还原型谷胱甘肽对大鼠力竭运动后自由基和心肌细胞凋亡的影响

目的:探讨还原型谷胱甘肽(GSH)对大鼠力竭运动后自由基和心肌细胞凋亡的影响。方法:30只SD大鼠随机分为对照组、力竭组和GSH组,每组10只。对照组大鼠安静状态下饲养,腹腔注射生理盐水1周后取样;力竭组大鼠腹腔注射生理盐水1周后进行一次性力竭运动,运动后即刻取样;GSH组大鼠给予GSH腹腔注射1周后,进行一次性力竭运动,运动后即刻取样。检测各组大鼠血清及心肌丙二醛(MDA)含量和心肌超氧化物歧化酶(SOD)活性,并采用TUNEL法检测心肌细胞凋亡率。结果:力竭组大鼠心肌SOD活性显著低于对照组和GSH组(P<0.05);力竭组大鼠血清和心肌MDA含量均显著高于对照组和GSH组(P<0.05)。力竭组大鼠心肌细胞凋亡率均显著高于对照组和GSH组(P<0.05)。结论:氧自由基生成增加和(或)SOD活性降低导致大鼠力竭运动后心肌细胞凋亡增加;GSH可减轻力竭运动后大鼠心肌细胞凋亡程度。     

补锌对耐力训练、力竭运动大鼠腓肠肌超氧化物岐化酶、谷胱甘肽过氧化物酶、总抗氧化能力以及Bax、Bcl-2 mRNA的影响

目的:探讨补锌对耐力训练、力竭运动大鼠腓肠肌超氧化物岐化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px,简称GPX)、总抗氧化能力(T-AOC)、细胞凋亡诱导和阻遏分子(Bax,Bcl-2)mRNA的影响。方法:将32只雄性SD大鼠随机分成对照组、耐力训练组、耐力训练补锌组和一次性力竭运动补锌组4组,每组8只。补锌方法为在饮用水中溶入ZnSO4.7H2O,[Zn]=227 mg/L。耐力训练组和耐力训练补锌组进行6周游泳耐力训练,每周6天。6周训练结束24小时后取材。一次性力竭运动组取材当日在同一泳池每隔15 min被放入2只大鼠,进行无负重游泳至力竭后取材。测定大鼠腓肠肌SOD、GPX、T-AOC活性,Bax、Bcl-2 mRNA表达。结果:耐力训练补锌组大鼠腓肠肌SOD活性显著高于对照组(P<0.05),GPX活性显著高于对照组和耐力训练组(P<0.05)。力竭运动补锌组大鼠腓肠肌GPX和T-AOC活性显著高于对照组(P<0.05)。耐力训练补锌组大鼠腓肠肌Bax mRNA表达显著低于对照组和耐力训练组(P<0.05),Bcl-2 mRNA表达显著高于对照组和耐力训练组(P<0.05)。力竭运动补锌组大鼠腓肠肌Bax,Bcl-2 mRNA表达均显著高于对照组(P<0.05)。结论:耐力训练提高大鼠腓肠肌GPX活性,增加Bcl-2 mRNA表达,降低大鼠腓肠肌Bax mRNA表达,补锌效果更明显。一次性力竭运动补锌提高大鼠腓肠肌GPX活性和T-AOC活性,增加腓肠肌Bcl-2 mRNA表达,但对Bax mRNA表达影响不明显。     

耐力训练和一次性力竭运动对大鼠心肌和骨骼肌硫氧还蛋白还原酶的影响

目的:观察运动对大鼠心肌、骨骼肌硫氧还蛋白还原酶(TR)的影响.方法:SD大鼠30只,随机分成安静对照组、耐力训练组和力竭组.训练组进行6周渐增游泳训练,5次/周.最后1次训练后24小时,断头处死安静对照组和训练组大鼠,力竭组大鼠在一次性力竭运动后即刻处死.DTNB法检测心肌、骨骼肌TR活性,RT-PCR法观察TR mRNA水平变化.结果:6周耐力训练大鼠心肌TR活性显著高于安静对照组,一次性力竭组大鼠心肌TR活性显著低于安静对照组.各组大鼠心肌TR mRNA均无显著性差异.结论:6周耐力训练显著提高大鼠心肌TR活性,而在mRNA水平上无显著性差异.     

