Lack of alpha-1-adrenergic receptor-mediated downregulation of angiotensin II receptors in neuronal cultures from spontaneously hypertensive rat brain

Neuronal cells from Wistar Kyoto (WKY) and spontaneously hypertensive (SH) rat brains were established in culture to compare the expression of angiotensin II (Ang II) specific receptors and their regulation by norepinephrine (NE). Neurons from SH rat brains possess twice more Ang II specific receptors and expressed a proportional increase in Ang II stimulated [³H]-NE uptake compared with WKY neurons. NE caused a dose-dependent decrease in¹²⁵I-Ang II binding in WKY neurons, an effect not observed when neurons from SH rat brains were incubated with NE. These observations suggest that the lack of NE-induced downregulation of Ang II receptors in neuronal cultures is genetically regulated.     

Effects of angiotensin II on the spontaneous activity of rostral ventrolateral medullary cardiovascular neurons and blood pressure in spontaneously hypertensive rats

The interactive role of rostral ventrolateral medulla (RVL) cardiovascular neurons and brain angiotensin II (Ang II) in regulating the arterial blood pressure was examined by recording simultaneously the spontaneous activity of these spinal projecting neurons and the arterial blood pressure in the pentobarbital-anesthetized spontaneously hypertensive rat (SHR) and its normotensive control, the Wistar Kyoto rat (WKY). It was found that Ang II elicited dose-dependent excitatory responses in a subpopulation of RVL cardiovascular neurons, followed by a subsequent increase in blood pressure. These effects of Ang II were significantly greater in SHR than in WKY. The effects were attenuated or abolished by co-administration of Ang II antagonist, [Sar¹, Ile⁸]-Ang II. Pre-administration of [Sar¹, Ile⁸]-Ang II to RVL using bilateral microinjection attenuated the blood pressure effects of intracerebroventricularly administered Ang II by as much as 70%. These results indicated that spinal projecting RVL cardiovascular neurons are important in mediating the pressor action of Ang II. The enhanced sensitivity and responsiveness of RVL cardiovascular neurons to Ang II may be pertinent to the genesis of hypertension in adult SHR.     

Protein Kinase C Agonists Increase the Expression of Angiotensin II Receptors in Neuronal Cultures

Previous studies have shown that norepinephrine is important in the regulation of central angiotensin II receptors, an effect mediated by alpha 1-adrenergic receptors. Because alpha 1-adrenergic stimulation leads to inositol phospholipid hydrolysis and activation of protein kinase C, we have examined a possible role of this enzyme in the regulation of central angiotensin II (Ang II) receptors. In the present study, we have examined the effects of protein kinase C activators, phorbol esters, on the expression of Ang II receptors in neuronal cultures prepared from 1-day-old rat brains. The active phorbol ester phorbol-12-myristate-13-acetate (TPA) caused time- and concentration-dependent increases in the specific binding of [125I]Ang II to its receptors in neuronal cultures of normotensive and spontaneously hypertensive rat brains. The stimulatory effect of TPA on Ang II receptors was apparent within 15 min and reached a maximum between 1 and 2 h. Ang II specific binding had returned to control levels by 24 h. Various phorbol esters increased [125I]Ang II binding in accordance with their order of potency in stimulating protein kinase C activity. Saturation and Scatchard analysis revealed that the phorbol ester-induced increase in [125I]Ang II binding was due to an increase in the number of Ang II receptors. These observations indicate that activation of protein kinase C results in an increased expression of Ang II receptors in neuronal cultures from both normotensive and spontaneously hypertensive rat brains. The results suggest a possible role of phosphorylation in Ang II receptor expression in neuronal cultures.     

Oxidative Inactivation of Mitochondrial Aconitase Results in Iron and H2O2-Mediated Neurotoxicity in Rat Primary Mesencephalic Cultures

Background

Mitochondrial oxidative stress is a contributing factor in the etiology of numerous neuronal disorders. However, the precise mechanism(s) by which mitochondrial reactive oxygen species (ROS) modify cellular targets to induce the death of neurons remains unknown. The goal of this study was to determine if oxidative inactivation of mitochondrial aconitase (m-aconitase) resulted in the release of redox-active iron (Fe²⁺) and hydrogen peroxide (H₂O₂) and whether this contributes to cell death.

