晶状体生化的研究——亚硒酸钠诱发大鼠白内障晶状体中巯基、二硫键及维生素C的含量

我们测定了正常及亚硒酸钠诱发的白内障大鼠晶状体中非蛋白质巯基、蛋白质巯基、蛋白质结合巯基和维生素C的含量,发现随着白内障的进展非蛋白质巯基及蛋白质巯基均减少,蛋白质结合巯基在核混浊时增加,而在整个晶状体混浊时下降到与正常对照组相近,在白内障形成过程中二硫交联的蛋白质含量明显增加,而维生素C含量似乎无明显变化。     

AC1及AC3抗白内障形成中晶状体谷胱甘肽代谢相关酶活性的变化

观察了亚硒酸钠,AC1,AC3对大鼠晶状体中谷胱甘肽过氧化物酶(GSH-Px),谷胱甘肽还原酶(GR)及谷胱甘肽硫转移酶(GST)的影响。结果表明,亚硒酸钠组大鼠的晶状体尚未混浊前已出现GSH-Px活性增高及GR和GST的活性降低。GR活性下降随白内障进展而加重。AC1及AC3均可使亚硒酸钠所致的酶活性变化逆转,但对正常晶状体的酶活性没有影响。     

比较不同类型白内障形成过程中非蛋白质巯基与蛋白质巯基的动态变化及关系

本文报道了在亚硒酸钠、平阳霉素及半乳糖诱发大鼠产生白内障过程中晶状体中非蛋白质巯基及蛋白质巯基的动态变化,并探讨了其变化机理及相互关系。在亚硒酸钠诱发白内障过程中,给药24h后晶状体中非蛋白质巯基减少到正常的二分之一,以后又逐渐回升,但始终未达到正常水平,至第7天,非蛋白质巯基又再度减少。在平阳霉素及半乳糖诱发白内障过程中,晶状体中非蛋白质巯基分别在给药后的第7天及第3天开始大量减少,以后继续减少,至第15天时,其含量分别为正常的十分之一及五分之一。在体外,亚硒酸钠有促进还原型谷胱甘肽自氧化的作用,半乳糖对此作用无影响,而平阳霉素可阻止其进行,但能加强亚硒酸钠的促进作用。在三种白内障晶状体中,蛋白质巯基开始减少的时间均较非蛋白质巯基为晚,这表明只有非蛋白质巯基减少到一定程度后蛋白质巯基才会被大量氧化,同时也说明非蛋白质巯基具有保护蛋白质巯基免受氧化的作用。只有这种保护作用减弱后,才会使蛋白质巯基遭受氧化而导致白内障。     

亚硒酸钠诱导的晶状体蛋白质聚合作用

不仅在体内,而且在体外亚硒酸钠可引起晶状体蛋白质聚合。将亚硒酸钠加到pH7.4的晶状体蛋白质溶液中,在37℃保温30min后观察到蛋白质溶液变混浊,随时间的延长混浊程度加重并有沉淀形成。经SDS聚丙烯酰胺凝胶电泳发现,加硒保温后形成的不溶性蛋白质中有大量的高分子聚合物。当加入二硫苏糖醇后混浊的蛋白质溶液变清,其中的高分子聚合物也基本消失,我们还发现;在亚硒酸钠使晶状体蛋白质变混浊的同时,蛋白质巯基减少,而蛋白质结合的硒量增加,且二者之间有较固定的比例关系,即蛋白质上每增加一个硒原子,蛋白质巯基就减少4.26个。当用二硫苏糖醇还原后,68％的硒从蛋白质中释放出来。这些结果表明,亚硒酸钠可引起大鼠晶状体水溶性蛋白质聚合,其可能方式如下:4PSH+SeO_3~-→PSSP+PS-Se-SP+H_2O+2OH~-这可能是亚硒酸钠诱发白内障的主要原因。     

中药合剂CB在晶状体白内障形成中对巯基及二硫键的作用

用白内障诱发剂三硝基甲苯、平阳霉素、亚硒酸钠和半乳糖分别加入大鼠晶状体培养基中,共同培养２４ｈ,同时在各培养基中分别加入中药合剂ＣＢ,以观察其药效,用维生素Ｃ作对照。结果表明中药合剂ＣＢ能够保护非蛋白质巯基免致氧化,抑制蛋白质巯基交联,降低晶状体不溶性蛋白质中二硫键含量,故中药合剂ＣＢ有可能作为抗白内障药物应用于临床。     

