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Introduction: Cardiovascular diseases are the leading cause of morbidity and mortality worldwide. Ischemic heart disease is one of the most harmful conditions to cellular structure and function. After reperfusion treatment, a spectrum of adverse effects becomes evident, encompassing altered cell viability, heightened oxidative stress, activated autophagy, and increased apoptosis. Photobiomodulation (PBM) has been utilized in experimental models of cardiac hypoxia to enhance mitochondrial response and ameliorate biochemical changes in injured tissue. However, the effects of PBM on cultured cardiomyocytes subjected to hypoxia/reoxygenation are not yet well established., Method: H9C2 cardiomyocytes were exposed to hypoxia with concentrations of 300 μM CoCl 2 for 24 h, followed by 16 h of reoxygenation through incubation in a normoxic medium. Treatment was conducted using GaAIAs Laser (850 nm) after hypoxia at an intensity of 1 J/cm 2 . Cells were divided into three groups: Group CT (cells maintained under normoxic conditions), Group HR (cells maintained in hypoxia and reoxygenation conditions without treatment), Group HR + PBM (cells maintained in hypoxia and reoxygenation conditions that underwent PBM treatment). Cell viability was analyzed using MTT, and protein expression was assessed by western blot. One-way ANOVA with the Tukey post hoc test was used for data analysis. Differences were significant when p < 0.05., Results: PBM at an intensity of 1 J/cm 2 mitigated the alterations in cell survival caused by hypoxia/reoxygenation. Additionally, it significantly increased the expression of proteins Nrf2, HSP70, mTOR, LC3II, LC3II/I, and Caspase-9, while reducing the expression of PGC-1α, SOD2, xanthine oxidase, Beclin-1, LC3I, and Bax., Conclusion: PBM at intensities of 1 J/cm 2 reverses the changes related to oxidative stress, mitochondrial biogenesis, autophagy, and apoptosis caused by hypoxia and reoxygenation in a culture of cardiomyocytes., Competing Interests: Declaration of competing interest The authors declare that no conflict of interest is relevant to the content of this article., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Although our previous studies have established the crucial role of RP105 in myocardial ischemia/reperfusion injury (MI/RI), its involvement in regulating oxidative stress induced by MI/RI remains unclear. To investigate this, we conducted experiments using a rat model of ischemia/reperfusion (I/R) injury. Adenovirus carrying RP105 was injected apically at multiple points, and after 72 h, the left anterior descending coronary artery was ligated for 30 min followed by 2 h of reperfusion. In vitro experiments were performed on H9C2 cells, which were transfected with recombinant adenoviral vectors for 48 h, subjected to 4 h of hypoxia, and then reoxygenated for 2 h. We measured oxidative stress markers, including superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities, as well as malondialdehyde (MDA) concentration, using a microplate reader. The fluorescence intensity of reactive oxygen species (ROS) in myocardial tissue was measured using a DHE probe. We also investigated the upstream and downstream components of the signal transducer and activator of transcription 3 (STAT3). Upregulation of RP105 increased SOD and GSH-Px activities, reduced MDA concentration, and inhibited ROS production in response to I/R injury in vivo and hypoxia reoxygenation (H/R) stimulation in vitro. The overexpression of RP105 led to a decrease in the myocardial enzyme LDH in serum and cell culture supernatant, as well as a reduction in infarct size. Additionally, left ventricular fraction (LVEF) and fractional shortening (LVFS) were improved in the RP105 overexpression group compared to the control. Upregulation of RP105 promoted the expression of Lyn and Syk and further activated STAT phosphorylation, which was blocked by PP2 (a Lyn inhibitor). Our findings suggest that RP105 can inhibit MI/RI-induced oxidative stress by activating STAT3 via the Lyn/Syk signaling pathway., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Objectives: To investigate the regulatory role of miRNA-224-5p in hypoxia/reoxygenation (H/R) -induced H9c2 cardiomyocyte injury., Methods: Plasma samples were collected from 160 patients with acute myocardial infarction and 80 healthy controls(HC) to measure miRNA-224-5p levels and other biochemical parameters. In cultured H9c2 cells with H/R injury, the effects of transfection with miR-224-5p mimics or a negative control sequence on cell viability, malondialdehyde (MDA) content, and superoxide dismutase 2 (SOD2) and lactate dehydrogenase (LDH) activities were tested. Dual luciferase reporter gene assay was performed to verify the targeting relationship between miR-224-5p and PTEN. Bioinformatics methods were used to analyze the potential mechanisms of the target genes. The expression of miRNA-224-5p in the treated cells was detected with qRT-PCR, the protein expressions of PTEN, Bcl-2, Bax, cleaved caspase-3, SOD2, p-PI3K/PI3K, p-Akt/Ak and p-FoxO1/FoxO1 were determined using Western blotting, and cell apoptosis was analysed with flow cytometry., Results: The levels of blood glucose, C-reactive protein, CK, CK-MB and cTnI were significantly higher in the AMI group compared with the HC group ( P < 0.05). The expression level of miR-224-5p was significantly lowered in patients with STEMI and NSTEMI and in H9c2 cells with H/R injury. The viability of H9c2 cells decreased time-dependently following H/R injury. PTEN was a target gene of miR-224-5p, and the PI3K/Akt pathway was the most significantly enriched pathway. H9c2 cells with H/R injury showed significantly decreased SOD2 activity, increased LDH activity and MDA content, increased cell apoptosis, decreased protein expression levels of p-PI3K, p-Akt, p-FoxO1, SOD2, and Bcl-2, and increased expressions of PTEN, Bax, and cleaved caspase-3. These changes were obviously attenuated by trasnfection of the cells with miR-224-5p mimics prior to H/R exposure., Conclusion: MiR-224-5p overexpression upregulates the expression of the antioxidant gene SOD2 through the PI3K/Akt/FoxO1 axis to relieve H/R-induced oxidative stress and reduce apoptosis of H9c2 cells.

Tumor necrosis factor α-induced protein 1 (TNFAIP1) has been documented as a vital regulator of apoptosis and oxidative stress under various pathological conditions. However, whether TNFAIP1 plays a role in myocardial ischemia/reperfusion (I/R) injury has not been well investigated. This work aimed to evaluate the possible role of TNFAIP1 in mediating myocardial I/R injury. Firstly, we demonstrated that TNFAIP1 expression was dramatically increased in rat cardiomyocytes following hypoxia/reoxygenation (H/R) in vitro, and in rat myocardial tissues following I/R treatment in vivo. Silencing of TNFAIP1 alleviated H/R-induced apoptosis, oxidative stress and inflammatory response in rat cardiomyocytes in vitro. Moreover, knockdown of TNFAIP1 ameliorated I/R-induced myocardial injury, infarction size, cardiac apoptosis, oxidative stress and inflammatory response in vivo. Further investigation elucidated that knockdown of TNFAIP1 enhanced the activation of nuclear factor erythroid 2-related factor 2 (Nrf2) signaling associated with modulation of the Akt/glycogen synthase kinase-3β (GSK-3β) pathway in vitro and in vivo. Inhibition of Akt markedly abrogated TNFAIP1-knockdown-mediated Nrf2 activation in cardiomyocytes following H/R injury. In addition, suppression of Nrf2 significantly diminished TNFAIP1-knockdown-induced cardioprotective effects in H/R-exposed cardiomyocytes. In summary, this work elucidates that inhibition of TNFAIP1 ameliorates myocardial I/R injury by potentiating Nrf2 signaling via the modulation of the Akt/GSK-3β pathway. Our study highlights a vital role of the TNFAIP1/Akt/GSK-3β/Nrf2 pathway in mediating myocardial I/R injury and suggests TNFAIP1 as an attractive target for treatment of this disease., (Copyright © 2021 Elsevier B.V. All rights reserved.)

We have focused on the underlying role of miR-1224 in cardiomyocyte injury stimulated by hypoxia/reoxygenation (H/R). In the current study, the rat cardiomyocyte cell line H9C2 was used to construct a H/R cell model to validate the cardioprotective effects of miR-1224. Data from the dual-luciferase assay revealed that the glutathione peroxidase 4 (GPX4) was a direct target of miR-1224. Expression of miR-1224, determined using qRT-PCR, was remarkably increased while that of GPX4 protein, evaluated via western blotting, was significantly decreased in cardiomyocytes in response to H/R exposure. ROS generation, superoxide dismutase (SOD) activity, concentrations of malondialdehyde (MDA) and 4-hydroxy aldehydes (4-HNE), and H9C2 cell apoptosis were further evaluated following overexpression of miR-1224 or silencing of GPX4 in H9C2 cells. H9C2 cells under H/R conditions displayed increased synthesis of ROS, along with overexpression of miR-1224 and downregulation of GPX4. SOD activity was significantly decreased while concentrations of MDA and 4-HNE were markedly increased under H/R injury conditions. In addition, miR-1224 mimic or GPX4 siRNA plasmids dramatically enhanced H/R-mediated apoptosis, Bax expression and caspase-3 activity, with a concomitant reduction in Bcl-2 expression. Conversely, inhibition of miR-1224 exerted suppressive effects on oxidative stress and apoptosis in H9C2 cells under H/R conditions. Interestingly, silencing of GPX4 attenuated the negative effects of miR-1224 inhibition. Our results suggested that inhibition of miR-1224 caused resistance to H/R and diminished oxidative stress in vitro through targeting of GPX4., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Propofol (PRO) protects against hepatic ischemia/reperfusion (I/R) injury. Bnip3 is involved in the I/R-induced injury. This study investigated whether the effect of PRO on hepatic hypoxia/reoxygenation (H/R) injury was realized through regulating Bnip3. After establishing a hepatic ischemia reperfusion (I/R ) injury model in mice, the serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were determined by an automatic biochemical analyzer. The histopathology and apoptosis of liver tissues were detected by hematoxylin-eosin and TUNEL staining. After the H/R liver cells were cultured and treated with PRO, the viability, apoptosis, reactive oxygen species (ROS) production, and the levels of lactate dehydrogenase (LDH), malondialdehyde (MDA), TNF-α, and IL-6 were detected by MTT, flow cytometry, colorimetry, and ELISA. The expressions of Bnip3 and apoptosis-related factors in I/R mouse liver tissues and H/R cells were determined by immunohistochemical assay, immunofluorescence, Western blot, or RT-qPCR. PRO ameliorated the abnormal histopathology, reduced cell apoptosis and the levels of AST, ALT, Bnip3, Cleaved Caspase-3, and Bax, but upregulated the Bcl-2 level in the liver tissues of I/R mice. In H/R liver cells, PRO promoted the cell viability, downregulated the levels of LDH, MDA, TNF-α, IL-6, and reduced ROS production. Moreover, PRO promoted the downregulated expressions of cytosolic Bnip3, total Bni3p, Cleaved Caspase-3, and Bax and upregulated the Bcl-2 level. siBnip3 reversed the effect of H/R on the liver cells, and its overexpression also reversed the effect of PRO on H/R-induced liver cells. PRO protects against hepatic I/R injury via inhibiting Bnip3., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC part of Springer Nature.)

