Inhibition Of Glycogen Synthase Kinase-3β Research Articles

Phytochemical natural killer cells reprogram tumor microenvironment for potent immunotherapy of solid tumors

Natural killer cells (NKs) hold great promise in cancer treatment, but their application in solid tumors remains a great challenge because current solutions hardly can overcome various difficulties that faced. Herein, we endow NKs with the phytochemical feature for effective immunotherapy of solid tumors. NKs are decorated with natural thylakoid (Tk) membranes through an efficient and convenient membrane fusion strategy. Tk engineering effectively activates NKs, because the antioxidase on Tk induce glycogen synthase kinase-3β inhibition, and subsequently increase the expression of activating receptor and cytotoxic effector molecules in NKs. After systemic administration, the phytochemical NKs (PC-NKs) can target tumor tissues, and then profoundly reprogram tumor microenvironment (TME) with the help of catalase on Tk, resulting in significantly enhanced direct killing of PC-NKs and immune activated TME. Therefore, potent therapeutic effects with few abnormalities are achieved, providing a novel idea for the development of highly efficient NKs for solid tumors.

Synthesis and biological evaluation of thieno[3,2-c]pyrazol-3-amine derivatives as potent glycogen synthase kinase 3β inhibitors for Alzheimer’s disease

Glycogen synthase kinase 3β (GSK-3β) catalyses the hyperphosphorylation of tau protein in the Alzheimer’s disease (AD) pathology. A series of novel thieno[3,2-c]pyrazol-3-amine derivatives were designed and synthesised and evaluated as potential GSK-3β inhibitors by structure-guided drug rational design approach. The thieno[3,2-c]pyrazol-3-amine derivative 16b was identified as a potent GSK-3β inhibitor with an IC50 of 3.1 nM in vitro and showed accepted kinase selectivity. In cell levels, 16b showed no toxicity on the viability of SH-SY5Y cells at the concentration up to 50 μM and targeted GSK-3β with the increased phosphorylated GSK-3β at Ser9. Western blot analysis indicated that 16b decreased the phosphorylated tau at Ser396 in a dose-dependent way. Moreover, 16b effectively increased expressions of β-catenin as well as the GAP43, N-myc, and MAP-2, and promoted the differentiated neuronal neurite outgrowth. Therefore, the thieno[3,2-c]pyrazol-3-amine derivative 16b could serve as a promising GSK-3β inhibitor for the treatment of AD.

Baicalin Coadministration with Lithium Chloride Enhanced Neurogenesis via GSK3β Pathway in Corticosterone Induced PC-12 Cells.

Accumulating studies suggest that hippocampal neurogenesis plays a crucial role in the pathological mechanism of depression. As a classic antidepressant, lithium chloride can play an antidepressant role by inhibiting glycogen synthase kinase 3β (GSK3β) and promoting neurogenesis. Correspondingly, baicalin is a compound extracted from natural plants, which shows potential antidepressant effect, however, whether baicalin exerts antidepressant effects by promoting neurogenesis still needs further investigation. In the current study, we established an in vitro depression model through corticosterone induced PC-12 cells, and explored the potential mechanism of baicalin's antidepressant effect by comparing it with lithium chloride alone and the coadministration with lithium chloride. We used Cell Counting Kit-8 (CCK-8) assay, 5-ethynil-2'-deoxyuridine (EdU) staining and cell cycle analysis to evaluate the state of cell survival and cell proliferation. The protein expression levels of neurodevelopmental related factors Doublecortin (DCX), brain-derived neurotrophic factor (BDNF), and the GSK3β pathway-related proteins and mRNA were detected by Western blot and Real-time PCR. The results showed that baicalin could decrease the expression level of GSK3β, while upregulate the expression level of DCX, BDNF, Cyclin D1-cyclin dependent kinase 4/6 (CDK4/6), thus promoted cell proliferation and survival in corticosterone (CORT) induced PC-12 cells. Moreover, this effect was enhanced when baicalin and lithium chloride were coadministration. Taking the above results together, we conclude that baicalin can promote the proliferation and development of PC-12 cells by regulating GSK3β pathway, so as to reverse the depressive-like pathological changes induced by corticosterone.