曲美他嗪对力竭运动后大鼠心肌组织的保护作用研究

目的 观察曲美他嗪对力竭运动后大鼠心肌组织氧化应激、凋亡水平及能量代谢的影响,探讨曲美他嗪对力竭运动后大鼠心肌组织的保护作用.方法 30只雄性Wistar大鼠随机分为安静对照组(C组)、反复力竭运动组(E组)、反复力竭运动+曲美他嗪组(TE组),每组10只.通过游泳运动建立大鼠力竭运动模型,TE组加用曲美他嗪干预(10mg/kg),各组均于最后一次力竭运动后留取心肌组织.用分光光度法检测大鼠心肌细胞线粒体超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)和活性丙二醛(MDA)含量;用RT-PCR和免疫组化法分别检测心肌组织Bax、Bcl-2和解偶连蛋白2(UCP2)的mRNA和蛋白表达水平;TUNEL法检测心肌细胞凋亡率.结果 与C组比较,E组和TE组心肌线粒体SOD及GSH-Px活性均显著降低,其中E组降低更明显;MDA含量均升高,其中E组升高更明显(P＜0.01).与C组比较,E组和TE组Bax表达量均明显升高,Bcl-2表达量均明显降低(P＜0.01);与E组比较,TE组Bax表达量明显降低,Bcl-2表达量明显升高(P＜0.01).TUNEL检测结果显示,与C组比较,E组和TE组大鼠心肌细胞凋亡率明显升高(P＜0.01);与E组比较,TE组心肌细胞凋亡率明显降低(P＜0.01).与C组比较,E组和TE组UCP2表达量均明显升高(P＜0.01);与E组比较,TE组UCP2表达量明显降低(p＜0.01).结论 曲美他嗪可减轻力竭运动后大鼠心肌组织损伤,对大鼠心肌具有保护作用.     

烧伤脓毒症早期心肌内泛素mRNA的表达变化及意义

目的　观察烧伤脓毒症大鼠心肌内泛素mRNA表达的变化及意义。方法　采用大鼠 30 %Ⅲ度烫伤复合腹腔注射内毒素(6mg/kg)动物模型 ,分为伤后 2h和 6h组 ,同时设对照组 ,通过核糖核酸印迹法检测大鼠心肌内泛素mRNA表达的变化。结果　伤后 2h和 6h大鼠心肌内泛素mRNA 2 4kb条带和 1 2kb条带的表达均较对照组均显著升高 ,其中 2 4kb条带的升高更明显 (P<0 0 1)。提示烧伤脓毒症早期心肌内重要的蛋白降解途径———泛素系统活性呈持续增强现象 ,预示蛋白降解代谢可能显著增加。结论　该发现对进一步从代谢角度研究烧伤脓毒症时心肌结构的变化及其对心功能的影响有重要意义。     

牛磺酸对大鼠力竭运动后肝组织自由基损伤的对抗作用

以大鼠力竭运动为模型 ,观察牛磺酸对肝脏组织丙二醛 (MDA)含量、谷胱甘肽 (GSH)含量、超氧化物歧化酶 (SOD)和血清谷丙转氨酶 (GPT)的影响。结果显示 :力竭运动后大鼠肝组织MDA含量和血清GPT活性显著上升 ,肝组织GSH含量和SOD活性显著下降 ,而牛磺酸可显著地抑制力竭运动后大鼠肝组织MDA和血清GPT的升高以及肝组织中GSH含量和SOD活性的下降。提示牛磺酸对肝组织具有直接抗氧化损伤的作用。     

