过氧化心肌线粒体产生的脂类自由基及（—）—EGCG的抑制…

本文利用ＥＳＲ技术研究了心肌线粒体酶修饰下的脂过氧化和脂类自由基,以及（－）－ＥＧＣＧ的抑制作用,结果表明：４－ＰＯＢＮ能捕集ｌｉｐｏｘｙｇｅｎａｓｅ诱发心肌线粒体产生的自由基,得到６条线谱的脂类自由基和４条线谱捕集物,（－）－ＥＧＣＧ对该本系中使用的ｌｉｐｏｘｙｇｅｎａｓｅ活性无影响,对所产生有明显的消除作用,并呈量效关系,对脂质过氧化的抑制作用,在本试验浓度范围内随浓度增加变化不大,最大抑制率     

钙过负荷和丹参酮对线粒体脂质过氧化的影响

用ＥＳＲ自旋捕集技术捕捉到Ｆｅ２＋启动的心肌线粒体膜脂质过氧化过程中产生的脂类自由基Ｌ·（ａＮ＝１５．６Ｇ,ａＨ＝２．９７Ｇ）。钙过负荷时,钙离子使线粒体产生的脂类自由基增加,当钙浓度为３０μｍｏｌ／Ｌ·ｍｇＰｒ时,产生的脂类自由基约增加３２％。丹参酮可清除Ｆｅ３＋启动的线粒体膜脂质过氧化过程中产生的脂类自由基。当丹参酮浓度为０．８ｍｇ／ｍｇＰｒ时,清除率达到７０％左右。钙过负荷时丹参酮仍能有效清除脂类自由基。另外,还发展了用自旋探针ＣＴＰＯ测量线粒体氧消耗的方法,检测氧消耗所需样品量比Ｃｌａｒｋ电极少几十倍。用此方法测量小鼠心肌线粒体不同状态的氧消耗和呼吸控制率,证明一定浓度的Ｃａ２＋和Ｆｅ２＋影响线粒体的呼吸功能．     

丹参酮对心肌肌质网脂质过氧化过程中脂类自由基的清除作用

应用自旋捕集方法和化学发光方法研究天然抗氧化剂丹参酮（Ｔａｎｓｈｉｎｏｎｅ）对心肌肌质网膜脂质过氧化过程中产生的脂类自由基的清除作用。发现在一定的浓度范围内,丹参酮对脂质过氧化有较好的保护作用,在丹参酮浓度大于１ｍｇ／ｍｇ蛋白时,对脂类自由基清除率可达６０％。丹参酮对肌质网膜脂质过氧化的保护机理可能是通过清除脂类自由基而阻断脂质过氧化的链式反应,而不是清除氧自由基而防止脂质过氧化的启动。     

GPX－abzyme对受XO／HX系统损伤的心肌线粒体的保护作用

探讨了研制的具有谷胱甘肽过氧化物酶活力的含硒抗体酶（ＧＰＸ－ａｂｚｙｍｅ）对于受损心肌线粒体的保护作用,利用牛的心肌线粒体为实验材料,通过线粒体的膨胀度、脂质过氧化物含量、ＣＣＯ活力变化及电镜观察等几个方面证明ＧＰＸ－ａｂｚｙｍｅ能抵抗ＸＯ／ＨＸ系统产生的自由基的损伤作用,ＥＳＲ研究也表明ＧＰＸ－ａｂｚｙｍｅ能明显降低ＸＯ／ＨＸ损伤系统中的自由基含量。     

