巯基参与胃粘膜防御机制

本工作研究了胃粘膜非蛋白质巯基物质(NPSH)在粘膜防御功能中的作用。结果表明,酸性乙醇灌胃或冷冻加束缚应激引起大鼠胃粘膜 NPSH 含量显著下降;补充含-SH 的化合物半胱胺或还原型谷胱甘肽可防止酸性乙醇引起的胃粘膜损伤;在酸性乙醇灌胃或应激后,胃粘膜谷胱甘肽还原酶活性明显降低,并与 NPSH 含量的下降在时间上一致;丙二醛含量在酸性乙醇灌胃后显著升高,自由基清除剂二甲亚砜可减轻胃粘膜损伤。上述结果提示,胃粘膜NPSH 可能通过对自由基的清除作用参与粘膜的局部防御机制;谷胱甘肽还原酶活性下降和自由基生成增加所导致的粘膜 NPSH 含量下降可能是损伤发生过程的重要环节。     

生长抑素对氧自由基引起的离体胃粘膜细胞损伤的保护作用及其机制

采用pronase—EDTA法分离大鼠胃粘膜细胞,将黄嘌呤氧化酶(xanthineoxidase,XO)—黄嘌呤(xanthine,X)氧自由基生成系统加入培养液诱发细胞的损伤。观察到预先加入生长抑素可以剂量依赖性地减轻XO—X引起的细胞死亡和乳酸脱氢酶的漏出;同时抑制XO—X引起的细胞脂质过氧化水平的升高,并翻转细胞膜流动性及溶血性卵磷脂与卵磷脂比值的变化。上述结果提示,生长抑素对氧自由基引起的胃粘膜细胞损伤可能具有直接的保护作用,其机制似与防止质膜的脂质过氧化并从而保护细胞膜免受损伤有关。     

氧自由基在应激性胃溃疡中的发病学意义

本工作研究了氧自由基在大鼠冷冻束缚应激性胃溃疡中的发病学意义。实验结果如下:(1)以超氧自由基清除剂超氧化物歧化酶(SOD)或羟自由基清除剂二甲亚砜和甘露醇预先处理大鼠,均可显著地减轻胃粘膜损伤;(2)应激时,胃粘膜内的脂质过氧化分解产物丙二醛的含量显著升高;(3)组织化学的研究显示,胃粘膜层含有丰富的黄嘌呤氧化酶,其活性在应激时明显升高,预先用别嘌呤醇处理大鼠以抑制黄嘌呤氧化酶的活性,可使胃粘膜损伤显著减轻。上述结果提示,氧自由基是应激性胃溃疡的重要致病因子,而黄嘌呤氧化酶活性的升高似可能为应激时氧自由基生成增加的重要原因。     

I组mGluRs反义寡核苷酸抗谷氨酸对小鼠大脑皮层神经元的兴奋毒作用

目的:研究I组代谢型谷氨酸受体(mGluRs)反义寡核苷酸对谷氨酸钠(Glu)引起的小鼠大脑皮层神经元损伤的保护作用。方法:以细胞乳酸脱氢酶(LDH)漏出、光镜下细胞形态变化为指标,观察培养液中加入Glu引起的神经元损伤及mGluRl反义寡核苷酸或mGluR5反义寡核苷酸的保护作用;用免疫细胞化学检测神经元mGlulα仪和mGluR5表达。结果:实验显示0.1mmol.L-1的谷氨酸钠可明显造成神经元损伤,使LDH漏出增加(P<0.01),mGluRl反义寡核苷酸或mGluR5反义寡核苷酸6μmol.L-1和8μmol.L-1可明显拮抗Glu引起的神经元损伤,使LDH漏出显著减少(P<0.01);免疫组化实验证实体外培养神经元mGluRlα和mGluR5阳性表达。结论:mGluRl反义寡核苷酸和mGluR5反义寡核苷酸可对抗Glu引起的皮层神经元损伤。     