We aimed to investigate the protective role and mechanism of dexmedetomidine (DEX) on H9c2 cardiomyocytes after hypoxia/reoxygenation (H/R) injury. Six experimental groups were designed as follows: normal control group (group C), H/R group, H/R + DEX group, H/R + gastrodin group, H/R + Ex527 (SIRT1 inhibitor) group, and H/R + DEX + Ex527 group. Lactate dehydrogenase (LDH) activity and the levels of oxidative stress-related enzymes such as malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT) and glutathione (GSH) were measured using corresponding commercial kits. Cell counting kit (CCK)-8 assay was used to detect cell survival rate while flow cytometry and caspase 3/7 activity were used to determine cell apoptosis, respectively. Western blot was used to detect the expression of silent information regulator 1 (SIRT1), C/EBP homologous protein (CHOP), cleaved-caspase-12/3 and pro-caspase-12/3 in each group. From our findings, when compared with H/R, H/R + Ex527 and H/R + DEX + Ex527 groups, DEX pretreatment of cells in H/R + DEX group significantly increased cell survival rate, and simultaneously reduced LDH activity, oxidative stress and the apoptosis rate of H9c2 cells with H/R injury. Moreover, DEX up-regulated SIRT1 expression level and down-regulated the levels of endoplasmic reticulum (ER) stress-related markers such as CHOP, cleaved-caspase-12 and cleaved-caspase-3, respectively. Ex527 could completely block DEX-induced upregulated expression of SIRT1, and partially blocked the DEX-induced downregulated expression levels of CHOP, cleaved-caspase-12 and cleaved-caspase-3. These results proved that DEX reversed H/R injury-induced oxidative stress and ER stress-dependent apoptosis of cardiomyocytes via SIRT1/CHOP signaling pathway.

Homeodomain interacting protein kinase-2 (HIPK2) has emerged as a crucial stress-responsive kinase that plays a critical role in regulating cell survival and apoptosis. However, whether HIPK2 participates in regulating cardiomyocyte survival during myocardial ischemia/reperfusion injury remains unclear. Here, we investigated the regulatory effect of HIPK2 on hypoxia/reoxygenation (H/R)-induced cardiomyocyte injury and its potential underlying molecular mechanism. We found that HIPK2 expression was induced in response to H/R exposure. HIPK2 depletion by small interfering RNA (siRNA)-mediated gene silencing significantly decreased the viability and exacerbated H/R-induced apoptosis and reactive oxygen species (ROS) production in cardiomyocytes. Comparatively, HIPK2 overexpression effectively rescued H/R-impaired viability and repressed H/R-induced apoptosis and ROS production in cardiomyocytes. HIPK2 overexpression significantly increased the nuclear expression of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and enhanced Nrf2-mediated transcriptional activity. Moreover, HIPK2 overexpression significantly increased the transcription of Nrf2/ARE target genes. Additionally, Nrf2 inhibition partially reversed the HIPK2-mediated protective effect. Overall, these results demonstrate that HIPK2 overexpression protects cardiomyocytes from H/R-induced injury by enhancing Nrf2/ARE antioxidant signaling, data that suggest HIPK2 is a potential target for cardioprotection., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2019 Elsevier B.V. All rights reserved.)

This study aimed to investigate the cytoprotective effects of flavonoids, their metabolites alone or in combination against hypoxia/reoxygenation induced oxidative stress in the transformed human first trimester trophoblast cell line (HTR-8/SVneo). Oxidative stress was achieved with hypoxia followed by reoxygenation and the following assays were performed: MTT, CellTox™ Green Cytotoxicity, CellTiter-Glo®, NADP/NADPH-Glo™, ROS-Glo™/H 2 O 2 , GSH/GSSG-Glo™ and Caspase-Glo® 3/7 assays. HTR-8/SVneo cells, pre-treated for 24 h with flavonoids or their metabolites were protected significantly from oxidative stress. Flavonoids were associated with ROS modulation, reducing the generation of superoxide/hydrogen peroxide. The activities of caspases 3/7 were also significantly reduced significantly in HTR-8/SVneo cells pre-treated with flavonoids. This study has shown for the first time that 24 h pre-treatment with flavonoids, their metabolites alone or in combination, protected against HR-induced oxidative stress in the trophoblast cell line. These data indicate that dietary flavonoids may be beneficial to placental health and invasion during early gestation., (Crown Copyright © 2018. Published by Elsevier Ltd. All rights reserved.)

DJ-1 was recently reported to mediate the cardioprotection of delayed hypoxic preconditioning (DHP) by suppressing hypoxia/reoxygenation (H/R)-induced oxidative stress, but its mechanism against H/R-induced oxidative stress during DHP is not fully elucidated. Here, using the well-established cellular model of DHP, we again found that DHP significantly improved cell viability and reduced lactate dehydrogenase release with concurrently up-regulated DJ-1 protein expression in H9c2 cells subjected to H/R. Importantly, DHP efficiently improved mitochondrial complex I activity following H/R and attenuated H/R-induced mitochondrial reactive oxygen species (ROS) generation and subsequent oxidative stress, as demonstrated by a much smaller decrease in reduced glutathione/oxidized glutathione ratio and a much smaller increase in intracellular ROS and malondialdehyde contents than that observed for the H/R group. However, the aforementioned effects of DHP were antagonized by DJ-1 knockdown with short hairpin RNA but mimicked by DJ-1 overexpression. Intriguingly, pharmacological inhibition of mitochondria complex I with Rotenone attenuated all the protective effects caused by DHP and DJ-1 overexpression, including maintenance of mitochondria complex I and suppression of mitochondrial ROS generation and subsequent oxidative stress. Taken together, this work revealed that preserving mitochondrial complex I activity and subsequently inhibiting mitochondrial ROS generation could be a novel mechanism by which DJ-1 mediates the cardioprotection of DHP against H/R-induced oxidative stress damage., (Copyright © 2018 John Wiley & Sons, Ltd.)

Background: Vascular endothelial dysfunction is the closely related determinant of ischemic heart disease (IHD). Endothelial dysfunction and ischemia/reperfusion injury (IRI) have been associated with an increase in microvesicles (MVs) in vivo. However, the potential contribution of endothelial microvesicles (EMVs) to myocardial damage is unclear. Here we aimed to investigate the role of EMVs derived from hypoxia/reoxygenation (H/R) -treated human umbilical vein endothelial cells (HUVECs) on cultured H9c2 cardiomyocytes., Results: H/R injury model was established to induce HUVECs to release H/R-EMVs. The H/R-EMVs from HUVECs were isolated from the conditioned culture medium and characterized. H9c2 cardiomyocytes were then incubated with 10, 30, 60 μg/mL H/R-EMVs for 6 h. We found that H9c2 cells treated by H/R-EMVs exhibited reduced cell viability, increased cell apoptosis and reactive oxygen species (ROS) production. Moreover mechanism studies demonstrated that H/R-EMVs could induce the phosphorylation of p38 and JNK1/2 in H9c2 cells in a dose-dependent manner. In addition, H/R-EMVs contained significantly higher level of ROS than EMVs generated from untreated HUVECs, which might be a direct source to trigger a cascade of myocardial damage., Conclusion: We showed that EMVs released during H/R injury are pro-apoptotic, pro-oxidative and directly pathogenic to cardiomyocytes in vitro. EMVs carry ROS and they may impair myocardium by promoting apoptosis and oxidative stress. These findings provide new insights into the pathogenesis of IRI.

To meet the challenges presented by dive-derived hypoxia/reoxygenation transition, the aquatic mammals possess multi-level adaptations. However, the adjustments of the semiaquatic animals as modern analogs of evolutionary intermediates between ancestral terrestrial mammals and their fully aquatic descendants are still not fully elucidated. The aim of this study was to analyze the total lactate dehydrogenase (LDH) activity (in the lactate to pyruvate direction), the LDH patterns and the antioxidant defense in the tissues (heart, kidney, liver, lung, muscle, spleen) of semiaquatic rodents such as Eurasian beaver (Castor fiber), muskrat (Ondatra zibethicus) and nutria (Myocastor coypus). Samples from Wistar rat were used for comparison. Semiaquatic rodents had higher catalase activity compared to rats. The superoxide dismutase activity was higher and the catalase activity was lower in almost all tissues of muskrat than of both beaver and nutria. Comparing beaver and nutria, no significant differences in the antioxidant enzyme activities were found for the heart, kidney and liver. In beaver, most of the examined tissues (heart, kidney, lung and spleen) use lactate as preference to glucose as a substrate but in muskrat the heart, liver and skeletal muscle showed the increased LDH activity. Nutria had the unusual LDH properties that are needed to be further investigated. Our results suggest that beaver, nutria and muskrat have distinct mechanisms of adaptation to diving hypoxia/reoxygenation and support the hypothesis that semiaquatic mammals are the intermediate animals that help to define which potential selection factors and mechanical constraints may have directed the evolution of the aquatic forms., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Angiotensin1-7 (Ang1-7) is a biologically active member of the renin-angiotensin system, which has been reported to exhibit protective effect in myocardial ischemia reperfusion-induced injury. However, the molecular basis of this effect is not well understood. It has been proposed that oxidative stress-induced cardiomyocyte apoptosis is a major consequence of hypoxia/reoxygenation (H/R) injury. This study investigates the protective effect of Ang1-7 against H/R-induced oxidative stress in rat H9C2 cells. Our results showed that Ang1-7 (80nM) treatment significantly protected cells from H/R-induced oxidative injury via improving cell viability and reducing cell apoptosis. The protective effect of Ang1-7 was associated with the inhibition of ROS-associated mitochondrial dysfunction as well as the induction of Akt phosphorylation. These findings may significantly contribute to better understanding the protective effect of Ang1-7, particularly in hypoxia/reoxygenation-induced heart diseases and form the basis in the therapeutic development in treating cardiovascular diseases., (Copyright © 2015. Published by Elsevier GmbH.)

