Effect of melatonin on production of hydroxyl radical and lactate dehydrogenase during hypoxia in rat cortical slices 1

目的：研究褪黑激素对大鼠大脑皮层脑片在缺氧后羟自由基及乳酸脱氢酶生成的影响．方法：通过水杨酸捕获法来观察缺氧再给氧中羟自由基含量的变化．脑片通以９１６％Ｎ２＋８４％Ｏ２造成缺氧．ＬＤＨ用细胞毒检测盒测定．结果：ＤＨＢＡ水平在缺氧及再给氧后显著升高;缺氧时给予褪黑激素可以浓度依赖性地降低再给氧１５ｍｉｎ时ＤＨＢＡ的含量,但对缺氧３０ｍｉｎ时ＤＨＢＡ的含量没有显著作用．ＬＤＨ的含量在缺氧后１ｈ内持续升高;褪黑激素可以显著降低缺氧后ＬＤＨ的释放．结论：褪黑激素降低大鼠缺氧后的损伤以及羟自由基的产生．     

Melatonin decreases production of hydroxyl radical during cerebral ischemia-reperfusion

目的：研究褪黑激素（ｍｅｌａｔｏｎｉｎ）对大鼠脑缺血再灌注中羟自由基生成的影响．方法：采用栓线法阻塞左侧ＭＣＡ３０ｍｉｎ再灌注模型．通过水杨酸捕获法与微透析技术结合来观察缺血再灌中羟自由基含量的变化．结果：ＤＨＢＡ水平在缺血１５ｍｉｎ后显著升高，持续到再灌注后３０ｍｉｎ仍维持较高水平．缺血前３０ｍｉｎ给予ｍｅｌａｔｏｎｉｎ（４ｍｇ·ｋｇ－１，ｓｃ）显著降低缺血１６－３０ｍｉｎ及再灌注１－３０ｍｉｎ时ＤＨＢＡ的含量．结论：Ｍｅｌａｔｏｎｉｎ对大鼠缺血再灌中羟自由基的产生有抑制作用．     

褪黑激素降低脑缺血再灌注中羟自由基的生成

目的：研究褪黑激素对大鼠脑缺血再灌注中羟自由基生成的影响。方法：采用栓线法阻塞左侧ＭＣＡ３０ｍｉｎ再灌注模型。通过水杨酸捕获法与微透析技术结合来观察缺血再灌中羟自由基含量的变化。结果：ＤＨＢＡ水平在缺血１５ｍｉｎ后显升高，持续到再灌注后３０ｍｉｎ仍维持较高水平。缺血前３０ｍｉｎ给予ｍｅｌａｔｏｎｉｎ（４ｍｇ·ｋｇ＾－１，ｓｃ）显降低缺血１６￣３０ｍｉｎ及再灌注１－３０ｍｉｎ时ＤＨＢＡ的含量。结     

Protective effects of ditiocarb sodium against anoxic damage of primary cortical neurons in culture

取体外原代培养１４ｄ左右的大鼠皮层神经元，换上无血清除氧培养基并置通９５％Ｎ２＋５％ＣＯ２的缺氧罐中造成神经元缺氧．以研究缺氧对皮层神经元的损伤及二硫卡钠（ＤＴＣ）的保护作用．用存活神经元数目及神经元线粒体活性来评价神经元活力．乳酸脱氢酶（ＬＤＨ）活性及脂质过氧化产物丙二醛（ＭＤＡ）含量分别用紫外分光光度法和硫代巴比酸法测定，作为评价损伤的指标．原代培养皮层神经元分别缺氧４，５和６ｈ后，活细胞数显著减少，线粒体活性明显降低；ＬＤＨ释放及ＭＤＡ产生显著增加；而缺氧２ｈ后，仅活细胞数无明显变化．超氧阴离子清除剂超氧化物歧化酶（ＳＯＤ）可逆转上述变化，抑制缺氧对神经元的损伤，表明培养神经元缺氧损伤可能与自由基产生有关．类似ＳＯＤ作用，ＤＴＣ可显著对抗缺氧对神经元的损伤，剂量依赖地抑制缺氧引起的ＬＤＨ释放和ＭＤＡ形成的增加．结果表明：ＤＴＣ对神经元缺氧损伤具保护作用，其作用可能与清除自由基有关．     