核糖核酸酶抑制因子对H_2O_2损伤的大鼠神经胶质瘤细胞系C6的影响

核糖核酸酶抑制因子 (ribonucleaseinhibitor,RI)是广泛存在于哺乳动物细胞浆中的一种酸性糖蛋白 .为了进一步了解RI的功能 ,根据RI分子结构富含巯基的特点 ,研究了RI对过氧化氢(H2 O2 )损伤的大鼠神经胶质瘤细胞 (C6 )的影响 .用不同浓度的H2 O2 分别作用于转染有RIcDNA并且RI过表达的C6细胞和正常C6细胞 ,对比损伤前后 2者的细胞存活率、LDH漏出量、细胞内GSH和MDA含量差别 ,以及细胞内抗氧化酶类GPX、CAT和GST活性的差别 .结果表明 ,与正常C6细胞相比 ,RI过表达的C6细胞在H2 O2 作用下存活率高 ,LDH漏出量、MDA含量明显减少 ,而细胞内GSH较多 ;RI过表达的C6细胞在损伤前后均表现出更强的CAT和GST活性 .提示RI具有抗氧化功能 ,能够减轻H2 O2 所致的细胞过氧化损伤 .     

Ⅰ组mGluRs反义寡核苷酸抗谷氨酸对小鼠大脑皮层神经元的兴奋毒作用

目的研究Ⅰ组代谢型谷氨酸受体(mGluRs)反义寡核苷酸对谷氨酸钠(Glu)引起的小鼠大脑皮层神经元损伤的保护作用.方法以细胞乳酸脱氢酶(LDH)漏出、光镜下细胞形态变化为指标,观察培养液中加入Glu引起的神经元损伤及mGluRl反义寡核苷酸或mGluR5反义寡核苷酸的保护作用;用免疫细胞化学检测神经元mGlulα仪和mGluR5表达.结果实验显示0.1 mmol·L-1的谷氨酸钠可明显造成神经元损伤,使LDH漏出增加(P＜0.01),mGluRl反义寡核苷酸或mGluR5反义寡核苷酸6 μmol·L-1和8μmol·L-1可明显拮抗Glu引起的神经元损伤,使LDH漏出显著减少(P＜0.01);免疫组化实验证实体外培养神经元mGluRlα和mGluR5阳性表达.结论mGluRl反义寡核苷酸和mGluR5反义寡核苷酸可对抗Glu引起的皮层神经元损伤.     

厨房油烟引起AL细胞遗传损伤和胞内自由基形成

为了解厨房油烟(cooking oil fumes,COF)对细胞的危害及其遗传毒性机制,以A细胞为实验模型,对细胞活力水平、细胞基因突变率、细胞非巯基蛋白化合物水平(NPSH)以及细胞内活性氧(ROS)产生的变化规律进行了研究。实验发现：用厨房油烟(COF)处理后,A细胞的活力下降,细胞CD59基因的突变率随着处理浓度的增加而增加,NPSH水平随着处理浓度的增加而降低,且存在一定的剂量一效应关系。检测胞内产生的ROS,发现400μg／ml COF处理30min后,细胞内ROS含量高于对照3倍多。实验结果表明：COF可引起细胞氧化胁迫,诱导哺乳动物细胞基因突变。     

葛根素对大鼠星形胶质细胞的体外保护作用

目的：研究葛根素（Pue)对缺氧缺糖（OGD）、谷氨酸钠（Glu)或反式－氨基－环戊基－ 1,3－二羧酸（trans-ACPD)引起的体外培养大鼠星形胶质细胞损伤的保护作用。方法：用乳酸脱氢酶（LDH）试剂盒测定细胞LDH漏出,用3－氧－甲基[1-^3H]－D－葡萄糖摄取法测定细胞体积。结果：缺氧缺糖5h、Glu 0.5mmol/L或trans-ACPD 1mmol/L作用星形胶质细胞1h,细胞体积及LDH漏出明显增加;当细胞在缺氧缺糖、Glu或trans-ACPD损伤的同时,加Pue 0.1mmol/L,能明显减少细胞的体积及LDH的漏出。结论：Pue对OGD、谷氨酸或trans-ACPD致大鼠星形胶质细胞损伤有保护作用。     

