Neuroprotective effects of glyceryl nonivamide against microglia-like cells and 6-hydroxydopamine-induced neurotoxicity in SH-SY5Y human dopaminergic neuroblastoma cells

Glyceryl nonivamide (GLNVA), a vanilloid receptor (VR) agonist, has been reported to have calcitonin gene-related peptide-associated vasodilatation and to prevent subarachnoid hemorrhage-induced cerebral vasospasm. In this study, we investigated the neuroprotective effects of GLNVA on activated microglia-like cell mediated- and proparkinsonian neurotoxin 6-hydroxydopamine (6-OHDA)-induced neurotoxicity in human dopaminergic neuroblastoma SH-SY5Y cells. In coculture conditions, we used lipopolysaccharide (LPS)-stimulated BV-2 cells as a model of activated microglia. LPS-induced neuronal death was significantly inhibited by diphenylene iodonium (DPI), an inhibitor of NADPH oxidase. However, capsazepine, the selective VR1 antagonist, did not block the neuroprotective effects of GLNVA. GLNVA reduced LPS-activated microglia-mediated neuronal death, but it lacked protection in DPI-pretreated cultures. GLNVA also decreased LPS activated microglia induced overexpression of neuronal nitric-oxide synthase (nNOS) and glycoprotein 91 phagocyte oxidase (gp91(phox)) on SH-SY5Y cells. Pretreatment of BV-2 cells with GLNVA diminished LPS-induced nitric oxide production, overexpression of inducible nitric-oxide synthase (iNOS), and gp91(phox) and intracellular reactive oxygen species (iROS). GLNVA also reduced cyclooxygenase (COX)-2 expression, inhibitor of nuclear factor (NF)-kappaB (IkappaB)alpha/IkappaBbeta degradation, NF-kappaB activation, and the overproduction of tumor necrosis factor-alpha, interleukin (IL)-1beta, and prostaglandin E2 in BV-2 cells. However, GLNVA augmented anti-inflammatory cytokine IL-10 production on LPS-stimulated BV-2 cells. Furthermore, in 6-OHDA-treated SH-SY5Y cells, GLNVA rescued the changes in condensed nuclear and apoptotic bodies, prevented the decrease in mitochondrial membrane potential, and reduced cells death. GLNVA also suppressed accumulation of iROS and up-regulated heme oxygenase-1 expression. 6-OHDA-induced overexpression of nNOS, iNOS, COX-2, and gp91(phox) was also reduced by GLNVA. In summary, the neuroprotective effects of GLNVA are mediated, at least in part, by decreasing the inflammation- and oxidative stress-associated factors induced by microglia and 6-OHDA.     

A novel neurotrophic agent, T-817MA [1-{3-[2-(1-benzothiophen-5-yl) ethoxy] propyl}-3-azetidinol maleate], attenuates amyloid-beta-induced neurotoxicity and promotes neurite outgrowth in rat cultured central nervous system neurons

Progressive neuronal loss in Alzheimer's disease (AD) is considered to be a consequence of the neurotoxic properties of amyloid-beta peptides (A beta). T-817MA (1-{3-[2-(1-benzothiophen-5-yl) ethoxy] propyl}-3-azetidinol maleate) was screened as a candidate therapeutic agent for the treatment of AD based on its neuroprotective potency against A beta-induced neurotoxicity and its effect of enhancing axonal regeneration in the sciatic nerve axotomy model. The neuroprotective effect of T-817MA against A beta(1-42) or oxidative stress-induced neurotoxicity was assessed using a coculture of rat cortical neurons with glia. T-817MA (0.1 and 1 microM) was strongly protective against A beta(1-42)-induced (10 microM for 48 h) or H2O2-induced (100 microM for 24 h) neuronal death. T-817MA suppressed the decrease of GSH levels induced by H2O2 exposure (30 microM for 4 h) in cortical neuron culture; therefore, T-817MA was likely to alleviate oxidative stress. Besides the neuroprotective effect, T-817MA (0.1 and 1 microM) promoted neurite outgrowth in hippocampal slice cultures and reaggregation culture of rat cortical neurons. T-817MA also increased the growth-associated protein 43 content in the reaggregation culture of cortical neurons. These findings suggest that T-817MA exerts neuroprotective effect and promotes neurite outgrowth in rat primary cultured neurons. Based on these neurotrophic features, T-817MA may have a potential for disease modification and be useful for patients with neurodegenerative diseases, such as AD.     

