NAD(P)H:醌氧化还原酶1对多巴胺能细胞的保护作用

目的: 研究多巴胺(DA)对多巴胺能细胞的毒性作用、NAD(P)H:醌氧化还原酶1(NQO1)的保护作用及其机制。方法: 运用MTT法检测多巴胺对神经母细胞瘤细胞(SH-SY5Y)活性的影响;运用脂质体转染或Ⅱ相酶诱导剂预处理的方法对细胞进行预处理,并用免疫荧光方法检测转染效率;运用蛋白质印迹(Western blotting)方法检测细胞经过不同处理后胞内NQO1蛋白的表达情况;运用醌蛋白检测方法(硝基四氮唑蓝/甘氨酸法)检测细胞经不同处理后胞内醌化蛋白含量的变化。结果: 多巴胺对细胞具有剂量依赖性毒性,且与细胞内醌化蛋白的含量相关;脂质体转染和Ⅱ相酶诱导剂萝卜硫素(SF)均能使细胞内NQO1表达量增高;NQO1高表达能缓解多巴胺对细胞造成的毒性;细胞内NQO1表达量增高能减少多巴胺引起的细胞内醌化蛋白含量。结论: 细胞内NQO1的过表达可以减轻多巴胺对细胞产生的毒性作用。     

Regional NAD(P)H:quinone oxidoreductase activity in Alzheimer's disease

Converging evidence supports the role of oxidative stress in the pathology of Alzheimer's disease (AD). This notion is further supported by recent findings of increased NAD(P)H:quinone oxidodreductase (NQO1) activity, a potent antioxidant system, in association with hippocampal AD pathology. If increased NQO1 activity is truly related to the AD process, however, we would expect to see regional co-localization of NQO1 activity with AD pathology throughout affected brain regions and the absence of NQO1 activity in regions unaffected by AD. We examined this hypothesis by measuring NQO1 enzymatic activity and NQO1 immunohistochemical staining in regions commonly affected by the AD process such as frontal cortex and compared this to regions generally unaffected by the AD process such as occipital cortex, cerebellum, and substantia nigra for a group of AD patients and controls. The ratio of frontal to cerebellar NQO1 enzymatic activity was significantly increased in patients with AD (2.07 +/- 1.90) versus controls (0.60 +/- 0.31; P < 0.03). Moreover, regional immunohistochemical staining revealed specific localization of NQO1 staining to astrocytes and neurites surrounding senile plaques. The extent of immunohistochemical staining also closely correlated with the extent of local AD pathology across the various brain regions examined. Neuronal NQO1 staining seen in frontal cortex of AD patients was absent in frontal cortex of controls, but was found to the same extent in neurons of the substantia nigra of both AD patients and controls. We conclude that NQO1 activity co-localizes closely with AD pathology supporting a presumed role as an antioxidant system upregulated in response to the oxidative stress of the AD process. The antioxidant role for NQO1 is further supported by finding increased neuronal NQO1 activity in substantia nigra neurons of both AD patients and controls as this neuronal population is known to be under constant oxidative stress. While requiring further study, these findings, in conjunction with previous work, suggest that increased NQO1 activity may be neuroprotective, may offer novel insights into the pathophysiology of AD and may also provide possible avenues for future treatment.     

食管癌发病风险与NAD(P)H:醌氧化还原酶1C609T基因多态性

目的 研究NAD(P)H：醌氧化还原酶l[NAD(P)H：quinone oxidoreductase l，NQO1]C609T基因多态性与食管鳞状上皮癌(esophageal squamous cell carcinoma，ESCC)发病风险的关系。方法 应用聚合酶链反应-限制性片段长度多态性方法检测193例ESCC患者及141名正常对照的NQOl C609T多态性位点的基因型。结果 ESCC患者的突变型(T)等位基因频率明显高于健康对照组(X^2=4．86，P=0．028)。ESCC患者的NQO1C／C和C／T基因型频率与健康对照组相比差异无显著性(X^2值分别为2．27和0．127；P值分别为0．132和0．721)，而ESCC患者的T／T基因型频率明显高于对照组(X^2=4．39，P=0．036)。与NQOlC／C及C／T基因型相比，T／T基因型可明显增加患ESCC的风险性(校正OR=1．8l，95％CI：1．04～3．15)，且在有上消化道肿瘤家族史的患者中尤为明显(校正OR=2．22，95％CI：1．18～4．17)。结论 对NQO1 C609T多态性位点的基因型检测可能对判断ESCC高危个体具有指导意义。     

