Mitochondrial Rac1 GTPase import and electron transfer from cytochrome c are required for pulmonary fibrosis

The generation of reactive oxygen species, particularly H(2)O(2), from alveolar macrophages is causally related to the development of pulmonary fibrosis. Rac1, a small GTPase, is known to increase mitochondrial H(2)O(2) generation in macrophages; however, the mechanism by which this occurs is not known. This study shows that Rac1 is localized in the mitochondria of alveolar macrophages from asbestosis patients, and mitochondrial import requires the C-terminal cysteine of Rac1 (Cys-189), which is post-translationally modified by geranylgeranylation. Furthermore, H(2)O(2) generation mediated by mitochondrial Rac1 requires electron transfer from cytochrome c to a cysteine residue on Rac1 (Cys-178). Asbestos-exposed mice harboring a conditional deletion of Rac1 in macrophages demonstrated decreased oxidative stress and were significantly protected from developing pulmonary fibrosis. These observations demonstrate that mitochondrial import and direct electron transfer from cytochrome c to Rac1 modulates mitochondrial H(2)O(2) production in alveolar macrophages pulmonary fibrosis.     

Romo1 expression contributes to oxidative stress-induced death of lung epithelial cells

Oxidant-mediated death of lung epithelial cells due to cigarette smoking plays an important role in pathogenesis in lung diseases such as idiopathic pulmonary fibrosis (IPF). However, the exact mechanism by which oxidants induce epithelial cell death is not fully understood. Reactive oxygen species (ROS) modulator 1 (Romo1) is localized in the mitochondria and mediates mitochondrial ROS production through complex III of the mitochondrial electron transport chain. Here, we show that Romo1 mediates mitochondrial ROS production and apoptosis induced by oxidative stress in lung epithelial cells. Hydrogen peroxide (H₂O₂) treatment increased Romo1 expression, and Romo1 knockdown suppressed the cellular ROS levels and cell death triggered by H₂O₂ treatment. In immunohistochemical staining of lung tissues from patients with IPF, Romo1 was mainly localized in hyperplastic alveolar and bronchial epithelial cells. Romo1 overexpression was detected in 14 of 18 patients with IPF. TUNEL-positive alveolar epithelial cells were also detected in most patients with IPF but not in normal controls. These findings suggest that Romo1 mediates apoptosis induced by oxidative stress in lung epithelial cells.     

HIP/PAP protects against bleomycin‐induced lung injury and inflammation and subsequent fibrosis in mice

Hepatocarcinoma‐intestine‐pancreas/pancreatitis‐associated protein (HIP/PAP), a C‐type lectin, exerts anti‐oxidative, anti‐inflammatory, bactericidal, anti‐apoptotic, and mitogenic functions in several cell types and tissues. In this study, we explored the role of HIP/PAP in pulmonary fibrosis (PF). Expression of HIP/PAP and its murine counterpart, Reg3B, was markedly increased in fibrotic human and mouse lung tissues. Adenovirus‐mediated HIP/PAP expression markedly alleviated bleomycin (BLM)‐induced lung injury, inflammation, and fibrosis in mice. Adenovirus‐mediated HIP/PAP expression alleviated oxidative injury and lessened the decrease in pulmonary superoxide dismutase (SOD) activity in BLM‐treated mice, increased pulmonary SOD expression in normal mice, and HIP/PAP upregulated SOD expression in cultured human alveolar epithelial cells (A549) and human lung fibroblasts (HLF‐1). Moreover, in vitro experiments showed that HIP/PAP suppressed the growth of HLF‐1 and ameliorated the H₂O₂‐induced apoptosis of human alveolar epithelial cells (A549 and HPAEpiC) and human pulmonary microvascular endothelial cells (HPMVEC). In HLF‐1, A549, HPAEpiC, and HPMVEC cells, HIP/PAP did not affect the basal levels, but alleviated the TGF‐β1‐induced down‐regulation of the epithelial/endothelial markers E‐cadherin and vE‐cadherin and the over‐expression of mesenchymal markers, such as α‐SMA and vimentin. In conclusion, HIP/PAP was found to serve as a potent protective factor in lung injury, inflammation, and fibrosis by attenuating oxidative injury, promoting the regeneration of alveolar epithelial cells, and antagonizing the pro‐fibrotic actions of the TGF‐β1/Smad signaling pathway.     

