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To investigate the effects of Cirsilineol on cell viability, pyroptosis, and inflammation in hypoxia/reoxygenation-induced renal tubular cells, we utilized the renal tubular epithelial cell line HK2. The in vitro renal injury model was induced by hypoxia/reoxygenation. Cell viability in hypoxia/reoxygenation-induced HK2 cells was determined using a methylthiazolyldiphenyltetrazolium bromide assay and lactate dehydrogenase releasing kit. The effects of Cirsilineol on inflammation and pyroptosis in HK2 cells were evaluated using enzyme-linked immunosorbent assay, flow cytometry, and immunoblot assays. Our results demonstrated that Cirsilineol promoted survival in hypoxia/reoxygenation-induced tubular cells, inhibited inflammation, and reduced pyroptosis in these cells. Further investigation revealed that the protective effects of Cirsilineol are mediated by the suppression of the Toll-like receptor 4/nuclear factor kappa B pathway, which mitigates the cellular response to hypoxia/ reoxygenation injury. Our study suggests that Cirsilineol protects renal tubular epithelial cells from hypoxia/reoxygenation injury by inhibiting inflammation and pyroptosis, highlighting its potential therapeutic value in renal conditions characterized by ischemic injury. [ABSTRACT FROM AUTHOR]

A new benzofuran, named as (2S, 3S)-5,6-dimethoxy-3-methyl-2-(3'-hydroxyphenyl)-2,3-dihydrobenzofuran (1), along with the six known isoflavonoids, 2'-hydroxy-4',7-dimethoxyisoflavone (2), 2' ²-methoxybiochanin A (3), tectorigenin (4), calycosin (5), 7-hydroxy-2',4',5'-trimethoxyisoflavone (6), orobol (7) were isolated from the heartwood of Dalbergia odorifera T.Chen. The structure of compounds was characterised by NMR spectroscopic data and comparisons with relevant literature data. The absolute structural configuration of compound 1 was determined through X-ray single crystal diffraction. Moreover, compounds 1-7 have no significant cytotoxic effects on H9c2 cells (IC50 > 200 μM). Compound 1-7 exhibit a significant protective effect against H/R (hypoxia/reoxygenation) induced H9c2 cell damage at 10~40, 5~40, 5~40, 5~40, 5~40, 5~40 and 5~10 μM (P≩0.05). [ABSTRACT FROM AUTHOR]

This research is concentrated on investigating the role and mechanism of miR-652-3p in the protective effects of isoflurane (ISO) against myocardial ischemia–reperfusion (I/R) injury. H9c2 cells underwent pretreatment with varying concentrations of ISO, and subsequently, a hypoxia/reoxygenation (H/R) model was constructed. The levels of miR-652-3p, ISL LIM homeobox 1 (ISL1), and inflammatory cytokines interleukin (IL)-6 and tumor necrosis factor-alpha (TNF-α) were evaluated through reverse transcription polymerase chain reaction (RT-qPCR). Enzyme-linked immunosorbent assay was employed to investigate concentrations of myocardial injury markers, such as creatine kinase-MB (CK-MB) and cardiac troponin I (cTnI). Cell counting kit-8 was used to evaluate cell viability, while flow cytometry was utilized to measure apoptosis. Additionally, a dual luciferase reporter assay was conducted to validate the targeting relationship between ISL1 and miR-652-3p. Herein, we confirmed that the level of miR-652-3p was gradually increased with prolonged hypoxia; nevertheless, this increase was suppressed by ISO pretreatment (P < 0.05). Additionally, ISO pretreatment prevented the decrease in cell viability, increase in apoptosis, and overproduction of IL-6, TNF-α, CK-MB, and cTnI induced by H/R (P < 0.05). However, the inhibitory effects of ISO were counteracted by the increased levels of miR-652-3p (P < 0.05). ISL1 is a potential target of miR-652-3p. H/R induction suppressed ISL1 levels compared to the control, but ISO treatment increased its expression (P < 0.05). Overexpression of ISL1 inhibited the elimination of the protective effect of ISO on myocardial damage induced by the elevation of miR-652-3p (P < 0.05). The findings of this research confirm that miR-652-3p attenuated the protective effect of ISO on cardiomyocytes in myocardial ischemia by targeting ISL1. [ABSTRACT FROM AUTHOR]

Aims: Ischaemic heart disease remains a significant cause of mortality globally. A pharmacological agent that protects cardiac mitochondria against oxygen deprivation injuries is welcome in therapy against acute myocardial infarction. Here, we evaluate the effect of large‐conductance Ca2+‐activated K+ channels (BKCa) activator, Compound Z, in isolated mitochondria under hypoxia and reoxygenation. Methods: Mitochondria from mice hearts were obtained by differential centrifugation. The isolated mitochondria were incubated with a BKCa channel activator, Compound Z, and subjected to normoxia or hypoxia/reoxygenation. Mitochondrial function was evaluated by measurement of O2 consumption in the complexes I, II, and IV in the respiratory states 1, 2, 3, and by maximal uncoupled O2 uptake, ATP production, ROS production, transmembrane potential, and calcium retention capacity. Results: Incubation of isolated mitochondria with Compound Z under normoxia conditions reduced the mitochondrial functions and induced the production of a significant amount of ROS. However, under hypoxia/reoxygenation, the Compound Z prevented a profound reduction in mitochondrial functions, including reducing ROS production over the hypoxia/reoxygenation group. Furthermore, hypoxia/reoxygenation induced a large mitochondria depolarization, which Compound Z incubation prevented, but, even so, Compound Z created a small depolarization. The mitochondrial calcium uptake was prevented by the BKCa activator, extruding the mitochondrial calcium present before Compound Z incubation. Conclusion: The Compound Z acts as a mitochondrial BKCa channel activator and can protect mitochondria function against hypoxia/reoxygenation injury, by handling mitochondrial calcium and transmembrane potential. [ABSTRACT FROM AUTHOR]

Renal ischemia/reperfusion is a serious condition that not only causes acute kidney injury, a severe clinical syndrome with high mortality, but is also an inevitable part of kidney transplantation or other kidney surgeries. Alterations of oxygen levels during ischemia/reperfusion, namely hypoxia/reoxygenation, disrupt mitochondrial metabolism and induce structural changes that lead to cell death. A signature mitochondrial phospholipid, cardiolipin, with many vital roles in mitochondrial homeostasis, is one of the key players in hypoxia/reoxygenation-induced mitochondrial damage. In this study, we analyze the effect of hypoxia/reoxygenation on human renal proximal tubule epithelial cell (RPTEC) cardiolipins, as well as their metabolism and mitochondrial functions. RPTEC cells were placed in a hypoxic chamber with a 2% oxygen atmosphere for 24 h to induce hypoxia; then, they were replaced back into regular growth conditions for 24 h of reoxygenation. Surprisingly, after 24 h, hypoxia cardiolipin levels substantially increased and remained higher than control levels after 24 h of reoxygenation. This was explained by significantly elevated levels of cardiolipin synthase and lysocardiolipin acyltransferase 1 (LCLAT1) gene expression and protein levels. Meanwhile, hypoxia/reoxygenation decreased ADP-dependent mitochondrial respiration rates and oxidative phosphorylation capacity and increased reactive oxygen species generation. Our findings suggest that hypoxia/reoxygenation induces cardiolipin remodeling in response to reduced mitochondrial oxidative phosphorylation in a way that protects mitochondrial function. [ABSTRACT FROM AUTHOR]