巯基物质在消炎痛诱发大鼠胃粘膜损伤中的作用

本工作研究了巯基物质在消炎痛引起大鼠胃粘膜损伤中的可能作用。在胃粘膜损伤发生过程中、胃粘膜内非蛋白及蛋白结合的巯基物质含量均无明显降低。虽然半胱胺灌胃(132或264μmol)或皮下注射(132μmol)后均明显抑制消炎痛溃疡的发生,其抑制率分别为82％,92％和75％,但同样具有巯基的半胱氨酸却无保护作用。半胱胺(132μmol)皮下注射可使消炎痛大鼠胃酸分泌抑制46％,而灌胃则无此作用。两种途径给予的半胱胺均不影响胃壁结合粘液的分泌。这些结果表明,胃粘膜内巯基物质似不参与消炎痛的致溃疡过程。半胱胺在此种模型上虽有强烈的细胞保护作用,但似乎不是由于其分子上所带的巯基所致。因此,巯基物质在消炎痛引起的胃粘膜损伤模型上没有细胞保护作用。     

美洲大蠊油脂对过氧化氢诱导SH-SY5Y细胞氧化损伤的保护作用

采用人神经母细胞瘤(SH-SY5Y)细胞,建立过氧化氢(H2O2)诱导的氧化损伤模型,加入H2O2前用美洲大蠊油脂(100、200、400、800、1000μg/m L)预处理,通过MTS法检测细胞存活率,比色法检测细胞乳酸脱氢酶(LDH)漏出率,相关试剂盒检测细胞内超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)系列抗氧化酶的活性,以及脂质过氧化物丙二醛(MDA)含量的变化,从细胞水平研究美洲大蠊油脂对氧化损伤细胞的保护作用,并初步探讨其作用机制。实验结果显示,与对照组相比,模型组细胞存活率明显降低,LDH漏出率明显增加,胞内SOD、GSH-Px活性显著降低,MDA含量增多。与模型组相比,美洲大蠊油脂(800、1000μg/m L)能显著增强细胞SOD(P0.01)、GSH-Px(P0.05)活性,降低MDA含量(P0.05),使得细胞存活率显著提高(P0.01),LDH漏出率降低(P0.01)。其中油脂的浓度与细胞存活率、SOD活性之间为正相关,与MDA含量为负相关,均呈现浓度依赖性。结果提示美洲大蠊油脂对H2O2所致SH-SY5Y细胞氧化损伤具有明显的保护作用,其作用机制可能与提高细胞SOD、GSH-Px活性,降低MDA含量从而增强细胞自身抗氧化能力有关。     

Injury to primary cultures of rat heart endothelial cells by hypoxia and glucose deprivation

Primary cultures of rat heart endothelial cells were subjected to simulated conditions of ischemia: hyposia and glucose deprivation for 4 and 24 hr. Cellular injury was evaluated by measuring changes in viability, total protein, cellular morphology, and leakage of cytoplasmic enzymes from the cells into the culture medium. Deprivation of oxygen and glucose for 4 or 24 hr did not lethally injure the cells as noted by no change in cell viability, morphology, and total protein when compared to controls. However, reversible or non-lethal cellular injury was produced as reflected by a significant release of lactate dehydrogenase (LDH) from the cells into the medium after treatment with hypoxia and glucose deprivation for 4 or 24 hr. When the cultures were deprived of glucose, but were oxygenated, cellular injury was not evident after 24 hr. Deprivation of oxygen but not glucose resulted in significant loss of LDH after 4 or 24 hr. When the cultures were allowed to recover after oxygen and glucose deprivation in complete medium containing 1000 mg glucose per 1 and a normal atmosphere of 20% O2, they had levels of LDH leakage comparable to those of control cultures.     

