活性氧和线粒体ATP敏感钾通道介导肿瘤坏死因子α对缺氧/复氧心肌的保护作用

在培养的乳鼠心肌细胞上,研究肿瘤坏死因子α(TNF-α)对缺氧/复氧损伤心肌的保护作用的机制。结果发现：(1)用TNF-α(10—500U／ml)预处理,缺氧/复氧后心肌细胞内锰超氧化物歧化酶(Mn-SOD)活性增高、乳酸脱氢酶(LDH)释放量减少(P＜0．05);(2)用抗氧化剂N-乙酰半既氨酸(NAC,1mmol/L)、抗霉素A(antimycin A,50μmol/L)、2-巯基丙酰氨基乙酸(2-MPG,400μmol/L)和铜／锌超氧化物歧化酸(Cu/Zn,SOD)抑制剂二乙基二硫代氨基甲酸盐(DDC,100nmol/L)预处理,可取消TNF-α的抑制缺氧/复氧心肌细胞LDH释放和诱导Mn-SOD活性增高的作用;(3)mitoKATP通道抑制剂5-羟基癸酸(5-HD)预处理可阻断TNF-α对缺氧/复氧心肌细胞的保护作用;选择性mitoKATP通道开放剂diazoxide(50μmol/L)预处理可减少复氧后心肌细胞LDH的释放(P＜0．01),其作用可被5-HD(100μmol/L)和NAC所抑制。上述结果表明,活性氧和线粒体ATP敏感钾通道参与介导TNF-α对缺氧/复氧损伤的心肌保护作用。     

Recombinant PTD-Cu/Zn SOD attenuates hypoxia–reoxygenation injury in cardiomyocytes

Abstract

Background. Oxidative stress plays a pivotal role in myocardial ischemia–reperfusion injury. Increasing the protein expression of intracellular Cu/Zn SOD, which is the major endogenous antioxidant enzyme, may attenuate or prevent hypoxia–reoxygenation injury (HRI) in cultured cardiomyocytes. However, ectogenic Cu/Zn-SOD can hardly be transferred into cells to exert biological effects. In this study, we constructed PTD-Cu/Zn SOD plasmid with a kind of translocation structure-Protein transduction domain (PTD) and detected its transmembrane ability and antioxidant effects in H9c2 rat cardiomyocytes subjected to hypoxia/reoxygenation injury (HRI). Methods. We constructed the pET-PTD-Cu/Zn SOD (CDs) prokaryotic expression vectors in plasmid that were inserted into E. coli BL21 to induce the protein expression of PTD-Cu/Zn SOD. H9c2 cardiomyocyte HRI was achieved by exposing cardiomyocytes to 12 h hypoxia followed by 2 h reoxygenation. Protein expression of PTD-Cu/Zn SOD in cardiomyocytes was assayed by Western blot and their enzyme activities were investigated by immunohistochemistry and flow cytometry. Results. In cultured cardiomyocytes hypoxia–reoxygenation injury model, exogenous PTD-Cu/Zn SOD could penetrate cell membrane to clear superoxide anion and decrease hydrogen peroxide level in H9c2 cardiomyocytes subjected to HRI. The level of mitochondrial membrane potential was restored to normal, and the cell apoptosis was reduced in cardiomyocytes with PTD-Cu/Zn SOD treatment during HRI. Conclusion. Recombinant PTD-Cu/Zn SOD could scavenge intracellular-free superoxide anion, protect mitochondria from damages, and attenuate the hypoxia–reoxygenation injury in cultured cardiomyocytes.     