Gas-loaded nanocarriers (G-LN) show promise in improving heart transplantation (HTx) outcomes. Given their success in reducing cell death during normothermic hypoxia/reoxygenation (H/R) in vitro, we tested their integration into cardioplegic solutions and static cold storage (SCS) during simulated HTx. Wistar rat hearts underwent four hours of SCS with four G-LN variants: O2- or N2-cyclic-nigerosyl-nigerose-nanomonomers (CNN), and O2- or N2-cyclic-nigerosyl-nigerose-nanosponges (CNN-NS). We monitored physiological-hemodynamic parameters and molecular markers during reperfusion to assess cell damage/protection. Hearts treated with nanomonomers (N2-CNN or O2-CNN) showed improvements in left ventricular developed pressure (LVDP) and a trend towards faster recovery of the rate pressure product (RPP) compared to controls. However, nanosponges (N2-CNN-NS or O2-CNN-NS) did not show similar improvements. None of the groups exhibited an increase in diastolic left ventricular pressure (contracture index) during reperfusion. Redox markers and apoptosis/autophagy pathways indicated an increase in Beclin 1 for O2-CNN and in p22phox for N2-CNN, suggesting alterations in autophagy and the redox environment during late reperfusion, which might explain the gradual decline in heart performance. The study highlights the potential of nanomonomers to improve early cardiac performance and mitigate cold/H/R-induced stunning in HTx. These early improvements suggest a promising avenue for increasing HTx success. Nevertheless, further research and optimization are needed before clinical application. [ABSTRACT FROM AUTHOR]

Neurodegeneration is linked to the progressive loss of neural function and is associated with several diseases. Hypoxia is a hallmark in many of these diseases, and several therapies have been developed to treat this disease, including gene expression therapies that should be tightly controlled to avoid side effects. Cells experiencing hypoxia undergo a series of physiological responses that are induced by the activation of various transcription factors. Modulation of microRNA (miRNA) expression to alter transcriptional regulation has been demonstrated to be beneficial in treating multiple diseases, and in this study, we therefore explored potential miRNA candidates that could influence hypoxia-induced nerve cell death. Our data suggest that in mouse neuroblasts Neuro-2a cells with hypoxia/reoxygenation (H/R), miR-337-3p is downregulated to increase the expression of Potassium channel tetramerization domain containing 11 (KCTD11) and subsequently promote apoptosis. Here, we demonstrate for the first time that KCTD11 plays a role in the cellular response to hypoxia, and we also provide a possible regulatory mechanism by identifying the axis of miR-337-3p/KCTD11 as a promising candidate modulator of nerve cell survival after H/R exposure. [ABSTRACT FROM AUTHOR]

Hypoxia (20 min) and reoxygenation (30 min) were simulated on isolated rat cardiomyocytes to evaluate the cytoprotective effect of selective δ2-opioid receptor agonist deltorphin II, opioid receptor antagonist naloxone methiodide, μ-opioid receptor antagonist CTAP, κ-opioid receptor antagonist nor-binaltorphimine, ε1-opioid receptor antagonist BNTX, and δ2-opioid receptors naltriben. Deltorphin II was administered 5 min before reoxygenation, antagonists were administered 10 min before reoxygenation. The cytoprotective effect of deltorphin II was assessed by the number of cardiomyocytes survived after hypoxia/reoxygenation, as well as by the lactate dehydrogenase content in the incubation medium. It has been established that the cytoprotective effect of deltorphin II occurs at a concentration of 64 nmol/liter and is associated with activation of δ2-opioid receptors. [ABSTRACT FROM AUTHOR]

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]

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]

In patients undergoing interventional or surgical coronary revascularization, subclinical hypothyroidism is common and associated with worse outcome, including the need for postoperative inotropic support. In isolated rat hearts with global ischemia/reperfusion, exogenous triiodothyronine (T3) reduces infarct size. Aim of this study was, to investigate whether or not exogenous T3 protects human myocardium from ischemia/reperfusion injury. Right atrial trabeculae from patients undergoing routine coronary artery bypass grafting were isolated and transferred to Tyrode's buffer. Electrically initiated (1 Hz) contractile stress (mN/mm2) was recorded for 10 min at baseline (95% O 2 / 5% CO 2 , glucose). Sixty min hypoxia were induced by changing buffer gas and increasing stimulation rate (95% N 2 / 5% CO 2 , choline chloride, 3 Hz) before return to reoxygenation for 30 min. T3 (500 μg/l) vs. NaOH (solvent control) was administered A) throughout (n = 11 vs. n = 9) or B) only 15 min before and during reoxygenation (n = 12 vs. n = 13). Western blot analyses of established cardioprotective signaling proteins were performed. At baseline, contractile stress was comparable. T3 improved the cumulative recovery of contractile stress during reoxygenation from 41 ± 16 with NaOH to 55 ± 11% of baseline with T3, when given continuously in A or from 52 ± 13 with NaOH to 63 ± 11% of baseline with T3 when given just before and during reoxygenation in B. The ratio of mitochondrial complex I matrix arm to membrane NADH:ubiquinone oxidoreductase subunits (NDUF)V2 to NDUFA9 was reduced, reflecting increased complex I activity. T3 increases contractile recovery of human right atrial trabeculae from hypoxia/reoxygenation. • Triiodothyronine improves the contractile recovery of human right atrial trabeculae from hypoxia/reoxygenation. • Better contractile recovery with triiodothyronine goes along with improved mitochondrial function. • Use of triiodothyronine is recommended for inotropic support if needed in patients after coronary bypass surgery. [ABSTRACT FROM AUTHOR]

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]

Tripartite motif (TRIM) 38 is a ubiquitin E3 protein ligase that is involved in various intracellular physiological processes. However, the role of TRIM38 in myocardial ischaemia/reperfusion (I/R) injury remains to be elucidated. We aimed to establish an in vitro cellular hypoxia/reperfusion (H/R) model to explore the role and potential mechanisms of TRIM38 in H9c2, a rat cardiomyoblast cell line. Recombinant ade-noviruses for silencing or overexpressing TRIM38 were constructed and transfected into H9c2 cells. Western blotanalysisshowed that TRIM38 expression was significantly decreased after H/R injury. Functionally, TRIM38 expression relieved inflammatory responses and oxidative stress, and inhibited H/R-induced apoptosis in H9c2 cells. Mechanistically, TRIM38 overexpression inhibited H/R-induced transforming growth factor beta-activated kinase 1 (TAK1)/nuclear factor-kappa B (NF-κB) pathway activity in H9c2 cells. The opposite results were observed after TRIM38 knockdown. Furthermore, H/R-induced injury aggravated by TRIM38 deficiency in H9c2 cells was reversed upon treatment with 5Z-7-oxozeaenol, a TAK1 inhibitor. Therefore, TRIM38 reduction attenuated the anti-apoptotic capacity and anti-inflammatory potential of H/R-stimulated H9c2 cells by activating the TAK1/NF-κB signalling pathway. Specifically, TRIM38 alleviated H/R-induced H9c2 cell injury by promoting TNF receptor-associated factor 6 degradation, which led to the inactivation of the TAK1/NF-κB signalling pathway. Thus, our study provides new insights into the molecular mechanisms underlying H/R-induced myocardial injuries. [ABSTRACT FROM AUTHOR]