Dietary flavonoid apigenin is a potential inducer of intracellular oxidative stress: the role in the interruptive apoptotic signal

Apigenin is a representative dietary flavone (2-phenyl-4H-1-benzopyran-4-one) inhibiting cancer cell growth both in cell culture systems and in vivo. The prooxidant potential of apigenin was confirmed by the observations using flowcytometric and immunoblotting techniques that the intracellular accumulations of reactive oxygen species (ROS) and protein carbonyls were detected in the cells treated with apigenin in a dose-dependent manner. Conversely, chrysin (5,7-dihydroxyflavone) did not show any prooxidant effect. A structure-activity relationship data thus indicated that a 4'-monohydroxyl group, which can be oxidized to semiquinone radical but not up to quinone-like metabolite, is essential for prooxidant effect. When HL-60 cells were treated with not only a heme synthesis inhibitor succinyl acetone (SA) but also myeloperoxidase (MPO) inhibitors, the ROS level enhanced by apigenin was significantly reduced. The gathered data suggested that peroxidase-catalyzed production of apigenin B-ring phenoxyl radicals might be responsible for the prooxidant effect. This is supported by the observation that MPO is able to catalyze production of apigenin phenoxyl radicals, detected by an electron spin resonance-spin trapping technique. We also reveal that both SA and alpha-tocopherol enhance cellular susceptibility to apoptosis-inducing stimuli by apigenin. In conclusion, the prooxidant effect of apigenin is likely to oxidize a variety of thiols through the formation of phenoxyl radicals and thus seems to play a significant role in the abortive apoptotic pathway switching to necrotic cell death.     

Enzyme release and mitochondrial activity in reoxygenated cardiac muscle: relationship with oxygen-induced lipid peroxidation

The aim of this work was to precisely determine the sites of the peroxidative action on unsatured lipids by oxygen-derived free radicals and the lytic cell damage on reoxygenated perfused hearts. The cellular load of lipid peroxidation products (malondialdehyde) during the reoxygenation was dependent on PO2. This unfavorable biochemical response was linked to creatine kinase leakage, alteration of coronary flow and mitochondrial injury. When an enzymatic (superoxide dismutase, 290 IU/minute) or tripeptide scavenger of oxygen radicals (reduced glutathione, 0.5 mmol/l) was administered at the end of hypoxia and during reoxygenation, the abnormal intolerance of hypoxic heart to molecular oxygen was significantly weakened; the load of lipid peroxides load, enzyme release, and vascular alteration were all reduced. Moreover, mitochondrial activity was enhanced and the oxygen-induced uncoupling of mitochondrial remained limited: both the respiratory control ratio (RCR) and the ADP/O ratio were higher than in control reoxygenated hearts. The inhibition by rotenone (100 mumol/l) of reoxidation of electron chain transfer during oxygen readmission also reduced the unfavorable cardiac accumulation of lipid peroxidation products and the release of creatine kinase. These data demonstrate that in the oxygen paradox, the peroxidative attack on lipids plays an important role in inducing alterations of sarcolemmal permeability and mitochondrial activity. An uncontrolled reactivation of oxidative function of mitochondria during reoxygenation enhances the synthesis of oxygen-derived free radicals and triggers the peroxidation of cardiac lipids resulting in irreversible injury to cellular and intracellular membranes.     

Effects by doxorubicin on the myocardium are mediated by oxygen free radicals

We hypothesized that doxorubicin (DOX) induces cardiotoxicity of myocardium via oxygen radicals. The present study is aimed at examining the membrane alterations by oxygen radicals generated by DOX in adult rats and cultured neonatal myocytes. Our results showed that DOX 1) decreased beta-adrenoceptor (BAR) density in the cell membrane, 2) increased the membrane permeability of cultured neonatal rat myocytes and 3) altered the ultrastructure of myofibrils and subplasmalemmal actin networks. These effects were reproducible by exogenous hydrogen peroxide. The antioxidant melatonin (MLT) inhibited enzyme leakage and peroxidation in a concentration-dependent manner. It is concluded that DOX induces cardiotoxicity through lipid peroxidation and melatonin is an effective antioxidant against the reactive oxygen intermediates generated by DOX.     

