BIO alleviates inflammation through inhibition of GSK-3β in a rat model of intracerebral hemorrhage

2020 ◽  
Vol 133 (2) ◽  
pp. 383-391 ◽  
Author(s):  
Sha Zhao ◽  
Zhen Liu ◽  
Zihan Yu ◽  
Xinran Wu ◽  
Rui Li ◽  
...  
Keyword(s):  
Intracerebral Hemorrhage ◽  
Western Blotting ◽  
Rat Model ◽  
Glycogen Synthase ◽  
Specific Inhibitor ◽  
Microglia Activation ◽  
Enzyme Linked Immunosorbent Assay ◽  
Optimal Dosage ◽  
Autologous Whole Blood ◽  
Gsk 3Β

OBJECTIVEInflammation plays a key role in secondary brain damage following intracerebral hemorrhage (ICH). Glycogen synthase kinase–3β (GSK-3β) plays a strong proinflammatory role in many CNS diseases, including stroke. The present study was undertaken to examine the effects of 6-bromoindirubin-3ʹ-oxime (BIO), a specific inhibitor of GSK-3β, on inflammation in ICH rats.METHODSAn ICH rat model was induced by autologous whole-blood injection into the striatum. First, 10, 20, 40, 60, 80, or 100 μg/kg BIO was applied to ICH animals to determine an optimal dosage for producing sufficient GSK-3β inhibition in rat ipsilateral hippocampus by Western blotting. Second, 40 μg/kg BIO was applied to ICH rats for 1, 3, 7, or 14 days, respectively, to determine a suitable intervention time course of BIO by Western blotting analysis on GSK-3β. Third, Western blotting and enzyme-linked immunosorbent assay were used for quantification of inflammation-related factors upstream or downstream of GSK-3β in rat ipsilateral hippocampus. Then, immunohistochemical staining was applied to detect activated microglia and apoptotic cells in rat ipsilateral hippocampus. Last, neurobehavioral tests were performed to assess the sensorimotor impairments in the ICH rats.RESULTSThe results show that BIO 1) blocked GSK-3βTyr216 phosphorylation/activation, thus stabilizing β-catenin, increasing upstream brain-derived neurotrophic factor and downstream heat shock protein 70 levels, and decreasing the levels of nuclear factor–κB p65 and cyclooxygenase 2; 2) decreased the levels of the proinflammatory cytokines tumor necrosis factor–α and interleukin (IL)–1β and IL-6 and elevated the level of antiinflammatory cytokine IL-10; 3) inhibited microglia activation and cell apoptosis; and 4) improved the sensorimotor deficits of ICH rats.CONCLUSIONSBIO posttreatment inhibited microglia activation, prevented inflammation and hippocampal cell death, and ameliorated functional and morphological outcomes in a rat ICH model through inactivation of GSK-3β.

scholarly journals MiR-1906 attenuates neuropathic pain in rats by regulating the TLR4/mTOR/ Akt signaling pathway

2019 ◽  
Vol 10 (1) ◽  
pp. 175-179 ◽  
Author(s):  
Xianhai Fang ◽  
Huacheng Zhou ◽  
Shaopeng Huang ◽  
Jinfeng Liu
Keyword(s):  
Neuropathic Pain ◽  
Western Blotting ◽  
Rat Model ◽  
Latency Period ◽  
Enzyme Linked Immunosorbent Assay ◽  
Withdrawal Latency ◽  
Proinflammatory Mediators ◽  
Neuropathic Pain Model ◽  
Pathway Regulation

Abstract Background This study determined the role of miR-1906 in neuropathic pain and proliferation in neuronal cells using a chronic constriction injury (CCI)-induced neuropathic pain (NP) rat model. Methodology NP was induced by CCI. Animals were divided into a sham group, an NP group, and a miR-1906 mimic group, which received 500 nmol/kg of a miR-1906 mimic intrathecally for 10 consecutive days following surgery. The effect of miR-1906 agomir was determined by estimating the thermal and mechanical withdrawal latency; an enzyme-linked immunosorbent assay (ELISA) was used to determine the concentration of proinflammatory mediators. Western blotting and reverse-transcription polymerase chain reaction (RT-PCR) were used to determine protein expression in the spinal tissues of the CCI-induced neuropathic pain rat model. Results Administration of miR-1906 agomir increased the mechanical and thermal withdrawal latency period and the levels of inflammatory mediators compared with the NP group. Western blotting showed that treatment with miR-1906 agomir attenuated the levels of Akt, mTOR, TLR-4, and PI3K proteins in the spinal tissues of the CCI-induced neuropathic pain model. TLR-4 and NF-κB gene expression was lower in the miR-1906 agomir group than in the NP group. Conclusion miR-1906 gene stimulation reduced neuropathic pain by enhancing Akt/nTOR/PI3K and TLR-4/NF-κB pathway regulation.

