大鼠酒精性肝损伤模型及蛋白表达指纹图谱的建立

目的:建立大鼠酒精性肝损伤模型,以蛋白质芯片技术为实验手段对损伤过程中的大鼠血清及肝脏蛋白质变化进行观察,以期获得损伤过程中的蛋白标志物,建立损伤过程蛋白表达指纹图谱;观察有机硒对大鼠肝脏损伤的防护作用。方法:用食用酒精辅以橄榄油对大鼠进行肝损伤,动态检测大鼠肝重比、血清生化指标,并进行肝组织病理切片观察;应用表面加强激光电离解吸飞行时间质谱技术对大鼠肝损伤过程进行跟踪检测,获得蛋白标志物,同时证实有机硒对肝损伤具有一定的防护作用。结果:肝重比、生化指标及病理切片均表明损伤模型已基本建立,同时验证了有机硒的肝损伤保护作用。在12周的损伤过程中,发现大鼠血清中有4个具有统计学意义的蛋白标志物,其相对分子质量(Mr)分别为7010、8307、11624和14041;在肝组织中发现了2个有统计意义的蛋白标志物,其Mr分别为5931.2和7104.6。结论:建立了大鼠酒精性肝损伤模型,获得了血清及肝脏蛋白标志物,为后期进行临床实验提供了可行的研究方法;同时为有机硒应用于临床进行肝损伤防护奠定了良好的实验基础。     

硒酸精氨酸对小鼠酒精性肝损伤的保护作用

目的探讨硒酸精氨酸对小鼠酒精性肝损伤的保护作用。方法用连续灌服酒精的方法建立小鼠酒精性肝损伤模型。将60只雄性小鼠随机分为四组：对照组,模型组,硒酸精氨酸处理组和二氧化硒处理组。连续灌胃8周后,测定小鼠血清中天冬氨酸氨基转移酶（AST）,丙氨酸转移酶（ALT）,肝匀浆中超氧化物歧化酶（SOD）、谷胱甘肽过氧化物酶（GPx）、谷胱甘肽（GSH）的含量。对肝脏切片做HE染色和Masson染色,在光镜下观察肝脏病理学变化。结果硒酸精氨酸能显著降低酒精性肝损伤引起的AST的升高（P〈0．01）,有效保持了肝脏中GPx和SOD的活性（P〈0．01）。病理学观察结果与酶学变化相一致,硒酸精氨酸对肝脏的保护作用明显优于二氧化硒。结论硒酸精氨酸对小鼠酒精性肝损伤具有显著的保护作用,效果也优于二氧化硒。     

小鼠骨骼肌电转移hPON1 Q基因可以减轻CCl4诱导的急性肝损伤

目的 研究人Q型对氧磷酶1（human paraoxonase 1 Q,hPON1Q）转基因表达对小鼠四氯化碳（carbon tetrachloride,CCl4）诱导急性肝损伤的缓解效果,为防治肝脏疾病寻找新的途径.方法 小鼠骨骼肌直接注射含hPON1Q的真核表达质粒裸DNA并用电刺激介导表达,测量血清芳香酯酶的活性变化显示hPON1Q转基因表达效果,并使用血清谷丙转氨酶（ALT）、谷草转氨酶（AST）为指标及肝组织病理切片检测肝损伤的程度.结果 hPON1Q转基因表达小鼠血清中芳香酯酶活性提高约50%,并可持续到16 d以后.使用PON1裸DNA电刺激治疗组比对照组小鼠在用CCl4诱导24 h后血清芳香酯酶活性高60%,两种血清转氨酶指标及肝组织切片的病理学分析表明肝脏损伤程度有明显的减轻.结论 电刺激介导的重组人PON1Q基因裸DNA在小鼠体内的表达对CCl4诱导的肝损伤具有显著的防护作用.     

海珠益肝胶囊对免疫性肝损伤模型小鼠的保护作用

目的：观察海珠益肝胶囊对卡介苗（BCG）加脂多糖（LPS）诱导的小鼠免疫性肝损伤的防护作用。方法：采用卡介苗（BCG）加脂多糖（LPS）诱导小鼠免疫性肝损伤,通过检测小鼠的血清谷丙转氨酶（ALT）和谷草转氨酶（AST）活性及肝脏病理变化来研究海珠益肝胶囊的保肝功能。结果：海珠益肝胶囊防治组小鼠血清ALT及AST活性比模型组显著降低,两组比较,差异有统计学意义。海珠益肝胶囊可明显减轻肝组织病理损伤,以大剂量组作用最佳;海珠益肝胶囊的使用使免疫性肝损伤小鼠肝细胞凋亡减少,且有剂量依赖关系。结论：海珠益肝胶囊对BCG加LPS诱导小鼠产生免疫性肝炎的模型免疫性肝损伤具有显著的保护作用。     