The objective of this study was to investigate the role of miR-744 and its target genes in ISO protection against hypoxia/reoxygenation (H/R) induced myocardial injury. Rat cardiomyocytes H9c2 was used to establish an H/R model in vitro, and the level of miR-744 mRNA was detected by fluorescence quantitative PCR. CCK-8 and flow cytometry was used to detected cell viability and apoptosis. Myocardial injury markers CK-MB, cTnI, and LDH were detected by enzyme-linked immunosorbent assay (ELISA). Online bioinformatics software miRDB and miRWalk predicts miR-744 target and its potential binding site, and verifies the target by luciferase reporter assay. After H/R induction, miR-744 mRNA level was remarkedly increased, cell viability was deceased, and apoptosis was increased (p < 0.05). Myocardial injury markers CK-MB, cTnI, and LDH expressions were also increased (p < 0.05). However, ISO pretreatment can significantly alleviate the decrease in cell viability induced by H/R, the increase of cell apoptosis, and the increase of myocardial injury markers, and it play a cardioprotective effect (p < 0.05). More importantly, elevated miR-744 remarkedly weakened the protective effect of ISO on H/R-induced myocardial injury, resulting in decreased cell viability, increased apoptosis, and elevated concentration of myocardial injury indicators (p < 0.05). Luciferase reporter assay confirmed that Sirtuins6 (SIRT6) is a potential target of miR-744 and decreased in H/R-induced myocardial injury, and ISO exposure can reverse its level (p < 0.05). Our findings provide new insights that ISO pretreatment can remarkedly regulate miR-744 and its downstream target SIRT6 to mitigate myocardial injury induced by H/R. [ABSTRACT FROM AUTHOR]

Microcirculation is important for regulating ischemia-reperfusion (I/R) injury associated with skin flap transplantation surgery. We investigated whether co-culture with adipose-derived stem cells (ADSCs) could protect human dermal microvascular endothelial cells (HDMECs) from I/R injury by inhibiting cell apoptosis and enhancing cell proliferation. We also investigated the effects of hypoxic preconditioning on ADSCs. HDMECs were divided into four groups, control, HDMECs in normoxic culture conditions; hypoxia/reoxygenation (H/R), HDMECs in a hypoxic incubator for 8 h then in saturated aerobic culture medium for 24 h; H/R + ADSC(N), HDMECs treated similar to the H/R group then co-cultured with normoxic ADSCs; and H/R + ADSC(H), HDMECs treated similar to the H/R group then co-cultured with hypoxia preconditioned ADSCs. The rate of HDMECs apoptosis significantly increased in the H/R group, but decreased upon co-culture with ADSCs for 24 h, especially in the H/R + ADSC(H) group. Co-culture with ADSCs, especially hypoxia preconditioned ADSCs, significantly enhanced cell proliferation ability compared with that of the H/R group after 48 h and 72 h, but not after 24 h. Vascular endothelial growth factor levels were significantly higher in the H/R + ADSC(N) and H/R + ADSC(H) groups than in the H/R group. ADSCs attenuated H/R injury in endothelial cells by promoting proliferation ability and reducing apoptosis, with an increase in Vascular endothelial growth factor level, especially in the context of hypoxic preconditioning. This approach suggests the potential for an easy and safe method to reduce I/R injury associated with skin flap transplantation surgery. [ABSTRACT FROM AUTHOR]

The protective effect and mechanism of isoflurane on myocardial injury was investigated by constructing in vitro hypoxia/reoxygenation (HR) cell model. HR cell models were established in vitro and treated with isoflurane (ISO). qRT-PCR was used to detect the relative expression of miR-18a-5p. CCK-8 kit and flow cytometry were performed to evaluate cell proliferation and apoptosis. The myocardial injury related markers, such as Cκ-MB, cTnI and LDH were detected by ELISA. Luciferase reporter gene assay was used to verify the interaction between miR-18a-5p and target genes. The expression of miR-18a-5p was significantly increased in hypoxic cardiomyocytes compared with control group (P < 0.001). Meanwhile, cardiomyocytes in the HR group showed inhibition of proliferation, a significant increase in cell apoptosis and in myocardial injury indicators, such as Cκ-MB, cTnI and LDH (P < 0.001). However, 1% ISO treatment alleviated myocardial cell injury induced by HR. Transfection of miR-18a-5p under ISO reduced the protective effect of 1% ISO against myocardial cell damage. Luciferase report gene assay confirmed that CCND2 might be the target gene of miR-18a-5p. In the in vitro cell model of myocardium, ISO alleviated cardiomyocyte injury caused by hypoxia/reoxygenation by down-regulating the expression of miR-18a-5p. [ABSTRACT FROM AUTHOR]

Myocardial ischemia/reperfusion injury (MIRI) is the major cause of myocardial cell damage in acute myocardial infarction, and its treatment remains a clinical challenge. Ginsenoside Rb1 showed protective effects on the cardiovascular system; however, the underlying mechanism remains largely unclear. Effects of Ginsenoside Rb1 on rat MIRI-induced myocardial infarct size were evaluated through TTC staining. TUNEL assay and flow cytometry analysis were employed to estimate cell apoptosis. Apoptosis, autophagy and PI3K/Akt/mTOR pathway-related proteins were estimated via western blot. Expression of Beclin1 in myocardial tissues were examined by immunohistochemical analysis. Expression levels of IL-1 β , TNF- α and IL-6 were tested by enzyme-linked immunosorbent assay (ELISA). Here, we found that Ginsenoside Rb1 treatment not only alleviated MIRI in rats but also protected H9C2 cells against hypoxia/reoxygenation induced damage. Ginsenoside Rb1 abolished the MIRI-induced activation of autophagy. Meanwhile, we found that treatment of 3-MA (autophagy inhibitor) could enhance the protective effects of Ginsenoside Rb1 on H9C2 cells during H/R. Moreover, Ginsenoside Rb1 treatment resulted in the activation of the PI3K/Akt/mTOR pathway, and treatment of LY294002 (PI3K/Akt pathway repressor) abolished the protective effects of Ginsenoside Rb1 on myocardial in vitro and in vivo. Our results suggest that Ginsenoside Rb1 functions as a protector against MIRI by repressing cardiomyocyte autophagy through the PI3K/Akt/mTOR signaling pathway. [ABSTRACT FROM AUTHOR]