Signals of oxidant-induced cardiomyocyte hypertrophy: key activation of p70 S6 kinase-1 and phosphoinositide 3-kinase

Cardiomyocytes in culture can survive low or mild doses of oxidants but later increase cell volume and protein content. To understand the mechanism, we determined the early signaling events of oxidative stress. With 200 microM H2O2, the activity of p70 S6 kinase-1 (p70S6K1) increased at 30 min and reached a plateau at 90 min. Dose-response studies at the 60 min time point show that p70S6K1 activity reached its highest level with 150 microM H2O2. Increased p70S6K1 activity correlated with phosphorylation of Thr389 and Thr421/Ser424 residues, suggesting the involvement of an upstream kinase. Phosphoinositide 3-kinase (PI3K) activity was elevated by 5 min, reached a plateau at 10 min, and remained more than 6-fold induced for at least 60 min after 200 microM H2O2 exposure. The dose-response studies at 10 min found that 150 microM H2O2 induced the highest PI3K activity. Increased PI3K activity correlated with tyrosine phosphorylation of the 85-kDa regulatory subunit. Inactivating PI3K with wortmannin prevented H2O2 from inducing Thr389 phosphorylation and p70S6K1 activation. Wortmannin and rapamycin prevented H2O2 from inducing increases in cell volume and protein content. The antineoplastic drugs doxorubicin and daunorubicin also induced significant enlargement of cardiomyocytes at 10 to 100 nM dose range. Although the glutathione synthesis inhibitor buthionine sulfoximine potentiated the effect of doxorubicin and H2O2, the antioxidant N-acetylcysteine prevented induction of cell enlargement. Our data suggest that oxidative stress induces activation of PI3K, which leads to p70S6K1 activation and enlargement of cell size.     

Butein,isoliquiritigenin, and scopoletin attenuate neurodegeneration via antioxidant enzymes and SIRT1/ADAM10 signaling pathway

Neuronal cell death is a key feature of neurodegenerative disorders such as Parkinson''s and Alzheimer''s diseases. Plant polyphenols, namely butein, isoliquiritigenin, and scopoletin, have been shown to exhibit various biological activities including anti-inflammatory, antimicrobial, and antioxidant activities. Herein, butein, isoliquiritigenin, and scopoletin were explored for their neuroprotective properties against oxidative stress-induced human dopaminergic SH-SY5Y cell death. The cells exposed to hydrogen peroxide (H₂O₂) revealed a reduction in cell viability and increases in apoptosis and levels of reactive oxygen species (ROS). Interestingly, pretreatment of SH-SY5Y cells with 5 μM of butein, isoliquiritigenin, or scopoletin protected against the cell death induced by H₂O₂, and decreased the levels of apoptotic cells and ROS. In addition, the levels of SIRT1, FoxO3a, ADAM10, BCL-2, and antioxidant enzymes (catalase and SOD2) were maintained in the cells pretreated with butein, isoliquiritigenin, or scopoletin before H₂O₂ treatment compared to cells without pretreatment and the reference (resveratrol). Molecular docking analysis revealed that the interactions between the activator-binding sites of SIRT1 and the phenolic compounds were similar to those of resveratrol. Taken together, the data suggest that these polyphenolic compounds could be potential candidates for prevention and/or treatment of neurodegeneration.

Neuronal cells exposed to H₂O₂ may undergo increase ROS, reduction in cell viability and cell death. Butein, isoliquiritigenin, and scopoletin ameliorated H₂O₂-induced neurotoxicity by reducing ROS, balancing antioxidants and activating SIRT1-FoxO3a-ADAM10 pathway.     