急性长时间运动对大鼠心肌线粒体NOS/NO,COX和HIF-1mRNA表达的影响

目的:探讨急性长时间运动对大鼠心肌线粒体一氧化氮合酶/一氧化氮(NOS/NO)、细胞色素c氧化酶(COX)活性、COXⅣ亚基和低氧诱导因子(HIF-1)α亚基mRNA表达的影响.方法:23只8周龄的雌性SD大鼠,体重215±23g,随机分为对照组(7只)、运动即刻组(8只)和运动后4小时组(8只).游泳时间平均为249±54min.运动后分别测定大鼠心肌线粒体NOS活力和NO含量、COX活性、COXⅣ和HIF-1α mRNA的表达.结果:对照组、运动即刻组和运动后4小时组NOS活力无显著差异(P>0.05);运动即刻组和运动后4小时组心肌线粒体NO含量低于对照组,有显著性差异(P<0.01);运动即刻组COX活性高于对照组,有显著性差异(P<0.01),运动后4小时略有下降,但仍高于对照组(P<0.05).COXⅣ和HIF-1α mRNA表达3组之间无显著性差异.结果表明,急性长时间游泳运动大鼠心肌线粒体NOS活力相对稳定,但运动后即刻线粒体NO含量明显降低,COX活性明显增高,COXⅣ和HIF-1α mRNA表达无明显变化.结果提示,一次长时间运动后大鼠心肌线粒体呼吸链功能相对完好,该运动模型对COXⅣ和HIF-1α mRNA表达诱导作用不明显.     

运动性心肌肥大大鼠背根神经节降钙素基因相关肽基因表达的变化

目的:探讨运动性心肌肥大大鼠背根神经节降钙素基因相关肽基因表达的变化。方法:雄性SD大鼠随机分为对照组(n=33)和耐力训练组(n=33)。耐力训练组进行10周的跑台耐力训练,建立运动性心肌肥大模型。建模后48h,从两组随机抽取17只大鼠,连续3天进行力竭运动,最后一次力竭运动后即刻处死全部实验动物。记录力竭运动距离和大鼠体重,取大鼠心脏和腰段背根神经节,记录心脏湿重,采用荧光定量聚合酶链式反应技术测定大鼠背根神经节降钙素基因相关肽mRNA的表达量。结果:10周后,耐力训练组大鼠心脏重量与体重之比和力竭运动距离显著高于对照组(P<0.01)。与对照组比较,力竭运动前耐力训练组背根神经节CGRPmRNA表达显著下降(P<0.01),力竭运动后耐力训练组背根神经节CGRPmRNA表达与力竭运动前相比无明显变化,而对照组较力竭运动前显著下降(P<0.01)。结论:运动性心肌肥大大鼠背根神经节CGRP基因表达下调,力竭运动后表达保持稳定。     

力竭运动后不同时相大鼠心肌低氧诱导因子1α表达的变化

目的:探讨力竭运动对心肌低氧诱导因子1α(HIF-1α)表达的影响。方法:90只健康成年雄性SD大鼠,分为安静对照组(n=10)和一次力竭游泳运动组(包括一次力竭后即刻组、6小时组、12小时组和24小时组,每组10只大鼠)、2周反复力竭游泳运动组(包括反复力竭后即刻组、6小时组、12小时组和24小时组,每组10只大鼠),建立一次力竭游泳和反复力竭游泳运动后心肌微损伤运动实验模型。两力竭运动组分别于最后一次力竭运动后即刻、6小时、12小时及24小时取材,安静对照组同期安静状态下取材。应用免疫荧光技术和图像分析方法测定各组大鼠心肌HIF-1α表达。结果:一次力竭游泳运动后即刻、6小时、12小时组大鼠左室、右室及室间隔HIF-1α蛋白表达均显著高于相同部位对照组蛋白表达(P<0.05);反复力竭运动后即刻组大鼠左心室HIF-1α蛋白表达显著高于对照组、6小时组及24小时组(P<0.05),即刻组、12小时及24小时组右心室HIF-1α蛋白表达均显著高于对照组(P<0.05)。结果提示,力竭运动后心肌低氧诱导因子-1α表达与心肌受损有关。     

