Protein Levels Of GSK3β Research Articles

Nifedipine Improves the Ketogenic Diet Effect on Insulin-Resistance-Induced Cognitive Dysfunction in Rats.

Insulin resistance, induced by high fructose consumption, affects cognitive function negatively. Nifedipine may be suggested for neurological disorders. This study aimed to assess the effect of nifedipine with either a normal diet (ND) or a ketogenic diet (KD) in cognitive dysfunction. Male Wistar rats received 10% fructose in drinking water for 8 weeks to induce insulin resistance. Rats received nifedipine (5.2 mg/kg/day; p.o.) later with ND or KD for an additional five weeks. One and two-way ANOVAs were used in analyzing the data. Reversion to the ND improved insulin resistance and lipid profile, besides brain-derived neurotrophic factor (BDNF), glycogen synthase kinase-3 beta (GSK3β), and insulin-degrading enzyme (IDE) levels. Rats fed KD alone and those that received nifedipine with KD did not show similar improvement in the previously mentioned parameters as the ND group. However, nifedipine-ND rats showed improvement in cognitive behavior and insulin resistance. Treatment with nifedipine-KD ameliorated GSK3β, amyloid β (Aβ), and tau protein levels. As the nifedipine-KD combination succeeded in diminishing the accumulated Aβ and tau protein, KD may be used for a while due to its side effects, then nifedipine treatment could be continued with an ND. This conclusion is based on the finding that this combination mitigated insulin resistance with the associated improved behavior.

Multi-modal mechanisms of the metastasis suppressor, NDRG1: Inhibition of WNT/β-catenin signaling by stabilization of protein kinase Cα

The metastasis suppressor, N-myc downstream regulated gene-1 (NDRG1), inhibits pro-oncogenic signaling in pancreatic cancer (PC). This investigation dissected a novel mechanism induced by NDRG1 on WNT/β-catenin signaling in multiple PC cell-types. NDRG1 overexpression decreased β-catenin and down-regulated glycogen synthase kinase-3β (GSK-3β) protein levels and its activation. However, β-catenin phosphorylation at Ser33, Ser37, and Thr41 that are classically induced by GSK-3β were significantly increased after NDRG1 overexpression, suggesting a GSK-3β-independent mechanism. Intriguingly, NDRG1 overexpression up-regulated protein kinase Cα (PKCα), with PKCα silencing preventing β-catenin phosphorylation at Ser33, Ser37, and Thr41, and decreasing β-catenin expression. Further, NDRG1 and PKCα were demonstrated to associate, with PKCα stabilization occurring after NDRG1 overexpression. In fact, PKCα half-life increased from 1.5 ± 0.8 h (3) in control cells to 11.0 ± 2.5 h (3) after NDRG1 overexpression. Thus, NDRG1 overexpression leads to the association of NDRG1 with PKCα and PKCα stabilization, resulting in β-catenin phosphorylation at Ser33, Ser37, and Thr41. In fact, the association between PKCα, NDRG1, and β-catenin was identified, with the formation of a potential metabolon that promotes the latter β-catenin phosphorylation. This anti-oncogenic activity of NDRG1 was multi-modal, with the above mechanism accompanied by the down-regulation of the nucleo-cytoplasmic shuttling protein, p21-activated kinase 4 (PAK4), that is involved in β-catenin nuclear translocation, inhibition of AKT phosphorylation (Ser473), and decreased β-catenin phosphorylation at Ser552 that suppresses its transcriptional activity. These mechanisms of NDRG1 activity are important to dissect to understand the marked anti-cancer efficacy of NDRG1-inducing thiosemicarbazones that up-regulate PKCα and inhibit WNT signaling.

MiR-575/RIPK4 axis modulates cell cycle progression and proliferation by inactivating the Wnt/β-catenin signaling pathway through inhibiting RUNX1 in colon cancer.

Receptor interacting protein serine/threonine kinase 4 (RIPK4) is widely involved in human cancer development. Nevertheless, its role in colon cancer (COAD) has not been elucidated till now. Our research aimed at exploring the function and underlying molecular mechanism of RIPK4 in COAD progression. Through bioinformatic analyses and RT-qPCR, RIPK4 was discovered to be increased in COAD cells and tissues, and its high level predicted poor prognosis. Loss-of-function assays revealed that RIPK4 silencing suppressed COAD cell growth, induced cell cycle arrest, and enhanced cell apoptosis. In vivo experiments also proved that tumor growth was inhibited by silencing of RIPK4. Luciferase reporter assay validated that RIPK4 was targeted and negatively regulated by miR-575. Western blotting demonstrated that Wnt3a, phosphorylated (p)-GSK-3β, and cytoplasmic and nuclear β-catenin protein levels, β-catenin nuclear translocation, and Cyclin D1, CDK4, Cyclin E, and c-Myc protein levels were reduced by RIPK4 knockdown, which however was reversed by treatment with LiCl, the Wnt/β-catenin pathway activator. LiCl also offset the influence of RIPK4 knockdown on COAD cell growth, cell cycle process, and apoptosis. Finally, RIPK4 downregulation reduced RUNX1 level, which was upregulated in COAD and its high level predicted poor prognosis. RIPK4 is positively associated with RUNX1 in COAD. Overexpressing RUNX1 antagonized the suppression of RIPK4 knockdown on RUNX1, Wnt3a, p-GSK-3β, cytoplasmic β-catenin, nuclear β-catenin, Cyclin D1, CDK4, Cyclin E, and c-Myc levels. Collectively, miR-575/RIPK4 axis repressed COAD progression via inactivating the Wnt/β-catenin pathway through downregulating RUNX1.

