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Objective: The molecular pathways underlying hypoxemia-induced alterations in neurodevelopment of infants with congenital heart disease have not been delineated. We used transcriptome analysis to investigate differential gene expression induced by hypoxemia in an ovine artificial-womb model. Methods: Mid-gestation fetal sheep (median [interquartile range] 109 [107-112] days' gestation) were cannulated via the umbilical vessels, attached to a pumpless, low-resistance oxygenator circuit, and incubated in a sterile, fluid environment for 22 [21-23] days. Fetuses were maintained with an oxygen delivery of 20-25 mL/kg/min (normoxemia, n = 3) or 14-16 mL/kg/min (hypoxemia, n = 4). Transcriptional profiling by RNA sequencing was carried out on left frontal brains and hypoxemia-regulated genes were identified by differential gene expression analysis. Results: A total of 228 genes whose expression was up or down regulated by ≥1.5-fold (false discovery rate ≤0.05) were identified. The majority of these genes were induced in hypoxemic animals compared to normoxemic controls, and functional enrichment analysis identified respiratory electron transport as a pathway strongly upregulated in the brain during chronic hypoxemia. Further examination of hypoxemia-induced genes showed robust induction of all 7 subunits of the mitochondrial NADH:ubiquinone oxidoreductase (complex I). Other hypoxemia-induced genes included cytochrome B, a component of complex III, and ATP6, ATP8, both of which are components of complex V. Conclusions: Chronic fetal hypoxemia leads to upregulation of multiple mitochondrial respiratory complex genes critical for energy production and reactive oxygen species generation, including complex I. These data provide valuable insight into potential pathways involved in chronic hypoxemia-induced neuropathology and offers potential therapeutic targets for fetal neuroprotection in fetuses with congenital heart defects.

The coronary arteries mainly function to perfuse the myocardium. When coronary artery resistance increases, myocardial perfusion decreases and myocardial remodeling occurs. Mitochondrial damage has been regarded as the primary cause of microvascular dysfunction. In the present study, we explored the effects of mitophagy activation on microvascular damage. Hypoxia/reoxygenation injury induced mitochondrial oxidative stress, thereby promoting mitochondrial dysfunction in endothelial cells. Mitochondrial impairment induced apoptosis, reducing the viability and proliferation of endothelial cells. However, supplementation with the mitophagy inducer urolithin A (UA) preserved mitochondrial function by reducing mitochondrial oxidative stress and stabilizing the mitochondrial membrane potential in endothelial cells. UA also sustained the viability and improved the proliferative capacity of endothelial cells by suppressing apoptotic factors and upregulating cyclins D and E. In addition, UA inhibited mitochondrial fission and restored mitochondrial fusion, which reduced the proportion of fragmented mitochondria within endothelial cells. UA enhanced mitochondrial biogenesis in endothelial cells by upregulating sirtuin 3 and peroxisome proliferator-activated receptor gamma coactivator 1-alpha. These results suggested that activation of mitophagy may reduce hypoxia/reoxygenation-induced cardiac microvascular damage by improving mitochondrial quality control and increasing cell viability and proliferation.

Ischemia results in neuronal damage via alterations in gene transcription and protein expression. Long noncoding RNAs (LncRNAs) are pivotal in the regulation of target protein expression in hypoxia/reoxygenation (H/R). In this study, we observed the function of exosomes-carried lncRNA UCA1 in H/R-induced injury of cardiac microvascular endothelial cells (CMECs). In H/R cell model, CMECs were co-cultured with human umbilical cord mesenchymal stem cell-derived exosomes (hUCMSC-ex). The loss-of-function experiments were conducted to assess the effect of lncRNA UCA1 on H/R injury by assessing the biological behaviors of CMECs. The relationship among lncRNA UCA1, miR-143 and Bcl-2 were verified. An ischemia-reperfusion (I/R) rat model was established. Then hUCMSC-ex was injected into I/R rats to identify its effects on apoptosis and autophagy. Functional rescue experiments were performed to verify the sponge system. In vitro and in vivo experiments showed that hUCMSC-ex protected I/R rats and H/R CMECs against injury. Silencing UCA1 in hUCMSC-ex or miR-143 overexpression aggravated H/R injury in CMECs. LncRNA UCA1 competitively bound to miR-143 to upregulate Bcl-2. And hUCMSCs-ex/si-UCA1+inhi-miR-143 treatment protected CMECs against H/R injury and inhibited hyperautophagy. Together, hUCMSC-ex-derived lncRNA UCA1 alleviates H/R injury through the miR-143/Bcl-2/Beclin-1 axis. Hence, this study highlights a stem cell-based approach against I/R injury.

Patients with heart disease often suffer from ischemia, which can be treated by reperfusion. However, this treatment can lead to the development of ischemia/reperfusion (I/R) injury, an inflammatory condition that can cause further heart damage. Dexmedetomidine (Dex), an α2-adrenoceptor agonist, and the microRNA (miR)-17-3p, have both been suggested to alleviate I/R injury and cardiac tissue inflammation. The aim of the present study was to investigate whether Dex and miR-17-3p could act together to prevent I/R injury. H9C2 cells, a myoblast cell line used as a model of rat cardiomyocytes, were cultured in a hypoxic environment for 3 h, and then reoxygenated for 3 h. This hypoxia/reoxygenation (H/R) was used to model I/R. Cell Counting kit-8 was used to determine cell viability and an annexin V-FITC/propidium iodide apoptosis kit used to analyze cell apoptosis. A dual luciferase reporter assay was used to determine the interaction between miR-17-3p and toll-like receptor 4 (TLR4). Western blotting and reverse transcription-quantitative PCR were used to determine protein levels and mRNA expression of TLR4 and galectin-3. A concentration of 0.1-10 µmol/l Dex attenuated H/R injury, which was accompanied by increased miR-17-3p levels. Additionally, the inhibition of miR-17-3p exacerbated H/R injury and reduced the effect of Dex on H/R injury. H/R led to an increased galectin-3 level compared with that in control cells, and Dex or miR-17-3p inhibitor did not markedly affect the level of galectin-3, indicating that Dex alleviated the effects of I/R injury through other pathways. Inhibition of miR-17-3p in Dex-induced H9C2 cells during H/R increased the expression of inflammatory mediators including tumor necrosis factor-α, interleukin (IL)-6, IL-1β and phosphorylated NFκB subunit p65, while Dex reduced the H/R-induced expression of these inflammatory mediators. Inhibition of TLR4 also attenuated H/R injury. In summary, the findings of the present study indicated that Dex reduced H/R injury in H9C2 cell via the modulation of inflammatory signaling pathways, and these inflammatory factors could be regulated by miR-17-3p.

Hypoxia/reoxygenation (H/R) may play an important role via senescence in the mechanism of osteoarthritis (OA) development. The synovial membrane is highly sensitive to H/R due to its oxygen consumption feature. Excessive mechanical loads and oxidative stress caused by H/R induce a senescence‑associated secretory phenotype (SASP), which is related to the development of OA. The aim of the present study was to investigate the differences of SASP manifestation in synovial tissue masses between tissues from healthy controls and patients with OA. The present study used tumor necrosis factor‑α (TNF‑α) to pre‑treat synovial tissue and fibroblast‑like synoviocytes (FLS) to observe the effect of inflammatory cytokines on the synovial membrane before H/R. It was determined that H/R increased interleukin (IL)‑1β and IL‑6 expression levels in TNF‑α‑induced cell culture supernatants, increased the proportion of SA‑β‑gal staining, and increased the expression levels of high mobility group box 1, caspase‑8, p16, p21, matrix metalloproteinase (MMP)‑3 and MMP‑13 in the synovium. Furthermore, H/R opened the mitochondrial permeability transition pore, caused the loss of mitochondrial membrane potential (ΔΨm) and increased the release of reactive oxygen species (ROS). Moreover, H/R caused the expansion of the mitochondrial matrix and rupture of the mitochondrial extracorporeal membrane, with a decrease in the number of cristae. In addition, H/R induced activation of the JNK signaling pathway in FLS to induce cell senescence. Thus, the present results indicated that H/R may cause inflammation and escalate synovial inflammation induced by TNF‑α, which may lead to the pathogenesis of OA by increasing changes in synovial SASP and activating the JNK signaling pathway. Therefore, further studies expanding on the understanding of the pathogenesis of H/R etiology in OA are required.

Lung ischemia-reperfusion injury (LIRI) is one of the complications that can occur after lung transplantation and may lead to morbidity and mortality. Mangiferin (MAF) is a naturally occurring glucosyl xanthone that has been documented to possess anti-inflammatory, immunomodulatory and potent antioxidant effects. The purpose of the present study was to investigate the effect of MAF on LIRI using a hypoxia-reoxygenation (H/R) cell model. In the present study, the viability of lung alveolar epithelial cells (A549) and H/R-A549 were detected by MTT assay. ELISA was used to evaluate the expression levels of IL-6 and IL-1β. TUNEL assay and western blotting were used to evaluate the apoptosis. In addition, H/R-A549 cells were treated with sirtinol, which is known inhibitor of sirtuin 1 (SIRT1) activity, to determine the effects of MAF on proteins associated with the SIRT1/5'AMP-activate protein kinase (AMPK) signaling pathway using western blotting. The results showed that 20 µM MAF exerted a protective effect on A549 cells against H/R mediating no clear cytotoxic effects. In terms of inflammation, MAF reduced IL-6, IL-1β, cyclooxygenase-2 and inducible nitric oxide synthase expression, which was accompanied by activation of the SIRT1/AMPK signaling pathway. In addition, compared with those in the group treated with sirtinol, expression of SIRT1, Bcl-2 and AMPK activity were elevated in MAF-treated H/R-A549 cells, whereas the expression of Bax, cleaved caspase-3 and cleaved caspase-9 was suppressed. TUNEL analysis of H/R-A549 cells treated with MAF in combination with sirtinol revealed that treatment with sirtinol blocked the SIRT1/AMPK signaling pathway and increased the apoptosis rate compared with the MAF group. Taken together, results of the present study revealed that MAF could inhibit lung H/R cell injury through the SIRT1/AMPK signaling pathway.