Astaxanthin inhibits apoptosis in alveolar epithelial cells type II in vivo and in vitro through the ROS‐dependent mitochondrial signalling pathway

Oxidative stress is an important molecular mechanism underlying lung fibrosis. The mitochondrion is a major organelle for oxidative stress in cells. Therefore, blocking the mitochondrial signalling pathway may be the best therapeutic manoeuver to ameliorate lung fibrosis. Astaxanthin (AST) is an excellent antioxidant, but no study has addressed the pathway of AST against pulmonary oxidative stress and free radicals by the mitochondrion‐mediated signalling pathway. In this study, we investigated the antioxidative effects of AST against H₂O₂‐ or bleomycin (BLM)‐induced mitochondrial dysfunction and reactive oxygen species (ROS) production in alveolar epithelial cells type II (AECs‐II) in vivo and in vitro. Our data show that AST blocks H₂O₂‐ or BLM‐induced ROS generation and dose‐dependent apoptosis in AECs‐II, as characterized by changes in cell and mitochondria morphology, translocation of apoptotic proteins, inhibition of cytochrome c (Cyt c) release, and the activation of caspase‐9, caspase‐3, Nrf‐2 and other cytoprotective genes. These data suggest that AST inhibits apoptosis in AECs‐II cells through the ROS‐dependent mitochondrial signalling pathway and may be of potential therapeutic value in lung fibrosis treatment.     

Deregulation of selective autophagy during aging and pulmonary fibrosis: the role of TGFβ1

Aging constitutes a significant risk factor for fibrosis, and idiopathic pulmonary fibrosis (IPF) is characteristically associated with advancing age. We propose that age‐dependent defects in the quality of protein and cellular organelle catabolism may be causally related to pulmonary fibrosis. Our research found that autophagy diminished with corresponding elevated levels of oxidized proteins and lipofuscin in response to lung injury in old mice and middle‐aged mice compared to younger animals. More importantly, older mice expose to lung injury are characterized by deficient autophagic response and reduced selective targeting of mitochondria for autophagy (mitophagy). Fibroblast to myofibroblast differentiation (FMD) is an important feature of pulmonary fibrosis in which the profibrotic cytokine TGFβ1 plays a pivotal role. Promotion of autophagy is necessary and sufficient to maintain normal lung fibroblasts’ fate. On the contrary, FMD mediated by TGFβ1 is characterized by reduced autophagy flux, altered mitophagy, and defects in mitochondrial function. In accord with these findings, PINK1 expression appeared to be reduced in fibrotic lung tissue from bleomycin and a TGFβ1‐adenoviral model of lung fibrosis. PINK1 expression is also reduced in the aging murine lung and biopsies from IPF patients compared to controls. Furthermore, deficient PINK1 promotes a profibrotic environment. Collectively, this study indicates that an age‐related decline in autophagy and mitophagy responses to lung injury may contribute to the promotion and/or perpetuation of pulmonary fibrosis. We propose that promotion of autophagy and mitochondrial quality control may offer an intervention against age‐related fibrotic diseases.     

Nanoparticle-mediated catalase delivery protects human neurons from oxidative stress

Several neurodegenerative diseases and brain injury involve reactive oxygen species and implicate oxidative stress in disease mechanisms. Hydrogen peroxide (H₂O₂) formation due to mitochondrial superoxide leakage perpetuates oxidative stress in neuronal injury. Catalase, an H₂O₂-degrading enzyme, thus remains an important antioxidant therapy target. However, catalase therapy is restricted by its labile nature and inadequate delivery. Here, a nanotechnology approach was evaluated using catalase-loaded, poly(lactic co-glycolic acid) nanoparticles (NPs) in human neuronal protection against oxidative damage. This study showed highly efficient catalase encapsulation capable of retaining∼99% enzymatic activity. NPs released catalase rapidly, and antioxidant activity was sustained for over a month. NP uptake in human neurons was rapid and nontoxic. Although human neurons were highly sensitive to H₂O₂, NP-mediated catalase delivery successfully protected cultured neurons from H₂O₂-induced oxidative stress. Catalase-loaded NPs significantly reduced H₂O₂-induced protein oxidation, DNA damage, mitochondrial membrane transition pore opening and loss of cell membrane integrity and restored neuronal morphology, neurite network and microtubule-associated protein-2 levels. Further, catalase-loaded NPs improved neuronal recovery from H₂O₂ pre-exposure better than free catalase, suggesting possible applications in ameliorating stroke-relevant oxidative stress. Brain targeting of catalase-loaded NPs may find wide therapeutic applications for oxidative stress-associated acute and chronic neurodegenerative disorders.     