Involvement of Na+-Ca2+ exchanger in intracellular Ca2+ increase and neuronal injury induced by polychlorinated biphenyls in human neuroblastoma SH-SY5Y cells

In SH-SY5Y, a human neuroblastoma cell line, Aroclor 1254 (A1254), induced a dose-dependent (10-50 microg/ml) intracellular calcium concentration ([Ca2+]i) increase. Two rather specific sodium-calcium (Na+-Ca2+) exchanger (NCX) inhibitors, bepridil (10 microM) and KB-R7943 [2-[2-[4-(4-nitrobenzyloxy) phenyl]ethyl]isothiourea methanesulfonate] (10 microM), reduced A1254-induced [Ca2+]i increase. A 24-h exposure to 30 microg/ml A1254 caused remarkable SH-SY5Y neuroblastoma cell damage. It is noteworthy that both bepridil and KB-R7943 counteracted A1254-induced neuronal injury. These results indicate that NCX contributes to [Ca2+]i increase and neuronal injury induced by A1254. RT-PCR experiments revealed in SH-SY5Y neuroblastoma cells the expression of NCX1 and NCX3 isoforms. To investigate which isoform was involved in [Ca2+]i increase and neuronal damage induced by A1254, we used specific antisense oligodeoxynucleotides (ODNs) to reduce NCX1 or NCX3 protein expression. The results showed that only NCX1 ODN reduced [Ca2+]i increase and neuronal injury induced by A1254. In conclusion, these results indicate that NCX1 may participate to [Ca2+]i increase and neurotoxicity evoked by A1254 in SH-SY5Y neuroblastoma cells.     

Pogostemon cablin as ROS Scavenger in Oxidant-induced Cell Death of Human Neuroglioma Cells

Reactive oxygen species (ROS) have been implicated in the pathogenesis of a wide range of acute and long-term neurodegenerative diseases. This study was undertaken to examine the efficacy of Pogostemon cablin, a well-known herb in Korean traditional medicine, on ROS-induced brain cell injury. Pogostemon cablin effectively protected human neuroglioma cell line A172 against both the necrotic and apoptotic cell death induced by hydrogen peroxide (H(2)O(2)). The effect of Pogostemon cablin was dose dependent at concentrations ranging from 0.2 to 5?mg ml(-1). Pogostemon cablin significantly prevented depletion of cellular ATP and activation of poly ADP-ribose polymerase induced by H(2)O(2). The preservation of functional integrity of mitochondria upon the treatment of Pogostemon cablin was also confirmed by 3-(4,5-dimethyl-2-thiazyl)-2,5-diphenyl-2-H-tetrazolium bromide assay. Furthermore, Pogostemon cablin significantly prevented H(2)O(2)-induced release of cytochrome c into cytosol. Determination of intracellular ROS showed that Pogostemon cablin might exert its role as a powerful scavenger of intracellular ROS. The present study suggests the beneficial effect of Pogostemon cablin on ROS-induced neuroglial cell injury. The action of Pogostemon cablin as a ROS-scavenger might underlie the mechanism.     

Unequal neuroprotection afforded by the acetylcholinesterase inhibitors galantamine, donepezil, and rivastigmine in SH-SY5Y neuroblastoma cells: role of nicotinic receptors

