GSK-3β Signaling Pathway Research Articles

Sappanone A alleviates hypoxia/reoxygenation-induced cardiomyocytes injury through inhibition of mitochondrial apoptosis and activation of PI3K-Akt-Gsk-3β pathway.

Myocardial ischemia reperfusion injury (MIRI) is a complex pathophysiological process involved with the activation of oxidative stress, inflammation and apoptosis. Sappanone A (SA), a homoisoflavanone isolated from the heartwood of Caesalpinia sappan L., could exhibit antioxidant, anti-inflammatory and anti-apoptotic activities. Therefore, we assumed that SA has a potential use for preventing against MIRI. The present study aimed to investigate the effect of SA treatment on MIRI and its mechanism. Cardiomyocytes (H9c2 cells) were treated with SA for 1 h, followed by 6 h of hypoxia/3 h of reoxygenation. Cell viability assay was detected by CCK-8 assay. Apoptosis was measured by flow cytometry and Hoechst staining. Mitochondrial permeability transition pore (mPTP) opening and mitochondrial transmembrane potential (ΔΨm) were measured by spectrophotometry and JC-1 staining. The changes of mitochondrial apoptosis-related proteins and PI3K–Akt–Gsk-3β signaling pathway were evaluated by Western blotting. The results showed that SA pretreatment enhanced the cell viability and decreased the activity of myocardial enzyme in a dose-dependent manner. Moreover, SA pretreatment significantly inhibited apoptosis, blocked mPTP opening, suppressed the release of ΔΨm, prevented the cytochrome c releasing from mitochondria into cytoplasm, and repressed the cleavage of caspase-9 and caspase-3. Furthermore, SA pretreatment increased the phosphorylation levels of Akt and Gsk-3β but not of Stat-3. Meanwhile, the protective effect of SA was abrogated by PI3K inhibitor (LY294002). In conclusion, our results demonstrate that SA could prevent hypoxia/reoxygenation-induced cardiomyocytes injury through inhibition of mitochondrial apoptosis and activation of PI3K–Akt–Gsk-3β pathway. Thus, SA may have a potential use for the prevention of MIRI.

Lappaconitine Sulfate Inhibits Proliferation and Induces Apoptosis in Human Hepatocellular Carcinoma HepG2 Cells through the Reactive Oxygen Species-Dependent Mitochondrial Pathway

Background: Hepatocellular carcinoma (HCC) is the third leading cause of tumor-related deaths in the word. Lappaconitine (LA), a diterpenoid alkaloid, exerts antitumor activities. However, the effects and mechanisms of LA sulfate (LS) on HCC remain unclear. This study evaluated the activities and explored the underlying mechanisms of LS in HCC cell line HepG2 cells. Materials and Methods: The cell viability and proliferation were evaluated using the Cell Counting Kit-8 (CCK-8) and 5-ethynyl-2′-deoxyuridine (EdU) assay, respectively. The cell cycle distribution was detected by propidium iodide (PI) staining assay. The apoptosis was detected by Annexin ­V-fluorescein isothiocyanate (FITC)/PI double staining assay. The cell cycle arrest and apoptosis-related proteins were estimated by western blot analysis. The mitochondrial membrane potential (MMP) was ­determined through the 5, 5′, 6, 6′-tetrachloro-1, 1′, 3, 3′-tetraethylbenzimi-dazolyl carbocyanine iodide (JC-1) staining assay. The reactive oxygen species (ROS) was monitored by 20–70-dichlorofluorescein diacetate (DCFH-DA) staining assay. In vivo antitumor activities were investigated by HepG2 xenograft model. Results: Our results showed that LS significantly ­inhibited the viability and proliferation of HepG2 cells. LS triggered G0/G1 cell cycle arrest, apoptosis and caspase activation. Furthermore, LS induced MMP loss and ROS accumulation. Additionally, LS suppressed the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT)/glycogen synthase kinase 3β (GSK3β) signaling pathway. An in vivo assay showed that LS exhibited a pronounced antitumor effect in nude mice bearing HepG2 xenografts. Conclusions: Our results demonstrated that LS is a promising therapeutic agent for HCC directed ­toward the proliferation inhibition and the induction of apoptosis.