Background Catechin protects heart from myocardial ischaemia/reperfusion (MI/R) injury. However, whether catechin inhibits H/R‐induced myocardial cell apoptosis is largely unknown. Objective This study aims to investigate the underlying mechanism of catechin in inhibiting the apoptosis of H/R‐induced myocardial cells. Methods LncRNA MIAT expression was detected by qRT‐PCR. Cell viability of H9C2 cells was detected using CCK‐8 assay. The apoptosis of H9C2 cells was detected by flow cytometry. The interaction between CREB and MIAT promoter regions was confirmed by dual‐luciferase reporter gene assay and ChIP assay. Results In MI/R rats, catechin improved heart function and down‐regulated lncRNA MIAT expression in myocardial tissue. In H/R‐induced H9C2 cells, catechin protected against cell apoptosis, and lncRNA MIAT overexpression attenuated this protective effect of catechin. We confirmed that transcription factor CREB could bind to MIAT promoter region, and catechin suppressed lncRNA MIAT expression through up‐regulating CREB. Catechin improved mitochondrial function and relieved apoptosis through promoting Akt/Gsk‐3β activation. In addition, MIAT inhibited Akt/Gsk‐3β activation and promoted cell apoptosis in H/R‐induced H9C2 cells. Finally, we found catechin promoted Akt/Gsk‐3β activation through inhibiting MIAT expression in H/R‐induced H9C2 cells. Conclusion Catechin relieved H/R‐induced myocardial cell apoptosis through regulating CREB/lncRNA MIAT/Akt/Gsk‐3β pathway.

Hypoxia‐inducible factor 1α (HIF‐1α) plays a critical role in the apoptotic process during cardiac ischaemia/reperfusion (I/R) injury. This study aimed to investigate whether post‐treatment with dexmedetomidine (DEX) could protect against I/R‐induced cardiac apoptosis in vivo and in vitro via regulating HIF‐1α signalling pathway. Rat myocardial I/R was induced by occluding the left anterior descending artery for 30 minutes followed by 6‐hours reperfusion, and cardiomyocyte hypoxia/reoxygenation (H/R) was induced by oxygen‐glucose deprivation for 6 hours followed by 3‐hours reoxygenation. Dexmedetomidine administration at the beginning of reperfusion or reoxygenation attenuated I/R‐induced myocardial injury or H/R‐induced cell death, alleviated mitochondrial dysfunction, reduced the number of apoptotic cardiomyocytes, inhibited the activation of HIF‐1α and modulated the expressions of apoptosis‐related proteins including BCL‐2, BAX, BNIP3, cleaved caspase‐3 and cleaved PARP. Conversely, the HIF‐1α prolyl hydroxylase‐2 inhibitor IOX2 partly blocked DEX‐mediated cardioprotection both in vivo and in vitro. Mechanistically, DEX down‐regulated HIF‐1α expression at the post‐transcriptional level and inhibited the transcriptional activation of the target gene BNIP3. Post‐treatment with DEX protects against cardiac I/R injury in vivo and H/R injury in vitro. These effects are, at least in part, mediated via the inhibition of cell apoptosis by targeting HIF‐1α signalling.

Supplemental Digital Content is available in the text, Sirtuin1 (Sirt1) and Sirtuin3 (Sirt3) are known to participate in regulating mitochondrial function. However, whether Total Salvianolic Acid Injection (TSI) protects against myocardial ischemia/reperfusion (I/R) injury through regulating Sirt1, Sirt3, and mitochondrial respiratory chain complexes is unclear. The aim of this study was to explore the effects of TSI on I/R-induced myocardial injury and the underlying mechanism. Male Sprague–Dawley rats were subjected to 30 min occlusion of the left anterior descending coronary artery followed by 90 min reperfusion with or without TSI treatment (8 mg/kg/h). The results demonstrated that TSI attenuated I/R-induced myocardial injury by the reduced infarct size, recovery of myocardial blood flow, and decreased cardiac apoptosis. Moreover, TSI protected heart from oxidative insults, such as elevation of myeloperoxidase, malondialdehyde, hydrogen peroxide, ROS, as well as attenuated I/R-elicited downregulation of Sirt1, Sirt3, NADH dehydrogenase [ubiquinone] 1 alpha subcomplex 10 (NDUFA10), succinate dehydrogenase complex, subunit A, flavoprotein variant (SDHA), and restoring mitochondrial respiratory chain complexes activity. The in vitro study in H9c2 cells using siRNA transfection further confirmed the critical role of Sirt1 and Sirt3 in the effect of TSI on the expression of NDUFA10 and SDHA. These results demonstrated that TSI attenuated I/R-induced myocardial injury via inhibition of oxidative stress, which was related to the activation of NDUFA10 and SDHA through the upregulation of Sirt1 and Sirt3.

Objective MiR-499 has been reported to be expressed only in cardiomyocytes, and its expression would increase after acute myocardial infarction (AMI). miR-499 plays a role in the process of cardiomyocytes injury induced by hypoxia/reoxygenation (H/R), however, it still remains unclear. Results Hypoxia inhibited miR-499-5p expression and H/R induced apoptosis. SOX6 was a target gene of miR-499-5p, and high expression of miR-499-5p inhibited the expression of SOX6. MiR-499-5p reduced H9c2 cells injury by inhibiting the expression of SOX6, overexpression of which could reverse the effect of miR-499-5p on H9c2 cells. MiR-499-5p inhibited the levels of LDH and MDA, while overexpression of miR-499-5p inhibited H/R-induced cell apoptosis. MiR-499-5p could up-regulate the level of Bcl-2 and down-regulate the expression levels of Bax and caspase-3. However, SOX6 partially reversed these effects of miR-499-5p. Conclusion We proved that miR-499-5p inhibited H/R-induced cardiomyocytes injury by targeting SOX6. Our results suggested that miR-499-5p/SOX6 pathway may present a potential therapeutic target for the treatment of AMI.

Mitochondrial transplantation emerges as a novel therapeutic solution for ischemia/reperfusion injury (IRI) in various tissues. Platelets have recently been used in mitochondrial transplantation as readily-available donors of small-size platelet mitochondria (plt-mito). Interestingly, FUN14 Domain Containing 2 (FUNDC2), a protein highly-expressed in the outer membrane (OMM) of plt-mito, has been identified to maintain platelet survival under hypoxic condition. The current study determined whether and how FUNDC2 contributed to the therapeutic effect of plt-mito transplantation for hypoxia/reoxygenation (HR) injury. The results showed that incorporation of human plt-mito into SH-SY5Y cells rescued HR-induced mitochondrial malfunction and mitochondrial apoptotic pathway. Mechanistically, plt-mito transplantation led to an increased expression of FUNDC2 in the recipient cells. This protein induced mitochondrial translocation of phosphatidylinositol-3,4,5-trisphosphate (PIP3) via its N-term, resulting in the stimulation of the protein kinase B (Akt)/forkhead box O3a (FOXO3a) pathway, which inhibited HR-induced mitochondrial accumulation of a mitochondrial target of FOXO3a, Bim, also known as a pro-apoptotic protein. Therefore, the FUNDC2/PIP3/Akt/FOXO3a axis may facilitate the incorporated plt-mito to restore mitochondrial function and cell viability of the recipient cells, and platelets may serve as readily-available sources of donor mitochondria that afford therapeutic benefits against IRI.

Safranal (SFR), an active ingredient extracted from saffron, exhibits a protective effect on the cardiovascular system. However, the mechanism of SFR against hypoxia/reoxygenation (H/R)-induced cardiomyocyte injury has previously not been investigated in vitro. The aim of the present study was therefore to observe the protective effects of SFR on H/R-induced cardiomyocyte injury and to explore its mechanisms. A H/R injury model of H9c2 cardiac myoblasts was established by administering 800 µmol/l CoCl2 to H9c2 cells for 24 h and reoxygenating the cells for 4 h to induce hypoxia. H9c2 cardiac myoblasts were pretreated with SFR for 12 h to evaluate the associated protective effects. A Cell Counting Kit-8 assay was used for cell viability detection, and the expression levels of lactate dehydrogenase (LDH), creatine kinase-MB (CK-MB), glutathione peroxidase (GSH-px), catalase (CAT), superoxide dismutase (SOD), malondialdehyde (MDA) and caspase-3, and the intracellular Ca2+ concentration were measured using the corresponding commercial kits. Levels of reactive oxygen species (ROS) in the cells were detected using 2,7-dichlorodihydrofluorescein diacetate. Flow cytometry was used to determine the degree of apoptosis and the level of mitochondrial membrane potential (MMP). Moreover, the expression levels of phosphorylated (p-)PI3K, AKT, p-AKT, glycogen synthase kinase 3β (GSK3β), p-GSK3β, Bcl-2, Bax, caspase-3 and cleaved caspase-3 were measured using western blot analysis. Results of the present study demonstrated that the H9c2 cardiac myoblasts treated with SFR exhibited significantly improved levels of viability and significantly reduced levels of ROS, compared with the H/R group. Furthermore, compared with the H/R group, SFR treatment significantly increased the MMP levels and antioxidant enzyme levels, including CAT, SOD and GSH-px; whereas the levels of CK-MB, LDH, MDA and intracellular Ca2+ concentration were significantly decreased. Moreover, the results of the present study demonstrated that SFR significantly reduced caspase-3, cleaved caspase-3 and Bax protein expression levels, but upregulated the Bcl-2 protein expression levels. SFR also increased the protein expressions of PI3K/AKT/GSK3β. In summary, the results suggested that SFR may exert a protective effect against H/R-induced cardiomyocyte injury, which occurs in connection with the inhibition of oxidative stress and apoptosis via regulation of the PI3K/AKT/GSK3β signaling pathway.

