对照出现HE症状后,血清中3-NT的含量较造模前显著增加(P<0.01)；ALT的活性明显增高(P<0.01)；SOD的活性降低(P<0.05),MDA的含量显著增加(P<0.01)。
3.2 HE大鼠与正常大鼠血清生化指标的比较出现HE症状后,Ⅱ组、Ⅲ组和Ⅳ组大鼠各血清指标与Ⅰ组相比：3-NT和MDA的含量均显著高于Ⅰ组(P<0.01)；ALT的活性明显高于Ⅰ组(P<0.01)；SOD的活性显著低于Ⅰ组(P<0.05),排除了饮食及腹腔注射刺激等因素对实验结果的影响。
3.3药物处理后各组大鼠血清生化指标的比较药物处理后,Ⅱ组和Ⅳ组3-NT、ALT和MDA的含量显著高于Ⅰ组和ⅢⅡ组(P<0.01)；Ⅱ组和Ⅳ组、Ⅰ组和Ⅲ组之间各指标的差异无统计学意义(P>0.05)。Ⅱ组和Ⅳ组SOD的活性显著低于Ⅰ组和Ⅲ组(P<0.01),Ⅱ组和Ⅳ组、Ⅰ组和Ⅲ组之间大鼠血清中SOD活性的差异不具有统计学意义(P>0.05)。
3.4各组大鼠不同时期血清生化指标的比较Ⅰ组大鼠在不同HE时期血清生化指标的比较：Ⅰ组大鼠在各种处理前后各血清指标的变化均无统计学意义(P>0.05)；Ⅱ组和Ⅳ组大鼠在不同HE时期血清生化指标分析结果的比较：Ⅱ组和Ⅳ组大鼠在出现HE症状后血清中各生化指标的含量及活性与腹腔注射TAA前比较：3-NT和MDA的含量显著升高(P<0.01),ALT的活性显著增强(P<0.01),SOD的活性降低(P<0.05)；用药后血清各生化指标的含量及活性与用药前比较：3-NT和MDA的含量显著升高(P<0.01),ALT的活性明显增强(P<0.01),SOD的活性降低(P<0.05)；Ⅲ组大鼠在不同HE时期血清生化指标检测结果的比较：Ⅲ组大鼠出现HE症状后血清中各生化指标的含量及活性与腹腔注射TAA前相比：3-NT和MDA的含量显著升高(P<0.01),ALT的活性显著增强(P<0.01),SOD的活性降低(P<0.05)；用药后血清中各生化指标的含量及活性与用药前相比：3-NT和MDA的含量显著降低(P<0.01),ALT的活性明显降低(P<0.01),SOD的活性增强(P<0.05)。
4.大鼠病理学观察结果Ⅱ组和Ⅳ组大鼠肝细胞有不同程度的坏死、崩解及消失,未坏死细胞也有严重变性,坏死组织中有较多的炎性细胞浸润。随HE分期越高,肝脏的病理改变越明显。Ⅲ组肝细胞形态与Ⅰ组相比,肝细胞体积增大,肝小叶结构未见明显改变。
结论：
本次实验从临床和动物实验两个方面,从生化和组织病理学角度初步探讨了ONOO在HE发病中的作用,ONOO的异常增多导致蛋白质硝基化可能是HE的发病机制之一,提示ONOO-的清除剂如依布硒啉等有益于控制病情。
Objective
The 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.
Methods
1. Clinical study of patients with liver injury
1.1 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 rats
2.1. 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 indicator
1.1 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 rats
3.1 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.
Conclusions
This 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.