Age-related neurological disorders are of growing concern among the elderly, and natural products with neuroprotective properties have been attracting increasing attention as candidates for the prevention or treatment of neurological disorders induced by oxidative stress. In an effort to explore natural resources, we collected some common marine seaweed from the Korean peninsula and Indonesia and screened them for neuroprotective activity against hypoxia/reoxygenation (H/R)-induced oxidative stress. Of the 23 seaweeds examined, the ethanol extract of Gracilariopsis chorda (GCE) provided maximum neuroprotection at an optimum concentration of 15 μg/mL, followed by Undaria pinnatifida. GCE increased cell viability after H/R, decreased the formation of reactive oxygen species (measured by 2',7'-dichlorodihydrofluorescein diacetate [DCF-DA] staining), and inhibited the double-stranded DNA breaks (measured by H2AX immunocytochemistry), apoptosis (measured by Annexin V/propidium iodide staining), internucleosomal DNA fragmentation (measured by DNA laddering), and dissipation of mitochondrial membrane potential (measured by JC-1 staining). Using reverse-phase high-pressure liquid chromatography, we quantitated the arachidonic acid (AA) in GCE, which provides neuroprotection against H/R-induced oxidative stress. This neuroprotective effect of AA was comparable to that of GCE. These findings suggest that the neuroprotective effect of GCE against H/R-induced neuronal death is due, at least in part, to the AA content that suppresses neuronal apoptosis.

Oxidative stress induced by reactive oxygen species (ROS) is associated with various neurological disorders including aging, neurodegenerative diseases, as well as traumatic and ischemic insults. Astrocytes have an important role in the anti-oxidative defense in the brain. The gap junction protein connexin43 (Cx43) forms intercellular channels as well as hemichannels in astrocytes. In the present study, we investigated the contribution of Cx43 to astrocytic death induced by the ROS hydrogen peroxide (H2O2) and the mechanism by which Cx43 exerts its effects. Lack of Cx43 expression or blockage of Cx43 channels resulted in increased ROS-induced astrocytic death, supporting a cell protective effect of functional Cx43 channels. H2O2 transiently increased hemichannel activity, but reduced gap junction intercellular communication (GJIC). GJIC in wild-type astrocytes recovered after 7 h, but was absent in Cx43 knock-out astrocytes. Blockage of Cx43 hemichannels incompletely inhibited H2O2-induced hemichannel activity, indicating the presence of other hemichannel proteins. Panx1, which is predicted to be a major hemichannel contributor in astrocytes, did not appear to have any cell protective effect from H2O2 insults. Our data suggest that GJIC is important for Cx43-mediated ROS resistance. In contrast to hypoxia/reoxygenation, H2O2 treatment decreased the ratio of the hypophosphorylated isoform to total Cx43 level. Cx43 has been reported to promote astrocytic death induced by hypoxia/reoxygenation. We therefore speculate the increase in Cx43 dephosphorylation may account for the facilitation of astrocytic death. Our findings suggest that the role of Cx43 in response to cellular stress is dependent on the activation of signaling pathways leading to alteration of Cx43 phosphorylation states.

Human placenta produces melatonin and expresses its receptors. We propose that melatonin, an antioxidant, protects the human placenta against hypoxia/reoxygenation (H/R)-induced damage. Primary term villous cytotrophoblasts were cultured under normoxia (8% O2) with or without 1mM melatonin for 72h to induce differentiation into the syncytiotrophoblast. The cells were then cultured for an additional 22h under normoxia or subjected to hypoxia (0.5% O2) for 4h followed by 18h reoxygenation (8% O2) with or without melatonin. H/R induced oxidative stress, which activated the Bax/Bcl-2 mitochondrial apoptosis pathway and the downstream fragmentation of DNA. Villous trophoblast treatment with melatonin reversed all the negative effects induced by H/R to normoxic levels. This study shows that melatonin protects the villous trophoblast against H/R-induced oxidative stress and apoptosis and suggests a potential preventive and therapeutic use of this indolamine in pregnancy complications characterized by syncytiotrophoblast survival alteration., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Nuclear factor-κB (NF-κB) is a transcription factor that plays a crucial role in coordinating innate and adaptive immunity, inflammation, and apoptotic cell death. NF-κB is activated by various inflammatory stimuli including peptide factors and infectious microbes. It is also known as a redox-sensitive transcription factor activated by reactive oxygen species (ROS). Over the past decades, various investigators focused on the role of ROS in the activation of NF-κB by cytokines and lipopolysaccharides. However, recent studies also suggested that ROS have the potential to repress NF-κB activity. Currently, it is not well addressed how ROS regulate activity of NF-κB in a bidirectional fashion. In this paper, we summarize evidence for positive and negative regulation of NF-κB by ROS, possible redox-sensitive targets for NF-κB signaling, and mechanisms underlying biphasic and bidirectional influences of ROS on NF-κB, especially focusing on a role of ROS-mediated induction of endoplasmic reticulum stress., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Calcium influx into neurons triggers neuronal death during cerebral ischemia/reperfusion injury. Various calcium channels are involved in cerebral ischemia/reperfusion injury. Cav3.2 channel is a main subtype of T-type calcium channels. T-type calcium channel blockers, such as pimozide and mibefradil, have been shown to prevent cerebral ischemia/reperfusion injury-induced brain injury. However, the role of Cav3.2 channels in cerebral ischemia/reperfusion injury remains unclear. Here, in vitro and in vivo models of cerebral ischemia/reperfusion injury were established using middle cerebral artery occlusion in mice and high glucose hypoxia/reoxygenation exposure in primary hippocampal neurons. The results showed that Cav3.2 expression was significantly upregulated in injured hippocampal tissue and primary hippocampal neurons. We further established a Cav3.2 gene-knockout mouse model of cerebral ischemia/reperfusion injury. Cav3.2 knockout markedly reduced infarct volume and brain water content, and alleviated neurological dysfunction after cerebral ischemia/reperfusion injury. Additionally, Cav3.2 knockout attenuated cerebral ischemia/reperfusion injury-induced oxidative stress, inflammatory response, and neuronal apoptosis. In the hippocampus of Cav3.2-knockout mice, calcineurin overexpression offset the beneficial effect of Cav3.2 knockout after cerebral ischemia/reperfusion injury. These findings suggest that the neuroprotective function of Cav3.2 knockout is mediated by calcineurin/nuclear factor of activated T cells 3 signaling. Findings from this study suggest that Cav3.2 could be a promising target for treatment of cerebral ischemia/reperfusion injury. [ABSTRACT FROM AUTHOR]

Objective To explore the role and mechanism of pumilio homolog 1 （PUM1） in hypoxia/reoxygenation induced cell injury in human renal tubular epithelial cells （HK-2）. Methods A hypoxia/reoxygenation （H/R） model of HK-2 cells was established in vitro. PUM1 expression was knocked down through small interfering RNA （siRNA）. Cells were randomized into three groups of control， hypoxia/reoxygenation （H/R） and H/R+siRNA. Western blot （WB） method was used for detecting the expression level of PUM1 protein. Cell Count Kit 8 （CCK-8） was employed for detecting cell viability. Hydrogen peroxide （H2O2）， malondialdehyde （MDA） and superoxide dismutase （SOD） were used for evaluating the levels of oxidative stress. Flow cytometry was utilized for detecting the level of cell apoptosis. Results As compared with control group， protein expression level of PUM1 in H/R 3 h group （1.76 ± 0.11 vs 0.98 ± 0.05）， H/R 6 h group （2.89 ± 0.14 vs 0.98 ± 0.05） and H/R 12 h group （3.78 ± 0.08 vs 0.98 ± 0.05） gradually spiked with the prolongation of hypoxic time. As compared with H/R group， knocking down the expression of PUM1 significantly improved the cell viability （73.67 ± 3.42 vs 29.60 ± 2.94）， oxidative stress [H2O2:（13.53 ± 0.85）μmol/L vs （22.43 ± 1.12）μmol/L， MDA: （16.03 ± 0.70）μmol/L vs （31.20 ± 1.50）μmol/L， SOD: （34670 ± 1800）U/L vs （5730 ± 1220）U/L] and apoptotic level [（14.89 ± 1.65）% vs （39.71 ± 1.94）%] after H/R in H/R+si-PUM1 group. Conclusion PUM1 is up-regulated in H/R induced HK-2 cells and its inhibition may alleviate H/R injury through reducing oxidative stress and lowering cell apoptosis levels.