Heat shock protein 70 (HSP70) can regulate astrocyte viability under hypoxic and ischemic conditions. However, the protective mechanism involved is not completely clear. This study aimed to investigate whether HSP70 protects U87 glioma cells against hypoxic damage via the extracellular signal-regulated kinases 1/2 (ERK1/2) and phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) signaling pathways. Lentivirus-mediated HSP70-siRNA was used for HSP70 silencing. U87 glioma cells with lentiviral infection were exposed to hypoxia for 4, 8, 12, and 24 h, respectively, followed by a 24-h reoxygenation treatment. A Cell-Counting Kit-8 was then used to evaluate the viability of the U87 glioma cells. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blotting were performed to determine the mRNA and protein levels, respectively. The expression of HSP70, p-ERK1/2, p-AKT, and U87 cell viability were increased after 8 h of hypoxia/24 h of reoxygenation (P < 0.01). However, HSP70 silencing significantly decreased the U87 cell viability after the hypoxia/reoxygenation treatment (P < 0.01). The protein expressions of p-ERK1/2 and p-AKT also decreased in HSP70-silenced U87 cells (P < 0.01). In conclusion, HSP70 inhibition suppressed the viability of U87 glioma cells during hypoxia/reoxygenation (at least partially) by inhibiting the ERK1/2 and PI3K/AKT signaling pathways. This study may help to understand the molecular mechanisms underlying the progression and development of cerebral hypoxia–ischemia. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Objective: To investigate to the expression effect of hypoxia and hypoxia/reoxygenation on ROS, MAPKs and cell apoptosis in H9c2 cardiomyocytes. Methods: H9c2 cells were treated with cobalt chloride (CoCl2) to establish the chemical hypoxia and hypoxia/reoxygenation-induced cardiomyocyte injury model. CoCl2 was used to process cells at different concentrations from 150-2400µmol/L and different time from4-24 h; H9c2 cells viability was detected by MTT, and the intracellular ROS level was measured by 2', 7'-dichlorflµoresceindiacetate (DCFH-DA) and dihydroethidiµm (DHE) staining and photoflurography. The active expression level of mitogen-activated protein kinases (MAPKs) (including JNK, ERK and p38) and caspase-3. Results: At the concentration from 300 to 1200µmol/L, CoCl2 does/time-dependently inhibited the cell viability in H9c2 cells (P<0.01). Compared with control group, the ROS levels in hypoxia group were significantly increased (P<0.05). In hypoxia group, the active expression levels of p-JNK,p-p38 and caspase-3 was higher than those in control group (P<0.05). However, the expression of p-ERK wasn't significant differernce. Furthermore, all the expression levels of ROS, p-JNK, p-ERK, p-p38 and caspase-3 in H/R group were significantly raised compared with hypoxia group (P<0.01). Conclusions: Reoxygenation further aggravate chemical hypoxia induced cardiomyocyte oxidative stress injury by activating ROS/MAPKs signals, suggesting the role of myocardial ischemia/reperfusion injury in the pathogenesis of ischemic heart disease. [ABSTRACT FROM AUTHOR]

Myocardial ischemia-reperfusion injury (MIRI) has been confirmed to induce endoplasmic reticulum stress (ERS) during downstream cascade reactions after the sufficient deterioration of cardiomyocyte function. However, clinically outcomes have been inconsistent with experimental findings because the mechanism has not been entirely elucidated. Dexmedetomidine (DEX), an α2 adrenergic receptor agonist with anti-inflammatory and organ-protective activity, has been shown to attenuate IRI in the heart. The present study aimed to determine whether DEX is able to protect injured cardiomyocytes under in vitro hypoxia/reoxygenation (H/R) conditions and evaluate the conditions under which ERS is efficiently ameliorated. The cytotoxicity of DEX in H9c2 cells was evaluated 24 h after treatment with several different concentrations of DEX. The most appropriate H/R model parameters were determined by the assessment of cell viability and injury with Cell Counting Kit-8 and lactate dehydrogenase (LDH) release assays after incubation under hypoxic conditions for 3 h and reoxygenation conditions for 3, 6, 12 and 24 h. Additionally, the aforementioned methods were used to assess cardiomyocytes cultured with various concentrations of DEX under H/R conditions. Furthermore, the degree of apoptosis and the mRNA and protein expression levels of glucose-regulated protein 78 (GRP78), C/EBP homologous protein (CHOP) and caspase-12 were evaluated in all groups. The addition of 1, 5 and 10 μM DEX to the cell culture significantly increased the proliferation of H9c2 cells by >80% under normal culture conditions. In the H/R model assessment, following 3 h of anoxia exposure, H9c2 cell viability decreased to 62.67% with 3 h of reoxygenation and to 36% with 6 h of reoxygenation compared with the control. The viability of H9c2 cells subjected to hypoxia for 3 h and reoxygenation for 3 h increased by 61.3% when pretreated with 1 μM DEX, and the LDH concentration in the supernatant was effectively decreased by 13.7%. H/R significantly increased the percentage of apoptotic cells, as detected by flow cytometry, and increased the expression levels of GRP78, CHOP and caspase-12, while treatment with either DEX or 4-phenylbutyric acid (4-PBA) significantly attenuated these effects. Additionally, despite the protective effect of DEX against H/R injury, 4-PBA attenuated the changes induced by DEX and H/R. In conclusion, treatment with 1 μM DEX alleviated cell injury, apoptosis and the increases in GRP78, CHOP and caspase-12 expression levels in H9c2 cells induced by 3 h of hypoxia and 3 h of reoxygenation. [ABSTRACT FROM AUTHOR]

The high‐mortality rate of cardiovascular diseases (CVDs) is associated with the myocardial ischemia and reperfusion (I/R). Recent investigations have revealed that microRNAs (miRNAs) exert vital functions in the apoptosis of cardiomyocyte cell. Nevertheless, the potential role of miR‐30a‐5p in the regulation of cardiomyocyte cell apoptosis needs to be illuminated. In the current study, we observed that hypoxia/reoxygenation (H/R) remarkably raised the level of miR‐30a‐5p but reduced the expression of E2F transcription factor 3 (E2F3) in H9c2 cardiomyocytes. In vivo, miR‐30a‐5p was found to be significantly upregulated in the hearts of rats following I/R. Downregulation of miR‐30a‐5p using anti‐miR‐30a‐5p decreased H9c2 cardiomyocytes apoptosis caused by H/R and promoted the proliferation of H9c2 inhibited by H/R. Moreover, E2F3 was a possible target gene of miR‐30a‐5p and upregulation of miR‐30a‐5p reduced the expression level of E2F3 in H9c2 cardiomyocytes. We further identified that E2F3 silencing reversed the effect of anti‐miR‐30a‐5p on the proliferation and apoptosis in H/R treated H9c2 cells. These studies suggested that downregulation of miR‐30a‐5p attenuated the impact of H/R on H9c2 cardiomyocytes through targeting E2F3. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Morphological and functional abnormalities of vascular endothelial cells (VECs) are risk factors of ischemiareperfusion in skin flaps. Signaling pathway mediated by interleukin-1 receptor (IL-1R) is essential to hypoxia/reoxygenation (H/R) injury of VECs. While the TIR/BB-loop mimetic (AS-1) disrupts the interaction between IL-1R and myeloid differentiation primary-response protein 88 (MyD88), its role in the VECs dysfunction under H/R is unclear. In this study, we first showed that there was an infiltration of inflammatory cells and the apoptosis of VECs by using a skin flap section from patients who received flap transplantation. We then showed that the H/R treatment induced apoptosis and loss of cell migration of endothelial cell line H926 were attenuated by AS-1. Furthermore, our data suggested that AS-1 inhibits the interaction between IL-1R and MyD88, and subsequent phosphorylation of IB and p38 pathway, as well as the nuclear localization of NF-KB subunit p65/p50. Thus, this study indicated that the protective role of AS-1 in H/R induced cellular injury may be due to the AS-1 mediated down-regulation of IL-1R signaling pathway. [ABSTRACT FROM AUTHOR]