Methodology/Principal Findings

Incubation of rat primary mesencephalic cultures with the redox cycling herbicide paraquat (PQ²⁺) resulted in increased production of H₂O₂ and Fe²⁺ at times preceding cell death. To confirm the role of m-aconitase as a source of Fenton reagents and death, we overexpressed m-aconitase using an adenoviral construct thereby increasing the target available for inactivation by ROS. Co-labeling studies identified astrocytes as the predominant cell type expressing transduced m-aconitase although neurons were identified as the primary cell type dying. Oxidative inactivation of m-aconitase overexpressing cultures resulted in exacerbation of H₂O₂ production, Fe²⁺ accumulation and increased neuronal death. Increased cell death in m-aconitase overexpressing cultures was attenuated by addition of catalase and/or a cell permeable iron chelator suggesting that neuronal death occurred in part via astrocyte-derived H₂O₂.

Conclusions

These results suggest a role of ROS-sensitive m-aconitase as a source of Fe²⁺ and H₂O₂ and as a contributing factor to neurotoxicity.     

Icariside II,a novel phosphodiesterase 5 inhibitor,protects against H2O2‐induced PC12 cells death by inhibiting mitochondria‐mediated autophagy

Oxidative stress is a major cause of cellular injury in a variety of human diseases including neurodegenerative disorders. Thus, removal of excessive reactive oxygen species (ROS) or suppression of ROS generation may be effective in preventing oxidative stress‐induced cell death. This study was designed to investigate the effect of icariside II (ICS II), a novel phosphodiesterase 5 inhibitor, on hydrogen peroxide (H₂O₂)‐induced death of highly differentiated rat neuronal PC12 cells, and to further examine the underlying mechanisms. We found that ICS II pre‐treatment significantly abrogated H₂O₂‐induced PC12 cell death as demonstrated by the increase of the number of metabolically active cells and decrease of intracellular lactate dehydrogenase (LDH) release. Furthermore, ICS II inhibited H₂O₂‐induced cell death through attenuating intracellular ROS production, mitochondrial impairment, and activating glycogen synthase kinase‐3β (GSK‐3β) as demonstrated by reduced intracellular and mitochondrial ROS levels, restored mitochondrial membrane potential (MMP), decreased p‐tyr216‐GSK‐3β level and increased p‐ser9‐GSK‐3β level respectively. The GSK‐3β inhibitor SB216763 abrogated H₂O₂‐induced cell death. Moreover, ICS II significantly inhibited H₂O₂‐induced autophagy by the reducing autophagosomes number and the LC3‐II/LC3‐I ratio, down‐regulating Beclin‐1 expression, and up‐regulating p62/SQSTM1 and HSP60 expression. The autophagy inhibitor 3‐methyl adenine (3‐MA) blocked H₂O₂‐induced cell death. Altogether, this study demonstrated that ICS II may alleviate oxidative stress‐induced autophagy in PC12 cells, and the underlying mechanisms are related to its antioxidant activity functioning via ROS/GSK‐3β/mitochondrial signalling pathways.     

Evidence for a pro-apoptotic phenotype in skeletal muscle of hypertensive rats

In this report, we demonstrate that soleus muscle of spontaneously hypertensive rats (SHR) had significantly lower protein levels of apoptosis repressor with caspase recruitment domain (ARC) and X-linked inhibitor of apoptosis protein (XIAP) as well as significantly higher protein levels of second mitochondria-derived activator of caspase (Smac) and procaspase-8 compared to normotensive Wistar-Kyoto (WKY) rats. In addition, soleus muscle from hypertensive rats had significantly increased caspase-8 proteolytic enzyme activity as well as significantly elevated reactive oxygen species (ROS) generation and higher hydrogen peroxide (H₂O₂) content. There was no change in the protein levels of the antioxidant enzymes, catalase, copper-zinc superoxide dismutase (CuZnSOD), and manganese superoxide dismutase (MnSOD). Interestingly, ARC protein migrated at approximately 32 kDa in SHR but at 30 kDa in WKY rat muscle; possibly indicating a post-translational modification. These results demonstrate that soleus muscle of hypertensive rats display a pro-apoptotic phenotype and augmented ROS generation.     