单胺氧化酶B和NAD(P)H醌氧化还原酶基因多态性与帕金森病的关系

目的 探讨参与多巴胺代谢的单胺氧化酶B（monoamine oxidase B,MAO-B)内含子13A／G多态性,NAD（P）H醌氧化还原酶基因[NAD(P)H:quinone oxidoreductase,NQO1]cDNA609C/T多态性与帕金森病（Parkinson's disease,PD)遗传易感性的关系。方法 应用等位基因特异PCR扩增分析MAO－B基因的多态性,用PCR－RFLP分析NQO1基因多态性。结果 MAO－BA／G等位基因频率、各基因型频率PD组与对照组差异无显著性。NQO1基因T等位基因频率PD组（52％）高于正常对照组（43％）带T碱基的NQO1基因型频率PD组显著高于正常对照组（P＜0．05）,其患PD的相对危险度（odds ratio,OR)为3．8。在带有A等位基因MAO－B基因型的个体,带有T碱基因的T等位基因是PD的危险因素。MAO－B基因的A等位基因型与NQO1基因的T等位基因的基因型可相互协同,增加PD发生的风险。     

Expression of NAD(P)H:quinone oxidoreductase in the normal and Parkinsonian substantia nigra

Dopamine (DA) autooxidation, and consequent formation of neurotoxic DA-derived quinones and reactive oxygen species, has been implicated in dopaminergic cell death and, hence, in the pathogenesis of Parkinson's disease (PD). Stimulation of pathways involved in the detoxication of DA-quinones in the brain is hypothesized to be an effective means to limit oxidative stress and to confer neuroprotection in PD. In this respect, the inducible flavoprotein NAD(P)H:quinone oxidoreductase (NQO1) is of particular interest as it is directly implicated in the detoxication of DA-quinones and, in addition, has broad spectrum anti-oxidant properties. To study the potential pathophysiological role of NQO1 in PD, the cellular expression of NQO1 was examined in the mesencephalon of PD patients and age-matched controls. In the substantia nigra pars compacta (SNpc), NQO1 was found to be expressed in astroglial and endothelial cells and, albeit less frequently, also in dopaminergic neurons. Moreover, while overt NQO1 immunoreactivity was absent in the surrounding nervous tissue, in the Parkinsonian SNpc a marked increase in the astroglial and neuronal expression of NQO1 was consistently observed.     

Acrolein induces heme oxygenase-1 through PKC-delta and PI3K in human bronchial epithelial cells

Heme oxygenase-1 (HO-1) catalyzes the rate limiting reaction of heme metabolism and plays critical roles in resistance to oxidative stress and other cellular functions. It is well known that HO-1 is induced in response to various stresses; however, the signaling pathways involved remain incompletely elucidated. Acrolein is an alpha,beta-unsaturated aldehyde present in cigarette smoke and also a product of lipid peroxidation. In this investigation we studied HO-1 induction in response to acrolein and determined the signaling pathways involved in human bronchial epithelial cells (HBE1 cells). We demonstrated that acrolein significantly increased the HO-1 mRNA content and promoter activity. Acrolein-mediated HO-1 induction was significantly attenuated by pan-protein kinase C (PKC) inhibitors RO318220, staurosporine, and PKC-delta selective inhibitor rottlerin and PKC-delta small interfering RNA. The HO-1 induction was also decreased by phosphatidylinositol 3-kinase (PI3K) inhibitors LY294002 and wortmannin. No significant effects on HO-1 induction were observed with the pretreatment of mitogen-activated protein kinase pathway inhibitors PD98059 (ERK), SB203580 (p38MAPK) and JNKi, and conventional and atypical PKC inhibitors. Furthermore, Nrf2 silencing significantly attenuated the HO-1 induction by acrolein. Inhibition of PKC-delta significantly decreased acrolein-mediated Nrf2 nuclear translocation, though inhibition of PI3K had no effect. Taken together, our results indicate that acrolein up-regulates HO-1 expression through both PKC-delta and PI3K pathways in HBE1 cells; PKC-delta appears to regulate HO-1 induction via modulating Nrf2 nuclear translocation, while PI3K may work through targeting on downstream signaling molecules other than Nrf2.     

