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ABSTRACT: It has been well accepted that increased reactive oxygen species (ROS) and the subsequent oxidative stress is one of the major causes of ischemia/reperfusion (I/R) injury. DJ-1 protein, as a multifunctional intracellular protein, plays an important role in regulating cell survival and antioxidant stress. Here, we wondered whether DJ-1 overexpression attenuates simulated ischemia/reperfusion (sI/R)-induced oxidative stress. A rat cDNA encoding DJ-1 was inserted into a mammalian expression vector. After introduction of this construct into H9c2 myocytes, stable clones were obtained. Western blot analysis of the derived clones showed a 2.6-fold increase in DJ-1 protein expressing. Subsequently, the DJ-1 gene-transfected and control H9c2 cells were subjected to sI/R, and then cell viability, lactate dehydrogenase, malondialdehyde, intracellular ROS and antioxidant enzymes (superoxide dismutase, catalase and glutathione peroxidase) were measured appropriately. The results showed that stable overexpression of DJ-1 efficiently attenuated sI/R-induced viability loss and lactate dehydrogenase leakage. Additionally, stable overexpression of DJ-1 inhibited sI/R-induced the elevation of ROS and MDA contents followed by the increase of antioxidant enzymes (superoxide dismutase, catalase and glutathione peroxidase) activities and expression. Our data indicate that overexpression of DJ-1 attenuates ROS generation, enhances the cellular antioxidant capacity and prevents sI/R-induced oxidative stress, revealing a novel mechanism of cardioprotection. Importantly, DJ-1 overexpression may be an important part of a protective strategy against ischemia/reperfusion injury. Copyright © 2012 John Wiley & Sons, Ltd.
Cell Biochemistry and Function 01/2013; · 1.77 Impact Factor
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ABSTRACT: Myocardial preconditioning is a powerful phenomenon that can attenuate ischemia/reperfusion-induced oxidant stress and elicit delayed cardioprotection. Its mechanisms involve activation of intracellular signaling pathways and up-regulation of the protective antioxidant proteins. DJ-1 protein, as a multifunctional intracellular protein, plays an important role in attenuating oxidant stress and promoting cell survival. In the present study, we investigated whether DJ-1 is up-regulated during the late phase of hypoxic preconditioning (HP) and the up-regulation of DJ-1 is mediated by extracellular-regulated kinase 1/2 (ERK1/2) signaling pathway. Rat heart-derived H9c2 cells were exposed to HP. Twenty-four hours later cells were subjected to hypoxia/reoxygenation (H/R) and then cell viability, lactate dehydrogenase (LDH), intracellular reactive oxygen species (ROS), ERK1/2 phosphorylation, and DJ-1 protein were measured appropriately. The results showed that HP efficiently attenuated H/R-induced viability loss and LDH leakage. In addition, HP promoted ERK1/2 activation, up-regulated DJ-1 protein expression, inhibited H/R induced the elevation of ROS. However, when ERK1/2 phosphorylation was specifically inhibited by U0126, the increase in DJ-1 expression occurring during HP was almost completely abolished and, as a result, the delayed cardioprotection induced by HP was abolished, and the inhibitory effect of HP on H/R-induced oxidant stress was also reversed. Furthermore, knocking down DJ-1 by siRNA attenuated the delayed cardioprotection induced by HP. Our data indicate that HP can up-regulate DJ-1 protein expression through the ERK1/2-dependent signaling pathway. Importantly, DJ-1 might be involved in the delayed cardioprotective effect of HP against H/R injury.