Multi-target neuroprotective effects of herbal medicines for Alzheimer's disease

Ethnopharmacological relevanceAlzheimer's disease is the most common form of dementia, but its treatment options remain few and ineffective. To find new therapeutic strategies, natural products have gained interest due to their neuroprotective potential, being able to target different pathological hallmarks associated with this disorder. Several plant species are traditionally used due to their empirical neuroprotective effects and it is worth to explore their mechanism of action. Aim of the studyThis study intended to explore the neuroprotective potential of seven traditional medicinal plants, namely Scutellaria baicalensis, Ginkgo biloba, Hypericum perforatum, Curcuma longa, Lavandula angustifolia, Trigonella foenum-graecum and Rosmarinus officinalis. The safety assessment with reference to pesticides residues was also aimed. Materials and methodsDecoctions prepared from these species were chemically characterized by HPLC-DAD and screened for their ability to scavenge four different free radicals (DPPH•, ABTS•+, O2•‒ and •NO) and to inhibit enzymes related to neurodegeneration (cholinesterases and glycogen synthase kinase-3β). Cell viability through MTT assay was also evaluated in two different brain cell lines, namely non-tumorigenic D3 human brain endothelial cells (hCMEC/D3) and NSC-34 motor neurons. Furthermore, and using GC, 21 pesticides residues were screened. ResultsRegarding chemical composition, chromatographic analysis revealed the presence of several flavonoids, phenolic acids, curcuminoids, phenolic diterpenoids, one alkaloid and one naphthodianthrone in the seven decoctions. All extracts were able to scavenge free radicals and were moderate glycogen synthase kinase-3β inhibitors; however, they displayed weak to moderate acetylcholinesterase and butyrylcholinesterase inhibition. G. biloba and L. angustifolia decoctions were the less cytotoxic to hCMEC/D3 and NSC-34 cell lines. No pesticides residues were detected. ConclusionsThe results extend the knowledge on the potential use of plant extracts to combat multifactorial disorders, giving new insights into therapeutic avenues for Alzheimer's disease.

Glycogen synthase kinase 3β inhibition and insulin-receptor binding enhancement of compounds isolated from wild leafy vegetable Acalypha alnifolia

BackgroundThe traditional information of Acalypha alnifolia is captivate towards its therapeutic efficacy against diabetes along with the scientific confirmation were leads to an instinct to extract phytocompounds and to evaluate antidiabetic-molecular mechanism. MethodsColumn chromatographic compound isolation were adopted to extract the pure compounds from bioactive leaf extract and interactions of the compounds with diabetic metabolic proteins were accomplished through in-silico docking studies. By the direction, the anti-diabetic property was valued by the expressions of Glycogen Synthase Kinase 3β(GSK 3β) and Insulin on insulin-Receptor on Hep G2 cells. ResultsThe flavone compound - 5,7-dihydroxy-2-(4-hydroxyphenyl)-3-methoxy-4H-chromen-4-one and pyridine alkaloid - (2,6-dihydroxypyridin-4-yl)(3,5-dihydroxytetrahydro-2H-pyran-4-yl) acetic acid were isolated. Both the compounds reduced the GSK 3β expression and the flavone contributed also to enhance insulin binding ability expediently. ConclusionsThe results concluded that the compounds from A. alnifolia having potent to maintain glucose homeostasis in liver insulin-metabolism. By exploring pharmaceutical importance of the isolated compounds, this study will be an important breakthrough in developing new drug for Diabetes.

Anti-fibrotic effect of 6-bromo-indirubin-3'-oxime (6-BIO) via regulation of activator protein-1 (AP-1) and specificity protein-1 (SP-1) transcription factors in kidney cells.