利用竞争型ELISA法鉴定硒诱导的金属硫蛋白

利用竞争型ELISA法鉴定硒诱导的金属硫蛋白 (MT) ,发现对照组小鼠肝脏MT含量为 (2 .47± 0 .90 )μg/g湿重组织 ,硫酸锌组小鼠肝脏MT含量为 (8.15± 2 .2 0 ) μg/ g湿重组织 ,硒麦芽组小鼠肝脏MT含量为(12 .80± 1.44 ) μg/ g湿重组织。锌组和硒组的MT含量与对照组相比有显著性差异 (P <0 .0 5 ,P <0 .0 5 )。硒组MT含量要显著高于锌组的MT含量 (P <0 .0 5 )。     

冬虫夏草菌丝提取物对化学性肝损伤的辅助保护作用

为明确冬虫夏草菌丝提取物对急性肝损伤小鼠谷丙转氨酶（ALT）、谷草转氨酶（AST）、肝细胞变性及坏死程度的影响,采用四氯化碳（CCl4）诱导小鼠急性化学性肝损伤模型,将动物随机分成5组,分别是空白对照组、模型组、冬虫夏草菌丝提取物低剂量组（1.11g/kg BW）、中剂量组（3.33g/kg BW）、高剂量组（10.00g/kg BW）,检测血清ALT、AST值,并取肝脏作病理切片,观察肝脏的病理损伤情况。冬虫夏草菌丝提取物高剂量组能明显降低CCl4急性肝损伤小鼠血清ALT值,减轻肝细胞坏死程度,表明冬虫夏草菌丝提取物对化学性肝损伤有辅助保护功能。     

不同应激因子对小鼠肝脏金属硫蛋白诱导合成的影响

目的筛选出小鼠肝脏金属硫蛋白（MT）合成量最大的诱导方式。方法从时间-效应和剂量-效应两方面研究了重金属元素（Cd）、微量元素（Zn）、重金属与微量元素的组合（Cd＋Zn）、生理因子（饥饿）及创伤因子等五大类组合应激因子、19种诱导方式对小鼠肝脏中MT诱导合成的影响及效果。结果生理因子诱导MT量最小,饥饿诱导小鼠肝脏MT的量随饥饿程度的加重而增加,但各组间差异不显著（P〉0.05）;创伤因子诱导产生MT的量最高,其诱导量随创伤恢复时间的增加而降低,各组之间差异显著（P〈0.01）,本实验诱导峰值（9.0241±0.6441μmol/g）出现在创伤后6 h;重金属元素和微量元素诱导量居中,且两者混合诱导量比单独诱导量之和要大。结论成功筛选出诱导小鼠肝脏MT合成最有效的因子和最佳时间,为进一步大量合成MT及研究其功能等奠定基础。     

硒酸赖氨酸对四氧嘧啶诱发的小鼠肝损伤的防护作用

目的:研究硒酸赖氨酸对四氧嘧啶诱发的小鼠肝损伤的防护作用。方法:选取昆明小鼠50只,雌雄各半,随机分成五组,即对照组、模型组、低剂量组、中剂量组、高剂量组。采用四氧嘧啶致急性肝损伤模型,检测各组小鼠血清中谷丙转氨酶(ALT)、谷草转氨酶(AST)、碱性磷酸酶(AKP)活性,并对各组小鼠肝脏进行组织病理学观察。结果:硒酸赖氨酸能降低小鼠血清中ALT、AST、AKP活性(P<0.05或P<0.01),明显减轻四氧嘧啶致肝损伤小鼠肝细胞的病变及炎症反应。结论:硒酸赖氨酸具有对四氧嘧啶诱发小鼠肝损伤的保护作用。     