The occurrence of myocardial ischemia/reperfusion injury is commonly observed during cardiac surgery; however, there remains a dearth of effective therapeutic strategies to mitigate this injury. The a disintegrin and metallopeptidase domain 10 (ADAM10) is a transmembrane protein anchored on the cell membrane surface, and its precise mechanism of action in myocardial ischemia/reperfusion injury remains incompletely understood. This study aims to investigate the impact of ADAM10 on cardiomyocyte injury induced by hypoxia/reoxygenation (H/R) and elucidate the underlying mechanisms. The ADAM10 overexpression plasmid was transfected into H9c2 cells, which were subsequently treated with the Notch signaling pathway inhibitor DAPT and cultured under H/R conditions. Cell proliferation activity was assessed using the CCK-8 assay. The levels of LDH, SOD, and MDA were quantified through colorimetric analysis. The levels of ROS and the rate of apoptosis were measured using flow cytometry. The morphological changes in the nucleus of H9c2 cells were observed by employing Hoechst 33258 staining. The mRNA expression levels of ADAM10, Notch1, NICD, and Hes1 in H9c2 cells were determined using qRT-PCR. The expressions of Notch signaling pathway and apoptosis-related proteins were analyzed by Western blot. Overexpression of ADAM10 provided protection to H9c2 cells against injury induced by H/R, leading to an increase in SOD levels and alleviation of oxidative stress caused by the accumulation of ROS and the decrease of SOD activity. Meanwhile, overexpression of ADAM10 inhibited apoptosis in H9c2 cells exposed to H/R by regulating the expression of apoptosis-related proteins, such as Bax, Bcl-2 and Cleaved-caspase-3. Additionally, overexpression of ADAM10 facilitated the activation of the Notch1 signaling pathway in H9c2 cells exposed to H/R by upregulating the protein expression of Notch1, NICD, and Hes1. However, the protective effect of ADAM10 on H/R-induced H9c2 cells was partially reversed by DAPT. Our findings demonstrate that ADAM10 exerts protective effects in H/R-induced H9c2 cells by suppressing oxidative stress and apoptosis via the activation of the Notch signaling pathway. [ABSTRACT FROM AUTHOR]

Calcium influx into neurons triggers neuronal death during cerebral ischemia/reperfusion injury. Various calcium channels are involved in cerebral ischemia/reperfusion injury. Cav3.2 channel is a main subtype of T-type calcium channels. T-type calcium channel blockers, such as pimozide and mibefradil, have been shown to prevent cerebral ischemia/reperfusion injury-induced brain injury. However, the role of Cav3.2 channels in cerebral ischemia/reperfusion injury remains unclear. Here, in vitro and in vivo models of cerebral ischemia/reperfusion injury were established using middle cerebral artery occlusion in mice and high glucose hypoxia/reoxygenation exposure in primary hippocampal neurons. The results showed that Cav3.2 expression was significantly upregulated in injured hippocampal tissue and primary hippocampal neurons. We further established a Cav3.2 gene-knockout mouse model of cerebral ischemia/reperfusion injury. Cav3.2 knockout markedly reduced infarct volume and brain water content, and alleviated neurological dysfunction after cerebral ischemia/reperfusion injury. Additionally, Cav3.2 knockout attenuated cerebral ischemia/reperfusion injury-induced oxidative stress, inflammatory response, and neuronal apoptosis. In the hippocampus of Cav3.2-knockout mice, calcineurin overexpression offset the beneficial effect of Cav3.2 knockout after cerebral ischemia/reperfusion injury. These findings suggest that the neuroprotective function of Cav3.2 knockout is mediated by calcineurin/nuclear factor of activated T cells 3 signaling. Findings from this study suggest that Cav3.2 could be a promising target for treatment of cerebral ischemia/reperfusion injury.

AIM: To investigate the regulatory role of retinoid X receptor （RXR） in oxidative stress response of rat type II alveolar epithelial cells（ AECII） induced by hypoxia/reoxygenation（ HR）. METHODS: The AECII were divided into control （C） group, HR group, HR+solvent dimethyl sulfoxide （DMSO） group （HD group）, HR+RXR agonist 9-cis-retinoic acid （9-RA） group （RA group）, and HR+RXR antagonist HX531 group （HX group）. Cell Counting Kit-8 （CCK-8） method was used to measure the cell viability. Immunofluorescence staining was used to detect the expression of surfactant protein A （SP-A） and RXRα in AECII. Kits were detected to the levels of superoxide dismutase （SOD） and malondialdehyde （MDA） in cells. Transmission electron microscopy was used to observe the ultrastructural changes of the cells. Western blot was used to detect the protein level of nuclear factor E2-related factor 2 （Nrf2）. RT-PCR was used to detect the expression level of Nrf2 mRNA. RESULTS: Compared with C group, the cell viability and SOD activity in HR, HD, RA and HX groups were decreased significantly （P<0. 05）, the MDA content were increased significantly （P<0. 05）, the Nrf2 mRNA and protein expression levels were decreased significantly （P<0. 05 or P<0. 01）, and the immunofluorescence expression of RXRα was significantly increased （P<0. 01）. Compared with HR and HX groups, the cells in RA group showed significantly increased cell viability （P<0. 05）, increased SOD activity（ P<0. 05）, decreased MDA content （P<0. 05）, increased Nrf2 mRNA and protein expression levels （P<0. 01）, and significantly increased immunofluorescence expression of RXRα （P<0. 01）. CONCLUSION: Hypoxia/reoxygenation can aggravate the oxidative stress response of rat AECII, and RXR agonist intervention can alleviate HR-induced rat AECII injury by inhibiting oxidative stress. [ABSTRACT FROM AUTHOR]

Background: Anisodine hydrobromide (AT3), an anti-cholinergic agent, could be delivered to the brain across the blood-brain barrier and has been used clinically for the treatment of cerebral ischemia/reperfusion injury. Endothelial dysfunction can be caused by hypoxia/reoxygenation (H/R) via oxidative stress and metabolic alterations. The present study investigated whether AT3 regulates the production of nitric oxide (NO) and reactive oxygen species (ROS), and the HIF-1α pathway via regulation of muscarinic acetylcholine receptors (mAChRs) in brain microvascular endothelial cells after H/R exposure. Methods: Under H/R conditions, hCMEC/D3 cerebral microvascular endothelial cells were treated with AT3. Specific inhibitors of M2- and M4- mAChRs were used to explore the mechanism by which AT3 influences oxidative stress in endothelial cells. Then, mAChRs expression was detected by western blotting and NO production was detected by Greiss reaction. The intracellular ROS level was measured using DCFH-DA probes. The expression of hypoxia-inducible transcription factor 1α (HIF-1α) was also detected. Results: While H/R induced the expression of M2- and M4-mAChRs, AT3 suppressed the H/R-upregulated M2- and M4-mAChRs. H/R also induced the production of NO, ROS, and apoptosis. AT3 and M4-mAChR inhibitors inhibited the H/R-induced production of NO and ROS and apoptosis. HIF-1α was induced by H/R, but was suppressed by AT3. Conclusion: Thus, the in vitro evidence shows that AT3 protects against H/R injury in cerebral microvascular endothelial cells via inhibition of HIF-1α, NO and ROS, predominantly through the downregulation of M4-mAChR. The findings offer novel understandings regarding AT3-mediated attenuation of endothelial cell apoptosis and cerebral ischemia/ reperfusion injury. [ABSTRACT FROM AUTHOR]

Background: Myocardial infarction is one of the leading causes of mortality worldwide; hence, there is an urgent need to discover novel cardioprotective strategies. Kynurenic acid (KYNA), a metabolite of the kynurenine pathway, has been previously reported to have cardioprotective effects. However, the mechanisms by which KYNA may be protective are still unclear. The current study addressed this issue by investigating KYNA's cardioprotective effect in the context of myocardial ischemia/reperfusion. Methods: H9C2 cells and rats were exposed to hypoxia/reoxygenation or myocardial infarction, respectively, in the presence or absence of KYNA. In vitro, cell death was quantified using flow cytometry analysis of propidium iodide staining. In vivo, TTC-Evans Blue staining was performed to evaluate infarct size. Mitochondrial respiratory chain complex activities were measured using spectrophotometry. Protein expression was evaluated by Western blot, and mRNA levels by RT-qPCR. Results: KYNA treatment significantly reduced H9C2-relative cell death as well as infarct size. KYNA did not exhibit any effect on the mitochondrial respiratory chain complex activity. SOD2 mRNA levels were increased by KYNA. A decrease in p62 protein levels together with a trend of increase in PARK2 may mark a stimulation of mitophagy. Additionally, ERK1/2, Akt, and FOXO3α phosphorylation levels were significantly reduced after the KYNA treatment. Altogether, KYNA significantly reduced myocardial ischemia/reperfusion injuries in both in vitro and in vivo models. Conclusion: Here we show that KYNA-mediated cardioprotection was associated with enhanced mitophagy and antioxidant defense. A deeper understanding of KYNA's cardioprotective mechanisms is necessary to identify promising novel therapeutic targets and their translation into the clinical arena. [ABSTRACT FROM AUTHOR]

Acute myocardial infarction is regarded as myocardial necrosis resulting from myocardial ischemia/reperfusion (I/R) damage and retains a major cause of mortality. Neferine, which was extracted from the green embryos of mature seeds of Nelumbo nucifera Gaertn., has been reported to possess a broad range of biological activities. However, its underlying mechanism on the protective effect of I/R has not been fully clarified. A hypoxia/reoxygenation (H/R) model with H9c2 cells closely simulating myocardial I/R injury was used as a cellular model. This study intended to research the effects and mechanism underlying neferine on H9c2 cells in response to H/R stimulation. Cell Counting Kit‐8 and lactate dehydrogenase (LDH) release assays were employed to measure cell viability and LDH, respectively. Apoptosis and reactive oxygen species (ROS) were determined by flow cytometry analysis. Oxidative stress was evaluated by detecting malondialdehyde, superoxide dismutase, and catalase. Mitochondrial function was assessed by mitochondrial membrane potential, ATP content, and mitochondrial ROS. Western blot analysis was performed to examine the expression of related proteins. The results showed that hypoxia/reoxygenation (H/R)‐induced cell damage, all of which were distinctly reversed by neferine. Moreover, we observed that neferine inhibited oxidative stress and mitochondrial dysfunction induced by H/R in H9c2 that were concomitant with increased sirtuin‐1 (SITR1), nuclear factor erythroid 2‐related factor 2 (Nrf2), and heme oxygenase‐1 expression. On the contrary, silencing the SIRT1 gene with its small interferingRNA eliminated the beneficial effects of neferine. It is concluded that neferine preconditioning attenuated H/R‐induced cardiac damage via suppressing apoptosis, oxidative stress, and mitochondrial dysfunction, which may be partially ascribed to the activation of SIRT1/Nrf2 signaling pathway. [ABSTRACT FROM AUTHOR]