Abstract WP320: Neuronal ROCK2 Plays Important Role in Ischemic Stroke: GSK-3beta and Tau Signaling Pathways

Introduction: The role of neuronal cell specific ROCK2 in ischemic stroke in vivo has been poorly investigated. We investigated the pharmacological effect of selective ROCK2 inhibition by selective ROCK2 inhibitor, KD025 and the genetic effect using neuronal specific ROCK2 knockout (ROCK2Eno2-/-) mice. Methods: Isoflurane-anesthetized littermate control (LC) and ROCK2Eno2-/- mice (20-22 g) underwent ischemia (2h middle cerebral artery occlusion (MCAO)/reperfusion (22h after 2h MCAO) injury model. An 8.0 nylon monofilament coated with a silicone was introduced into the left internal carotid artery up to the anterior cerebral artery. To assess the mechanism of ROCK2 deficiency in neuronal cells, we seeded hippocampal neuronal (HT22) cells in 6-well plates and transfected ROCK2 siRNA. Phosphorylated glycogen synthase kinase 3 beta (pGSK-3β) and Tau proteins which were involved in neuroprotection were measured by western blotting from brain lysates and neuronal cells. Infarction volume was measured by TTC staining and evaluation of behavioral was assessed by neurological deficit score (NDS) at 22h after MCAO. Results: In WT mice, ROCK2 inhibition by KD025 (100 mg/kg, p.o.) decreased infarct volumes and improved the NDS in ischemic stroke. ROCK activity (p-MYPT1 to MYPT1 ratio) of the brain lysates by KD025 was decreased dose-dependently in ischemic mice brain. In genetic modified mice, infarction volume (64.6 ± 10.4 vs. LC (102.3 ± 12.4), n=5) of ROCK2Eno2-/- mice was significantly decreased by 37% and the NDSs (1.8 ± 0.4 vs. LC (2.8 ± 0.5), n=5) were improved by 36% compared to the LC groups. Both pGSK-3β and Tau in HT22 cells with ROCK2 siRNA transfection and ROCK2Eno2-/- mice brain was significantly decreased compared to each respective controls. Conclusion: These findings indicate that neuronal ROCK2 plays important role in ischemic stroke via GSK-3β and Tau signaling pathways and inhibition of neuronal specific ROCK2 may have therapeutic benefits in ischemic stroke.

Tolerance Induced by (S)-3,5-Dihydroxyphenylglycine Postconditioning is Mediated by the PI3K/Akt/GSK3β Signalling Pathway in an In Vitro Model of Cerebral Ischemia

Ischemic postconditioning (PostC) is an endogenous neuroprotective strategy for cerebral ischemia induced by low activation of glutamate receptors. We have previously shown that the application of the mGluR1/5 agonist (S)-3,5-dihydroxyphenylglycine (DHPG) 5 min after 30 min of oxygen and glucose deprivation (OGD) reduces CA1 damage in organotypic hippocampal slices by activating the PI3K–Akt signalling pathway. In order to extend these data, we analysed the production of reactive oxygen species (ROS) and the glycogen synthase kinase 3β (GSK3β) signalling pathway. Our results show that DHPG PostC was associated with a reduction in the formation of ROS that is massively increased 24 h after OGD exposure. This reduction was prevented by the PI3K inhibitor LY294002, indicating that there is a link between the PI3K/Akt pathway and the formation of ROS in the protective mechanisms of PostC. DHPG PostC also induces a transient increased in GSK3β phosphorylation and inactivation that is followed by nuclear accumulation of β-catenin, that probably lead to the up-regulation of neuroprotective genes. Our results propose GSK3β as new target for neuroprotection, therefore, we verified that the two GSK3β inhibitors N-(3-Chloro-4-methylphenyl)-5-(4-nitrophenyl)-1,3,4-oxadiazol-2-amine (TC-G 24) and LiCl are neuroprotective agents in OGD and also can be used as PostC agents.

Effects of mild intrauterine hypoperfusion in the second trimester on memory and learning function in rat offspring.