维生素E和微量元素Se对人肝癌细胞生长、分化及其癌基因表达的影响

本工作观察了体外环境中不同水平的维生素Ｅ和微量元素Ｓｅ对人肝癌细胞株（ＳＭＭＣ－７７２１）生长、分化和其癌基因（Ｎ－ｒａｓ、ｃ－ｍｙｃ）表达水平的影响。实验结果表明：高水平维生素Ｅ（２．４、９．２、２４．０ｎｍｏｌ／Ｌ）和Ｓｅ（０．１５、０．３０、０．６０ｎｍｏｌ／Ｌ）对肝癌细胞的集落形成率具有明显的抑制作用;生化分析显示高水平维生素Ｅ和微量元素Ｓｅ均可明显抑制环境中脂质过氧化的水平,Ｓｅ对癌细胞甲胎蛋白的分泌有明显的抑制作用,而维生素Ｅ作用不明显。细胞原位杂交发现维生素Ｅ浓度为２．４和９．２ｎｍｏ１／Ｌ时对细胞癌基因Ｎ－ｒａｓ的表达具有明显抑制作用;Ｓｅ浓度为０．１５和０．３０ｎｍｏｌ／Ｌ时对癌基因ｃ－ｍｙｃ的表达明显抑制。实验还观察了维生素Ｅ和Ｓｅ之间的叠加效应,结果显示除对环境中脂质过氧化的抑制作用具有叠加效果外,对其他指标没有明显作用。     

化学修饰单克隆抗体模拟谷胱甘肽过氧化物酶

化学修饰具有底物谷胱甘肽（ＧＳＨ）结合部位的单克隆抗体（４Ａ４）,使其结合部位上的丝氨酸（Ｓｅｒ）转变成谷胱甘肽过氧化物酶（ＧＰＸ）的催化基团硒代半胱氨酸（Ｓｅ－Ｃｙｓ）,因而产生高活力的含硒抗体酶（Ｓｅ－ａｂｚｙｍｅ）．突变的４Ａ４（ｍ４Ａ４）的ＧＰＸ活力达到了天然酶活力的１９％,并对ｍ４Ａ４的酶学性质和动力学性质进行了研究;硒代谷胱甘肽（ＧＳｅＨ）连到４Ａ４结合部位,其ＧＰＸ活力由３．８６Ｕ／μｍｏｌ提高到５９８．９Ｕ／μｍｏｌ用黄嘌呤氧化酶／次黄嘌呤为中心的心肌线粒体自由基损伤模型证明Ｓｅ－ａｂｚｙｍｅ（ｍ４Ａ４）可减轻活性氧对线粒体的损伤。     

GPX—abzyme对受XO／HX系统损伤的心肌线粒体的保护作用

探讨了研制的具有谷胱甘肽过氧化物酶活力的含硒抗体酶（ＧＰＸ－ａｂｚｙｍｅ）对于受损心肌线粒体的保护作用。利用牛的心肌线粒体为实验材料,通过线粒体的膨胀度、脂质过氧化物含量、ＣＣＯ活力变化及电镜观察等几个方面证明ＧＰＸ－ａｂｚｙｍｅ能抵抗ＸＯ／ＨＸ系统产生的自由基的损伤作用,ＥＳＲ研究也表明ＧＰＸ－ａｂｚｙｍｅ能明显降低ＸＯ／ＨＸ损伤系统中的自由基含量。     

羟自由基对心肌线粒体膜的影响及硒的效应

由Ｈ２Ｏ２和ＦｅＳＯ４体系产生的羟自由基（ＯＨ·）作用于大鼠心肌线粒体后,其膜脂双层内产生了脂类自由基。在观测时间内,其脂类自由基与自旋捕捉剂形成的自旋加合物的电子自旋共振（ＥＳＲ）信号强度随着孵育时间的延长而加强。一定浓度的硒代蛋氨酸（Ｓｅ—Ｍｅｔ）或Ｎａ２ＳｅＯ３可明显清除ＯＨ·并抑制脂类自由基的产生。在ＯＨ·的影响下,荧光探针ＤＰＨ在心肌线体膜脂中的荧光寿命和膜脂流动性的测试结果发生了明显变化。与此同时,心肌线粒体膜的能量转换过程（氧化磷酸化效率和呼吸控制率）也发生了显著的改变。１．０μｍｏｌ／Ｌ的Ｓｅ—Ｍｅｔ或２．３μｍｏｌ／Ｌ的Ｎａ２ＳｅＯ３可明显拮抗ＯＨ·的上述影响。前者的作用更为显著。     

