Activated PKB/GSK-3β synergizes with PKC-δ signaling in attenuating myocardial ischemia/reperfusion injury via potentiation of NRF2 activity: Therapeutic efficacy of dihydrotanshinone-I

@#Disrupted redox status primarily contributes to myocardial ischemia/reperfusion injury (MIRI). NRF2, the endogenous antioxidant regulator, might provide therapeutic benefits. Dihydrotanshinone-I (DT) is an active component in Salvia miltiorrhiza with NRF2 induction potency. This study seeks to validate functional links between NRF2 and cardioprotection of DT and to investigate the molecular mechanism particularly emphasizing on NRF2 cytoplasmic/nuclear translocation. DT potently induced NRF2 nuclear accumulation, ameliorating post-reperfusion injuries via redox alterations. Abrogated cardioprotection in NRF2-deficient mice and cardiomyocytes strongly supports NRF2-dependent cardioprotection of DT. Mechanistically, DT phosphorylated NRF2 at Ser40, rendering its nuclear-import by dissociating from KEAP1 and inhibiting degradation. Importantly, we identified PKC-δ-(Thr505) phosphorylation as primary upstream event triggering NRF2-(Ser40) phosphorylation. Knockdown of PKC-δ dramatically retained NRF2 in cytoplasm, convincing its pivotal role in mediating NRF2 nuclear-import. NRF2 activity was further enhanced by activated PKB/GSK-3β signaling via nuclear-export signal blockage independent of PKC-δ activation. By demonstrating independent modulation of PKC-δ and PKB/GSK-3β/Fyn signaling, we highlight the ability of DT to exploit both nuclear import and export regulation of NRF2 in treating reperfusion injury harboring redox homeostasis alterations. Coactivation of PKC and PKB phenocopied cardioprotection of DT in vitro and in vivo, further supporting the potential applicability of this rationale. Graphical abstract

Anti-hepatocellular carcinoma effects of Grifola frondosa extract and its underlying mechanisms / 中国药学杂志

OBJECTIVE: To investigate the anti-hepatocellular carcinoma effect and underlying mechanisms. METHODS: In PLC/PRF/5 and HepG2, after treatment with Grifola frondosa extract, MTT method, chemical method, JC-1 staining and Western Blot were applied to determine cell viability, caspase 3 activity, mitochondrial membrane potential, the expression of Bcl-2 and Bax, and the phosphorylation of Akt/GSK3β. The anti-tumor activity of Grifola frondosa extract was further confirmed in PLC/PRL/5-xengrafted mice model. RESULTS: Grifola frondosa extract significantly reduced cell viability, mitochondrial membrane potential, the expression of Bcl-2 and the phosphorylation of Akt/GSK3β, and enhanced LDH release, caspase 3 activity and the expression of Bax in both PLC/PRF/5 and HepG2 cells. 12-day Grifola frondosa extract treatment significantly inhibited the PLC/PRF/5-xenografted tumor growth without influence the body weight of mouse. CONCLUSION: All these data indicate that Grifola frondosa extract-mediated anti-hepatocellular carcinoma effects are related to its modulation of the activations of Akt/GSK3β and mitochondrial pathway.

Advances of anti-diabetic drugs based on new targets / 中国药科大学学报

@#With the deepening of research, different new anti-diabetic drug targets have been discovered and reported, several categories of anti-diabetic drugs(linagliptin, exenatide, dapagliflozin, etc. )have been brought to the market and some new drugs acting on different targets are in late-stage clinical trials. All these progresses provide new means for overcoming diabetes. GPR119, GPR120, GPR40, AMPK, apelin receptor and GSK3β are anti-diabetic targets with great research values in current days and the future. This article reviews the mechanisms, drugs and research advances with respect to the above-mentioned targets.

Ferulic acid regulates the AKT/GSK-3beta/CRMP-2 signaling pathway in a middle cerebral artery occlusion animal model / 한국실험동물학회지

Ferulic acid, a component of the plants Angelica sinensis (Oliv.) Diels and Ligusticum chuanxiong Hort, exerts a neuroprotective effect by regulating various signaling pathways. This study showed that ferulic acid treatment prevents the injury-induced increase of collapsin response mediator protein 2 (CRMP-2) in focal cerebral ischemia. Glycogen synthase kinase-3beta (GSK-3beta) regulates CRMP-2 function through phosphorylation of CRMP-2. Moreover, the pro-apoptotic activity of GSK-3beta is inactivated by phosphorylation by Akt. This study investigated whether ferulic acid modulates the expression of CRMP-2 and its upstream targets, Akt and GSK-3beta, in focal cerebral ischemia. Male rats were treated immediately with ferulic acid (100 mg/kg, i.v.) or vehicle after middle cerebral artery occlusion (MCAO), and then cerebral cortices were collected 24 hr after MCAO. MCAO resulted in decreased levels of phospho-Akt and phospho-GSK-3beta, while ferulic acid treatment prevented the decrease in the levels of these proteins. Moreover, phospho-CRMP-2 and CRMP-2 levels increased during MCAO, whereas ferulic acid attenuated these injury-induced increases. These results demonstrate that ferulic acid regulates the Akt/GSK-3beta/CRMP-2 signaling pathway in focal cerebral ischemic injury, thereby protecting against brain injury.