Molecular and Cellular Biochemistry 08/2012; 370(1-2):231-40. · 2.06 Impact Factor
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ABSTRACT: We have shown recently that sasanquasaponin (SQS) can inhibit ischemia/reperfusion-induced elevation of intracellular Cl(-) concentration ([Cl(-)](i)) and elicit cardioprotection by up-regulating anion exchanger 3 (AE(3)) expression. In the present study, we futher analysed the intracellular signal transduction pathways by which SQS up-regulates AE(3) expression and elicits cardioprotection. Cardiomyocytes were incubated for 24 h with or without 10 µmol/L SQS, followed by simulated ischemia/reperfusion (sI/R). NO formation, Ras activity, and extracellular-regulated kinase 1/2 (ERK1/2) phosphorylation were measured appropriately. We showed that SQS pretreatment efficiently attenuated viability loss and lactate dehydrogenase leakage induced by sI/R in cardiomyocytes. Moreover, SQS induced NO production and promoted Ras activation, which futher promoted extracellular-regulated kinase 1/2 (ERK1/2) phosphorylation. These effects were paralleled by an increase in AE(3) expression. However, when the cardiomyocytes were treated with 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-l-oxyl-3-oxide (c-PTIO; an NO scavenger), S-trans-trans-farnesylthiosalicylic acid (FTS) (a Ras inhibitor), U0126 (an ERK1/2 inhibitor), respectively, the increase in AE(3) expression occurring during SQS pretreatment was almost completely abolished and, as a result, SQS-induced cardioprotection was prevented. Our findings indicate that SQS might up-regulate AE(3) expression through NO/Ras/ERK1/2 signal pathway to elicit cardioprotection in cultured cardiomyocytes.
Canadian Journal of Physiology and Pharmacology 06/2012; 90(7):873-80. · 1.95 Impact Factor
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ABSTRACT: Recent studies have shown that the cardioprotection of sasanquasaponin (SQS) against ischemia/reperfusion injury is related to inhibiting ischemia/reperfusion-induced elevation of intracellular Cl(-) concentration ([Cl(-) ](i)). However, the mechanism of inhibition remains unclear. Anion exchanger 3 (AE(3)) is an important regulatory protein for [Cl(-)](i). This study investigated whether AE(3) plays the critical role in the inhibitory effect of SQS on elevation of [Cl(-)](i) induced by ischemia/reperfusion and mediates the cardioprotection of SQS in H9c2 cells. Normal and AE(3) -knockdown H9c2 cells were incubated for 24 h with or without various concentrations of SQS (0.1, 1, or 10 µM) followed by simulated ischemia/reperfusion (sI/R). AE(3) expression was detected by Western blot. Flow cytometer analysis was employed to determine [Cl(-)](i,) [Ca(2+)](i) , reactive oxygen species (ROS) production, and cell apoptosis. The results showed that SQS pretreatment concentration-dependently attenuated sI/R-induced viability loss and lactate dehydrogenase leakage in normal H9c2 cells. Additionally, SQS concentration-dependently up-regulated AE(3) protein expression, and inhibited sI/R-induced the elevation of [Cl(-)](i) followed by the attenuation of Ca(2+) overload, ROS production, and cell apoptosis. However, the dose-dependent cardioprotection induced by SQS was abolished in AE(3) -knockdown H9c2 cells, and the inhibitory effects of SQS on [Cl(-)](i), Ca(2+) overload, ROS production, and cell apoptosis were also reversed. Our data indicate that AE(3) mediates the cardioprotective effect of SQS against sI/R injury. Importantly, AE(3) is required for SQS to inhibit sI/R-induced elevation of [Cl(-)](i), which subsequently inhibited sI/R-induced Ca(2+) overload, ROS production, and cell apoptosis.
Journal of Cellular Biochemistry 05/2011; 112(10):2803-12. · 2.87 Impact Factor
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ABSTRACT: Excess apoptosis of endothelial cells (EC) plays crucial roles in the onset and progression of vasculopathy in diabetes mellitus. Anion exchanger-2 (AE2) might be involved in the vasculopathy. However, little is known about the molecular mechanisms that AE2 mediated the apoptosis of EC. The purpose of this study was to explore the role of AE2 in the apoptosis of HUVECs induced by high glucose (HG) and its possible mechanisms. First, HUVECs were exposed to different glucose concentrations (5.5, 17.8, 35.6, 71.2 and 142.4 mmol/l, respectively, pH = 7.40) for different time points (12, 24, 48, 72, 120, and 168 h, respectively). Intracellular Cl(-) concentration ([Cl(-)]i), AE2 expression and the apoptosis were assayed. Then, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), Cl(-)-free media or specific RNA interference (RNAi) for AE2 was used to confirm whether AE2 could mediate the apoptosis induced by HG. Finally, the mechanisms of the AE2-mediated apoptosis were investigated by detecting mitochondrial permeability transition pore (mPTP, DeltaPsim) openings, reactive oxygen species (ROS) levels and Caspase-3 activity. We found that HG upregulated the AE2 expression and activity, increased [Cl(-)]i and induced the apoptosis in a time- and concentration-dependent manner. The apoptosis of HUVECs by HG was possibly mediated by AE2 through an mPTP-ROS-Caspase-3 dependent pathway. These findings suggested that AE2 was likely to be a glucose-sensitive transmembrane transporter and a novel potential therapeutic target for diabetic vasculopathy.