Our previous study reported that microRNA (miR)-30a-5p upregulation under hypoxia postconditioning (HPostC) exert a protective effect on aged H9C2 cells against hypoxia/reoxygenation injury via DNA methyltransferase 3B-induced DNA hypomethylation at the miR-30a-5p gene promoter. This suggests that miR-30a-5p may be a potential preventative and therapeutic target for ischemic heart disease in aged myocardium. The present study aimed to investigate the underlying mechanisms of miR-30a-5p transcription in aged myocardium in ischemic heart disease. Cardiomyocytes were treated with 8 mg/ml D-galactose for 9 days, and then exposed to hypoxic conditions. Cell viability was determined using a cell viability assay. Expression levels of histone deacetylase 2 (HDAC2), LC3B-II/I, beclin-1 and p62 were detected via reverse transcription-quantitative PCR and western blotting. Chromatin immunoprecipitation-PCR and luciferase reporter assays were performed to evaluate the effect of c-Myc binding and activity on the miR-30a-5p promoter in senescent cardiomyocytes following HPostC. It was found that HPostC enhanced the acetylation levels of H3K14 at the miR-30a-5p gene promoter in senescent cardiomyocytes, which attributed to the decreased expression of HDAC2. In addition, c-Myc could positively regulate miR-30a-5p transcription to inhibit senescent cardiomyocyte autophagy. Mechanically, it was observed that increased H3K14 acetylation level exposed to romidepsin facilitated c-Myc binding to the miR-30a-5p gene promoter region, which led to the increased transcription of miR-30a-5p. Taken together, these results demonstrated that HDAC2-mediated H3K14 hyperacetylation promoted c-Myc binding to the miR-30a-5p gene promoter, which contributed to HPostC senescent cardioprotection.

Endoplasmic reticulum stress (ERS)-induced apoptosis serves a crucial role in the pathogenesis of myocardial ischemia/reperfusion injury (MIRI). Previous studies have confirmed that pleckstrin homology-like domain family A member 3 (PHLDA3) is an important mediator in ERS-associated apoptosis. The aim of the current study focused on whether PHLDA3 served protective effects on hypoxia/reoxygenation (H/R)-injured cardiomyocytes by inhibiting ERS-induced apoptosis. Furthermore, the molecular mechanisms associated with the PI3K/AKT signaling pathway were investigated. Primary neonatal rat cardiomyocytes were isolated and randomized into four groups: i) Control + adenovirus encoding scrambled short hairpin RNA (AdshRNA); ii) control + adenoviral vectors encoding PHLDA3 shRNA (AdshPHLDA3); iii) H/R+ AdshRNA and iv) H/R+AdshPHLDA3. AdshPHLDA3 was used to knock down PHLDA3. An H/R injury model was constructed by treatment with hypoxia for 4 h followed by reoxygenation for 6 h. A PI3K/AKT inhibitor, LY294002, was supplemented in mechanistic studies. Cell viability and LDH/CK releases were detected to evaluate myocardial damage. Flow cytometry assays were used to assess apoptotic response. Western blotting assays were used to detect protein expression. The results demonstrated that H/R induced myocardial damage and increased PHLDA3 expression. ERS-induced apoptosis was significantly increased following H/R injury, as indicated by increased apoptotic rates and ERS-associated protein expression, including those of CHOP, 78 kDa glucose-regulated protein and caspase-12. However, PHLDA3 inhibition following AdshPHLDA3 transfection reversed cell damage and ERS-associated apoptosis on H/R injury. Studies for molecular mechanisms concluded that the apoptosis-inhibition effects and cardioprotective roles of PHLDA3 inhibition were induced partly by the activation of the PI3K/AKT pathway, which was verified by LY294002 treatment. In conclusion, in the process of H/R injury, PHLDA3 inhibition reduced ERS-induced apoptosis and H/R injury by activating the PI3K/AKT pathway. PHLDA3 may be a therapeutic target for the treatment of MIRI.

BackgroundAcute myocardial infarction (AMI) is one of the leading causes of morbidity and death worldwide. Studies have indicated that microRNAs in mesenchymal stem cell (MSC)-derived exosomes are crucial for treating various diseases.MethodsHuman umbilical cord MSC (hucMSC)-derived exosomes (hucMSC-exo) were isolated and used to treat cardiomyocytes that underwent hypoxia/reoxygenation (H/R) injury. Bioluminescence assessment was used to study binding of miRNA to its targeting gene.ResultsWe found that H/R decreased the viability of AC16 cells, increased the expression of NLRP3, and activated caspase-1(p20) and GSDMD-N as well as release of IL-1β and IL-18, and such effects were abolished by administration of hucMSC-exo. Administration of exosomes from negative scramble miRNA (NC)-transfected hucMSCs blocked H/R-caused lactate dehydrogenase release, pyroptosis, and over-regulation of NLRP3 and activated caspase-1(p20) and GSDMD-N as well as release of IL-1β and IL-18. More importantly, in comparison to exsomes from NC-transfected hucMSCs, exsomes from miR-100-5p-overexpressing hucMSCs had more obvious effects, and those from miR-100-5p-inhibitor-transfected hucMSCs showed fewer effects. Functional study showed that miR-100-5p bound to the 3’-untranslated region (3’-UTR) of FOXO3 to suppress its transcription. Moreover, overexpression of FOXO3 abolished the protective effects of miR-100-5p.ConclusionEnriched miR-100-5p in hucMSC-exo suppressed FOXO3 expression to inhibit NLRP3 inflammasome activation and suppress cytokine release and, therefore, protected cardiomyocytes from H/R-induced pyroptosis and injury.

Background: Poloxamer 188 (P188) possesses anti-inflammatory properties and can help to maintain plasma membrane function. P188 has been reported to exert beneficial effects in the treatment of various disorders. However, the effects of P188 in ischemia/reperfusion (IR)-induced acute lung injury have not been examined.Methods: We investigated the ability of P188 to attenuate IR-induced acute lung injury in rats and hypoxia/reoxygenation (HR) injury in murine epithelial cells. Isolated perfused rat lungs were exposed to 40 min ischemia followed by 60 min reperfusion to induce IR injury.Results: IR led to lung edema, increased pulmonary arterial pressure, promoted lung tissue inflammation and oxidative stress, and upregulated the levels of TNF-α, IL-6 and CINC-1, and increased Lactic dehydrogenase (LDH) activity in bronchoalveolar lavage fluid. IR also downregulated the levels of inhibitor of κB (IκB-α), upregulated nuclear factor (NF)-κB (NF-κB), and promoted apoptosis in lung tissues. P188 significantly suppressed all these effects. In vitro, P188 also exerted a similar effect in murine lung epithelial cells exposed to HR. Furthermore, P188 reduced the number of propidium iodide-positive cells, maintained cell membrane integrity, and enhanced cell membrane repair following HR.Conclusion: We conclude that P188 protects against lung IR injury by suppressing multiple signaling pathways and maintaining cell membrane integrity.

Hesperidin (HDN) has been reported to have hydrogen radical- and hydrogen peroxide-removal activities and to serve an antioxidant role in biological systems. However, whether HDN protects hepatocytes (HCs) against hypoxia/reoxygenation (H/R)-induced injury remains unknown. The present study aimed to explore the role of HDN in H/R-induced injury. HCs were isolated and cultured under H/R conditions with or without HDN treatment. HC damage was markedly induced under H/R, as indicated by cell viability, supernatant lactate dehydrogenase levels and alanine aminotransferase levels; however, HDN treatment significantly reversed HC injury. Oxidative stress markers (malondialdehyde, superoxide dismutase, glutathioneand reactive oxygen species) were increased markedly during H/R in HCs; however, this effect was significantly attenuated after exposure to HDN. Compared with those of the control group, the mRNA expression levels of IL-6 and TNF-α in HCs and the concentrations of IL-6 and TNF-α in the supernatants increased significantly following H/R, and HDN significantly ameliorated these effects. Western blotting demonstrated that microtubule-associated protein 1 light chain 3α (MAP1LC3A, also known as LC3) and Beclin-1 protein expression levels increased, while sequestosome 1 levels decreased during H/R following exposure to HDN. The number of GFP-LC3 puncta in HCs following exposure to HDN was increased compared with that observed in HCs without HDN exposure under the H/R conditions after bafilomycin A1 treatment. In summary, the present study demonstrated that HDN attenuated HC oxidative stress and inflammatory responses while enhancing autophagy during H/R. HDN may have a potential protective effect on HCs during H/R-induced injury.

The significance of circular RNAs (circRNAs) is reported in various kidney diseases including acute kidney injury (AKI). Specific circRNAs have the capacity to function as novel indicators of AKI. Circ_0023404 exhibits an important role in several diseases. Nevertheless, the detailed biological role of circ_0023404 in AKI remains poorly known. The present study aimed to investigate the effect of circ_0023404 on renal ischaemia/reperfusion (I/R) injury in vitro. Here, we evaluated the function of circ_0023404 in HK‐2 cells in response to hypoxia/reoxygenation (H/R). We established a cell AKI model induced by H/R in HK‐2 cells. We found circ_0023404 was significantly increased in AKI. Then, we found loss of circ_0023404 increased cell growth, repressed apoptosis, reduced inflammatory factors secretion and oxidative stress generation in vitro. Besides, circ_0023404 sponged miR‐136. miR‐136 overturned the effects of circ_0023404 on HK‐2 cell injury. We assumed IL‐6 receptor (IL‐6R) as a target of miR‐136 and IL‐6R was activated by circ_0023404 via sponging miR‐136. In conclusion, we revealed circ_0023404 contributed to HK‐2 cells injury stimulated by H/R via sponging miR‐136 and activating IL‐6R.