Mild intrauterine hypoperfusion (MIUH) is a serious pathological event that affects the growth and development of fetuses and offspring. MIUH can lead to growth restriction, low birth weight, neurodevelopmental disorders, and other adverse clinical outcomes. To study the effects of MIUH on learning and memory function in offspring, a model of MIUH was established by placing a coil (length 2.5 mm, diameter 0.24 mm) on the uterine artery and ovarian uterine artery of Sprague-Dawley rats in the second trimester of pregnancy (day 17). Next, 120 mg/kg lithium chloride (the MIUH + Li group) or normal saline (the MIUH group) was injected intraperitoneally into these rats. In addition, 120 mg/kg lithium chloride (the Li group) or normal saline (the SHAM group) was injected intraperitoneally into pregnant rats without coil placement. The Morris water maze was used to detect changes in learning and memory ability in the offspring at 4 weeks after birth. In the MIUH group, the escape latency and journey length before reaching the platform were both increased, and the number of times that the platform was crossed and the activity time in the target quadrant within 90 seconds were both decreased compared with the SHAM group. Immunofluorescence double staining and western blot assays demonstrated that hippocampal nestin and Ki67 (both cell-proliferation-related proteins) expression was significantly downregulated in the MIUH group compared with the SHAM group. Furthermore, western blot assays were conducted to investigate changes in related signaling pathway proteins in the brains of offspring rats, and revealed that glycogen synthase kinase 3β (GSK3β) expression was upregulated and β-catenin expression was downregulated in the MIUH group compared with the SHAM group. In addition, compared with the MIUH group, the expression levels of p-GSK3β and β-catenin were upregulated in the MIUH + Li group. These results suggest that MIUH may affect learning and memory function in rat offspring by regulating the GSK3β signaling pathway. The experimental procedures were approved by Animal Ethics Committee of Shengjing Hospital of China Medical University (approval No. 2018PS07K) in June 2018.

Glycyrrhizic acid promotes neural repair by directly driving functional remyelination.

Natural compounds are a rich source of effective candidate drugs for the treatment of neurological disorders. Glycyrrhizic acid (GA), the major water-soluble ingredient isolated from Glycyrrhiza glabra, is reported to show anti-inflammatory and immunomodulatory activities. However, its effect on CNS demyelinating disease is unclear. In this study, we showed that GA ameliorated the clinical disease severity of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS), especially at the chronic stage of clinical EAE. Histological evaluation demonstrated that, in the prophylactic treatment regimen, GA significantly inhibited inflammatory demyelination in the CNS. During the chronic stage when myelin and axon damage has already occurred, GA induced oligodendrocyte progenitor cell (OPC) differentiation into mature oligodendrocytes, thus effectively accelerating remyelination. Evidence from the cuprizone-induced mouse model of de- and remyelination, ex vivo organotypic slice cultures, and in vitro OPC maturation experiments indicated that the observed efficacy of this compound resulted directly from enhanced remyelination rather than immune suppression. Furthermore, we found that GA promoted oligodendrocyte maturation through modulating GSK-3β signaling pathways. Our data led to the conclusion that GA could be used as a potential therapeutic candidate for the treatment of demyelinating diseases such as MS, which remains refractory to available treatments.

Sonchus oleraceus Linn extract enhanced glucose homeostasis through the AMPK/Akt/ GSK-3β signaling pathway in diabetic liver and HepG2 cell culture

The extracts of S. oleraceus Linn (SOL) and its main phenolic compounds have shown anti-diabetic effects, but their underlying mechanisms for glucose homeostasis remain unclear. The aim of this study is to evaluate the anti-diabetic mechanism of SOL by using the streptozocin (STZ) induced diabetic rat model. When diabetic rats were fed with SOL at a dose of 400 mg/kg/day for 6 weeks, the concentrations of triglycerides (TG), total cholesterol (TC), and low-density lipoprotein cholesterol (LDL-C) were reduced by 43%, 22%, and 16%, respectively. Meanwhile, it was also found that daily feeding of SOL to diabetic rats led to a decrease in plasma glucose level by approximately 23%. Positive effects were observed on glucose homeostasis due to the down-regulation of AMPK/Akt/GSK-3β pathway, as indicated by the suppressions of adenosine 5‘-monophosphate (AMP)-activated protein kinase (AMPK), protein kinase (Akt) phosphorylation, glycogen synthase kinase 3 beta (GSK-3β), and the hepatic insulin resistance. In HepG2 cells, AMPK, Akt and GSK-3β showed a consistent transcript regulation. SOL at dose of 400 mg/kg/day feeding for 6 weeks showed a positive effect comparable to metformin.