自噬抑制剂氯喹对急性酒精诱导小鼠肝损伤的影响

目的：探讨自噬抑制剂氯喹（CQ）对急性酒精诱导肝损伤的影响及其作用机制。方法：将雄性C57BL/6小鼠随机分为3组：正常对照组、酒精组、氯喹干预组（n=7）,其中酒精组按4.5 g/kg剂量给予33%（V/V）酒精灌胃。HE和油红O染色检测各组小鼠肝组织脂滴变化;检测肝组织甘油三酯（TG）含量变化;检测血清谷草转氨酶（AST）和谷丙转氨酶（ALT）活性;免疫荧光法检测微管相关蛋白轻链3（LC3）蛋白变化;Western blot法检测LC3蛋白和核蛋白P65表达的变化;ELISA法检测促炎因子TNF-α、IL-6的变化。结果：与对照组比较,酒精组脂滴形成、TG含量、血清AST和ALT活性明显增高。与对照组比较,酒精组LC3-Ⅱ蛋白表达明显增加;与酒精组比较,氯喹干预组使酒精诱导的LC3-Ⅱ蛋白表达增强进一步加剧,使酒精诱导的TG含量、血清AST和ALT活性进一步增高,同时增加了酒精诱导的p65入核及TNFα、IL-6释放。结论：急性酒精能引起小鼠肝脏脂肪变化及炎症,而自噬抑制剂氯喹抑制自噬进程,加剧酒精诱导的肝损伤,说明自噬在酒精诱导肝损伤中可能具有保护效应。     

虫草素对四氯化碳所致小鼠急性肝损伤的保护作用

本文探究蛹虫草活性成分虫草素对四氯化碳（CCl₄）造成的小鼠急性肝损伤的保护作用及其分子机制。首先建立四氯化碳致小鼠急性肝损伤的动物模型,通过检测血清生化指标、肝功指标的变化及HE染色观察组织切片病理的病变情况,评价虫草素的保肝效果,进一步通过Western blot检测虫草素能否通过激活Nrf-2/Keap1信号通路及其下游抗氧化因子（HO-1、NQO-1）的表达来提高机体抗氧化损伤能力以及抑制炎症因子（TNFα、TNFβ、IL-6、IL-10）的表达。对比模型组结果显示,虫草素能极显著降低（P<0.01）小鼠血清中ALT、AST及肝脏中MDA水平,并能极显著提高肝脏中SOD水平（P<0.01）;HE染色结果显示虫草素能有效降低改善受损肝组织中的炎细胞浸润及纤维组织增生;Western blot结果表明虫草素能够通过激活Nrf-2信号通路,促进下游抗氧化因子及抗炎因子的表达,从而降低炎症反应。虫草素对CCl₄致小鼠急性肝损伤具有一定的保护作用,其机制与Nrf-2信号通路相关,实验结果为后续蛹虫草及虫草素的开发应用奠定基础。     

Nickel-selenium interaction-time dependent biochemical alterations and metal decorporation in rats

The influence of selenium on the disposition of nickel and on Ni induced metallothionein levels was studied in female rats. Concomitant administration of Se (6.3 mumol/kg, intraperitoneally) and 63Ni (0.12 mmol/kg subcutaneously) lowered the Ni burden of all the soft organs and the plasma ceruloplasmin levels. Selenium caused no potentiating effect on the Ni induced hepatic MT. However, 3 days later, the lowered MT levels appeared related to the corresponding decrease in hepatic Ni content at day 6. The Ni selenide excretable complex and Ni-selenide protein complex appear to be probable mechanisms of the Ni-Se interaction in the present study.     

Tissue Metallothionein Concentrations in Mice and Humans with Hyperglycemia

Besides participating in tissue zinc homeostasis and protecting against heavy metal toxicities, metallothionein (MT) is known as an antioxidant. Increased MT activity can ameliorate diabetic hyperglycemia, and subjects with less MT synthesis are more prone to diabetic complications. However, whether tissue MT status is varied in the subjects with diabetes mellitus remains unclear. This study was undertaken to measure tissue MT levels in laboratory mice (serum, liver, and epididymal adipose tissue) and humans (serum) with hyperglycemia. Tissue MT levels were measured by enzyme-linked immunosorbent assay. The results showed that MT status in serum and adipose tissue did not markedly differ between the subjects with and without hyperglycemia. In addition, streptozotocin- and high-fat-diet-induced hyperglycemic mice had higher while ob/ob mice had lower liver MT levels than that of normal control mice. Furthermore, serum MT levels tended to correlate with glycemia values in mice. The results of this study indicate that serum MT value does not differ in subjects with hyperglycemia and cannot be used as an index to evaluate the susceptibility or progress of diabetes mellitus.     

