Differential susceptibility of human skeletal muscle proteins to free radical induced oxidative damage: a histochemical,immunocytochemical and electron microscopical study in vitro

In this study we describe a novel experimental approach to quantify the relative susceptibility of (membrane-associated, contractile and mitochondrial) proteins in normal human muscle tissue sections to oxidative damage by the reactive oxygen species (ROS), hydroxyl (OH·) or superoxide (O₂.^–) radicals. The latter species were generated under controlled experimental conditions in vitro using a ⁶⁰Co gamma radiation source, with subsequent analysis of damage to target proteins (dystrophin, β-dystroglycan, β-spectrin, fast and slow myosin heavy chain, NADH tetrazolium reductase, succinate dehydrogenase and cytochrome oxidase) via standard histochemistry, immunocytochemistry and electron microscopy of muscle tissue sections. In general terms, each of the proteins listed above was more susceptible to oxidative damage by OH·, compared to O₂·^–. Different proteins (differing in structure, function or intracellular localisation) showed different susceptibility to oxidative damage, with certain mitochondrial proteins (succinate dehydrogenase, cytochrome oxidase) showing particular susceptibility. In addition, the use of monoclonal antibodies to four different regions of dystrophin showed the latter to contain both resistant and susceptible regions to ROS induced oxidative damage. At the ultrastructural level of subcellular organelle damage, mitochondria were identified as being particularly susceptible to ROS induced oxidative damage. We therefore speculate that oxidative damage to mitochondria and/or mitochondrial proteins may represent the principal initial route of free radical-induced damage within skeletal muscle tissue. Received:31 October 1995 / Revised, accepted: 19 February 1996     

大鼠颈总动脉闭塞性休克时对海马结构SOD、MDA、AChE水平影响的研究

目的：观察乙酰酸胆硷酯酶在脑缺血损伤中的作用及作用机理。方法：采用大鼠颈总动脉闭塞性休克模型，测定大鼠海马结构内MDA的含量及SOD、AChE的活性。统计学处理采用t检验。结果：休克组与对照组比较，海马组织MDA含量升高，SOD活性降低，AChE光密度明显升高。结论：①脑缺血后迅速发生了组织低灌流及神经细胞膜功能损害。②AChE不仅参与胆硷能神经递质的传递，还可能参与了脑缺血早期对神经元损伤的保护作用。     

Prefabrication of free flaps on a base of fascia

A method of performing free fasciocutaneous flaps on a base of vascularized fascia from the scapula area (dorsal thoracic fascia of scapula) and fascia covering the serratus anterior muscle (lateral thoracic or serratus) is presented. Prefabrication allows customization of flaps, with the ability to choose a satisfactory donor area to have good vascular pedicle length and to choose skin which is better suited to the recipient region. When using these flaps, it may be necessary to perform a one- or two-stage microvascular anastomosis. The prefabrication method is presented, together with the clinical use of the two different flaps.     

Effects of nicotine on hydroxyl free radical formation in vitro and on MPTP-induced neurotoxicity in vivo

