Abstract Resveratrol (Res) has cardioprotective, anti-inflammatory, anti-aging and antioxidant effects; however, its mechanism remains unclear. Here we explored the protective effects of resveratrol on cardiomyocytes, focusing on the role of Zn 2+ and mitophagy. Using the MTT/LDH assay, we found that addition of a zinc chelator TPEN for 4h induced mitophagy and significantly reduced cell viability, increased cytotoxicity and apoptosis in H9c2, while Res significantly inhibited these effects of TPEN. Similarly, Res inhibited TPEN-induced expression of mitophagy-associated proteins P62, LC3, PINK1, Parkin and TOM20. The mitophagy inhibitor 3-MA blocked the function of Res. At the same time, we found that Mfn2 siRNA could reverse the inhibition of mitophagy by Res through AMPK (adenosine 5'-monophosphate (AMP)-activated protein kinase) and prevent the effect of the mitochondrial permeability transition pore (mPTP) opening. Thus, our data suggest that Res can exert mitochondrial protection by inhibiting mitophagy and preventing mPTP opening through AMPK-Mfn2 axis in myocardial cells.
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.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.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.
With the development of economic level and growth in the living standard, how to enhance the accuracy and efficiency of remote sepsis diagnosis has become the key issue and hot topic in the current medical research. As the traditional BP neural network is poor in the generalization ability and need s a large number of samples, we proposed the study on remote sepsis diagnosis by using the FOA-optimized BP neural network. Besides, based on the wireless sensor network and ZigBee network, we constructed the mathematical model of FOA_BP remote sepsis diagnosis based on wireless sensor network. The sepsis data set of School of Medicine and Public Health, University of Wisconsin was used as the object of study. The FOA-BP algorithm is significantly superior to the BP neural network algorithm in the diagnosis accuracy, verifying the validity and reliability of making a sepsis diagnosis with FOA_BP. Therefore, the method can be popularized to other fields to solve other similar problems.
While the role of endoplasmic reticulum stress (ERS) in myocardial ischemia/reperfusion (I/R) injury has been extensively investigated, the precise mechanism by which inhibition of ERS induces cardioprotection remains unclear. We aimed to explore the mechanism of ERS inhibition-induced cardioprotection against I/R injury, focusing on the role of Zn 2+ and the mitochondrial permeability transition pore (mPTP). Exposure of H9c2 cells to 800 μM H 2 O 2 for 20 min increased GRP78 and GRP94 expressions (296.2 ± 41.0 and 150.6 ± 13.5 %, respectively), suggesting that H 2 O 2 can induce ERS. Cells treated with H 2 O 2 showed a significant decrease (40.6 ± 7.4 %) in TMRE fluorescence compared to the normal group (92.6 ± 0.1 %), indicating that H 2 O 2 can induce the mPTP opening. In contrast, ERS inhibitor TUDCA (30 μM) prevented the loss of TMRE fluorescence (77.8 ± 6.8 %), implying that inhibition of ERS can prevent the mPTP opening. This effect of TUDCA was blocked by zinc chelator TPEN (37.7 ± 13.0 %), indicating a role of Zn 2+ in the action of TUDCA on the mPTP opening. In support, TUDCA increased intracellular free zinc, as indicated by a marked increase in Newport Green DCF fluorescence intensity. In isolated rat hearts, GRP78 expression was not increased during ischemia but was increased upon reperfusion (1.3, 1.5, 1.9, and 1.6-fold increases at 10, 30, 60, and 120 min of reperfusion). Hearts treated with TUDCA showed a significant reduction of GRP78 expression 30 and 60 min after the onset of reperfusion, an effect that was reversed by TPEN. The immunofluorescence study also showed that the effect of TUDCA on GRP78 expression was reversed by TPEN. TUDCA reduced infarct size, and this was reversed by the mPTP opener atractyloside, indicating that ERS inhibition may protect the heart by modulating the mPTP opening. Experiments with transmission electron microscopy and hematoxylin-eosin staining revealed that TUDCA prevented endoplasmic reticulum and mitochondrial damages at reperfusion, which was blocked by TPEN. In conclusion, reperfusion but not ischemia initiates ERS and inhibition of ERS protects the heart from reperfusion injury through prevention of the mPTP opening. Increased intracellular free Zn 2+ accounts for the cardioprotective effect of ERS inhibition.
