心肌细胞缺氧复氧模型的建立方法及能量代谢相关检测指标研究进展

在心肌梗死、缺血再灌注损伤或心力衰竭等多种心血管疾病的发生发展过程中多伴有心肌细胞损伤。心肌细胞缺氧复氧其损伤模型可直接观测药物对损伤心肌细胞的形态结构、生理生化指标,并可用于后续药物作用机制的深入探讨,是体外评价抗心肌缺血、抗治疗心衰等活性成分药物的优选模型,在心血管药物研究和开发中应用极为广泛。同时心脏作为高度耗能耗氧的器官,其收缩和舒张都需要消耗能量,故给药后能量代谢相关指标的检测通常作为评价此类药物得到改善可作为药物发挥疗效的基本依据。本文通过查阅近年国内外有关心肌细胞缺氧复氧模型建立与指标检测的相关文献,来论述从心肌细胞种类的选择、不同模型建立方法的优缺点、代表性能量代谢指标的检测等方面的研究和应用进展进行综述,并就模型建立过程中注意事项进行探讨,为心血管药物研究中有效复制该模型提供参考。     

荭草苷对缺氧模型小鼠的抗缺氧作用研究

目的:研究荭草苷对缺氧模型小鼠的抗缺氧作用。方法:通过常压耐缺氧、亚硝酸钠中毒、氰化钾中毒、利多卡因中毒、夹闭气管及断头等建立小鼠缺氧模型,于造模前20min给予相应药物,观察药物的抗缺氧作用。结果:与生理盐水空白对照组比较,荭草苷可明显延长模型小鼠在常压缺氧、亚硝酸钠中毒、氰化钾中毒、利多卡因中毒时的存活时间,延长夹闭气管后的心电图消失时间及断头后的喘气时间。结论:荭草苷对缺氧模型小鼠具有抗缺氧作用。     

七氟醚对培养心肌细胞缺氧损伤的保护作用

<正>七氟醚学作为新型的吸入麻醉药,其对心肌缺血再灌注动物及冠心患者有着明显的心肌保护作用[1,2]。本课题选用氰化钠造成培养心肌细胞缺氧(细胞内缺氧)模型,排除了血流动力学及其因素在缺氧损伤的影响,研究七氟醚对细胞缺氧损伤的直接作用,从而提示七氟醚对机体的非麻醉作用即抗损伤作用     

抗缺氧药物的研究进展

社会竞争的增大和生活节奏的加快使得现代人承担的压力越来越大,脑力工作的负担也不断增加,需更多的血液和氧气才能保证大脑的顺畅工作。当进行高空、潜水等作业或患某些疾病时,同样会遇到各种情况的缺氧。缺氧可引起机体一系列生理代偿反应,如心率增加,血流动力学改变,肺动脉高压、能量代谢障碍等,尤其是其可致心肌受损、脑功能下降。因此采用一些抗缺氧的药物改善机体供氧和能量状态,对提高机体在缺氧条件下的生活质量和活动能力有非常重要的意义。     

乙酰唑胺对PC12细胞缺氧损伤的保护作用及其机制研究

目的研究乙酰唑胺对缺氧PC12细胞的保护作用及其机制。方法用三气培养箱建立缺氧模型培养PC12细胞,筛选乙酰唑胺作用于缺氧PC12细胞的最佳浓度,观察细胞形态,测定细胞活力、LDH的含量、细胞周期,实时荧光定量PCR法检测HIF-1α及Caspase-3 mRNA的表达。结果与缺氧模型组相比,乙酰唑胺可明显提高缺氧PC12细胞存活率、降低培养基中LDH的含量,降低G1期、提高S期细胞百分比、提高HIF-1αmRNA的表达、降低Caspase-3 mRNA的表达水平。结论乙酰唑胺具有保护PC12细胞缺氧损伤的作用。     