Glycogen Synthase Kinase-3\u03b2 Inhibition with 9-ING-41 Attenuates the Progression of Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) is a progressive interstitial lung disease with a median survival of 3 years after diagnosis. Although the etiology of IPF is unknown, it is characterized by extensive alveolar epithelial cell apoptosis and proliferation of myofibroblasts in the lungs. While the origins of these myofibroblast appear to be diverse, fibroblast differentiation contributes to expansion of myofibroblasts and to disease progression. We found that agents that contribute to neomatrix formation and remodeling in pulmonary fibrosis (PF); TGF-β, Factor Xa, thrombin, plasmin and uPA all induced fibroblast/myofibroblast differentiation. These same mediators enhanced GSK-3β activation via phosphorylation of tyrosine-216 (p-Y216). Inhibition of GSK-3β signaling with the novel inhibitor 9-ING-41 blocked the induction of myofibroblast markers; α-SMA and Col-1 and reduced morphological changes of myofibroblast differentiation. In in vivo studies, the progression of TGF-β and bleomycin mediated PF was significantly attenuated by 9-ING-41 administered at 7 and 14 days respectively after the establishment of injury. Specifically, 9-ING-41 treatment significantly improved lung function (compliance and lung volumes; p < 0.05) of TGF-β adenovirus treated mice compared to controls. Similar results were found in mice with bleomycin-induced PF. These studies clearly show that activation of the GSK-3β signaling pathway is critical for the induction of myofibroblast differentiation in lung fibroblasts ex vivo and pulmonary fibrosis in vivo. The results offer a strong premise supporting the continued investigation of the GSK-3β signaling pathway in the control of fibroblast-myofibroblast differentiation and fibrosing lung injury. These data provide a strong rationale for extension of clinical trials of 9-ING-41 to patients with IPF.

Sepsis impairs aggregation of nicotinic acetylcholine receptors on murine skeletal muscle cell membranes by inhibiting AKT/GSK3β phosphorylation

To investigate the role of the protein-serine-threonine kinase (AKT)/glycogen synthase kinase 3β (GSK3β) signaling pathway in nicotinic acetylcholine receptors (nAChRs) aggregation disorder on skeletal muscle cell membranes induced by sepsis. Mouse C2C12 myoblasts were differentiated into myotubes by horse serum, and then C2C12 myotubes were randomly divided into four groups: the Sham group treated with serum from sham-operated mice, the Sepsis group treated with serum from septic mice, the Sepsis+D group treated with serum from septic mice and dimethyl sulfoxide (DMSO), the Sepsis+SB group treated with serum from septic mice and GSK3β inhibitor SB216763. Agrin was added into the cell culture to induce nAChRs aggregation before the treatment. After serum treatment for 5.5 h, the myotubes were examined for nAChRs clusters using Alexa Fluor 594-conjugated α-bungarotoxin (α- BTX). The expression levels of AKT, GSK3β and CLIP- associated protein 2 (CLASP2) and the phosphorylation of AKT, GSK3β were examined with Western blotting. The phosphorylation of CLASP2 and the interaction between CLASP2 and α-tubulin were detected with co-immunoprecipitation (Co-IP) assay. Compared with the serum from sham-operated mice, the serum from septic mice caused significant reduction in the area and density of nAChRs clusters on C2C12 myotubes, lowered the levels of phosphorylated AKT (p-AKT) and phosphorylated GSK3β (p-GSK3β), increased the expression of phosphorylated CLASP2 (p-CLASP2), and obviously reduced the binding between CLASP2 and α-tubulin. Compared with DMSO, SB216763 significantly increased the area and density of nAChRs clusters on C2C12 myotubes treated with serum from septic mice, decreased the expression of p-CLASP2, and enhanced the interaction between CLASP2 and α-tubulin. Septic mouse serum impairs nAChRs aggregation on C2C12 myotubes possibly by suppressing AKT/GSK3β phosphorylation to cause reduced interaction between CLASP2 and α-tubulin.

Metformin Ameliorates Testicular Damage in Male Mice with Streptozotocin-Induced Type 1 Diabetes through the PK2/PKR Pathway.