Background Liver ischemia/reperfusion (I/R) injury is an inevitable pathological phenomenon in various clinical conditions, such as liver transplantation, resection surgery, or shock, which is the major cause of morbidity and mortality after operation. Ginkgo Biloba Dropping Pill (GBDP) is a unique Chinese Ginkgo Biloba leaf extract preparation that exhibits a variety of beneficial biological activities. The aim of this study is to investigate the protective effects of GBDP on the liver I/R injury both in the in vitro and in vivo. Methods Hypoxia/reoxygenation (H/R) experiments were performed in alpha mouse liver 12 (AML-12) cells and primary hepatocytes, which were pretreated with GBDP (60 or 120 µg/mL) followed by incubation in a hypoxia chamber. Cell viability was detected by 3-(4,5-dimethylthiazol-2-yl)-2.5-diphenyltetrazolium bromide (MTT) assay. Annexin V staining as well as western blot analysis of apoptosis-related proteins was performed to detect the protective effect of GBDP on cell apoptosis induced by H/R injury. C57BL/6 mice were used to establish the liver I/R injury model, and were pretreated with GBDP (100 or 200 mg/kg/day, i.g.) for two weeks. The liver damage was evaluated by detection of plasma levels of alanine transaminase (ALT) and aspartate transaminase (AST), as well as histopathological examinations. Liver inflammation was determined by detecting the secretion of pro-inflammatory cytokines and neutrophil infiltration through enzyme-linked immunosorbent assay (ELISA) and myeloperoxidase (MPO) immunohistochemistry staining. Finally, Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick and labeling (TUNEL) staining and western blot analysis of apoptosis-related proteins were used to investigate the anti-apoptotic effect of GBDP in mice. Results In the in vitro study, GBDP pretreatment improved the cell viability of AML-12 cells in the H/R injury model. Similarly, the same result was found in the primary hepatocytes isolated from C57BL/6 mice. Moreover, GBDP decreased the number of apoptotic cells and reduced the expression of apoptosis-related proteins induced by H/R injury. In the in vivo study, oral administration of GBDP ameliorated liver injury evidenced by a significant decline in the levels of ALT and AST. Furthermore, the result of hematoxylin and eosin (H&E) staining showed that GBDP reduced the size of necrosis area in the liver tissue. In addition, the decreased infiltration of neutrophils and secretion of pro-inflammatory cytokines indicated that GBDP may play an anti-inflammatory effect. More importantly, GBDP reduced TUNEL-positive cells and the expression of apoptosis-related proteins in the liver indicating GBDP has anti-apoptotic effects. Conclusions Our findings elucidated that GBDP has potential effects for protecting against liver I/R injury characterized by its anti-apoptotic, anti-necrotic, and anti-inflammatory properties, which would promisingly make contributions to the exploration of therapeutic strategies in the liver I/R injury.

Objective This study aimed to determine whether proinflammatory cytokines have an effect on myocardial cells (MCs) and hepatocytes during myocardial ischemia to induce cyclic AMP-responsive element-binding protein H (CREBH) cleavage, activate the acute phase response in the liver, and cause a superimposed injury in MCs. Methods In this study, a hepatocyte–MC transwell co-culture system was used to investigate the relationship between myocardial hypoxia/reperfusion injury and CREBH cleavage. MCs and hepatocytes of neonatal rats were obtained from the ventricles and livers of Sprague–Dawley rats, respectively. MCs were inoculated into the lower chamber of transwell chambers for 12 hours under hypoxia. Levels of the endoplasmic reticulum stress protein glucose-regulated protein 78 in MCs, CREBH in hepatocytes, inflammatory factor (tumor necrosis factor-α and interleukin-6) levels, and cell viability were evaluated. The effect of CREBH knockdown was also studied using a CREBH-specific short hairpin RNA (Ad-CREBHi). Results We found that proinflammatory cytokines affect MCs and hepatocytes during myocardial ischemia to induce CREBH cleavage, activate the acute phase response in the liver, and cause superimposed injury in MCs. Conclusions Expression of CREBH aggravates myocardial injury during myocardial ischemia.

Ischemia-reperfusion (I/R) plays an important role in myocardial damage, which has been widely recognized as a key procedure in the cardiovascular disease. A hypoxia/reoxygenation (H/R) model was established using H9c2 cardiomyocytes to investigate the possible positive effect of oxymatrine (OMT), an alkaloid originating from the traditional Chinese herb Sophora flavescens Aiton, on cardiomyocytes exposed to H/R injury and the underlying molecular mechanisms. Cell viability was measured using the MTT assay, lactate dehydrogenase release measurements and hematoxylin and eosin staining. Oxidative stress was detected by measuring cellular malondialdehyde (MDA) content, as well as superoxide dismutase (SOD) and catalase (CAT) activities. Apoptosis was detected using TUNEL staining and flow cytometric analysis, and the underlying mechanism was investigated using reverse transcription-quantitative PCR and western blot analyses. The results revealed that OMT increased the viability of H9c2 cardiomyocytes exposed to H/R. The OMT pretreatment decreased the production of MDA by reactive oxygen species and increased the activities of SOD and CAT. Furthermore, the OMT pretreatment reduced the expression of Bax and caspase-3, while inducing Bcl-2 expression. In addition, the protective effect of OMT was shown to be associated with the PI3K/Akt signaling pathway, and the PI3K inhibitor LY294002 attenuated the effects of OMT on the H9c2 cardiomyocytes exposed to H/R. These findings indicate that OMT could be a potential therapeutic candidate for the treatment of myocardial ischemia/reperfusion injury.

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.

Liver injury is often observed in various pathological conditions including posthepatectomy state and cancer chemotherapy. It occurs mainly as a consequence of the combined necrotic and apoptotic types of cell death. In order to study liver/hepatocyte injury by the necrotic type of cell death, we studied signal-regulated necrosis (necroptosis) by developing a new optic probe for detecting receptor-interacting protein kinase 1 (RIP)/RIP3 binding, an essential process for necroptosis induction. In the mouse hepatocyte cell line, TIB-73 cells, TNF-/cycloheximide (T/C) induced RIP1/3 binding only when caspase activity was suppressed by the caspase-specific inhibitor z-VAD-fmk (zVAD). T/C/zVAD-induced RIP1/3 binding was inhibited by necrostatin-1 (Nec-1), an allosteric inhibitor of RIP1. The reduced cell survival by T/C/zVAD was improved by Nec-1. These facts indicate that T/C induces necroptosis of hepatocytes when the apoptotic pathway is inhibited/unavailable. FasL also induced cell death, which was only partially inhibited by zVAD, indicating the possible involvement of necroptosis rather than apoptosis. FasL activated caspase 3 and, similarly, induced RIP1/3 binding when the caspases were inactivated. Interestingly, FasL-induced RIP1/3 binding was significantly suppressed by the antioxidants Trolox and N-acetyl cysteine (NAC), suggesting the involvement of reactive oxygen species (ROS) in FasL-induced necroptotic cellular processes. H2O2, by itself, induced RIP1/3 binding that was suppressed by Nec-1, but not by zVAD. Hypoxia induced RIP1/3 binding after reoxygenation, which was suppressed by Nec-1 or by the antioxidants. Cell death induced by hypoxia/reoxygenation (H/R) was also improved by Nec-1. Similar to H2O2, H/R did not require caspase inhibition for RIP1/3 binding, suggesting the involvement of a caspase-independent mechanism for non-ligand-induced and/or redox-mediated necroptosis. These data indicate that ROS can induce necroptosis and mediate the FasL- and hypoxia-induced necroptosis via a molecular mechanism that differs from a conventional caspase-dependent pathway. In conclusion, necroptosis is potentially involved in liver/hepatocyte injury induced by oxidative stress and FasL in the absence of apoptosis.

Systemic administration of dexmedetomidine provides cardioprotection against ischemia/reperfusion (I/R) injury; however, the direct effects of dexmedetomidine on cardiomyocytes have not been clarified. The present study investigated the effects of dexmedetomidine on primary neonatal rat cardiomyocytes under hypoxic/reoxygenation (H/R) conditions. In order to simulate in vivo I/R injury, primary neonatal rat cardiomyocytes were cultured under hypoxic conditions for 1 h and subsequently reoxygenated for 24 h. The effects of preconditioning with dexmedetomidine 2 h before hypoxia and postconditioning during reoxygenation were also examined. Cellular viability and activity were analyzed by monitoring the dynamic response profile of living cells using a real-time cell analyzer system. A special scaled index, defined as the normalized cell index (NCI), was used to minimize the influence of inter-experimental variations. The dose-effect curve was generated from the area under the time-course curve values of NCI. H/R exposure markedly decreased cell viability and activity. Furthermore, no cytotoxicity was associated with a clinically relevant concentration of dexmedetomidine. Preconditioning with dexmedetomidine concentration-dependently ameliorated the reductions in NCI in cardiomyocytes following H/R injury. Additionally, postconditioning with dexmedetomidine improved the reductions in NCI at concentrations between 3 and 200 nM. Finally, the effect of 3–40 nM dexmedetomidine postconditioning was greater than preconditioning. These results indicated that preconditioning and postconditioning with dexmedetomidine attenuated H/R injury in primary neonatal rat cardiomyocytes at the cellular level.

