The Akt/GSK-3β pathway mediates flurbiprofen-induced neuroprotection against focal cerebral ischemia/reperfusion injury in rats

Apoptosis is one of the major mechanisms of cell death during cerebral ischemia and reperfusion injury. Flurbiprofen has been shown to reduce cerebral ischemia/reperfusion injury in both focal and global cerebral ischemia models, but the mechanism remains unclear. This study aimed to investigate the potential association between the neuroprotective effect of flurbiprofen and the apoptosis inhibiting signaling pathways, in particularly the Akt/GSK-3β pathway. A focal cerebral ischemia rat model was subjected to middle cerebral artery occlusion (MCAO) for 120 min and then treated with flurbiprofen at the onset of reperfusion. The infarct volume and the neurological deficit scores were evaluated at 24 h after reperfusion. Cell apoptosis, apoptosis-related proteins and the levels of p-Akt and p-GSK-3β in ischemic penumbra were measured using TUNEL and western blot. The results showed that administration of flurbiprofen at the doses of 5 and 10 mg/kg significantly attenuated brain ischemia/reperfusion injury, as shown by a reduction in the infarct volume, neurological deficit scores and cell apoptosis. Moreover, flurbiprofen not only inhibited the expression of Bax protein and p-GSK-3β, but also increased the expression of Bcl-2 protein, the ratio of Bcl-2/Bax as well as the P-Akt level. Taken together, these results suggest that flurbiprofen protects the brain from ischemia/reperfusion injury by reducing apoptosis and this neuroprotective effect may be partly due to the activation of Akt/GSK-3β signaling pathway.     

The Akt/GSK-3β pathway mediates flurbiprofen-induced neuroprotection against focal cerebral ischemia/reperfusion injury in rats

The advent of infectious molecular clones of Hepatitis C virus (HCV) has unlocked the understanding of HCV life cycle. However, packaging of the genomic RNA, which is crucial to generate infectious viral particles, remains poorly understood. Molecular interactions of the domain 1 (D1) of HCV Core protein and HCV RNA have been described in vitro. Since compaction of genetic information within HCV genome has hampered conventional mutational approach to study packaging in vivo, we developed a novel heterologous system to evaluate the interactions between HCV RNA and Core D1. For this, we took advantage of the recruitment of Vpr fusion-proteins into HIV-1 particles. By fusing HCV Core D1 to Vpr we were able to package and transfer a HCV subgenomic replicon into a HIV-1 based lentiviral vector. We next examined how deletion mutants of basic sub-domains of Core D1 influenced HCV RNA recruitment. The results emphasized the crucial role of the first and third basic regions of D1 in packaging. Interestingly, the system described here allowed us to mobilise full-length JFH1 genome in CD81 defective cells, which are normally refractory to HCV infection. This finding paves the way to an evaluation of the replication capability of HCV in various cell types.     

AKT/GSK3β Signaling in Glioblastoma

Glioblastoma (GBM) is the most aggressive of primary brain tumors. Despite the progress in understanding the biology of the pathogenesis of glioma made during the past decade, the clinical outcome of patients with GBM remains still poor. Deregulation of many signaling pathways involved in growth, survival, migration and resistance to treatment has been implicated in pathogenesis of GBM. One of these pathways is phosphatidylinositol-3 kinases (PI3K)/protein kinase B (AKT)/rapamycin-sensitive mTOR-complex (mTOR) pathway, intensively studied and widely described so far. Much less attention has been paid to the role of glycogen synthase kinase 3 β (GSK3β), a target of AKT. In this review we focus on the function of AKT/GSK3β signaling in GBM.     