Dehydrocorydaline (DHC), an active component of Corydalis yanhusuo (Y.H. Chou & Chun C. Hsu) W.T. Wang ex Z.Y. Su & C.Y. Wu (Papaveraceae), exhibits protective and pain-relieving effects on coronary heart disease, but the underlying mechanism still remains unknown. Network pharmacology and experimental validation both in vivo and in vitro were applied to assess whether DHC can treat myocardial ischemia-reperfusion injury (MIRI) by regulating the forkhead box O (FoxO) signalling pathway to inhibit apoptosis. DHC and MIRI targets were retrieved from various databases. Molecular docking and microscale thermophoresis (MST) determined potential binding affinity. An in vivo mouse model of MIRI was established by ligating the left anterior descending coronary artery. C57BL/6N mice were divided into sham, MIRI, and DHC (intraperitoneal injection of 5 mg/kg DHC) groups. Haematoxylin and eosin, Masson, and immunohistochemical stainings verified DHC treatment effects and the involved signalling pathways. In vitro , H9c2 cells were incubated with DHC and underwent hypoxia/reoxygenation. TUNEL, JC-1, and reactive oxygen species stainings and western blots were used to explore the protective effects of DHC and the underlying mechanisms. Venny analysis identified 120 common targets from 121 DHC and 23,354 MIRI targets. DHC exhibited high affinity for CCND1, CDK2, and MDM2 (<−7 kcal/mol). In vivo, DHC attenuated decreases in left ventricular ejection fraction and fractional shortening, reduced infarct sizes, and decreased cTnI and lactate dehydrogenase levels. In vitro , DHC alleviated apoptosis and oxidative stress in the hypoxia/reoxygenation model by attenuating ΔΨm disruption; reducing the production of reactive oxygen species; upregulating Bax and CCND1 via the FoxO signalling pathway, as well as cleaved-caspase 8; downregulating the apoptosis-associated proteins Bcl-2, Bid, cleaved-caspase 3, and cleaved-caspase 9; and promoting the phosphorylation of FOXO1A and MDM2. By upregulating the FoxO signaling pathway to inhibit apoptosis, DHC exerts a cardioprotective effect, which could serve as a potential therapeutic option for MIRI. [Display omitted] [ABSTRACT FROM AUTHOR]

Myocardial infarction (MI) is caused by the formation of plaques in the arterial walls, leading to a decrease of blood flow to the heart and myocardium injury as a result of hypoxia. Ferroptosis is a crucial event in myocardial injury, and icariin (ICA) exerts protective effects against myocardial injury. Here, we investigated the protective mechanism of ICA in hypoxia/reoxygenation (H/R)‐induced ferroptosis of cardiomyocytes. H9C2 cells were subjected to H/R induction. The content of lactate dehydrogenase and the levels of oxidative stress and intracellular ferrous ion Fe2+ were measured. The levels of ferroptosis markers (ACSL4 and GPX4) were detected. H/R‐induced H9C2 cells were cultured with ICA in the presence or absence of ferroptosis inducer (erastin). Znpp (an HO‐1 inhibitor) was added to ICA‐treated H/R cells to verify the role of the Nrf2/HO‐1 pathway. H/R‐induced H9C2 cells showed reduced viability, enhanced oxidative stress and lactate dehydrogenase content, increased levels of Fe2+ and ACSL4, and decreased levels of GPX4. ICA inhibited H/R‐induced ferroptosis and oxidative stress in cardiomyocytes. Erastin treatment reversed the inhibitory effect of ICA on ferroptosis in H/R cells. The expression of Nrf2 and HO‐1 in H/R‐induced H9C2 cells was reduced, whereas ICA treatment reversed this trend. Inhibition of the Nrf2/HO‐1 pathway reversed the protective effect of ICA on H/R‐induced ferroptosis. Collectively, our results suggest that ICA attenuates H/R‐induced ferroptosis of cardiomyocytes by activating the Nrf2/HO‐1 signaling pathway.

Purpose: To investigate the protective role of tectorigenin in myocardial ischaemia/reperfusion. Methods: Myocardial cells (H9c2) were treated with different concentrations of tectorigenin and exposed to hypoxia/reoxygenation. Cell viability and apoptosis were determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) and TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) staining, respectively. Oxidative stress and inflammation were evaluated using enzyme-linked immunosorbent assay (ELISA), while autophagy and the underlying mechanisms of action were evaluated by Western blot. Results: Tectorigenin enhanced the proliferative activity of H9c2 under hypoxia/reoxygenation conditions, and significantly reduced the apoptotic activity (p < 0.001) through decrease in Bax and increase in Bcl-2. Tectorigenin also significantly up-regulated SOD (superoxide dismutase) and GSH (glutathione) levels (p < 0.01), and down-regulated MDA (malondialdehyde) and MPO (myeloperoxidase) in hypoxia/reoxygenation-induced H9c2. TNF-a (tumor necrosis factor-a), IL(interleukin)-1ß, and IL-6 levels were also inhibited by tectorigenin by down-regulating p-p65. Hypoxia/reoxygenation-induced increase in p62 and decrease in Beclin-1 and LC3-II/LC3-I were reversed by tectorigenin. Protein expressions of p-mTOR, p-AKT, and p-PI3K in hypoxia/reoxygenationinduced H9c2 were elevated by tectorigenin. Conclusion: Tectorigenin exerts anti-oxidant, anti-inflammatory, and anti-autophagic effects on hypoxia/reoxygenation-induced H9c2 through the activation of PI3K/AKT/mTOR pathway, thus suggesting that it is a potential agent for the management of myocardial ischaemia/reperfusion. [ABSTRACT FROM AUTHOR]

Preeclampsia (PE) is a pregnancy-specific disorder that affects 3 to 5% of pregnancies worldwide and is one of the leading causes of maternal and fetal morbidity and mortality. Nevertheless, how these events occur remains unclear. We hypothesized that the induction of hypoxic conditions in vitro in primary human trophoblast cells would mimic several characteristics of PE found in vivo. We applied and characterized a model of primary cytotrophoblasts isolated from healthy pregnancies that were placed under different oxygen concentrations: ambient O2 (5% pCO2, 21%pO2, 24 h, termed "normoxia"), low O2 concentration (5% pCO2, 1.5% pO2, 24 h, termed "hypoxia"), or "hypoxia/reoxygenation" (H/R: 6 h intervals of normoxia and hypoxia for 24 h). Various established preeclamptic markers were assessed in this cell model and compared to placental tissues obtained from PE pregnancies. Seventeen PE markers were analyzed by qPCR, and the protein secretion of soluble fms-like tyrosine kinase 1 (sFlT-1) and the placenta growth factor (PlGF) was determined by ELISA. Thirteen of seventeen genes associated with angiogenesis, the renin–angiotensin system, oxidative stress, endoplasmic reticulum stress, and the inflammasome complex were susceptible to H/R and hypoxia, mimicking the expression pattern of PE tissue. In cell culture supernatants, the secretion of sFlT-1 was increased in hypoxia, while PlGF release was significantly reduced in H/R and hypoxia. In the supernatants of our cell models, the sFlT-1/PlGF ratio in hypoxia and H/R was higher than 38, which is a strong indicator for PE in clinical practice. These results suggest that our cellular models reflect important pathological processes occurring in PE and are therefore suitable as PE in vitro models. [ABSTRACT FROM AUTHOR]

The search for new products to treat ischemic heart diseases has interested scientists for years as it is the main cause of cardiovascular disease mortality. In this study, we evaluated the protective effects of Panax bipinnatifidus Seem. extracts (PSE) on hypoxia/reoxygenation (HR)-treated rat H9C2 cardiomyocytes. Rat H9C2 cardiomyocytes were either grown under normal condition or subjected to HR conditions. NecroX-5 (10 μM) was used as positive control for cardiac protective effect. The PSE (5-500 µg/ml) was employed to culture media at the onset of reoxygenation. Cell viability and mitochondrial function were tested using a Cell Counting Kit-8 and suitable fluorescence kits. The obtained data shows that HR conditions dramatically induced H9C2 cell death (p

• PARP plays a critical role in liver ischemia/reperfusion-induced injury. • PARP is more critical than caspases in post-hypoxic cell injury. • PARP and caspases induce post-hypoxic cell death, independent of each other. • Hypoxia-induced PARP activation is redox-dependent, more than caspases. • PARP induces necrotic programmed cell death, parthanatos and necroptosis. Poly(ADP-ribose) polymerase (PARP) is a DNA-repairing enzyme activated by extreme genomic stress, and therefore is potently activated in the remnant liver suffering from ischemia after surgical resection. However, the impact of PARP on post-ischemic liver injury has not been elucidated yet. We investigated the impact of PARP on murine hepatocyte/liver injury induced by hypoxia/ischemia, respectively. PJ34, a specific inhibitor of PARP, markedly protected against hypoxia/reoxygenation (H/R)-induced cell death, though z-VAD-fmk, a pan-caspase inhibitor similarly showed the protective effect. PJ34 did not affect H/R-induced caspase activity or caspase-mediated cell death. z-VAD-fmk also did not affect the production of PAR (i.e., PARP activity). Therefore, PARP- and caspase-mediated cell death occurred in a mechanism independent of each other in H/R. H/R immediately induced activation of PARP and cell death afterwards, both of which were suppressed by PJ34 or Trolox, an antioxidant. This suggests that H/R-induced cell death occurred redox-dependently through PARP activation. H/R and OS induced nuclear translocation of apoptosis inducing factor (AIF, a marker of parthanatos) and RIP1-RIP3 interaction (a marker of necroptosis), both of which were suppressed by PJ34. H/R induced PARP-mediated parthanatos and necroptosis redox-dependently. In mouse experiments, PJ34 significantly reduced serum levels of AST, ALT & LDH and areas of hepatic necrosis after liver ischemia/reperfusion, similar to z-VAD-fmk or Trolox. PARP, activated by ischemic damage and/or oxidative stress, may play a critical role in post-ischemic liver injury by inducing programmed necrosis (parthanatos and necroptosis). PARP inhibition may be one of the promising strategies against post-ischemic liver injury. [ABSTRACT FROM AUTHOR]

Introduction and Objective: Dexmedetomidine (Dex) and a number of miRNAs contribute to ischemia/reperfusion injury. We aimed to explore the role of Dex and miR-146a on myocardial cells injured by hypoxia/reoxygenation (H/R). Method: H9C2 cells were injured by H/R. Cell viability was tested using the cell counting kit-8. Lactate dehydrogenase (LDH) activity, superoxide dismutase (SOD) activity, and malondialdehyde (MDA) levels were determined using commercial kits. Flow cytometry was performed to determine apoptosis rate and reactive oxygen species (ROS) level. Protein and mRNA levels were assessed using Western blot and qPCR. Results: miR-146a expression and cell viability of H9C2 cells were downregulated under the circumstance of H/R injury. The tendency could be reversed by Dex, which could also upregulate SOD activity and decrease apoptosis, LDH activity, MDA, 78-kDa glucose-regulated protein (GRP78), and C/EBP homologous protein (CHOP) levels of H9C2 cells. GRP78, CHOP levels, and cell viability were negatively modulated by miR-146a. Dex elevated cell viability, catalase, MnSOD, and NAD(P)H dehydrogenase (NQO1) levels but suppressed apoptosis rate, GRP78, and CHOP levels by increasing miR-146a expression and downregulating ROS, phosphorylation of p38, and extracellular signal-regulated kinases 1/2 levels. By using SB203580 (SB), the p38 mitogen-activated protein kinase (MAPK) inhibitor, Dex or the inhibition of miR-146 upregulated cell viability but downregulated GRP78 and CHOP levels. Conclusion: Dex might regulate miR-146a expression, which could further modulate the endoplasmic reticulum stress and oxidative stress and eventually affect the cell viability and apoptosis of myocardial cells injured by H/R via the MAPK signal pathway. [ABSTRACT FROM AUTHOR]