Myocardial ischemia/reperfusion (I/R) injury is a major pathological process in coronary heart disease and cardiac surgery, and is associated with aberrant microRNA (miR) expression. Previous studies have demonstrated that inhibition of miR-15a expression may ameliorate I/R‑induced myocardial injury. In the present study, the potential role and underlying mechanism of miR‑15a in hypoxia/reoxygenation‑induced apoptosis of cardiomyocytes was investigated. Myocardial I/R was simulated in cultured H9c2 cells by 24 h hypoxia followed by 24 h reoxygenation. Using recombinant lentivirus vectors, the inhibition of miR‑15a was indicated to significantly reduce cardiomyocyte apoptosis and release of lactate dehydrogenase and malondialdehyde. Conversely, upregulated miR‑15a expression was pro‑apoptotic. Mothers against decapentaplegic homolog 7 (SMAD7) was identified by bioinformatics analysis as a potential target of miR‑15a. Luciferase reporter assays and western blotting for endogenous SMAD7 protein indicated that miR‑15a inhibited SMAD7 expression via its 3'‑untranslated region. Nuclear levels of nuclear factor‑κB (NF‑κB) p65 were increased by miR‑15a expression and decreased by miR‑15a inhibition, which is consistent with the possibility that the inhibition of SMAD7 by miR-15a results in NF‑κB activation. These findings suggested that the therapeutic effects of miR‑15a inhibition on I/R injury may potentially be explained by its ability to release SMAD‑7‑dependent NF‑κB inhibition. This may provide evidence for miR‑15a as a potential therapeutic target for the treatment of cardiac I/R injury.

Previous studies have reported that lipoxin A4 (LXA4) may exert a renoprotective effect on ischemia/reperfusion injury in various animal models. The underlying mechanism of LXA4-induced renoprotection during ischemia/reperfusion injury remains to be elucidated. The present study investigated LXA4-induced protection on renal tubular cells subjected to hypoxia/reoxygenation (H/R) injury, and determined the effects of peroxisome proliferator-activated receptor-γ (PPARγ) and heme oxygenase-1 (HO-1) on LXA4 treatment. HK-2 human tubular epithelial cells exposed to H/R injury were pretreated with LXA4, signal molecule inhibitors or the HO-1 inhibitor zinc protoporphyrin-IX, or were transfected with PPARγ small interfering RNA (siRNA) or nuclear factor E2-related factor 2 (Nrf2) siRNA. The protein and mRNA expression levels of PPARγ and HO-1 were analyzed using western blotting and reverse transcription-quantitative polymerase chain reaction. Binding activity of Nrf2 to the HO-1 E1 enhancer was determined using chromatin immunoprecipitation. Nrf2 binding to the HO-1 antioxidant responsive element (ARE) was assessed using electrophoretic mobility shift assay. Preincubation of cells with LXA4 exposed to H/R injury led to a decreased production of inducible nitrogen oxide synthase, malondialdehyde, γ-glutamyl transpeptidase, leucine aminopeptidase and N-acetyl-β-glucosaminidase. In addition, LXA4 pretreatment increased cell viability, protein and mRNA expression levels of PPARγ and HO-1 and PPARγ and HO-1 promoter activity. SB20358 is a p38 mitogen-activated protein kinase (p38 MAPK) pathway inhibitor, which reduced LXA4-induced PPARγ expression levels. LXA4 treatment upregulated p38 MAPK activation, Nrf2 nuclear translocation and increased binding activity of Nrf2 to HO-1 ARE and E1 enhancer in cells exposed to H/R injury. Transfection of the cells with PPARγ siRNA reduced the LXA4-induced Nrf2 translocation. Transfection of the cells with PPARγ siRNA or Nrf2 siRNA also reduced the LXA4-induced increase in HO-1 expression. In conclusion, LXA4-induced protection of renal tubular cells against H/R injury was associated with the induction of PPARγ and HO-1, via activation of the p38 MAPK pathway, as well as Nrf2 nuclear translocation and binding to HO-1 ARE and E1 enhancer. Therefore, LXA4-induced renoprotection is associated with activation of the p38 MAPK/PPARγ/Nrf2-ARE/HO-1 pathway.

Myocardial ischemia/reperfusion (I/R) injury is a major complication of reperfusion therapy in myocardial infarction. Ischemic myocardium produces a variety of peptides. We recently identified PDRPS7 as a novel peptide in cardiomyocytes that can be induced by hypoxia. However, the role of PDRPS7 is unknown. Here, we investigated the effects of PDRPS7 on hypoxia/reoxygenation (H/R)‐induced injury in rat cardiomyoblast H9c2 cells and NRCMs. We found that PDRPS7 improved cell survival and attenuated lactate dehydrogenase leakage following H/R in H9c2 cells and NRCMs. PDRPS7 also alleviated H/R‐induced pulsation reduction in NRCMs. Moreover, H/R‐induced cell apoptosis was decreased in the presence of PDRPS7. H/R‐induced reactive oxygen species generation was reduced by PDRPS7; in addition, PDRPS7 did not impact H2O2‐induced cell injury. Signaling analysis demonstrated that H/R increased the phosphorylation levels of JNKs, ERKs, and p38 mitogen‐activated protein kinases. However, PDRPS7 only attenuated H/R‐induced JNK phosphorylation, but not phosphorylation of ERKs and p38. PDRPS7 protected cardiomyocytes from apoptosis by inhibiting JNK phosphorylation and c‐Jun phosphorylation pathways, markedly upregulating anti‐apoptotic Bcl‐2 expression and inhibiting that of pro‐apoptotic Bax and cleaved caspase‐3. Importantly, pharmacological activation of JNKs diminished the protective effect of PDRPS7 in terms of cell survival against H/R stimulation. In summary, our study identified PDRPS7 as a novel cardioprotective peptide against H/R challenge and this action was mediated, at least in part, through inactivation of JNKs., Novel inducible peptide PDRPS7 protects cardiomyocytes from H/R injury by reducing intracellular reactive oxygen species content and inhibiting the activation of JNKs and c‐Jun, specifically altering the expression of related molecules in the downstream apoptotic signaling pathway, including marked upregulation of anti‐apoptotic Bcl‐2 expression and inhibition of pro‐apoptotic Bax and cleaved caspase‐3.

Background Cardiovascular diseases are currently the leading cause of death in humans. The high mortality of cardiac diseases is associated with myocardial ischemia and reperfusion (I/R). Recent studies have reported that microRNAs (miRNAs) play important roles in cell apoptosis. However, it is not known yet whether miR-141-3p contributes to the regulation of cardiomyocyte apoptosis. It has been well established that in vitro hypoxia/reoxygenation (H/R) model can follow in vivo myocardial I/R injury. This study aimed to investigate the effects of miR-141-3p and CHD8 on cardiomyocyte apoptosis following H/R. Results We found that H/R remarkably reduces the expression of miR-141-3p but enhances CHD8 expression both in mRNA and protein in H9c2 cardiomyocytes. We also found either overexpression of miR-141-3p by transfection of miR-141-3p mimics or inhibition of CHD8 by transfection of small interfering RNA (siRNA) significantly decrease cardiomyocyte apoptosis induced by H/R. Moreover, miR-141-3p interacts with CHD8. Furthermore, miR-141-3p and CHD8 reduce the expression of p21. Conclusion MiR-141-3p and CHD8 play critical roles in cardiomyocyte apoptosis induced by H/R. These studies suggest that miR-141-3p and CHD8 mediated cardiomyocyte apoptosis may offer a novel therapeutic strategy against myocardial I/R injury-induced cardiovascular diseases.

Hypoxia/reoxygenation (H/R) is one of the main causes of coronary artery disease (CAD), which is primarily induced by damage to coronary artery endothelial cells (CAECs). Glycyrrhizic acid (GA) is a natural and abundant pentacyclic triterpenoid glycoside of the licorice root extract, and it has been reported to elicit protective effects against hypoxia, inflammation and apoptosis in ischemic myocardium; therefore, GA may serve as a promising therapeutic agent for ischemia-associated CAD. In the present study, the protective effects of GA against H/R-induced injury in CAECs were investigated. Treatment with GA during H/R maintained cell viability and decreased H/R-induced cell apoptosis in human CAECs. In addition, H/R-mediated induction of intracellular and mitochondrial reactive oxygen species (ROS) was significantly decreased by GA exposure. Similar to ROS scavengers, GA treatment not only exhibited protective effects, but also maintained the mitochondrial membrane potential after H/R and inhibited H/R-induced mitochondrial dysfunction, including deficits in ATP synthesis, mitochondrial DNA copy number and mitochondrial transcriptional activity. Furthermore, GA decreased autophagy/mitophagy, and its protective effect against H/R was abolished by autophagy promotion. Collectively, the results suggested that GA exhibited protective effects against H/R-induced CAEC injury by decreasing ROS accumulation and maintaining mitochondrial homeostasis. Further investigation into the precise mechanisms underlying the decrease in ROS accumulation induced by GA is required.

Liraglutide (Lir) is a glucagon-like peptide-1 receptor agonist that lowers blood sugar and reduces myocardial infarct size by improving endothelial cell function. However, its mechanism has not yet been clarified. Unfolded protein response (UPR) plays an important role in the pathogenesis of myocardial ischemia-reperfusion injury. It determines the survival of cells. Endoplasmic reticulum position protein homologue 2 (CNPY2) is a novel initiator of UPR that also participates in angiogenesis. To this extent, the current study further explored whether Lir regulates angiogenesis through CNPY2. In our article, a hypoxia/reoxygenation (H/R) injury model of human umbilical vein endothelial cells (HUVECs) was established and the effect of Lir on HUVECs was first evaluated by the Cell Counting Kit-8. Endothelial tube formation was used to analyze the ability of Lir to induce angiogenesis. Subsequently, the effect of Lir on the concentrations of hypoxia-inducible factor 1α (HIF1α), vascular endothelial growth factor (VEGF), and CNPY2 was detected by enzyme-linked immunosorbent assay. To assess whether Lir regulates angiogenesis through the CNPY2-initiated UPR pathway, the expression of UPR-related pathway proteins (CNPY2, GRP78, PERK, and ATF4) and angiogenic proteins (HIF1α and VEGF) was detected by reverse transcription-polymerase chain reaction and Western blot. The results confirmed that Lir significantly increased the expression of HIF1α and VEGF as well as the expression of CNPY2-PERK pathway proteins in HUVECs after H/R injury. To further validate the experimental results, we introduced the PERK inhibitor GSK2606414. GSK2606414 was able to significantly decrease both the mRNA and protein expression of ATF4, HIF1α, and VEGF in vascular endothelial cells after H/R injury. The effect of Lir was also inhibited using GSK2606414. Therefore, our study suggested that the CNPY2-PERK pathway was involved in the mechanism of VEGF expression after H/R injury in HUVECs. Lir increased the expression of VEGF through the CNPY2-PERK pathway, which may promote endothelial cell angiogenesis and protect HUVEC from H/R damage.