PAI-1 and CTGF are overexpressed in kidney diseases and cause fibrosis of the lungs, liver, and kidneys. We used a rat model of unilateral ureteral obstruction (UUO) to investigate whether 6-BIO, a glycogen synthase kinase-3β inhibitor, attenuated fibrosis by inhibiting PAI-1 and CTGF in vivo. Additionally, TGFβ-induced cellular fibrosis was observed in vitro using the human kidney proximal tubular epithelial cells (HK-2), and rat interstitial fibroblasts (NRK49F). Expression of fibrosis-related proteins and signaling molecules such as PAI-1, CTGF, TGFβ, αSMA, SMAD, and MAPK were determined in HK-2 and NRK49F cells using immunoblotting. To identify the transcription factors that regulate the expression of PAI-1 and CTGF the promoter activities of AP-1 and SP-1 were analyzed using luciferase assays. Confocal microscopy was used to observe the co-localization of AP-1 and SP-1 to PAI-1 and CTGF. Expression of PAI-1, CTGF, TGFβ, and α-SMA increased in UUO model as well as in TGFβ-treated HK-2 and NRK49F cells. Furthermore, UUO and TGFβ treatment induced the activation of P-SMAD2/3, SMAD4, P-ERK 1/2, P-P38, and P-JNK MAPK signaling pathways. PAI-1, CTGF, AP-1 and SP-1 promoter activity increased in response to TGFβ treatment. However, treatment with 6-BIO decreased the expression of proteins and signaling pathways associated with fibrosis in UUO model as well as in TGFβ-treated HK-2 and NRK49F cells. Moreover, 6-BIO treatment attenuated the expression of PAI-1 and CTGF as well as the promoter activities of AP-1 and SP-1, thereby regulating the SMAD and MAPK signaling pathways, and subsequently exerting anti-fibrotic effects on kidney cells.

The Effect of CHIR 99021, a Glycogen Synthase Kinase-3β Inhibitor, on Transforming Growth Factor β-Induced Tenon Fibrosis.

PurposeThis study investigated the effect of glycogen synthase kinase-3β (GSK-3β) inhibition on the fibrosis of human Tenon's fibroblasts (HTFs) induced by transforming growth factor-β (TGF-β).MethodsQuantitative real-time PCR and Western blot analyses were performed to determine the expression levels of molecules associated with the fibrosis of HTFs by TGF-β (fibronectin, collagen Iα, and α-smooth muscle actin) and GSK-3β. The levels of phosphorylated Smad2 and Smad3 were also analyzed in the presence of the GSK-3β inhibitor CHIR 99021. The wound healing assay was performed to determine the effect of CHIR 99021 on the migration of HTFs. All experiments were conducted using primary cultured HTFs or human tenon tissues obtained from normal subjects and patients with glaucoma.ResultsTreatment with TGF-β resulted in an increase in the levels of molecules associated with the fibrosis of HTFs. The expression levels of these molecules were higher in the tenon tissues obtained from patients with glaucoma than those from normal subjects. When the HTFs were treated with TGF-β, a significant increase in the active form of GSK-3β (Y216) was observed. A significant decrease in the active form of GSK-3β and molecules associated with fibrosis by TGF-β was noted in HTFs treated with CHIR 99021. CHIR 99021 treatment reduced the phosphorylated Smad2/Smad2 and phosphorylated Smad3/Smad3 ratios in HTFs and attenuated HTF migration.ConclusionsOur results demonstrated the effect of GSK-3β inhibition on the regulation of TGF-β–mediated fibrosis of HTFs, suggesting GSK-3β to be a potential target for maintaining bleb function after glaucoma filtration surgery.

Structure-Based design of Marine-derived Meridianin C derivatives as glycogen synthase kinase 3β inhibitors with improved oral bioavailability: From aminopyrimidyl-indoles to the sulfonyl analogues

Glycogen synthase kinase 3β (GSK-3β) has become an attractive target for the treatment of diabetes. Compound I is an indole-based GSK-3β inhibitor designed from the Meridianin C, a marine natural product (MNP) isolated from Aplidium meridianum. However, this compound has a moderate inhibitory activity toward GSK-3β (IC50 = 24.4 μM), moderate glucose uptake (38%), and especially, a low oral bioavailability (F = 11.4%). In the present study, applying the structure-based design strategy, a series of derivatives modified on the indole moiety were synthesized based on the lead compound I, followed by evaluating their cytotoxic activity, antihyperglycemic activity, and kinase inhibitory activity. Among this series, compound 6x with a sulfonyl group displayed the highest glucose uptake (83.5%) in muscle L6 cells, showing much higher inhibitory activity against GSK-3β (IC50 = 5.25 μM). Molecular docking indicated that compound 6x was properly inserted into the ATP-binding binding pocket of GSK-3β with a higher docking score (-8.145 kcal/mol) compared with that of compound I (-6.950 kcal/mol), interpreting the higher kinase inhibitory activity toward GSK-3β. Remarkably, compound 6x showed favorable drug-like properties, including significantly better oral bioavailability (F = 47.4%) and no two-week acute toxicity at a dose of 1 g/kg. Our findings suggest that these MNP-derived sulfonyl indole derivatives could be used as lead compounds for the development of anti-hyperglycemic drugs.