Zinc pyrithione salvages reperfusion injury by inhibiting NADPH oxidase activation in cardiomyocytes

Zinc pyrithione (ZPT), has a strong anti-apoptotic effect when administered just before reperfusion. Because oxidative stress has been proposed to contribute to myocardial reperfusion injury, we tested whether ZPT can reduce the production of reactive oxygen species during reoxygenation in cultured neonatal rat cardiac myocytes and evaluated the role of NADPH oxidase in hypoxia/reoxygenation (H/R) injury. The cells were subjected to 8 h of simulated ischemia, followed by either 30 min or 16 h of reoxygenation. ZPT when started just before reoxygenation significantly reduced superoxide generation, LDH release and improved cell survival compared to H/R. Attenuation of the ROS production by ZPT paralleled its capacity to prevent pyknotic nuclei formation. In addition, ZPT reversed the H/R-induced expression of NOX2 and p47^phox phosphorylation indicating that ZPT directly protects cardiomyocytes from reperfusion injury by a mechanism that attenuates NADPH oxidase mediated intracellular oxidative stress.     

G_(i/o)蛋白介导的信息转导通路在低氧预处理心肌保护中的作用

实验以低氧 3h后复氧期间心肌细胞的生存率和LDH的释放量为指标 ,观察Gi/o蛋白及其下游成分在低氧预处理 (hypoxicpreconditioning ,HP)心肌保护中的作用。与单纯低氧组相比 ,HP组 ( 2 5min低氧 30min复氧作为HP)细胞生存率增高 ,LDH释放减少 (P <0 0 1)。用NEM预处理 ,能完全模拟HP的心肌细胞保护作用 ;而用PTX阻断Gi/o蛋白 ,或Forskolin和 8 Br cAMP预处理后 ,再给予HP及低氧 3h/复氧 1h ,则细胞生存率降低 ,LDH释放增加 (P <0 0 1) ;U 7312 2预处理后 ,细胞生存率和LDH释放量无差异 (P >0 0 5 )。结果提示 :Gi/o蛋白通过抑制AC ,减少第二信使cAMP的生成介导了HP的心肌保护作用。PLC可能不参与HP的心肌保护作用     

Green tea protection of hypoxia/reoxygenation injury in cultured cardiac cells

Antioxidant-rich diets exert a protective effect in diseases involving oxidative damage. Among dietary components, green tea is an excellent source of antioxidants. In this study, cultured neonatal rat cardiomyocytes were used to clarify the protective effect of a green tea extract on cell damage and lipid peroxidation induced by different periods of hypoxia followed by reoxigenation. Cultures of neonatal rat cardiomyocytes were exposed to 2--8 hr hypoxia, eventually followed by reoxygenation, in the absence or presence of alpha-tocopherol or green tea. LDH release and the production of conjugated diene lipids were measured, and appeared linearly related to the duration of hypoxia. During hypoxia, both LDH release and conjugated diene production were reduced by alpha-tocopherol and, in a dose dependent manner, by green tea, the 50 &mgr;g/ml being the most effective dose. Reoxygenation caused no further increase in LDH leakage, while it caused a significant increase in conjugate dienes, which absolute value was lower in antioxidant supplemented cells. Anyway, the ratio between conjugated diene production after hypoxia and after reoxygenation was similar in all groups, indicating that the severity of free radical-induced reoxygenation injury is proportional to the severity of previous hypoxic injury. Since hypoxic damage is reduced by alpha-tocopherol and green tea, our data suggest that any nutritional intervention to attenuate reoxygenation injury must be directed toward the attenuation of the hypoxic injury. Therefore, recommendations about a high dietary intake of antioxidants may be useful not only in the prevention, but also in the reduction of cardiac injury following ischemia.     