Abstract Resveratrol displays cardioprotective activity; however, its mechanism of action remains unclear. In the current study, resveratrol-induced myocardial protection from endoplasmic reticulum stress (ERS) was investigated, focusing on the roles of Zn2+ and the mitochondrial permeability transition pore (mPTP). We found, using the MTT/LDH kit, that 2-DG-induced ERS significantly decreased H9c2 cell viability. Resveratrol markedly inhibited the expression of endoplasmic reticulum chaperone GRP 78/94 and ERS-related apoptosis proteins CHOP, Caspase12, and JNK induced by 2-DG. The zinc ion chelator TPEN, and ERK/GSK-3β inhibitors PD98059 and SB216763 and their siRNAs blocked resveratrol function. The AKT inhibitor LY294002 and siRNA did not alter the action of resveratrol. In addition, resveratrol significantly increased the phosphorylation of ERK and GSK-3β. Resveratrol prevented 2-DG-induced mPTP opening and increased intracellular Zn2+ concentration indicated by TMRE and Newport Green DCF fluorescence intensity, which were further abrogated by ERK/GSK-3β inhibitors and siRNAs. Our data suggested that resveratrol protected cardiac cells from ERS by mobilizing intracellular Zn2+ and preventing mPTP opening through the ERK/GSK-3β but not PI3K/AKT signaling pathway.
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 3 β (GSK‐3 β ) and the mitochondrial permeability transition pore (mPTP) through inhibition of endoplasmic reticulum stress (ERS). Methods and Results . H9c2 cells were exposed to H 2 O 2 for 20 minutes. NECA significantly prevented H 2 O 2 ‐induced TMRE fluorescence reduction, indicating that NECA inhibited the mPTP opening. NECA blocked H 2 O 2 ‐induced GSK‐3 β phosphorylation and GRP94 expression. NECA increased GSK‐3 β 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‐3 β and cGMP/PKG via ERS. In isolated rat hearts, both NECA and the ERS inhibitor TUDCA decreased myocardial infarction, increased GSK‐3 β phosphorylation, and reversed GRP94 expression at reperfusion, suggesting that NECA protected the heart by inhibiting GSK‐3 β 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‐3 β via ERS inhibition. The cGMP/PKG signaling pathway is responsible for GSK‐3 β inactivation by NECA.
This study investigates whether inhibition of endoplasmic reticulum (ER) stress prevents opening of the mitochondrial permeability transition pore (mPTP) and evaluates the corresponding signaling pathways involved in this process. Exposure of cardiac H9c2 cells to 800 µM H2O2 for 20 min opened mPTP in response to oxidative stress, as demonstrated by quenching of tetramethylrhodamine ethyl ester (TMRE) fluorescence. Oxidative stress-induced mPTP opening was rescued by the ER stress inhibitor tauroursodeoxycholic acid (TUDCA) in a dose-dependent manner at low concentrations. The PI3K and PKG inhibitors LY294002 and KT5823 inhibited the effect of TUDCA on mPTP opening, suggesting the involvement of PI3K/Akt and PKG signaling pathways. TUDCA significantly increased glycogen synthase kinase 3 (GSK-3β) phosphorylation at Ser-9, with peak effect at 30 µM TUDCA. The level of GRP78 (ER chaperone) expression was significantly upregulated by 30 µM TUDCA. TUDCA-induced increases in Akt and GSK-3β phosphorylation were inhibited by LY294002, whereas KT5823 suppressed TUDCA-induced increases in VASP and GSK-3β phosphorylation. Oxidative stress severely affected cell morphology and ultrastructure. TUDCA prevented H2O2-induced ER swelling and mitochondrial damage. TUDCA boosted the viability of cells disrupted by ischemia/reperfusion (I/R), indicating that TUDCA eased reperfusion injury. However, TUDCA did not improve the viability of cells expressing the constitutively active GSK-3β mutant (GSK-3β-S9A-HA) that were subjected to I/R, suggesting an essential role of GSK-3β inactivation in TUDCA-mediated cardioprotection against reperfusion damage. These data indicate that ER stress inhibition prevents mPTP opening and attenuates reperfusion injury through GSK-3β inactivation. The PI3K/Akt and PKG pathways may mediate GSK-3β inactivation.
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