From crypts to enteroids: establishment and characterization of avian intestinal organoids

Intestinal organoids (IO), known as “mini-guts”, derived from intestinal crypts, are self-organizing three-dimensional (3D) multicellular ex vivo models that recapitulate intestine epithelial structure and function and have been widely used for studying intestinal physiology, pathophysiology, molecular mechanisms of host-pathogen interactions, and intestinal disease in mammals. However, studies on avian IO are limited and the development of long-term cultures of IO model for poultry research is lacking. Therefore, the objectives of this study were to generate crypt-derived organoids from chicken intestines and to optimize conditions for cell growth and enrichments, passages, and cryopreservation. Crypts were collected from the small intestines of birds at embryonic d-19 and ceca from layer and broiler chickens with ages ranging from d 1 to 20 wk, embedded in a basement membrane matrix, and cultured with organoid growth media (OGM) prepared in house. The crypt-derived organoids were successfully grown and propagated to form 3D spheres like structures that were cultured for up to 3 wk. Organoids were formed on d one, budding appeared on d 3, and robust budding was observed on d 7 and beyond. For cryopreservation, dissociated organoids were resuspended in a freezing medium. The characteristics of IO upon extended passages and freeze-thaw cycles were analyzed using reverse transcription (RT)-PCR, immunoblotting, and live cell imaging. Immunoblotting and RT-PCR using E-cadherin (the marker for epithelial cells), leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5, the marker for stem cells), chromogranin A (the marker for enteroendocrine cells), lysozyme (the marker for Paneth cells), and mucin (the biomarker for goblet cells) confirmed that IO were composed of heterogeneous cell populations, including epithelial cells, stem cells, enteroendocrine cells, Paneth cells, and goblet cells. Furthermore, OGM supplemented with both valproic acid and CHIR99021, a glycogen synthase kinase 3β inhibitor and a histone deacetylase inhibitor, increased the size of the avian IO (P < 0.001). To the best of our knowledge, this is the first comprehensive report for establishing long-term, organoid culture models from small intestines and ceca of layer and broiler chickens. This model will facilitate elucidation of the mechanisms impacting host-pathogen interactions, eventually leading to the discovery of pathogen intervention strategies in poultry.

Treatment With Glycogen Synthase Kinase 3β Inhibitor Decreases Apoptotic and Autophagic Reactions in Rat Rotator Cuff Tears.

Background:Apoptosis and autophagy are known to be correlated with the extent of damage in torn rotator cuffs, and there is no biological evidence for self-recovery or healing of the rotator cuff tear.Purpose:To establish in a rat model of partial- and full-thickness rotator cuff tears how a glycogen synthase kinase 3β (GSK-3β) inhibitor affects the expression of apoptotic and autophagic markers.Study Design:Controlled laboratory study.Methods:Twelve-week-old Sprague Dawley rats were divided into 3 groups (n = 16 per group). Group 1 acted as the control, with no treatment; group 2 received partial-thickness (right side) and full-thickness (left side) rotator cuff tears only; and group 3 received the same rotator cuff injuries, with GSK-3β inhibitor injected afterward. The tendons from each group were harvested 42 days after surgery. Evaluation of gene expression, immunohistochemistry, and TUNEL staining (terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling) were performed for the following markers: caspases 3, 8, and 9 as well as Bcl-2 (B-cell lymphoma 2); BAX (Bcl-2-associated X protein); beclin 1; p53; and GSK-3β; which represented apoptotic and autophagic reactions. Statistical analysis was performed using 1-way analysis of variance.Results:In the group 2 rats with partial- and full-thickness tears, there were significant increases in the mRNA levels (fold changes) of all 8 markers as compared with group 1 (control). All these increased markers showed significant downregulation by the GSK-3β inhibitor in partial-thickness tears. However, the response to the GSK-3β inhibitor in full-thickness tears was not as prominent as in partial-thickness tears. The number of TUNEL-positive cells in group 2 (partial, 35.08% ± 1.625% [mean ± SE]; full, 46.92% ± 1.319%) was significantly higher than in group 1 (18.02% ± 1.036%; P < .01) and group 3 (partial, 28.04% ± 2.607% [P < .01]; full, 38.97% ± 2.772% [P < .01]), and immunohistochemistry revealed increased expression of all the markers in group 2 as compared with control.Conclusion:The apoptotic and autophagic activity induced in a rat model of an acute rotator cuff tear was downregulated after treatment with a GSK-3β inhibitor, particularly with partial-thickness rotator cuff tears.Clinical Relevance:A GSK-3β inhibitor may be able to modulate deterioration in a torn rotator cuff.