Approximately 90% of male diabetes mellitus patients have varying degrees of testicular dysfunction. The molecular mechanism underlying diabetes-induced testicular damage has not been thoroughly elucidated. In this research, we sought to determine the influence of metformin (Met) on diabetes-induced testicular injury and the mechanism involved with a focus on testicular dysfunction, apoptosis, autophagy, and prokineticin 2 (PK2) signalling. In our study, C57BL/6J mice were randomly divided into the normal control group, the diabetes group, and the Met-treated group. Streptozotocin (50 mg·kg−1·d−1) was injected intraperitoneally into the mice for 5 days in a row to induce type 1 diabetes, which was diagnosed by a blood glucose level ≥ 16.7 mmol/L after 7 days. The experimental animals were orally administered Met (250 mg·kg−1·d−1) for 16 weeks. Properties of testicular function, including sperm motility and the total concentration of epididymal sperm, were assessed. Changes in testicular structure, such as the blood-testis barrier, histological pathology, and organelles, were observed. The levels of apoptosis and expression of related proteins, such as Bax and Bcl-2, were measured. Moreover, autophagy-related proteins, including Beclin-1, p62, and LC3B, as well as the PK2/PKR pathway, which consists of PK2, PKR1, PKR2, AKT, and GSK3β, were analysed. Upon the induction of diabetes, reproductive capacity was significantly impaired and a disordered arrangement of testicular seminiferous tubules and destroyed organelles in spermatogenic cells was observed. Met administration preserved testicular function and structure. In addition, in mice with diabetes, the levels of PK2, PKR2, p-Akt, and p-GSK3β were significantly decreased at different times, while that of PKR1 was markedly increased, and these changes were normalized by Met. Furthermore, diabetic mice showed increased apoptosis and decreased autophagy in the testes, the effects of which were nullified by Met. These results suggest that Met rescues diabetes-induced testicular damage by attenuating apoptosis and inducing autophagy. This effect is likely mediated by the PK2/PKR/AKT/GSK3β signalling pathway.

Apigenin exhibits anti-inflammatory effects in LPS-stimulated BV2 microglia through activating GSK3β/Nrf2 signaling pathway

Objective: Apigenin is a natural flavonoid compound extracted from Matricaria chamomilla. We evaluated the anti-inflammatory effects of apigenin in this study using the Lipopolysaccharide (LPS)-stimulated BV2 microglia.Methods: BV2 cells were treated with apigenin for 1 h and then treated with LPS. The inflammatory cytokine productions were tested by qRT-PCR and ELISA. The expression of GSK3β, Nrf2, and NF-κB signaling pathways were measured by western blot analysis.Results: Apigenin significantly attenuated LPS-induced TNF-α, IL-1β, and IL-6 production. Apigenin suppressed LPS-induced NF-κB activation. Furthermore, GSK3β, Nrf2, and HO-1 were concentration-dependently increased by apigenin. The suppression of apigenin on LPS-induced inflammatory response and NF-κB activation were prevented when Nrf2 was knocked out or by GSK3β inhibitor.Conclusions: Collectively, apigenin suppressed LPS-induced microglia activation via activating GSK3β/Nrf2 signaling pathway.

A botanical drug composed of three herbal materials attenuates the sensorimotor gating deficit and cognitive impairment induced by MK-801 in mice.

A botanical drug derived from the ethanolic extract composed of Clematis chinensis Osbeck (Ranunculaceae), Trichosanthes kirilowii Maximowicz (Cucurbitaceae) and Prunella vulgaris Linné (Lamiaceae) has been used to ameliorate rheumatoid arthritis as an ethical drug in Korea. In our study, we investigated the effect of this herbal complex extract (HCE) on schizophrenia-like behaviours induced by MK-801. HCE (30, 100 or 300mg/kg, p.o) was orally administered to male ICR mice to a schizophrenia-like animal model induced by MK-801. We conducted an acoustic startle response task, an open-field task, a novel object recognition task and a social novelty preference task. We found that a single administration of HCE (100 or 300mg/kg) ameliorated MK-801-induced abnormal behaviours including sensorimotor gating deficits and social or object recognition memory deficits. In addition, MK-801-induced increases in phosphorylated Akt and GSK-3β expression levels in the prefrontal cortex were reversed by HCE (30, 100 or 300mg/kg). These results imply that HCE ameliorates MK-801-induced dysfunctions in prepulse inhibition, social interactions and cognitive function, partly by regulating the Akt and GSK-3β signalling pathways.

Inhibition of Histone Deacetylase 6 Protects Hippocampal Cells Against Mitochondria-mediated Apoptosis in a Model of Severe Oxygen-glucose Deprivation.