Apoptosis 02/2010; 15(6):693-704. · 4.07 Impact Factor
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ABSTRACT: To evaluate the roles of 8 short tandem repeats (STR) loci as STR panel in quantitative analysis of chimerism following transplantation, the primers were synthesized and marked with different dyes for D3S3045, D4S2366, D4S2639, D5S818, D13S317, D18S1002, D20S481 and D22S689. The blood samples of 15 cases received allogeneic stem cell transplantation were collected before and after transplantation, then DNA was extracted and amplified with these primers, and was further analysed under ABI Genetic Analyser 3100 to select suitable informative STR locus. Donor/recipient dilution series were prepared to get standard curves in selected loci, the DNAs extracted at different days after transplantation were used to quantitatively analyze the chimerism in patients according to the values of peak area or peak height of fluorescent signals. The standard curves can be used to calculate the chimerism by plotting the respective R/D quotient value against the percentage of recipient DNA. The results indicated that the calculated chimerism was in concordance with the donor/recipient dilution. The STR panel succeeded in identifying at least one informative marker and quantitative monitoring the chimerism after HSCT in 15 donor-recipient pairs and a relapsed case was diagnosed. It is concluded that the STR panel and its detection method can accurately and quantitatively monitor the chimerism after allogeneic HSCT, which is more economical and flexible than using commercial kits.
Zhongguo shi yan xue ye xue za zhi / Zhongguo bing li sheng li xue hui = Journal of experimental hematology / Chinese Association of Pathophysiology 07/2009; 17(3):706-12.
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ABSTRACT: Development of intracellular calcium overload is an important pathophysiological factor in myocardial ischemia/reperfusion or anoxia/reoxygenation injury. Recent studies have shown that Sodium Ferulate (SF) stimulates nitric oxide (NO) production and exerts a cardioprotective effect in the ischemia-reperfused heart. However, it has not been determined whether the cardioprotection of SF is associated with suppression of Ca(2+) overload via NO/cyclic GMP (cGMP)/cGMP-dependent protein kinase (PKG) pathway. In this work, after cardiomyocytes were incubated with 100, 200, 400, or 800 microM SF for 3 h, anoxia/reoxygenation injury was induced and intracellular Ca(2+) concentration, NO synthase (NOS) activity, guanylate cyclase activity, NO, and cGMP formation were measured appropriately. The results showed that treatment with SF concentration-dependently inhibited calcium overload induced by anoxia/reoxygenation. We also demonstrated that SF (100-800 microM) concentration dependently enhanced NO and cGMP formation through increasing NOS activity and guanylate cyclase activity in the cardiomyocytes. On the contrary, inhibition of calcium overload by SF was markedly attenuated by addition of an NOS inhibitor, an NO scavenger, an soluble guanylate cyclase inhibitor, and a PKG inhibitor: N(G)-nitro-l-arginine methyl ester (L-NAME, 100 microM), 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazole-1-oxyl-3-oxide (c-PTIO, 1.0 microM), 1H-[1, 2, 4] oxadiazolo [4, 3-alpha] quinoxalin-1-one (ODQ, 20 microM) and KT5823 (0.2 microM), respectively. Our findings indicate that SF significantly attenuates anoxia/reoxygenation-induced Ca(2+) overload and improves cell survival in cultured cardiomyocytes through NO/cGMP/PKG signal pathway.