维生素E和微量元素Se对人肝癌细胞生长,分化及其癌基因…

本工作观察了体外环境中不同水平的维生素Ｅ和微量元素Ｓｅ对人肝癌细胞株（ＳＭＭＣ－７７２１）生长,分经和其癌基因（Ｎ－ｒａｓ,ｃ－ｍｙｃ）表达水平的影响,实验结果表明：高水平维生素Ｅ（２．４、９．２、２４：０ｎｍｏｌ／Ｌ）和Ｓｅ（０．１５,０．３０,０．６０ｎｍｏｌ／Ｌ）对肝癌细胞的集落形成率具有明显的抑制作用;生化分析显示高水平维生素Ｅ微量元素Ｓｅ均可明显抑制环境中脂质过氧化的水平,Ｓｅ对癌细胞甲     

硒拮抗超氧阴离子导致的心肌线粒体膜损伤

由黄嘌呤和黄嘌呤氧化酶体系产生的超氧阴离子作用于心肌线粒体后,其膜脂双层内产生了脂类自由基。在一定时间内,脂类自由基与自旋捕捉剂形成的加合物的ＥＳＲ信号强度随着孵育时间的增加而加强。１．０μｍｏｌ／Ｌ硒代蛋氨酸（Ｓｅ－Ｍｅｔ）或２．３μｍｏｌ／ＬｍＮａ２ＳｅＯ３可显著清除并抑制脂类自由基的产生。在的影响下,荧光探针ＤＰＨ在膜脂双层中的荧光寿合和膜脂流动性发生了明显改变。一定浓度的Ｓｅ－Ｍｅｔ或Ｎａ２ＳｅＯ３可明显拮抗的上述影响,前者的作用更为显著。     

A potential prodrug for a green tea polyphenol proteasome inhibitor: evaluation of the peracetate ester of (-)-epigallocatechin gallate [(-)-EGCG

Green tea has been shown to have many biological effects, including effects on metabolism, angiogenesis, oxidation, and cell proliferation. Unfortunately, the most abundant green tea polyphenol (-)-epigallocatechin gallate or (-)-EGCG is very unstable in neutral or alkaline medium. This instability leads to a low bioavailability. In an attempt to enhance the stability of (-)-EGCG, we introduced peracetate protection groups on the reactive hydroxyls of (-)-EGCG (noted in text as 1). HPLC analysis shows that the protected (-)-EGCG analog is six times more stable than natural (-)-EGCG under slightly alkaline conditions. A series of bioassays show that 1 has no inhibitory activity against a purified 20S proteasome in vitro, but exhibits increased proteasome-inhibitory activity in intact leukemic cells over natural (-)-EGCG, indicating an intercellular conversion. Inhibition of cellular proteasome activity by 1 is associated with induction of cell death. Therefore, our results indicate that the protected analog 1 may function as a prodrug of the green tea polyphenol proteasome inhibitor (-)-EGCG.     

Methylation suppresses the proteasome-inhibitory function of green tea polyphenols

Under physiological conditions, biotransformation reactions, such as methylation, can modify green tea polyphenols (GTPs) and therefore limit their in vivo cancer-preventive activity. Although a recent study suggested that methylated polyphenols are less cancer-protective, the molecular basis is unknown. We previously reported that ester bond-containing GTPs, for example (-)-epigallocatechin-3-gallate [(-)-EGCG] or (-)-epicatechin-3-gallate [(-)-ECG], potently and specifically inhibit the proteasomal chymotrypsin-like activity. In this study, we hypothesize that methylated GTPs have decreased proteasome-inhibitory abilities. To test this hypothesis, methylated (-)-EGCG and (-)-ECG analogs that can be found in vivo were synthesized and studied for their structure-activity relationships (SARs) using a purified 20S proteasome. The addition of a single methyl group on (-)-EGCG or (-)-ECG led to decreased proteasome inhibition and, as the number of methyl groups increased, the inhibitory potencies further decreased. These SARs were supported by our findings from in silico docking analysis published recently. Previously, we synthesized a peracetate-protected (-)-EGCG molecule, Pro-EGCG (1), to enhance its cellular permeability and stability, and current HPLC analysis confirms conversion of Pro-EGCG (1) to (-)-EGCG in cultured human leukemic Jurkat T cells. Furthermore, in this study, peracetate-protected forms of methylated GTPs were added in intact Jurkat T cells to observe the intracellular effects of methylation. Peracetate-protected, monomethylated (-)-EGCG induced greater cellular proteasome inhibition and apoptosis than did peracetate-protected, trimethylated (-)-EGCG, consistent with the potencies of the parent methylated analogs against a purified 20S proteasome. Therefore, methylation on GTPs, under physiological conditions, could decrease their proteasome-inhibitory activity, contributing to decreased cancer-preventive effects of tea consumption.     

