Mechanism for resveratrol-induced cardioprotection against reperfusion injury involves glycogen synthase kinase 3β and mitochondrial permeability transition pore

Resveratrol pretreatment can protect the heart by inducing pharmacological preconditioning. Whether resveratrol protects the heart when applied at reperfusion remains unknown. We examined the effect of resveratrol on myocardial infarct size when given at reperfusion and investigated the mechanism underlying the effect. Isolated rat hearts were subjected to 30 min ischemia followed by 2 h of reperfusion, and myocardial samples were collected from the risk zone for Western blot analysis. Mitochondrial swelling was spectrophotometrically measured as a decrease in absorbance at 520 nm (A520). Resveratrol reduced infarct size and prevented cardiac mitochondrial swelling. Resveratrol enhanced GSK-3β phosphorylation upon reperfusion, an effect that was mediated by the cyclic guanosine monophosphate (cGMP)/protein kinase G (PKG) pathway. Resveratrol translocated GSK-3β from cytosol to mitochondria via the cGMP/PKG pathway. Further studies showed that mitochondrial GSK-3β was co-immunoprecipitated with cyclophilin D but not with VDAC (voltage dependent anion channel) or ANT (adenine nucleotide translocator). These data suggest that resveratrol prevents myocardial reperfusion injury presumably by targeting the mPTP through translocation of GSK-3β from cytosol to mitochondria. Translocated GSK-3β may ultimately interact with cyclophilin D to modulate the mPTP opening

NO mobilizes intracellular Zn2+ via cGMP/PKG signaling pathway and prevents mitochondrial oxidant damage in cardiomyocytes

Our aim was to determine if NO prevents mitochondrial oxidant damage by mobilizing intracellular free zinc (Zn2+)

The critical role of intracellular zinc in adenosine A2 receptor activation induced cardioprotection against reperfusion injury

Exogenous zinc can protect cardiac cells from reperfusion injury, but the exact roles of endogenous zinc in the pathogenesis of reperfusion injury and in adenosine A2 receptor activation-induced cardioprotection against reperfusion injury remain unknown. Adenosine A1/A2 receptor agonist 5′-(N-ethylcarboxamido) adenosine (NECA) given at reperfusion reduced infarct size in isolated rat hearts subjected to 30 min ischemia followed by 2 h of reperfusion. This effect of NECA was partially but significantly blocked by the zinc chelator N,N,N′,N′-tetrakis-(2-pyridylmethyl) ethylenediamine (TPEN), and ZnCl2 given at reperfusion mimicked the effect of NECA by reducing infarct size. Total tissue zinc concentrations measured with inductively coupled plasma optical emission spectroscopy (ICPOES) were decreased upon reperfusion in rat hearts and this was reversed by NECA. NECA increased intracellular free zinc during reperfusion in the heart. Confocal imaging study showed a rapid increase in intracellular free zinc in isolated rat cardiomyocytes treated with NECA. Further experiments revealed that NECA increased total zinc levels upon reperfusion in mitochondria isolated from isolated hearts. NECA attenuated mitochondrial swelling upon reperfusion in isolated hearts and this was inhibited by TPEN. Similarly, NECA prevented the loss of mitochondrial membrane potential (ΔΨm) caused by oxidant stress in cardiomyocytes. Finally, both NECA and ZnCl2 inhibited the mitochondrial metabolic activity. NECA-induced cardioprotection against reperfusion injury is mediated by intracellular zinc. NECA prevents reperfusion-induced zinc loss and relocates zinc to mitochondria. The inhibitory effects of zinc on both the mPTP opening and the mitochondrial metabolic activity may account for the cardioprotective effect of NECA

Astragaloside IV Inhibits Oxidative Stress-Induced Mitochondrial Permeability Transition Pore Opening by Inactivating GSK-3β via Nitric Oxide in H9c2 Cardiac Cells