冠心苏合丸系列组方药物血清对心肌细胞缺氧复氧损伤的影响*

目的:观察冠心苏合丸系列组方药物血清对大鼠心肌细胞缺氧复氧损伤的影响,阐明系列组方抗缺氧复氧损伤作用机制上的异同。方法:用体外细胞培养方法,培养乳鼠心肌细胞,复制心肌细胞缺氧复氧模型;运用血清药理学方法,将心肌细胞与含药血清共培养,测定细胞活性、上清液中肌酸激酶(CK)活性、乳酸脱氢酶(LDH)活性、丙二醛(MDA)含量以及细胞超氧化物歧化酶(SOD)活性、Na ,K -ATP酶和Ca2 ,Mg2 -ATP酶活性。结果:含青木香的冠心苏合丸、含土木香的冠心苏合丸以及不含木香的冠心苏合丸给药后30~90 m in的药物血清均能不同程度地增强缺氧复氧损伤的心肌细胞活性;显著抑制缺氧复氧损伤所致心肌细胞LDH和CK的外漏;显著增强缺氧复氧损伤的心肌细胞SOD活性,降低MDA含量;显著增强缺氧复氧损伤的心肌细胞Na ,K -ATP酶和Ca2 ,Mg2 -ATP酶活性;含土木香及不含木香的冠心苏合丸方在个别指标上的作用优于含青木香的冠心苏合丸。结论:冠心苏合丸方系列组方对缺氧复氧心肌细胞损伤均有明显保护作用,作用机制相似;原方中青木香被土木香替换或者去掉青木香,不影响其抗缺氧复氧损伤的作用。     

麝香保心丸抗心肌细胞缺氧-复氧损伤活性成分筛选

目的：建立原代心肌细胞缺氧-复氧模型,并对麝香保心丸及其20种血中活性成分进行抗缺氧-复氧损伤的活性筛选。方法取新生（1～3 d）SD乳鼠的心脏,建立原代心肌细胞的缺氧-复氧损伤模型,并采用四甲基偶氮唑盐（MTT）比色法进行麝香保心丸及其入血成分的活性筛选。结果麝香保心丸在50μg/ml浓度下具有较好的保护原代心肌细胞缺氧-复氧损伤的作用;人参皂苷Rb1、人参皂苷Rb2、蟾毒灵和麝香酮具有较好的保护原代心肌细胞缺氧-复氧损伤的作用。结论麝香保心丸具有抗心肌细胞缺氧-复氧损伤的作用,人参皂苷Rb1、人参皂苷Rb2、蟾毒灵和麝香酮是其主要的有效成分。该研究为麝香保心丸深入的药效和机制研究奠定了基础。     

丁苯酞对缺氧人脐静脉血管内皮细胞Ang-2蛋白表达的影响

目的:探讨药物丁苯酞(NBP)对人脐静脉血管内皮细胞(HUVEC)缺氧损伤的保护作用及其对血管生成素2(Ang-2)表达的影响。方法:体外培养HUVEC,建立稳定的内皮细胞缺氧损伤模型。试验分为正常对照组、缺氧损伤模型组、NBP组,按要求分别给予药物处理后,用CCK-8法检测细胞活力,采用免疫细胞化学方法和图像定量分析技术检测平均吸光度,比较各组Ang-2蛋白表达的变化;Western Blot法检测各组Ang-2蛋白的表达情况。结果:NBP可提高HUVEC的存活率;与正常组比较,缺氧组Ang-2蛋白表达量明显升高(P<0.05);与缺氧组比较,NBP组Ang-2蛋白表达进一步增高(P<0.05)。结论:NBP可以上调Ang-2蛋白的表达,对HUVEC缺氧损伤有一定的保护作用。     

丹参川芎嗪注射液抗脑缺血缺氧损伤的药效物质基础研究

目的从注射液、组分、成分3个层面,探究丹参川芎嗪注射液抗脑缺血缺氧损伤作用及其药效物质基础。方法首先采用SH-SY5Y神经细胞构建氧糖剥夺/复氧(oxygen-glucosedeprivation/reoxygenation,OGD/R)损伤模型,以细胞活力为指标,考察丹参川芎嗪注射液对神经细胞的保护作用。其次,采用制备液相色谱等技术制备丹参川芎嗪注射液标准组分,然后在SH-SY5Y神经细胞OGD/R模型上,系统筛选丹参川芎嗪注射液组分和成分中抗脑缺血缺氧损伤的药效物质,最后进一步通过LC-MS对有效组分进行分析,并对有效成分进行归属,初步明确丹参川芎嗪注射液抗脑缺血缺氧损伤的药效物质基础。结果与模型组相比,丹参川芎嗪注射液在2.5%~15%的稀释浓度下,具有显著的抗SH-SY5Y细胞OGD/R损伤的作用;组分I、J、L、M、O、P、Q以及丹酚酸C、丹酚酸A、丹参素和盐酸川芎嗪均具有显著的抗SH-SY5Y细胞OGD/R损伤的作用。LC-MS分析结果表明,组分O、P中均含有丹酚酸C,组分O中含有丹酚酸A、盐酸川芎嗪,组分P中含有丹参素。结论丹参川芎嗪注射液具有显著的抗脑缺血缺氧损伤作用,而丹酚酸C、丹酚酸A、丹参素和盐酸川芎嗪可能是其抗脑缺血缺氧损伤的主要药效物质。     