European journal of pharmacology 01/2009; 603(1-3):86-92. · 2.59 Impact Factor
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ABSTRACT: Reactive oxygen species can play an important role in the pathogenesis of anoxia-reoxygenation injury. Sasanquasaponin (SQS) is a biologically active ingredient extracted from the Chinese medicinal plant Camellia oleifera Abel. Some studies have shown that SQS possesses potent antioxidant activities. However, it has not been elucidated whether SQS diminishes reactive oxygen species stress induced by anoxia-reoxygenation injury in cardiomyocytes. In this work, neonatal rat cardiomyocytes pretreated with the test compound were subjected to anoxia-reoxygenation. The extent of cellular damage was accessed by cell viability and the amount of released lactate dehydrogenase (LDH). Superoxide dismutase, catalase and glutathione peroxidase activities, reduced (GSH) and oxidized glutathione (GSSG) levels, and malondialdehyde contents were measured by a colorimetric method. The levels of intracellular reactive oxygen species and calcium were determined by flow cytometry. The results showed that SQS reduced LDH release and increased cell viability in a dose-dependent manner up to 10 microM and concomitantly decreased malondialdehyde and GSSG contents, while significantly increased GSH contents and the activities of superoxide dismutase, catalase and glutathione peroxidase. Moreover, treatment with SQS decreased intracellular reactive oxygen species levels and alleviated calcium accumulation in cardiomyocytes undergoing anoxia-reoxygenation. It is suggested that SQS could protect cardiomyocytes against oxidative stress induced by anoxia-reoxygenation by attenuating reactive oxygen species generation and increasing activities of endogenous antioxidants.
European Journal of Pharmacology 01/2008; 575(1-3):21-7. · 2.52 Impact Factor
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ABSTRACT: Anoxic preconditioning (APC) attenuates myocardial injury caused by ischemia/reperfusion. The protective mechanisms of APC involve up-regulation of the protective proteins and inhibition of apoptosis. 14-3-3 protein, as a molecular chaperone, plays an important role in regulating cell survival and apoptosis. However, the role of 14-3-3 protein in cardioprotection of APC and the pathways determining 14-3-3 protein expression during APC are not clear. In this work, Western blotting analysis was used to detect the 14-3-3 protein expression and activity of extracellular signal-regulated protein kinase 1/2 (ERK1/2) in cardiomyocytes subjected to anoxia-reoxygenation injury with and without APC and control. The cardiomyocytes from APC group were more resistant to injury induced by anoxia-reoxygenation and had much stronger phosphorylation of ERK1/2 than the control. The 14-3-3 protein expression was positively correlated with the phosphorylation of ERK1/2. Furthermore, inhibition of the ERK1/2 with PD98059 abolished the 14-3-3 protein up-regulation in cardiomyocytes induced by APC. The results indicate that APC up-regulates 14-3-3 protein expression through the ERK1/2 signaling pathways.
Life Sciences 08/2007; 81(5):372-9. · 2.53 Impact Factor
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ABSTRACT: The aim of this study was to investigate the cardioprotective effects and the possible mechanisms of delayed preconditioning induced by tetramethylpyrazine (TMP) in cultured neonatal rat cardiomyocytes subjected to anoxia-reoxygenation injury. Cultured neonatal rat cardiomyocytes were preconditioned using TMP at different concentrations (100, 200 and 500 microM). Cell viability, lactate dehydrogenase release, malondialdehyde formation, superoxide dismutase activity and glutathione peroxidase activity were measured to determine the protective effects against anoxia-reoxygenation injury. The expression of heat shock protein 70 (Hsp70) was measured 24 hr after TMP preconditioning by Western blot analysis. The results showed that TMP decreased lactate dehydrogenase release, increased cell viability, suppressed malondialdehyde formation and augmented activities of superoxide dismutase and glutathione peroxidase in a concentration-dependent manner. Moreover, the delayed protection was abolished by pre-treating with either protein kinase C inhibitor chelerythrine chloride or PD98059, a selective inhibitor of extracellular signal-regulated protein kinase 1/2, respectively, and the expression of Hsp70 was significantly increased in 24 hr after TMP preconditioning that was also suppressed by chelerythrine chloride or PD98059. These results suggest that TMP can induce delayed cardioprotective effects by activation of protein kinase C and extracellular signal-regulated protein kinase 1/2 signalling pathways and subsequent increased expression of Hsp70 in rat neonatal cardiomyocytes.