Donepezil, rivastigmine, and galantamine are three drugs with acetylcholinesterase (AChE)-inhibiting activity that are currently being used to treat patients suffering from Alzheimer's disease. We have studied the neuroprotective effects of these drugs, in comparison with nicotine, on cell death caused by beta-amyloid (Abeta) and okadaic acid, two models that are relevant to Alzheimer's pathology, in the human neuroblastoma cell line SH-SY5Y. Galantamine and donepezil showed a U-shaped neuroprotective curve against okadaic acid toxicity; maximum protection was achieved at 0.3 microM galantamine and at 1 microM donepezil; at higher concentrations, protection was diminished. Rivastigmine showed a concentration-dependent effect; maximum protection was achieved at 3 microM. When apoptosis was induced by Abeta25-35, galantamine, donepezil, and rivastigmine showed maximum protection at the same concentrations: 0.3, 1, and 3 microM, respectively. Nicotine also afforded protection against Abeta- and okadaic acid-induced toxicity. The neuroprotective effects of galantamine, donepezil, and nicotine were reversed by the alpha7 nicotinic antagonist methyllycaconitine but not by the alpha4beta2 nicotinic antagonist dihydro-beta-erythroidine. The phosphoinositide 3-kinase (PI3K)-Akt blocker 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride (LY294002) reversed the protective effects of galantamine, donepezil, and nicotine but not that of rivastigmine. In contrast, the bcl-2 antagonist ethyl[2-amino-6-bromo-4-(1-cyano-2-ethoxy-2-oxoethyl)]-4H-chromene-3-carboxylate (HA 14-1) reversed the protective effects of the three AChE inhibitors and that of nicotine. Our results show that galantamine, donepezil, and rivastigmine afford neuroprotection through a mechanism that is likely unrelated to AChE inhibition. Such neuroprotection seemed to be linked to alpha7 nicotinic receptors and the PI3K-Akt pathway in the case of galantamine and donepezil but not for rivastigmine.     

Retracted Article: Sauchinone inhibits high glucose-induced oxidative stress and apoptosis in retinal pigment epithelial cells

Diabetic retinopathy (DR) is a common complication of diabetes mellitus and results in acquired blindness among working-age adults. It has been demonstrated that high glucose (HG)-induced oxidative stress and cell apoptosis in retinal pigment epithelial (RPE) cells are major factors for the pathogenesis of DR. Sauchinone, one of the active lignan isolated from Saururus chinensis, was reported to possess anti-oxidant and anti-apoptosis effects. In the present study, we investigated the effects of sauchinone on HG-induced oxidative stress and apoptosis in ARPE-19 cells. Our results proved that sauchinone improved the cell viability of HG-induced ARPE-19 cells. Moreover, sauchinone treatment caused a decrease in intracellular reactive oxygen species (ROS) generation and an increase in the activities of superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT). Besides, flow cytometry showed that the apoptotic rate in sauchinone-treated ARPE-19 cells obviously decreased as compared in the HG-treated cells. Western blot indicated that sauchinone treatment caused a significant decrease in bax expression and increase in bcl-2 expression in HG-treated ARPE-19 cells. Sauchinone treatment enhanced the HG-caused induction of p-Akt, nuclear factor erythroid 2-related factor (Nrf2), and heme oxygenase-1 (HO-1) expressions in ARPE-19 cells. However, the inhibitor of Akt, LY294002, reversed the effects of sauchinone on cell viability, oxidative stress, and cell apoptosis in HG-treated ARPE-19 cells. These findings suggested that sauchinone treatment prevented HG-induced oxidative stress and apoptosis via regulating the Akt/Nrf2/HO-1 pathway in HG-induced RPE cells. These findings suggested that sauchinone might be a therapeutic agent for the treatment and prevention of DR.

Diabetic retinopathy (DR) is a common complication of diabetes mellitus and results in acquired blindness among working-age adults.     