Cardioprotective Effect of ‘Methylamine Irisolidone’, a New Compound,in Hypoxia/Reoxygenation Injury in Cultured Rat Cardiac Myocytes

‘Methylamine irisolidone’ (=5,7‐dihydroxy‐6‐methoxy‐3‐(4‐methoxyphenyl)‐8‐[(methylamino)methyl]‐4H‐[1]benzopyran‐4‐one), a new compound, is a structurally modified kakkalide with good water solubility. In this study, we investigated its effect on hypoxia/reoxygenation (H/R) injury in cultured rat cardiac myocytes. The results showed that methylamine irisolidone could significantly inhibit lactate dehydrogenase (LDH) release, enhance the mitochondrial membrane potential, decrease intracellular calcium (Ca²⁺) associated with the attenuation of reactive oxygen species (ROS) generation, reduce contents of malondialdehyde (MDA), and increase the activity of superoxide dismutase (SOD) after H/R in a dose‐dependent manner. The present study demonstrated that methylamine irisolidone can directly protect cardiomyocytes against H/R injury, primarily as a result of reduction of the intracellular Ca²⁺ overload coincident with an attenuation of ROS generation and ROS‐mediated lipid peroxidation, which may contribute to the preservation of mitochondrion function and antioxidant against H/R injury.     

Gi／0蛋白介导的信息转导通路在低氧预处理心肌保护中的作用

实验以低氧３ｈ后复氧期间心肌细胞的生存率和ＬＤＨ的释放量为指标,观察Ｇｉ／ｏ蛋白及其下游成分在低氧预处理（ｈｙｐｏｘｉｃ ｐｒｅｃｏｎｄｉｔｉｏｎｉｎｇ,ＨＰ）心肌保护中的作用。与单纯低氧组相比,ＨＰ组（２５ｍｉｎ低氧＋３０ｍｉｎ复氧作为ＨＰ）细胞生存率增高,ＬＤＨ释放减少（Ｐ＜０．０１）。用ＮＥＭ预处理,能完全模拟ＨＰ的心肌细胞保护作用;而用ＰＴＸ阻断Ｇｉ／ｏ蛋白,或Ｆｏｒｓｋｏｌｉｎ和８－Ｂｒ     

Mixed tocopherol preparation is superior to alpha-tocopherol alone against hypoxia-reoxygenation injury

Hypoxia-reoxygenation (H-R) is associated with alterations in oxidant-antioxidant balance and L-arginine-nitric oxide system. Tocopherols decrease the activity of reactive oxygen species (ROS) and yet are not beneficial in clinical trials. It has been proposed that mixed tocopherols as found in nature may be more tissue protective than alpha-tocopherol alone found in commercial preparations. We compared the effect of a mixed tocopherol preparation with that of alpha-tocopherol alone on superoxide dismutase (SOD) activity and iNOS expression in cultured myocytes exposed to H-R. Myocytes from Sprague-Dawley rat hearts were subjected to hypoxia for 24 h followed by reoxygenation for 3 h H-R. Parallel groups of myocytes were pretreated with alpha-tocopherol alone or a mixed-tocopherol preparation (containing alpha-, gamma-, and delta-tocopherols) (50 microM) for 30 min. H-R resulted in myocyte injury (determined by LDH release), a decrease in SOD activity and an upregulation of iNOS expression/activity. Both tocopherol preparations attenuated cell injury and markedly decreased the effects of H-R on SOD activity and iNOS expression/activity (all P < 0.05 vs H-R group, n = 5). However, mixed-tocopherol preparation was much superior to alpha-tocopherol in terms of myocyte protection from the adverse effect of H-R (P < 0.05). Lack of efficacy of commercial tocopherol preparations in clinical trials may reflect absence of gamma- and delta-tocopherols.     