抗氧化剂与自由基清除剂对硒性白内障形成的影响

本文观察了8种化合物(抗氧化剂及自由基清除剂)对大鼠亚硒酸钠性白内障的影响。实验分为正常对照组,亚硒酸钠组及药物对抗组。亚硒酸钠组系给13日龄大鼠皮下注射亚硒酸钠(6μmoles/kg体重),间日一次,逐次递增1μmole/kg体重,连续5次,药物对抗组则同时腹腔注射抗氧化剂或自由基清除剂,每日观察并记录白内障的发生频率及程度,实验表明,一些抗氧化剂及自由基清除剂能够有效的对抗亚硒酸钠性白内障的发生发展,其中AC1、AC3及AC3的效果尤为明显。本文的结果为探讨白内障形成机理及防治提供了实验依据。     

亚硒酸钠诱发大鼠白内障晶状体前列腺素生物合成的研究

 用亚硒酸钠诱发大鼠产生白内障后,将晶状体微粒体与外源性花生四烯酸共同孵育,用放射免疫方法测定白内障晶状体前列腺素E_2(PGE_2)及前列腺素F_2α(PG-F_2α)的生物合成情况,并与正常晶状体进行了比较,结果表明大鼠晶状体具有酶促合成PGs的能力。正常晶状体及白内障晶状体合成PGE_2的能力分别为687.75±113.97及1095.00±79.39pg/100mg晶状体湿重/15分钟,PGE_2α则分别为51.45±36.72及158.83±115.94pg/100mg晶状体湿重/15分钟(平均数±S.D.)。这说明大鼠白内障晶状体合成PGs的能力明显增高,与正常晶状体相比有显著性差异(PGE_2P<0.001,PGF_2αP<0.02)。在前2次注射亚硒酸钠后,大鼠白内障晶状体PGs的合成能力逐渐高于正常晶状体,并随注射亚硒酸钠的次数增加和白内障晶状体混浊程度加重,PGs在晶状体内的含量增加。     

四种中草药对大鼠半乳糖性白内障氧化还原物质及糖类含量的影响

对正常和半乳糖性白内障及给中草药的大鼠晶状体中某些吡啶核苷酸成分、糖类、非蛋白质巯基的含量进行了比较。结果表明,在白内障晶状体中,NADPH及非蛋白质巯基的含量明显低于正常晶状体的,而NADP、半乳糖及半乳糖醇的含量明显高于正常晶状体的;当注射半乳糖的同时分别用黄岑、石斛、菟丝子及玉蝴蝶四种中草药水煎剂灌胃,上述变化为基本恢复至正常晶状体的水平。表明四种中草药对晶状体中的异常生化变化具有阻止及纠正作用。     

三硝基甲苯对大鼠晶状体中可溶性蛋白质、巯基及二硫键含量的影响

我们采用三硝基甲苯（ＴＮＴ）与大鼠晶状体体外培养的方法,动态观察了晶状体中可溶性蛋白质、非蛋白质巯基、蛋白质巯基、蛋白质结合巯基及二硫键含量的变化,发现随着三硝基甲苯作用时间的延长,可溶性蛋白质、非蛋白质巯基及蛋白质巯基均减少,蛋白质结合巯基及二硫键交联的蛋白质含量增加,其中可溶性蛋白质、非蛋白质巯基及二硫键含量的变化皆达到了统计学上显著意义水平（Ｐ＜０．０５）。     

抗白内障药物滴眼对亚硒酸钠性白内障的预防作用

 观察了三种化合物(抗氧化剂与自由基清除剂)对大鼠亚硒酸钠性白内障的滴眼预防作用。实验分为正常对照组、亚硒酸钠组及滴眼预防组。亚硒酸钠组及滴眼预防组系给12─13日龄的大鼠皮下注射亚硒酸钠,首次剂量为6μmol/kg体重,间日一次,逐次递增1μmol/kg体重,连续六次。预防组则为大鼠开眼后同时滴眼抗氧化剂与自由基清除剂。结果表明,三种化合物通过滴眼均能有效的防止亚硒酸钠性白内障的发生,白内障的发生率从95.8％降低至15％～43.5％。同时测定了各组晶状体中谷胱甘肽过氧化物酶(GSH-Px)、谷胱甘肽还原酶(GSSG-R)及谷胱甘肽硫转移酶(GSH-S)的活性,结果表明,凡注射硒的大鼠晶状体中GSH-Px及GSSG-R的活性均比正常晶状体的高,接受抗氧化剂与自由基清除剂预防的大鼠晶状体中这两种酶的活性比未接受预防的大鼠晶状体中的低。单独注射硒的大鼠晶状体中GSH-S的活性比正常晶状体的高。接受预防的大鼠晶状体中此酶的活性和正常晶状体无差异,但比单独注射硒的大鼠晶状体中的低。     