GSK3β phosphorylates Six1 transcription factor and regulates its APC/CCdh1 mediated proteosomal degradation

Sine oculis homeobox homolog 1 (Six1) is a developmentally important transcription factor that regulates cellular proliferation, apoptosis, and dissemination during embryogenesis. Six1 overexpression as reported in multiple cancers modulates expression of a repertoire of its target genes causing an increase in proliferation, metastasis and survival of cancer cells. Six1 exists as a cell cycle regulated nuclear phosphoprotein and its cellular turnover is regulated by APC/C (Anaphase promoting complex / Cyclosome) complex mediated proteolysis. However, the kinases that regulate Six1 proteolysis have not been identified and the mechanistic details that cause its overproduction in various cancers are lacking. Here, we report that Six1 is a physiological GSK3β substrate. GSK3β interacts with Six1 and phosphorylates it at Ser221 within the conserved consensus sequence in its carboxy terminus. Using pharmacological inhibition, siRNA mediated knockdown and protein overexpression of GSK3β; we show that GSK3β regulates Six1 protein stability. Pulse chase analysis of Six1 revealed that GSK3β regulates its ubiquitin proteolysis such that Six1 phosphomimicking mutant (Six1S221E) for Ser221 site had dramatically increased half-life than its phosphodeficient (Six1S221A) and wild type variants. Furthermore, we demonstrate that GSK3β rescues Six1 from APC dependent proteolysis by regulating its binding with APC/C co-activator protein Cdh1. Importantly, strong positive correlation exists between GSK3β and Six1 protein levels throughout the cell cycle and in multiple cancers indicating that GSK3β activation may in part contribute to Six1 overproduction in a subset of human cancers.

Ginseng promotes the function of intestinal stem cells through the Wnt/β-catenin signaling pathway in D-galactose-induced aging mice

BackgroundIntestinal stem cells (ISCs) are the reservoir source of various types of intestinal cells, and the decline of stem cell function in the gut may be a potential factor for aging-related disease. The present study aimed to explore the regulatory mechanisms of Panax ginseng C.A.Meyer (Araliaceae, Panax genus) that could restore gut aging by enhancing intestinal function and regulating ISCs in aging mice based on the Wnt/β-catenin signaling pathway. MethodsA total of 60 ICR male mice were randomly divided into control, model, metformin, and ginseng water decoction (GWD) 3.6, 1.8, and 0.9 g/kg groups. The aging model was induced by 1 % D-galactose (s.c. 0.1 mL/10 g) for 28 days. Moreover, GWD was given to aging mice intragastrically (i.g.) once a day for 28 successive days. The learning memory ability, pathological status, and function in the ileum tissue, the activity of digestive enzymes, and short-chain fatty acid (SCFA) content in the colon were evaluated, and the related mechanism was investigated. ResultsGinseng can decrease the escape latency time and increase the swimming speed and the number of crossing platforms in aging mice. Moreover, the pathology of ileum tissue improved, the length of the intestinal villi increased, and the width of the villi and the depth of the crypts decreased. The activities of trypsin, α-amylase, and lipase increased in duodenal content and intestinal mucosa. In the colon, the content of SCFA, such as acetic acid, propionic acid and butyric acid, increased, indicating that ginseng significantly improves intestinal function impairment. The mRNA expressions and protein levels of β-catenin, C-myc, GSK-3β, Lgr5, and Olfm4 were upregulated in the ginseng group. ConclusionsGinseng improves intestinal function and regulates the function of ISCs in order to protect intestinal health by activating the Wnt/β-catenin signaling pathway in aging mice.

Curcumin suppresses the Wnt/β-catenin signaling pathway by inhibiting NKD2 methylation to ameliorate intestinal ischemia/reperfusion injury.