Purpose: To explore the effects of octreotide (OCT) on oxidative stress, inflammation and apoptosis in hypoxia/reoxygenation (H/R)-induced cerebral infarction. Methods: The in vitro model of cerebral infarction was established by treating N2A cells with hypoxia for 4 h and reoxygenation for 24 h. The viability of N2A cells was determined by CCK-8 assay. The cells were divided into 3 groups: control group, H/R group, and H/R+OCT group. The cells in H/R+OCT group were pretreated with OCT (60 ng/mL) before H/R treatment. The oxidative stress of N2A cells were assessed by determining the levels of superoxide dismutase (SOD), glutathione peroxidase (GSHPx), catalase (CAT), reactive oxygen species (ROS) and malondialdehyde (MDA). Inflammation of N2A cells was evaluated by evaluating the levels of TNF-α, IL-1β, IL-6, and IL-8, while the apoptosis of N2A cells was assessed by flow cytometry. Western blot analysis was used to determine the expression of Bcl-2, Bax, TLR4, MyD88, and NF-κB. Results: Octreotide treatment significantly reduced the level of oxidative stress. The inflammation of N2A cells caused by hypoxia/reoxygenation was inhibited by treatment with octreotide. Apoptosis of N2A cells was also inhibited by octreotide treatment. Hypoxia/reoxygenation activated TLR4/MyD88/NF-κB signaling pathway, while octreotide inhibits the activation of this pathway. Conclusion: The results reveal that octreotide inhibits hypoxia/reoxygenation-induced oxidative stress, as well as the inflammation, and apoptosis of N2A cells by inhibiting TLR4/MyD88/NF-κB signaling pathway. Thus, these findings may provide new insights into the treatment of cerebral infarction. [ABSTRACT FROM AUTHOR]

Myocardial infarction (MI) is caused by the formation of plaques in the arterial walls, leading to a decrease of blood flow to the heart and myocardium injury as a result of hypoxia. Ferroptosis is a crucial event in myocardial injury, and icariin (ICA) exerts protective effects against myocardial injury. Here, we investigated the protective mechanism of ICA in hypoxia/reoxygenation (H/R)‐induced ferroptosis of cardiomyocytes. H9C2 cells were subjected to H/R induction. The content of lactate dehydrogenase and the levels of oxidative stress and intracellular ferrous ion Fe2+ were measured. The levels of ferroptosis markers (ACSL4 and GPX4) were detected. H/R‐induced H9C2 cells were cultured with ICA in the presence or absence of ferroptosis inducer (erastin). Znpp (an HO‐1 inhibitor) was added to ICA‐treated H/R cells to verify the role of the Nrf2/HO‐1 pathway. H/R‐induced H9C2 cells showed reduced viability, enhanced oxidative stress and lactate dehydrogenase content, increased levels of Fe2+ and ACSL4, and decreased levels of GPX4. ICA inhibited H/R‐induced ferroptosis and oxidative stress in cardiomyocytes. Erastin treatment reversed the inhibitory effect of ICA on ferroptosis in H/R cells. The expression of Nrf2 and HO‐1 in H/R‐induced H9C2 cells was reduced, whereas ICA treatment reversed this trend. Inhibition of the Nrf2/HO‐1 pathway reversed the protective effect of ICA on H/R‐induced ferroptosis. Collectively, our results suggest that ICA attenuates H/R‐induced ferroptosis of cardiomyocytes by activating the Nrf2/HO‐1 signaling pathway. [ABSTRACT FROM AUTHOR]

Objective To investigate the targeted regulation of long noncoding RNA (lncRNA)FGD5-AS1 on miRNA-421 (miR-421)and its effect on cardiomyocyte injury induced by hypoxia/reoxygenation (H/R). Methods H/R was used to induce the damage of cardiomyocytes H9C2. Real-time fluorescence quantitative PCR (qRT-PCR)was used to detect the expression of lncRNA FGD5-AS1 and miR-421. We transfected pcDNA-FGD5-AS1, anti-miR-421, or co-transfected pcDNA-FGD5-AS1 and miR-421 into cardiomyocytes, and then performed H/R injury. The kits were used to evaluat content of malondialdehyde (MDA)and activity of superoxide dismutase (SOD)and glutathione peroxidase (GSH-Px). Flow cytometry was used to measure the apoptosis rate of cells, and Western blotting was applied to determine the protein expression of B cell lymphoma/lewkmia-2 (Bcl-2)and Bcl-2 associated X protein (Bax). Bioinformatics prediction and dual luciferase report experiment verified the targeted regulation of lncRNA FGD5-AS1 on miR-421. Results Hypoxia/ reoxygenation reduced the expression of lncRNA FGD5-AS1, activity of SOD and GSH-Px and expression of Bcl-2 protein, while increased the expression of miR-421, MDA content, apoptosis rate, expression of Bax protein in the cardiomyocyte, and the difference was statistically significant (all P<0. 05). Overexpression of lncRNA FGD5-AS1 decreased the MDA content, apoptosis rate, and Bax protein expression of cardiomyocytes induced by hypoxia/reoxygenation, while increased activity of SOD, GSH-Px, and expression of Bcl-2 protein, and the difference was statistically significant (all P< 0. 05), and it was the same as the result of inhibiting the expression of miR-421. lncRNA FGD5-AS1 targeted and regulated the expression of miR-421. Co-transfection of pcDNA-FGD5-AS1 and miR-421 increased the MDA content, apoptosis rate, Bax protein expression of cardiomyocytes induced by hypoxia/reoxygenation, and decreased SOD, GSH-Px activity, and Bcl-2 protein expression. The differences were statistically significant (all P<0. 05). Conclusion In cardiomyocytes induced by hypoxia/reoxygenation, lncRNA FGD5-AS1 can reduce oxidative stress and inhibit apoptosis of cardiomyocytes by targeting miR-421, thereby reducing injury of cardiomyocytes induced by hypoxia/reoxygenation. [ABSTRACT FROM AUTHOR]

Background: Nerol was reported as a natural anti-oxidant product and its protective effects against cardiovascular diseases have been documented. Our current study was designed to explore the cardioprotective effect of Nerol on hypoxia/reoxygenation (H/R)-induced production of reactive oxygen species (ROS) and cell apoptosis in H9c2 cells. The potential molecular mechanisms were further investigated. Methods: The cells were treated with 2.5 or 5 µM Nerol before or after H/R. Lactate dehydrogenase (LDH) release, cell viability, oxidative stress markers, and apoptotic proteins were assessed by cell counting kit-8, LDH release assay, commercial kits, and Western blot, respectively. To explore the underlying mechanism, the phosphorylation of p85 and p38, regulatory subunits of phosphoinositide-3-kinase (PI3K)/protein kinase B (AKT) and mitogen-activated protein kinase (MAPK), was evaluated by Western blot. To further confirm that the PI3K/AKT signaling pathway participated in the cardiomyocyte protection, H9c2 cells were treated with 5 µM Nerol in the presence or absence of 5 µM BEZ235 or LY294002 followed by H/R treatment. Results: H/R remarkably induced apoptosis, LDH release and ROS production. The cell viability was suppressed via inhibiting the PI3K/AKT signaling pathway activation. By contrast, pretreatment with Nerol can neutralize these effects by activating the PI3K/AKT signaling pathway. With the addition of BEZ235 or LY294002, the inhibitory effects of Nerol were abolished. Conclusion: Nerol provided promising cardioprotective effect against H/R-induced injuries in H9c2 cells by activating the PI3K/AKT pathway.

BACKGROUND: Ischemic heart disease has become one of the important diseases that threaten human health, but the molecular mechanism of its occurrence and development has not yet been elucidated. OBJECTIVE: To investigate the effects of isoliquiritigenin (ISL) on oxidative stress and apoptosis in cardiomyocytes after hypoxia/reoxygenation (H/R) injury and its possible mechanism. METHODS: Rat cardiomyocytes H9C2 were cultured in vitro and randomly divided into normal control group, H/R group, H/R+ISL 10, 20, 40, 80 μmol/L groups, H/R+si-con group, H/R+si-SETD7 group, H/R+ISL+pcDNA group, H/R+ISL+pcDNA-SETD7 group. Cell viability was measured using methylthiazolyl tetrazolium (MTT) assay. Apoptosis was detected by flow cytometry. The levels of reactive oxygen species (ROS), glutathione peroxidase (GSH-Px), and malondialdehyde (MDA) in the cells were measured. The expression of apoptosis-related protein Cleaved Caspase-3 was detected by western blot assay. RESULTS AND CONCLUSION: Compared with the normal control group, the cell survival rate of H/R group was significantly decreased (P < 0.05), the apoptosis rate was significantly increased (P < 0.05), Cleaved caspase-3 expression and ROS and MDA levels were significantly increased (P < 0.05), while the GSH-Px content was significantly reduced (P < 0.05). Compared with the H/R group, the cell survival rate of H/R+ISL 10, 20, 40, 80 μmol/L groups was significantly increased (P < 0.05), and the apoptosis rate was significantly decreased (P < 0.05), and the expression of SETD7 and Cleaved caspase-3 was significantly decreased (P < 0.05), and the levels of ROS and MDA were significantly decreased (P < 0.05), while the content of GSH-Px was significantly increased (P < 0.05). Compared with the H/R+si-con group, the cell survival rate of H/R+si-SETD7 group was significantly increased (P < 0.05), the apoptosis rate was significantly decreased (P < 0.05), the expression of SETD7 and Cleaved caspase-3 was significantly decreased (P < 0.05), and the levels of ROS and MDA were significantly decreased (P < 0.05), while the content of GSH-Px was significantly increased (P < 0.05). Compared with the H/R+ISL+pcDNA group, the cell survival rate of H/R+ISL+pcDNA-SETD7 group was significantly decreased (P < 0.05), the apoptosis rate was significantly increased (P < 0.05), the expression of SETD7 and Cleaved caspase-3 was significantly increased (P < 0.05), and the levels of ROS and MDA were significantly increased (P < 0.05), while the content of GSH-Px was significantly decreased (P < 0.05). These findings indicate that ISL can inhibit the expression of SETD7 to reduce the apoptosis of cardiomyocytes induced by H/R and enhance the cell antioxidant capacity, thereby protecting cardiomyocytes against H/R injury. [ABSTRACT FROM AUTHOR]