力竭运动后不同时相大鼠心脏窦房结ADAMTS-1的变化

目的:探讨力竭运动后不同时相心脏窦房结带有血小板凝血酶敏感蛋白样模体的解整连蛋白金属蛋白酶-1(ADAMTS-1)mRNA和蛋白表达的变化特点。方法:100只健康成年雄性SD大鼠随机分为10组,每组10只。其中一次力竭游泳运动4组,2周反复力竭游泳运动4组,相应的安静对照组2组。安静对照组不运动。反复力竭各组大鼠尾部负重约3%体重,进行每周6天、每天1次、每次2小时左右、共2周的力竭游泳运动。一次力竭运动各组大鼠在正常喂养2周后进行一次性力竭游泳运动,方案同反复力竭组。分别于力竭运动后0、4、12及24小时取材,应用激光显微切割技术定位并收集窦房结细胞,通过实时荧光定量PCR、免疫荧光组化和图像分析技术测试大鼠心脏窦房结ADAMTS-1 mRNA和蛋白表达的变化。结果:一次力竭和反复力竭运动后4小时、12小时、24小时大鼠心脏窦房结ADAMTS-1 mRNA含量均显著低于其安静对照组(P<0.05);一次力竭和反复力竭运动后4小时、12小时、24小时心脏窦房结ADAMTS-1蛋白含量均显著低于其安静对照组(P<0.05),且力竭运动后即刻ADAMTS-1蛋白表达显著高于对照组(P<0.01)。结论:力竭运动后即刻心脏窦房结ADAMTS-1蛋白水平呈高表达,力竭运动后其它时相窦房结ADAMTS-1在mRNA和蛋白水平呈低表达,可能导致窦房结损伤。     

Reduction of Intracellular Glutathione Content and Radiosensitivity

Summary

The intracellular glutathione (GSH) content of HeLa, CHO and V79 cells was reduced by incubating the cells in growth medium containing buthionine sulphoximine or diethyl maleate (DEM). Clonogenicity, single-strand DNA breaks (ssb) and double-strand DNA breaks (dsb) were used as criteria for radiation-induced damage after X- or γ-irradiation. In survival experiments, DEM gave a slightly larger sensitization although it gave a smaller reduction of the intracellular GSH. In general, sensitization was larger for dsb than for ssb, also the reduction of the o.e.r. was generally larger for dsb than for ssb. This may be due to the higher dose rate in case of dsb experiments resulting in a higher rate of radiochemical oxygen consumption. In general, no effect was found on post-irradiation repair of ssb and dsb.     

Survival Curves of Glutathione Synthetase Deficient Human Fibroblasts: Correlation between Radiosensitivity in Hypoxia and Glutathione Synthetase Activity

Summary

The role of intracellular non-protein bound sulphydryl compounds (NPSH), and in particular that of glutathione (GSH), in the response of cells to ionizing radiation under different O₂ concentrations has been assessed using cell strains deficient in glutathione synthetase and exhibiting different NPSH levels. The cell strains used originated from patients with 5-oxoprolinuria and from their relatives (heterozygotes and proficient homozygotes). No correlation has been found between NPSH and GSH concentrations and radiosensitivity under oxic, aerobic and hypoxic conditions. However, a highly significant correlation has been observed between radiosensitivity under hypoxic conditions (and therefore the oxygen enhancement ratio) and the glutathione synthetase activity, suggesting that synthesis of GSH is required after irradiation. In order to explain our results we postulated, beside radical processes, the existence of a GSH-dependent enzymatic repair mechanism for N₂ type damage. Hypoxic radio-sensitivity measured with survival curves would result from the interaction of both competition and biochemical repair processes.     

The Migration of Radiation Damage in Reduced Glutathione

The migration of radiation induced unpaired spins in reduced glutathione has been followed by qualitative and quantitative electron-spin-resonance (EPR) measurements. Polycrystalline glutathione was irradiated with 220 kv x-rays and kept in vacuum at room temperature. Ten minutes after exposure the spectrum resembled that of an equimolar mechanical mixture of the three constituent amino acids. In the course of days the spectrum changed to become finally almost identical with that of irradiated cysteine.