Intestinal ischemia/reperfusion (I/R) injury is a life-threatening condition with no effective treatment currently available. Curcumin (CCM), a polyphenol compound in Curcuma Longa, reportedly has positive effects against intestinal I/R injury. However, the mechanism underlying the protective effect of CCM against intestinal I/R injury has not been fully clarified. To determine whether the protective effect of CCM was mediated by epigenetic effects on Wnt/β-catenin signaling, the effect of CCM was examined in vivo and in vitro. An intestinal I/R model was established in Sprague-Dawley (SD) rats with superior mesenteric artery occlusion, and Caco-2 cells were subjected to hypoxia/reoxygenation (H/R) for in vivo simulation of I/R. The results showed that CCM significantly reduced inflammatory, cell apoptosis, and oxidative stress induced by I/R insult in vivo and in vitro. Western blot analysis showed that CCM preconditioning reduced the protein levels of β-catenin, p-GSK3β, and cyclin-D1 and increased the protein level of GSK3β compared with the I/R group. Overexpressing β-catenin aggravated H/R injury, and knocking down β-catenin relieved H/R injury by improving intestinal permeability and reducing the cell apoptosis. Moreover, Naked cuticle homolog 2(NKD2) mRNA and protein levels were upregulated in the CCM-pretreated group. 5-aza-2'-deoxycytidine (5-AZA) treatment improved intestinal epithelial barrier impairment induced by H/R. Besides, the protein levels of total β-catenin, phosphor-β-catenin and cyclin-D1 were reduced after overexpressing NKD2 in Caco-2 cells following H/R insult. In conclusion, Our study suggests that CCM could attenuate intestinal I/R injury in vitro and in vivo by suppressing the Wnt/β-catenin signaling pathway via inhibition of NKD2 methylation.

Escin Activates Canonical Wnt/β-Catenin Signaling Pathway by Facilitating the Proteasomal Degradation of Glycogen Synthase Kinase-3β in Cultured Human Dermal Papilla Cells.

Abnormal inactivation of the Wnt/β-catenin signaling pathway is involved in skin diseases like androgenetic alopecia, vitiligo and canities, but small-molecule activators are rarely described. In this study, we investigated the stimulatory effects of escin on the canonical Wnt/β-catenin signaling pathway in cultured human dermal papilla cells (hDPCs). Escin stimulated Wnt/β-catenin signaling, resulting in increased β-catenin and lymphoid enhancer-binding factor 1 (LEF1), the accumulation of nuclear β-catenin and the enhanced expression of Wnt target genes in cultured hDPCs. Escin drastically reduced the protein level of glycogen synthase kinase (GSK)-3β, a key regulator of the Wnt/β-catenin signaling pathway, while the presence of the proteasome inhibitor MG-132 fully restored the GSK-3β protein level. The treatment of secreted frizzled-related proteins (sFRPs) 1 and 2 attenuated the activity of escin in Wnt reporter assays. Our data demonstrate that escin is a natural agonist of the canonical Wnt/β-catenin signaling pathway and downregulates GSK-3β protein expression by facilitating the proteasomal degradation of GSK-3β in cultured hDPCs. Our data suggest that escin likely stimulates Wnt signaling through direct interactions with frizzled receptors. This study underscores the therapeutic potential of escin for Wnt-related diseases such as androgenetic alopecia, vitiligo and canities.

The potential role of GSK-3β signaling pathway for amelioration actions of ketamine on the PTSD rodent model

RationalePost-traumatic stress disorder (PTSD) is a complex, chronic psychiatric disorder typically triggered by life-threatening events and, as yet, lacks a specialized pharmacological treatment. The potential therapeutic role of ketamine, an N-methyl-D-aspartate receptor antagonist, in mitigating PTSD has been the subject of investigation. ObjectiveThe aim of this study was to elucidate alterations in the glycogen synthase kinase-3β (GSK-3β) signaling pathway in response to ketamine intervention, using the single prolonged stress (SPS) model of PTSD at a molecular level. MethodsPTSD-like symptoms were simulated using the SPS model. Ketamine (10 mg/kg) and GSK-3β antagonist SB216763 (5 mg/kg) were then administered intraperitoneally. Stress-related behavior was evaluated through the open field test (OFT) and the elevated plus maze test (EMPT). Additionally, brain activity was analyzed using quantitative electroencephalography (qEEG). Changes in protein and mRNA expressions of glucocorticoid receptor (GR), brain-derived neurotrophic factor (BDNF), GSK-3β, phosphorylated ser-9 GSK-3β (p-GSK-3β), FK506 binding protein 5 (FKBP5), and corticotropin-releasing hormone (CRH) were assessed in the hypothalamus via western blot and qPCR. ResultsSPS-exposed rats exhibited reduced distance and time spent in the center of the open arms, a pattern divergent from control rats. qEEG readings revealed SPS-induced increases in alpha power, low gamma and high gamma power. Furthermore, SPS triggered an upregulation in the protein and gene expression of GSK-3β, GR, BDNF, p-GSK-3β, and FKBP5, and downregulated CRH expression in the hypothalamus. Ketamine administration following the SPS procedure counteracted these changes by increasing the time spent in the center of the OFT, the distance traversed in the open arms of the EMPT, and mitigating SPS-induced alterations in cerebral cortex oscillations. Moreover, ketamine reduced the protein levels of GSK-3β, GR, p-GSK-3β, and altered the ratio of p-GSK-3β to GSK-3β. Gene expression of GSK-3β, GR, BDNF, and FKBP5 decreased in the SPS-Ket group compared to the SPS-Sal group. ConclusionsKetamine appeared to remediate the abnormal GSK-3β signaling pathway induced by SPS. These findings collectively suggest that ketamine could be a promising therapeutic agent for PTSD symptoms, working through the modulation of the GSK-3β signaling pathway.