Study of the mechanisms of hydrogen peroxide and hydroxyl free radical-induced cellular injury and calcium overload in cardiac myocytes.

There is evidence that myocardial injury, as would occur on post-ischemic reperfusion, may be caused by the generation of oxygen radicals, as well as by the induction of intracellular calcium overload; however, the relationship between these two mechanisms of injury is not known. To test the hypothesis that oxidants and oxygen radicals can cause cardiac myocyte injury and intracellular calcium overload, isolated adult rat ventricular myocytes were exposed to H2O2 (1-10 mM) and Fe3(+)-nitrilotriacetate. EPR measurements confirmed the production of the highly reactive .OH radical by this system. The oxygen radical generating system initially caused a transient augmentation of twitch amplitude in single field stimulated myocytes. This was followed by contractile oscillations occurring during the twitch prior to full cell relaxation, and spontaneous mechanical oscillations occurring between electrically stimulated contractions. Eventually, cells became inexcitable and abruptly underwent contracture. In the presence of lower bathing calcium concentrations, these oxidant-induced alterations were prevented or delayed. However, cells exposed to the radical generating system in the absence of extracellular calcium still eventually underwent contracture but stimulated contractions or mechanical oscillations were not seen. Measurements in single myocytes loaded with the fluorescent probe of intracellular calcium, Indo-1, demonstrated a rise in both systolic and diastolic fluorescence ratio, as well as oscillations and widening of the fluorescence transient, suggestive of cellular calcium loading, following exposure to the radical generating system. Injured myocytes did not take up trypan blue dye. Contractile dysfunction and calcium channel blocker, nitrendipine. NMR measurements of cellular [ATP] demonstrated that these alterations in cellular calcium preceded the depletion of ATP. Subsequent depletion of ATP was accompanied by the appearance of increased concentrations of sugar phosphates indicative of a block in glycolysis and ATP depletion correlated with cellular rigor. Thus, oxygen free radicals can cause cardiac myocyte injury with contractile abnormalities which occur due to myocyte calcium loading. The mechanism of oxidant-induced calcium loading is not due to nonspecific membrane damage, or energy depletion, but rather due to increased calcium influx through voltage gated calcium channels. This early calcium overload state as well as oxidant induced block of glycolysis result in cellular energy depletion and cell death with the induction of contracture.     

Effect of superoxide and lipid peroxide on cytosolic free calcium concentration in cultured pig aortic endothelial cells

The effect of reactive oxygen on cytosolic free calcium concentration [( Ca++]i) in pig aortic endothelial cells (ECs) was studied. Linoleate hydroperoxide (LHO) and superoxide radicals generated from xanthine with xanthine oxidase (X-XO) were used as sources of reactive oxygen. [Ca++]i in ECs was measured with quin 2 and the value for quiescent ECs was 112 +/- 11 nM. Both LHO and X-XO increased [Ca++]i in a dose-dependent manner without accompanying the significant cellular damage. Nifedipine suppressed the increase in [Ca++]i provoked by LHO and X-XO. Thus, the biological effects of reactive oxygen might be mediated, at least in part, by the activation of voltage-dependent calcium channels in ECs.     