Mitochondrial uncoupling protein‐2 reprograms metabolism to induce oxidative stress and myofibroblast senescence in age‐associated lung fibrosis

Mitochondrial dysfunction has been associated with age‐related diseases, including idiopathic pulmonary fibrosis (IPF). We provide evidence that implicates chronic elevation of the mitochondrial anion carrier protein, uncoupling protein‐2 (UCP2), in increased generation of reactive oxygen species, altered redox state and cellular bioenergetics, impaired fatty acid oxidation, and induction of myofibroblast senescence. This pro‐oxidant senescence reprogramming occurs in concert with conventional actions of UCP2 as an uncoupler of oxidative phosphorylation with dissipation of the mitochondrial membrane potential. UCP2 is highly expressed in human IPF lung myofibroblasts and in aged fibroblasts. In an aging murine model of lung fibrosis, the in vivo silencing of UCP2 induces fibrosis regression. These studies indicate a pro‐fibrotic function of UCP2 in chronic lung disease and support its therapeutic targeting in age‐related diseases associated with impaired tissue regeneration and organ fibrosis.     

Involvement of Kv1.5 Protein in Oxidative Vascular Endothelial Cell Injury

Endothelial injury related to oxidative stress is a key event in cardiovascular diseases, such as hypertension and atherosclerosis. The activation of the redox-sensitive Kv1.5 potassium channel mediates mitochondrial reactive oxygen species (ROS)-induced apoptosis in vascular smooth muscle cells and some cancer cells. Kv1.5 channel is therefore taken as a new potential therapeutic target for pulmonary hypertension and cancers. Although Kv1.5 is abundantly expressed in vascular endothelium, there is little knowledge of its role in endothelial injury related to oxidative stress. We found that DPO-1, a specific inhibitor of Kv1.5, attenuated H₂O₂-evoked endothelial cell apoptosis in an in vivo rat carotid arterial model. In human umbilical vein endothelial cells (HUVECs) and human pulmonary arterial endothelial cells (HPAECs), angiotensin II and oxLDL time- or concentration-dependently enhanced Kv1.5 protein expression in parallel with the production of intracellular ROS and endothelial cell injury. Moreover, siRNA-mediated knockdown of Kv1.5 attenuated, whereas adenovirus-mediated Kv1.5 cDNA overexpression enhanced oxLDL–induced cellular damage, NADPH oxidase and mitochondria-derived ROS production and restored the decrease in protein expression of mitochondria uncoupling protein 2 (UCP2). Collectively, these data suggest that Kv1.5 may play an important role in oxidative vascular endothelial injury.     

Molecular mechanisms of asbestos-induced lung epithelial cell apoptosis

Asbestos causes pulmonary fibrosis (asbestosis) and malignancies (bronchogenic lung cancer and mesothelioma) by mechanisms that are not fully elucidated. Accumulating evidence show that alveolar epithelial cell (AEC) apoptosis is a crucial initiating and perpetuating event in the development of pulmonary fibrosis following exposure to a wide variety of noxious stimuli, including asbestos. We review the important molecular mechanisms underlying asbestos-induced AEC apoptosis. Specifically, we focus on the role of asbestos in augmenting AEC apoptosis by the mitochondria- and p53-regulated death pathways that result from the production of iron-derived reactive oxygen species (ROS) and DNA damage. We summarize emerging evidence implicating the endoplasmic reticulum (ER) stress response in AEC apoptosis in patients with idiopathic pulmonary fibrosis (IPF), a disease with similarities to asbestosis. Finally, we discuss a recent finding that a mitochondrial oxidative DNA repair enzyme (8-oxoguanine DNA glycosylase; Ogg1) acts as a mitochondrial aconitase chaperone protein to prevent oxidant (asbestos and H₂O₂)-induced AEC mitochondrial dysfunction and intrinsic apoptosis. The coupling of mitochondrial Ogg1 to mitochondrial aconitase is a novel mechanism linking metabolism to mitochondrial DNA that may be important in the pathophysiologic events resulting in oxidant-induced toxicity as seen in tumors, aging, and respiratory disorders (e.g. asbestosis, IPF). Collectively, these studies are illuminating the molecular basis of AEC apoptosis following asbestos exposure that may prove useful for developing novel therapeutic strategies. Importantly, the asbestos paradigm is elucidating pathophysiologic insights into other more common pulmonary diseases, such as IPF and lung cancer, for which better therapy is required.     