Parkinson’s disease (PD) is one of the most frequent disorders of the basal ganglia. From epidemiological studies there is a controversial discussion on the question whether tobacco smoking is correlated with a decreased incidence of PD. The present study aimed to elucidate the role of nicotine and its potential neuroprotective effects in a rodent model of PD. These effects may be related to an altered hydroxyl radical formation; this possibility was studied in vitro. Nicotine and α-phenyl-N-tert-butyl nitrone (PBN) were examined in a cell-free in vitro Fenton system (Fe³⁺/EDTA + H₂O₂) for their radical scavenging properties using the salicylate trapping method. Salicylic acid (0.5 mM) was incubated in the presence and absence of nicotine or PBN and the main products of the reaction of hydroxyl radicals with salicylic acid, namely 2,3- and 2,5-dihydroxybenzoic acid, were immediately determined using HPLC in combination with electrochemical detection. Nicotine and PBN were both able to significantly reduce hydroxyl radical levels at concentrations of 1, 2.5 and 5 mM. Interestingly, at 5 mM nicotine was able to reduce hydroxyl radical levels significantly more than the radical scavenger PBN (5 mM). To investigate the in vivo effects of nicotine, male C57BL/6 mice were used in the MPTP mouse model of PD. Nicotine (0.1 or 0.4 mg/kg s.c.) was administered twice daily for a period of 14 days. On day 8 a single injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP, 30 mg/kg s.c.) was given as well as an enhanced protocol of nicotine treatment (0.1 or 0.4 mg/kg s.c., 30 min before MPTP and 30, 90, 210, 330, 450, 570 min after MPTP) for a total of seven injections of nicotine. High dosage nicotine treatment significantly increased the MPTP-induced loss of body weight and resulted in a significantly decreased striatal dopamine content and an increased dopamine turnover in comparison with the MPTP-treated controls at day 15. However, the lower dosage of nicotine did not significantly alleviate the MPTP-induced effects, although some parameters showed a slight tendency in this direction. These results demonstrate that in vitro nicotine has radical scavenging properties which might suggest neuroprotective effects. In vivo experiments with nicotine, however, showed that a low dosage of nicotine did not alleviate the MPTP-induced dopamine depletion, but a large dosage even enhanced it. Received: 20 March 1998 / Accepted: 23 June 1998     

大鼠缺血心肌线粒体损伤及其意义

选用Wistar大鼠皮下注射异丙肾上腺素（ISP，5mg/kg)造成心肌缺血损伤模型。结果证实缺血心肌线粒体膜有明显损伤，表现在线粒体膜磷脂含量降低，胆固醇含量及胆固醇与磷脂比值增大，Ca^2 浓度显著增加，这些变化与氧自由基引发的脂质过氧化作用增强相关。     

葛根素对羟自由基培养 HUVECs分泌NO和ACE活性的影响

目的采用羟自由基条件体外培养人脐静脉血管内皮细胞(HUVECs),观察中药葛根素(Puerarin)对内皮细胞产生一氧化氮(NO)和血管紧张素转移酶(ACE)活性的影响.方法利用胶原酶消化人脐静脉获得血管内皮细胞(VECs),传至3～5代用于实验,分成羟自由基和无羟自由基两组,每组又分为对照组、葛根素Ⅰ(0.5 mg/ml)和葛根素Ⅱ(1.0 mg/ml)三小组;按细胞密度为1×105/ml接种于24孔培养板中,待细胞长成融合状态后追加葛根素进行干预,继续培养48 h后检测其上清液中NO和ACE的含量.结果与无羟自由基对照组相比,羟自由基对照组中VECs产生NO减少,而分泌ACE的活性增加(P＜0.01);羟自由基组中的葛根素Ⅰ和Ⅱ组与羟自由基对照组相比,其VECs产生NO的量明显减少,分泌ACE活性则增加,且随剂量的增加,差别越显著,葛根素Ⅰ P＜0.05,葛根素ⅡP＜0.01.结论羟自由基促使VECs产生NO减少,使VECs分泌ACE活性增加;葛根素能减轻羟自由基对VECs功能的影响,使VECs产生NO增加和ACE活性降低.     

Brain glutathione reductase induction increases early survival and decreases lipofuscin accumulation in aging frogs

Brain catalase was continuously depleted throughout the life span starting with a large population of initially young and old frogs. Free radical-related parameters were measured in the brain tissue once per year after 2.5, 14.5, and 26.5 months of experimentation. Brain lipofuscin accumulation was observed after 14.5 and 26.5 months, and survival was continuously followed during 33 months. The age of the animal did not decrease endogenous antioxidants nor increase tissue peroxidation either in cross-sectional or longitudinal comparisons. Continuous catalase depletion similarly affected young and old animals, inducing glutathione reductase, tending to decrease oxidized glutathione/reduced glutathione (GSSG/GSH) ratio, decreasing lipofuscin accumulation in the brain, and increasing survival from 46% to 91% after 14.5 months. At 26.5 months of experimentation the loss of the glutathione reductase induction in catalase-depleted animals was accompanied by the presence of higher lipofuscin deposits than in controls and was followed by a great increase in mortality rate. Even though the maximal life span (7 years) was the same in the control and treated animals which were already old (4.2 years) at the beginning of the experiment, the treated animals showed a strong reduction in the rates of early death. It is proposed that the maintenance of a high antioxidant/prooxidant balance in the vertebrate brain greatly increases the probability of the individual to reach the final segments of its species-specific life span. © 1993 Wiley-Liss, Inc.     