Glycogen synthase kinase-3β controls autophagy during myocardial ischemia and reperfusion

Autophagy is a catabolic process that degrades long-lived proteins, pathogens and damaged organelles. Autophagy is active in the heart at baseline and is further stimulated by stresses, such as nutrient starvation, ischemia/reperfusion (I/R) and heart failure. Baseline autophagy plays an adaptive role in the heart, and contributes to the maintenance of cardiac structure and function and the inhibition of age-associated abnormalities, by achieving quality control of proteins and organelles. Activation of autophagy during ischemia is beneficial because it improves cell survival and cardiac function. However, excessive autophagy with robust upregulation of BECN1 during reperfusion appears to enhance cell death, which is detrimental to the heart. We have shown recently that autophagy during prolonged ischemia and I/R is critically regulated by glycogen synthase kinase-3β (GSK-3β), a ubiquitously expressed serine/threonine kinase, in a phase-dependent manner. Here we discuss the role of GSK-3β in mediating autophagy in the heart.     

Akt1 is involved in tubular apoptosis and inflammatory response during renal ischemia–reperfusion injury

Molecular Biology Reports - Renal ischemia–reperfusion injury (IRI) is one of the major causes of acute kidney injury (AKI). Although Akt is involved in renal IRI, it is unclear as to which...     

Preproendothelin-1 expression is negatively regulated by IFNγ during hepatic stellate cell activation

Endothelin-1 (ET-1), a powerful vasoconstrictor peptide, is produced by activated hepatic stellate cells (HSC) and promotes cell proliferation, fibrogenesis, and contraction, the latter of which has been thought to be mechanistically linked to portal hypertension in cirrhosis. Interferon-γ (IFNγ), a Th1 cytokine produced by T cells, inhibits stellate cell proliferation, fibrogenesis, and muscle-specific gene expression. Whether IFNγ-induced inhibitory effects are linked to regulation of ET-1 expression in activated stellate cells remains unknown. Here we examined IFNγ's effects on preproET-1 mRNA expression and the signaling pathways underlying this process. We demonstrated that preproET-1 mRNA expression in HSCs was prominently increased during cell culture-induced activation; IFNγ significantly inhibited both preproET-1 mRNA expression and ET-1 peptide production. Similar results were found in an in vivo model of liver injury and intraperitoneal administration of IFNγ. PreproET-1 promoter analysis revealed that IFNγ-induced inhibition of preproET-1 mRNA expression was closely linked to the AP-1 and Smad3 signaling pathways. Furthermore, IFNγ reduced JNK phosphorylation, which tightly was associated with decreased phosphorylation of downstream factors c-Jun and Smad3 and decreased binding activity of c-Jun and Smad3 in the preprpET-1 promoter. Importantly, IFNγ reduced both c-Jun mRNA and protein levels. Given the important role of ET-1 in wound healing, our results suggest a novel negative signaling network by which IFNγ inhibits preproET-1 expression, highlighting one potential molecular mechanism for IFNγ-induced host immunomodulation of liver fibrogenesis.     

Aesculin suppresses the NLRP3 inflammasome-mediated pyroptosis via the Akt/GSK3β/NF-κB pathway to mitigate myocardial ischemia/reperfusion injury

BackgroundAesculin (AES), an effective component of Cortex fraxini, is a hydroxycoumarin glucoside that has diverse biological properties. The nucleotide-binding domain leucine-rich repeat-containing receptor, pyrin domain-containing 3 (NLRP3) inflammasome has been heavily interwoven with the development of myocardial ischemia/reperfusion injury (MIRI). Nevertheless, it remains unclear whether AES makes a difference to the changes of the NLRP3 inflammasome in MIRI.PurposeWe used rats that were subjected to MIRI and neonatal rat cardiomyocytes (NRCMs) that underwent oxygen-glucose deprivation/restoration (OGD/R) process to investigate what impacts AES exerts on MIRI and the NLRP3 inflammasome activation.MethodsThe establishment of MIRI model in rats was conducted using the left anterior descending coronary artery ligation for 0.5 h ischemia and then untying the knot for 4 h of reperfusion. After reperfusion, AES were administered intraperitoneally using 10 and 30 mg/kg doses. We evaluated the development of reperfusion ventricular arrhythmias, hemodynamic changes, infarct size, and the biomarkers in myocardial injury. The inflammatory mediators and pyroptosis were also assessed. AES at the concentrations of 1, 3, and 10 μM were imposed on the NRCMs immediately before the restoration process. We also determined the cell viability and cell death in the NRCMs exposed to OGD/R insult. Furthermore, we also analyzed the levels of proteins that affect the NLRP3 inflammasome activation, pyroptosis, and the AKT serine/threonine kinase (Akt)/glycogen synthase kinase 3 beta (GSK3β)/nuclear factor-kappa B (NF-κB) signaling pathway via western blotting.ResultsWe found that AES notably attenuated reperfusion arrhythmias and myocardia damage, improved the hemodynamic function, and ameliorated the inflammatory response and pyroptosis of cardiomyocytes in rats and NRCMs. Additionally, AES reduced the NLRP3 inflammasome activation in rats and NRCMs. AES also enhanced the phosphorylation of Akt and GSK3β, while suppressing the phosphorylation of NF-κB. Moreover, the allosteric Akt inhibitor, MK-2206, abolished the AES-mediated cardioprotection and the NLRP3 inflammasome suppression.ConclusionsThese findings indicate that AES effectively protected cardiomyocytes against MIRI by suppressing the NLRP3 inflammasome-mediated pyroptosis, which may relate to the upregulated Akt activation and disruption of the GSK3β/NF-κB pathway.     