Objective. This study aimed to investigate whether astragaloside IV modulates the mitochondrial permeability transition pore (mPTP) opening through glycogen synthase kinase 3β (GSK-3β) in H9c2 cells. Methods. H9c2 cells were exposed to astragaloside IV for 20 min. GSK-3β (Ser9), Akt (Ser473), and VASP (Ser239) activities were determined with western blot. The mPTP opening was evaluated by measuring mitochondrial membrane potential (ΔΨm). Nitric oxide (NO) generation was measured by 4-amino-5-methylamino-2′, 7′-difluorofluorescein (DAF-FM) diacetate. Fluorescence images were obtained with confocal microscopy. Results. Astragaloside IV significantly enhanced GSK-3β phosphorylation and prevented H2O2-induced loss of ΔΨm. These effects of astragaloside IV were reversed by the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002, the NO sensitive guanylyl cyclase selective inhibitor ODQ, and the PKG inhibitor KT5823. Astragaloside IV activated Akt and PKG. Astragaloside IV was also shown to increase NO production, an effect that was reversed by L-NAME and LY294002. Astragaloside IV applied at reperfusion reduced cell death caused by simulated ischemia/reperfusion, indicating that astragaloside IV can prevent reperfusion injury. Conclusions. These data suggest that astragaloside IV prevents the mPTP opening and reperfusion injury by inactivating GSK-3β through the NO/cGMP/PKG signaling pathway. NOS is responsible for NO generation and is activated by the PI3K/Akt pathway

Neuroprotection Effect of Astragaloside IV from 2-DG-Induced Endoplasmic Reticulum Stress

Objective. Astragaloside IV shows neuroprotective activity, but its mechanism remains unclear. To investigate whether astragaloside IV protects from endoplasmic reticulum stress (ERS), we focus on the regulation of glycogen synthase kinase-3β (GSK-3β) and mitochondrial permeability transition pore (mPTP) by astragaloside IV in neuronal cell PC12. Methods and Results. PC12 cells treated with different concentrations of ERS inductor 2-deoxyglucose (2-DG) (25-500 μM) showed a significant increase of glucose-regulated protein 78 (GRP 78) and GRP 94 expressions and a decrease of tetramethylrhodamine ethyl ester (TMRE) fluorescence intensity and mitochondrial membrane potential (∆Ψm), with the peak effect seen at 50 μM, indicating that 2-DG induces ERS and the mPTP opening. Similarly, 50 μM of astragaloside IV increased the GSK-3β phosphorylation at Ser9 most significantly. Next, we examined the neuroprotection of astragaloside IV by dividing the PC12 cells into control group, 2-DG treatment group, astragaloside IV plus 2-DG treatment group, and astragaloside IV only group. PC12 cells treated with 50 μM 2-DG for different time courses (0-36 hr) showed a significant increase of Cleaved-Caspase-3 with the peak at 6 hr. 2-DG significantly induced cell apoptosis and increased the green fluorescence intensity of Annexin V-FITC, and these effects were reversed by astragaloside IV. Such a result indicates that astragaloside IV protected neural cell survival from ERS. 2-DG treatment significantly increased the expressions of inositol-requiring ER-to-nucleus signal kinase 1 (IRE1), phosphor-protein kinase R-like ER kinase (p-PERK), but not affect the transcription factor 6 (ATF6) expression. 2-DG treatment significantly decreased the phosphorylation of GSK-3β and significantly reduced the TMRE fluorescence intensity and ∆Ψm, following mPTP open. Astragaloside IV significantly inhibited the above effects caused by 2-DG, except the upregulation of ATF6 protein. Taken together, astragaloside IV significantly inhibited the ERS caused by 2-DG. Conclusion. Our data suggested that astragaloside IV protects PC12 cells from ERS by inactivation of GSK-3β and preventing the mPTP opening. The GRP 78, GRP 94, IRE1, and PERK signaling pathways but not ATF6 are responsible for GSK-3β inactivation and neuroprotection by astragaloside IV

Application of three-dimensional visualization modeling technology of ore bodies in metallogenic mode analysis

Objective Copper is an important strategic metal resource for national economic and social development. Therefore, the exploration and resource assessment of copper mines are of great significance. Methods Based on collected geological profiles and drilling data, this study constructed a three-dimensional geological model for a copper ore deposit in western Yunnan, which was applied to estimate the resource reserve in the mining area. Results The ore resource is estimated to be 48.93 million tons, including 0.543 million tons of copper. Through comparative analysis, the model and resource estimation established by our newly proposed three-dimensional geological modelling system shows high credibility, in which multiple analysis modules and dynamic update function have a wide range of applications and it can be used for future drilling engineering and resources estimation. Conclusion This study provides the basis for further exploration work in this region, and it can also be applied to the exploration and mining of related polymetallic deposits

Roles of Endoplasmic Reticulum Stress in NECA-Induced Cardioprotection against Ischemia/Reperfusion Injury