Endoplasmic reticulum (ER) oxidase 1α (ERO1α) is a glycosylated flavoenzyme that is located on the luminal side of the ER membrane, which serves an important role in catalyzing the formation of protein disulfide bonds and ER redox homeostasis. However, the role of ERO1α in myocardial hypoxia/reoxygenation (H/R) injury remains largely unknown. In the present study, ERO1α expression levels in H9C2 cardiomyocytes increased following H/R, reaching their highest levels following 3 h of hypoxia and 6 h of reoxygenation. In addition, H/R induced apoptosis, and significantly increased expression levels of ER stress (ERS) markers 78 kDa glucose-regulated protein and C/EBP homologous protein. Moreover, the genetic knockdown of ERO1α using short hairpin RNA suppressed cell apoptosis, caspase-3 activity, expression levels of cleaved caspase-12 and cytochrome c in the cytoplasm. Overall, this suggested that ERO1α knockdown may protect against H/R injury. The ERS activator tunicamycin (TM) was used to counteract the ERO1α-induced reduction in ERS; however, the percentage of apoptotic cells and the level of mitochondrial damage did not change. In conclusion, the results from the present study suggested that ERO1α knockdown may protect H9C2 cardiomyocytes from H/R injury through inhibiting intracellular ROS production and increasing intracellular levels of Ca2+, suggesting that ERO1α may serve an important role in H/R. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Neuregulin‐1 (NRG‐1)/erythroblastic leukaemia viral oncogene homologues (ErbB) pathway activation plays a crucial role in regulating the adaptation of the adult heart to physiological and pathological stress. In the present study, we investigate the effect of recombined human NRG‐1 (rhNRG‐1) on mitochondrial biogenesis, mitochondrial function, and cell survival in neonatal rat cardiac myocytes (NRCMs) exposed to hypoxia/reoxygenation (H/R). The results of this study showed that, in the H/R‐exposed NRCMs, mitochondrial biogenesis was impaired, as manifested by the decrease of the expression of peroxisome proliferator‐activated receptor gamma coactivator‐1 alpha (PGC‐1α) and mitochondrial membrane proteins, the inner membrane (Tim23), mitofusin 1 (Mfn1), and mitofusin 2 (Mfn2). RhNRG‐1 pretreatment effectively restored the expression of PGC‐1α and these membrane proteins, upregulated the expression of the anti‐apoptosis proteins Bcl‐2 and Bcl‐xL, preserved the mitochondrial membrane potential, and attenuated H/R‐induced cell apoptosis. Blocking PGC‐1 expression with siRNA abolished the beneficial role of rhNRG‐1 on mitochondrial function and cell survival. The results of the present study strongly suggest that NRG‐1/ErbB activation enhances the adaption of cardiomyocytes to H/R injury via promoted mitochondrial biogenesis and improved mitochondrial homeostasis. Significance of the Study: The results of this research revealed for the first time the relationship between neuregulin‐1 (NRG‐1)/erythroblastic leukaemia viral oncogene homologues (ErbB) activation and mitochondrial biogenesis in neonatal cardiomyocytes and verified the significance of this promoted mitochondrial biogenesis in attenuating hypoxia/reoxygenation injury. This finding may open a new field to further understand the biological role of NRG‐1/ErbB signalling pathway in cardiomyocyte. [ABSTRACT FROM AUTHOR]

Background: miR-377 is closely related to myocardial regeneration. miR-377-adjusted mesenchymal stem cells abducted ischemic cardiac angiogenesis. Nevertheless, there were rarely reports about the impact of miR-377 on myocardial ischemia injury. The purpose of this work is that whether miR-377 can protect against myocardial injury caused by hypoxia/reoxygenation (H/R). Methods: Gene expression omnibus database (http://www.ncbi.nlm.nih.gov/geo/; no. GSE53211) was utilized to study the differential expression of miR-377 in patients with an acute ST-segment elevation myocardial infarction and healthy controls. The luciferase activity was determined utilizing the dual-luciferase reporter system. Quantitative real-time polymerase chain reaction and Western blotting were used to measure the messenger RNA and protein level. Results: Low expression of miR-377 and high expression of leukocyte immunoglobulin-like receptor B2 (LILRB2) were identified in patients with myocardial infarction from analyzing the Gene Expression Omnibus data set. Besides, miR-377 expression was downregulated in cardiomyocyte exposed to H/R. Additionally, overexpression of miR-377 could visibly improve cardiomyocyte injury by regulating cell activity and apoptosis. Conclusions: In short, our findings suggested that miR-377/LILRB2 might regard as a hopeful therapeutic target for myocardial ischemic. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Ferulic acid protects against cardiac injury by scavenging free radicals. However, the role of mitophagy in ferulic acid-induced cardioprotection remains obscure. In the present study, H9c2 cells were exposed to hypoxia/reoxygenation and ferulic acid treatment during hypoxia. We illustrated the impact of ferulic acid on oxidative damage in H9c2 cells. Our results showed that ferulic acid significantly attenuated apoptosis induced by hypoxia/reoxygenation injury and reduced mitochondrial dysfunction, evidenced by a decline in the overproduction of reactive oxygen species and ATP depletion and recovery of the membrane potential. We also found that mitophagy, a selective form of autophagy, was excessively activated in H9c2 cells subjected to hypoxia/reoxygenation. Ferulic acid reduced the binding of mitochondria to lysosomes, down-regulated the PINK1/Parkin pathway, and was accompanied by increased p62 and decreased LC3-II/LC3-I levels. Ferulic acid also antagonistically reduced the activation of mitophagy by rapamycin. These findings suggest that ferulic acid may protect H9c2 cells against ischemia/reperfusion injury by suppressing PINK1/Parkin-dependent mitophagy. Accordingly, our findings may provide a potential target and powerful reference for ferulic acid in clinical prevention and treatment of hypoxia/reoxygenation injury. [ABSTRACT FROM AUTHOR]

Background and Purpose: Intracellular calcium concentration ([Ca2+]i) overload occurs in myocardial ischemia and -reperfusion. The augmentation of the late sodium current (INaL) causes intracellular Na+ accumulation and subsequent [Ca2+]i overload via the reverse mode of the Na+/Ca2+ exchange current (reverse-INCX), which can lead to arrhythmia and cardiac dysfunction. Thus, inhibition of INaL is a potential therapeutic approach for ischemic heart disease. The aim of this study was to investigate the effects of thyroid hormone on augmented INaL, reverse-INCX, altered action potential duration (APD), and [Ca2+]i concentration in hypoxia/reoxygenation (H/R)-induced ventricular myocytes in vitro. Methods: The transient Na+ current (INaT), INaL, reverse-INCX, and APs were recorded using a whole-cell patch-clamp technique in neonatal mouse ventricular myocytes. [Ca2+]i concentration alteration were, respectively, observed by confocal microscopy and flow cytometry. Results: Triiodothyronine (T3) pretreatment decreased the INaL in a concentration-dependent manner. H/R injury aggravated the INaL, INaT, and reverse-INCX in cardiomyocytes and increased the continuous accumulation of [Ca2+]i (p < 0.05). The application of T3 prior to H/R injury significantly decreased the increased INaL, INaT, and reverse-INCX and blunted the [Ca2+]i increase. Furthermore, T3 pretreatment shortened the APD induced by H/R injury. Conclusion: T3 inhibited H/R-increased INaL and reverse INCX augmentation, shortened the APD, and diminished [Ca2+]i overload, indicating a potential therapeutic use of T3 as an INaL inhibitor to maintain Ca2+ homeostasis and protect cardiomyocytes against H/R injury. [ABSTRACT FROM AUTHOR]