Mechanism of in vitro heme-induced LDL oxidation: effects of antioxidants

BACKGROUND: Heme protein toxicity, owing to generation of reactive oxygen species most likely by direct interaction between heme iron and hydrogen peroxide (H2O2), may be involved in various pathologies, including atherogenesis and pigmentary acute renal failure. The aim of this study was to investigate the mechanism of heme cytotoxicity and the effects of antioxidant therapies in an in vitro model of heme-induced low-density lipoprotein (LDL) oxidation. MATERIALS AND METHODS: Human LDLs were exposed to heme, iron (Fe), protoporphyrin (PPIX) and PPIX-Zinc (Zn) with or without H2O2. Lipid peroxidation was monitored by measurement of conjugated diene formation (at the 234-nm absorbance). The effect of various antioxidants, such as vitamin E and vitamin C, reduced glutathione (GSH), and oxidized glutathione (GSSG), mannitol and desferoxamine (DFO) was further investigated in the established in vitro model of LDL oxidation. RESULTS: Incubation of LDLs in the presence of heme/H2O2 induced lipid peroxidation with the optimal oxidation rate being at 5 microm heme/100 microm H2O2 doses. By contrast, incubation of LDL with H2O2, Fe, Fe/H2O2, PPIX, PPIX/H2O2, heme or PPIX-Zn did not initiate any LDL oxidation. In vitro, the vitamin E load protected native LDLs against heme/H2O2 oxidative modifications. Incubation of LDLs with increasing doses of vitamin C, GSH and DFO conferred a dose-dependent protection, while mannitol and GSSG had no effect. CONCLUSIONS: Initiation and propagation of heme-induced lipid peroxidation is not mediated by a Fenton reaction but depends on specific interactions between heme and H2O2. It may result from the generation of ferryl and perferryl radicals derived from hemic Fe and H2O2 interactions. A protective effect of vitamins E, C, GSH and DFO was demonstrated in this model.     

米贝地尔对罗哌卡因诱导SH-SY5Y细胞凋亡的抑制作用研究

目的:观察T型钙通道拮抗剂米贝地尔对罗哌卡因致SH-SY5Y细胞凋亡的影响,探讨罗哌卡因诱发神经细胞毒性是否与T型钙通道相关。方法:将SH-SY5Y细胞随机分为4组:SH-SY5Y细胞正常培养组(A组);SH-SY5Y细胞5μmol/L米贝地尔培养组(B组);SH-SY5Y细胞3 mmol/L罗哌卡因培养组(C组);SH-SY5Y细胞5μmol/L米贝地尔+3 mmol/L罗哌卡因培养组(D组)。各组细胞分别在有或无3 mmol/L罗哌卡因处理开始时(T0)、处理后1 h(T1)、6 h(T2)、12 h(T3)、24 h(T4),MTT法检测细胞活力、流式细胞术和Hoechst33258染色法检测细胞凋亡率。结果:与A组相比,C组、D组在T1、T2、T3、T4时点细胞活力明显降低(P<0.05);但C组降低幅度更明显,C组与D组比较,两组在T1、T2、T3、T4时点差异有统计学意义(P<0.05)。C组随时间的递增其细胞凋亡率逐渐增加,在T4时凋亡率达到最高;D组在T1、T2、T3、T4时点的细胞凋亡率明显比C组降低,两组差异有统计学意义(P<0.05)。结论:罗哌卡因对SH-SY5Y细胞有毒性作用,米贝地尔可减轻罗哌卡因诱发的神经细胞损伤,提示罗哌卡因诱发神经毒性可能与T型钙通道有关。     

Differential effects of heme oxygenase isoforms on heme mediation of endothelial intracellular adhesion molecule 1 expression.