Respiration-dependent H2O2 Removal in Brain Mitochondria via the Thioredoxin/Peroxiredoxin System

Mitochondrial reactive oxygen species (ROS) play an important role in both physiological cell signaling processes and numerous pathological states, including neurodegenerative disorders such as Parkinson disease. While mitochondria are considered the major cellular source of ROS, their role in ROS removal remains largely unknown. Using polarographic methods for real-time detection of steady-state H₂O₂ levels, we were able to quantitatively measure the contributions of potential systems toward H₂O₂ removal by brain mitochondria. Isolated rat brain mitochondria showed significant rates of exogenous H₂O₂ removal (9–12 nmol/min/mg of protein) in the presence of substrates, indicating a respiration-dependent process. Glutathione systems showed only minimal contributions: 25% decrease with glutathione reductase inhibition and no effect by glutathione peroxidase inhibition. In contrast, inhibitors of thioredoxin reductase, including auranofin and 1-chloro-2,4-dinitrobenzene, attenuated H₂O₂ removal rates in mitochondria by 80%. Furthermore, a 50% decrease in H₂O₂ removal was observed following oxidation of peroxiredoxin. Differential oxidation of glutathione or thioredoxin proteins by copper (II) or arsenite, respectively, provided further support for the thioredoxin/peroxiredoxin system as the major contributor to mitochondrial H₂O₂ removal. Inhibition of the thioredoxin system exacerbated mitochondrial H₂O₂ production by the redox cycling agent, paraquat. Additionally, decreases in H₂O₂ removal were observed in intact dopaminergic neurons with thioredoxin reductase inhibition, implicating this mechanism in whole cell systems. Therefore, in addition to their recognized role in ROS production, mitochondria also remove ROS. These findings implicate respiration- and thioredoxin-dependent ROS removal as a potentially important mitochondrial function that may contribute to physiological and pathological processes in the brain.     

Sonic hedgehog promotes neurite outgrowth of cortical neurons under oxidative stress: Involving of mitochondria and energy metabolism

Oxidative stress has been demonstrated to be involved in the etiology of several neurobiological disorders. Sonic hedgehog (Shh), a secreted glycoprotein factor, has been implicated in promoting several aspects of brain remodeling process. Mitochondria may play an important role in controlling fundamental processes in neuroplasticity. However, little evidence is available about the effect and the potential mechanism of Shh on neurite outgrowth in primary cortical neurons under oxidative stress. Here, we revealed that Shh treatment significantly increased the viability of cortical neurons in a dose-dependent manner, which was damaged by hydrogen peroxide (H₂O₂). Shh alleviated the apoptosis rate of H₂O₂-induced neurons. Shh also increased neuritogenesis injuried by H₂O₂ in primary cortical neurons. Moreover, Shh reduced the generation of reactive oxygen species (ROS), increased the activities of SOD and and decreased the productions of MDA. In addition, Shh protected mitochondrial functions, elevated the cellular ATP levels and amelioratesd the impairment of mitochondrial complex II activities of cortical neurons induced by H₂O₂. In conclusion, all these results suggest that Shh acts as a prosurvival factor playing an essential role to neurite outgrowth of cortical neuron under H₂O₂ -induced oxidative stress, possibly through counteracting ROS release and preventing mitochondrial dysfunction and ATP as well as mitochondrial complex II activities against oxidative stress.     

Analysis of Dichlorodihydrofluorescein and Dihydrocalcein as Probes for the Detection of Intracellular Reactive Oxygen Species

Dihydrocalcein (H₂-calcein) is recommended as a superior probe for intracellular radical (ROS) detection as different to dichlorodihydrofluorescein (H₂-DCF), its oxidation product calcein is thought not to leak out of cells. We determined whether H₂-calcein is a useful tool to measure ROS in vascular smooth muscle cells. In vitro, both compounds were oxidized by peroxynitrite, hydroxyl radicals and peroxidase, but not hydrogen peroxide or nitric oxide. The intracellular half-life of calcein was several hours whereas that of DCF was approximately 5?min. Intracellular ROS, as generated by the angiotensin II (Ang II)-activated NADPH oxidase, did not increase the oxidation of H₂-calcein but increased the oxidation of H₂-DCF by approximately 50%. Similar changes were detected using electron spin resonance spectroscopy. Inhibition of the NADPH oxidase using gp91ds-tat prevented the Ang II-induced increase in DCF fluorescence, without affecting cells loaded with H₂-calcein. Diphenylene iodonium (DPI), which inhibits all flavin-dependent enzymes, including those in the respiratory chain, had little effect on the basal but prevented the Ang II-induced oxidation of H₂-DCF. In contrast, DPI inhibited H₂-calcein oxidation in non-stimulated cells by almost 50%. Blockade of respiratory chain complex I inhibited H₂-calcein oxidation, whereas inhibitors of complex III were without effect. Calcein accumulated in the mitochondria, whereas DCF was localized in the cytoplasm. In submitochondrial particles, H₂-calcein, but not H₂-DCF inhibited complex I activity.