lncUCA1 is abundantly expressed in the heart, indicating it may be important in maintaining normal myocardial function. However, the underlying mechanism of lncUCA1 in heart disease, particularly myocardial infarction (MI), is still in its infancy. LncUCA1 and miR-143 expression were measured in hearts of MI models. Overexpression and knockdown of lncUCA1 in neonatal rat cardiomyocytes were performed to confirm the effects of lncUCA1 in hypoxia-induced apoptosis. The expression of lncUCA1 decreased but miR-143 increased inversely in MI heart. Overexpressing lncUCA1 protected cardiomyocytes from H/R induced apoptosis via inhibiting miR-143, which regulates apoptosis by targeting MDM2/p53 pathway. While silencing lncUCA1 caused miR-143 upregulation and H/R-induced apoptosis increase. Moreover, miR-143 was proved to be a competitive target of lncUCA1. lncUCA1 might protect cardiomyocyte against H/R induced apoptosis by suppressing miR-143 and modulated the following downstream MDM2/p53 signaling pathway, indicating the therapeutic potential of targeting lncUCA1 for MI. • UCA1 was downregulated and miR-143 was upregulated in MIRI, which promoted cell apoptosis. • UCA1 reciprocal interacted with miR-143 by direct binding to each other. • UCA1 protects cardiomyocytes against H/R-induced apoptosis by targeting miR-143/MDM2/p53 signaling axis. [ABSTRACT FROM AUTHOR]

MicroRNA (miR) plays an integral role in cardiovascular diseases. M‐iR‐423‐5p is aberrantly expressed in patients with myocardial infarction and heart failure. The aim of the present study was to study the roles and mechanisms of miR‐423‐5p in hypoxia/reoxygenation (H/R) mediated cardiomyocytes injury. H9C2 cells were transfected with negative control, miR‐423‐5p mimic, and inhibitor for 48 hr, followed by exposed to H/R condition. Cell apoptosis rate, caspase 3/7 activities, Bax and cleaved‐caspase 3 (c‐caspase 3) protein levels were assayed by flow cytometry, Caspase‐Glo 3/7 Assay kit, western blot analysis, respectively. Furthermore, the mitochondrial membrane potential, adenosine triphosphate (ATP) content, reactive oxygen species (ROS) production, and Drp1 expression were also investigated. Furthermore, the dual‐luciferase reporter assay was used to evaluate the relationship between miR‐423‐5p and Myb‐related protein B (MYBL2). The roles of miR‐423‐5p in wnt/β‐catenin were assessed by western blot analysis. The results revealed that H/R triggered miR‐423‐5p expression. Overexpression of miR‐423‐5p promoted cardiomyocyte apoptosis, enhanced the activities of caspase 3/7, upregulated the expression of Bax and c‐caspase 3. miR‐423‐5p upregulation caused the loss of mitochondrial membrane potential and the reduction of ATP content, the augment of ROS production and Drp1 expression. However, the opposite trends were observed upon suppression of miR‐423‐5p. In addition, miR‐423‐5p could target the 3′ untranslated region of MYBL2. miR‐423‐5p depletion led to the activation of the wnt/β‐catenin signaling pathway via targeting MYBL2. Knockdown of MYBL2 was obviously reversed the roles of miR‐423‐5p in apoptosis and mitochondrial dysfunction. Taken together, miR‐423‐5p suppression reduced H/R‐induced cardiomyocytes injury through activation of the wnt/β‐catenin signaling pathway via targeting MYBL2 in cardiomyocytes. [ABSTRACT FROM AUTHOR]

The aim of the present study was to determine the function of microRNA-16 (miR-16) in myocardial hypoxia/reoxygenation (H/R)-induced cardiomyocyte injury and the possible mechanism underlying its involvement. An H/R model was constructed using H9c2(2-1) cells in vitro. The results of reverse transcription-quantitative PCR demonstrated that the expression levels of miR-16 were significantly upregulated in H9c2(2-1) cells in the H/R group compared with the sham group (1.53±0.09 vs. 1.0±0.08; P=0.0019). Cell Counting Kit-8 assays revealed that the relative proliferative ability of H9c2(2-1) cells was significantly decreased in the H/R + negative control (NC) group compared with the sham group (0.53±0.05 vs. 1.0±0.08; P=0.00005). Upregulation of miR-16 using miR-16 mimics further decreased the proliferative ability of cells (0.31±0.03 vs. 0.53±0.05; P=0.0097), whereas downregulation of miR-16 using an miR-16 inhibitor increased the proliferative ability of cells compared with the H/R+NC group (0.89±0.08 vs. 0.53±0.05; P=0.000385). Flow cytometric analysis found that the apoptotic rate of H9c2(2-1) cells was increased significantly following H/R compared with the sham group (25.86±2.62% vs. 9.29±0.82%, P=0.000014). Upregulation of miR-16 further increased the apoptotic rate (38.62±2.04% vs. 25.86±2.62%; P=0.000099), whereas downregulation of miR-16 decreased the apoptotic rate compared with the H/R+NC group (15.14±0.92% vs. 25.86±2.62%; P=0.000343). miR-16 directly bound to the 3′-untranslated region of cytokine-induced apoptosis inhibitor 1 (CIAPIN1) and negatively modulated CIAPIN1 expression. Overexpression of CIAPIN1 reversed the changes in the expression of apoptosis-associated proteins caused by H/R. Western blot analysis revealed that the levels of phospho-(p-)nuclear factor-κB (NF-κB) and p-NF-κB inhibitor α (IκBα) were upregulated following H/R (1.82±0.11 vs. 1.0±0.08; P=0.000152; and 1.77±0.07 vs. 1.0±0.00; P=0.000024, respectively), and these changes were further enhanced when miR-16 expression levels were increased (3.10±0.14 vs. 1.82±0.11; P=0.000006; and 2.19±0.10 vs. 1.77±0.07; P=0.0017, respectively). Downregulation of miR-16 exhibited the opposite effect on p-NF-κB and p-IκBα expression levels. The present study illustrates that downregulation of miR-16 may protect against H/R-induced injury partially by targeting CIAPIN1 and the NF-κB signaling pathway. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Cardiovascular disease including cerebral ischemic stroke is the major complication that increases the morbidity and mortality in patients with diabetes mellitus as much as four times. It has been well established that irisin, with its ability to regulate glucose and lipid homeostasis as well as anti-inflammatory and anti-apoptotic properties, has been widely examined for its therapeutic potentials in managing metabolic disorders. However, the mechanism of irisin in the regulation of cerebral ischemic stroke remains unclear. Using PC12 cells as a model, we have shown that hypoxia/reoxygenation inhibits cell viability and increases lactic dehydrogenase. Irisin, in a dose-dependent manner, reversed these changes. The increase in inflammatory mediators (IL-1β, IL-6, and TNF-α) by hypoxia/reoxygenation was reversed by irisin. Furthermore, the cell apoptosis promoted by hypoxia/reoxygenation was also inhibited by irisin. Irisin suppressed TLR4/MyD88 signaling pathway leading to amelioration of inflammation and apoptosis in PC12 cells. Thus, inhibition of TLR4/MyD88 signaling pathway via irisin could be an important mechanism in the regulation of hypoxia/reoxygenation-induced inflammation and apoptosis in PC12 cells. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Remifentanil postconditioning (RPC) has been shown to provide potent cardioprotection against ischemia/reperfusion (I/R) injury, but the underlying mechanism has not been fully elucidated. The current study was designed to investigate whether RPC protects cardiomyocytes against I/R injury through enhancement of autophagic flux. H9c2 cardiomyocytes were exposed to hypoxia/reoxygenation (H/R) to mimic myocardial I/R injury in vitro. Autophagosome formation was evaluated by detecting of light chain 3 (LC3) puncta number and LC3Ⅱ levels using immunofluorescence and western blotting, respectively. Additionally, dual fluorescent staining of LC3 and lysosomal-associated membrane protein 2, a lysosomal marker protein, were used to detect autolysosome formation. Moreover, autophagic flux integrity was tracked using changes in LC3Ⅱ and p62 levels. Lastly, myocardial injury was detected by Hoechst 33342 and propidium iodide staining and MTT assay. The results showed that RPC increased autophagosome formation and promoted autophagosome-lysosome fusion, thereby improving autophagic flux in H9c2 cells. Reversal of these effect by bafilomycin A1 or chloroquine co-administration at reoxygenation onset indicated that RPC improved the impaired autophagic flux following H/R injury. Induction of autophagy was associated with increased cell viability and decreased apoptosis. Autophagy inhibition with bafilomycin A1 or chloroquine and ATG7shRNA significantly abolished RPC-induced cardioprotection. In conclusion, our finding that RPC can protect cardiomyocytes against H/R injury through enhancement of autophagic flux suggests a new mechanism for myocardial protection of opioid postconditioning. • Autophagosome formation increased but its clearance impaired following H/R insult. • RPC increased autophagosome generation and promoted autophagosome-lysosome fusion. • RPC restored autophagic flux and ameliorated myocardial injury after H/R injury. • Protective autophagy of RPC provides a new therapeutic strategy for heart disease. [ABSTRACT FROM AUTHOR]