缓激肽对新生大鼠心肌细胞缺氧：再给氧损伤的保护作用

缓激肽能使培养乳鼠心肌细胞缺氧再给氧损伤时，ＬＤＨ释放减少和ＴＢＡＲＳ含量降低，此作用能被缓激肽拮抗剂Ｋ８６／４３２１所取消，心肌细胞Ａ／Ｒ损伤后，ＨＰＬＣ－ＥＤ法测定培养液中有羟自由基生成，ＢＫ能减少其产生。ＢＫ保护作用的机理可能与其抗脂质过氧化和减少羟自由基生成有关。     

Ebselen对自由基诱发的皮层神经元和皮层线粒体脂质过氧化损伤的保护作用

观察了ｅｂｓｅｌｅｎ对超氧阴离子Ｏ·－２和羟自由基·ＯＨ诱发的体外培养大鼠皮层神经元乳酸脱氢酶（ｌａｃｔｉｃｄｅｈｙｄｒｏｇｅｎａｓｅ，ＬＤＨ）释放，和ＴＢＡＲＳ（ｔｈｉｏｂａｒｂｉｔｕｒｉｃａｃｉｄｒｅａｃｔｉｖｅｓｕｂｓｔａｎｃｅ）含量升高及制备的皮层线粒体ＴＢＡＲＳ含量升高的影响。结果表明：超氧阴离子和羟自由基引起了培养神经元和线粒体明显的损伤。浓度在５～５０μｍｏｌ·Ｌ－１之间，ｅｂｓｅｌｅｎ能剂量依赖性地抑制ＬＤＨ释放和ＴＢＡＲＳ含量的增加，对线粒体的ＴＢＡＲＳ含量升高也有显著的抑制作用。但浓度为０．２～５０μｍｏｌ·Ｌ－１时，药物无直接清除超氧阴离子和羟自由基的活性。因此，ｅｂｓｅｌｅｎ对氧自由基诱发的神经元脂质过氧化损伤有拮抗作用，这种作用与直接清除自由基无关。     

Inhibitory effects of α-methyl-4-(3-oxo-2H-1,2-benzoisoselenazol-2-yl)benzeneacetic acid on Cu~(2+) and Fe~(2+) induced oxidative modification of human low density lipoprotein

为观察α－甲基－４－（３－氧－２Ｈ－１，２－苯并异硒唑－２－基）苯乙酸（ＭＢＢＡ）对Ｃｕ２＋及Ｆｅ２＋氧化修饰低密度脂蛋白（ＬＤＬ）的保护作用及其作用机理，采用分光光度法测定ＬＤＬ中丙二醛（ＭＤＡ）和共轭双烯（ＣＤ）的产生量．ＭＢＢＡ（０．２－２μｍｏｌ·Ｌ－１）能以剂量依赖性抑制Ｃｕ２＋及Ｆｅ２＋诱导的ＭＤＡ和ＣＤ生成．２μｍｏｌ·Ｌ－１的ＭＢＢＡ对Ｃｕ２＋诱导ＬＤＬ产生ＭＤＡ和ＣＤ的抑制率分别为８９．７％和６０．３％．０．５ｍｍｏｌ·Ｌ－１ＧＳＨ对ＬＤＬ产生ＭＤＡ无影响，但能显著增强ＭＢＢＡ对ＭＤＡ生成的抑制作用．上述结果表明ＭＢＢＡ对ＬＤＬ氧化修饰的抑制作用可能依赖于其ＧＳＨ－Ｐｘ样活性的作用和（或）直接还原脂质氢过氧化物的作用．     

Efflux transport of[~3H]GABA across blood-brain barrier after cerebral ischemia-reperfusion in rats