CCL27 expression is regulated by both p38 MAPK and IKKβ signalling pathways

The skin-specific chemokine CCL27 is believed to play a pivotal role in establishing the inflammatory infiltrate characteristic for common inflammatory skin diseases. Through binding to the chemokine receptor 10 (CCR10), CCL27 mediates inflammation by promoting lymphocyte migration into the skin. Little is known about the regulation of CCL27 gene expression. The purpose of our study was to investigate the regulation of the IL-1β-induced CCL27 gene expression in normal human keratinocytes (NHEK). Preincubation of NHEK with the inhibitory κB (IκB) kinase (IKK) inhibitor, SC-514, or the p38 mitogen-activated protein kinase (MAPK) inhibitor, SB202190, revealed a profound reduction in both CCL27 mRNA and CCL27 protein expression indicating the significance of these pathways in the regulation of CCL27 expression. Furthermore, the impact of inhibitors of mitogen- and stress-activated kinase 1 (MSK1) or the mitogen-activated protein kinase-interacting kinases (Mnk1+2), downstream kinases of p38 MAPK, on IL-1β-induced CCL27 expression in NHEK were investigated. We identified seven NF-κB binding elements upstream from the CCL27 gene start codon using electrophoretic mobility shift assay (EMSA). Supershift analyses demonstrated the involvement of the p50/p65 NF-κB heterodimer. We conclude that IL-1β-induced CCL27 gene expression in NHEK is regulated through the p38 MAPK/MSK1/Mnk1+2 as well as the IKKβ/NF-κB signalling pathways.     

USP4 is regulated by AKT phosphorylation and directly deubiquitylates TGF-β type I receptor

The stability and membrane localization of the transforming growth factor-β (TGF-β) type I receptor (TβRI) determines the levels of TGF-β signalling. TβRI is targeted for ubiquitylation-mediated degradation by the SMAD7-SMURF2 complex. Here we performed a genome-wide gain-of-function screen and identified ubiquitin-specific protease (USP) 4 as a strong inducer of TGF-β signalling. USP4 was found to directly interact with TβRI and act as a deubiquitylating enzyme, thereby controlling TβRI levels at the plasma membrane. Depletion of USP4 mitigates TGF-β-induced epithelial to mesenchymal transition and metastasis. Importantly, AKT (also known as protein kinase B), which has been associated with poor prognosis in breast cancer, directly associates with and phosphorylates USP4. AKT-mediated phosphorylation relocates nuclear USP4 to the cytoplasm and membrane and is required for maintaining its protein stability. Moreover, AKT-induced breast cancer cell migration was inhibited by USP4 depletion and TβRI kinase inhibition. Our results uncover USP4 as an important determinant for crosstalk between TGF-β and AKT signalling?pathways.     