Shenmai injection (SMI) is a CFDA-approved and widely prescribed herbal medicine injection in China for treating cardiac dysfunction, especially myocardial ischemia and reperfusion (I/R) injury. However, despite of its known clinical efficacy, the cardioprotective mechanisms of SMI remain to be established. The present study aimed to investigate the role of SMI on mitophagy and mitochondrial dynamics in cardiomyocytes with a hypoxia/reperfusion (H/R) injury setting. H9c2 cardiomyocytes were subjected to 12 h of hypoxia followed by 2 h of reoxygenation to induce cellular injury. Multi-parameter imaging analysis was performed using Operetta High Content Imaging System to detect changes in mitochondrial function and morphological texture. The mPTP opening was directly assessed by analyzing mitochondrial calcein release in H9c2 and by Ca2+-induced swelling of isolated cardiac mitochondria. Mitochondrial respiration was measured by XF 24 analyzer of Seahorse Bioscience. RT-PCR and Western blotting analyses were used to detect mitophagy, mitochondrial fusion and fission biomarkers at the gene and protein levels. Pretreatment of SMI significantly improved myocardial cell survival and protected against H/R-induced deterioration of mitochondrial structure and function, as evidenced by decreased mitochondrial mass and cytosolic Ca2+, increased mitochondrial membrane potential (ΔΨ m) and mitochondrial morphology by SER Texture analysis, inhibited mPTP opening in H9c2 cells and isolated cardiac mitochondria, and alleviated severely impaired mitochondrial respiration. Mechanistically, SMI attenuated H/R injury by inducing mitophagy and then modulated mitochondrial dynamics as indicated by a significantly increased expression of LC3, Beclin 1, Parkin and Pink, and the inhibition of excessive mitochondria fission and increased mitochondrial fusion. Finally, the cardioprotective effect of SMI was confirmed in a LAD-induced cardiac dysfunction model in vivo. We found that alleviation of H/R injury by pretreatment with SMI may be attributable to inducing mitophagy and modulating mitochondrial dynamics in cardiomyocytes, thereby providing a rationale for future clinical applications and potential mitoprotective therapy for MI/R injury. Image 1 [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]

Abstract Our previous study showed that Sphingosine-1-phosphate (S1P) could protect cardiomyocytes against hypoxia/reoxygenation (H/R) injury via the JAK-STAT pathway and maintain normal myocardial mitochondria integrity in vivo. However, it is not known yet whether S1P can relieve mitochondrial dysfunction via the mitochondrial apoptotic pathway and its detailed mechanism remains to be investigated. The aim of this study was to demonstrate the mitochondrial protective effects of S1P in a cardiomyocyte H/R injury model. In the present study, we established a H/R model in H9c2 cells. Cell viability was determined by the MTT assay, and apoptosis was evaluated by annexin V-FITC/PI staining. Mitochondrial calcium ion concentration, mitochondrial membrane potential (ΔΨm), opening of the mitochondrial permeability transition pore (mPTP), and release of cytochrome C were detected by laser confocal microscopy. The results showed that S1P inhibited the decrease in cell viability induced by H/R injury and reduced apoptosis. Confocal microscopy showed that S1P prevented loss of ΔΨm, relieved mitochondrial calcium overload, and inhibited opening of the mPTP and release of cytochrome C. The STAT3 inhibitor STATTIC can reverse the antiapoptotic effects of S1P and block the effect of S1P on mitochondria. Taken together, our results indicate that S1P protects cardiomyocytes against H/R injury by relieving mitochondrial dysfunction via the STAT3 pathway. Highlights • S1P protects cardiomyocytes against hypoxia/reoxygenation injury. • S1P relieves mitochondria dysfunction induced by H/R injury. • S1P protects cardiomyocytes against H/R injury by relieving mitochondrial dysfunction via the STAT3 pathway. [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]

Abstract Hypoxia/reoxygenation (H/R) plays an important role in the pathogenesis of osteoarthritis. Fibroblast-like synoviocytes (FLS), which are highly sensitive to H/R, are thought to be associated with cartilage degradation during osteoarthritis development. In this study, we investigated the biological effects of insulin-like growth factor (IGF) system and the expression of inflammatory mediators in FLS. We also pretreated FLS with tumor necrosis factor-α (TNF-α) before H/R in order to observe the response of FLS with the background of inflammatory cytokines. H/R increased the levels of TNF-α-induced C-C chemokine ligand 5 (CCL5), interleukin-1β (IL-1β) and interleukin-6 (IL-6) in cell-free culture supernatants; H/R also increased the expression of TNF-α-induced insulin-like growth factor binding protein 3 (IGFBP-3), downregulated the expression of insulin-like growth factor 1 (IGF-1), promoted the loss of mitochondrial membrane potential (MMP), the openness of mitochondrial permeability transition pore (MPTP), the release of intracellular reactive oxygen species (ROS), and mitochondrial matrix swelling, outer membrane rupture and decrease in cristae. Furthermore, H/R induced the expression of catabolic factors and activated the NF-κB signaling pathway in FLS. We therefore concluded that H/R may play a role in inducing inflammation and increase the TNF-α-induced inflammatory effect in FLS, contributing to osteoarthritis pathogenesis. Highlights • H/R affects the expression of IGF system and may associate with synovitis. • H/R induces mitochondrial dysfunction and the release of ROS. • NF-κB signaling pathway are activated under H/R conditions. [ABSTRACT FROM AUTHOR]