Prior increase in metallothionein levels is required to prevent doxorubicin cardiotoxicity

Many studies have shown that metallothionein (MT) can be increased significantly by different oxidative insults in multiple organ systems. However, the increase in MT production often fails to protect against oxidative tissue injury. On the other hand, recent studies using a cardiac-specific, MT-overexpressing, transgenic mouse model have shown that MT protects against oxidative heart injury. Thus, the present study was undertaken to test the hypothesis that prior increase in MT levels is required to prevent oxidative injury. Oxidative heart injury was induced by doxorubicin (DOX), an important anticancer drug that causes severe cardiotoxicity through oxidative stress. Cardiac-specific, MT-overexpressing, transgenic mice and wild-type (WT) FVB mice were treated with DOX at 20 mg/kg. Four days after the treatment, MT concentrations were markedly elevated in the WT mouse heart. The elevated MT concentrations were comparable with those found in the transgenic mouse heart, which did not show further MT elevation in response to DOX challenge. Severe oxidative injury occurred in the heart of WT mice, including myocardial lipid peroxidation, morphological changes as examined by electron microscopy, high levels of serum creatine kinase activity, and decreased total glutathione concentrations in the heart. However, all of these pathological changes were significantly inhibited in the MT-transgenic mice. Therefore, this study demonstrates that there is a correlation between MT induction and oxidative stress in the DOX-treated mouse heart. However, MT can protect the heart from oxidative injury only if it is present prior to induction of oxidative stress.     

Evaluation of comparative effect of pre‐ and posttreatment of selenium on mercury‐induced oxidative stress,histological alterations,and metallothionein mRNA expression in rats

To evaluate the effect of pre‐ or posttreatment of selenium (6 μmol/kg b.w., single intraperitoneal injection) in mercury intoxication, rats were exposed to mercury (12 μmol/kg b.w., single intraperitoneal injection). Exposure to mercury resulted in induced oxidative stress in liver, kidney, and brain tissues. Marked changes in serum biochemical parameters together with alterations in histopathology and an induction in metallothionein‐I and metallothionein‐II mRNA expression in the liver and kidney were observed. Pretreatment with selenium to mercury‐exposed animals had protective effect on the liver, whereas posttreatment had partial protection on restoration of altered oxidative stress parameters. In the kidney, pretreatment with selenium showed partial protection on restoration of altered biochemical parameters, whereas no protection was observed in posttreatment. The pretreatment with selenium resulted in restoration of mercury‐induced metallothionein‐I and metallothionein‐II mRNA expression, which was completely restored in the liver whereas partial restoration was observed in the kidney. Posttreatment with selenium resulted in further induction in metallothionein‐I and metallothionein‐II mRNA expression in the liver and kidney. In the brain, selenium showed partial protection on alerted biochemical parameters. Results indicate that pretreatment with selenium is beneficial in comparison to posttreatment in mercury intoxication. Thus, dietary intake of selenium within safe limit may, therefore, enable us in combating any foreseen effects due to mercury exposure. © 2010 Wiley Periodicals, Inc. J Biochem Mol Toxicol 24:123–135, 2010; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20320     

Sex and restraint stress differences in rat metallothionein and Zn levels

Sex differences in serum and liver metallothionein (MT) levels were studied in adult male and female rats. Whereas it was found that female rats had higher hepatic MT levels than male rats in basal, unstressed conditions, no significant differences were found in serum MT levels. Restraint stress increased both serum and liver MT in both sexes. The increase in serum MT was greater in male than in female rats, whereas no significant differences between sexes were found in liver MT content after restraint stress. It is suggested that MT regulation might be sex-dependent and that MT might play some extrahepatic function during stress.     

电针对大鼠胃黏膜损伤修复的血清蛋白质差异表达研究

目的：观察电针胃经穴对大鼠胃黏膜损伤修复的血清蛋白质差异表达,为进一步阐明针刺效应的体液机理提供理论依据。方法：用表面增强激光解吸离子化飞行时间质谱（SELDI—TOF—MS）技术和WCX2（弱阳离子交换芯片）对正常大鼠血清和电针大鼠血清进行蛋白质指纹图谱检测分析,通过Biomarker Wizard和Biomarker Patterns System软件判别分析处理数据并结合生物信息学方法筛选差异表达蛋白质。结果：与正常大鼠血清比较,电针大鼠血清蛋白质在质荷比为2000-50000有25个蛋白质峰差异有显著意义,其中有19个标志蛋白在电针胃经大鼠血清中呈现高表达,6个标志蛋白在电针胃经大鼠血清中呈现低表达。结论：电针可促进胃黏膜损伤大鼠血清蛋白质差异表达,这种差异蛋白质可能与电针促进胃黏膜损伤修复效应密切相关。     

Endotoxin-induced zinc accumulation by liver cells is mediated by metallothionein synthesis

Endotoxin induces a decrease in zinc concentration in the serum and an increase in zinc levels in the liver. We have studied whether metallothionein (MT), which is a heavy metal-binding protein, is associated with this phenomenon in vitro. When MT of liver cells is induced by a factor secreted by endotoxin-stimulated macrophages, the cells accumulate zinc from the medium. The temporal accumulation of zinc is correlated with the induction of MT, and the accumulated zinc binds to MT. These results suggest that zinc accumulation by liver cells is mediated by metallothionein produced in response to a macrophage factor, which is elicited by endotoxin.     