Inhibitory effects of green tea catechins on the activity of human matrix metalloproteinase 7 (matrilysin)

Inhibitory effects of green tea catechins and their derivatives on the matrilysin-catalyzed hydrolysis of a synthetic substrate, (7-methoxycoumarin-4-yl)acetyl-L-Pro-L-Leu-Gly-L-Leu-[N(3)-(2,4-dinitrophenyl)-L-2,3-diamino-propionyl]-L-Ala-L-Arg-NH(2) [MOCAc-PLGL(Dpa)AR], were examined. The 10 catechins examined were classified into three groups according to their inhibition potency. Catechins with a galloyl group at the 3 position, including a major component of green tea catechin, (-)-epigallo-3-catechin gallate [(-)-EGCG], were the most potent inhibitors and inhibited matrilysin in a non-competitive manner with K(i) values of 0.47-1.65 micro M. The inhibitory potency of (-)-EGCG was not influenced by the presence of an inhibitor, ZnCl(2), suggesting that the inhibitions of matrilysin by (-)-EGCG and by ZnCl(2) might be independent of each other. The inhibitory effects of green tea catechins suggest that a high intake of green tea might be effective for the prevention of tumor metastasis and invasion in which matrilysin is concerned.     

Antioxidative activities of galloyl glucopyranosides from the stem-bark of Juglans mandshurica

Two phenolics, 1,2,6-trigalloylglucose (1) and 1,2,3,6-tetragalloylglucose (2), isolated from the stem-bark of Juglans mandshurica were evaluated for their antioxidative activities. The results showed that compounds 1 and 2 exhibited strong scavenging activities against 1,1'-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azino-bis-(3-ethylbenzenthiazoline-6-sulphonic) acid (ABTS(*+)), and superoxide radicals (O(2)(*-)), and also had a significant inhibitory effect on lipid peroxidation and low-density lipoprotein (LDL) oxidation. The strong superoxide radical scavenging of 1 and 2 resulted from the potential competitive inhibition with xanthine at the active site of xanthine oxidase (OX). In addition, compounds 1 and 2 displayed significant lipoxygenase inhibitory activity, the mode of inhibition also being identified as competitive. In comparison, the antioxidative activities of compounds 1 and 2, together with gallic acid, indicated that the number of galloyl moieties could play an important role in the antioxidative activity.     

A para-amino substituent on the D-ring of green tea polyphenol epigallocatechin-3-gallate as a novel proteasome inhibitor and cancer cell apoptosis inducer

Analogs of (-)-EGCG containing a para-amino group on the D-ring in place of the hydroxyl groups have been synthesized and their proteasome inhibitory activities were studied. We found that, the O-acetylated (-)-EGCG analogs possessing a p-NH(2) or p-NHBoc (Boc; tert-butoxycarbonyl) D-ring (5 and 7) act as novel tumor cellular proteasome inhibitors and apoptosis inducers with potency similar to natural (-)-EGCG and similar to (-)-EGCG peracetate. These data suggest that the acetylated amino-GTP analogs have the potential to be developed into novel anticancer agents.     