scholarly journals Molecular Mechanism for PACAP 38-Induced Neurite Outgrowth in PC12 Cells

2021 ◽  
Vol 2021 ◽  
pp. 1-12
Author(s):  
Junko Shibato ◽  
Fumiko Takenoya ◽  
Takahiro Hirabayashi ◽  
Ai Kimura ◽  
Michio Yamashita ◽  
...  
Keyword(s):  
Neurite Outgrowth ◽  
Pc12 Cells ◽  
Molecular Mechanism ◽  
Glycogen Synthase ◽  
Specific Inhibitor ◽  
Two Dimensional Gel Electrophoresis ◽  
Downstream Signaling ◽  
Mediator Protein ◽  
Gsk 3Β ◽  
Signaling Components

The present research investigates the molecular mechanism of neurite outgrowth (protrusion elongation) under pituitary adenylate cyclase-activating polypeptide (PACAP) 38 treatments using a rat adrenal-derived pheochromocytoma cell line—PC12. This study specifically looks into the regulation of PACAP38-induced collapsing response mediator protein 2 (CRMP2) previously identified in a mouse brain ischemia model and which could be recovered by PACAP38 treatment. Previously, DNA microarray analysis revealed that PACAP 38-mediated neuroprotection involved not only CRMP2 but also pathways related to glycogen synthase kinase-3β (GSK-3β) and other signaling components. Thus, to clarify whether CRMP2 acts directly on PACAP38 or through GSK-3β as part of the mechanism of PACAP38-induced neurite outgrowth, we observed neurite outgrowth in the presence of GSK-3β inhibitors and activators. PC12 cells were treated with PACAP38 being added to the cell culture medium at concentrations of 10−7 M, 10−8 M, and 10−9 M. Post PACAP38 treatment, immunostaining was used to confirm protrusion elongation of the PC12 cells, while RT-PCR, two-dimensional gel electrophoresis in conjunction with Western blotting, and inhibition experiments were performed to confirm the expression of the PACAP gene, its receptors, and downstream signaling components. Our data show that neurite protrusion elongation by PACAP38 (10−7 M) in PC12 cells is mediated through the PAC1-R receptor as demonstrated by its suppression by a specific inhibitor PA-8. Inhibitor experiments suggested that PACAP38-triggered neurite protrusion follows a GSK-3β-regulated pathway, where the AKT and cAMP/ERK pathways are involved and where the inhibition of Rho/Roc could enhance neurite protrusion under PACAP38 stimulation. Although we could not yet confirm the exact role and position of CRMP2 in PACAP38-mediated PC12 cell elongation, it appears that its phosphorylation and dephosphorylation have a correlation with the neurite protrusion elongation through the interplay of CDK5, which needs to be investigated further.

AT7519, a Novel Small Molecule Multi-Cyclin Dependent Kinase Inhibitor, Induces Apoptosis in Multiple Myeloma VIA GSK3β

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 251-251 ◽  
Author(s):  
Loredana Santo ◽  
Sonia Vallet ◽  
Teru Hideshima ◽  
Diana Cirstea ◽  
Samantha Pozzi ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Small Molecule ◽  
Glycogen Synthase ◽  
Group Versus ◽  
Specific Inhibitor ◽  
Control Group ◽  
Rna Pol Ii ◽  
Pol Ii ◽  
Gsk 3Β