肝性脑病(hepatic encephalopathy, HE)是严重危害人类健康的疾病,有关其发病机制尚未完全明确。众所周知,HE病人血液中内毒素、肿瘤坏死因子升高,这两者均是一氧化氮合酶(nitric oxide synthase, NOS)的促进剂。NOS被激活,促进一氧化氮(nitric oxide, NO)的合成,使体内NO大量增加,而且由于抗氧化防御能力下降,体内超氧阴离子自由基(superoxide anion,O2-·)也常见升高。NO与O2-·会迅速反应生成过氧亚硝酸阴离子(peroxynitrite, ONOO-),该反应几乎不可逆,因而HE病人体内ONOO可能异常增高。由于异常增高的ONOO可特异性使蛋白质酪氨酸(tyrosine, Tyr)线基硝基化而直接影响到蛋白质的功能；可使神经元细胞膜氧化从而导致神经传导功能障碍；可能造成肝、脑细胞线粒体破坏及能量合成障碍等,所以,在HE发病过程中,ONOO的作用不能排除。因此本研究通过对临床肝损伤病人及大鼠HE模型血清氧化／硝基化指标的检测,探讨ONOO在HE的发生发展中的作用,揭示ONOO-的致病机制,为HE病因的研究及其治疗提供新的思路和科学依据。
研究方法：
1.对临床肝损伤病人的研究
1.1研究对象的选取与分组选取同一时期根据卫生部病毒性肝炎诊断标准明确诊断的病毒性肝炎伴HE患者45例和病毒性肝炎非HE患者30例以及同一时期的健康查体者30例,根据疾病严重程度将其分为3组：病毒性肝炎伴HE组,病毒性肝炎非HE组,健康对照组。HE的诊断：病毒性肝炎患者除肝功能严重损伤外,临床上出现一系列无其他原因可解释的神经精神症状,并按下列条件分期：Ⅰ期：前驱期,Ⅱ期：昏迷前期,Ⅲ期：昏睡期,Ⅳ期：昏迷期。
1.2各种生化指标的测定空腹采血,离心分离后取血清。用高效液相色谱-荧光检测法检测血清中3-硝基酪氨酸(3-NT)含量；硫代巴比妥酸法检测血清中丙二醛(MDA)含量；黄嘌呤氧化物酶法检测血清中超氧化物歧化酶(SOD)的活力。
2.对大鼠HE模型的研究
2.1.大鼠HE模型的建立及分组用硫代乙酰胺(Thioacetamide, TAA)建立大鼠HE模型。雄性Wistar大鼠随机分为4组(每组15只)：空白对照组(Ⅰ)、HE模型组(Ⅱ)、依布硒啉处理组(Ⅲ)、溶剂对照组(Ⅳ)。除Ⅰ组外,其它各组第1 d均腹腔注射TAA 300 mg/kg,第2-4 d,每日分别注射TAA 150 mg/kg,直至出现HE症状；Ⅰ组给予等量生理盐水腹腔注射。TAA处理组出现HE症状后,Ⅲ组每天经口灌胃依布硒啉1 mL/100g,Ⅰ组和Ⅱ组灌胃等量生理盐水,Ⅳ组灌胃等量0.5%二甲基亚砜(DMSO)。
2.2.各种生化指标的测定各组大鼠于TAA处理前断尾法取血；采用TAA处理后,TAA处理组大鼠出现Ⅱ期HE症状时,各组大鼠断尾取血；灌胃处理后,在Ⅱ组大鼠出现Ⅲ期或Ⅳ期HE症状后,各组大鼠均断头处死,取血。用高效液相色谱-荧光检测法检测血清中3-NT含量；赖氏法检测血清中ALT的活力；硫代巴比妥酸法检测血清中MDA含量；黄嘌呤氧化物酶法检测血清中SOD的活力。
2.3.组织病理切片的制作及染色各组大鼠断头处死后取肝组织,制作石蜡切片,进行苏木素-伊红染色观察组织病理改变。
结果：
1.临床生化指标分析
1.1 HE病人血清中3-NT、SOD和MDA的检测结果病毒性肝炎伴HE组病人血清生化指标与病毒性肝炎非HE组比较：病毒性肝炎伴HE组病人血清中3-NT的含量明显升高(P<0.01),SOD的活性显著降低(P<0.01),MDA的含量明显升高(P<0.01)。病毒性肝炎非HE组病人血清生化指标与健康对照组比较：病毒性肝炎非HE组病人血清中3-NT的含量显著升高(P<0.01),SOD活性明显降低(P<0.01),MDA的含量显著升高(P<0.01)。
1.2不同时期HE病人血清中3-NT、SOD和MDA的检测结果病毒性肝炎伴HE组病人Ⅰ-Ⅱ期与Ⅲ-Ⅳ期血清中各生化指标的差异均具有显著性(P<0.01)。随HE病情加重,病人血清中3-NT的含量升高(P<0.01),SOD的活性降低(P<0.01),MDA的含量升高(P<0.01)。