靶向PUMA的siRNA对缺氧/复氧诱导大鼠心肌细胞凋亡的保护作用

为探讨p53上调凋亡调制物(p53 up-regulated modulator of apoptosis,PUMA)在大鼠心肌细胞缺氧/复氧(hypoxia/reoxygenatio,H/R)损伤中的作用,本研究将靶向PUMA的siRNA(si-PUMA)转染大鼠心肌细胞以建立PUMA沉默表达模型,观察其对心肌细胞H/R损伤的影响.RT-PCR和Western印迹结果表明,最适转染浓度50 nmol/L si-PUMA能靶向抑制H/R损伤心肌细胞的PUMA表达;MTT法检测心肌细胞存活率及培养基乳酸脱氢酶(lactate dehydrogenase,LDH)活性测定结果发现,si-PUMA组细胞存活率较H/R 6 h模型组明显提高,培养液中LDH活性显著降低(P<0.01);分光光度法及Annexin V-FITC/PI联合染色流式细胞凋亡检测结果显示,si-PUMA组caspase-3活性较H/R 6h组明显下调,细胞凋亡率明显降低(P<0.01);RT-PCR结果提示,与H/R6 h组相比,si-PUMA组Bax及Bcl-2表达分别出现显著下调及上调(P<0.05).以上结果表明,靶向PUMA的siRN...     

神经元缺氧复氧损伤时氧自由基的毒性作用及其机制

在原代分离培养Ｗｉｓｔａｒ乳鼠大脑皮质神经元上研究了缺氧复氧损伤（Ｈ／Ｒ）对神经细胞乳酸脱氢酶（ＬＤＨ）,漏出率,死亡率和脂质过氧化物含量的影响,并选用一氧化氮（ＮＯ）合酶抑制剂Ｌ－ＮＧ－硝基－精氨酸（Ｌ－ＮＮＡ）巯基供体Ｎ－乙酰半胱氨酸（ＮＡＣ）和超氧化物歧化酶（Ｃｕ,Ｚｎ－ＳＯＤ）三种自由基清除剂进行预保护等方法来探讨机制。结果表明 Ｈ／Ｒ损伤引起ＬＤＨ漏出率,细胞死亡率和脂过氧化物含量极显著     

Neuroprotective MK801 is associated with nitric oxide synthase during hypoxia/reoxygenation in rat cortical cell cultures.

The neuroprotective effect of MK801 against hypoxia and/or reoxygenation-induced neuronal cell injury and its relationship to neuronal nitric oxide synthetase (nNOS) expression were examined in cultured rat cortical cells. Treatment of cortical neuronal cells with hypoxia (95% N(2)/5% CO(2)) for 2 h followed by reoxygenation for 24 h induced a release of lactate dehydrogenase (LDH) into the medium, and reduced the protein level of MAP-2 as well. MK801 attenuated the release of LDH and the reduction of the MAP-2 protein by hypoxia, suggesting a neuroprotective role of MK801. MK801 also diminished the number of nuclear condensation by hypoxia/reoxygenation. The NOS inhibitors 7-nitroindazole (7-NI) and N (G)-nitro-L-arginine methyl ester (L-NAME), as well as the Ca(2+) channel blocker nimodipine, reduced hypoxia-induced LDH, suggesting that nitric oxide (NO) and calcium homeostasis contribute to hypoxia and/or the reoxygenation-induced cell injury. The levels of nNOS immunoactivities and mRNA by RT-PCR were enhanced by hypoxia with time and, down regulated following 24 h reoxygenation after hypoxia, and were attenuated by MK801. In addition, the reduction of nNOS mRNA levels by hypoxia/reoxygenation was also diminished by MK801. Further delineation of the mechanisms of NO production and nNOS regulation are needed and may lead to additional strategies to protect neuronal cells against hypoxic/reoxygenation insults.     

Hypoxia followed by reoxygenation induces secretion of cyclophilin A from cultured rat cardiac myocytes

We previously reported that hypoxia followed by reoxygenation (hypoxia/reoxygenation) rapidly activated intracellular signaling such as mitogen-activated protein kinases (MAPKs) including extracellular signal-regulated protein kinase (ERK) 1/2, p38MAPK, and stress-activated protein kinases (SAPKs). To investigate the humoral factors which mediate cardiac response to hypoxia/reoxygenation, we analyzed the conditioned media from cardiac myocytes subjected to hypoxia/reoxygenation by two-dimensional electrophoresis and mass spectrometry. We identified cyclophilin A (CyPA) as one of the proteins secreted from cardiac myocytes in response to hypoxia/reoxygenation. Hypoxia/reoxygenation induced the expression of CyPA and its cell surface receptor CD147 on cardiac myocytes in vitro. This was also confirmed by ischemia/reperfusion in vivo. Recombinant human (rh) CyPA activated ERK1/2, p38MAPK, SAPKs, and Akt in cultured cardiac myocytes. Furthermore, CyPA significantly increased Bcl-2 in cardiac myocytes. These data strongly suggested that CyPA is released from cardiac myocytes in response to hypoxia/reoxygenation and may protect cardiac myocytes from oxidative stress-induced apoptosis.     