Immobilization of Glycogen Synthase Kinase-3β Inhibitor on 316L Stainless Steel via Polydopamine to Accelerate Endothelialization.

The coverage of stents with healthy endothelium is crucial to the success of cardiovascular stent implantation. Immobilizing bioactive molecules on stents is an effective strategy to generate such stents. Glycogen synthase kinase-3β inhibitor (GSKi) is a bioactive molecule that can effectively accelerate vascular endothelialization. In this work, GSKi was covalently conjugated on 316L stainless steel through polydopamine to develop a stable bioactive surface. Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM) and water contact angle results revealed the successful introduction of GSKi onto 316L stainless steel. The GSKi coating did not obviously affect the hemocompatibility of plates. The adhesion and proliferation of human coronary artery endothelial cells (HCAECs) on stainless steel was significantly promoted by the addition of GSKi. In summary, this work provides a universal and stable strategy of immobilizing GSKi on the stent surface. This method has the potential for widespread application in the modification of vascular stents.

The cardioprotective actions of statins in targeting mitochondrial dysfunction associated with myocardial ischaemia-reperfusion injury

During cardiac reperfusion after myocardial infarction, the heart is subjected to cascading cycles of ischaemia reperfusion injury (IRI). Patients presenting with this injury succumb to myocardial dysfunction resulting in myocardial cell death, which contributes to morbidity and mortality. New targeted therapies are required if the myocardium is to be protected from this injury and improve patient outcomes. Extensive research into the role of mitochondria during ischaemia and reperfusion has unveiled one of the most important sites contributing towards this injury; specifically, the opening of the mitochondrial permeability transition pore. The opening of this pore occurs during reperfusion and results in mitochondria swelling and dysfunction, promoting apoptotic cell death. Activation of mitochondrial ATP-sensitive potassium channels (mitoKATP) channels, uncoupling proteins, and inhibition of glycogen synthase kinase-3β (GSK3β) phosphorylation have been identified to delay mitochondrial permeability transition pore opening and reduce reactive oxygen species formation, thereby decreasing infarct size. Statins have recently been identified to provide a direct cardioprotective effect on these specific mitochondrial components, all of which reduce the severity of myocardial IRI, promoting the ability of statins to be a considerate preconditioning agent. This review will outline what has currently been shown in regard to statins cardioprotective effects on mitochondria during myocardial IRI.

Abstract 10237: Activated Protein C Protects Neurons from Alzheimer’s Disease-Related Changes Through Reduction of Tau Hyperphosphorylation

Introduction: Activated protein C (APC) is a blood serine protease that has been shown to have neuroprotective function via its downstream signaling cascade. However, there is no study has investigated how the lack of APC signaling is associated with Alzheimer’s disease (AD)-related neuronal changes. Hypothesis: Diminished APC signaling is related to AD-associated biomarkers and behavioral symptoms. APC ameliorates these symptoms via the activation of the protein kinase B (Akt) signaling pathway which inhibits glycogen synthase kinase 3β (GSK3β) and tau hyperphosphorylation. Methods: Immunofluorescence imaging and proteomic analysis were performed on EPCR R84A/R84A mice, which had blunted APC activity due to mutated endothelial protein C receptor (EPCR), for examining amyloid β (Aβ) deposition. A rodent model of AD, 5xFAD mice, were subjected to either one-time or chronic daily APC injection (100 μg/kg/d i.p.). Morris's water maze test was performed to assess spatial memory and learning. The tissue of the hippocampus and cortex were collected for transcriptomic and proteomic analysis. Results: EPCR R84A/R84A mice showed greater hippocampal and cortical deposition of Aβ comparing to EPCR WT/WT counterparts. This result indicates defective APC signaling might contribute to the development of Aβ accumulation and insoluble plaque formation. A one-time injection of APC significantly activated AKT through phosphorylating the Ser 473 site, as well as inhibited AKT’s downstream target, GSK3β, via phosphorylation at Ser 9 in both wild-type and 5 x FAD mice. Downregulation of GSK3β activity is associated with decreased tau phosphorylation at Ser 202 /Thr 205 but not Ser 396 , indicating a reduction in the hyperphosphorylated form of tau that is prone to neurofibrillary tangle formation. Additionally, chronic APC administration for 3 months was observed to attenuate the cognitive deficits seen in 5 x FAD mice. Conclusions: APC has a crucial neuroprotective role because dampened APC signaling is related to AD-associated neuronal changes. APC has the downstream effect of Akt activation, which in turn inhibits GSK3β and the hyperphosphorylation of tau, ameliorating both the histological and behavioral markers seen in AD.