Background/Aims: Myocardial ischemia/reperfusion (I/R) or hypoxia/reoxygenation (H/R) injury is always characterized by Ca2+ overload, energy metabolism disorder and necrocytosis of cardiomyocytes. We showed previously that chronic intermittent hypobaric hypoxia (CIHH) improves cardiac function during I/R through improving cardiac glucose metabolism. However, the underlying cellular and molecular mechanisms of CIHH treatment improving energy metabolism in cardiomyocytes are still unclear. In this study, we determined whether and how CIHH protects cardiomyocytes from Ca2+ overload and necrocytosis through energy regulating pathway. Methods: Adult male Sprague-Dawley rats were randomly divided into two groups: control (CON) and CIHH group. CIHH rats received a hypobaric hypoxia simulating 5,000-m altitude for 28 days, 6 hours each day, in hypobaric chamber. Rat ventricular myocytes were obtained by enzymatic dissociation. The intracellular calcium concentration ([Ca2+]i) and cTnI protein expression were used to evaluate the degree of cardiomyocytes injury during and after H/R. The mRNA and protein expressions involved in cardiac energy metabolism were determined using quantitative PCR and Western blot techniques. PGC-1α siRNA adenovirus transfection was used to knock down PGC-1α gene expression of cardiomyocytes to determine the effect of PGC-1α in the energy regulating pathway. Results: H/R increased [Ca2+]i and cTnI protein expression in cardiomyocytes. CIHH treatment decreased [Ca2+]i (p<0.01) and cTnI protein expression (p<0.01) in cardiomyocytes after H/R. Both mRNA and protein expression of PGC-1α increased after CIHH treatment, which was reversed by PGC-1α siRNA adenovirus transfection. Furthermore, CIHH treatment increased the expression of HIF-1α, AMPK and p-AMPK in cardiomyocytes, and pretreatment with AMPK inhibitor dorsomorphin abolished the enhancement of PGC-1α protein expression in cardiomyocytes by CIHH (p<0.01). In addition, PGC-1α knock down also abolished the increased protein level of GLUT4 (p<0.01) and decreased the protein level of CPT-1b (p<0.05) in cardiomyocytes by CIHH treatment. Conclusion: CIHH treatment could reduce the calcium overload and H/R injury in cardiomyocytes by up-regulating the expression of PGC-1α and regulating the energy metabolism of glucose and lipid. The HIF-1α-AMPK signaling pathway might be involved in the process. [ABSTRACT FROM AUTHOR]

The sonic hedgehog (Shh) signaling pathway has been reported to protect cells against hypoxia/reoxygenation (H/R) injury; however, the role of Shh and relevant molecular mechanisms remain unclear. In the present study, the rat cardiomyoblast cell line H9C2 was subjected to hypoxia and serum-starvation for 4 h. Cells were subsequently reoxygenated using 95% O2 and 5% CO2. Reverse transcription-quantitative polymerase chain reaction was performed to quantify the expression of Shh mRNA, while cell apoptosis was assessed using flow cytometry. Caspase-3 activity and p53 expression were measured by western blotting and an MTT assay was subsequently used to assess cell viability. In addition, reactive oxygen species levels were measured using dichlorofluorescein and H/R-induced changes in the activation of superoxide dismutase, catalase, phosphorylated-endothelial nitric oxide synthase, phosphorylated-protein kinase B (Akt) and mammalian target of rapamycin activation were assessed using western blotting. H/R treatment decreased the cell viability of H9C2 cells, but activated endogenous Shh signaling. The activation of Shh signaling protected H9C2 myocardial cells from H/R-induced apoptosis and restored cell viability. In the present study, Shh signaling was demonstrated to serve a protective role against H/R by activating the phosphoinositol 3-kinase (PI3K)/Akt pathway and promoting the expression of anti-oxidant enzymes to ameliorate oxidative stress. In summary, Shh signaling attenuated H/R-induced apoptosis through via the PI3K/Akt pathway. [ABSTRACT FROM AUTHOR]

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]

Abstract Fibroblast growth factor 19 (FGF19) has emerged as a crucial cytoprotective regulator that antagonizes cell apoptosis and oxidative stress under adverse conditions. However, whether FGF19 plays a cytoprotective role in preventing myocardial damage during myocardial ischemia/reperfusion injury remains unknown. In this study, we aimed to investigate the potential role of FGF19 in regulating hypoxia/reoxygenation (H/R)-induced injury of cardiomyocytes in vitro. We found that FGF19 expression was upregulated in response to H/R treatment in cardiomyocytes. Silencing of FGF19 significantly inhibited viability and increased apoptosis and reactive oxygen species (ROS) generation in cardiomyocytes with H/R treatment. In contrast, overexpression of FGF19 improved viability and inhibited apoptosis and ROS generation induced by H/R treatment, showing a cardioprotective effect. Moreover, we found that FGF19 regulated the phosphorylation of glycogen synthase kinase-3β (GSK-3β) and the nuclear translocation of nuclear factor-E2-related factor 2 (Nrf2). In addition, FGF19 promoted the activation of Nrf2-mediated antioxidant response element (ARE) antioxidant signaling. Notably, treatment with a GSK-3β inhibitor significantly abrogated the adverse effects of FGF19 silencing on H/R-induced injury, whereas silencing of Nrf2 partially blocked the FGF19-mediated cardioprotective effect against H/R-induced injury in cardiomyocytes. Taken together, our findings demonstrate that FGF19 alleviates H/R-induced apoptosis and oxidative stress in cardiomyocytes by inhibiting GSK-3β activity and promoting the activation of Nrf2/ARE signaling, providing a potential therapeutic target for prevention of myocardial injury. Graphical abstract Image 1 Highlights • FGF19 is induced by H/R treatment in cardiomyocytes. • FGF19 knockdown augments H/R-induced injury. • FGF19 overexpression protects cardiomyocytes from H/R-induced injury. • FGF19-mediated GSK-3β/Nrf2 signaling contributes to cardioprotection. [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 Background Ischemia/reperfusion injury is characterized by excess generation of reactive oxygen species (ROS). The purpose of this study is to test the effect of reoxygenation speed on ROS production and the cellular injury responses in hypoxic macrophages RAW 264.7 cells and its potential mechanisms for the generation of ROS. Materials and methods After hypoxic exposure of RAW 264.7 cells for 20 h, reoxygenation was performed for 6 h by stepwise increase in oxygen concentration (0.8% increase of oxygen every 15 min) in the slow reoxygenation (SRox) group or by moving the culture flasks quickly to a normoxic incubator in the rapid reoxygenation (RRox) group. To identify the potential effect of reoxygenation speed on the generation of ROS, the cells were pretreated with apocynin, VAS2870, and MitoTEMPO before the induction of hypoxia. Results SRox significantly decreased cell death and cytotoxicity compared with RRox ( P < 0.05). RRox resulted in significantly more generation of ROS, interleukin-1β, interleukin-6, and nitric oxide than SRox ( P < 0.05). SRox also increased the expression of prosurvival proteins and decreased apoptosis. In cells pretreated with VAS2870 or MitoTEMPO, the reduced ROS generation by SRox was maintained. However, pretreatment with apocynin abolished the effect of reoxygenation speed on ROS generation. Conclusions SRox compared with RRox decreased cellular injury in hypoxic RAW 264.7 cells by decreasing ROS and inflammatory cytokine production and decreasing apoptosis. [ABSTRACT FROM AUTHOR]