三七皂苷对缺氧复氧心肌细胞损伤保护作用的研究

目的：研究三七皂苷对纯化培养大鼠乳鼠心肌细胞缺氧/复氧损伤的保护作用及机制。方法：采用纯化培养的心肌细胞建立缺氧/复氧损伤模型,测定细胞凋亡率、caspase-3、乳酸脱氢酶（LDH）、丙二醛（MDA）、超氧化物歧化酶（SOD）含量。结果：与正常组比较,模型组LDH、MDA含量、caspase-3活性及细胞凋亡率明显升高（P＆lt;0.01）,SOD活性明显降低（P＆lt;0.01）;三七皂苷组降低LDH、MDA含量、caspase-3活性和细胞凋亡率,提高SOD活性,与缺氧/复氧组比较各实验指标差异均具有显著性（P＆lt;0.05）。结论：三七皂苷对缺氧/复氧心肌细胞损伤有保护作用,作用机制与清除氧自由基,抗脂质过氧化及降低细胞凋亡率有关。     

Phenylarsine oxide induces mitochondrial permeability transition, hypercontracture, and cardiac cell death

The mitochondrial permeability transition (MPT) is implicated in cardiac reperfusion/reoxygenation injury. In isolated ventricular myocytes, the sulfhydryl (SH) group modifier and MPT inducer phenylarsine oxide (PAO) caused MPT, severe hypercontracture, and irreversible membrane injury associated with increased cytoplasmic free [Ca(2+)]. Removal of extracellular Ca(2+) or depletion of nonmitochondrial Ca(2+) pools did not prevent these effects, whereas the MPT inhibitor cyclosporin A was partially protective and the SH-reducing agent dithiothreitol fully protective. In permeabilized myocytes, PAO caused hypercontracture at much lower free [Ca(2+)] than in its absence. Thus PAO induced hypercontracture by both increasing myofibrillar Ca(2+) sensitivity and promoting mitochondrial Ca(2+) efflux during MPT. Hypercontracture did not directly cause irreversible membrane injury because lactate dehydrogenase (LDH) release was not prevented by abolishing hypercontracture with 2,3-butanedione monoxime. However, loading myocytes with the membrane-permeable Ca(2+) chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA-AM) prevented PAO-induced LDH release, thus implicating the PAO-induced rise in cytoplasmic [Ca(2+)] as obligatory for irreversible membrane injury. In conclusion, PAO induces MPT and enhanced susceptibility to hypercontracture in isolated cardiac myocytes, both key features also implicated in cardiac reperfusion and reoxygenation injury.     

Exploration on Mechanism of a New Type of Melatonin Receptor Agonist Neu-p11 in Hypoxia–Reoxygenation Injury of Myocardial Cells