Using MT(-/-) mice to study metallothionein and oxidative stress

Mice with null mutations for metallothionein genes MT-1 and MT-2 were used to study the role that metallothionein plays in protecting cellular targets in vivo from oxidative stress. Wild-type (MT(+/+)) and MT-null (MT(-/-)) mice were treated with either saline or zinc and exposed to two types of oxidative stress: gamma-irradiation or 2-nitropropane. There was no alteration in the antioxidant defense system (superoxide dismutase, catalase, or glutathione peroxidase and glutathione levels) to compensate for the lack of the metallothionein in the MT(-/-) mice. The amount of oxidative damage to liver DNA, lipids, and proteins were similar for the MT(-/-) and MT(+/+) mice even though the levels of metallothionein in the livers of the saline- or zinc-pretreated MT(+/+) mice were 5- to 100-fold greater than found in the MT(-/-) mice. To determine if metallothionein can protect mice from the lethal effects of ionizing radiation, the mean survivals of MT(-/-) and MT(+/+) mice exposed to whole body gamma-irradiation were measured and found to be similar. However, the mean survival increased significantly after zinc pretreatment for both the MT(-/-) and MT(+/+) mice. These results demonstrate that tissue levels of metallothionein do not protect mice in vivo against oxidative stress.     

Involvement of Oxidative Stress in Paraquat-Induced Metallothionein Synthesis Under Glutathione Depletion

The inhibition of glutathione (GSH) synthesis by -buthionine-SR-sulfoximine (BSO) causes aggravation of hepatotoxicity of paraquat (PQ), an oxidative-stress inducing substance, in mice. On the other hand, synthesis of metallothionein (MT), a cysteine-rich protein having radical scavenging activity, is induced by PQ, and the induction by PQ is significantly enhanced by pretreatment of mice with BSO. The purpose of present study is to examine whether generation of reactive oxygens is involved in the induction of MT synthesis by PQ under inhibition of GSH synthesis. Administration of PQ to BSO-pretreated mice increased hepatic lipid peroxidation and frequency of DNA single strand breakage followed by manifestation of the liver injury and induction of MT synthesis. Both vitamin E and deferoxamine prevented MT induction as well as lipid peroxidation in the liver of mice caused by administration of BSO and PQ. In cultured colon 26 cells, both cytotoxicity and the increase in MT mRNA level caused by PQ were significantly enhanced by pretreatment with BSO. Facilitation of PQ-induced reactive oxygen generation was also observed by BSO treatment. These results suggest that reactive oxygens generated by PQ under inhibition of GSH synthesis may stimulate MT synthesis. GSH depletion markedly increased reactive oxygen generation induced by PQ, probably due to the reduced cellular capability to remove the radical species produced.     

Serum selenium,glutathione peroxidase,lipids, and human liver microsomal enzyme activity

This study evaluated selenium status in relation to lipid peroxidation, liver microsomal function, and serum lipids in humans. Serum selenium concentration, glutathione peroxidase (GSH-Px) activity, liver microsomal enzyme activity, assessed by plasma antipyrine clearance (AP-CL) rate, and serum lipids were determined in 23 healthy subjects in a double-blind placebo-controlled trial of selenium supplementation. The low selenium concentration (74.0±14.2 μg/L, mean±SD) is attributable to the low selenium content of the diet. Subjects with the lowest selenium levels (n=11) had reduced serum GSH-Px activity, AP-CL rate, high-density lipoprotein cholesterol (HDL-C), and total cholesterol (T-C) as compared with subjects with higher selenium concentrations (n=12). Low AP-CL rates were associated with low HDL-C: T-C ratios. Selenium supplementation, 96 μg/d for 2 wk, increased serum selenium, GSH-Px activity, and the HDL-C: T-C ratio. The results suggest that a low serum selenium level is associated with a decrease in liver microsomal enzyme activity and serum HDL-C and T-C concentrations. Selenium supplementation in subjects with low serum selenium may favorably influence relations between serum lipoproteins connected with the development of atherosclerotic vascular disease.