Histone deacetylase (HDAC) 6 inhibitors have demonstrated significant protective effects in traumatic injuries. However, their roles in neuroprotection and underlying mechanisms are poorly understood. This study sought to investigate the neuroprotective effects of Tubastatin A (Tub-A), an HDAC6 inhibitor, during oxygenglucose deprivation (OGD) in HT22 hippocampal cells. HT22 hippocampal cells were exposed to OGD. Cell viability and cytotoxicity were assessed by cell counting kit-8 (CCK-8) and lactate dehydrogenase (LDH) release assay. Cellular apoptosis was assessed by Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Mitochondria membrane potential was detected using JC-1 dye. Expressions of acetylated α-tubulin, α-tubulin, cytochrome c, VDAC, Bax, Bcl- 2, cleaved caspase 3, phosphorylated Akt, Akt, phosphorylated GSK3β and GSK3β were analyzed by Western blot analysis. Tub-A induced acetylation of α-tubulin, demonstrating appropriate efficacy. Tub-A significantly increased cell viability and attenuated LDH release after exposure to OGD. Furthermore, Tub-A treatment blunted the increase in TUNEL-positive cells following OGD and preserved the mitochondrial membrane potential. Tub-A also attenuated the release of cytochrome c from the mitochondria into the cytoplasm and suppressed the ratio of Bax/Bcl-2 and cleaved caspase 3. This was mediated, in part, by the increased phosphorylation of Akt and GSK3β signaling pathways. HDAC 6 inhibition, using Tub-A, protects against OGD-induced injury in HT22 cells by modulating Akt/GSK3β signaling and inhibiting mitochondria-mediated apoptosis.

Caspase-2 promotes AMPA receptor internalization and cognitive flexibility via mTORC2-AKT-GSK3\u03b2 signaling

Caspase-2 is the most evolutionarily conserved member in the caspase family of proteases and is constitutively expressed in most cell types including neurons; however, its physiological function remains largely unknown. Here we report that caspase-2 plays a critical role in synaptic plasticity and cognitive flexibility. We found that caspase-2 deficiency led to deficits in dendritic spine pruning, internalization of AMPA receptors and long-term depression. Our results indicate that caspase-2 degrades Rictor, a key mTOR complex 2 (mTORC2) component, to inhibit Akt activation, which leads to enhancement of the GSK3β activity and thereby long-term depression. Furthermore, we found that mice lacking caspase-2 displayed elevated levels of anxiety, impairment in reversal water maze learning, and little memory loss over time. These results not only uncover a caspase-2–mTORC2–Akt–GSK3β signaling pathway, but also suggest that caspase-2 is important for memory erasing and normal behaviors by regulating synaptic number and transmission.

Inhibition of PDE5 attenuates streptozotocin-induced neuroinflammation and tau hyperphosphorylation in a streptozotocin-treated rat model

Intracerebroventricular (icv) streptozotocin (STZ) injection decreases cerebral insulin signal pathway function and produces multiple effects that resemble the molecular, pathological, and behavioural features of Sporadic Alzheimer’s disease (SAD). We previously reported that yonkenafil (yonk), the analogue of sildenafil and a novel PDE5 inhibitor exerts an anti-amyloidogenesis effect by regulating the Aβ level and inhibiting the expression of β-amyloid precursor protein in the APP/PS1 transgenic mice model. In this study, the effects of yonk on cognitive behaviors as well as the pathological features in streptozotocin-induced SAD rat model were investigated. The results demonstrated that administration of yonk at doses of 3 and 10 mg/kg for three weeks significantly improved cognitive deficits, attenuated STZ-induced neuronal death, inhibited the over-activation of microglia and astrocytes and the levels of pro-inflammatory markers, as well as decreased PDE5 protein expression in the hippocampus. Furthermore, yonk (3 mg/kg) notably prevented changes in tau hyperphosphorylation, decreased IRS-1and JNK phosphorylation and increased the GSK3β (ser9) phosphorylation induced by STZ. In summary, these data suggested that yonk significantly reversed STZ-induced memory deficits by inhibiting the over-activation of microglia and astrocytes, as well as ameliorated the levels of pro-inflammatory makers and tau hyperphosphorylation through regulating GSK3β signalling pathway.

GSK3β and MCL-1 mediate cardiomyocyte apoptosis in response to high glucose.