Effects of Treadmill Exercise on the Recovery of Dopaminergic Neuron Loss and Muscle Atrophy in the 6-OHDA Lesioned Parkinson's Disease Rat Model

This study was to determine the effect of exercise on the recovery of dopaminergic neuron loss and muscle atrophy in 6-OHDA-induced hemi Parkinson's disease model. Exercise was loaded twice per day for 30 minutes each time, at 5 days after 6-OHDA lesioning and continued for 16 days using a treadmill. Exercise significantly increased the number of tyrosine hydroxylase positive neuron in the lesioned substantia nigra and the expression level of tyrosine hydroxylase in the striatum compared with the control group. To examine which signaling pathways may be involved in the exercise, the phosphorylation of GSK3beta and ERK were observed in the striatum. In the control group, basal level of GSK3beta phosphorylation was less than in both striatum, but exercise increased it. ERK phosphorylation decreased in the lesioned striatum, but exercise recovered it. These findings suggest that exercise inactivates GSK3beta by phosphorylation which may be involved in the neuroprotective effect of exercise on the 6-OHDA-induced cell death. In the exercise group, weight, and Type I and II fiber cross-sectional area of the contralateral soleus significantly recovered and expression of myosin heavy chain and Akt and ERK phosphorylation significantly increased by exercise. These results suggest that exercise recovers Parkinson's disease induced dopaminergic neuron loss and contralateral soleus muscle atrophy.

The Regulation of ERK, GSK3beta and AKT after Acute Ethanol Exposure and Withdrawal in SH-SY5Y Human Neuroblastoma Cell-line / 신경정신의학

OBJECTIVES: This study aimed to demonstrate the activities and phosphorylation changes induced by acute ethanol treatment and withdrawal conditions in the intracellular signal transduction molecules [such as extracellular signal-regulated kinase (ERK), glycogen synthase kinase 3beta (GSK3beta), and Akt] of the SH-SY5Y neuroblastoma cell line. METHODS: The acute treatment exposed SH-SY5Y cells to 100 mM ethanol, and we took samples 30 minutes, 60 minutes, and 24 hours after initiating this treatment. After 24 hours' continuous ethanol treatment, we initiated ethanol withdrawal, taking samples at 30 minutes and 60 minutes. We assayed the kinase phosphorylations via an immunoblot analysis using phosphorspecific antibodies, quantified by optical densitometry. RESULTS: Ethanol treatment induced a transient increase in phosphorylation of GSK3beta and Akt at 30 minutes but failed to change the phosphorylation level of ERK. Ethanol withdrawal induced a transient ERK phosphorylation increase at 30 minutes, but it had no effect on the phosphorylation of GSK3beta or Akt. CONCLUSION: The results indicate that the ethanol-induced cellular response includes the ERK, GSK3beta, and Akt systems. In particular, the ERK pathway may play a role in the acute withdrawal response. This also suggests that a relatively short exposure to ethanol, such as the 24-hour exposure in this study, can induce functional adaptation within a cell.

GSK3beta Inhibitor Peptide Protects Mice from LPS-induced Endotoxin Shock

BACKGROUND: Glycogen synthase kinase 3beta (GSK3beta) is a ubiquitous serine/threonine kinase that is regulated by serine phosphorylation at 9. Recent studies have reported the beneficial effects of a number of the pharmacological GSK3beta inhibitors in rodent models of septic shock. Since most of the GSK3beta inhibitors are targeted at the ATP-binding site, which is highly conserved among diverse protein kinases, the development of novel non-ATP competitive GSK3beta inhibitors is needed. METHODS: Based on the unique phosphorylation motif of GSK3beta, we designed and generated a novel class of GSK3beta inhibitor (GSK3i) peptides. In addition, we investigated the effects of a GSK3i peptide on lipopolysaccharide (LPS)-stimulated cytokine production and septic shock. Mice were intraperitoneally injected with GSK3i peptide and monitored over a 7-day period for survival. RESULTS: We first demonstrate its effects on LPS-stimulated pro-inflammatory cytokine production including interleukin (IL)-6 and IL-12p40. LPS-induced IL-6 and IL-12p40 production in macrophages was suppressed when macrophages were treated with the GSKi peptide. Administration of the GSK3i peptide potently suppressed LPS-mediated endotoxin shock. CONCLUSION: Collectively, we present a rational strategy for the development of a therapeutic GSK3i peptide. This peptide may serve as a novel template for the design of non-ATP competitive GSK3 inhibitors.