1.3 HE病人血清中3-NT和SOD、MDA的相关性分析HE病人血清中3-NT和SOD、3-NT和MDA之间存在明显相关性(P<0.05),其中3-NT含量和SOD活性呈负相关。
2大鼠一般状态及解剖观察腹腔注射TAA 2 d后,即可出现HEⅠ期或Ⅱ期症状。根据每只大鼠的具体情况减量注射TAA后,在3-7 d内,可继续发展至Ⅲ期或Ⅳ期症状直至死亡。Ⅲ组大鼠在出现HE症状后灌胃依布硒啉,发现HE的发展速度减缓、症状减轻,存活时间延长。Ⅳ组大鼠在出现HE症状后灌胃DMSO,其病情进展基本同Ⅱ组。
3大鼠血清生化指标分析
3.1 HE大鼠血清生化指标的自身前后对照出现HE症状后,血清中3-NT的含量较造模前显著增加(P<0.01)；ALT的活性明显增高(P<0.01)；SOD的活性降低(P<0.05),MDA的含量显著增加(P<0.01)。
3.2 HE大鼠与正常大鼠血清生化指标的比较出现HE症状后,Ⅱ组、Ⅲ组和Ⅳ组大鼠各血清指标与Ⅰ组相比：3-NT和MDA的含量均显著高于Ⅰ组(P<0.01)；ALT的活性明显高于Ⅰ组(P<0.01)；SOD的活性显著低于Ⅰ组(P<0.05),排除了饮食及腹腔注射刺激等因素对实验结果的影响。
3.3药物处理后各组大鼠血清生化指标的比较药物处理后,Ⅱ组和Ⅳ组3-NT、ALT和MDA的含量显著高于Ⅰ组和ⅢⅡ组(P<0.01)；Ⅱ组和Ⅳ组、Ⅰ组和Ⅲ组之间各指标的差异无统计学意义(P>0.05)。Ⅱ组和Ⅳ组SOD的活性显著低于Ⅰ组和Ⅲ组(P<0.01),Ⅱ组和Ⅳ组、Ⅰ组和Ⅲ组之间大鼠血清中SOD活性的差异不具有统计学意义(P>0.05)。
3.4各组大鼠不同时期血清生化指标的比较Ⅰ组大鼠在不同HE时期血清生化指标的比较：Ⅰ组大鼠在各种处理前后各血清指标的变化均无统计学意义(P>0.05)；Ⅱ组和Ⅳ组大鼠在不同HE时期血清生化指标分析结果的比较：Ⅱ组和Ⅳ组大鼠在出现HE症状后血清中各生化指标的含量及活性与腹腔注射TAA前比较：3-NT和MDA的含量显著升高(P<0.01),ALT的活性显著增强(P<0.01),SOD的活性降低(P<0.05)；用药后血清各生化指标的含量及活性与用药前比较：3-NT和MDA的含量显著升高(P<0.01),ALT的活性明显增强(P<0.01),SOD的活性降低(P<0.05)；Ⅲ组大鼠在不同HE时期血清生化指标检测结果的比较：Ⅲ组大鼠出现HE症状后血清中各生化指标的含量及活性与腹腔注射TAA前相比：3-NT和MDA的含量显著升高(P<0.01),ALT的活性显著增强(P<0.01),SOD的活性降低(P<0.05)；用药后血清中各生化指标的含量及活性与用药前相比：3-NT和MDA的含量显著降低(P<0.01),ALT的活性明显降低(P<0.01),SOD的活性增强(P<0.05)。
4.大鼠病理学观察结果Ⅱ组和Ⅳ组大鼠肝细胞有不同程度的坏死、崩解及消失,未坏死细胞也有严重变性,坏死组织中有较多的炎性细胞浸润。随HE分期越高,肝脏的病理改变越明显。Ⅲ组肝细胞形态与Ⅰ组相比,肝细胞体积增大,肝小叶结构未见明显改变。
结论：
本次实验从临床和动物实验两个方面,从生化和组织病理学角度初步探讨了ONOO在HE发病中的作用,ONOO的异常增多导致蛋白质硝基化可能是HE的发病机制之一,提示ONOO-的清除剂如依布硒啉等有益于控制病情。
Objective
The 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.
Methods
1. Clinical study of patients with liver injury
1.1 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 rats
2.1. 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 indicator
1.1 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 rats
3.1 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.