Enriched environment for offspring improves learning and memory impairments induced by sevoflurane exposure during the second trimester of pregnancy.

Studies in animals indicate that sevoflurane exposure in the second trimester of pregnancy has harmful effects on the learning and memory of offspring. Whether an enriched environment can reverse the damage of sevoflurane exposure in the second trimester of pregnancy on the learning and memory of rat offspring remains unclear. In this study, rats at 14 days of pregnancy were exposed to 3.5% sevoflurane for 2 hours and their offspring were treated with an enriched environment for 20 successive days. We found that the enriched environment for offspring increased nestin and Ki67 levels in hippocampal tissue, increased hippocampal neurogenesis, inhibited glycogen synthase kinase 3β activity, and increased the expression of cell proliferation-related β-catenin and apoptosis-related Bcl-2, indicating that an enriched environment reduces sevoflurane-induced damage by increasing the proliferation of stem cells in the hippocampus. These findings suggest that an enriched environment can reverse the effects of sevoflurane inhaled by rats during the second trimester of pregnancy on learning and memory of offspring. This study was approved by the Animal Ethics Committee of Shengjing Hospital of China Medical University (approval No. 2018PS07K) on January 2, 2018.

Keratin 19 interacts with GSK3β to regulate its nuclear accumulation and degradation of cyclin D3.

Cyclin D3 regulates the G1/S transition and is frequently overexpressed in several cancer types including breast cancer, where it promotes tumor progression. Here we show that a cytoskeletal protein keratin 19 (K19) physically interacts with a serine/threonine kinase GSK3β and prevents GSK3β-dependent degradation of cyclin D3. The absence of K19 allowed active GSK3β to accumulate in the nucleus and degrade cyclin D3. Specifically, the head (H) domain of K19 was required to sustain inhibitory phosphorylation of GSK3β Ser9, prevent nuclear accumulation of GSK3β, and maintain cyclin D3 levels and cell proliferation. K19 was found to interact with GSK3β and K19–GSK3β interaction was mapped out to require Ser10 and Ser35 residues on the H domain of K19. Unlike wildtype K19, S10A and S35A mutants failed to maintain total and nuclear cyclin D3 levels and induce cell proliferation. Finally, we show that the K19–GSK3β-cyclin D3 pathway affected sensitivity of cells toward inhibitors to cyclin-dependent kinase 4 and 6 (CDK4/6). Overall, these findings establish a role for K19 in the regulation of GSK3β-cyclin D3 pathway and demonstrate a potential strategy for overcoming resistance to CDK4/6 inhibitors.

Glycogen synthase kinase-3β inhibitor promotes the migration and osteogenic differentiation of rat dental pulp stem cells via the β-catenin/PI3K/Akt signaling pathway

Background/purposeGlycogen synthase kinase-3β (GSK3β) inhibitor enhances bone formation, while dental pulp stem cells (DPSC) are potentially used to repair bone defects. The present study aimed to investigate the effect of AR-A014418 (AR, a specific glycogen synthase kinase-3β inhibitor) on the migration and osteogenic differentiation of rat-derived dental pulp stem cells (rDPSCs), and further explore the underlying mechanism. Materials and methodsrDPSCs were isolated from rats, and then cultured with different concentrations of AR with or without LY294002 (a PI3K inhibitor). Then, cell viability, migration, osteogenic differentiation, and the involvement of PI3K pathway were detected by CCK-8 assay, Transwell assay, Alizarin Red S Staining, Alkaline phosphatase (ALP) assay, Western blot, and RT-PCR, respectively. ResultsOur present study demonstrated that AR of various concentrations (1 μM, 2.5 μM, and 5 μM) not only promoted the rDPSC proliferation and migration, but also increased calcium deposition, the activity of alkaline phosphatase (ALP), and levels of osteogenic markers (RUNX2, OPN, OCN, and OSX) in rDPSCs. It was also found that the administration of AR resulted in an increase in the expression level of p-GSK3β (Ser), β-catenin, p-PI3K, and p-Akt, and a reduction in p-GSK3β (Tyr216). Furthermore, PI3K inhibitor LY294002 abrogated the enhanced cell migration and osteogenic differentiation of rDPSCs induced by AR. ConclusionOur results provide evidence that AR significantly promotes migration and osteogenic differentiation of rDPSCs by activating β-catenin/PI3K/Akt signaling pathway.