This study aims to investigate the role of targeting lncRNA myocardial infarction-associated transcript (MIAT) in protection against hypoxia/reoxygenation (H/R) injury in H9c2 cells in vitro and myocardial ischemia/reperfusion (I/R) injury in vivo by regulating expression of NF-kB and p53 upregulated modulator of apoptosis (PUMA). H9C2 cells were infected with lentivirus expressing the short-hairpin RNA direct against human MIAT gene (Lv-MIAT shRNA) or lentivirus expressing scrambled control (Lv-NC shRNA) or PUMA siRNA or p65 siRNA or their control siRNA respectively. Then the H9c2 cells were infected with Lv-shRNA to 2 hours of hypoxia (H) and 24 hour of reoxygenation (R). 100 ul of Lv-MIAT shRNA (1 × 108 PFU) or Lv-NC shRNA was transfected into mouse hearts, then the hearts were subjected to I/R (1h/72 h). We discovered targeting MIAT remarkably enhanced H9c2 cell viability, decreased H/R-induced cell apoptosis and LDH leakage and significantly decreased I/R-induced myocardial infarct size, reduced myocardial apoptosis and enhanced the heart function. Targeting MIAT downregulated p65 nuclear translocation, NF-κB activity and anti-apoptotic protein cleaved-caspase-3, Bax, and upregulated anti-apoptotic protein Bcl-2 induced by H/R or I/R. Our study suggests that targeting MIAT may protect against H9c2 cardiomyoblasts H/R injury or myocardial I/R injury via inhibition of cell apoptosis, mediated by NF-κB and PUMA signal pathway.

Over the past 30 years concerted campaigns have been undertaken to reverse nutrient-driven eutrophication in coastal waters in Europe, North America, Asia and Australia. Typically, eutrophication abatement has proven a more recalcitrant challenge than anticipated, with ecosystem improvements only recently beginning to emerge or falling short of goals. Reduction in nutrient loads has come mainly from advanced treatment of wastewaters and has lagged targets set for diffuse agricultural sources. Synthesis of the major campaigns—varying in terms of physical settings, ecosystem characteristics, nutrient sources, socio-economic drivers, and governance—identified barriers inhibiting eutrophication abatement and potential bridges to overcome them. Actionable science can be advanced by: application of the well-established and emerging knowledge and experience around the globe, client-responsive strategic research, and timely and conclusive adjudication of scientific controversies. More accountable governance requires: enduring engagement of high-level officials of the responsible governments; effective communication of the causes, risks and benefits to the public and stakeholders; quantitative and accountable allocation of responsibility for nutrient load reductions; and binding requirements, as opposed to simply voluntary actions. Effective reduction in nutrient loads requires: reduction strategies for both nitrogen and phosphorus; inclusion of actions that reduce atmospheric emissions of nitrogen in addition to direct inputs to waterways; efficacious regulations; public subsidies based on performance; limitations on biofuel production that increases nutrient loads; and enhancing the sinks and losses for legacy nutrients retained in soils and groundwater. Outcomes must be measured and strategies appropriately adjusted through: sustained monitoring of essential indicators and processes, the use of multiple models, truly adaptive management, and the precautious interventions within the coastal ecosystem. The changing climate must be taken into account by reassessing achievable future conditions and seeking alternatives for mitigating and adapting to climate change that also reduce nutrient loads.

MicroRNAs and autophagy play critical roles in cardiac hypoxia/reoxygenation (H/R)‐induced injury. Here, we investigated the function of miR‐21 in regulating autophagy and identified the potential molecular mechanisms involved. To determine the role of miR‐21 in regulating autophagy, H9c2 cells were divided into the following six groups: control group, H/R group, (miR‐21+ H/R) group, (miR‐21‐negative control + H/R) group, (BEZ235+ H/R) group and (miR‐21+ BEZ235+ H/R) group. The cells underwent hypoxia for 1 hr and reoxygenation for 3 hrs. Cell count kit‐8 was used to evaluate cell function and apoptosis was analysed by Western blotting. Western blotting and transmission electron microscopy were used to investigate autophagy. We found that miR‐21 expression was down‐regulated, and autophagy was remarkably increased in H9c2 cells during H/R injury. Overexpression of miR‐21 with a miR‐21 precursor significantly inhibited autophagic activity and decreased apoptosis, accompanied by the activation of the AKT/mTOR pathway. In addition, treatment with BEZ235, a novel dual Akt/mTOR inhibitor, resulted in a significant increase in autophagy and apoptosis. However, we found that miR‐21‐mediated inhibition of apoptosis and autophagy was partly independent of Akt/mTOR activation, as demonstrated in cells treated with both miR‐21 and BEZ235. We showed that miR‐21 could inhibit H/R‐induced autophagy and apoptosis, which may be at least partially mediated by the Akt/mTOR signalling pathway.

Myocardial ischemia/reperfusion (I/R) injury may cause the apoptosis of cardiomyocytes as well as cardiac fibrosis, which is characterized as the transdifferentiation of fibroblasts to myofibroblasts and collagen deposition. MicroRNAs (miRNAs or miRs) have been demonstrated to be involved in myocardial I/R injury. However, the underlying molecular mechanism remains largely unclear. In the present study, mouse cardiomyocyte M6200 cells were treated with hypoxia/reoxygenation (H/R). Our data indicated that H/R treatment led to cell apoptosis, the increased expression of fibrosis‑related proteins, namely collagen I, II, III, and fibronectin, as well as the downregulation of miR-142-3p in M6200 cells. Overexpression of miR-142-3p suppressed the H/R-induced apoptosis and fibrosis of M6200 cells. Bioinformatics analysis and a Dual‑Luciferase reporter assay further identified high mobility group box 1 (HMGB1) as a direct target gene of miR-142-3p, and miR-142-3p negatively regulated the protein level of HMGB1 in M6200 cells. Furthermore, knockdown of HMGB1 enhanced cell proliferation whereas it inhibited the apoptosis and fibrosis of M6200 cells. In addition, TGF-β1/Smad3 signaling was suggested to be involved in the miR-142-3p/HMGB1-mediated apoptosis and fibrosis of M6200 cells treated with H/R. Taken together, the findings of the present study demonstrate that miR-142-3p inhibits H/R-induced apoptosis and fibrosis of cardiomyocytes, partly at least, by the direct inhibition of HMGB1 expression. Therefore, these findings have increased our understanding of the pathogenesis of H/R-induced myocardial injury.

Ischemia-reperfusion injury due to hypoxia/reoxygenation (H/R) is one of the main causes of liver damage during liver surgery. Donor interleukin-6 (IL-6) rs1800796 single nucleotide polymorphisms (SNPs) affect the metabolism of tacrolimus following liver transplantation-related hepatic H/R. This study investigated the response of IL-6 and its promoter polymorphisms to hepatic H/R in liver parenchymal cells. The association between IL-6 rs1800796 SNPs and IL-6 expression was measured in 84 disease-free liver tissues using tissue microarrays and immunohistochemistry. Subsequently, LO2G, LO2C and NC-LO2 cells were successfully constructed via stable lentivirus-mediated transfection. The effects of IL-6 and its SNPs on the biological function of LO2 cells were examined using a cell model of H/R. Our results revealed that IL-6 was mainly expressed in hepatocytes. The intermediate IL-6 expression rate in genotype CC carriers was higher than that in genotype CG/GG carriers (P=0.006), which was subsequently verified at the IL-6 mRNA level (P=0.002). The concentrations of alanine aminotransferase in the LO2G cells were significantly higher than those in the LO2C cells following H/R for 6 h and H/R for 24 h (P

Endothelial cells are highly sensitive to hypoxia and contribute to myocardial ischemia/reperfusion injury. We have reported that N-n-butyl haloperidol iodide (F2) can attenuate hypoxia/reoxygenation (H/R) injury in cardiac microvascular endothelial cells (CMECs). However, the molecular mechanisms remain unclear. Neonatal rat CMECs were isolated and subjected to H/R. Pretreatment of F2 leads to a reduction in H/R injury, as evidenced by increased cell viability, decreased lactate dehydrogenase (LDH) leakage and apoptosis, together with enhanced AMP-activated protein kinase (AMPK) and liver kinase B1 (LKB1) phosphorylation in H/R ECs. Blockade of AMPK with compound C reversed F2-induced inhibition of H/R injury, as evidenced by decreased cell viability, increased LDH release and apoptosis. Moreover, compound C also blocked the ability of F2 to reduce H/R-induced reactive oxygen species (ROS) generation. Supplementation with the ROS scavenger N-acetyl-L-cysteine (NAC) reduced ROS levels, increased cell survival rate, and decreased both LDH release and apoptosis after H/R. In conclusion, our data indicate that F2 may mitigate H/R injury by stimulating LKB1/AMPK signaling pathway and subsequent suppression of ROS production in CMECs.