Basic & Clinical Pharmacology & Toxicology 07/2007; 100(6):366-71. · 2.18 Impact Factor
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Zhonghua gan zang bing za zhi = Zhonghua ganzangbing zazhi = Chinese journal of hepatology 04/2006; 14(3):230-2.
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ABSTRACT: This is a study on the allele composing of ABO, FUT1 and FUT2 gene loci of 10 para-Bombay individuals in China.
Ten samples coming from different districts of China were suspected of para-Bombay phenotype by primary serology tests. Routine and absorb-elution tests were conducted to identify their ABO type, and duplex polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was applied to getting their ABO genotype. Most of them were submitted to a test of their Lewis type as well. Then through direct DNA sequencing with PCR products of FUT1 and FUT2 genes, the genotypes of their H and SE gene loci were analyzed.
It can be confirmed that the 10 samples are para-Bombay. All of their ABO genotypes are consistent with the serological absorb-elution results and the substances detected results in saliva. Seven out of 10 have recessive homozygous gene at their H locus. Each phenotype of h1h1 (nt547-552Deltaag), h2h2 (nt880-882Deltatt) and h4h4 (nt35 t-->c) are ascertained in 2 individuals; moreover, h3h3 (nt 658 c-->t) is identified in one individual. The rest are hh heterozygous individuals: one is h3/h(new-1); the other is h2/h(new-2); the last one is h1/h2. The h(new-1) (nt586 c-->t) allele has a point mutation at nt 586 C to T, which leads a nonsense mutation Gln(CAG) to stop (TAG).The second h (new-2) (nt328 g-->a) has an nt328 G to A missense mutation,which leads Ala (GCC),was replaced by Thr (ACC) at 110 amino acid position. All the 10 samples have Se (nt357 c-->t) synonymous mutation. One Bm(h) (B/O) individual with h4h4 phenotype has a Se(w)(nt357 c-->t; nt385 a-->t) allele, whose Lewis type is Le(a+b+). Moreover, the authors detected a (nt716 g-->a) mutation in two samples' Se gene.
Four kinds of known h alleles (h1-h4), 2 kinds of novel non-functional FUT1 alleles, a Se(w) allele, and a novel SeG716A polymorphism in Chinese para-Bombay individuals were detected. At the same time, the authors noticed that all the 10 samples have the nt357 c-->t mutation in their FUT2 gene.
Zhonghua yi xue yi chuan xue za zhi = Zhonghua yixue yichuanxue zazhi = Chinese journal of medical genetics 11/2004; 21(5):417-21.
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ABSTRACT: This is a study on some ABO subgroup samples which show discordant results of serological and molecular blood typing, the aim is to clarify their true ABO type by means of nucleotide analysis on exons 6 and 7 of their ABO gene.
Absorb-elution test and family investigation were conducted to study 7 samples which were involved in ABO grouping discrepancies. Duplex polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP) method was used to identify their ABO genotypes. PCR products of exons 6 and 7 were cloned and sequenced.
All the 7 ABO subgroup samples with the discordant results of serological and molecular blood typing were found to have the normal O gene. Four out of them were typed as ABsub by serology, they were all of the A*102/O genotype. Sequencing analysis found all their A gene having the nt467 (C-->T) and nt803 (G-->C) mutation by comparison with the A*101 allele, i.e. their real type should be CisAB/O. Three out of 7 were typed as AsubB by serology and as BO by genotype; and point mutation was detected in all of their B gene. One of them had the nt700 (C-->G) mutation, the other 2 unrelated individuals had the novel nt640 (A-->G) mutation in their B alleles.
Through nucleotide analysis, 7 samples have been typed as AB subgroup in serology with the normal O gene, their real ABO type being CisAB in 4 cases and B(A) in 3 cases. At the same time, a kind of novel B (A)640 allele has been uncovered in this study.
Zhonghua yi xue yi chuan xue za zhi = Zhonghua yixue yichuanxue zazhi = Chinese journal of medical genetics 08/2004; 21(4):321-4.