Free oxygen radicals in restraint-induced stress gastritis in the rat

In this experimental study, the role of free oxygen radicals (FOR) in stress gastritis (SG) was investigated in a restraint stress model for rats. Allopurinol, dimethyl sulfoxide (DMSO), and superoxide dismutase (SOD) were tested as single agents in controlled groups. The portal blood pH values, the ratio of the mucosal erosion area to the gastric mucosal area (EA/MA), the ratio of the depth of mucosal erosions to the concomitant gastric mucosal wall (ED/MD), and concentrations of malondialdehyde (MDA) — a lipid peroxidation coproduct — in the gastric mucosa were used as parameters in the experiment. The EA/MA between the treated and untreated control groups were found to have no statistically significant difference (P > 0.05). ED/MD, a crucial determinant for bleeding probability, was found to be decreased in the SOD group (P < 0.05). SOD, allopurinol, and DMSO reduced the mucosal MDA concentration to lower values than the untreated group (P < 0.05). We concluded that although FOR may not play a dominant role in stress-induced gastric lesions, SOD may be a good candidate for a clinical trial on SG prophylaxis.     

心可舒对心肌缺血再灌注损伤的保护作用

目的 :探讨心可舒对心肌缺血再灌注损伤的保护作用 ,并初步探讨其作用机制。方法 :19只家兔随机分为 3组 :①假手术组 (SH组 ,n =6 ) :开胸 ,左冠状动脉前降支 (LAD)只穿线不结扎 ,旷置 2 0min ,再观察 6h ;②缺血 再灌注组 (IR组 ,n =7) ;③心可舒处理组 (XKS组 ,n =6 ) :两组均开胸结扎兔LAD 2 0min ,然后松扎 6h。以电镜下心肌超微结构、心率、再灌注心律失常、左室功能、右室血清SOD活性和MDA的含量及心肌组织中SOD、MDA、ATP水平为观察指标。结果 :3组心率均呈进行性下降趋势 ,其中IR组下降幅度最大。XKS组左室内压力上升和下降速率及左室内压峰值均显著高于IR组 (P <0 .0 1) ,血清及心肌组织中SOD活性和心肌组织中ATP含量显著高于IR组 (P <0 .0 1) ,而MDA含量显著低于IR组 (P <0 .0 1) ,再灌注心律失常发生率低于IR组 (P <0 .0 1) ,XKS组心肌超微结构损害较轻。与SH组比较 ,XKS组上述各指标均无显著差异 (P >0 .0 5 )。结论 :心可舒可促进心肌缺血 再灌注损伤心功能的恢复 ,其作用机制可能与清除氧自由基、改善心肌能量代谢等有关。     

Ginkgo biloba modulates glutathione redox cycle in vascular endothelial cells

We recently reported that Ginkgo biloba extract (GBE) suppressed oxidative burst in macrophages and protected bovine pulmonary artery endothelial cells (PAEC) from oxidant injury. In this study the effects of GBE on glutathione (GSH) redox cycle and activity of antioxidant enzymes were investigated. Confluent monolayers of PAEC were incubated with GBE for 8–48 h, washed, and then lysed with Triton X-100. Biochemical assays were performed with the lysate. GBE caused both dose- and time-dependent increase in GSH level and glutathione disulfide (GSSG) reductase activity while GSH peroxidase and superoxide dismutase activity remained unaffected. Exposure of PAEC to an organic oxidant tert-butyl hydroperoxide (tBHP) resulted in decreased GSH level, increased lipid peroxidation, and elevated leakage of intracellular lactate dehydrogenase. Preincubation or simultaneous treatment of PAEC with GBE prevented these changes induced by tBHP. Our data suggest that the antioxidant effect of GBE may be due to its modulation of the GSH redox cycle in PAEC as well as direct scavenging of the oxidant.