Heme oxygenase (HO), by catabolizing heme to bile pigments, down-regulates cellular hemoprotein, hemoglobin, and heme; the latter generates pro-oxidant products, including free radicals. Two HO isozymes, the products of distinct genes, have been described; HO-1 is the inducible isoform, whereas HO-2 is suggested to be constitutively expressed. We studied the inducing effect of several metal compounds (CoCl(2), stannic mesoporphyrin, and heme) on HO activity. Additionally, we studied HO-1 expression in experimental models of adhesion molecule expression produced by heme in endothelial cells, and the relationship of HO-1 expression to the induced adhesion molecules. Flow cytometry analysis showed that heme induces intracellular adhesion molecule 1 (ICAM-1) expression in a concentration (10-100 microM)- and time (1-24 h)-dependent fashion in human umbilical vein endothelial cells. Pretreatment with stannic mesoporphyrin, an inhibitor of HO activity, caused a 2-fold increase in heme-induced ICAM-1 expression. In contrast, HO induction by CoCl(2) decreased heme-induced ICAM-1 expression by 33%. To examine the contribution of HO-1 and HO-2 to endothelial HO activity, specific antisense oligonucleotides (ODNs) of each isoform were tested for their specificity to inhibit HO activity in cells exposed to heme. Endothelial cells exposed to heme elicited increased HO activity, which was prevented (70%) by HO-1 antisense ODNs. HO-2 antisense ODN inhibited heme-induced HO activity by 21%. Addition of HO-1 antisense ODNs prevented heme degradation and resulted in elevation of microsomal heme. Western blot analysis showed that HO-1 antisense ODNs selectively inhibited HO-1 protein and failed to inhibit HO-2 protein. Incubation of endothelial cells with HO-1 antisense enhanced heme-dependent increase of ICAM-1. In contrast, addition of HO-2 antisense to endothelial cells failed to increase adhesion molecules. The role of glutathione, an important antioxidant, was examined on heme-induced ICAM-1 expression. Endothelial cells pretreated with a glutathione precursor, N-acetylcysteine, or glutathione ester, showed a decrease in heme-induced ICAM-1 expression of 37 and 44%, respectively, suggesting that the mechanism of ICAM-1 induction by heme may be partly dependent on the levels of antioxidant. It is possible that amelioration of the heme-induced oxidative stress and expression of ICAM-I is due, in part, to the induction of HO-1 activity. Regulation of HO activity in this manner may have clinical applications.     

Serofendic acid, a sulfur-containing diterpenoid derived from fetal calf serum, attenuates reactive oxygen species-induced oxidative stress in cultured striatal neurons

We previously identified a novel endogenous substance, serofendic acid, from a lipophilic extract of fetal calf serum. Serofendic acid protects cultured cortical neurons against the cytotoxicity of glutamate and nitric oxide. Here, we reported the protective effect of serofendic acid on reactive oxygen species-induced oxidative stress using primary rat striatal cultures. In addition, we compared the neuroprotective effect and the radical-scavenging activity of serofendic acid with those of dimethyl sulfoxide (DMSO), because serofendic acid possesses a DMSO structure. Paraquat caused neuronal death, which was inhibited by a cell-permeable superoxide dismutase (SOD) mimetic, Mn(III)tetrakis(4-benzoic acid)porphyrin chloride (Mn-TBAP); a cell-permeable SOD/catalase mimetic, EUK-134 [manganese 3-methoxy N,N'-bis(salicylidene)ethylenediamine chloride]; and a ferrous ion chelator, 2,2'-dipyridyl, in rat striatal cultures. Serofendic acid (10-100 microM) suppressed the neurotoxicity of paraquat, whereas DMSO (10-100 microM) did not. By contrast, higher concentrations (30-300 mM) of DMSO ameliorated the paraquat-induced cell death. Furthermore, H(2)O(2) induced neurotoxicity, which was prevented by EUK-134 and 2,2'-dipyridyl. Serofendic acid (10-100 microM) also protected striatal neurons against the H(2)O(2)-induced toxicity. Higher concentrations (30-300 mM) of DMSO ameliorated H(2)O(2)-induced neuronal death, whereas lower concentrations (10-100 microM) did not. Electron spin resonance spectrometry with a spin-trapping technique revealed that serofendic acid and DMSO had approximately the same ability to inhibit the formation of the hydroxyl radical (.OH). These results suggest that the.OH-scavenging activity of serofendic acid is attributable to its DMSO structure and that the remaining components such as the atisane structure play an important role in eliciting neuroprotection at a concentration range of 10 to 100 microM.     