Gastrodin ameliorates microvascular reperfusion injury–induced pyroptosis by regulating the NLRP3/caspase-1 pathway

Journal of Physiology and Biochemistry - Inflammation is a pivotal feature of myocardial reperfusion–induced microvascular injury and dysfunction. However, the molecular mechanisms by which...     

Reduction of apoptosis and preservation of mitochondrial integrity under ischemia/reperfusion injury is mediated by estrogen receptor β

Background

Estrogen improves cardiac recovery after ischemia/reperfusion (I/R) by yet incompletely understood mechanisms. Mitochondria play a crucial role in I/R injury through cytochrome c-dependent apoptosis activation. We tested the hypothesis that 17β-estradiol (E2) as well as a specific ERβ agonist improve cardiac recovery through estrogen receptor (ER)β-mediated mechanisms by reducing mitochondria-induced apoptosis and preserving mitochondrial integrity.

Methods

We randomized ovariectomized C57BL/6N mice 24h before I/R to pre-treatment with E2 or a specific ERβ agonist (ERβA). Isolated hearts were perfused for 20min prior to 30min global ischemia followed by 40min reperfusion.

Results

Compared with controls, ERβA and E2 treated groups showed a significant improvement in cardiac recovery, i.e. an increase in left ventricular developed pressure, dP/dtmax and dP/dtmin. ERβA and E2 pre-treatment led to a significant reduction in apoptosis with decreased cytochrome c release from the mitochondria and increased mitochondrial levels of anti-apoptotic Bcl2 and ACAA2. Protein levels of mitochondrial translocase inner membrane (TIM23) and mitochondrial complex I of respiratory chain were increased by ERβA and E2 pre-treatment. Furthermore, we found a significant increase of myosin light chain 2 (MLC2) phosphorylation together with ERK1/2 activation in E2, but not in ERβA treated groups.

Conclusions

Activation of ERβ is essential for the improvement of cardiac recovery after I/R through the inhibition of apoptosis and preservation of mitochondrial integrity and can be a achieved by a specific ERβ agonist. Furthermore, E2 modulates MLC2 activation after I/R independent of ERβ.

     

Activation of Akt/GSK-3β signaling pathway is involved in intermedin1-53 protection against myocardial apoptosis induced by ischemia/reperfusion

Intermedin (IMD) is a novel member of the calcitonin/calcitonin gene-related peptide family. We investigated the cardioprotective mechanism of IMD_1-53 in the in vivo rat model of myocardial ischemia/reperfusion (I/R) injury and in vitro primary neonatal cardiomyocyte model of hypoxia/reoxygenation (H/R). Myocardial infarct size was measured by 2,3,5-triphenyl tetrazolium chloride staining. Cardiomyocyte viability was determined by trypan blue staining, cell injury by lactate dehydrogenase (LDH) leakage, and cardiomyocyte apoptosis by terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labeling assay, Hoechst staining, gel electrophoresis and caspase 3 activity. The translocation of mitochondrial cytochrome c of myocardia and expression of apoptosis-related factors Bcl-2 and Bax, phosphorylated Akt and phosphorylated GSK-3β were determined by western blot analysis. IMD_1-53 (20 nmol/kg) limited the myocardial infarct size in rats with I/R; the infarct size was decreased by 54%, the apoptotic index by 30%, and caspase 3 activity by 32%; and the translocation of cytochrome c from mitochondria to cytosol was attenuated. IMD_1-53 increased the mRNA and protein expression of Bcl-2 and ratio of Bcl-2 to Bax by 81 and 261%, respectively. IMD_1-53 (1 × 10⁻⁷ mol/L) inhibited the H/R effect in cardiomyocytes by reducing cell death by 43% and LDH leakage by 16%; diminishing cellular apoptosis; decreasing caspase 3 activity by 50%; and increasing the phosphorylated Akt and GSK-3β by 41 and 90%, respectively. The cytoprotection of IMD_1-53 was abolished with LY294002, a PI3K inhibitor. In conclusion, IMD_1-53 exerts cardioprotective effect against myocardial I/R injury through the activation of the Akt/GSK-3β signaling pathway to inhibit mitochondria-mediated myocardial apoptosis. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.