GSK3β-driven SOX2 overexpression is a targetable vulnerability in esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is one of the deadliest forms of human malignancy that currently lacks approved targeted therapeutics. Accumulating evidence suggests that SOX2 overexpression is a key driving factor for ESCC and various squamous cell carcinoma. Here, through screening a small-molecule kinase inhibitor library, we identified GSK3β as a kinase that is critically required for robust SOX2 expression in ESCC cells. GSK3β did not promote SOX2 transcriptionally but was required for SOX2 protein stability. We demonstrated that GSK3β interacts with and phosphorylates SOX2 at residue S251, which blocks SOX2 from ubiquitination and proteasome-dependent degradation instigated by ubiquitin E3 ligase CUL4ADET1-COP1. Pharmacological inhibition or knockdown of GSK3β by RNA interference selectively impaired SOX2-positive ESCC cell proliferation, cancer stemness, and tumor growth in mouse xenograft model, suggesting that GSK3β promotes ESCC tumorigenesis primarily by driving SOX2 overexpression. GSK3β was found to be frequently overexpressed in clinical esophageal tumors, and there was a positive correlation between GSK3β and SOX2 protein levels. Notably, we found that SOX2 enhanced GSK3β expression transcriptionally, suggesting the existence of a vicious cycle that drives a coordinated GSK3β and SOX2 overexpression in ESCC cells. Finally, we demonstrated in tumor xenograft model that GSK3β inhibitor AR-A014418 was effective in suppressing SOX2-positive ESCC tumor progression and inhibited tumor progression cooperatively with chemotherapeutic agent carboplatin. In conclusion, we uncovered a novel role for GSK3β in driving SOX2 overexpression and tumorigenesis and provided evidence that targeting GSK3β may hold promise for the treatment of recalcitrant ESCCs.

Therapeutic effects of shikonin on adjuvant-induced arthritis in rats and cellular inflammation, migration and invasion of rheumatoid fibroblast-like synoviocytes via blocking the activation of Wnt/β-catenin pathway

BackgroundShikonin (SKN), the main bioactive component isolated from Lithospermum erythrorhizon Sieb et Zucc, has multiple activities including anti-rheumatic effect, but its specific roles and the precise mechanisms in regulating biological properties of rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) are unclear and need further clarification. PurposeThis study explored the therapeutic roles of SKN on rat adjuvant-induced arthritis (AIA) and cellular inflammation, migration and invasion of TNF-α-induced RA FLS (MH7A cells), and further demonstrated the involved mechanisms. MethodsSKN was intraperitoneally given to AIA rats and its therapeutic role was valued. The effects of SKN in vivo and in vitro on the production of pro-inflammatory factors were examined by ELISA and western blot. Wound-healing, transwell and phalloidin staining assay were carried out to evaluate the effects of SKN on TNF-α-induced migration and invasion in RA FLS. The involvement of Wnt/β-catenin pathway was checked by immunohistochemistry or immunofluorescence assay for β-catenin and western blot for pathway-related proteins. ResultsSKN treatment in AIA rats reduced paw swelling, arthritis index and pathological damage of ankle joints, indicating its anti-arthritic effect in vivo. SKN had anti-inflammatory roles in vivo and in vitro, evidenced by inhibiting the production of pro-inflammatory factors (like IL-1β, IL-6, IL-8, TNF-α, MMP-2 and MMP-9) in sera and synovium of AIA rats, and in TNF-α-induced MH7A cells. Gelatin zymography result revealed the suppression of SKN on TNF-α-induced MMP-2 activity in vitro. Moreover, SKN inhibited TNF-α-induced migration, invasion and cytoskeletal reorganization in MH7A cells. Mechanistically, SKN suppressed the activation of Wnt/β-catenin signaling in AIA rat synovium and in TNF-α-induced MH7A cells, indicated by the reduced protein levels of Wnt1, p-GSK-3β (Ser9) and β-catenin, the raised protein level of GSK-3β and the decreased nuclear translocation of β-catenin. Interestingly, the combination of LiCl (Wnt/β-catenin agonist) canceled the therapeutic functions of SKN on cellular inflammation, migration and invasion in TNF-α-induced MH7A cells, whereas XAV939 (Wnt/β-catenin inhibitor) enhanced the therapeutic roles of SKN. ConclusionSKN showed therapeutic effects on rat AIA and cellular inflammation, migration and invasion of TNF-α-stimulated RA FLS via interrupting Wnt/β-catenin pathway.