To explore the mechanism of a new type of melatonin receptor agonist Neu-p11 in hypoxia–reoxygenation injury of myocardial cells. Hypoxia/reoxygenation (H/R) model of H9c2 myocardial cells was established, and the cells were divided into control group, H/R group, and Neu-p11 group. Apoptosis rates of myocardial cells in different groups, the contents of creatinine kinase (CK), lactic dehydrogenase (LDH), superoxide dismutase (SOD), and malondialdehyde (MDA) in cell culture media were compared. Myocardial cells in control group showed diverse shape, and the refractivity of cells were high and the pulse was strong with synchronous rhythm of 60–80/min; The refractivity of myocardial cells in H/R group decreased, the pseudopodium was thinner, and the rhythm was reduced to 30–40/min; The morphology and refractivity of myocardial cells in Neu-p11 group were significantly improved with rhythm of 50–60/min. The apoptosis rates in the control group, the H/R group, and the Neu-p11 group were 2.48, 39.66, and 17.94 %, respectively. Levels of CK, LDH, and MDA were significantly decreased in Neu-p11 compared with H/R group, yet, both of which were significantly higher than that in control group. The SOD level was significantly lower in H/R group compared to that in control group, and Neu-p11 group with no statistical difference between the Neu-p11 group and the control group. Neu-p11 has protective effects on hypoxia–reoxygenation injury of myocardial cells. It inhibits cell apoptosis and improves the morphology and rhythm of myocardial cells; It alleviates injury of cell membrane by reducing its permeability, which can stabilize myocardial cell membrane; It also alleviates lipid peroxidation and protects mitochondria from myocardial ischemia/reperfusion injury.     

Development of an in vitro model for study of the efficacy of ischemic preconditioning in human skeletal muscle against ischemia-reperfusion injury.

Ischemia-reperfusion (I/R) injury causes skeletal muscle infarction and ischemic preconditioning (IPC) augments ischemic tolerance in animal models. To date, this has not been demonstrated in human skeletal muscle. This study aimed to develop an in vitro model to investigate the efficacy of simulated IPC in human skeletal muscle. Human skeletal muscle strips were equilibrated in oxygenated Krebs-Henseleit-HEPES buffer (37 degrees C). Aerobic and reperfusion phases were simulated by normoxic incubation and reoxygenation, respectively. Ischemia was simulated by hypoxic incubation. Energy store, cell viability, and cellular injury were assessed using ATP, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), and lactate dehydrogenase (LDH) assays, respectively. Morphological integrity was assessed using electron microscopy. Studies were designed to test stability of the preparation (n = 5-11) under normoxic incubation over 24 h; the effect of 1, 2, 3, 4, or 6 h hypoxia followed by 2 h of reoxygenation; and the protective effect of hypoxic preconditioning (HPC; 5 min of hypoxia/5 min of reoxygenation) before 3 h of hypoxia/2 h of reoxygenation. Over 24 h of normoxic incubation, muscle strips remained physiologically intact as assessed by MTT, ATP, and LDH assays. After 3 h of hypoxia/2 h of reoxygenation, MTT reduction levels declined to 50.1 +/- 5.5% (P < 0.05). MTT reduction levels in HPC (82.3 +/- 10.8%) and normoxic control (81.3 +/- 10.2%) groups were similar and higher (P < 0.05) than the 3 h of hypoxia/2 h of reoxygenation group (45.2 +/- 5.8%). Ultrastructural morphology was preserved in normoxic and HPC groups but not in the hypoxia/reoxygenation group. This is the first study to characterize a stable in vitro model of human skeletal muscle and to demonstrate a protective effect of HPC in human skeletal muscle against hypoxia/reoxygenation-induced injury.     

MsrA protects cardiac myocytes against hypoxia/reoxygenation induced cell death

Reactive oxygen species (ROS) are critical in tissue responses to ischemia-reperfusion. The enzyme methionine sulfoxide reductase-A (MsrA) is capable of protecting cells against oxidative damage by reversing damage to proteins caused by methionine oxidation or by decreasing ROS through a scavenger mechanism. The current study employed adenovirus mediated over-expression of MsrA in primary neonatal rat cardiac myocytes to determine the effect of this enzyme in protecting against hypoxia/reoxygenation in this tissue. Cells were transduced with MsrA encoding adenovirus and subjected to hypoxia/reoxygenation. Apoptotic cell death was decreased by greater than 45% in cells over-expressing MsrA relative to cells transduced with a control virus. Likewise total cell death as determined by levels of LDH release was dramatically decreased by MsrA over-expression. These observations indicate that MsrA is protective against hypoxia/reoxygenation stress in cardiac myocytes and point to MsrA as an important therapeutic target for ischemic heart disease.     