Association analysis of NAD(P)H:quinone oxidoreductase gene 609 C/T polymorphism with Alzheimer's disease

Alterations of the NAD(P)H:quinone oxidoreductase (NQO1) activity are associated with Alzheimer's disease (AD). A polymorphism consisting of a single nucleotide (C-->T) change at position 609 of NQO1 influences the NQO1 activity. Therefore the NQO1 C609T polymorphism may confer susceptibility for AD developing. To test the hypothesis, we have performed an association study between the NQO1 gene polymorphism C609T and late-onset Alzheimer's disease (LOAD) in Chinese population. Totally 104 LOAD patients and 128 controls were enrolled in our data set. All subjects were genotyped for NQO1 and Apolipoprotein E (APOE). There were no significant differences in NQO1 genotype or allele frequencies between cases and controls. Likewise, with the stratification of APOE psilon4 status, no statistical difference was observed between cases and controls. Our findings suggested that this polymorphism might not represent additional genetic risk factor for LOAD. However, the present study cannot exclude NQO1 as a possible candidate for LOAD. Further study in a larger population and biological functional analysis of NQO1 gene is required to verify the role of NQO1 in LOAD.     

NAD(P)H:quinone oxidoreductase activity is increased in hippocampal pyramidal neurons of patients with Aalzheimer's disease

NAD(P)H:quinone oxidoreductase (QR) catalyzes the two-electron reduction of quinones, preventing their participation in redox cycling and subsequent generation of reactive oxygen species. Pretreatment of neuroblastoma cells with compounds, such as tert-butylhydroquinone and dimethyl fumarate, that increase QR expression protect cells from oxidative stress-induced cell death by glutamate, H(2)O(2,) and dopamine. The potential neuroprotective role of QR as well as the evidence for oxidative stress-induced neuronal cell death in Alzheimer's disease (AD) led us to examine the expression pattern of QR from AD and control patients. Histochemical staining of hippocampal sections from AD patients revealed QR activity in pyramidal neurons. The presence of QR protein in these neurons also was confirmed by immunoreactivity. In control patients, hippocampal pyramidal neurons were negative for both QR enzymatic activity and QR immunoreactivity. In addition, the QR positive neurons of AD patients were selectively located in areas where neuronal populations exhibited tau immunostaining. Our data demonstrate that QR is up-regulated in hippocampal pyramidal neurons of AD patients. We hypothesize that this is part of a neuroprotective system up-regulated in response to the AD process. Understanding this system may lead to further insights into the pathogenesis and potential new avenues of treatment for AD.     

中国人NAD(P)H:醌氧化还原酶基因多态性与帕金森病遗传易感性的关系

目的　探讨依赖还原型辅酶 / 醌氧化还原酶 [NAD(P) H:quinone oxidoreductase,NQO1]c DNA6 0 9位点 C→ T多态性与帕金森病 (Parkinson'sdisease,PD)遗传易感性的关系。方法　用聚合酶链反应 -变性高效液相技术 (polymerase chain reaction- denaturing high performance liquid chromatog-raphy,PCR- DHPL C)分析了 NQO1基因c DNA6 0 9位点C→T多态性在 PD患者与正常对照之间分布频率的差异。结果　PD组和对照组的 TT基因型频率分别为 2 2 .6 %和 11.8% (P=0 .0 0 4 ) ,TT基因型使患 PD的危险度提高 2 .186倍 (P=0 .0 0 5 ) ;根据发病年龄分组后 ,这种差异主要存在于晚发性 PD和对照组之间 ,TT基因型使患 PD的危险度提高 2 .6 2 7倍 (P=0 .0 0 1)。等位基因在总体 PD组、早发 PD组、晚发 PD组和对照组中的频率分布差异无显著性。结论　NQO1基因c DNA6 0 9位点C→T多态性在对照组和PD患者之间的分布差异有显著性 ,突变基因型 (TT基因型 )频率在 PD组中较高 ,研究结果支持 NQO1基因多态性与 PD相关的假说 ,而且与 PD发病年龄有关。     

The NAD(P)H:quinone oxidoreductase I C609T polymorphism modifies the risk of Barrett esophagus and esophageal adenocarcinoma.