Activation of mitogen-activated protein kinases is essential for hydrogen peroxide -induced apoptosis in retinal pigment epithelial cells

Retinal pigment epithelial (RPE) cells are constantly exposed to oxidative injury while clearing byproducts of photoreceptor turnover, a circumstance thought to be responsible for degenerative retinal diseases. The mechanisms of hydrogen peroxide (H₂O₂)-induced apoptosis in RPE cells are not fully understood. We studied signal transduction mechanisms of H₂O₂-induced apoptosis in the human RPE cell line ARPE-19. Activation of two stress kinases (JNK and p38) occurs during H₂O₂ stimulation, and H₂O₂-mediated cell death was significantly reduced by their specific inhibition. Exposure to a lethal dose of H₂O₂ elicited Bax translocation to the mitochondria and release of apoptosis-inducing factor (AIF) from the mitochondria, both of which were abolished by either JNK- or p38-specific inhibitors. Both H₂O₂-induced cell death and JNK/p38 phosphorylation were partially inhibited by C. difficile toxin B, inhibitor of Rho, Rac, and cdc42. Use of pull-down assays revealed that the small GTPase activated by H₂O₂ is Rac1. This study is the first to demonstrate that H₂O₂ induces a Rac1/JNK1/p38 signaling cascade, and that JNK and p38 activation is important for H₂O₂-induced apoptosis as well as AIF/Bax translocation of RPE cells. Y.-C. Yang and T.-C. Ho contributed equally to the work described herein.     

NDUFV1 attenuates renal ischemia–reperfusion injury by improving mitochondrial homeostasis

Impaired mitochondrial function and dysregulated energy metabolism have been shown to be involved in the pathological progression of kidney diseases such as acute kidney injury (AKI) and diabetic nephropathy. Hence, improving mitochondrial function is a promising strategy for treating renal dysfunction. NADH: ubiquinone oxidoreductase core subunit V1 (NDUFV1) is an important subunit of mitochondrial complex I. In the present study, we found that NDUFV1 was reduced in kidneys of renal ischemia/reperfusion (I/R) mice. Meanwhile, renal I/R induced kidney dysfunction as evidenced by increases in BUN and serum creatinine, severe injury of proximal renal tubules, oxidative stress, and cell apoptosis. All these detrimental outcomes were attenuated by increased expression of NDUFV1 in kidneys. Moreover, knockdown of Ndufv1 aggravated cell insults induced by H₂O₂ in TCMK-1 cells, which further confirmed the renoprotective roles of NDUFV1. Mechanistically, NDUFV1 improved the integrity and function of mitochondria, leading to reduced oxidative stress and cell apoptosis. Overall, our data indicate that NDUFV1 has an ability to maintain mitochondrial homeostasis in AKI, suggesting therapies by targeting mitochondria are useful approaches for dealing with mitochondrial dysfunction associated renal diseases such as AKI.     

A novel fine tuning scheme of miR-200c in modulating lung cell redox homeostasis

Oxidative stress induces miR-200c, the predominant microRNA (miRNA) in lung tissues; however, the antioxidant role and biochemistry of such induction have not been clearly defined. Therefore, a lung adenocarcinoma cell line (A549) and a normal lung fibroblast (MRC-5) were used as models to determine the effects of miR-200c expression on lung antioxidant response. Hydrogen peroxide (H₂O₂) upregulated miR-200c, whose overexpression exacerbated the decrease in cell proliferation, retarded the progression of cells in the G2/M-phase, and increased oxidative stress upon H₂O₂ stimulation. The expression of three antioxidant proteins, superoxide dismutase (SOD)-2, haem oxygenase (HO)-1, and sirtuin (SIRT) 1, was reduced upon H₂O₂ stimulation in miR-200c-overexpressed A549 cells. This phenomenon of increased oxidative stress and antioxidant protein downregulation also occurs simultaneously in miR-200c overexpressed MRC-5 cells. Molecular analysis revealed that miR-200c inhibited the gene expression of HO-1 by directly targeting its 3′-untranslated region. The downregulation of SOD2 and SIRT1 by miR-200c was mediated through zinc finger E-box-binding homeobox 2 (ZEB2) and extracellular signal-regulated kinase 5 (ERK5) pathways, respectively, where knockdown of ZEB2 or ERK5 decreased the expression of SOD2 or SIRT1 in A549 cells. LNA anti-miR-200c transfection in A549 cells inhibited the endogenous miR-200c expression, resulting in increased expressions of antioxidant proteins, reduced oxidative stress and recovered cell proliferation upon H₂O₂ stimulation. These findings indicate that miR-200c fine-tuned the antioxidant response of the lung cells to oxidative stress through several pathways, and thus this study provides novel information concerning the role of miR-200c in modulating redox homeostasis of lung.     