Gestational diabetes mellitus is a risk factor for congenital heart defects. Our previous results indicated that a decrease in myocardial cells and an increase in apoptotic cells leads to heart defects under hyperglycemia, but much work remains to elucidate this important mechanism of myocardial cell apoptosis induced by high glucose (HG). In this study, we found that a decrease in GSK3β phosphorylation on Ser9 occurred concomitantly with HG-induced cardiomyocyte apoptosis and in the heart tissues of the offspring of diabetic rats in vitro and in vivo. Decreases in GSK3β (Ser9) phosphorylation in response to HG were remarkably restored after treatment with SC79, an activator of the Akt signaling pathway. SB216763, an effective inhibitor of the GSK3β signaling pathway, suppressed HG-induced apoptosis in cardiomyocytes. Further studies showed a decrease in the expression of the anti-apoptotic protein MCL-1 was associated with GSK3β-mediated apoptosis. MCL-1 overexpression partly inhibits HG-induced apoptosis in cardiomyocytes. Herein, this study revealed the roles of GSK3β and MCL-1 in modulating HG-induced cardiomyocyte apoptosis and maternal diabetes-induced abnormalities.

NGL-3 in the regulation of brain development, Akt/GSK3b signaling, long-term depression, and locomotive and cognitive behaviors.

Netrin-G ligand-3 (NGL-3) is a postsynaptic adhesion molecule known to directly interact with the excitatory postsynaptic scaffolding protein postsynaptic density-95 (PSD-95) and trans-synaptically with leukocyte common antigen-related (LAR) family receptor tyrosine phosphatases to regulate presynaptic differentiation. Although NGL-3 has been implicated in the regulation of excitatory synapse development by in vitro studies, whether it regulates synapse development or function, or any other features of brain development and function, is not known. Here, we report that mice lacking NGL-3 (Ngl3−/− mice) show markedly suppressed normal brain development and postnatal survival and growth. A change of the genetic background of mice from pure to hybrid minimized these developmental effects but modestly suppressed N-methyl-D-aspartate (NMDA) receptor (NMDAR)-mediated synaptic transmission in the hippocampus without affecting synapse development, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor (AMPAR)-mediated basal transmission, and presynaptic release. Intriguingly, long-term depression (LTD) was near-completely abolished in Ngl3−/− mice, and the Akt/glycogen synthase kinase 3β (GSK3β) signaling pathway, known to suppress LTD, was abnormally enhanced. In addition, pharmacological inhibition of Akt, but not activation of NMDARs, normalized the suppressed LTD in Ngl3−/− mice, suggesting that Akt hyperactivity suppresses LTD. Ngl3−/− mice displayed several behavioral abnormalities, including hyperactivity, anxiolytic-like behavior, impaired spatial memory, and enhanced seizure susceptibility. Among them, the hyperactivity was rapidly improved by pharmacological NMDAR activation. These results suggest that NGL-3 regulates brain development, Akt/GSK3β signaling, LTD, and locomotive and cognitive behaviors.

Plantamajoside attenuates isoproterenol-induced cardiac hypertrophy associated with the HDAC2 and AKT/ GSK-3β signaling pathway

As a compensatory response to cardiac overload, cardiac hypertrophy is closely associated with the occurrence and development of a variety of cardiovascular diseases, in which histone deacetylase 2 (HDAC2) has been reported to play an important role. Plantamajoside (PMS) is an active component extracted from Herba Plantaginis, which is a traditional Chinese medicine, and many biological activities of PMS have been reported. Here, we investigated the effects and mechanism of PMS on isoproterenol (ISO)-induced cardiac hypertrophy. ISO at 10 μmol/L was used in vitro to induce H9c2 cardiomyocyte hypertrophy. Cell viability and cell surface area were determined by MTT assay and immunocytochemistry, respectively. Furthermore, an in vivo, cardiac hypertrophy model was established by subcutaneous injection of ISO. Pathological alterations and fibrosis in the myocardium were studied by H&E and Masson's trichrome staining, respectively. Myocardial injury-related genes and proteins were detected by real-time PCR and western blotting. HDAC2 and its downstream proteins, AKT and GSK3β, were analyzed by western blotting. Our results showed that, in vitro, PMS inhibited the ISO-induced increase in H9c2 cell surface area and the mRNA expression of ANP, BNP and Myh7. In vivo, PMS improved the ISO-induced decrease in cardiac function, inhibited the increase in cardiac anatomical parameters and alleviated the histopathological changes in cardiac tissues. Moreover, PMS inhibited the mRNA and protein expression of ANP, BNP, Myh7, COL1 and COL3. Furthermore, PMS suppressed the activity of HDAC2 and down-regulated the expression of the downstream proteins p-AKT and p-GSK3β both in vitro and in vivo. Overall, our results indicated that PMS exerts significant cardioprotective effects against ISO-induced cardiac hypertrophy, and this protective effect may be mediated by inhibition of the HDAC2 and AKT/GSK-3β signaling pathway.