Phillyrin ameliorates diabetic nephropathy through the PI3K/Akt/GSK-3β signalling pathway in streptozotocin-induced diabetic mice.

Diabetic nephropathy is a progressive kidney disease resulting from long-term hyperglycaemia in diabetic patients, and the underlying mechanism is complex and lacks effective treatments. Various active ingredients in Chinese herbs have been shown to alleviate renal injury and improve DN in recent years. Phillyrin, a natural medicinal active compound extracted from the Oleaceae family, has various pharmacological effects, including antioxidative, antiapoptotic and antiobesity effects. However, the role of phillyrin and its underlying mechanism in DN have not yet been explored. To investigate the effects of phillyrin on DN and its potential mechanisms of action, we performed experiments using streptozotocin (STZ)-induced DN mice as models. Phillyrin significantly reduced the levels of fasting blood glucose (FBG) and glycosylated haemoglobin A1c (HbA1c), downregulated the levels of serum blood urea nitrogen (BUN), serum creatinine (Scr), serum and urine β2-microglobulins (β2-MG) and improved the pathological changes of the kidney in a DN mouse model. Phillyrin also increased the level of antioxidants and attenuated oxidative damage in DN model mice. In addition, phillyrin inhibited Glycogen synthase kinase-3β (GSK-3β) activity by activating the PI3K/Akt signalling pathway, increased the Bcl-2/Bax ratio, reduced the release of cytochrome c from the mitochondria to the cytoplasm, subsequently inhibited the activation of caspase-3 and ultimately suppressed renal cell apoptosis. These findings suggested that phillyrin could be a new promising therapeutic strategy for DN, and this protective effect might be related to suppressing oxidative stress and apoptosis via the PI3K/Akt/GSK-3β pathway.

Massive expansion of human induced pluripotent stem cells resulting in efficient biobanking and functional 3D tissue analysis of genetic cardiomyopathies

Abstract Introduction Over the past decade, various protocols were established to ensure efficient differentiation of hiPSC into cardiomyocytes (CMs). A major limitation, however, remained the batch-to-batch variability of hiPSC-CM efficiency and cell number. Here, we suggest an approach in which concomitant GSK-3β inhibition and removal of cell-cell contact inhibition, resulted in a massive proliferative response of hiPSC-CMs1–3. This efficient method allows expansion and passaging of functional hiPSC-CMs, that routinely can be cryopreserved and subsequently used as a stable cell source for the downstream applications, such 3D in vitro models for the disease modelling of dilated cardiomyopathy (DCM). We focussed on the deletion of arginine 14 in the PLN gene (R14del), which is associated with severe heart failure in DCM patients, associated with arrhythmias, cardiac fibrosis and premature death. Methods Subsequent expansion of hiPSC-CM cultures is generally modest (&amp;lt;10 fold). Here, we describe a cost-effective strategy for massive expansion (up to 250-fold) of high-purity hiPSC-CMs relying on two aspects; 1) inhibition of cell-cell contact via low-density seeding and serial passaging in culture flask-format, 2)small molecular glycogen synthase kinase-3β inhibition with CHIR99021 (CHIR). Patient-specific hiPSC-CMs harbouring a PLNR14del mutation were generated and used for EHT formation and functional follow-up. Results We observed that proliferating hiPSC-CMs, especially within the first 2 passages, can routinely be cryopreserved and subsequently further expanded or utilized in downstream applications. Moreover, using this strategy, it is possible to produce ultimately &amp;gt;1 billion CMs within 3–5 weeks starting with one differentiation batch of day 11 hiPSC-CMs, without the need for cell sorting or selection. Expanded hiPSC-CMs retain their capacity to mature and allows fibrin-based engineered heart tissues (EHTs) formation. Previously expanded CMs from PLNR14del patient-specific hiPSC were used to generate EHT and displayed a reduced force phenotype (0.137±0.012 mN) vs healthy control (0.229±0.030 mN) and isogenic control (0.224±0.008 mN) in previously expanded CMs. Conclusion We provpresent a novel strategy for the massive expansion of functional hiPSC-CMs with concomitant GSK-3β inhibition and low cell density culture that ultimately generates up to a 250-fold increase in hiPSC-CM numbers. Expansion healthy control hiPSC-CMs does not limit the subsequent maturation process, and moreover cells remain fully functional such as required for downstream tissue engineering approaches. Therefore, CM expansion forms a well-controlled platform for upscaling hiPSC-CM production for functional 3-dimensionale PLN cardiac disease models, large drug screenings and multiple translational/regenerative applications. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): PLN Foundation