Mitochondrial quality control protects photoreceptors against oxidative stress in the H2O2-induced models of retinal degeneration diseases

Retinal degeneration diseases (RDDs) are common and devastating eye diseases characterized by the degeneration of photoreceptors, which are highly associated with oxidative stress. Previous studies reported that mitochondrial dysfunction is associated with various neurodegenerative diseases. However, the role of mitochondrial proteostasis mainly regulated by mitophagy and mitochondrial unfolded protein response (mtUPR) in RDDs is unclear. We hypothesized that the mitochondrial proteostasis is neuroprotective against oxidative injury in RDDs. In this study, the data from our hydrogen peroxide (H₂O₂)-treated mouse retinal cone cell line (661w) model of RDDs showed that nicotinamide riboside (NR)-activated mitophagy increased the expression of LC3B II and PINK1, and promoted the co-localization of LC3 and mitochondria, as well as PINK1 and Parkin in the H₂O₂-treated 661w cells. However, the NR-induced mitophagy was remarkably reversed by chloroquine (CQ) and cyclosporine A (CsA), mitophagic inhibitors. In addition, doxycycline (DOX), an inducer of mtUPR, up-regulated the expression of HSP60 and CHOP, the key proteins of mtUPR. Activation of both mitophagy and mtUPR increased the cell viability and reduced the level of apoptosis and oxidative damage in the H₂O₂-treated 661w cells. Furthermore, both mitophagy and mtUPR played a protective effect on mitochondria by increasing mitochondrial membrane potential and maintaining mitochondrial mass. By contrast, the inhibition of mitophagy by CQ or CsA reversed the beneficial effect of mitophagy in the H₂O₂-treated 661w cells. Together, our study suggests that the mitophagy and mtUPR pathways may serve as new therapeutic targets to delay the progression of RDDs through enhancing mitochondrial proteostasis.Subject terms: Cell death, Diseases     

Isoflurane Anesthesia Initiated at the Onset of Reperfusion Attenuates Oxidative and Hypoxic-Ischemic Brain Injury

This study demonstrates that in mice subjected to hypoxia-ischemia (HI) brain injury isoflurane anesthesia initiated upon reperfusion limits a release of mitochondrial oxidative radicals by inhibiting a recovery of complex-I dependent mitochondrial respiration. This significantly attenuates an oxidative stress and reduces the extent of HI brain injury. Neonatal mice were subjected to HI, and at the initiation of reperfusion were exposed to isoflurane with or without mechanical ventilation. At the end of HI and isoflurane exposure cerebral mitochondrial respiration, H₂O₂ emission rates were measured followed by an assessment of cerebral oxidative damage and infarct volumes. At 8 weeks after HI navigational memory and brain atrophy were assessed. In vitro, direct effect of isoflurane on mitochondrial H₂O₂ emission was compared to that of complex-I inhibitor, rotenone. Compared to controls, 15 minutes of isoflurane anesthesia inhibited recovery of the compex I-dependent mitochondrial respiration and decreased H₂O₂ production in mitochondria supported with succinate. This was associated with reduced oxidative brain injury, superior navigational memory and decreased cerebral atrophy compared to the vehicle-treated HI-mice. Extended isoflurane anesthesia was associated with sluggish recovery of cerebral blood flow (CBF) and the neuroprotection was lost. However, when isoflurane anesthesia was supported with mechanical ventilation the CBF recovery improved, the event associated with further reduction of infarct volume compared to HI-mice exposed to isoflurane without respiratory support. Thus, in neonatal mice brief isoflurane anesthesia initiated at the onset of reperfusion limits mitochondrial release of oxidative radicals and attenuates an oxidative stress. This novel mechanism contributes to neuroprotective action of isoflurane. The use of mechanical ventilation during isoflurane anesthesia counterbalances negative effect of isoflurane anesthesia on recovery of cerebral circulation which potentiates protection against reperfusion injury.     