Conclusions
This 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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48. Gardner P. R., Fridovich L. Superoxide sensitivity of the Escherichia coli 6-phosphogluconate dehydratase. J. Biol. Chem.,1991,266:1478-1483.
49. Squadrito G. L., Pryor W. A. The formation of peroxynitrite invivo from nitricoxide and superoxide. Chem. Biol. Interact.,1995,96:203-206.
50. Beckman J. S., Beckman T. W., Chen J., et al.. Apparent hydroxyl radical production by peroxynitrite:implications for endothelial injury form nitric oxide and superoxide. Proc. Natl. Acad. Sci. USA,1990,87:1620-1624.
51. Merenyi G.,Lind J. Free radical formation in the peroxynitrous acid (ONOOH)/ peroxynitrite (ONOO") system. Chem. Res. Toxicol.,1998,11:243-246.
52. Kissner R., Koppenol W. H. Product distribution of peroxynitrite decay as a function of pH, temperature and concentration. J. Am. Chem. Soc.,2002,124: 234-239.
53. Khan A. U., Kovacic D., Kolbanovskiy A., et al.. The decomposition of peroxy-nitrite to nitroxyl anion (NO2) and singlet oxygen in aqueous solution. Proc. Natl. Acad. Sci. USA,2000,97:2984-2989.
54. Merenyi G., Lind J.,Czapski G., et al.. The decomposition of peroxynitrite does not yield nitroxyl anion and singlet oxygen. Proc. Natl. Acad. Sci. USA,2000,97: 8216-8218.
55. Martinez G. R., Mascio P. Di, Bonini M. G., et al. Peroxynitrite does not decomp-ose to singlet oxygen and nitroxyl. Proc. Natl. Acad. Sci.USA,2000,97:10307-10312.
56. Mascio P. Di, Bechara E. J. H., Medeiros M. H. G., Briviba K., Sies H. Singlet molecular oxygen production in the reaction of peroxynitrite with hydrogen peroxide. FEBS Lett.,1994,355:287-289.
57. Huang RF et al. J Nutr,2002,132(8):2151-2156.
58. Reiter RJ et al. Ann N Y Acad Sci,2001,939:200-215.
59. KohnenS.L., Mouithys, MickaladA.A., et al.. Investigation of the reaction of per-oxynitrite with propofolatacid pH:predominant production of oxidized, nitrated and halogenated derivatives. Nitric Oxide.2003,8:170-181.
60. Radi R, Cassina A, Hodara R. Nitric oxide and peroxynitrite interactions with mitochondria. BiolChem.2002,383:401-409.
61. Rhee, S. G., Chae, H. Z.& Kim, K. Peroxiredoxins:a historical overview and speculative preview of novel mechanisms and emerging concepts in cell signaling. Free Radic. Biol. Med.2005,38:1543-1552.
62.陈敏,李强,陈彬.过氧亚硝基阴离子的研究进展.《生命的化学》,2003,23(6)：465-467.
63. Virag L, Szabo E, Gergely P, et al.. Peroxynitrite-induced cytotoxicity:mechani-sm and opportunities for intervention. Toxicol Lett,2003,140/141:113-124.
64. Radi R., Cassina, A., HodaraR., et al.. Peroxynitrite reactions and formation in mitochondria. FreeRadic. Biol.Med.,2002,33:1451-1464.
65. Schopfer F., Riobo N., Carreras M. C., et al., Oxidation of ubiquinol by peroxy-nitrite:implications for protection of mitochondria against nitrosative damage. Biochem.J.,2000,349:35-42.
66. Brown G. C. Regulation of mitochondrial respiration by nitric oxide inhibition of cytochrome c oxidase. Biochim. Biophys. Acta,2001,1504:46-57.
67. PAL PACHER, JOSEPH S. BECKMAN, and LUCAS LIAUDET. Nitric Oxide and Peroxynitrite in Health and Disease. NIH Public Access Author Manuscript,2007, 87(1):315-424.
68. Moncada S., Erusalimsky J. D. Does nitricoxide modulate mitochondrial energy generation and apoptosis? Nat. Rev. Mol. Cell Biol.,2002,3:214-220.
69. SaekiM., MaedaS., WadaK., KamisakiY. Insulin-like growth factor-1 protects per-oxynitrite-induced cell death by preventing cytochrome c-induced caspase-3 activation. J. Cell. Biochem.,2002,84:708-716.