Cardiomyocyte apoptosis induced by hypoxia and ischemia plays important roles in heart dysfunction after acute myocardial infarction (AMI). However, the mechanism of apoptosis induction remains unclear. A previous study reported that Y‐box protein 1 (YB1) is upregulated after myocardial hypoxia/reoxygenation or ischemia/reperfusion (H/R or I/R, respectively) injury; however, whether YB1 is associated with H/R‐induced cardiomyocyte apoptosis is completely unknown. In the present study, we investigated the roles of YB1 in H/R‐induced cardiomyocyte apoptosis and the possible underlying molecular mechanisms. In vitro, H/R treatment upregulated the YB1 expression in H9C2 cells, whereas YB1 knockdown inhibited H/R‐induced cardiomyocyte apoptosis and induced H9C2 cell proliferation via Src homology region 2 domain‐containing phosphatase 1 (SHP‐1)‐mediated activation of signal transducer and activator of transcription 3 (STAT3). In vivo, YB1 knockdown ameliorated AMI, reducing infarct size, cardiomyocyte apoptosis, and oxidative stress, via SHP‐1‐mediated inactivation of STAT3. Additionally, YB1 knockdown inhibited H/R‐ or I/R‐induced oxidative stress in vitro and in vivo. H/R and I/R increase YB1 expression, and YB1 knockdown ameliorates AMI injury via SHP‐1‐dependent STAT3 inactivation. [ABSTRACT FROM AUTHOR]

Resveratrol (RES) is a naturally occurring antioxidant compound found in red wine. Although it has been demonstrated to have a cardioprotective effect, the mechanism underlying this effect remains to be fully elucidated. The aim of the present study was to determine whether RES exerts a protective effect against mitochondrial oxidative stress and apoptosis in neonatal rat cardiomyocytes (NRCMs) induced by hypoxia/reoxygenation (H/R) injury. Primary cultured NRCMs were used as a model system and were divided into four experimental groups: Control, H/R, H/R + DMSO (H/R with 0.2% DMSO) and H/R + RES (H/R with 100 µM RES) groups. Mitochondrial oxidative stress was determined by measuring the alteration in the mitochondrial membrane potential (ΔΨm) of NRCMs, the release of lactate dehydrogenase (LDH) and the ratio of B-cell lymphoma 2 (Bcl-2)/Bcl-2-associated X protein (Bax) from NRCMs. Cell apoptosis was assessed by measuring cell apoptotic rates and the activity of caspase 3. In the H/R+RES group, RES significantly alleviated structural impairment, including disordered α-actin and F-actin, in the NRCMs induced by H/R injury. RES attenuated H/R injury-induced mitochondria oxidative stress. RES also attenuated H/R injury-induced cell apoptosis; it decreased the NRCM apoptotic rate from 84.25±7.41% (H/R) to 46.39±5.43% (H/R+RES) (P<0.05, n=4), rescued the decrease in the Bcl2/Bax ratio induced by H/R from 0.53±0.08-fold (H/R) to 0.86±0.06-fold (H/R+RES) (P<0.05, n=5) and alleviated the increased activity of caspase 3 induced by H/R from 1.32±0.06-fold to 1.02±0.04-fold (P<0.05, n=5). Furthermore, RES significantly attenuated the increment of LDH release induced by H/R injury in NRCMs from 1.41±0.03-fold (H/R) to 1.02±0.06-fold (H/R+RES) (P<0.01, n=4) and alleviated the depolarization of ΔΨm induced by H/R, shifting the ratio of JC-1 monomer from 62.39±1.82% (H/R) to 35.31±8.63% (H/R+RES) (P<0.05, n=4). RES alleviated the decrease in sirtuin 1 induced by H/R injury from 0.61±0.06-fold (H/R) to 1.01±0.05-fold (H/R+RES) (P<0.05, n=5). In conclusion, the present study is the first, to the best of our knowledge, to demonstrate that RES provides cardioprotection against H/R injury through decreasing mitochondria-mediated oxidative stress injury and structural impairment in NRCMs. These results provide scientific evidence for the clinical application of RES in the treatment of cardiac conditions. [ABSTRACT FROM AUTHOR]

Rapid fluctuations of the oxygen content of both natural and anthropogenic origin are relatively common in freshwater environments. Fish adaptation to these conditions implies tolerance of both low levels of oxygen availability and reoxygenation. Hypoxia tolerance in fish has been widely studied, but the involvement of mitochondria in the response of fish to rapid hypoxia/reoxygenation stress is less known. Zebrafish, a floodplain species, is likely facing significant changes in dissolved oxygen in its natural environment and displays a moderate ability to tolerate hypoxia. In the present study, we report the effects of an acute hypoxia/reoxygenation stress (H/R) protocol on mitochondrial functionality (respiration, complex activities, rate of H2O2 release) and redox state (level of HPs and protein oxidation) of muscle tissue. In parallel, the animal metabolic performance (routine metabolism, nitrogen excretion and swimming performance) was measured. Additionally, the recovery from H/R was tested 20 h after treatment. A significant stimulation by H/R of muscle mitochondrial respiration and H2O2 release was observed, which was only in part counteracted by stimulation of the antioxidant system, resulting in an increased level of lipid peroxides and protein carbonyls. In parallel, H/R increased the animal oxygen consumption and urea excretion rate and reduced routine activity. A significant strong reduction of endurance at 80% Ucrit was also observed. Most of the altered parameter did not recover 20 h after reoxygenation. These data indicate a significant alteration of zebrafish muscle mitochondrial state after acute H/R, associated with changes in tissue redox state and locomotor performance. [ABSTRACT FROM AUTHOR]

Background & Aims: Deep hypothermic circulatory arrest (DHCA) is a cerebral protection technique that has been used in the operations involving the aortic arch and brain aneurysm for decades. We previous showed that DHCA treated rats developed a significant oxidative stress and apoptosis in neurons. We here intend to investigate the protective the effect of hydrogen against oxidative stress-induced cell injury and the involved mechanisms using an in vitro experimental model of hypoxia/reoxygenation (H/R) on HT-22 cells. Methods: The model of H/R was established using an airtight culture container and the anaeropack. Measurement of mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) production was used H2DCFDA and JC-1 staining. Western blot was used for the quantification of Akt, p-Akt, Bcl-2, Bax and cleaved caspase-3 proteins. The microRNA (miRNA) profile in hippocampal neurons from rat model of DHCA was determined by miRNA deep sequencing. Results: The elevation of ROS and reduction of MMP were significantly induced by the treatment with hypoxia for 18 h followed by reoxygenation for 6 h. Hydrogen treatment significantly reduced H/R-caused cell death. The levels of p-Akt (Ser 473) and Bcl-2 were significantly increased while Bax and cleaved caspase-3 were decreased by hydrogen treatment on the model of H/R. The expression of miR-200 family was significantly elevated in model of DHCA and H/R. Hydrogen administration inhibited the H/R-induced expression of miR-200 family in HT-22 cells. In addition, inhibition of miR-200 family suppressed H/R-caused cell death through reducing ROS production. Conclusions: These results suggest that H/R causes oxidative stress-induced cell death and that the hydrogen protects against H/R-induced cell death in HT22 cells, in part, due to reducing expression of miR-200 family.

Alternative redox stimuli such as pervanadate or hypoxia/reoxygenation, induce transcription factor NF-κB by phospho-tyrosine-dependent and proteasome-independent mechanisms. While considerable attention has been paid to the absence of proteasomal regulation of tyrosine phosphorylated IκBα, there is a paucity of information regarding proteasomal regulation of signaling events distinct from tyrosine phosphorylation of IκBα. To delineate roles for the ubiquitin-proteasome pathway in the phospho-tyrosine dependent mechanism of NF-κB induction, we employed the proteasome inhibitor, Aclacinomycin, and the phosphotyrosine phosphatase inhibitor, pervanadate (PV). Results from these studies demonstrate that phospho-IκBα (Tyr-42) is not subject to proteasomal degradation in a murine stromal epithelial cell line, confirming results previously reported. Correspondingly, proteasome inhibition had no discernable effect on the key signaling intermediaries, Src and ERK1/2, involved in the phospho-tyrosine mechanisms regulating PV-mediated activation of NF-κB. Consistent with previous reports, a significant redox imbalance leading to the activation of tyrosine kinases, as occurs with pervanadate, is required for the induction of NF-κB. Strikingly, our studies demonstrate that proteasome inhibition can potentiate oxidative stress associated with PV-stimulation without impacting kinase activation, however, other cellular implications for this increase in intracellular oxidation remain to be fully delineated.