Differential bone remodeling mechanism in hindlimb unloaded and hibernating Daurian ground squirrels: a comparison between artificial and natural disuse within the same species.

Loss of bone mass can occur in mammals after prolonged disuse but the situation for hibernators that are in a state of torpor for many months of the year is not yet fully understood. The present study assesses the bone remodeling mechanisms present in Daurian ground squirrels (Spermophilus dauricus) during hibernation as compared with a model of hindlimb disuse. Differences in microstructure, mechanical properties, bone remodeling-related proteins (Runx2, OCN, ALP, RANKL, CTK and MMP-9) and key proteins of Wnt/β-catenin signaling pathway (GSK-3β and phospho-β-catenin) were evaluated in ground squirrels under 3 conditions: summer active (SA) vs. hibernation (HIB) vs. hindlimb unloaded (HLU). The results indicated that the body weight in HLU ground squirrels was lower than the SA group, and the middle tibia diameter in the HLU group was lower than that in SA and HIB groups. The thickness of cortical and trabecular bone in femurs from HLU ground squirrels was lower than in SA and HIB groups. Most parameters of the tibia in the HLU group were lower than those in SA and HIB groups, which indicated cortical bone loss in ground squirrels. Moreover, our data showed that the changes in microscopic parameters in the femur were more obvious than those in the tibia in HLU and HIB ground squirrels. The levels of Runx2 and ALP were lower in HLU ground squirrels than SA and HIB groups. The protein levels of OCN were unchanged in the three groups, but the protein levels of ALP were lower in the HLU group than in SA and HIB groups. RANKL, CTK and MMP-9 protein levels were significantly decreased in tibia of HLU ground squirrels as compared with SA and HIB groups. In addition, the protein expression levels of RANKL, CTK and MMP-9 showed no statistical difference between SA and HIB ground squirrels. Thus, the mechanisms involved in the balance between bone formation and resorption in hibernating and hindlimb unloading ground squirrels may be different. The present study showed that in femur, the Wnt signaling pathway was inhibited, the protein level of GSK-3β was increased, and the protein expression of phospho-β-catenin was decreased in the HIB group as compared with the SA group, which indicates that the Wnt signaling pathway has a great influence on the femur of the HIB group. In conclusion, the natural anti-osteoporosis properties of Daurian ground squirrels are seasonal. The squirrels do not experience bone loss when they are inactive for a long time during hibernation, but the mechanisms of anti-osteoporosis did not work in HLU summer active squirrels.

A new gentiopicroside derivative improves cognitive deficits of AD mice via activation of Wnt signaling pathway and regulation of gut microbiota homeostasis

BackgroundIn our previous study, we found that gentiopicroside (GPS) isolated from Gentiana rigescens Franch has a significant antiaging activity via regulation of mitophagy and oxidative stress. In order to increase the anti-aging activity of GPS, several compounds based on the chemical structure of GPS were synthesized and evaluated for bioactivity with yeast replicative lifespan assay, and 2H-gentiopicroside (2H-GPS) as leading compound was selected for AD treatment. Purpose and methodsTo investigate whether 2H-GPS has anti- Alzheimer's disease effects, we used D-galactose (Dgal)-induced model mice to evaluate the effect of 2H-GPS on AD mice. Furthermore, we explored the action mechanism of this compound with RT-PCR, Western Blot, ELISA and 16S rRNA gene sequence analysis. ResultsMemory dysfunction and reduction in the number of neurons in the brain of mice were observed in Dgal treated group. These symptoms of AD mice were significantly relieved by administering 2H-GPS and donepezil (Done), respectively. In the Dgal only treated group, the protein levels of β-catenin, REST and phosphorylated GSK-3β, involved in the Wnt signaling pathway were significantly decreased, whereas the protein levels of GSK-3β, Tau, phosphorylated Tau, P35 and PEN-2 were significantly increased. Importantly, treatment with 2H-GPS resulted in restoration of memory dysfunction and levels of these proteins. Furthermore, the composition of the gut microbiota after 2H-GPS administration was explored through 16S rRNA gene sequence analysis. Moreover, the mice, in which depleted gut microbiota with antibiotic cocktail (ABX), were used for evaluation of whether the gut microbiota is involved to the effect of 2H-GPS. Significant changes in gut microbiota composition were observed between AD and 2H-GPS-treated AD mice, and ABX partially eliminated the AD-restoring effect of 2H-GPS. Conclusion2H-GPS improves the symptoms of AD mice through combination of the regulation of Wnt signaling pathway and the microbiota–gut–brain axis, and the mechanism of action of 2H-GPS is distinct from that of Done.