乳鼠与成年大鼠晶状体中某些代谢的比较

对乳鼠与10月龄成年大鼠的晶状体代谢进行比较。结果表明,年成大鼠晶状体脂类过氧化作用较乳鼠显著高(P<0.05),而非蛋白质就基含量则较乳鼠低约22％(P<0.01),谷胱甘肽过氧化物酶(GSH-Px)活性随鼠龄增长而逐渐下降,成年大鼠GSH-Px活性较18日龄乳鼠降低62％(P<0.01),成年大鼠的谷胱甘肽还原酶活性较18日龄乳鼠低47％(P<0.01),而谷胱甘肽硫转移酶则高14％(P<0.01)。据此结果推测,去年性白内障的发生可能与晶状体抗氧化遗伤的防御机制减弱与脂类过氧化作用增加有关。而乳鼠晶状体谷胱甘肽硫转移酶(GST)活性低于成年大鼠或许是某些中毒性白内障只可在幼年动物诱发成功的影响因素之一。     

三硝基甲苯中毒性白内障动物模型的建立及其发病机理的初步研究

反复多次给大鼠皮下注射20％三硝基甲苯(TNT)甘油:水混悬液,染毒15个月后21％动物发生白内障,其裂隙灯检查结果与人TNT性白内障基本相似,同时注射甘油:水溶剂的对照组大鼠无一例发生白内障。染毒10个月的大鼠,其晶状体LPO增高,GSH-P_X及GST活性降低,GR活性无变化,而GSH含量明显增加;注射TNT后肝脏LPO值、GSH含量、GSH-P_X、GR及GST活性均明显增高。本文结果提示,TNT中毒性白内障的形成可能系TNT及其代谢产物直接作用于晶状体,造成昌状体氧化损伤所致。TNT白内障大鼠模型的建立亦为深入探讨其发病机理及防治奠定了基础。     

Protective effects of selenium, vitamin C and vitamin E against oxidative stress of cigarette smoke in rats

Cataractous lenses have been found to have an altered distribution of the intracellular ionic environment: the concentrations of potassium and magnesium being decreased and the concentrations of sodium and calcium increased. These changes arise as a result of changes to lens membrane characteristics causing an increase in lens membrane permeability. In this study flame atomic absorption spectroscopy (AAS) was used for calcium, magnesium, iron and zinc determination, and flame atomic emission spectroscopy (AES) was used for sodium and potassium contents in normal and cigarette smoke-exposed rat lenses. The methods are sensitive enough to detect quantitatively all six cations in a single rat lenses. In this work, six elements, including Ca2+, K+, Na+, Zn2+, Fe2+ and Mg2+ in experimental rat eye lenses and normal transparent lenses were determined. It was found that the concentrations of Ca2+, Na+, Zn2+, and Fe2+ were increased dramatically while K+ and Mg2+ decreased in smoke-exposed rat lenses when compared to the control rat lenses. There were no significant changes between 'smoked' rats supplied with vitamin C and control groups. A positive correlation was found also in the other two groups of 'cigarette smoked' animals supplemented with selenium plus vitamin E and selenium when compared with 'cigarette smoked' without any supplements. These data provide support for the hypothesis that cigarette smoking increases the risk of cataract formation. We investigated whether vitamin C is the most important antioxidant in the body. The roles of diet with optimum amounts of antioxidant vitamins C and vitamin E and the antioxidant mineral selenium are discussed.     

The effect of selenium on the function of the anion transporter (Band 3) of erythrocyte membranes.

The transport activity of Band 3 of spectrin-stripped inside-out erythrocyte membrane vesicles (IOVs) or resealed ghosts was enhanced in the presence of trace amounts of Na2SeO3 (0.2-0.5 p.p.m.); however, at higher concentrations of Na2SeO3 (> 4.0 p.p.m.), an inverse result was obtained. Reassociation of spectrin with IOVs has no effect either on the transport activity of Band 3 or on the enhancement of its activity by Na2SeO3. Sulfhydryl reagents (p-chloromercuribenzoic acid and N-ethylmaleimide) could also inhibit Band 3 activity and eliminate the selenium effect. It is suggested that SH groups are involved in anion transport of Band 3 and that the selenium effect is based on the interaction of SH groups of Band 3 with Na2SeO3.     

Selenium: inhibition of microtubule formation and interaction with tubulin.