Study on the effect of cell proliferation and anti-oxidative damage of aldehyde dehydrogenase-2 gene transfected into K562 cells

OBJECTIVE: To construct a eukaryotic expression vector containing aldehyde dehydrogenase-2 (ALDH2) gene and investigate the effects and its possible mechanisms of ALDH2 gene on cell proliferation and anti-oxidative damage in the K562 cells. METHODS: An eukaryotic expression vector containing the ALDH2 gene cloned from human hepatocytes was constructed and transfected into K562 cells by liposome. RT-PCR and Western blot were used to evaluate the expression of ALDH2. MTT assay was used to check the cell proliferation and trypan blue exclusion to check K562 cells damage induced by hydrogen peroxide (H2O2). RT-PCR and fluorescence spectrophotometry were used to determine the expression of heme oxygenase-1 (HO-1) and the generation of intracellular reactive oxygen species (ROS) respectively. RESULTS: RT-PCR and Western blot analysis showed distinct higher ALDH2 protein expression in gene transfected group. The latter group had a higher cell proliferation (P < 0.05) and survival rate against H2O2 induced-oxidative damage, being increased by 7.8 times (IC(50) was 12.3 μmol/L and 1.4 μmol/L for K562-pcDNA3.1-ALDH2 and control cells, respectively, P < 0.01). The HO-1 mRNA expression and the generation of intracellular ROS were downregulated at a specific concentration of H2O2 in the ALDH2 gene transfected group. CONCLUSION: ALDH2 gene transfection can protect K562 cells against oxidative damage, and the downregulation of HO-1 expression and intracellular ROS may be involved in this process.     

Endonuclease-induced DNA damage and cell death in oxidant injury to renal tubular epithelial cells.

Hydrogen peroxide (H2O2)-induced DNA damage and cell death have been attributed to the direct cytotoxicity of H2O2 and other oxidant species generated from H2O2. We examined the possibility that oxidants activate endonucleases leading to DNA damage and cell death in renal tubular epithelial cells, similar to that described for apoptosis. Within minutes, H2O2 caused DNA strand breaks in a dose-dependent manner, followed by cell death. DNA fragmentation was demonstrated both by the release of [3H]thymidine in 27,000-g supernatant as well as the occurrence of low molecular weight DNA fragments on agarose gel electrophoresis, characteristic of endonuclease cleavage. Endonuclease inhibitors, aurintricarboxylic acid, Evans blue, and zinc ion prevented H2O2-induced DNA strand breaks, fragmentation, and cell death. Inhibitors of protein or mRNA synthesis had only minor protection against H2O2-induced DNA damage in contrast to complete protection reported in apoptotic thymocytes. Micrococcal endonuclease induced similar DNA strand breaks in LLC-PK1 cells, and the endonuclease inhibitors prevented the events confirming the ability of endonucleases to induce DNA damage. The protective effect of aurintricarboxylic acid was not due to the prevention of the rise in intracellular free calcium. We conclude that endonuclease activation occurs as an early event leading to DNA damage and cell death in renal tubular epithelial cells exposed to oxidant stress and, in contrast to apoptotic thymocytes, does not require macromolecular synthesis.     

ALDH2基因导入对K562细胞增殖和抗氧化损伤的影响

目的 克隆人乙醛脱氢酶2(ALDH2)基因,研究ALDH2基因导入慢性粒细胞白血病细胞系K562细胞后对其增殖和抗氧化损伤的影响.方法 从肝细胞中克隆人ALDH2基因,构建真核表达载体,用脂质体法将其导入K562细胞中,用RT-PCR和Western blot法检测ALD-H2基因的表达,锥虫蓝拒染法和MTT法检测转基因组细胞的增殖水平及对氧自由基引起的氧化损伤的反应;在此基础上,利用RT-PCR以及荧光分光光度法进一步检测氧自由基诱导后转基因组细胞中血红素加氧酶-1(HO-1)的表达和细胞内活性氧类(ROS)的产生.结果 成功克隆人ALDH2基因并将构建的真核表达载体转染K562细胞,RT-PCR和Western blot法检测到ALDH2的高表达.锥虫蓝拒染法和MTT结果显示转基因组细胞增殖水平明显高于对照组(P＜0.05),经基因修饰的细胞对H2O2耐受性增高,H2O2的IC50值提高了7.8倍(IC50值分别为12.3μnol/L和1.4 μmol/L,P＜0.01).HO-1的表达和ROS的产生随H2O2浓度的增大而增加,而一定浓度H2O2诱导后HO-1的表达和ROS的产生在转基因组中显著低于对照组(P＜0.05).结论 ALDH2基因导人K562细胞后可增加对氧自由基引起的细胞损伤的耐受性,起到保护作用,该过程伴随着ROS水平以及HO-1表达的降低.     