PURPOSE: The role of genetic susceptibility to esophageal adenocarcinoma and its precursor lesion Barrett esophagus has not been fully elucidated. This study investigated the effect of polymorphisms in the manganese superoxide dismutase (MnSOD) and NAD(P)H:quinone oxidoreductase 1 (NQO1) genes in modulating the risk of developing Barrett esophagus or esophageal adenocarcinoma. METHODS: A total of 584 patients (146 esophagitis, 200 Barrett esophagus, 144 esophageal adenocarcinoma, and 94 controls) were genotyped for the MnSOD C14T and NQO1 C609T polymorphisms using polymerase chain reaction and restriction fragment length polymorphism analysis. RESULTS: The NQO1 TT genotype was less common in Barrett esophagus (2.0%) and esophageal adenocarcinoma (1.4%) patients, compared with both esophagitis patients (7.6%) and controls (5.4%). After adjustment for sex, age, body mass index, reflux symptoms, and smoking status, patients with the homozygous TT genotype had a 4.5-fold decreased risk of developing Barrett esophagus (odds ratio = 0.22, 95% confidence interval = 0.07-0.76, P = 0.01) and a 6.2-fold decreased risk of esophageal adenocarcinoma (odds ratio = 0.16, 95% confidence intervals = 0.03-0.94, P = 0.04) compared with individuals with the TC and CC genotypes. No significant differences between groups were observed for the MnSOD polymorphism (P = 0.289). CONCLUSIONS: Overall, the results of this study suggest that the NQO1 TT genotype may offer protection from reflux complications such as Barrett esophagus and esophageal adenocarcinoma.     

NAD(P)H oxidase-derived reactive oxygen species as mediators of angiotensin II signaling

Angiotensin II has been shown to participate in both physiological processes, such as sodium and water homeostasis and vascular contraction, and pathophysiological processes, including atherosclerosis and hypertension. The effects of this molecule on vascular tissue are mediated at least in part by the modification of the redox milieu of its target cells. Angiotensin II has been shown to activate the vascular NAD(P)H oxidase(s) resulting in the production of reactive oxygen species, namely superoxide and hydrogen peroxide. In this article, we review what is known about the molecular steps that link angiotensin II and its receptor to production of reactive oxygen species and subsequent redox-mediated events, focusing on the structural and functional properties of the vascular NAD(P)H oxidases and their downstream mediators. As such, we provide a framework linking angiotensin II to crucial vascular pathologies, such as hypertension, atherosclerosis, and restenosis after angioplasty, by means of the NAD(P)H-dependent oxidases and their effector molecules.     

Upregulation of heme oxygenase-1 via PI3K/Akt and Nrf-2 signaling pathways mediates the anti-inflammatory activity of Schisandrin in Porphyromonas gingivalis LPS-stimulated macrophages

The lipopolysaccharide (LPS) of Porphyromonas gingivalis is thought to induce periodontitis. In this study, we isolated Schisandrin from the dried fruits of Schisandra chinensis and examined the anti-inflammatory effect of Schisandrin in macrophages stimulated with LPS from P. gingivalis. First, Schisandrin inhibited LPS-induced pro-inflammatory cytokines, including TNF-α, IL-1β, and IL-6. And Schisandrin suppressed the nuclear translocation and activity of NF-κB and phosphorylation of IκBα in LPS-stimulated RAW 264.7 cells. Next, the presence of a selective inhibitor of HO-1 (SnPP) and a siRNA specific for HO-1 inhibited Schisandrin-mediated anti-inflammatory activity. Furthermore, Schisandrin induced HO-1 expression of RAW 264.7 cells through Nrf-2, PI3K/Akt, and ERK activation. Therefore, these results suggest that the anti-inflammatory effects of Schisandrin on P. gingivalis LPS-stimulated RAW 264.7 cells may be due to a reduction of NF-κB activity and induction of the expression of HO-1, leading to TNF-α, IL-1β, and IL-6 down-regulation.     

High throughput genotyping for the detection of a single nucleotide polymorphism in NAD(P)H quinone oxidoreductase (DT diaphorase) using TaqMan probes.

AIMS: The two electron reduction of quinones to hydroquinones by NAD(P)H quinone oxidoreductase (NQO1) plays an important role in both activation and detoxification of quinone and similarly reactive compounds. A single nucleotide polymorphism at exon 6 leads to an amino acid change at codon 187 from proline to serine. The variant allele has been associated with decreased NQO1 enzyme activity and increased cancer risks. The aim of this study was to develop a rapid genotyping procedure for epidemiological and clinical research into the potential biological and toxicological implications associated with this genetic polymorphism. METHODS: A high throughput genotyping method using fluorogenic probes has been developed to screen this single nucleotide polymorphism. This assay utilises the 5' nuclease activity of Taq polymerase in conjunction with fluorogenic TaqMan probes. The TaqMan genotyping procedure was validated by a restriction fragment length polymorphism method and direct sequencing. RESULTS: This method can be used for the rapid screening of known polymorphisms in large populations. In a population of 143 unrelated individuals, Pro/Pro (wildtype), Pro/Ser (heterozygous), and Ser/Ser (mutant) genotypes were 69.2%, 26.6%, and 4.2%, respectively. CONCLUSIONS: This genotyping method is highly accurate and could be applied to automated large scale genotyping studies.     