Generation of Probucol Radicals and Their Reduction by Ascorbate and Dihydrolipoic Acid in Human Low Density Lipoproteins

Probucol, 4.4′-[(1-methylethylidene)bis(thio)]bis-[2,6-bis(1.1-dimethyl)phenol], is a lipid regulating drug whose therapeutic potential depends on its antioxidant properties. Probucol and x-tocopherol were quantitatively compared in their ability to scavenge peroxyl radicals generatcd by the thermal decomposition of the lipid-soluble azo-initiator 2,2′-azo-bis(2,4-dimethyl-valeronitrile), AMVN, in dioleoylphos-phatidylcholine (DOPC) liposomes. Probucol showed 15-times lower peroxyl radical scavenging efficiency than x-tocopherol as measured by the effects on AMVN-induced luminol-dependent chemiluminescence. We suggest that probucol cannot protect x-tocopherol against its loss in the course of oxidation, although probucol is known to prevent lipid peroxidation in membranes and lipoproteins. In human low density lipoproteins (LDL) ESR signals of the probucol phenoxyl radical were detected upon incubation with lipoxygenase + linolenic acid or AMVN. Ascorbate was shown to reduce probucol radicals. Dihydro-lipoic acid alone was not able to reduce the probucol radical but in the presence of both ascorbate and dihydrolipoic acid a synergistic effect of a stepwise reduction was observed. This resulted from ascorbate-dependent reduction of probucol radicals and dihydrolipoic acid-dependent reduction of ascorbyl radicals. The oxidized form of dihydrolipoic acid, thioctic acid, did not affect probucol radicals either in the presence or in the absence of ascorbate.     

Radical scavenger can scavenge lipid allyl radicals complexed with lipoxygenase at lower oxygen content

Lipoxygenases have been proposed to be a possible factor that is responsible for the pathology of certain diseases, including ischaemic injury. In the peroxidation process of linoleic acid by lipoxygenase, the E,Z-linoleate allyl radical-lipoxygenase complex seems to be generated as an intermediate. In the present study, we evaluated whether E,Z-linoleate allyl radicals on the enzyme are scavenged by radical scavengers. Linoleic acid, the content of which was greater than the dissolved oxygen content, was treated with soya bean lipoxygenase-1 (ferric form) in the presence of radical scavenger, CmP (3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-N-oxyl). The reaction rate between oxygen and lipid allyl radical is comparatively faster than that between CmP and lipid allyl radical. Therefore a reaction between linoleate allyl radical and CmP was not observed while the dioxygenation of linoleic acid was ongoing. After the dissolved oxygen was depleted, CmP stoichiometrically trapped linoleate-allyl radicals. Accompanied by this one-electron redox reaction, the resulting ferrous lipoxygenase was re-oxidized to the ferric form by hydroperoxylinoleate. Through the adduct assay via LC (liquid chromatography)-MS/MS (tandem MS), four E,Z-linoleate allyl radical-CmP adducts corresponding to regio- and diastereo-isomers were detected in the linoleate/lipoxygenase system, whereas E,E-linoleate allyl radical-CmP adducts were not detected at all. If E,Z-linoleate allyl radical is liberated from the enzyme, the E/Z-isomer has to reach equilibrium with the thermodynamically favoured E/E-isomer. These data suggested that the E,Z-linoleate allyl radicals were not liberated from the active site of lipoxygenase before being trapped by CmP. Consequently, we concluded that the lipid allyl radicals complexed with lipoxygenase could be scavenged by radical scavengers at lower oxygen content.     

Antioxidant properties of EPC-K1: a study on mechanisms.