目的：研究脑缺血／再灌后［３Ｈ］ＧＡＢＡ通过大鼠血脑屏障的外排转运是否增强及其机制．方法：将［３Ｈ］ＧＡＢＡ或ＧＡＢＡ（或丙磺舒）与其联合注射到缺血／再灌大鼠大脑皮层顶二区后，测定［３Ｈ］ＧＡＢＡ的脑外排指数（ＢＥＩ）及ｉｖ依文思蓝（ＥＢ）后ＥＢ的脑摄取量．结果：１０ｍｉｎ缺血／再灌３０ｍｉｎ、２ｈ、６ｈ和２４ｈ大鼠的ＢＥＩ分别为６７％，８３％，９２％和８７％，显著高于对照值（５８％），ＥＢ脑摄取量也显著增加；ＧＡＢＡ或丙磺舒明显降低正常及再灌６ｈ大鼠的ＢＥＩ，但对再灌５ｍｉｎ大鼠的ＢＥＩ无明显影响．结论：大鼠脑缺血／再灌后［３Ｈ］ＧＡＢＡ通过血脑屏障的外排转运显著增强．     

芸香甙对脑缺血再灌损伤的保护作用

目的研究芸香甙（Ｒｕ）对脑缺血再灌损伤的保护作用。方法小鼠脑缺血再灌模型通过结扎双侧颈总动脉并尾部放血０．３ｍｌ，再灌后，记录异常神经症状和断头后张口喘气时间，测定脑组织中乳酸脱氢酶（ＬＤＨ）含量；利用４血管模型，大鼠前脑缺血３０ｍｉｎ后再灌注４０ｍｉｎ。取脑组织测定ＬＤＨ、过氧化物歧化酶（ＳＯＤ）、丙二醛（ＭＤＡ）、一氧化氮（ＮＯ）及脑水肿。结果Ｒｕ（５０，１００ｍｇ·ｋｇ－１，ｉｖ），显著改善小鼠脑缺血再灌损伤后的异常神经症状和抑制断头后张口喘气时间的缩短及脑组织中ＬＤＨ的减少。Ｒｕ显著抑制大鼠脑缺血再灌损伤所致的脑水肿形成和脑组织中ＬＤＨ、ＳＯＤ、ＭＤＡ及ＮＯ的变化。结论Ｒｕ对脑缺血再灌损伤有显著的保护作用，其机制与抗自由基和ＮＯ有关。     

脂必妥与安慰剂治疗高脂血症的随机双盲多中心研究

ｃｈ，总有效率分别为８０％及１６％；８２％及１７％；６９％及２８％。２组间疗效对比有非常显著差异（Ｐ＜０．０１），对ＴＣ，ＴＧ，ＨＤＬ－ｃｈ水平及ＴＣ－ＨＤＬ－ｃｈ／ＨＤＬ－ｃｈ比值均有显著改变，与对照组相比差异非常显著。２组对ＬＤＬ－ｃｈ，ＡｐｏＡＩ，ＡｐｏＢ１００，ＡｐｏＡＩ／ＡｐｏＢ１００，ＬＰ（ａ）水平则改变不明显。未见不良反应。结论：脂必妥是一种安全有效的调脂药物。     

羟丁酸钠对大鼠原代培养皮层神经元缺氧复氧损伤的保护作用与GABAA受体的关系

目的 :探讨羟丁酸钠 (SO)对大鼠皮层神经元缺氧复氧损伤的保护作用与GABAA 受体的关系。方法 :采用原代培养大鼠皮层神经元建立缺氧复氧损伤模型 ,观察细胞的形态学 ,测定其LDH漏出率、MDA含量、SOD、GPx活力。结果 :缺氧复氧可引起神经元损伤 ,LDH漏出率增加 ,MDA含量升高 (P <0 .0 1) ,SOD、GPx活力降低 (P <0 .0 1)。SO能显著减少损伤神经元的LDH漏出率及MDA的生成 (P <0 .0 1) ,升高SOD、GPx(P <0 .0 1)的活力 ,这种作用可被GABAA 受体阻断剂seurinine减弱 (P <0 .0 1)。结论 :SO对缺氧复氧神经元损伤的保护作用可能与其激动GABAA 受体有关。     

Involvement of peroxynitrite and hydroxyradical generated from nitric oxide in hypoxia/reoxygenation injury in rat cerebrocortical slices