Abstract Cyclin dependent kinases (CDKs) and their cyclin complexes play a crucial role in cell cycle control and transcriptional regulation. In multiple myeloma (MM), the abnormal activation of different CDKs and their cyclin partners, especially CDK4/cyclin D1 and CDK6/Cyclin D2, mediate uncontrolled cell cycle progression. Therefore CDKs represent promising novel therapeutic targets for MM. Additionally the cytokine dependent PI3K/Akt signaling pathway mediates growth, survival, drug resistance, migration and cell cycle regulation in MM. Activated Akt in turn phosphorylates downstream target molecules like glycogen synthase kinase (GSK)-3 β impacting growth and survival. Here we investigated the preclinical activity of a novel small-molecule multi-CDK inhibitor, AT7519 in MM. In vitro kinase assays demonstrated more potent inhibition of CDK 1, 2, 4, 5 and 9 compared to CDK 3, 6, and 7. AT7519 also demonstrated potent inhibitory activity against GSK-3 β. No significant inhibitory effects against other kinases were observed. We next investigated the growth inhibitory effect of AT7519 on MM cell lines. Maximal cytotoxicity was observed in 48 hour culture with IC50 values ranging from 0.5μM (MM.1S, U266) to 4 μM (MM1R). AT7519 was also effective against primary tumor cells from MM patients with no significant cytotoxicity noted in peripheral blood mononuclear cells from healthy volunteers. To delineate the underlying mechanism of cytotoxicity induced by AT7519, cell cycle analysis using PI staining in MM.1S cell line was performed. No significant accumulation of cells in a particular phase of cell cycle was noted; however, AT7519 showed an increased sub-G1 population, indicative of apoptosis, which was confirmed by Annexin V+PI+ staining and associated with caspase-8-9 and -3 cleavage. Importantly, we found that AT7519 markedly inhibited phosphorylation (serine 2 and serine 5 sites) of the carboxyl terminal domain of RNA polymerase II (RNA pol II) within 6 hours of treatment. Non-cell cycle CDKs including CDK9 are responsible for phosphorylation and activation of RNA pol II. Similarly, AT7519 also inhibited phosphorylation of GSK-3β while no significant effects on CDK expression levels were evident at early time points. To investigate whether there was a correlation between inhibition of phosphorylation of GSK-3β and RNA pol II, MM.1S cells were cultured with α-amanitin, a specific inhibitor of RNA pol II. Although phosphorylation of RNA pol II was significantly inhibited, phosphorylation of GSK-3β was not altered by amanitin (10 μM for up to 24 hours). These results suggest that GSK-3β and RNA pol II dephosphorylation at serine 2 and serine 5 may be two independent mechanisms by which AT7519 induces apoptosis in MM cells. Ongoing studies are confirming the role of GSK-3 β in AT7519 induced cytotoxicity of MM cells. Finally, the in vivo efficacy of AT7519 was examined using a xenograft mouse model of human MM. Mice treated with AT7519 demonstrated slower tumor growth compared to the control group without adverse effects. Moreover, AT7519 resulted in a significant prolongation in median overall survival in treated mice (40 days in the treatment group versus 27.5 days in the control cohort, p = 0.0324). In conclusion, these results show significant anti-MM activity of AT7519, and provide the rationale for its clinical evaluation in MM.

Morphine prevents the mitochondrial permeability transition pore opening through NO/cGMP/PKG/Zn2+/GSK-3β signal pathway in cardiomyocytes

2010 ◽  
Vol 298 (2) ◽  
pp. H601-H607 ◽  
Author(s):  
Jinkun Xi ◽  
Wei Tian ◽  
Lei Zhang ◽  
Yulan Jin ◽  
Zhelong Xu
Keyword(s):  
Mitochondrial Permeability Transition Pore ◽  
Mitochondrial Permeability Transition ◽  
Glycogen Synthase ◽  
Specific Inhibitor ◽  
Permeability Transition Pore ◽  
Permeability Transition ◽  
Mitochondrial Permeability ◽  
Mptp Opening ◽  
Pore Opening ◽  
Gsk 3Β

The aim of this study was to test whether morphine prevents the mitochondrial permeability transition pore (mPTP) opening through Zn2+ and glycogen synthase kinase 3β (GSK-3β). Fluorescence dyes including Newport Green Dichlorofluorescein (DCF), 4-amino-5-methylamino-2′,7′-difluorofluorescein (DAF-FM), and tetramethylrhodamine ethyl ester (TMRE) were used to image free Zn2+, nitric oxide (NO), and mitochondrial membrane potential (ΔΨm), respectively. Fluorescence images were obtained with confocal microscopy. Cardiomyocytes treated with morphine for 10 min showed a significant increase in Newport Green DCF fluorescence intensity, an effect that was reversed by the NO synthase inhibitor N G-nitro-l-arginine methyl ester (l-NAME), indicating that morphine mobilizes Zn2+ via NO. Morphine rapidly produced NO. ODQ and NS2028, the inhibitors of guanylyl cyclase, prevented Zn2+ release by morphine, implying that cGMP is involved in the action of morphine. The effect of morphine on Zn2+ release was also abolished by KT5823, a specific inhibitor of protein kinase G (PKG). Morphine prevented oxidant-induced loss of ΔΨm, indicating that morphine can modulate the mPTP opening. The effect of morphine on the mPTP was reversed by KT5823 and the Zn2+ chelator N, N, N′, N′-tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN). The action of morphine on the mPTP was lost in cells transfected with the constitutively active GSK-3β mutant, suggesting that morphine may prevent the mPTP opening by inactivating GSK-3β. In support, morphine significantly enhanced phosphorylation of GSK-3β at Ser9, and this was blocked by TPEN. GSK-3β small interfering RNA prevented the pore opening in the control cardiomyocytes but failed to enhance the effect of morphine on the mPTP opening. In conclusion, morphine mobilizes intracellular Zn2+ through the NO/cGMP/PKG signaling pathway and prevents the mPTP opening by inactivating GSK-3β through Zn2+.