Inhibition of GSK_3β by Iridoid Glycosides of Snowberry (Symphoricarpos albus) Effective in the Treatment of Alzheimer's Disease Using Computational Drug Design Methods.

The inhibition of glycogen synthase kinase-3β (GSK-3β) activity prevents tau hyperphosphorylation and binds it to the microtubule network. Therefore, a GSK-3β inhibitor may be a recommended drug for Alzheimer’s treatment. In silico methods are currently considered as one of the fastest and most cost-effective available alternatives for drug/design discovery in the field of treatment. In this study, computational drug design was conducted to introduce compounds that play an effective role in inhibiting the GSK-3β enzyme by molecular docking and molecular dynamics simulation. The iridoid glycosides of the common snowberry (Symphoricarpos albus), including loganin, secologanin, and loganetin, are compounds that have an effect on improving memory and cognitive impairment and the results of which on Alzheimer’s have been studied as well. In this study, in the molecular docking phase, loganin was considered a more potent inhibitor of this protein by establishing a hydrogen bond with the ATP-binding site of GSK-3β protein and the most negative binding energy to secologanin and loganetin. Moreover, by molecular dynamics simulation of these ligands and GSK-3β protein, all structures were found to be stable during the simulation. In addition, the protein structure represented no change and remained stable by binding ligands to GSK-3β protein. Furthermore, loganin and loganetin have higher binding free energy than secologanin; thus, these compounds could effectively bind to the active site of GSK-3β protein. Hence, loganin and loganetin as iridoid glycosides can be effective in Alzheimer’s prevention and treatment, and thus, further in vitro and in vivo studies can focus on these iridoid glycosides as an alternative treatment.

Inactivation of the Wnt/β-catenin signaling contributes to the epithelial barrier dysfunction induced by sodium oxalate in canine renal epithelial cells.

High oxalate consumption has been recognized as a risk factor for renal calcium oxalate stones in companion animals (dogs and cats). However, the cellular signaling involved in oxalate-induced dysfunction in renal tubular epithelial cells remains not fully elucidated. In this study, Mardin-Darby canine kidney (MDCK) cells, an epithelial cell line derived from canine kidney tubule, were tested for cell proliferation activity and barrier function after being exposed to sodium oxalate (NaOx). Further, the involvement of Wnt/β-catenin in NaOx-induced renal epithelial barrier dysfunction was evaluated. MDCK cells treated with NaOx exhibited reduction in cell proliferation and migration. Besides, NaOx exposure led to a decrease in transepithelial electrical resistance and an increase in paracellular permeability. The deleterious effects of NaOx on epithelial barrier function were related to the suppressed abundance of tight junction proteins including zonula occludens, occludin, and claudin-1. Of note, protein levels of β-catenin and phosphorylated (p)-β-catenin (Ser552) in MDCK cells were repressed by NaOx, indicating inhibitory effects on Wnt/β-catenin signaling. An inhibition of glycogen synthase kinase-3β (GSK-3β) by SB216763 enhanced the abundance of β-catenin and p-β-catenin (Ser552), and protected against epithelial barrier dysfunction in NaOx-treated MDCK cells. The results revealed a critical role of Wnt/β-catenin signaling in the epithelial barrier function of MDCK cells. Activation of Wnt/β-catenin signaling might be a potential therapeutic target for the treatment of oxalate-linked renal stones.