Quince extract resists atherosclerosis in rats by down-regulating the EGFR/PI3K/Akt/GSK-3β pathway

We identified the effective components and the underlying mechanisms of Quince (Cydonia oblonga Mill, COM) extract against atherosclerosis. The effective components of COM extract were identified with UHPLC-Q-TOF-MS/MS. Network pharmacology was performed. A rat model of atherosclerosis induced by high-fat emulsion combined with vitamin D3 was established. The anti-atherosclerosis effect of COM extract was evaluated from various aspects such as blood lipid regulation, anti-oxidative stress, anti-inflammatory response, and vascular protection function. We identified 14 serum components of COM extract using UHPLC-Q-TOF-MS/MS. Through prediction, 573 targets were obtained, among which 224 targets were atherosclerosis specific targets. The key targets included GSK3β, ESR1, EGFR, and HSP90AA1. The key signaling pathway was PI3K-Akt signaling pathway. Pharmacodynamics analysis showed that COM extract reduced the levels of TC, TG, and LDL-C as well as ALT and AST, while increased the level of HDL-C. Mechanistically, COM extract significantly increased serum SOD and GSH-Px activities, but decreased MDA content in atherosclerosis rats, showing antioxidant effects. Meanwhile, COM extract significantly down-regulated the levels of pro-inflammatory factors IL-1β, IL-6, TNF-α and CRP, but up-regulated anti-inflammatory factor IL-10. Additionally, COM extract increased the levels of NO, eNOS, and 6-keto-PGF1α; whereas, decreased the levels of ET-1 and TXB2. Furthermore, COM extract significantly inhibited the mRNA and protein levels of EGFR, p-PI3K, p-AKT, GSK-3β, Bax, and Caspase-3 as well as the Bax/Bcl-2 ratio. Conclusively, COM extract exerts hypolipidemic, anti-oxidative, anti-inflammatory, anti-thrombotic and vascular endothelium protective effects on atherosclerosis rat model, which may be related to the inhibition of EGFR/PI3K/AKT/GSK-3β signaling pathway.

Epigallocatechin-3-gallate alleviates gestational stress-induced postpartum anxiety and depression-like behaviors in mice by downregulating semaphorin3A and promoting GSK3β phosphorylation in the hippocampus.

Postpartum depression (PPD) is a common neuropsychiatric disorder characterized by depression and comorbid anxiety during the postpartum period. PPD is difficult to treat because of its elusive mechanisms. Epigallocatechin-3-gallate (EGCG), a component of tea polyphenols, is reported to exert neuroprotective effects in emotional disorders by reducing inflammation and apoptosis. However, the effect of EGCG on PPD and the underlying mechanism are unknown. We used a mouse model of PPD established by exposing pregnant mice to gestational stress. Open field, forced swimming and tail suspension tests were performed to investigate the anxiety and depression-like behaviors. Immunohistochemical staining was used to measure the c-fos positive cells. The transcriptional levels of hippocampal semaphorin3A(sema3A), (glycogen synthase kinase 3-beta)GSK3β and collapsin response mediator protein 2(CRMP2) were assessed by RT-PCR. Alterations in protein expression of Sema3A, GSK3β, p-GSK3β, CRMP2 and p-CRMP2 were quantified by western blotting. EGCG was administrated to analyze its effect on PPD mice. Gestational stress induced anxiety and depression-like behaviors during the postpartum period, increasing Sema3A expression while decreasing that of phosphorylated GSK3β as well as c-Fos in the hippocampus. These effects were reversed by systemic administration of EGCG. Thus, EGCG may alleviate anxiety and depression-like behaviors in mice by downregulating Sema3A and increasing GSK3β phosphorylation in the hippocampus, and has potential application in the treatment of PPD.

Hastatoside attenuatescarbon tetrachloride-induced liver fibrosis by targeting glycogen synthase kinase-3β