Obacunone attenuates high glucose-induced oxidative damage in NRK-52E cells by inhibiting the activity of GSK-3β

Hyperglycemia-induced proximal tubule injury plays a critical role in the pathogenesis of diabetic nephropathy (DN). Attenuating high glucose (HG)-induced oxidative damage in renal tubular epithelial cells has been documented to ameliorate DN. Obacunone (OB), a natural bioactive compound isolated from the Rutaceae family, has been demonstrated to possess various pharmacological effects with low toxicity. However, the role of OB in DN has not yet been investigated. To explore the influence of OB on oxidative damage that is induced by HG and its potential mechanisms of action, we set up a high glucose model and induced oxidative damage in NRK-52E cells. OB could protect the NRK-52E cells from the HG-induced decrease of cell viability and the accumulation of ROS. The protective effects of OB were associated with its ability to increase the levels of antioxidants (SOD, GSH and CAT), inhibit the production of ROS, and stabilize the mitochondrial membrane potential. In addition, OB significantly downregulated the activity of GSK-3β, enhanced the nuclear translocation of Nrf2 and increased the mRNA expression of the Nrf2-driven genes NQO-1 and HO-1 in HG-treated cells. OB also decreased the release of cytochrome c from the mitochondria to the cytosol and inhibited the activation of caspase-3 in HG-treated cells. Pretreatment with a GSK-3β activator blocked the protective effects of OB, while pretreatment with a GSK-3β inhibitor yielded opposite results. These findings indicate that the renoprotective effects of OB against HG-induced oxidative damage in NRK-52E cells may be mediated by its ability to inhibit oxidative stress and mitochondrial dysfunction through the GSK-3β signaling pathway.

5′-Iodotubercidin represses insulinoma-associated-1 expression, decreases cAMP levels, and suppresses human neuroblastoma cell growth

Insulinoma-associated-1 (INSM1) is a key protein functioning as a transcriptional repressor in neuroendocrine differentiation and is activated by N-Myc in human neuroblastoma (NB). INSM1 modulates the phosphoinositide 3-kinase (PI3K)-AKT Ser/Thr kinase (AKT)-glycogen synthase kinase 3β (GSK3β) signaling pathway through a positive-feedback loop, resulting in N-Myc stabilization. Accordingly, INSM1 has emerged as a critical player closely associated with N-Myc in facilitating NB cell growth. Here, an INSM1 promoter-driven luciferase-based screen revealed that the compound 5'-iodotubercidin suppresses adenosine kinase (ADK), an energy pathway enzyme, and also INSM1 expression and NB tumor growth. Next, we sought to dissect how the ADK pathway contributes to NB tumor cell growth in the context of INSM1 expression. We also found that 5'-iodotubercidin inhibits INSM1 expression and induces an intra- and extracellular adenosine imbalance. The adenosine imbalance, which triggers adenosine receptor-3 signaling that decreases cAMP levels and AKT phosphorylation and enhances GSK3β activity. We further observed that GSK3β then phosphorylates β-catenin and promotes the cytoplasmic proteasomal degradation pathway. 5'-Iodotubercidin treatment and INSM1 inhibition suppressed extracellular signal-regulated kinase 1/2 (ERK1/2) activity and the AKT signaling pathways required for NB cell proliferation. The 5'-iodotubercidin treatment also suppressed β-catenin, lymphoid enhancer-binding factor 1 (LEF-1), cyclin D1, N-Myc, and INSM1 levels, ultimately leading to apoptosis via caspase-3 and p53 activation. The identification of the signaling pathways that control the proliferation of aggressive NB reported here suggests new options for combination treatments of NB patients.