scholarly journals Potential involvement of glycogen synthase kinase (GSK)-3β in a rat model of multiple sclerosis: evidenced by lithium treatment

2017 ◽  
Vol 50 (1) ◽  
pp. 48 ◽  
Author(s):  
Meejung Ahn ◽  
Jeongtae Kim ◽  
Changnam Park ◽  
Jinhee Cho ◽  
Youngheun Jee ◽  
...  
Keyword(s):  
Multiple Sclerosis ◽  
Rat Model ◽  
Glycogen Synthase ◽  
Lithium Treatment ◽  
Glycogen Synthase Kinase ◽  
Gsk 3Β

scholarly journals DOP68 Thiomyristoyl ameliorates colitis by blocking the differentiation of Th17 cells via STAT3/IL-6 signal pathway

2020 ◽  
Vol 14 (Supplement_1) ◽  
pp. S105-S106
Author(s):  
Y XU ◽  
M zhang ◽  
G Xu ◽  
X Zou
Keyword(s):  
Flow Cytometry ◽  
Western Blotting ◽  
Th17 Cells ◽  
Culture Supernatant ◽  
Specific Inhibitor ◽  
Signal Pathway ◽  
T Cell Subsets ◽  
Enzyme Linked Immunosorbent Assay ◽  
Control Group ◽  
Ifn Γ

Abstract Background The pathogenesis of inflammatory bowel disease (IBD), including Crohn’s disease (CD) and ulcerative colitis (UC), has not been fully elucidated, but a strong correlation between IBD and the immune dysregulation of the host has been persistently detected. Sirtuin 2 (SIRT2), a histone deacetylase, has recently been found to play an important role in inflammation, infection and immunomodulatory. As a potent SIRT2-specific inhibitor, thiomyristoyl (TM) has an extensive anticancer activity, but its anti-inflammatory property remains unclear. Methods Human peripheral blood mononuclear cells (PBMC) were differentiated into T helper 17 (Th17) cells in vitro and were treated with TM simultaneously. Interleukin (IL)-17A in the culture supernatant, the ratio of T cells, the levels of phosphorylated-signal transducer and activator of transcription (p-stat3) and phosphorylated-nuclear factor kappa-B (p-NF κB) were determined by enzyme-linked immunosorbent assay (ELISA), flow cytometry or western blotting, respectively. Then, colitis C57BL/6J mice induced by 2.5% dextran sulphate sodium salt (DSS) were treated with TM, and were evaluated by disease activity index (DAI) and haematoxylin-eosin (HE) staining. T-cell subsets in the mice spleen, IL-6, IL-10 and IL-17A in serum, related factors retinoic acid receptor-related orphan receptor-γ t (ROR-γt), forkhead box protein P3 (FOXP3), IL-17A, interferon γ γ (IFN-γ), IL-23, IL-10 and hypoxia-inducible factor (HIF)-1α in mice colon were measured by flow cytometry, ELISA, quantitative real-time polymerase chain reaction (Q-PCR) and western blotting, respectively. Results Compared with the positive control group, the levels of IL-17A in culture supernatant, the percentage of Th17, the levels of p-stat3 and p-NF kappa B in cell lysate were lower in the TM group. Compared with PBS-treated colitis mice, TM-treated colitis mice had longer colons, fewer weight-losses, lower DAI and histopathologic scores. Interestingly, although the expression of IFN-γ, IL-17A, and ROR-γt was inhibited in the colons of TM-treated mice, the level of FOXP3 did not change. Consistently, the percentage of spleen Th17 cells was decreased in the TM group while the percentage of Treg cells was not affected. In addition, the TM group had reduced levels of IL-23 and HIF-1α, and an increased level of IL-10 in the colon, compared with colitis group. Conclusion TM ameliorates DSS-induced experimental colitis by blocking the differentiation of Th17 cells, which may be associated with the STAT3/IL-6 signal pathway. SIRT2 may represent a potential target for the treatment of IBD.

scholarly journals Artesunate attenuates traumatic brain injury-induced impairments in rats