Purpose Activation of proteolytic enzymes, calpains and caspases, have been observed in many models of retinal disease. We previously demonstrated calpain activation in monkey retinal explants cultured under hypoxia. However, cellular responses are often species-specific. The purpose of the present study was to determine whether calpains or caspase-3 was involved in retinal ganglion cell (RGC) damage caused by hypoxia/reoxygenation in human retinal explants. The explant model was improved by use of an oxygen-controlled chamber. Methods Human and monkey retinal explants were cultured under hypoxic conditions in an oxygen-controlled chamber and then reoxygenated. Calpain inhibitor SNJ-1945 was maintained throughout the culture period. Immunohistochemistry and immunoblotting were performed for calpains 1 and 2, calpastatin, α-spectrin, calpain-specific α-spectrin breakdown product at 150 kDa (SBDP150), caspase-3, and apoptosis-inducing factor (AIF). Propidium iodide (PI) staining measured membrane disruption, and TUNEL staining detected DNA fragmentation. Results Activation of calpains in nerve fibers and increases of PI-positive RGCs were observed in retinal explants incubated for 16-hour hypoxia/8-hour reoxygenation. Except for autolysis of calpain 2, SNJ-1945 ameliorated these changes. In longer incubations under 24-hour hypoxia/16-hour reoxygenation, TUNEL-positive cells appeared, although activated caspase-3 and truncated AIF were not observed. DNA fragmentation was inhibited by SNJ-1945. Conclusions An improved human retinal explant model showed that calpains, not caspase-3, were involved in cell damage induced by hypoxia/reoxygenation. This finding could be relevant for patient treatment with a calpain inhibitor if calpain activation is documented in human retinal ischemic diseases.

MiR‐27 prevents atherosclerosis by inhibiting inflammatory responses induced by lipoprotein lipase. Overexpression of miR‐27b attenuates angiotensin‐induced atrial fibrosis. Nevertheless, studies have rarely investigated on the effect of miR‐27 in cardiomyocyte injury. H9c2 cells were transfected with miR‐27 mimic/inhibitor. Then the cell proliferation was tested by MTT assay and the cell apoptosis was detected by flow cytometry. The luciferase activity assay was utilized to analyze the relationship between miR‐27 and TGFBR1. Quantificational real‐time polymerase chain reaction and western blot were utilized to detect the cardiomyocyte differentiation marker and nuclear factor kappa B (NF‐κB) pathway. Our outcomes demonstrated that miR‐27 expression was downregulated cardiomyocyte injury subjected to hypoxia/reoxygenation (H/R). Additionally, overexpression of miR‐27 could significantly alleviate cardiomyocyte injury by regulating cell activity and apoptosis. The luciferase activity assay confirmed that transforming growth factor ß receptor 1 (TGFBR1) is a direct hallmark of miR‐27. Besides, overexpression of miR‐27 promoted the expression of TGFBR1 in H/R model. After transfection with miR‐27 mimic/inhibitor, the expression of NF‐κB pathway‐related proteins was decreased/increased. Taken together, our data manifested that miR‐27 repressed cardiomyocyte injury induced by H/R via mediating TGFBR1 and inhibiting NF‐κB signaling pathway. Furthermore, miR‐27/ TGFBR1 might be utilized as hopeful biomarkers for myocardial ischemia diagnosis and treatment.

Fibroblast growth factor (FGF)-21, a member of the family of FGFs, exhibits protective effects against myocardial ischemia and ischemia/reperfusion injury; it is also an enhancer of autophagy. However, the mechanisms underlying the protective role of FGF-21 against cardiomyocyte hypoxia/reoxygenation (H/R) injury remain unclear. The present study aimed to investigate the effect of FGF-21 on H9c2 cardiomyocyte injury induced by H/R and the mechanism associated with changes in autophagy. Cultured H9c2 cardiomyocytes subjected to hypoxia were treated with a vehicle or FGF-21 during reoxygenation. The viability of H9c2 rat cardiomyocytes was measured using Cell Counting Kit-8 and trypan blue exclusion assays. The contents of creatine kinase (CK) and creatine kinase isoenzymes (CK-MB), cardiac troponin I (cTnT), cardiac troponin T (cTnI) and lactate dehydrogenase (LDH) in culture medium were detected with a CK, CK-MB, cTnT, cTnI and LDH assay kits. The protein levels were examined by western blot analysis. Autophagic flux was detected by Ad-mCherry-GFP-LC3B autophagy fluorescent adenovirus reagent. The results indicated that FGF-21 alleviated H/R-induced H9c2 myocardial cell injury and enhanced autophagic flux during H/R, and that this effect was antagonized by co-treatment with 3-methyladenine, an autophagy inhibitor. Furthermore, FGF-21 increased the expression levels of Beclin-1 and Vps34 proteins, but not of mechanistic target of rapamycin. These data indicate that FGF-21 treatment limited H/R injury in H9c2 cardiomyocytes by promoting autophagic flux through upregulation of the expression levels of Beclin-1 and Vps34 proteins.

Changes in the oxygen partial pressure caused by a violation of uteroplacental perfusion are considered a powerful inducer of a cascade of reactions leading to the clinical manifestation of preeclampsia (PE). At the same time, the induction of oxygen-dependent molecule expression, in particular, miRNA and erythropoietin, is modulated. Therefore, the focus of our study was aimed at estimating the miRNA expression profile of placental tissue and blood plasma in pregnant women with preeclampsia using deep sequencing and quantitative RT-PCR, as well as determining the concentration of erythropoietin. The expression of miR-27b-3p, miR-92b-3p, miR-125b-5p, miR-181a-5p, and miR-186-5p, as regulated by hypoxia/reoxygenation, was significantly increased in blood plasma during early-onset preeclampsia. The possibility of detecting early PE according to the logistic regression model (miR-92b-3p, miR-125b-5p, and miR-181a-5p (AUC = 0.91)) was evaluated. Furthermore, the erythropoietin level, which is regulated by miR-125b-5p, was significantly increased. According to PANTHER14.1, the participation of these miRNAs in the regulation of pathways, such as the hypoxia&rsquo, s response via HIF activation, oxidative stress response, angiogenesis, and the VEGF signaling pathway, were determined.

Ghrelin is an endogenous ligand of growth hormone (GH) secretagogue receptor (GHSR) and has a number of biological effects, including heart protection. The present study aimed to reveal the positive effect of ghrelin on myocardium with hypoxia/reoxygenation (H/R) treatment and the involved molecular mechanisms. Successful construction of lentiviral expression vector (ghrelin-pLVX-Puro) was confirmed by colony polymerase chain reaction (PCR) verification. Primary rat cardiac myocytes were isolated and identified by immunofluorescence staining. Existence of red fluorescence of α-sarcomeric actinin indicated the successful isolation. Following ghrelin transfection and H/R treatment, primary cells were divided into four groups: Control, H/R, empty (empty pLVX-Puro + H/R) and ghrelin (ghrelin-pLVX-Puro + H/R). Cell viability and apoptosis were evaluated by Cell Counting Kit-8 (CCK-8) and Hoechst staining, respectively. The cell viability in the ghrelin group was significantly higher than that in the empty control group (P

Both calpain activation and endoplasmic reticulum (ER) stress are implicated in ischemic heart injury. However, the role of calpain in ER stress remains largely elusive. This study investigated whether calpain activation causes ER stress, thereby mediating cardiomyocyte apoptosis in an in vitro model of hypoxia/re-oxygenation (H/R). In neonatal mouse cardiomyocytes and rat cardiomyocyte-like H9c2 cells, up-regulation of calpain-1 sufficiently induced ER stress, c-Jun N-terminal protein kinase1/2 (JNK1/2) activation and apoptosis. Inhibition of ER stress or JNK1/2 prevented apoptosis induced by calpain-1. In an in vitro model of H/R-induced injury in cardiomyocytes, H/R was induced by a 24-hour hypoxia followed by a 24-hour re-oxygenation. H/R activated calpain-1, induced ER stress and JNK1/2 activation, and triggered apoptosis. Inhibition of calpain and ER stress blocked JNK1/2 activation and prevented H/R-induced apoptosis. Furthermore, blockade of JNK1/2 signaling inhibited apoptosis following H/R. The role of calpain in ER stress was also demonstrated in an in vivo model of ischemia/reperfusion using transgenic mice over-expressing calpastatin. In summary, calpain-1 induces ER stress and JNK1/2 activation, thereby mediating apoptosis in cardiomyocytes. Accordingly, inhibition of calpain prevents ER stress, JNK1/2 activation and apoptosis in H/R-induced cardiomyocytes. Thus, ER stress/JNK1/2 activation may represent an important mechanism linking calpain-1 to ischemic injury.

Extracellular (ex) HSP60 is increasingly recognized as an agent of cell injury. Previously, we reported that low endotoxin exHSP60 causes cardiac myocyte apoptosis. Our findings supported a role for Toll-like receptor (TLR) 4 in HSP60 mediated apoptosis. To further investigate the involvement of TLR4 in cardiac injury, we studied adult cardiac myocytes from C3H/HeJ (HeJ) mice, which have a mutant, nonfunctional TLR4, and compared the results with parallel studies using wild-type (WT) mice. Nuclear factor κB (NFκB) activation is an early step downstream of TLR4. NFκB was activated 1h after treatment with HSP60 in WT, but not HeJ mouse myocytes. ExHSP60 caused apoptosis in cardiac myocytes from WT mice, but not in myocytes from the HeJ mutants. To further elucidate the importance of exHSP60 in cardiac myocyte injury, both WT and HeJ mutant isolated mouse adult cardiac myocytes were exposed to hypoxia/reoxygenation. Anti-HSP60 antibody treatment reduced apoptosis in the WT group, but had no effect on the HeJ mutant myocytes. Unexpectedly, necrosis was also decreased in the HeJ mutants. Necrosis after hypoxia/reoxygenation in WT cardiac myocytes was mediated in part by TLR2 and TLR4 through rapid activation of PKCα, followed by increased expression of Nox2, and this was ameliorated by blocking antibodies to TLR2/4. These studies provide further evidence that TLR4 mediates exHSP60-associated apoptosis and that exHSP60 has an important role in cardiac myocyte injury, both apoptotic and necrotic.