The changes in nitric oxide (NO) formation during hypoxia and reoxygenation were measured in slices of rat cerebral cortex, and the possible involvement of NO and its decomposition products, including peroxynitrite and hydroxyradical, in the hypoxia/reoxygenation injury was subsequently investigated. NO formation estimated from cGMP accumulation in the extracellular fluids was enhanced during hypoxia and to a lesser extent in the reoxygenation period. The mRNA for inducible NO synthase (NOS) was detected 3-5 h after reoxygenation, although neuronal NOS mRNA decreased after reoxygenation. Several NOS inhibitors such as N(G)-monomethyl-L-arginine and N(G)-nitro-L-arginine blocked not only the NO formation but also the hypoxia/reoxygenation injury as determined by lactate dehydrogenase (LDH) leakage. The hypoxia/reoxygenation injury was prevented by peroxynitrite scavengers including deferoxamine and uric acid, or several hydroxyradical scavengers such as dimethylthiourea, 2-mercaptopropionylglycine and D(-) mannitol. In addition, the hypoxia/reoxygenation injury was attenuated by poly(ADP-ribose)synthetase inhibitors such as banzamide, 3-aminobenzamide and 1,5-isoquinolinediol. On the other hand, both N-morpholinosidnonimine, a peroxynitrite generator, and hydroxyradical-liberating solution containing FeCl(3)-ADP and dihydroxyfumarate caused a marked LDH leakage in normoxic slices. These findings suggest that the enhanced formation of NO causes hypoxia/reoxygenation injury after degradation to peroxynitrite and hydroxyradical and the resultant activation of poly(ADP-ribose)synthetase.     

Nimbolide, a neem limonoid abrogates canonical NF-κB and Wnt signaling to induce caspase-dependent apoptosis in human hepatocarcinoma (HepG2) cells

Reactive oxygen species production is necessary to induce cell death following hypoxia/reoxygenation but the effect of reactive oxygen species produced during hypoxia on mitochondrial permeability transition pore (mPTP) opening and cell death is not established. Here we designed a model of hypoxia/reoxygenation in isolated cardiomyocytes measuring simultaneously reactive oxygen species production, mPTP opening and cell death in order (i) to establish a causal relationship between them, and (ii) to investigate the roles of various reactive oxygen species in mPTP opening. The percentage of cardiomyocytes exhibiting mPTP opening during reoxygenation increased with the duration of hypoxia. Antioxidants increased the time to mPTP opening when present during hypoxia but not at reoxygenation. This was associated with a drop in hydroxyl radical and hydrogen peroxide during hypoxia and the first minutes of reoxygenation. The increase in time to mPTP opening was accompanied by an improvement in cell viability reflected by maintenance of superoxide production at reoxygenation. Cyclosporin A delayed both the time to mPTP opening and cell death despite maintenance of reactive oxygen species production during hypoxia. These findings demonstrate that reactive oxygen species production precedes mPTP opening and that reactive oxygen species produced during hypoxia, particularly hydroxyl radicals and hydrogen peroxide, are necessary to induce mPTP opening which depends on hypoxia duration.     

Effect of Trolox C on hypoxia/reoxygenation-induced injury in isolated perfused rat liver

Livers isolated from 18 hours fasted rats were subjected to N(2) hypoxia (for 45 min) followed by reoxygenation (for 45 min). The perfusion medium used was Krebs-Henseleit bicarbonate buffer (KHBB, pH 7.4). Lactate and alanine were added as gluconeogenic and ureagenic substrates and Trolox C was also added to perfusate. Oxygen consumption, lactate dehydrogenase (LDH), alanine transaminase (ALT), total glutathione, oxidized glutathione, bile flow, glucose and urea were measured. After hypoxia oxygen consumption significantly dropped but Trolox C had no influence on this decrease. ALT and LDH were significantly increased by hypoxia/reoxygenation. This increase was markedly attenuated in the presence of Trolox C. The total glutathione and oxidized glutathione efflux increased following hypoxia, which were prevented by the treatment of Trolox C. Bile flow rate decreased following hypoxia/reoxygenation but did not continue to decrease in the reoxygenation phase by Trolox C. Following hypoxia/reoxygenation glucose and urea releases decreased. Trolox C had no influence on inhibition of glucose and urea production. These results suggest that Trolox C protected the liver cells against hypoxia/reoxygenation injury, yielding further evidence for a causative role of oxidative stress in this model.     