Endothelial Cells as A Source of Oxygen-Free Radicals An Esr Study

Endothelial cells were subjected to anoxia/reoxygenation in order to simulate some of the free radical mechanisms occurring in ischaemialreperfusion. With ESR and spin trapping using the spin traps 5.5-dimethyl-l-pyrroline-l-oxide (DMPO) and 3,3,5,5-dimethyl-l-pyrroline-l-oxide (M₄PO), the results show that upon reoxygenation of endothelial cells, following a period of anoxia, these cells generate superoxide (0₂). Cytotoxicity of the spin traps was measured by standard trypan blue exclusion methods. Cell injury or death was measured at various times during reoxygenation by lactate dehydrogenase (LDH) release. Experiments using oxypurinol, SOD, CAT and a combination of SOD and CAT show that while oxypurinol partially prevents spin adduct formation. the combination of SOD and CAT is more effective in doing so. These results suggest that the majority of the oxygen radicals produced by endothelial cells are done so exogenously. The results also suggest that endothelial cells are not only a source of oxygen radicals but also a target.     

Endothelial Cells as A Source of Oxygen-Free Radicals An Esr Study

Endothelial cells were subjected to anoxia/reoxygenation in order to simulate some of the free radical mechanisms occurring in ischaemialreperfusion. With ESR and spin trapping using the spin traps 5.5-dimethyl-l-pyrroline-l-oxide (DMPO) and 3,3,5,5-dimethyl-l-pyrroline-l-oxide (M₄PO), the results show that upon reoxygenation of endothelial cells, following a period of anoxia, these cells generate superoxide (0₂). Cytotoxicity of the spin traps was measured by standard trypan blue exclusion methods. Cell injury or death was measured at various times during reoxygenation by lactate dehydrogenase (LDH) release. Experiments using oxypurinol, SOD, CAT and a combination of SOD and CAT show that while oxypurinol partially prevents spin adduct formation. the combination of SOD and CAT is more effective in doing so. These results suggest that the majority of the oxygen radicals produced by endothelial cells are done so exogenously. The results also suggest that endothelial cells are not only a source of oxygen radicals but also a target.     

Mitochondrial involvement in IGF-1 induced protection of cardiomyocytes against hypoxia/reoxygenation injury

Studies in animal models of myocardial ischemia-reperfusion revealed that the administration of insulin-like growth factor (IGF-1) can provide substantial cardioprotective effect. However, the mechanisms by which IGF-1 prevents myocardial ischemia-reperfusion injury are not fully understood. This study addresses whether mitochondrial bioenergetic pathways are involved in the cardioprotective effects of IGF-1. Single cardiomyocytes from adult rats were incubated in the absence or presence of IGF-1 for 60 min and subjected to 60 min hypoxia followed by 30 min reoxygenation at 37°C. Mitochondrial function was evaluated by assessment of enzyme activities of oxidative phosphorylation and Krebs cycle pathways. Hypoxia/reoxygenation (HR) caused significant inhibition of mitochondrial respiratory complex IV and V activities and of the Krebs cycle enzyme citrate synthase, whereas pretreatment with IGF-1 maintained enzyme activities in myocytes at or near control levels. Mitochondrial membrane potential, evaluated with JC-1 staining, was significantly higher in IGF-1 + HR- treated myocytes than in HR alone, with levels similar to those found in normal control cardiomyocytes. In addition, IGF-1 reduced both HR-induced lactate dehydrogenase (LDH) release and malondialdehyde production (an indicator of lipid peroxidation) in cardiomyocytes. These results suggest that IGF-1 protects cardiomyocytes from HR injury via stabilizing mitochondria and reducing reactive oxidative species (ROS) damage.