These observations indicate that H₂-DCF is an indicator for intracellular ROS, whereas the oxidation of H₂-calcein most likely occurs as a consequence of direct electron transfer to mitochondrial complex I.     

Nox4-dependent H2O2 production contributes to chronic glutamate toxicity in primary cortical neurons

Reactive oxygen species (ROS) can trigger neuronal cell death and has been implicated in a variety of neurodegenerative diseases as well as brain ischemia. Here, we demonstrate that chronic (but not acute) glutamate toxicity in primary cortical neuronal cultures is associated with hydrogen peroxide (H₂O₂) accumulation in the culture medium and that neurotoxicity can be eliminated by external catalase treatment. Neuronal cultures in Ca²⁺-free medium or treated with BAPTA showed reduced glutamate-induced H₂O₂ generation, indicating that H₂O₂ generation is Ca²⁺-dependent. Pharmacological and genetic approaches revealed that NADPH oxidase plays a role in glutamate-induced H₂O₂ generation and that activation of NMDA and AMPA receptors is involved in this H₂O₂ generation. The Nox4 siRNA reduced NMDA-induced H₂O₂ production by 54% and cytotoxicity in parallel, suggesting that Nox4-containing NADPH oxidase functions NMDA receptor-mediated H₂O₂ production resulting in neurotoxicity. These findings suggest that the modulation of NADPH oxidase can be used as a new therapeutic strategy for glutamate-induced neuronal diseases.     

Hydrogen peroxide-induced neuronal apoptosis is associated with inhibition of protein phosphatase 2A and 5, leading to activation of MAPK pathway

Oxidative stress-induced neuronal apoptosis is a prominent feature found in neurodegenerative disorders. However, how oxidative stress induces neuronal apoptosis is not well understood. To address this question, undifferentiated and differentiated neuronal cell lines (PC12 and SH-SY5Y) were exposed to hydrogen peroxide (H₂O₂), a major oxidant generated when oxidative stress occurs. We observed that H₂O₂ induced generation of reactive oxygen species (ROS), leading to apoptosis of the cells in a concentration- and time-dependent manner. H₂O₂ rapidly activated the mitogen-activated protein kinases (MAPK) including extracellular signal-regulated kinase 1/2 (Erk1/2), c-Jun N-terminal kinase (JNK) and p38. Inhibition of Erk1/2, JNK or p38 with kinase inhibitors (U0126, SP600125 or PD169316, respectively), downregulation of Erk1/2 or p38 using RNA interference, or expression of dominant negative c-Jun partially prevented H₂O₂-induced apoptosis. Pretreatment with N-acetyl-l-cysteine (NAC) scavenged H₂O₂-induced ROS, blocking activation of MAPKs and cell death. Furthermore, we found that H₂O₂-induced ROS inhibited serine/threonine protein phosphatases 2A (PP2A) and 5 (PP5), which was abrogated by NAC. Overexpression of PP2A or PP5 partially prevented H₂O₂-activation of Erk/12, JNK and p38, as well as cell death. Similar results were observed in primary murine neurons as well. The results suggest that H₂O₂-induction of ROS inhibit PP2A and PP5, leading to activation of Erk1/2, JNK and p38 pathways thereby resulting in neuronal apoptosis. Our findings suggest that inhibitors of MAPKs (JNK, Erk1/2 and p38), activators of phosphatases (PP2A and PP5) or antioxidants may have potentials to prevent and treat oxidative stress-induced neurodegenerative diseases.     