药物血清对缺氧/复氧心肌细胞Ca~(2+)及NOS-NO系统的相关性研究

目的探讨双参通冠方药物血清对培养心肌细胞缺氧/复氧损伤的Ca2+超载、NOS-NO系统的影响。方法培养心肌细胞,建立缺氧/复氧损伤模型。运用血清药理学方法研究双参通冠方药物血清对缺氧/复氧心肌Ca2+超载、NOS-NO系统的影响。荧光分光光度法测定心肌细胞胞质[Ca2+]i,比色法检测心肌细胞培养上清NOS活性、NO含量。结果缺氧/复氧后[Ca2+]i增高,总NOS(T-NOS)及诱导型NOS(iNOS)活性增高,双参通冠方药物血清能降低缺氧/复氧心肌细胞[Ca2+]i,并能降低T-NOS、iNOS活性和NO含量(P<0.05)。结论双参通冠方药物血清可抑制缺氧/复氧损伤时心肌细胞Ca2+超载及NOS-NO系统的激活。     

Brain glutathione and the anti-hypoxic effect of glutathione depletors in mice

The anti-hypoxic effect (assessed by a standard hypoxia survival test) 3 hr after 2-cyclohexene-1-one (CHX) was completely abolished by injections of L-cysteine after CHX. Under these conditions, brain GSH was depleted to about 50% of the control level in CHX-treated mice and recovered to about 80% of control level following L-cysteine post-treatment. However, D-cysteine could not cause such effects. Intracerebroventricular injection of CHX, which caused a selective depletion of brain GSH, produced an anti-hypoxic effect. Thus, the anti-hypoxic effect may be related to the decrease of brain GSH levels.     

沙苑子黄酮对D-半乳糖所致亚急性衰老模型小鼠的抗衰老作用研究

目的:研究沙苑子黄酮(FAC)对D-半乳糖致亚急性衰老模型小鼠的作用。方法:制备小鼠D-半乳糖亚急性衰老模型,检测FAC对模型小鼠衰老相关酶、免疫器官指数、耐缺氧时间、游泳时间等指标的影响。结果:FAC能明显提高衰老小鼠血中SOD活性,降低MDA含量,明显提高脾指数和胸腺指数,延长衰老模型小鼠缺氧条件下的存活时间和游泳时间。结论:FAC具有抗衰老作用,且能增强小鼠耐缺氧和耐疲劳能力,其作用可能与其增强小鼠非特异性免疫功能有关。     

Study on the anti-hypoxic effect of cinnarizine and its interaction with prostacyclin

The anti-hypoxic effect of cinnarizine was studied using the following experimental methods: hypobaric and anoxic hypoxia in mice, complete ischemia by decapitation in mice and hemic hypoxia in rats. Papaverine, xanthinol nicotinate and naftidrofuryl were used as reference drugs. In hypobaric and anoxic hypoxia the interaction of cinnarizine with the effect of prostacyclin (PGI2) was investigated. Cinnarizine showed an anti-hypoxic effect in all the methods used. It was more effective in hypobaric and anoxic hypoxia, in incomplete ischemia by decapitation, and less effective in hemic hypoxia. Cinnarizine potentiated the effect of PGI2 shifting the anti-hypoxic dose-response curve of PGI2 to the left. Suggestions as to the possible mechanism of anti-hypoxic action of cinnarizine are made.     

Protective effect of prostaglandins D2, E1 and I2 against cerebral hypoxia/anoxia in mice

The protective effect of prostaglandins (PGs) against cerebral hypoxia/anoxia was investigated with a variety of experimental models in relation to their CNS depressant effects in mice. Furthermore, the effect of PGs on the changes of cerebral energy metabolites and cyclic nucleotide was examined in hypoxic mice. Mice were given s.c. doses of PGs 30 min before tests. Among the PGs tested, treatment with PGD2, PGE1 and PGI2 Na showed a consistent and dose-dependent protection against cerebral anoxia induced by all models studied: histotoxic anoxia by KCN, hypobaric hypoxia, normobaric hypoxia and decapitation-induced gasping. However, PGA1, PGA2, PGB1, PGB2, PGE2, PGF1 alpha, PGF2 alpha and 6-keto-PGF1 alpha at a dose of 3 mg/kg were without effect against normobaric hypoxia and gasping duration. The three PGs, i.e. PGD2, PGE1 and PGI2 which showed anti-hypoxic effects decreased locomotor activity and potentiated hexobarbital-induced sleep. On the other hand, PGE2, PGA1, PGA2 and PGB2 also caused a decrease in locomotor activity. Similarly, PGE2 and PGA1 caused a potentiation of hexobarbital-induced sleep, but interestingly they did not cause clear-cut increase in cerebral resistance to hypoxia, in contrast with the former three PGs. Thus general depression of CNS function appears not to be responsible for the PGD2-, PGE1- and PGI2-induced increase in cerebral resistance to hypoxia. The levels of Cr-P and ATP were significantly reduced and those of ADP and AMP were markedly elevated in hypoxic brain, resulting in a decrease in a calculated energy charge potential. The lactate level and lactate/pyruvate ratio increased and the glucose level decreased markedly.(ABSTRACT TRUNCATED AT 250 WORDS)     