Hypoxia/reoxygenation (H/R) often results in cellular oxidative stress and the subsequent apoptosis of cardiac microvascular endothelial cells (CMECs). More recently, studies have highlighted the therapeutic effects of matrine on various cardiovascular diseases. Thus, the aim of the present study was to investigate the underlying mechanism and effects of matrine on hypoxia/reoxygenation (H/R)-induced apoptosis of CMECs in rats. CMECs from Sprague Dawley (SD) rats were primarily treated with H/R, ld (low-dose, 0.5 mg/mL)-Ma + H/R, md (middle-dose, 1 mg/mL)-Ma + H/R, hd (high-dose, 2 mg/mL)-Ma + H/R, Ma + AG490 + H/R (2 mg/mL matrine and 50 μmol/L AG490, a JAK2/STAT3 signaling pathway inhibitor), and AG490 + H/R in an attempt to identify the underlying regulatory mechanisms of matrine. MTT assay was applied to determine cell viability. Hoechst staining was performed to detect the morphology of apoptotic CMECs, while cell cycle and the rate of apoptosis rate were determined by flow cytometry means. The mRNA and protein expression of the JAK2/STAT3 signaling pathway and apoptosis related genes were determined through the use of RT-qPCR and western blot assay methods respectively. An in vitro angiogenesis assay was employed to evaluate the value of matrine in tube formation. CMECs treated with ld-Ma+H/R, md-Ma+H/R, hd-Ma+H/R and Ma + AG490+H/R exhibited higher cell viability, greater cell ratio at the S phase, higher expression levels of p-JAK2 and p-STAT3, increased tube formation ability, and a lower apoptosis rate, with a lower ratio of cells at the G1 phase and Bax/Bcl-2 ratio. Meanwhile, the rats treated with AG490+H/R exhibited opposite results. Taken together, the key findings of the present study suggest that matrine inhibits the H/R-induced apoptosis of CMECs in rats via the JAK2/STAT3 signaling pathway, highlighting its therapeutic potential for H/R injury. [ABSTRACT FROM AUTHOR]

To unveil the possible protective role of isorhamnetin, an immediate 3′- O -methylated metabolite of quercetin, in cardiomyocyte under hypoxia/reoxygenation (H/R) condition and the underlying mechanisms involved, H9c2 cardiomyocytes were exposed to the vehicle or H/R for 6 h (2 h of hypoxia following by 4 h of reoxygenation) with isorhamnetin (0, 3, 6, 12, 25, 50 μM for 4 h prior to H/R exposure). Apoptosis was evaluated by TUNEL staining, flow cytometry analysis and western blot assay for cleaved caspase-3. Myocardial injure in vivo was determined by infarct size using TTC staining, histological damage using H&E staining and myocardial apoptosis. Here, we found that isorhamnetin dose-dependently protected H9c2 cardiomyocytes against H/R-induced injure, as evidenced by the reduction in lactate dehydrogenase (LDH) levels, increases in cell viability, superoxide dismutase (SOD) and catalase (CAT) activity, with the maximal effects at 25 μΜ. In addition, isorhamnetin treatment significantly inhibited apoptosis in H/R-induced H9c2 cardiomyocytes and ameliorated H/R-induced myocardial injure in vivo, concomitant with the upregulation of sirtuin 1 (SIRT1) expression. Mechanism studies demonstrated that isorhamnetin pretreatment remarkably abolished H/R-induced downregulation of Nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) expressions and upregulation of NADPH oxidase-2/4 (NOX-2/4) expressions in cardiomyocytes. However, SIRT1 inhibition (Sirtinol) not only inhibited isorhamnetin-induced Nrf2/HO-1 upregulation and NOX-2/4 downregulation, but also alleviated its anti-apoptotic effects. Taken together, these data indicate that isorhamnetin can exhibit positive effect on H/R-induced injure by attenuating apoptosis and oxidative stress in H9c2 cardiomyocytes, which is partly attributable to the upregulation of SIRT1 and Nrf2/HO-1-mediated antioxidant signaling pathway. [ABSTRACT FROM AUTHOR]

Abstract: Trimetazidine (TMZ) has been demonstrated to have protective effects against myocardial ischemia/reperfusion (MI/R) injury. In the present study, we investigated the effects and the underlying mechanisms of TMZ on autophagy during MI/R in vivo and in vitro. In the in vivo study, an animal model of MI/R was induced by coronary occlusion. TMZ (20 mg/kg/day) protected the rat hearts from MI/R-induced heart failure by increasing ejection fraction and fractional shortening and decreasing end-systolic volume, end-diastolic volume, left ventricular (LV) internal diameter at systole, and LV internal diameter at diastole; it alleviated myocardial injury and oxidative stress by decreasing LDH, creatine kinase MB isoenzyme, ROS, and MDA levels and increasing SOD and glutathione peroxidase levels in plasma. TMZ also reduced myocardial infarct size and apoptosis. Moreover, TMZ markedly inhibited MI/R-induced autophagy by decreasing the protein and messenger RNA levels of LC3-II, Beclin1, ATG5, and ATG7 and the number of autophagosomes and by involving the AKT/mTOR pathway. Further, in the in vitro experiments, H9c2 cells were incubated with TMZ (40 μM) to explore the direct effects of TMZ following exposure to hypoxia and reoxygenation (H/R). TMZ increased cell viability and the concentration of intracellular SOD and inhibited H/R-induced cell apoptosis and ROS production. Moreover, TMZ decreased the number of autophagosomes and autophagy-related protein expression; it also upregulated p-AKT and p-mTOR expression. In addition, TMZ augmented Bcl-2 protein expression and diminished Bax protein expression, the Bax/Bcl-2 rate, and cleaved caspase-3 level. However, these effects on H9c2 cells were notably abolished by the PI3K inhibitor LY294002. In conclusion, our results showed that TMZ inhibited I/R-induced excessive autophagy and apoptosis, which was, at least partly, mediated by activating the AKT/mTOR pathway.Key messages: TMZ improved cardiac function, alleviated myocardial injury and oxidative stress, and reduced the myocardial infarct area and apoptosis.TMZ inhibited MI/R-induced myocardial autophagy, H/R-induced H9c2 cell apoptosis, and autophagy flux.The effect of TMZ on autophagy was repressed by LY294002.TMZ protected against MI/R injury by inhibiting excessive autophagy via activating the AKT/mTOR pathway. [ABSTRACT FROM AUTHOR]

Ischemic stroke is regarded one of the most common causes of brain vulnerability. Silibinin (SIL), extracted from the seeds of Silybinisus laborinum L., has been found to exhibit obvious therapeutic effects on neurodegenerative diseases. GAS6 has been proven to have significant neuroprotective effects; however, the role of SIL and GAS6 in ischemic stroke remains unclear. This study aimed to investigate the protective effects of SIL against cerebral ischemia-reperfusion injury in neuroblastoma N2a cells, as well as the mechanisms involved. Firstly, the toxicity of SIL was evaluated, and safe concentrations were chosen for subsequent experiments. Then, SIL exerts significant neuroprotection against hypoxia/reoxygenation (HR) injury in N2a cells, as manifested by increased cell viability, decreased apoptotic rate, LDH, and ROS generation. Additionally, SIL was found to inhibit HR-induced apoptosis, mitochondria dysfunction, and oxidative stress. However, silencing of GAS6 inhibited the neuroprotective effects of SIL. To sum up, these results suggest that SIL may be a promising therapeutic agent for the treatment of ischemic stroke. • Silibinin was found to inhibit hypoxia/reoxygenation-induced apoptosis, oxidative stress, and mitochondria dysfunction. • Silibinin treatment mitigated the hypoxia/reoxygenation injury in N2a cells, as evidenced by increased cell viability, and decreased apoptosis and reactive oxygen species accumulation. • GAS6 siRNA reversed the protective effect of silibinin on hypoxia/reoxygenation injury in N2a cells. • GAS6 plays a positive effect in the silibinin treatment on cerebral hypoxia/reoxygenation injury. [ABSTRACT FROM AUTHOR]

Background/Aims: Acetylcholine (ACh) is known to modulate the cardiac redox environment and thereby suppress reactive oxygen species (ROS) generation during oxidative stress. However, there is little information about its regulation on ROS clearance. Here we investigate the beneficial effects of ACh on superoxide dismutase (SOD) as key ROS-detoxifying enzyme system in cultured rat cardiomyoblasts. Methods: H9c2 cells were subjected to hypoxia/reoxygenation (H/R) to mimic oxidative stress. Western blot was used to detect the expression of SOD and related signaling molecules. Specific protein knockdown was performed with siRNA transfection. Results: ACh treatment on the beginning of reoxygenation decreased ROS and apoptosis. ACh increased ATP synthesis and mitochondrial DNA. Furthermore, ACh significantly reversed H/R-induced reduction in protein expressions and activities of SOD. ACh stimulated peroxisome proliferator activated receptor γ co-activator 1α (PGC-1α) and decreased forkhead box subfamily O3a (FoxO3a) phosphorylation. Atropine (muscarinic receptor antagonist) abolished the cytoprotection afforded by ACh. PGC-1α siRNA blocked ACh-induced invigorating effects on SOD2, whereas it did not alter SOD1 and FoxO3a phosphorylation. FoxOSa siRNA drastically decreased the expressions of SOD2 and PGC-1α, while it did not affect SOD1. Conclusion: ACh activates SOD2 within mitochondria through FoxO3a/PGC-1α pathway and up-regulates SOD1 in the cytoplasm, thus protecting against oxidative injury induced by H/R. Our findings provide new insights into mechanisms underlying the cardioprotection of ACh on ROS detoxifying. © 2014 S. Karger AG, Basel

DJ-1 was recently reported to be involved in the cardioprotection of hypoxic preconditioning (HPC) against hypoxia/reoxygenation (H/R)-induced oxidative stress damage, by preserving mitochondrial complex I activity and, subsequently, inhibiting mitochondrial reactive oxygen species (ROS) generation. However, the molecular mechanism by which HPC enables mitochondrial translocation of DJ-1, which has no mitochondria-targeting sequence, to preserve mitochondrial complex I, is largely unknown. In this study, co-immunoprecipitation data showed that DJ-1 was associated with glucose-regulated protein 75 (Grp75), and this association was significantly enhanced after HPC. Immunofluorescence imaging and Western blot analysis showed that HPC substantially enhanced the translocation of DJ-1 from cytosol to mitochondria in H9c2 cells subjected to H/R, which was mimicked by DJ-1 overexpression induced by pFlag-DJ-1 transfection. Importantly, knockdown of Grp75 markedly reduced the mitochondrial translocation of DJ-1 induced by HPC and pFlag-DJ-1 transfection. Moreover, HPC promoted the association of DJ-1 with mitochondrial complex I subunits ND1 and NDUFA4, improved complex I activity, and inhibited mitochondria-derived ROS production and subsequent oxidative stress damage after H/R, which was also mimicked by pFlag-DJ-1 transfection. Intriguingly, these effects of HPC and pFlag-DJ-1 transfection were also prevented by Grp75 knockdown. In conclusion, these results indicated that HPC promotes the translocation of DJ-1 from cytosol to mitochondria in a Grp75-dependent manner and Grp75 is required for DJ-1-mediated protection of HPC on H/R-induced mitochondrial complex I defect and subsequent oxidative stress damage.