Immunohistochemical Study on the Distribution of Glycogen Synthase Kinase (GSK) 3beta in the Central Nervous System of SOD1G93A Transgenic Mice / 체질인류학회지

In the present study, we investigated influences of glycogen synthase kinase (GSK) 3beta on the development and/or progression of amyotrophic lateral sclerosis (ALS). We used transgenic mice expressing a human Cu/Zn superoxide dismutase mutant (SOD1G93A) as an in vivo model of ALS and examined expressional changes of GSK3beta immunohistochemically in the spinal cord, brain stem and cerebellum. With these experiments we demonstrate that the neurons in these regions of symptomatic SOD1G93A transgenic mice showed increased GSK3beta immunoreactivities compared with wild-type SOD1 transgenic mice. In contrast to symptomatic SOD1G93A transgenic mice, few GSK3beta immunoreactivity changes were detected in 8w- and 13w-old presymptomatic SOD1G93A transgenic mice. These data suggest the possibility that GSK3 functions as a modulating factor of apoptosis-related alterations in ALS and that GSK3beta exert differential functions in the development and/or progression of ALS. But the exact functional significances of these changes require further elucidation.

PS-341-Induced Apoptosis is Related to JNK-Dependent Caspase 3 Activation and It is Negatively Regulated by PI3K/Akt-Mediated Inactivation of Glycogen Synthase Kinase-3beta in Lung Cancer Cells / 결핵및호흡기질환

BACKGROUND: PS-341 is a novel, highly selective and potent proteasome inhibitor, which showed cytotoxicity against some tumor cells. Its anti-tumor activity has been suggested to be associated with modulation of the expression of apoptosis-associated proteins, such as p53, p21(WAF/CIP1), p27(KIP1), NF-kappa, Bax and Bcl-2. c-Jun N-terminal kinase (JNK) and glycogen synthase kinase-3beta(GSK-3beta are important modulators of apoptosis. However, their role in PS-341-induced apoptosis is unclear. This study was undertaken to elucidate the role of JNK and GSK-3beta in the PS-341-induced apoptosis in lung cancer cells. METHOD: NCI-H157 and A549 cells were used in the experiments. The cell viability was assayed using the MTT assay and apoptosis was evaluated by proteolysis of PARP. The JNK activity was measured by an in vitro immuno complex kinase assay and by phosphorylation of endogenous c-Jun. The protein expression was evaluated by Western blot analysis. Dominant negative JNK1 (DN-JNK1) and GSK-3betawere overexpressed using plasmid and adenovirus vectors, respectively. RESULT: PS-341 reduced the cell viability via apoptosis, activated JNK and increased the c-Jun expression. Blocking of the JNK activation by overexpression of DN-JNK1, or pretreatment with SP600125, suppressed the apoptosis induced by PS-341. The activation of caspase 3 was mediated by JNK activation. Blocking of the caspase 3 activation suppressed PS-341-induced apoptosis. PS-341 activated the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, but its blockade enhanced the PS-341-induced cell death via apoptosis. GSK-3betawas inactivated by PS-341 via the PI3K/Akt pathway. Overexpression of constitutively active GSK-3beta enhanced PS-341-induced apoptosis; in contrast, this was suppressed by dominant negative GSK-3beta(DN-GSK-3beta. Inactivation of GSK-3beta by pretreatment with lithium chloride or the overexpression of DN-GSK-3beta suppressed both the JNK activation and c-Jun up-regulation induced by PS-341. CONCLUSION: The JNK/caspase pathway is involved in PS-341-induced apoptosis, which is negatively regulated by the PI3K/Akt-mediated inactivation of GSK-3beta in lung cancer cells.

Phosphorylation of Glycogen Synthase Kinase-3beta(S9) Induced Neuronal Cell Survival by Sulindac in the Rat Retina

PURPOSE: Inactivation of glycogen synthase kinase-3beta (GSK-3beta) by S9 phosphorylation is implicated in neuronal cell survival. In this study, we examined the involvement of GSK-3betaS9) phosphorylation on retina cell survival by sulindac (SLD) in model of retina ischemia. METHODS: Retinal ischemia was induced by increasing intraocular pressure to a range of 160 mm Hg to 180 mm Hg for 60 minutes in adult rats. SLD was treated pre and after (0.01 to 0.1 mM) ischemic injury. In vitro study, the retinas were isolated at postnatal 1-2day and were used to glutamate for ischemic injury. For morphological study, retinas were embedded in resin 24 hours after ischemic injury. The patterns of retinal cell were determined using light microscopy. Western blot analysis was performed using GSK-3beta(S9) and phospho-GSK-3beta(S9) antibodies. RESULTS: In ischemic animal model, cell death with necrosis and apoptosis was observed, treatment with SLD was reduced cell death. In vitro study, treatment of glutamate were reduce dose dependent manners, SLD treatment were decrease retina cell death. Western blot analysis of GSK-3beta(S9) phosphorylation known to induce neuronal cell survival, were increased in the SLD treated retina in ischemic injury. CONCLUSIONS: This study suggest that GSK-3beta(S9) is one of the effect by which SLD treatment protect retina from neuronal cell death.