Mono-(2-Ethylhexyl) Phthalate Induces Injury in Human Umbilical Vein Endothelial Cells

Mono-(2-ethylhexyl) phthalate (MEHP), the active metabolite of di-(2-ethylhexyl) phthalate (DEHP), is a widespread environmental contaminant and has been proved to have potential adverse effects on the reproductive system, carcinogenicity, liver, kidney and developmental toxicities. However, the effect of MEHP on vascular system remains unclear. The main purpose of this study was to evaluate the cytotoxic effects of MEHP on human umbilical endothelial cells (HUVEC) and its possible molecular mechanism. HUVEC cells were treated with MEHP (0, 6.25, 12.5, 25,50 and 100 µM), and the cellular apoptosis and mitochondrial membrane potential as well as intracellular reactive oxygen species were determined. In present study, MEHP induced a dose-dependent cell injury in HUVEC cell via an apoptosis pathway as characterized by increased percentage of sub-G1, activation of caspase-3, -8and -9, and increased ratio of Bax/bcl-2 mRNA and protein expression as well as cytochrome C releasing. In addition, there was obvious oxidative stress, represented by decreased glutathione level, increased malondialdehyde level and superoxide dismutase activity. N-Acetylcysteine, as an antioxidant that is a direct reactive oxygen species scavenger, could effectively block MEHP-induced reactive oxygen species generation, mitochondrial membrane potential loss and cell apoptosis. These data indicated that MEHP induced apoptosis in HUVEC cells through a reactive oxygen species-mediated mitochondria-dependent pathway.     

Polymorphonuclear leukocytes-induced injury in hypoxic cardiac myocytes

Growing evidence suggests that free radicals derived from polymorphonuclear leukocytes (PMNs) play an important role in myocardial ischemia-reperfusion injury. To elucidate the cellular mechanism by which activated PMNs exacerbate ischemic myocardial damage, we investigated the extent of cell injury, assessed by the morphological deterioration, free radical generation, and lipid peroxidation in mouse embryo myocardial cells coincubated with activated PMNs. The generation of PMN-derived free radicals was related to the extent of myocardial cell injury. When myocardial cell sheets were subjected to hypoxia and glucose-free media, myocardial cells were injured (cristalysis in the mitochondria and disruption of the sarcolemma) after adding various PMN activators, and the injury extended to the adjacent cells. Chemiluminescent emission and production of thiobarbituric acid-reactive substances in the coincubated cells increased markedly compared with myocardial cells or PMNs alone. The augmented lipid peroxidation coincided with the progression of myocardial cell injury. Catalase inhibited the myocardial cell injury by 52%, the chemiluminescence by 46%, and lipid peroxidation by 50%, whereas superoxide dismutase exhibited less pronounced inhibition. These results indicate that a chain reaction of lipid peroxidation in myocardial cells induced by PMN-derived free radicals closely correlates with membrane damage and contributes to the propagation of irreversible myocardial cell damage.     

Injury to primary cultures of rat heart endothelial cells by hypoxia and glucose deprivation

Summary Primary cultures of rat heart endothelial cells were subjected to simulated conditions of ischemia: hypoxia and glucose deprivation for 4 and 24 hr. Cellular injury was evaluated by measuring changes in viability, total protein, cellular morphology, and leakage of cytoplasmic enzymes from the cells into the culture medium. Deprivation of oxygen and glucose for 4 or 24 hr did not lethally injure the cells as noted by no change in cell viability, morphology, and total protein when compared to controls. However, reversible or nonlethal cellular injury was produced as reflected by a significant release of lactate dehydro-genase (LDH) from the cells into the medium after treatment with hypoxia and glucose deprivation for 4 or 24 hr. When the cultures were deprived of glucose, but were oxygenated, cellular injury was not evident after 24 hr. Deprivation of oxygen but not glucose resulted in significant loss of LDH after 4 or 24 hr. When the cultures were allowed to recover after oxygen and glucose deprivation in complete medium containing 1000 mg glucose per l and a normal atmosphere of 20% O₂, they had levels of LDH leakage comparable to those of control cultures. This study was supported by Research Grant HL 18647 from the National Heart, Lung, and Blood Institute and by a National Chicano Council on Higher Education Post-Doctoral Fellowship awarded to D. Acosta from the Ford Foundation. Additional support was provided to D. Acosta by a Faculty Research Assignment Award from the University of Texas Research Institute.