Objective. This study aimed to investigate whether the nonselective A2 adenosine receptor agonist NECA induces cardioprotection against myocardial ischemia/reperfusion (I/R) injury via glycogen synthase kinase 3β (GSK-3β) and the mitochondrial permeability transition pore (mPTP) through inhibition of endoplasmic reticulum stress (ERS). Methods and Results. H9c2 cells were exposed to H2O2 for 20 minutes. NECA significantly prevented H2O2-induced TMRE fluorescence reduction, indicating that NECA inhibited the mPTP opening. NECA blocked H2O2-induced GSK-3β phosphorylation and GRP94 expression. NECA increased GSK-3β phosphorylation and decreased GRP94 expression, which were prevented by both ERS inductor 2-DG and PKG inhibitor KT5823, suggesting that NECA may induce cardioprotection through GSK-3β and cGMP/PKG via ERS. In isolated rat hearts, both NECA and the ERS inhibitor TUDCA decreased myocardial infarction, increased GSK-3β phosphorylation, and reversed GRP94 expression at reperfusion, suggesting that NECA protected the heart by inhibiting GSK-3β and ERS. Transmission electron microscopy showed that NECA and TUDCA reduced mitochondrial swelling and endoplasmic reticulum expansion, further supporting that NECA protected the heart by preventing the mPTP opening and ERS. Conclusion. These data suggest that NECA prevents the mPTP opening through inactivation of GSK-3β via ERS inhibition. The cGMP/PKG signaling pathway is responsible for GSK-3β inactivation by NECA

The critical role of intracellular zinc in adenosine A(2) receptor activation induced cardioprotection against reperfusion injury

Exogenous zinc can protect cardiac cells from reperfusion injury, but the exact roles of endogenous zinc in the pathogenesis of reperfusion injury and in adenosine A(2) receptor activation-induced cardioprotection against reperfusion injury remain unknown. Adenosine A(1)/A(2) receptor agonist 5′-(N-ethylcarboxamido) adenosine (NECA) given at reperfusion reduced infarct size in isolated rat hearts subjected to 30 min ischemia followed by 2 h of reperfusion. This effect of NECA was partially but significantly blocked by the zinc chelator N,N,N′,N′-tetrakis-(2-pyridylmethyl) ethylenediamine (TPEN), and ZnCl(2) given at reperfusion mimicked the effect of NECA by reducing infarct size. Total tissue zinc concentrations measured with inductively coupled plasma optical emission spectroscopy (ICPOES) were decreased upon reperfusion in rat hearts and this was reversed by NECA. NECA increased intracellular free zinc during reperfusion in the heart. Confocal imaging study showed a rapid increase in intracellular free zinc in isolated rat cardiomyocytes treated with NECA. Further experiments revealed that NECA increased total zinc levels upon reperfusion in mitochondria isolated from isolated hearts. NECA attenuated mitochondrial swelling upon reperfusion in isolated hearts and this was inhibited by TPEN. Similarly, NECA prevented the loss of mitochondrial membrane potential (ΔΨm) caused by oxidant stress in cardiomyocytes. Finally, both NECA and ZnCl(2) inhibited the mitochondrial metabolic activity. NECA-induced cardioprotection against reperfusion injury is mediated by intracellular zinc. NECA prevents reperfusion-induced zinc loss and relocates zinc to mitochondria. The inhibitory effects of zinc on both the mPTP opening and the mitochondrial metabolic activity may account for the cardioprotective effect of NECA

Postconditioning prevents reperfusion injury by activating delta-opioid receptors. Anesthesiology 2008

Background: While postconditioning has been proposed to protect the heart by targeting the mitochondrial permeability transition pore (mPTP), the detailed mechanism underlying this action is unknown. The authors hypothesized that postconditioning stimulates opioid receptors, which in turn protect the heart from reperfusion injury by targeting the mPTP. Methods: Rat hearts (both in vivo and in vitro) were subjected to 30 min of ischemia and 2 h of reperfusion. Postconditioning was elicited by six cycles of 10-s reperfusion and 10-s ischemia. To measure nitric oxide concentration, cardiomyocytes loaded with 4-amino-5-methylamino-2=,7=-difluorofluorescein were imaged using confocal microscopy. Mitochondrial membrane potential was determined by loading cardiomyocytes with tetramethylrhodamine ethyl ester. Result