Loss of oxidative stress tolerance in hypertension is linked to reduced catalase activity and increased c-Jun NH2-terminal kinase activation

Hypertension is accompanied by increased levels of reactive oxygen species, which may contribute to progressive renal injury and dysfunction. Here we tested the hypothesis that sensitivity to exogenous hydrogen peroxide (H₂O₂) is enhanced in immortalized renal proximal tubular epithelial cells from spontaneously hypertensive rats (SHR) compared to normotensive control Wistar Kyoto rats (WKY). We found that SHR cells were more sensitive to H₂O₂-induced cell death than WKY cells. Lower survival in SHR cells correlated with increased DNA fragmentation, chromatin condensation, and caspase-3 activity, indicating apoptosis. H₂O₂ degradation was slower in SHR than in WKY cells, suggesting that reduced antioxidant enzyme activity might be the basis for their increased sensitivity. In fact, catalase activity was downregulated in SHR cells, whereas glutathione peroxidase activity was similar in both cell types. We next examined whether MAPK signaling pathways contributed to H₂O₂-mediated apoptosis. Inhibition of c-Jun NH₂-terminal kinase (JNK) with SP600125 partially rescued H₂O₂-induced apoptosis in WKY but not in SHR cells. In addition, p54 JNK2 isoform was robustly phosphorylated by H₂O₂, this effect being more pronounced in SHR cells. Together, these results suggest that the survival disadvantage of SHR cells upon exposure to H₂O₂ stems from impaired antioxidant mechanisms and activated JNK proapoptotic signaling pathways.     

AMPK-mediated increase of glycolysis as an adaptive response to oxidative stress in human cells: Implication of the cell survival in mitochondrial diseases

We report that the energy metabolism shifts to anaerobic glycolysis as an adaptive response to oxidative stress in the primary cultures of skin fibroblasts from patients with MERRF syndrome. In order to unravel the molecular mechanism involved in the alteration of energy metabolism under oxidative stress, we treated normal human skin fibroblasts (CCD-966SK cells) with sub-lethal doses of H₂O₂. The results showed that several glycolytic enzymes including hexokinase type II (HK II), lactate dehydrogenase (LDH) and glucose transporter 1 (GLUT1) were up-regulated in H₂O₂-treated normal skin fibroblasts. In addition, the glycolytic flux of skin fibroblasts was increased by H₂O₂ in a dose-dependent manner through the activation of AMP-activated protein kinase (AMPK) and phosphorylation of its downstream target, phosphofructokinase 2 (PFK2). Moreover, we found that the AMPK-mediated increase of glycolytic flux by H₂O₂ was accompanied by an increase of intracellular NADPH content. By treatment of the cells with glycolysis inhibitors, an AMPK inhibitor or genetic knockdown of AMPK, respectively, the H₂O₂-induced increase of NADPH was abrogated leading to the overproduction of intracellular ROS and cell death. Significantly, we showed that phosphorylation levels of AMPK and glycolysis were up-regulated to confer an advantage of survival for MERRF skin fibroblasts. Taken together, our findings suggest that the increased production of NADPH by AMPK-mediated increase of the glycolytic flux contributes to the adaptation of MERRF skin fibroblasts and H₂O₂-treated normal skin fibroblasts to oxidative stress.     

ROS Produced by NOX2 Controls In Vitro Development of Cerebellar Granule Neurons Development

Reactive oxygen species (ROS) act as signaling molecules that regulate nervous system physiology. ROS have been related to neural differentiation, neuritogenesis, and programmed cell death. Nevertheless, little is known about the mechanisms involved in the regulation of ROS during neuronal development. In this study, we evaluated the mechanisms by which ROS are regulated during neuronal development and the implications of these molecules in this process. Primary cultures of cerebellar granule neurons (CGN) were used to address these issues. Our results show that during the first 3 days of CGN development in vitro (days in vitro; DIV), the levels of ROS increased, reaching a peak at 2 and 3 DIV under depolarizing (25 mM KCl) and nondepolarizing (5 mM KCl) conditions. Subsequently, under depolarizing conditions, the ROS levels markedly decreased, but in nondepolarizing conditions, the ROS levels increased gradually. This correlated with the extent of CGN maturation. Also, antioxidants and NADPH-oxidases (NOX) inhibitors reduced the expression of Tau and MAP2. On the other hand, the levels of glutathione markedly increased at 1 DIV. We inferred that the ROS increase at this time is critical for cell survival because glutathione depletion leads to axonal degeneration and CGN death only at 2 DIV. During the first 3 DIV, NOX2 was upregulated and expressed in filopodia and growth cones, which correlated with the hydrogen peroxide (H₂O₂) distribution in the cell. Finally, NOX2 KO CGN showed shorter neurites than wild-type CGN. Taken together, these results suggest that the regulation of ROS is critical during the early stages of CGN development.     