2020 ◽  
Vol 11 (1) ◽  
pp. 309-318
Author(s):  
Zhike Zhou ◽  
Jun Hou ◽  
Qinghua Li
Keyword(s):  
Rat Model ◽  
Brain Injuries ◽  
Glycogen Synthase ◽  
Combined Treatment ◽  
Potential Candidate ◽  
Behavioral Experiments ◽  
Protective Function ◽  
Protein Levels ◽  
Gsk 3Β ◽  
The Impact

AbstractBackgroundBlood–brain barrier (BBB) dysfunction and neuroinflammation induced by traumatic brain injuries (TBIs) cause a succession of secondary brain damage events and finally lead to a massive and progressive cerebral neuronal destruction. Artesunate, a semisynthetic artemisinin derivative, is a potential candidate for the management of cerebral damage induced by TBI due to its protective function to BBB and cerebral neurons.MethodsTo demonstrate the effect of artesunate to TBI-induced BBB dysfunction and neural damage, TBI rat model was constructed by cortical impact injury. Behavioral experiments were used to estimate the impact of the combined treatment on rats. Western blotting was performed to demonstrate the protein levels in the brain tissues of rats. Quantitative real-time PCRs were utilized to investigate the alteration in the expression of various RNA levels. The chemokine levels were estimated by ELISA.ResultsArtesunate treatment attenuated the impact caused by TBI on rat brain and improved the long-term neurological recover. Artesunate treatment protected the integrity of BBB and inhibited neuroinflammation. Artesunate treatment promoted the phosphorylation of Akt and inhibited the phosphorylation of glycogen synthase kinase (GSK)-3β in TBI rat model.ConclusionArtesunate protected rats from TBI-induced impairments of BBB and improved longer-term neurological outcomes.

scholarly journals Phosphorylation and inactivation of glycogen synthase kinase-3 by soluble kit ligand in mouse oocytes during early follicular development

2007 ◽  
Vol 38 (1) ◽  
pp. 137-146 ◽  
Author(s):  
Lian Liu ◽  
Singareddy Rajareddy ◽  
Pradeep Reddy ◽  
Krishna Jagarlamudi ◽  
Chun Du ◽  
...  
Keyword(s):  
Glycogen Synthase ◽  
Specific Inhibitor ◽  
Follicular Development ◽  
Glycogen Synthase Kinase ◽  
Glycogen Synthase Kinase 3 ◽  
Oocyte Growth ◽  
Kit Ligand ◽  
Mouse Oocytes ◽  
Gsk 3Β

Communication between mammalian oocytes and their surrounding granulosa cells through the Kit–Kit ligand (KL, or stem cell factor, SCF) system has been shown to be crucial for follicular development. Our previous studies (Reddy et al. 2005, Liu et al. 2006) have indicated that the intra-oocyte KL–Kit–PI3 kinase (PI3K)–Akt–Foxo3a cascade may play an important role in follicular activation and early development. In the present study, using in situ hybridization and in vitro culture of growing oocytes from 8-day-old postnatal mice, we have demonstrated that another Akt substrate, glycogen synthase kinase-3 (GSK-3), is expressed in growing oocytes. Also, treatment of cultured mouse oocytes with soluble KL not only leads to increased Akt kinase activity in the oocytes, which can phosphorylate recombinant GSK-3 in vitro, but also leads to phosphorylation of oocyte GSK-3α and GSK-3β, which can result in the inactivation of GSK-3 function in oocytes. In addition, we have shown that the regulation of GSK-3α and GSK-3β in cultured oocytes by soluble KL is accomplished through PI3K, since the PI3K-specific inhibitor LY294002 completely abolished the KL-induced phosphorylation of GSK-3α and GSK-3β. Moreover, blockage of the Kit signaling pathway by a Kit function-blocking antibody, ACK2, resulted in reduced phosphorylation of GSK-3. Taken together, our data suggest that the cascade from granulosa cell-derived KL to Kit–PI3K–Akt–GSK-3 in oocytes may take part in regulation of oocyte growth and early ovarian follicular development.

Glycogen synthase kinase (GSK)-3β levels and activity in a neurodevelopmental rat model of schizophrenia

2003 ◽  
Vol 141 (1-2) ◽  
pp. 33-37 ◽  
Author(s):  
Carmit Nadri ◽  
Barbara K Lipska ◽  
Nitsan Kozlovsky ◽  
Daniel R Weinberger ◽  
Robert H Belmaker ◽  
...  
Keyword(s):  
Rat Model ◽  
Glycogen Synthase ◽  
Glycogen Synthase Kinase ◽  
Gsk 3Β
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