70. Radi R. Proc. Natl. Acad. Sci. USA,2004,101:4003-4008.
71. Radi R., Peluffo G., Alvarez M. N., et, al.. Unraveling peroxynitrite formation in biological systems, Free Radic. Biol. Med.,2001,30:463-488.
72.池泉,黄开勋.蛋白质酪氨酸硝化的研究.化学进展,2006,18(7／8)：1019-1025.
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80. King P A, Anders on V E, Edwards J O, et al.. A stable s olid that generates hydr oxyl radical diss oluti ons in aqueous s olutions:reaction with p r oteins and nucleic acid. J Am Chem Soc,1992,114:5430-5432.
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83. Zingarelli B, O'Connor M, Wong H, et al.. Peroxynitrite-mediated DNA strand breakage activates poly ADP-ribosyl synthetase and causes cellular energy depletion in macrophages stimulated with bacterial lipopolysaccharide. J Imm unol,1996,156 (1):350-358
84. Szabo C. Multiple pathways of peroxynitrite cytotoxicity. Toxicol. Lett.,2003, 140-141,105-112.
85. Jagtap, P.& Szabo, C. Poly (ADP-ribose) polymerase and the therapeutic effects of its inhibitors. Nature Rev. Drug Discov.,2005,4,421-440.
86. Virag, L., Szabo, E., Gergely, P. et al.. Peroxynitrite-induced cytotoxicity:mecha-nism and opportunities for intervention. Toxicol. Lett.,2003,140-141,113-124
87. Shacka, J. J. et al. Two distinct signaling pathways regulate peroxynitrite-induced apoptosis in PC12 cells. Cell Death Differ.,2006,13,1506-1514.
88. Vicente, S. et al. NO and peroxynitrite induce cellular death in bovine chromaffin cells:evidence for a mixed necrotic and apoptotic mechanism with caspases activation. J. Neurosci. Res.,2006,84,78-96.
89. Borutaite, V., Morkuniene, R.& Brown, G. C. Release of cytochrome c from heart mitochondria is induced by high Ca2+ and peroxynitrite and is responsible for Ca(2+)-induced inhibition of substrate oxidation. Biochim. Biophys. Acta,1999,1453, 41-48.
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92. Dickhout, J. G. et al. Peroxynitrite causes endoplasmic reticulum stress and apo-ptosis in human vascular endothelium:implications in atherogenesis. Arterioscler. Thromb. Vasc. Biol.,2005,25,2623-2629.
93. Redondo, P. C. et al. Hydrogen peroxide and peroxynitrite enhance Ca2+ mobili-zation and aggregation in platelets from type 2 diabetic patients. Biochem. Biophys. Res. Commun.,2005,333,794-802.
94. Whiteman, M. et al. Peroxynitrite mediates calcium-dependent mitochondrial dys-function and cell death via activation of calpains. FASEB J.,2004,18,1395-1397.
95. Zhang, X. et al. Intranuclear localization of apoptosis-inducing factor (AIF) and large scale DNA fragmentation after traumatic brain injury in rats and in neuronal cultures exposed to peroxynitrite. J. Neurochem.,2002,82,181-191.
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47. Popov D, Costache G, Georgescu A, et al. Beneficial effects of L-arginine supple-mentation in experimental hyperlipemia-hyperglycemia in the hamster. Cell Tissue Res,2002,308(1):109-120.
48. Gardner P. R., Fridovich L. Superoxide sensitivity of the Escherichia coli 6-phosphogluconate dehydratase. J. Biol. Chem.,1991,266:1478-1483.
49. Squadrito G. L., Pryor W. A. The formation of peroxynitrite invivo from nitricoxide and superoxide. Chem. Biol. Interact.,1995,96:203-206.
50. Beckman J. S., Beckman T. W., Chen J., et al.. Apparent hydroxyl radical production by peroxynitrite:implications for endothelial injury form nitric oxide and superoxide. Proc. Natl. Acad. Sci. USA,1990,87:1620-1624.
51. Merenyi G.,Lind J. Free radical formation in the peroxynitrous acid (ONOOH)/ peroxynitrite (ONOO") system. Chem. Res. Toxicol.,1998,11:243-246.
52. Kissner R., Koppenol W. H. Product distribution of peroxynitrite decay as a function of pH, temperature and concentration. J. Am. Chem. Soc.,2002,124: 234-239.