Redox regulation of p38 MAPK activation and expression of ICAM-1 and heme oxygenase-1 in human alveolar epithelial (A549) cells

We have explored the potential role of redox events in p38 mitogen-activated protein kinase (MAPK) activation and their relevance to the inducible expression of intercellular adhesion molecule-1 (ICAM-1) and heme oxygenase-1 (HO-1) in A549 cells. Tumor necrosis factor-alpha (TNFalpha) and hydrogen peroxide (H2O2) both activated p38, but only TNFalpha activated nuclear factor-kappaB (NF-kappaB). N-Acetyl-L-cysteine (20 mM) inhibited both H2O2- and TNFalpha-induced p38 phosphorylation (14 +/- 7 and 37 +/- 4% of control, respectively). The mitochondrial complex I and III inhibitors, rotenone and antimycin A, and allopurinol partially inhibited H2O2- but not TNFalpha-induced p38 activation. However, rotenone and antimycin A augmented intracellular oxidative stress measured by dichlorofluorescein fluorescence. TNFalpha, but not H2O2, induced ICAM-1 in A549 cells, which was attenuated by a proteasome inhibitor, but not by the p38 MAPK inhibitor SB203580. In contrast, hemin and hemoglobin, but neither TNFalpha nor H2O2, caused efficient HO-1 expression. However, hemin had no effect on p38 activation and SB203580 did not influence hemin-induced HO-1 protein expression. Collectively, these data suggest that p38 is a cytokine- and oxidative stress-responsive pathway in A549 cells. Whereas NF-kappaB appears crucial in ICAM-1 induction, p38 activation itself is not sufficient to confer HO-1 expression and may not be involved in HO-1 and ICAM-1 induction in A549 cells.     

Nodosin通过Apaf-1和caspase-3诱导HepG2细胞凋亡

目的:研究传统中药提取物nodosin对人肝细胞癌Hep G2细胞凋亡的影响,并探讨其作用机制。方法:将nodosin设置为1. 25μmol/L、2. 5μmol/L、5μmol/L、10μmol/L和20μmol/L不同浓度组,作用于Hep G2细胞24 h后,用Hoechst 33258染色和电镜观察不同浓度的药物对细胞形态学的影响,用流式细胞术检测细胞凋亡率,用RT-qPCR检测凋亡蛋白酶激活因子1 (Apaf-1) mRNA的表达,用Western blot检测caspase-3及其前体和活化体的蛋白水平。结果:形态学结果显示,随着用药剂量的增加,细胞皱缩和细胞核偏移越明显,凋亡小体在5μmol/L、10μmol/L和20μmol/L剂量组明显增多。Apaf-1 mRNA的表达增加,caspase-3及其前体的表达和cleaved caspase-3的蛋白水平随着用药剂量的增加逐渐增加(P 0. 01)。结论:Nodosin能够诱导Hep G2细胞凋亡。该作用可能是通过增加Apaf-1 mRNA的表达继之激活caspase-3实现的。     

DHA通过抑制PI3K通路和GLUT2表达而诱导NSCLC细胞毒性

目的:研究二氢青蒿素(dihydroartemisinin,DHA)诱导NSCLC细胞毒性的分子机制。方法:将不同浓度的DHA作用NSCLC细胞系A549和NCI-H1650细胞不同时间,运用MTT法、克隆形成实验、Annexin V染色和流式细胞术测定DHA对细胞活力、克隆形成能力和细胞凋亡的影响;同时测定DHA对细胞中葡萄糖水平、ATP和乳酸含量的影响;Western blot检测PI3K通路活性和GLUT2表达变化;通过细胞转染实现葡萄糖转运体2(GLUT2)和Rheb在A549和NCI-H1650细胞中高表达,测定和分析DHA作用下细胞活力、细胞凋亡、葡萄糖水平、ATP含量和PI3K通路活性的变化;分析葡萄糖缺乏对DHA诱导NSCLC细胞毒性的影响。结果:与对照组比较,DHA显著抑制A549和NCI-H1650细胞活力和克隆形成能力以及诱导细胞凋亡,同时降低ATP和乳酸含量以及抑制细胞对葡萄糖的摄取,具有时间和剂量依赖效应。Western blot结果显示,DHA能抑制PI3K通路活性和GLUT2的表达。上调GLUT2的表达和激活PI3K通路能减弱DHA对NSCLC细胞的毒性作用;葡萄糖缺乏能增强DHA对NSCLC细胞的毒性;相反,高浓度葡萄糖则抑制DHA对NSCLC细胞的毒性。结论:DHA能抑制NSCLC细胞活力和克隆形成,诱导细胞凋亡,该作用是通过降低PI3K活性和GLUT2的表达进而抑制细胞糖酵解代谢实现的。     