BackgroundHastatoside is an iridoid glycoside extracted from the herb, Verbena officinalis, that exerts various pharmacological effects, including anti-inflammatory, sleep-promoting, and analgesic effects. However, only a few studies have reported the efficacy of hastatoside in liver fibrosis. Liver fibrosis is a pathophysiological process, and its persistence can seriously affect the quality of life and well-being of the patients. Hypothesis/purposeThis study aimed to investigate the role of hastatoside on liver fibrosis and its possible underlying mechanisms. MethodsC57BL/6 J mice with carbon tetrachloride (CCl4)-induced hepatic fibrosis were used as the in vivo models. Histological features of the liver were observed using Masson's trichrome and hematoxylin–eosin staining. Alanine aminotransferase and aspartate aminotransferase levels and the hepatic fibrosis indices (type 3 procollagen, laminin, and hyaluronic acid) were measured using corresponding assay kits. LX-2 human hepatic stellate cells (HSCs) stimulated with the transforming growth factor β1 were used as the vitro models. Transfection of the glycogen synthase kinase (GSK)-3β small interfering RNA (siRNA) and β-catenin plasmids was also performed in vitro. Protein levels of GSK-3β, phospho-GSK-3β (Ser 9), α-smooth muscle actin, collagen type I alpha 1, c-Myc, cyclin D1, and β-catenin were determined via western blotting. Moreover, the p-GSK-3β:GSK-3β ratio was calculated to determine the GSK-3β activity. ResultsHastatoside prevented CCl4-induced liver injury and histological damage. It inhibited the upregulation of α-SMA and Col1α1 levels in a CCl4-induced mouse hepatic fibrosis model. In vitro, hastatoside inhibited the proliferation and activation of HSCs by decreasing the expression levels of cyclin D1 and c-Myc and the proportion of LX-2 cells activated in the G0/G1 phase. Molecular docking results showed that hastatoside bound to GSK-3β. Hastatoside significantly increased the GSK-3β activity and inhibited the downstream effector expression of β-catenin. ConclusionThese findings suggest that hastatoside can bind to GSK-3β and promote its activity, while inhibiting the GSK-3β downstream effector expression of β-catenin, thereby inhibiting the activation and proliferation of HSCs, which further prevents the development of liver fibrosis. These results provide innovative insights into the underlying liver fibrosis. Moreover, hastatoside is a potential anti-fibrosis monomer that can potentially be used for the treatment of liver fibrosis.

Feiyanning formula modulates the molecular mechanism of osimertinib resistance in lung cancer by regulating the Wnt/β-catenin pathway.

Feiyanning Formula (FYN), a Chinese herbal formula derived from summarized clinical experience, is proven to have anti-tumor effects in lung cancer patients. Osimertinib, a third-generation epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI), can improve progression-free survival and overall survival of patients but drug resistance is inevitable. The current study evaluated the effects of FYN in osimertinib-resistant HCC827OR and PC9OR cells. FYN preferentially inhibited the proliferation and migration of HCC827OR and PC9OR cells. Moreover, FYN and osimertinib exhibited synergistic inhibitory effects on proliferation and migration. Real-time qPCR (RT-qPCR) and western blotting results indicated that FYN downregulated gene and protein levels of GSK3β and SRFS1, which are enriched in the Wnt/β-catenin pathway. Besides, FYN inhibited tumor growth and exhibited synergistic effects with osimertinib in vivo. Collectively, the results suggested that FYN exerted an anti-osimertinib resistance effect via the Wnt/β-catenin pathway.

Imidazole-pyridine hybrids as potent anti-cancer agents

In the current investigation, fifteen novel imidazole-pyridine-based molecules were synthesized and tested against cell lines of the lung (H1299) and colon (HCT116) adenocarcinomas by proliferation assay. The results demonstrated that compounds 5a, 5d, 5e, and 5f were the most active (IC50<30 µM). Based on recent literature and the current results, the glycogen synthase kinase-3β (GSK-3β) protein was investigated in-silico as a possible target. The molecular docking and QSAR revealed an excellent binding affinity of the selected imidazole-pyridine compounds to GSK-3β. Notably, GSK-3β protein levels were significantly upregulated in hepatocellular liver carcinoma (LIHCs) tissues and negatively affected patient prognosis. Consequently, the compounds were evaluated on liver cancer cell lines (HepG2, HUH-7, and PLC/PRF/5) by the MTT assay, and 5d showed the highest antitumor activity. This study offers new compounds with interesting biological activity on GSK-3β as a target, exhibiting a potential therapeutic impact for hepatocellular carcinoma patients.

The Drosha-Independent MicroRNA6778-5p/GSK3β Axis Mediates the Proliferation of Gastric Cancer Cells.

Background Gastric cancer (GC) is a primary cause of cancer death around the world. Previous studies have found that Drosha plays a significant role in the development of tumor cells. Soon after, we unexpectedly found that the expression of microRNA6778-5p (miR6778-5p) is unconventionally high in the gastric cancer cells low-expressing Drosha. So, we designed the Drosha interference sequence and recombined it into a lentiviral vector to construct Drosha knockdown lentivirus and transfected the Drosha knockdown lentivirus into gastric cancer cells to establish Drosha knockdown gastric cancer cell lines. We aimed to explore the effect of microRNA6778-5p on the proliferation of gastric cancer cells with Drosha knockdown and its intrinsic mechanism. Methods We designed the Drosha interference sequence and recombined it into a lentiviral vector to construct Drosha knockdown lentivirus and transfected the Drosha knockdown lentivirus into gastric cancer cells to establish Drosha knockdown gastric cancer cell lines. After transfecting miR6778-5p mimics and inhibitor into gastric cancer cell lines with Drosha knockdown, the expression levels of miR6778-5p mimics in Drosha low-expressing gastric cancer cells increased, while miR6778-5p inhibitor decreased the expression levels of miR6778-5p. The Cell Counting Kit-8 (CCK-8) experiment was used to detect the proliferation ability of gastric cancer cells after overexpression or knockdown of miR6778-5p and bioinformatics predicted the relationship between miR6778-5p and glycogen synthase kinase-3β (GSK3β). Results After infection with the Drosha knockdown lentivirus, Drosha's mRNA and protein levels were significantly downregulated in gastric cancer cells. The expression levels of miR6778-5p mimics in Drosha low-expressing gastric cancer cells increased, while miR6778-5p inhibitor decreased the expression levels of miR6778-5p. Overexpression of miR6778-5p significantly enhanced the proliferation ability of Drosha low-expression gastric cancer cells; on the contrary, knocking down miR6778-5p weakened the proliferation ability of Drosha low-expression gastric cancer cells. Bioinformatics predicted that miR6778-5p targeted glycogen synthase kinase-3β (GSK3β) and the mRNA and protein levels of GSK3β decreased significantly after overexpression of miR6778-5p. Conclusion miR6778-5p promotes the proliferation of Drosha low-expressing gastric cancer cells by targeting GSK3β.