Detection and specific targeting of hypoxic regions within solid tumors: current preclinical and clinical strategies

Poor oxygenation of solid tumors is a major indicator of adverse prognosis after standard treatment, e.g. radiotherapy. This observation founded on intratumoral pO(2) electrode measurements has been supported more recently by studies of injected hypoxia markers (pimonidazole, EF5) or hypoxia-related proteins (hypoxia-inducible factor-1alpha, carbonic anhydrase IX) detected immunohistochemically. Alternative approaches include imaging of tumor hypoxia by nuclear medicine studies and the measurement of hypoxia-related proteins (osteopontin) in patient plasma. Low oxygen levels as found in tumors are rarely observed in normal tissues. The presence of hypoxic tumor cells is therefore regarded not only as an adverse prognostic factor but as an opportunity for tumor-specific treatment. Classic approaches to normalize tumor oxygenation involve the breathing of modified gas mixtures and pharmacologic modification of blood flow as in the "accelerated radiotherapy, carbogen, nicotinamide" (ARCON) scheme. Specific killing of hypoxic tumor cells can potentially be achieved by hypoxia-selective cytotoxins (model substance tirapazamine), which has shown promise in head and neck cancer. Direct targeting of hypoxia-related molecules such as hypoxia-inducible factor-1alpha, the central regulator of the hypoxic response in tumor cells, is an attractive approach currently tested in preclinical models. For clinical applications, the appropriate combination of hypoxia detection for patient selection with a hypoxia-specific treatment is essential. A therapeutic benefit has been suggested for the selection of patients by plasma osteopontin level and treatment with the hypoxic radiosensitizer nimorazole in addition to radiotherapy, for selection by F-misonidazole positron-emission tomography (PET) and treatment with tirapazamine in addition to chemoradiation and for selection by pimonidazole immunohistochemistry and ARCON treatment, all in head and neck cancer.     

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.     

Anti-hypoxic effect of piracetam and its interaction with prostacyclin

The anti-hypoxic effect of piracetam was studied using the following experimental methods: hypobaric and anoxic hypoxia in mice, complete ischemia by decapitation in mice, incomplete ischemia by bilateral carotid occlusion in rats and hemic hypoxia in rats. Cinnarizine and vinpocetine were used as reference drugs. In hypobaric hypoxia, anoxic hypoxia, and complete ischemia by decapitation the interaction of piracetam with the effect of prostacyclin (PGI2) was investigated. Piracetam showed anti-hypoxic effect in all the methods used. Its effect was greater than that of cinnarizine and similar to that of vinpocetine. Piracetam potentiated the effect of PGI2 shifting the anti-hypoxic dose-response curve of PGI2 to the left.     

脉络宁注射液对人血管内皮细胞缺氧损伤的保护作用

目的:研究脉络宁注射液对人脐静脉血管内皮细胞缺氧损伤的保护作用及其机制。方法:常规进行人血管内皮细胞(HUVECs)培养,将细胞随机分为正常对照组、缺氧组和脉络宁20 mg.L-1组。采用CCK-8法检测细胞存活率;Hochest33258荧光染料检测细胞凋亡率;RT-PCR法检测各组细胞中血管内皮生长因子(VEGF),血管生成素-2(Ang-2)mRNA表达水平。结果:脉络宁注射液可显著提高缺氧损伤细胞的存活率(P<0.05);与正常对照组比较,缺氧组细胞内VEGF和Ang-2 mRNA表达水平明显增高。与缺氧组比较,脉络宁组VEGF和Ang-2的表达进一步增强,各组间比较均有差异(P<0.05)。结论:脉络宁注射液可减轻血管内皮细胞缺氧损伤,上调缺氧血管内皮细胞中VEGF和Ang-2的表达,对血管内皮细胞缺氧损伤有一定的保护作用。