53. Khan A. U., Kovacic D., Kolbanovskiy A., et al.. The decomposition of peroxy-nitrite to nitroxyl anion (NO2) and singlet oxygen in aqueous solution. Proc. Natl. Acad. Sci. USA,2000,97:2984-2989.
54. Merenyi G., Lind J.,Czapski G., et al.. The decomposition of peroxynitrite does not yield nitroxyl anion and singlet oxygen. Proc. Natl. Acad. Sci. USA,2000,97: 8216-8218.
55. Martinez G. R., Mascio P. Di, Bonini M. G., et al. Peroxynitrite does not decomp-ose to singlet oxygen and nitroxyl. Proc. Natl. Acad. Sci.USA,2000,97:10307-10312.
56. Mascio P. Di, Bechara E. J. H., Medeiros M. H. G., Briviba K., Sies H. Singlet molecular oxygen production in the reaction of peroxynitrite with hydrogen peroxide. FEBS Lett.,1994,355:287-289.
57. Huang RF et al. J Nutr,2002,132(8):2151-2156.
58. Reiter RJ et al. Ann N Y Acad Sci,2001,939:200-215.
59. KohnenS.L., Mouithys, MickaladA.A., et al.. Investigation of the reaction of per-oxynitrite with propofolatacid pH:predominant production of oxidized, nitrated and halogenated derivatives. Nitric Oxide.2003,8:170-181.
60. Radi R, Cassina A, Hodara R. Nitric oxide and peroxynitrite interactions with mitochondria. BiolChem.2002,383:401-409.
61. Rhee, S. G., Chae, H. Z.& Kim, K. Peroxiredoxins:a historical overview and speculative preview of novel mechanisms and emerging concepts in cell signaling. Free Radic. Biol. Med.2005,38:1543-1552.
62.陈敏,李强,陈彬.过氧亚硝基阴离子的研究进展.《生命的化学》,2003,23(6)：465-467.
63. Virag L, Szabo E, Gergely P, et al.. Peroxynitrite-induced cytotoxicity:mechani-sm and opportunities for intervention. Toxicol Lett,2003,140/141:113-124.
64. Radi R., Cassina, A., HodaraR., et al.. Peroxynitrite reactions and formation in mitochondria. FreeRadic. Biol.Med.,2002,33:1451-1464.
65. Schopfer F., Riobo N., Carreras M. C., et al., Oxidation of ubiquinol by peroxy-nitrite:implications for protection of mitochondria against nitrosative damage. Biochem.J.,2000,349:35-42.
66. Brown G. C. Regulation of mitochondrial respiration by nitric oxide inhibition of cytochrome c oxidase. Biochim. Biophys. Acta,2001,1504:46-57.
67. PAL PACHER, JOSEPH S. BECKMAN, and LUCAS LIAUDET. Nitric Oxide and Peroxynitrite in Health and Disease. NIH Public Access Author Manuscript,2007, 87(1):315-424.
68. Moncada S., Erusalimsky J. D. Does nitricoxide modulate mitochondrial energy generation and apoptosis? Nat. Rev. Mol. Cell Biol.,2002,3:214-220.
69. SaekiM., MaedaS., WadaK., KamisakiY. Insulin-like growth factor-1 protects per-oxynitrite-induced cell death by preventing cytochrome c-induced caspase-3 activation. J. Cell. Biochem.,2002,84:708-716.
70. Radi R. Proc. Natl. Acad. Sci. USA,2004,101:4003-4008.
71. Radi R., Peluffo G., Alvarez M. N., et, al.. Unraveling peroxynitrite formation in biological systems, Free Radic. Biol. Med.,2001,30:463-488.
72.池泉,黄开勋.蛋白质酪氨酸硝化的研究.化学进展,2006,18(7／8)：1019-1025.
73. Pfeiffer S., Schmidt K., Mayer B. Dityrosine formation outcompetes tyrosine nitration at low steady-state concentrations of perox-ynitrite:implications for tyrosine modification by nitric oxide/superoxide in vivo. J. Biol. Chem.,2000,275:6346-6352.
74. Pfeiffer S., Lass A., Schmidt K., Mayer B. Protein tyrosine nitration in mouse peritoneal macrophages activated in vitro and in vivo:evidence against an essential role of peroxynitrite. FASEB J.,2001,15:2355-2364.