Alkaline ceramidase 3 promotes growth of hepatocellular carcinoma cells via regulating S1P/S1PR2/PI3K/AKT signaling

Objective

Hepatocellular carcinoma (HCC) is one of the cancer types with poor prognosis. To effectively treat HCC, new molecular targets and therapeutic approaches must be identified. Alkaline ceramidase 3 (Acer3) hydrolyzed long-chain unsaturated ceramide to produce free fatty acids and sphingosine. However, whether and how Acer3 modulates progression of HCC remains largely unknown.

The human carcinogen aristolochic acid i is activated to form DNA adducts by human NAD(P)H:quinone oxidoreductase without the contribution of acetyltransferases or sulfotransferases

Ingestion of aristolochic acid (AA) is associated with development of urothelial tumors linked with AA nephropathy and is implicated in the development of Balkan endemic nephropathy-associated urothelial tumors. We investigated the efficiency of human NAD(P)H:quinone oxidoreductase (NQO1) to activate aristolochic acid I (AAI) and used in silico docking, using soft-soft (flexible) docking procedure, to study the interactions of AAI with the active site of human NQO1. AAI binds to the active site of NQO1 indicating that the binding orientation allows for direct hydride transfer (i.e., two electron reductions) to the nitro group of AAI. NQO1 activated AAI, generating DNA adduct patterns reproducing those found in urothelial tissues from humans exposed to AA. Because reduced aromatic nitro-compounds are often further activated by sulfotransferases (SULTs) or N,O-acetlytransferases (NATs), their roles in AAI activation were investigated. Our results indicate that phase II reactions do not play a major role in AAI bioactivation; neither native enzymes present in human hepatic or renal cytosols nor human SULT1A1, -1A2, -1A3, -1E, or -2A nor NAT1 or NAT2 further enhanced DNA adduct formation by AAI. Instead under the in vitro conditions used, DNA adducts arise by enzymatic reduction of AAI through the formation of a cyclic hydroxamic acid (N-hydroxyaristolactam I) favored by the carboxy group in peri position to the nitro group without additional conjugation. These results emphasize the major importance of NQO1 in the metabolic activation of AAI and provide the first evidence that initial nitroreduction is the rate limiting step in AAI activation.     

PI3K/Akt signaling pathway-induced heme oxygenase-1 upregulation mediates the adaptive cytoprotection of hydrogen peroxide preconditioning against oxidative injury in PC12 cells

Both the phosphatidylinositol 3-kinase (PI3K)/Akt pathway and heme oxygenase-1 (HO-1) create a survival signal against oxidative stress-induced injuries. Although we have demonstrated that hydrogen peroxide (H2O2) preconditioning confers adaptive cytoprotection against oxidative stress-induced injury in PC12 cells, it remains unknown whether these defense systems are involved in the protective effect of H2O2 preconditioning. In the current study, PC12 cells were preconditioned with 100 μM H2O2 for 90 min, followed by 24 h recovery and subsequent exposure to 300 μM H2O2 for further 12 h. The findings showed that preconditioning with 100 μM H2O2 upregulated HO-1 expression. Zinc protoporphyrin IX (ZnPP), a selective inhibitor of HO-1, at a concentration of 15 μM, significantly attenuated H2O2 preconditioning-elicited cytotoxicity, apoptosis, oxidative stress and mitochondrial membrane potential (ΔΨm) loss in PC12 cells. In addition, H2O2 preconditioning enhanced phosphorylation of Akt. Treatment with 25 μM LY294002, a selective inhibitor of PI3K, for 20 min before H2O2 preconditioning blocked not only H2O2 preconditioning-induced HO-1 induction, but also the protective effect of H2O2 preconditioning against cytotoxicity. The present study provides novel evidence for the effect of preconditioning with H2O2 on the induction of HO-1, which contributes to the adaptive cytoprotection of H2O2 preconditioning against oxidative stress-induced cellular injury via a PI3K/Akt-dependent mechanism in PC12 cells.