α-Lipoic acid (ALA) is known as a powerful antioxidant, which has been reported to have protective effects against various cardiovascular diseases. The present study aimed to determine whether ALA pre- or post-treatment induced protective effects against hypoxia/reoxygenation-induced injury via inhibition of apoptosis and autophagy in human umbilical vein endothelial cells (HUVECs). In order to simulate the conditions of hypoxia/reoxygenation, HUVECs were subjected to 4 h of oxygen-glucose deprivation (OGD) followed by 12 h of reoxygenation. For the pre-treatment, ALA was added to the buffer 12 h prior to OGD, whereas for the post-treatment, ALA was added at the initiation of reoxygenation. The results demonstrated that ALA pre- or post-treatment significantly reduced lactate dehydrogenase (LDH) release induced through hypoxia/reoxygenation in HUVECs in a dose-dependent manner; of note, 1 mM ALA pre- or post-treatment exhibited the most potent protective effects. In addition, ALA significantly reduced hypoxia/reoxygenation-induced loss of mitochondrial membrane potential, apoptosis and the expression of cleaved caspase-3 in HUVECs. In the presence of the specific autophagy inhibitor 3-methyladenine, hypoxia/reoxygenation-induced apoptosis was significantly reduced. Furthermore, the formation of autophagosomes, cytosolic microtubule-associated protein 1A/1B-light chain 3 ratio and beclin1 levels significantly increased following hypoxia/reoxygenation injury; however, all of these effects were ameliorated following pre- or post-treatment with ALA. The results of the present study suggested that ALA may provide beneficial protection against hypoxia/reoxygenation-induced injury via attenuation of apoptosis and autophagy in HUVECs.

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.

Early growth response-1 (Egr-1), a transcription factor which often underlies the molecular basis of myocardial ischemia/reperfusion (I/R) injury, and oxidative stress, is key to myocardial I/R injury. Silent information regulator of transcription 1(SIRT1) not only interacts with and is inhibited by Egr-1, but also downregulates reactive oxygen species (ROS) via the Forkhead box O1(FOXO1)/manganese superoxide dismutase (Mn-SOD) signaling pathway. N-n-butyl haloperidol iodide (F2), a new patented compound, protects the myocardium against myocardial I/R injury in various animal I/R models in vivo and various heart-derived cell hypoxia/reoxygenation (H/R) models in vitro. In addition, F2 can regulate the abnormal ROS/Egr-1 signaling pathway in cardiac microvascular endothelial cells (CMECs) and H9c2 cells after H/R. We studied whether there is an inverse Egr-1/ROS signaling pathway in H9c2 cells and whether the SIRT1/FOXO1/Mn-SOD signaling pathway mediates this. We verified a ROS/Egr-1 signaling loop in H9c2 cells during H/R and that F2 protects against myocardial H/R injury by affecting SIRT1-related signaling pathways. Knockdown of Egr-1, by siRNA interference, reduced ROS generation, and alleviated oxidative stress injury induced by H/R, as shown by upregulated mitochondrial membrane potential, increased glutathione peroxidase (GSH-px) and total SOD anti-oxidative enzyme activity, and downregulated MDA. Decreases in FOXO1 protein expression and Mn-SOD activity occurred after H/R, but could be blocked by Egr-1 siRNA. F2 treatment attenuated H/R-induced Egr-1 expression, ROS generation and other forms of oxidative stress injury such as MDA, and prevented H/R-induced decreases in FOXO1 and Mn-SOD activity. Nuclear co-localization between Egr-1 and SIRT1 was increased by H/R and decreased by either Egr-1 siRNA or F2. Therefore, our results suggest that Egr-1 inhibits the SIRT1/FOXO1/Mn-SOD antioxidant signaling pathway to increase ROS and perpetuate I/R injury. F2 inhibits induction of Egr-1 by H/R, thereby activating SIRT1/FOXO1/Mn-SOD antioxidant signaling and decreasing H/R-induced ROS, demonstrating an important mechanism by which F2 protects against myocardial H/R injury.

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.

miRNAs have been implicated in processing of cardiac hypoxia/reoxygenation (H/R)-induced injury. Recent studies demonstrated that miR-19a might provide a potential cardioprotective effect on myocardial disease. However, the effect of miR-19a in regulating myocardial ischemic injury has not been previously addressed. The present study was to investigate the effect of miR-19a on myocardial ischemic injury and identified the potential molecular mechanisms involved. Using the H/R model of rat cardiomyocytes H9C2 in vitro, we found that miR-19a was in low expression in H9C2 cells after H/R treatment and H/R dramatically decreased cardiomyocyte viability, and increased lactate dehydrogenase (LDH) release and cardiomyocyte apoptosis, which were attenuated by co-transfection with miR-19a mimic. Dual-luciferase reporter assay and Western blotting assay revealed that PTEN was a direct target gene of miR-19a, and miR-19a suppressed the expression of PTEN via binding to its 3′-UTR. We further identified that overexpression of miR-19a inhibited the expression of PTEN at the mRNA and protein levels. Moreover, PTEN was highly expressed in H/R H9C2 cells and the apoptosis induced by H/R was associated with the increase in PTEN expression. Importantly, miR-19a mimic significantly increased p-Akt levels under H/R. In conclusion, our findings indicate that miR-19a could protect against H/R-induced cardiomyocyte apoptosis by inhibiting PTEN /PI3K/p-Akt signaling pathway.

It has been reported that salidroside (SAL), a natural dietary isothiocyanate, exhibits neuroprotective roles in cerebral ischemia-reperfusion injury. However, to the best of our knowledge, its underlying protective mechanism remains unknown. Sirtuin 1 (SIRT1) is a class III histone deacetylase involved in a variety of cellular functions. SIRT1 has been identified as a mediator of cerebral ischemia and may induce neuroprotection by activating various intracellular downstream targets, such as forkhead box protein O3α (FOXO3α). Therefore, the present study aimed to investigate whether SAL protects human brain vascular smooth muscle cells (HBVSMC) against hypoxia/reoxygenation (H/R) injury, which is a cell model of cerebral ischemia-reperfusion injury, through regulating the SIRT1-activited signaling pathway. The present study revealed that H/R treatment significantly reduced the expression of SIRT1 protein in HBVSMCs. Additionally, pretreatment with SAL reversed the H/R-induced decrease in cellular viability, increased caspase-3 activity, the appearance of apoptotic cells and the apoptosis rate in HBVSMCs. SAL attenuated the H/R-induced decrease in the expression of SIRT1 and phosphorylated FOXO3α protein in HBVSMCs, suggesting that the protective role of SAL in H/R injury occurs via the SIRT1/FOXO3α pathway. Furthermore, sirtinol, a SIRT1-specific inhibitor, suppressed the inhibitory effects of SAL on H/R-induced cytotoxicity and apoptosis as indicated by the downregulation of cell viability and upregulation of caspase-3 activity and apoptosis rate induced by sirtinol treatment in HBVSMCs. The reversal effects of SAL on H/R-induced alternation of B-cell lymphoma (Bcl-2) and Bcl-2 associated X protein expression were also attenuated by sirtinol. These results suggest that SAL exhibits neuroprotective effects against H/R injury by activating the SIRT1/FOXO3α pathway, which may become a novel potential therapeutic target for the treatment of cerebral ischemic disease.

Background/Aims: Hepatic ischemia-reperfusion (I/R) injury, which is mainly induced by inflammation and unstable intracellular ions, is a major negative consequence of surgery that compromises hepatic function. However, the exact mechanisms of liver I/R injury have not been determined. Positive crosstalk with the Ca2+/CaMKII pathway is required for complete activation of the TLR4 pathway and inflammation. We previously found that miR-148a, which decreased in abundance with increasing reperfusion time, targeted and repressed the expression of CaMKIIα. In the present study, we examined the role of the miR-148a machinery in I/R-induced Ca2+/CaMKII and TLR4 signaling changes, inflammation, and liver dysfunction in vivo and in vitro. Methods: Liver function was evaluated by serum aminotransferase levels and hematoxylin-eosin (HE) staining. Inflammatory factors were detected by enzyme-linked immunosorbent assay. Gene and protein expression were assessed by RT-PCR and western blot. Small interfering RNA was used to silence target gene expression. HE staining and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling were used to measure hepatic tissue apoptosis. These assays were performed to identify factors upregulated in hepatic I/R injury and downregulated by miR-148a. Results: We manifested that expression of CaMKIIα and phosphorylation of TAK1 and IRF3 were elevated in hypoxia/reoxygenation (H/R)-treated primary Kupffer cells (KCs) and liver tissue of I/R-treated mice, but these effects were attenuated by treatment with miR-148a mimic and were accompanied by the alleviation of liver dysfunction and hepatocellular apoptosis. Luciferase reporter experiments showed that miR148a suppressed luciferase activity by almost 60%. Moreover, knockdown of CaMKIIα in H/R KCs led to significant deficiencies in p-TAK1, P-IRF3, IL-6, and TNF-α, which was consistent with the effects of miR-148a overexpression. Otherwise, the same trend of activation of TAK1 and IRF3 and inflammatory factors in vitro was observed in the siTAK1 + siIRF3 group compared with the siCaMKIIα group. Conclusion: Taken together, we conclude that miR-148a may mitigate hepatic I/R injury by ameliorating TLR4-mediated inflammation via targeting CaMKIIα in vitro and in vivo.