We have studied the interaction of Na2SeO3 with microtubule proteins and tubulin. This selenium compound inhibits the polymerization of MTP (half-inhibition occurred for Na2SeO3 10 microM), and to a lesser that of tubulin. This effect of selenite is related to the formation of disulfide bridges between tubulin sulfhydryl groups, inducing a conformational change of the protein. This is corroborated by the modified binding of colchicine and vinblastine in presence of selenium. The selenite inhibitory concentrations are similar to the toxic blood levels of selenium (40 microM).     

The effects of selenium depletion and repletion on the metabolism of thyroid hormones in the rat

Rats were fed selenium-deficient (less than 0.005 mg selenium/kg) or selenium-supplemented diets (0.1 mg selenium/kg, as Na2SeO2) for up to five wks from weaning to assess the effects of developing selenium deficiency on the metabolism of thyroid hormones. Within two wks 3:5,3'-triiodothyronine (T3) production from thyroxine (T4) in liver homogenates from selenium-deficient rats was significantly lower compared with the activity in liver homogenates from selenium-supplemented rats. This decreased activity was probably responsible, in part, for the higher T4 and lower T3 concentrations in plasma from the selenium-deficient rats after 3, 4, and 5 weeks of experiment. Repletion of selenium-deficient rats with single intra-peritoneal injections of 200 micrograms selenium/kg body wt. (as Na2SeO3) 5 days before sampling reversed the effects of the deficiency on thyroid hormone metabolism and significantly increased liver and plasma glutathione peroxidase activities. However a dose of 10 micrograms selenium/kg body wt given to rats of similar low selenium status had no effect on thyroid hormone metabolism or glutathione peroxidase activity but did reverse the increase in hepatic glutathione S-transferase activity characteristic of severe selenium deficiency. Imbalances in thyroid hormone metabolism are an early consequence of selenium deficiency and are probably not related to changes in hepatic xenobiotic metabolizing enzymes associated with severe deficiency.     

Effects of inorganic selenium compounds on oxidative DNA damage

Exposure of Escherichia coli or mammalian cells to H2O2 results in cell death due to iron-mediated DNA damage. Since selenium compounds have been examined for their ability to act as antioxidants to neutralize radical species, and inorganic selenium compounds are used to supplement protein mixes, infant formula, and animal feed, determining the effect of these compounds on DNA damage under conditions of oxidative stress is crucial. In the presence of Fe(II) and H2O2, the effects of Na2SeO4, Na2SeO3, SeO2 (0.5-5000 microM), and Na2Se (0.5-200 microM) on DNA damage were quantified using gel electrophoresis. Both Na2SeO4 and Na2Se have no effect on DNA damage, whereas SeO2 inhibits DNA damage and Na2SeO3 shows antioxidant or pro-oxidant activity depending on H2O2 concentration. Similar electrophoresis experiments with [Fe(EDTA)](2-) (400 microM) and Na2SeO3 or SeO2 show that metal coordination by the selenium compound is required for antioxidant activity. In light of these results, Na2SeO4 may be safer than Na2SeO3 for nutritional supplements.     

Inhibition of type I and type II iodothyronine deiodinase activity in rat liver, kidney and brain produced by selenium deficiency.

Selenium deficiency for periods of 5 or 6 weeks in rats produced an inhibition of tri-iodothyronine (T3) production from added thyroxine (T4) in brain, liver and kidney homogenate. This inhibition was reflected in plasma T4 and T3 concentrations, which were respectively increased and decreased in selenium-deficient animals. Although plasma T4 levels increased in selenium-deficient animals, this did not produce the normal feedback inhibition on thyrotropin release from the pituitary. Selenium deficiency was confirmed in the animals by decreased selenium-dependent glutathione peroxidase (Se-GSH-Px) activity in all of these tissues. Administration of selenium, as a single intraperitoneal injection of 200 micrograms of selenium (as Na2SeO3)/kg body weight completely reversed the effects of selenium deficiency on thyroid-hormone metabolism and partly restored the activity of Se-GSH-Px. Selenium administration at 10 micrograms/kg body weight had no significant effect on thyroid-hormone metabolism or on Se-GSH-Px activity in any of the tissues studied. The characteristic changes in plasma thyroid-hormone levels that occurred in selenium deficiency appeared not to be due to non-specific stress factors, since food restriction to 75% of normal intake or vitamin E deficiency produced no significant changes in plasma T4 or T3 concentration. These data are consistent with the view that the Type I and Type II iodothyronine deiodinase enzymes are seleno-enzymes or require selenium-containing cofactors for activity.