Targeting Cpt1a-Bcl-2 interaction modulates apoptosis resistance and fibrotic remodeling

The mitochondrial calcium uniporter (MCU) regulates metabolic reprogramming in lung macrophages and the progression of pulmonary fibrosis. Fibrosis progression is associated with apoptosis resistance in lung macrophages; however, the mechanism(s) by which apoptosis resistance occurs is poorly understood. Here, we found a marked increase in mitochondrial B-cell lymphoma-2 (Bcl-2) in lung macrophages from subjects with idiopathic pulmonary fibrosis (IPF). Similar findings were seen in bleomycin-injured wild-type (WT) mice, whereas Bcl-2 was markedly decreased in mice expressing a dominant-negative mitochondrial calcium uniporter (DN-MCU). Carnitine palmitoyltransferase 1a (Cpt1a), the rate-limiting enzyme for fatty acid β-oxidation, directly interacted with Bcl-2 by binding to its BH3 domain, which anchored Bcl-2 in the mitochondria to attenuate apoptosis. This interaction was dependent on Cpt1a activity. Lung macrophages from IPF subjects had a direct correlation between CPT1A and Bcl-2, whereas the absence of binding induced apoptosis. The deletion of Bcl-2 in macrophages protected mice from developing pulmonary fibrosis. Moreover, mice had resolution when Bcl-2 was deleted or was inhibited with ABT-199 after fibrosis was established. These observations implicate an interplay between macrophage fatty acid β-oxidation, apoptosis resistance, and dysregulated fibrotic remodeling.Subject terms: Cell biology, Medical research     

Amniotic Fluid Stem Cells Inhibit the Progression of Bleomycin-Induced Pulmonary Fibrosis via CCL2 Modulation in Bronchoalveolar Lavage

The potential for amniotic fluid stem cell (AFSC) treatment to inhibit the progression of fibrotic lung injury has not been described. We have previously demonstrated that AFSC can attenuate both acute and chronic-fibrotic kidney injury through modification of the cytokine environment. Fibrotic lung injury, such as in Idiopathic Pulmonary Fibrosis (IPF), is mediated through pro-fibrotic and pro-inflammatory cytokine activity. Thus, we hypothesized that AFSC treatment might inhibit the progression of bleomycin-induced pulmonary fibrosis through cytokine modulation. In particular, we aimed to investigate the effect of AFSC treatment on the modulation of the pro-fibrotic cytokine CCL2, which is increased in human IPF patients and is correlated with poor prognoses, advanced disease states and worse fibrotic outcomes. The impacts of intravenous murine AFSC given at acute (day 0) or chronic (day 14) intervention time-points after bleomycin injury were analyzed at either day 3 or day 28 post-injury. Murine AFSC treatment at either day 0 or day 14 post-bleomycin injury significantly inhibited collagen deposition and preserved pulmonary function. CCL2 expression increased in bleomycin-injured bronchoalveolar lavage (BAL), but significantly decreased following AFSC treatment at either day 0 or at day 14. AFSC were observed to localize within fibrotic lesions in the lung, showing preferential targeting of AFSC to the area of fibrosis. We also observed that MMP-2 was transiently increased in BAL following AFSC treatment. Increased MMP-2 activity was further associated with cleavage of CCL2, rendering it a putative antagonist for CCL2/CCR2 signaling, which we surmise is a potential mechanism for CCL2 reduction in BAL following AFSC treatment. Based on this data, we concluded that AFSC have the potential to inhibit the development or progression of fibrosis in a bleomycin injury model during both acute and chronic remodeling events.     

Blockade of SOCE protects HT22 cells from hydrogen peroxide-induced apoptosis

Oxidative stress is an established event in the pathology of neurobiological diseases. Previous studies indicated that store-operated Ca²⁺ entry (SOCE) has been involved in oxidative stress. The present study was carried out to investigate the effects of SOCE inhibition on neuronal oxidative stress injury induced by hydrogen peroxide (H₂O₂) in HT22 cells, a murine hippocampal neuronal model. H₂O₂ insult induced significant intracellular Ca²⁺ overload, mitochondrial dysfunction and cell viability decrease. Inhibition of SOCE by pharmacological inhibitor and STIM1 RNAi significantly alleviated intracellular Ca²⁺ overload, restored the mitochondrial membrane potential (MMP), decreased cytochrome C release and eventually inhibited H₂O₂-induced cell apoptosis. These findings suggest that SOCE inhibition exhibited neuroprotection against oxidative stress induced by H₂O₂ and SOCE might be a useful therapeutic target in neurobiological disorders.