Scavenging effects of L-ascorbic acid 2-[3,4-dihydro-2,5,7,8- tetramethyl-2-(4,8,12-trimethytridecyl)-2H-1-benzopyran- 6-yl-hydrogen phosphate] potassium salt (EPC-K1) on hydroxyl radicals, alkyl radicals and lipid radicals were studied with ESR spin trapping techniques. The inhibition effects of EPC-K1 on lipid peroxidation were assessed by TBA assay. The kinetics of EPC-K1 reacting with hydroxyl radicals and linoleic acid radicals were studied by pulse radiolysis. The active site of EPC-K1 and the structure-antioxidative activity relationships were discussed. The superoxide radicals scavenging capacity of the brain homogenate of EPC-K1-treated rats was measured. The results revealed that in comparison with Trolox and vitamin C, EPC-K1 showed better overall antioxidative capacity in vitro and in vivo. EPC-K1 was a moderate scavenger on hydroxyl radicals and alkyl radicals, a potent scavenger on lipid radicals, and an effective inhibitor on lipid peroxidation. EPC-K1 could react with hydroxyl radicals with a rate constant of 7.1 x 10(8) dm3 mol-1 s-1 and react with linoleic acid radicals with a rate constant of 2.8 x 10(6) dm3 mol-1 s-1. The active site of EPC-K1 was the enolic hydroxyl group. After administration of EPC-K1, the ability of rat brain to scavenge superoxide radicals was significantly increased. The potent scavenging effects of EPC-K1 on both hydrophilic and hydrophobic radicals were relevant with its molecular structure, which consisted of both hydrophilic and hydrophobic groups.     

Generation of Probucol Radicals and Their Reduction by Ascorbate and Dihydrolipoic Acid in Human Low Density Lipoproteins

Probucol, 4.4'-[(1-methylethylidene)bis(thio)]bis-[2,6-bis(1.1-dimethyl)phenol], is a lipid regulating drug whose therapeutic potential depends on its antioxidant properties. Probucol and x-tocopherol were quantitatively compared in their ability to scavenge peroxyl radicals generatcd by the thermal decomposition of the lipid-soluble azo-initiator 2,2'-azo-bis(2,4-dimethyl-valeronitrile), AMVN, in dioleoylphos-phatidylcholine (DOPC) liposomes. Probucol showed 15-times lower peroxyl radical scavenging efficiency than x-tocopherol as measured by the effects on AMVN-induced luminol-dependent chemiluminescence. We suggest that probucol cannot protect x-tocopherol against its loss in the course of oxidation, although probucol is known to prevent lipid peroxidation in membranes and lipoproteins. In human low density lipoproteins (LDL) ESR signals of the probucol phenoxyl radical were detected upon incubation with lipoxygenase + linolenic acid or AMVN. Ascorbate was shown to reduce probucol radicals. Dihydro-lipoic acid alone was not able to reduce the probucol radical but in the presence of both ascorbate and dihydrolipoic acid a synergistic effect of a stepwise reduction was observed. This resulted from ascorbate-dependent reduction of probucol radicals and dihydrolipoic acid-dependent reduction of ascorbyl radicals. The oxidized form of dihydrolipoic acid, thioctic acid, did not affect probucol radicals either in the presence or in the absence of ascorbate.     

Natural killer cell-mediated lysis involves an hydroxyl radical-dependent step

The role of oxygen radicals in lysis of K562 target cells by human natural killer (NK) cells was determined by addition of scavengers of these free radicals. Lysis was greatly reduced under hypoxic conditions. Superoxide dismutase and cytochrome c, scavengers of superoxide anions, and catalase and scavengers of hypochlorite had no effect on lysis. Of 15 hydroxyl radical scavengers tested, 13 inhibited lysis. These were not toxic, because cell morphology and spontaneous chromium release were not affected and preculture with scavengers was not inhibitory. These scavengers differed widely in structure, but degree of inhibition of lysis correlated with their rate constants (k) for reaction with hydroxyl radical (k vs log inhibitor concentration required to decrease lysis by 50%: r = -0.9202, p less than 0.001), showing that inhibition was due to inactivation of the hydroxyl radical. Target cell binding was not reduced at concentrations that inhibited lysis. Inhibitors of the lipoxygenase pathway also decreased lysis, suggesting this pathway to be the source of hydroxyl radicals. In view of the reported requirements for hydroxyl radical-mediated lipid peroxidation for optimal secretory activity in a number of cell types, it appears that the generation of hydroxyl radicals by NK cells is required for delivery of cytotoxic factors.