75. Amici M., Lupidi G., Angeletti M., et al. Peroxynitrite induced oxidation and its effects on isolated proteasomal systems. Free Radic. Biol. Med.,2003,34:987-996.
76. Ford E., Hughes M. N., Wardman P. Kinetics of the reactions of nitrogen dioxide with glutathione cysteine and uric acid at physiological Ph. Free Radic. Biol. Med., 2002,32:1314-1323.
77. Csaba S, Hir oshi O. DNA damage induced by peroxynitrite subsequent biologi-cal effects. Nitric Oxide:Biology and Chemistry,1997,1 (5):373-385.
78. Steenken S, Jovanovic S V. How easily oxidizable is DNA? One-electron reduc-tion potentials of adenosine and guanosine radicals in aqueous solution. J Am Chem Soc,1997,119 (3):617-618.
79. Samar B, Jacquin C, Niles P C, et al, DNA damage in deoxynucleosides and olig-onucleotides treated with peroxynitrite. Chem Res Toxicol,1999,12 (6):513-520.
80. King P A, Anders on V E, Edwards J O, et al.. A stable s olid that generates hydr oxyl radical diss oluti ons in aqueous s olutions:reaction with p r oteins and nucleic acid. J Am Chem Soc,1992,114:5430-5432.
81. Salgo M G, Stone K, Squadrit o G L, et al.. Peroxynitrite causes DNA nicks in plasmid pBR322. B iochem B iophys Res Commun,1995,210 (3):1025-1030.
82. Maria G S, EliezerB, Giuseppe L, et al. DNA damage and oxidation of thiols peroxynitrite causes in rat thymocytes. A rchives B iochem B iophy,1995,322 (2): 500-505.
83. Zingarelli B, O'Connor M, Wong H, et al.. Peroxynitrite-mediated DNA strand breakage activates poly ADP-ribosyl synthetase and causes cellular energy depletion in macrophages stimulated with bacterial lipopolysaccharide. J Imm unol,1996,156 (1):350-358
84. Szabo C. Multiple pathways of peroxynitrite cytotoxicity. Toxicol. Lett.,2003, 140-141,105-112.
85. Jagtap, P.& Szabo, C. Poly (ADP-ribose) polymerase and the therapeutic effects of its inhibitors. Nature Rev. Drug Discov.,2005,4,421-440.
86. Virag, L., Szabo, E., Gergely, P. et al.. Peroxynitrite-induced cytotoxicity:mecha-nism and opportunities for intervention. Toxicol. Lett.,2003,140-141,113-124
87. Shacka, J. J. et al. Two distinct signaling pathways regulate peroxynitrite-induced apoptosis in PC12 cells. Cell Death Differ.,2006,13,1506-1514.
88. Vicente, S. et al. NO and peroxynitrite induce cellular death in bovine chromaffin cells:evidence for a mixed necrotic and apoptotic mechanism with caspases activation. J. Neurosci. Res.,2006,84,78-96.
89. Borutaite, V., Morkuniene, R.& Brown, G. C. Release of cytochrome c from heart mitochondria is induced by high Ca2+ and peroxynitrite and is responsible for Ca(2+)-induced inhibition of substrate oxidation. Biochim. Biophys. Acta,1999,1453, 41-48.
90. Richter, C., Schweizer, M.& Ghafourifar P. Mitochondria, NO, and peroxynitrite. Methods Enzymol.1999,301,381-393.
91. Virag, L. et al. Requirement of intracellular calcium mobilization for peroxyni-trite-induced poly (ADP-ribose) synthetase activation and cytotoxicity. Mol. Pharma-col.,1999,56,824-833.
92. Dickhout, J. G. et al. Peroxynitrite causes endoplasmic reticulum stress and apo-ptosis in human vascular endothelium:implications in atherogenesis. Arterioscler. Thromb. Vasc. Biol.,2005,25,2623-2629.
93. Redondo, P. C. et al. Hydrogen peroxide and peroxynitrite enhance Ca2+ mobili-zation and aggregation in platelets from type 2 diabetic patients. Biochem. Biophys. Res. Commun.,2005,333,794-802.
94. Whiteman, M. et al. Peroxynitrite mediates calcium-dependent mitochondrial dys-function and cell death via activation of calpains. FASEB J.,2004,18,1395-1397.
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