Blocking late sodium current reduces hydrogen peroxide-induced arrhythmogenic activity and contractile dysfunction

Reactive oxygen species (ROS), including H2O2, cause intracellular calcium overload and ischemia-reperfusion damage. The objective of this study was to examine the hypothesis that H2O2-induced arrhythmic activity and contractile dysfunction are the results of an effect of H2O2 to increase the magnitude of the late sodium current (late INa). Guinea pig and rabbit isolated ventricular myocytes were exposed to 200 microM H2O2. Transmembrane voltages and currents and twitch shortening were measured using the whole-cell patch-clamp technique and video edge detection, respectively. [Na+]i and [Ca2+]i were determined by fluorescence measurements. H2O2 caused a persistent late INa that was almost completely inhibited by 10 microM tetrodotoxin (TTX). H2O2 prolonged the action potential duration (APD), slowed the relaxation rate of cell contraction, and induced early afterdepolarizations (EADs) and aftercontractions. H2O2 also caused increases of [Na+]i and [Ca2+]i. Ranolazine (10 microM), a novel inhibitor of late INa, attenuated H2O2-induced late INa by 51+/-9%. TTX (2 microM) or 10 microM ranolazine attenuated H2O2-induced APD prolongation and suppressed EADs. Ranolazine accelerated the twitch relaxation rate in the presence of H2O2 and abolished H2O2-induced aftercontractions. Pretreatment of myocytes with ranolazine delayed and reduced the increases of APD, [Na+]i, and [Ca2+]i caused by H2O2. In conclusion, the results confirm the hypothesis that an increase in late INa during exposure of ventricular myocytes to H2O2 contributes to electrical and contractile dysfunction and suggest that inhibition of late INa may offer protection against ROS-induced Na+ and Ca2+ overload.     

Erythropoietin protects against doxorubicin-induced cardiomyopathy via a phosphatidylinositol 3-kinase-dependent pathway

Doxorubicin (DOX) is an effective antineoplastic agent whose use has been limited by its cardiotoxic side effects. Recent studies have established that erythropoietin (EPO), a cytokine essential for red blood cell production, protects against ischemic injury in the heart and other organs. The purpose of this study was to assess whether EPO protects the heart against cardiotoxicity induced by DOX. We found that DOX-induced apoptosis and impaired heart function in mice were largely prevented by EPO administration. To investigate the mechanism of protection by EPO, cultured neonatal mouse ventricular myocytes were treated with EPO at therapeutic levels (i.e., 1 U/ml), before application of DOX (0.1-1.0 microM). EPO protected against DOX-induced cardiomyocyte death (by approximately 50%) and apoptosis assessed by annexin-V labeling, DNA fragmentation, and caspase-3 activity. DOX-mediated increases in reactive oxygen species, which trigger cardiotoxicity, were also reversed by preconditioning with EPO. These functional effects of EPO correlated with increased Akt/protein kinase B ( approximately 2-fold) and glycogen synthase kinase 3 (GSK-3; approximately 1.3-fold) phosphorylations, suggesting protection by EPO was mediated by phosphatidylinositol 3-kinase activation. Indeed, preventing Akt and GSK-3beta phosphorylations by phosphatidylinositol 3-kinase (PI3K) inhibition abolished protection by EPO against cardiomyocyte loss, apoptosis, and oxidative stress. Thus, pretreatment with therapeutic levels of EPO can protect the myocardium against DOX-induced impaired heart function and cardiomyocyte apoptosis by activating PI3K-Akt cell survival pathways.