Epigallocatechin-3-gallate inhibits interleukin-6- and angiotensin II-induced production of C-reactive protein in vascular smooth muscle cells

AimsExtensive research suggests that atherosclerosis is an inflammatory disease and that epigallocatechin-3-gallate (EGCG) is able to inhibit the formation and development of atherosclerosis. However, the mechanisms of action of EGCG against atherosclerosis are still unclear. Therefore, the effect of EGCG on interleukin-6 (IL-6)- and angiotensin II (Ang II)-induced CRP production in vascular smooth muscle cells (VSMCs) was studied to provide experimental evidence for its anti-inflammatory and anti-atherosclerotic actions.Main methodsRat VSMCs were cultured, and IL-6 (10^? 7 M) and Ang II (10^? 7 M) were used as stimulants for CRP generation. The CRP concentration in the supernatant was measured with ELISA, and mRNA and protein expression of CRP was assayed with RT-qPCR and immunocytochemistry, respectively. The production of reactive oxygen species (ROS) and superoxide anion (O₂^?) was detected with ROS and O₂^? assay kits, respectively.Key findingsThe results showed that both IL-6 and Ang II increased CRP levels in the supernatant of VSMCs and induced mRNA and protein expression of CRP in VSMCs, whereas pretreatment of the cells with EGCG (1 × 10^? 6 M, 3 × 10^? 6 M, 10 × 10^? 6 M) significantly inhibited IL-6- and Ang II-induced production and expression of CRP in VSMCs in a concentration-dependent manner. Additionally, Ang II stimulated O₂^? and ROS generations in VSMCs, and EGCG decreased the Ang II-induced increase of O₂^? and ROS in a concentration-dependent fashion.SignificanceThese results suggest that EGCG plays an anti-inflammatory role via inhibiting IL-6- and Ang II-induced CRP secretion, as well as the Ang II-induced generation of O₂^? and ROS in VSMCs, which contributes to its anti-atherosclerotic action.     

Protective effect of salidroside against H2O2-induced cell apoptosis in primary culture of rat hippocampal neurons

Salidroside, a phenylpropanoid glycoside separated from a medicinal plant Rhodiola rosea, has been documented to have protective effects on neuronal cells in vitro. This study investigated whether salidroside was able to extend its unique neuroprotection to primary cultured rat hippocampal neurons against hydrogen peroxide (H₂O₂)-induced cell damage. Cell viability tests and cell apoptosis assays confirmed that salidroside pretreatment attenuated H₂O₂-stimulated apoptotic cell death in primary culture of hippocampal neurons in a concentration-dependent manner. The measurements of caspase-3 activity, nitric oxide (NO) production, and NO synthase (NOS) activity suggest that the protection of salidroside, shown in this study, might be mediated by inhibiting caspase-3 activity, and antagonizing NO production and NOS activity during H₂O₂ stimulation. Perhaps, this study might contribute to the development of salidroside as a broad-spectrum agent for preventing and/or treating neuronal damage in neurodegenerative disorders.     

Metformin protects PC12 cells and hippocampal neurons from H2O 2-induced oxidative damage through activation of AMPK pathway

Metformin, a first line anti type 2 diabetes drug, has recently been shown to extend lifespan in various species, and therefore, became the first antiaging drug in clinical trial. Oxidative stress due to excess reactive oxygen species (ROS) is considered to be an important factor in aging and related disease, such as Alzheimer's disease (AD). However, the antioxidative effects of metformin and its underlying mechanisms in neuronal cells is not known. In the present study, we showed that metformin, in clinically relevant concentrations, protected neuronal PC12 cells from H₂O₂-induced cell death. Metformin significantly ameliorated cell death due to H₂O₂ insult by restoring abnormal changes in nuclear morphology, intracellular ROS, lactate dehydrogenase, and mitochondrial membrane potential induced by H₂O₂. Hoechst staining assay and flow cytometry analysis revealed that metformin significantly reduced the apoptosis in PC12 cells exposed to H₂O₂. Western blot analysis further demonstrated that metformin stimulated the phosphorylation and activation of AMP-activated protein kinase (AMPK) in PC12 cells, while application of AMPK inhibitor compound C, or knockdown of the expression of AMPK by specific small interfering RNA or short hairpin RNA blocked the protective effect of metformin. Similar results were obtained in primary cultured hippocampal neurons. Taken together, these results indicated that metformin is able to protect neuronal cells from oxidative injury, at least in part, via the activation of AMPK. As metformin is comparatively cheaper with much less side effects in clinic, our findings support its potential to be a drug for prevention and treatment of aging and aging-related diseases.