Abstract P1062: Regional Differences In Sarcomeric Glycogen Synthase Kinase 3β Maintain Normal Chamber Level Function

The structure of the left ventricle is spatially and temporally heterogeneous. The heart’s electrical system helps synchronize these events, but mechanical events play an important role as well. However, experimental evidence of the regional mechanical processes that underpin a “normal contraction” remain largely unknown. Despite the differences in deformation between the anterior and posterior walls, sarcomere lengths during filling and ejection are the same in these regions. One explanation for this disconnect is the myocyte’s force production is different despite similar sarcomere lengths, a relationship known as Length Dependent Activation (LDA). Indeed, a modeling study suggested LDA is a critical regulator of mechanical synchrony in the left ventricle. We recently found the serine/threonine kinase GSK-3β localizes to the z-disc and can modulate LDA, and thus hypothesized it may play a role in maintaining normal cardiac synchrony. We used inducible cardiomyocyte specific GSK-3β KO mice and GSK-3βfl/fl/Cre- littermate Con treated with tamoxifen in which GSK-3β was KO for three weeks. We assessed regional differences in GSK-3β protein levels and found that Con mice had substantial regional heterogeneity: 50% more cytosolic GSK-3β in the posterior wall compared to the anterior wall; however, the myofilament-bound GSK-3β was the opposite, with 50% less GSK-3β in the posterior wall. This regional heterogeneity was lost in KO mice, in which protein levels were homogenously low. There were no differences detected by m-mode echo between Control and KO mice. However, strain analysis revealed a significant interaction (p=0.037) between genotype and region (Anterior vs. Posterior), in which regional differences in Systolic Strain Rate were ablated in the knockout. These data suggest that the regional differences in GSK-3β sarcomeric levels were important for maintaining regional functional heterogeneity. We further observed that sarcomeric GSK-3β levels (but not cytosolic) decreased in a mouse myocardial infarction model, which these findings suggest could worsen mechanical dyssynchrony post-infarct. Perhaps a possible therapy for mechanical dyssynchrony could involve regional restoration of sarcomeric GSK-3β levels to restore synchrony.

Quercetin Inhibits KBM7R Cell Proliferation through Wnt/β-Catenin Signaling.

Background Tyrosine kinase inhibitors could treat chronic myelogenous leukemia (CML) effectively, but they have no effect on patients with T315I mutation. It is necessary to find drugs to overcome the resistance. Quercetin (Qu) is a kind of bioflavonoid with an antitumor effect. In this study, we observed the effect of Qu on proliferation and Wnt/β-catenin pathway in KBM7R cells, an imatinib-resistant cell with T315I mutation. Methods The IC50 of Qu was detected by trypan blue staining. The KBM7R cell apoptosis and cycle were detected through the method of flow cytometry (FCM). The expression of the related mRNA and protein was evaluated by means of an RT-PCR assay and western blot in KBM7 (sensitive to IM) and KBM7R cells. Results These results showed that in the KBM7R cell, the proliferation inhibition effect was increased after 48 h administration with different Qu concentrations. The IC50 to Qu was 241.7 μmol/L. The different doses of Qu (50, 100, and 200 μmol/L) would raise apoptosis and depress the cell cycle at the G1 phase. Dealing with a median Qu concentration (100 μmol/L) for 48 h, the mRNA and the protein level of caspase-3, caspase-8, and caspase-9 along with p21 and p27 raised compared with the control. The median concentration of Qu could inhibit both the mRNA and protein levels of GSK-3β, β-catenin, and Lef-1 in the Wnt/β-catenin signal pathway and also the downstream targets PPAR-δ and cyclin D1 in both KBM7 and KBM7R cells. Conclusions Our findings suggest that Qu could inhibit proliferation, induce apoptosis, and arrest the cell cycle on IM-resistant KBM7R cells with T315I mutation. And this effect could be related with the inhibition of the Wnt/β-catenin signal pathway and downstream targets.