The spectrum of the mechanical mixture was shown to be a simple sum of those of the constituent amino acids in which the majority of EPR centres reside in the glutamic acid and glycine. The data indicate that in irradiated glutathione EPR centres are initially induced in all three amino acid residues. Subsequently, the EPR centres are slowly transferred to the cysteine part. This transfer may possibly involve an interaction between radiation-induced molecular fragments and intact neighbouring glutathione molecules.     

Non-protein Sulphydryl and Glutathione Content of Ehrlich Ascites Tumour Cells after Treatment with the Radioprotectors AET,Cysteamine and Glutathione

Summary

Treatment of Ehrlich ascites cells in vitro with increasing concentrations of cysteamine, AET or glutathione (GSH) resulted in a proportional increase of the intracellular level of non-protein-bound sulphydryl (NPSH). At equimolar concentrations, the increase in NPSH was greatest after AET-treatment, less after cysteamine and least after GSH. A considerable part of the increase in NPSH after AET- and cysteamine-treatment was due to an increase in content of cellular GSH. The increase in intracellular NPSH after GSH treatment was, in part, due to some compound(s) other than GSH.

After interruption of AET or cysteamine treatment, the total NPSH content of the cells decreased immediately, but the GSH level continued to increase for some time before it also started to fall.

The results were interpreted as indicating that intracellular release of GSH may be a major mechanism for the radioprotection afforded by the substances used in the investigation.     

高效液相色谱法测定人血浆中还原型谷胱甘肽浓度

目的建立HPLC方法测定人血浆中还原型谷胱甘肽（GSH）的浓度。方法采健康人体血浆样品，加入定量的二硫苏糖醇（DTT），用10％高氯酸沉淀蛋白，在硼酸缓冲液中与邻苯二甲醛（OPA）进行衍生化，取上清液20txl进样测定。C18色谱柱，流动相为0．02mol·L^-1KH2PO4缓冲盐-乙腈（V：V=94：6）；流速1ml／min；荧光检测波长λex 340nm，λem 420nm。结果GSH标准衄线在0．5～200mg·L^-1内，线性良好，r=0．9968（n=7）。日内和日间精密度的RSD均在9％以内，3种浓度下样品的方法回收率均〉95％。结论该方法准确，无干扰，可用于人体药动学及生物等效性研究。     

Glutathione and cysteine levels in human tumour biopsies

There is evidence that some human tumours could be treated with a combination of buthionine sulfoximine and hypoxic cell sensitizers. However, clinical application of this technique requires a prior knowledge of the level of non-protein bound sulfhydryl (NPSH) compounds in these tumours. The present study provides data on the levels of glutathione (GSH) and cysteine (CYS) in human tumour biopsies from the cervix and from the head and neck. The NPSH compounds were measured by high performance liquid chromatography. The median GSH values were 20.5 nmol/mg protein (cervix) and 23 nmol/mg protein (head and neck) while the median CYS values were 4.4 (cervix) and 4.2 nmol/mg protein (head and neck). The values varied widely from one patient to another.     

The Effect of Whole-body X-irradiation on the Glutathione Content of Rat Thymus

The reduced glutathione content of rat thymus has been measured by amperometric titration. During the rapid growth of the rat (2–16 weeks) the glutathione concentration remained constant. Whole-body x-irradiation with 200 rads and 900 rads caused a significant fall in glutathione concentration apparent after 110 min. The magnitude of this fall was little affected by increased doses of x-radiation, and it is unlikely that a direct radiochemical reaction was the cause of the lowered glutathione levels. These results are discussed with relation to the known sensitivity of thymus to x-rays, and it is suggested that glutathione levels fall before histological evidence of death is forthcoming. The normal value for reduced glutathione per thymocyte was found to be 413 × 10^?12 µ moles per cell.