风轮菜总黄酮联合miR-702-5p抑制物对缺氧/复氧诱导的心肌细胞损伤的影响

目的研究风轮菜总黄酮联合miR-702-5p抑制物对缺氧/复氧诱导的心肌细胞损伤的影响。方法实验设置对照组(Con,不作任何处理)、缺氧/复氧组(H/R,缺氧6 h,复氧3 h),H/R+药物-1组、H/R+药物-2组、H/R+药物-3组(6.25μg/ml、12.5μg/ml、25μg/ml的风轮菜总黄酮+缺氧/复氧处理)、H/R+anti-miR-NC组(转染anti-miR-NC+缺氧/复氧处理)、H/R+anti-miR-702-5p组(转染anti-miR-702-5p+缺氧/复氧处理)、H/R+药物-3+anti-miR-NC组(转染anti-miR-NC+25μg/ml风轮菜总黄酮+缺氧/复氧处理)、H/R+药物-3+anti-miR-702-5p组(转染anti-miR-702-5p+25μg/ml风轮菜总黄酮+缺氧/复氧处理)。蛋白质印迹(Western blot)法检测细胞周期蛋白依赖性激酶抑制剂1A(P21)、半胱氨酸天冬氨酸蛋白酶-3(Caspase-3)蛋白表达;细胞计数试剂盒8(CCK-8)检测细胞存活率;流式细胞术检测细胞凋亡;试剂盒检测丙二醛(MDA)含量及乳酸脱氢酶(LDH)、过氧化氢酶(CAT)、超氧化物歧化酶(SOD)活性;实时荧光定量PCR(RT-qPCR)检测miR-702-5p表达水平。结果缺氧/复氧诱导的心肌细胞中P21、Caspase-3表达水平升高,细胞存活率降低,细胞凋亡率升高,MDA含量升高,LDH活性升高,CAT、SOD活性降低。不同浓度风轮菜总黄酮处理后P21、Caspase-3表达水平降低,细胞存活率升高,细胞凋亡率降低,MDA含量降低,LDH活性降低,CAT、SOD活性升高。抑制miR-702-5p表达可抑制缺氧/复氧诱导的心肌细胞损伤。风轮菜总黄酮联合miR-702-5p抑制剂可显著提高细胞存活率,抑制细胞凋亡和氧化应激反应。结论风轮菜总黄酮联合miR-702-5p抑制物可保护心肌细胞免受缺氧/复氧引起的损伤。     

Efficacy and mechanism of hypoxic postconditioning in salvage of ex vivo human rectus abdominis muscle from hypoxia/reoxygenation injury

In reconstructive surgery, skeletal muscle may endure protracted ischemia before reperfusion, which can lead to significant ischemia/reperfusion injury. Ischemic postconditioning induced by brief cycles of reperfusion/reocclusion at the end of ischemia has been shown to salvage skeletal muscle from ischemia/reperfusion injury in several animal models. However, ischemic postconditioning has not been confirmed in human skeletal muscle. Using an established in vitro human skeletal muscle hypoxic conditioning model, we tested our hypothesis that hypoxic postconditioning salvages ex vivo human skeletal muscle from hypoxia/reoxygenation injury and the mechanism involves inhibition of opening of the mitochondrial permeability transition pore (mPTP) and preservation of ATP synthesis. Muscle strips (~0.5×0.5×15mm) from human rectus abdominis muscle biopsies were cultured in Krebs-Henseleit-HEPES buffer, bubbled with 95%N(2)/5%CO(2) (hypoxia) or 95%O(2)/5%CO(2) (reoxygenation). Samples were subjected to 3h hypoxia/2h reoxygenation. Hypoxic postconditioning was induced by one or two cycles of 5min reoxygenation/5min hypoxia after 3h hypoxia. Muscle injury, viability and ATP synthesis after 2h of reoxygenation were assessed by measuring lactate dehydrogenase (LDH) release, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) reduction and ATP content, respectively. Hypoxic postconditioning or treatment with the mPTP-opening inhibitors Cyclosporine A (CsA, 5×10(-6)M) or N-Methyl-4-isoleucine Cyclosporine (NIM811, 5×10(-6)M) 10min before reoxygenation decreased LDH release, increased MTT reduction and increased muscle ATP content (n=7 patients; P<0.05). Conversely, treatment with the mPTP opener Atractyloside (5×10(-6)M) 10min before hypoxic postconditioning abolished its protective effect (n=7 patients; P<0.05). We conclude that hypoxic postconditioning effectively salvages human skeletal muscle from hypoxia/reoxygenation injury by inhibition of mPTP opening and preservation of ATP synthesis during reoxygenation.     

Aniracetam,a Pyrrolidinone-type Cognition Enhancer,Attenuates the Hydroxyl Free Radical Formation in the Brain of Mice with Brain Ischaemia

We demonstrate here that aniracetam has the ability to block the formation of cytotoxic hydroxyl radicals (· OH) during ischaemia-reperfusion of mouse brain. The fact that brain ischaemia for 40 min followed by reperfusion increased ·OH was evidenced by detection of a peaked increase at 20 min after an ischaemic insult in the formation of 2,3-dihydroxy-benzoate (DHBA) from salicylate in cerebroventricular perfusate, a means of monitoring ·OH formation. A clearcut increase in dopamine was also observed during and after brain ischaemia. The ischaemia-reperfusion mice given aniracetam at an intraperitoneal dose of 30 or 100 mg kg^?1 showed a smaller increase in the formation of DHBA than those given the vehicle only. Aniracetam at 100 mg kg^?1 significantly suppressed the formation of DHBA by approximately 80%, becoming evident at 20 min after reperfusion and thereafter. Protection against death in mice insulted with a 40-min brain ischaemia (3/13 vs 13/25) was observed following 100 mg kg^?1 aniracetam. The increase in the dopamine levels was substantially reduced following aniracetam treatment and the reduction became significant at 20 min after reperfusion and thereafter in parallel with attenuation by aniracetam of DHBA formation. This finding suggests that the inhibitory activity of aniracetam in attenuating the hydroxyl free-radical formation in ischaemic mice is probably due, at least in part, to its palliative action on the dopaminergic neurons.     

二氢石蒜碱对大鼠乳鼠培养心肌细胞缺氧/复氧损伤的保护作用

目的观察二氢石蒜碱(dihydrolycorine,DL)对Wistar大鼠的乳鼠培养心肌细胞缺氧/复氧损伤(H/R)的保护作用。方法采用培养的Wistar乳鼠的心肌细胞,建立H/R损伤模型,检测指标包括:①心肌细胞存活率;②超氧化物歧化酶(SOD)活性;③丙二醛(MDA)含量;④乳酸脱氢酶(LDH)活性。结果模型组缺氧后心肌细胞存活率降低,复氧后进一步下降,各个时间点细胞内SOD活性下降,MDA含量升高,乳酸脱氢酶(LDH)活性升高,与正常组比较差异具有显著性(P<0.01);在DL1、10、100μmol·L-1组,随着给药浓度的增加,细胞存活率升高,LDH活性变低,MDA含量逐渐趋于恢复正常,SOD活性逐渐回升,表明经DL干预之后,心肌细胞抗损伤能力加强,发生损伤的程度减轻。结论DL对培养的乳鼠心肌细胞H/R损伤具有保护作用,其作用在一定范围内呈剂量依赖关系,机制可能与其阻断α、β受体、抑制心肌细胞脂质过氧化反应有关。     

羟丁酸钠对皮质神经元缺氧复氧损伤的保护作用(英文)

目的　研究羟丁酸钠 (GHB)对缺氧复氧脑损伤的保护机制。方法　用原代培养大鼠皮层神经元建立缺氧复氧损伤模型 ,缺氧 2h复氧 6h ;缺氧前30min在培养基内加入GHB(5 ,2 0 ,80mmol·L- 1) ,观察神经元的形态学变化 ,检测培养基中乳酸脱氢酶漏出率及细胞中丙二醛 (MDA)含量、超氧化物歧化酶 (SOD)和谷胱甘肽过氧化物酶 (GSH Px)的活力。结果　缺氧复氧降低神经元生存率 ,增加乳酸脱氢酶 (LDH)漏出及MDA含量 ,而导致SOD及GSH Px活力下降。GHB 2 0 ,80mmol·L- 1预处理能显著提高缺氧复氧神经元的生存率 ,减少LDH的漏出及MDA的生成 ,升高SOD和GSH Px的活力。结论　GHB对缺氧复氧引起的神经元损伤的保护作用与它保护抗氧化酶的作用有关。