Zinc Deficiency Decreases Neurite Extension via CRMP2 Signal Pathway.

Previous reports indicated that zinc deficiency could increase the risk of infectious diseases and developmental retardation in children. In experimental study, it has been reported that zinc deficiency during the embryonic period inhibited fetal growth, and disturbed neural differentiation and higher brain function later in adulthood. Although it has been suggested that zinc deficiency during development can have significant effects on neuronal differentiation and maturation, the molecular mechanisms of the effects of low zinc on neuronal differentiation during development have not been elucidated in detail. This study was performed to determine the effects of low zinc status on neurite outgrowth and collapsin response mediator protein 2 (CRMP2) signal pathway. Low zinc suppressed neurite outgrowth, and caused increase levels of phosphorylated CRMP2 (pCRMP2) relative to CRMP2, and decrease levels of phosphorylated glycogen synthase kinase 3ß (pGSK3ß) relative to GSK3ß in human neuroblastoma cell line (SH-SY5Y) cells on days 1, 2, and 3 of neuronal differentiation induction. Neurite outgrowth inhibited by low zinc was restored by treatment with the GSK3ß inhibitor CHIR99021. These results suggested that low zinc causes neurite outgrowth inhibition via phosphorylation of CRMP2 by GSK3ß. In conclusion, this study is the first to demonstrate that CRMP signaling is involved in the suppression of neurite outgrowth by low zinc.

Discovery of novel quinolin-2-one derivatives as potential GSK-3ß inhibitors for treatment of Alzheimer's disease: Pharmacophore-based design, preliminary SAR, in vitro and in vivo biological evaluation.

Recently, glycogen synthase kinase-3ß (GSK-3ß) has been considered as a critical factor implicated in Alzheimer's disease (AD). In a previous work, a 3D pharmacophore model for GSK-3ß inhibitors was created and the results suggested that derivative ZINC67773573, VIII, may provide a promising lead for developing novel GSK-3ß inhibitors for the AD's treatment. Consequently, in this work, novel series of quinolin-2-one derivatives were synthesized and assessed for their GSK-3ß inhibitory properties. In vitro screening identified three compounds: 7c, 7e and 7f as promising GSK-3ß inhibitors. Compounds 7c, 7e and 7f were found to exhibit superior inhibitory effect on GSK-3ß with IC50 value ranges between 4.68 ± 0.59 to 8.27 ± 0.60 nM compared to that of staurosporine (IC50 = 6.12 ± 0.74 nM). Considerably, compounds 7c, 7e and 7f effectively lowered tau hyperphosphorylated aggregates and proving their safety towards the SH-SY5Y and THLE2 normal cell lines. The most promising compound 7c alleviated cognitive impairments in the scopolamine-induced model in mice. Compound 7c's activity profile, while not highly selective, may provide a starting point and valuable insights into the design of multi-target inhibitors. According to the ADME prediction results, compounds 7c, 7e and 7f followed Lipinski's rule of five and could almost permeate through the BBB. Molecular docking simulations showed that these compounds are well accommodated in the ATP binding site interacting by its quinoline-2-one ring through hydrogen bonding with the key amino acids Asp133 and Val135 at the hinge region. The findings of this study suggested that these new compounds may have potential as anti-AD drugs targeting GSK-3ß.

[Banxia Xiexin Decoction inhibiting colitis-associated colorectal cancer infected with Fusobacterium nucleatum by regulating Wnt/ß-catenin pathway].

This paper investigates the intervention effect and mechanism of Banxia Xiexin Decoction(BXD) on colitis-associated colorectal cancer(CAC) infected with Fusobacterium nucleatum(Fn). C57BL/6 mice were randomly divided into a control group, Fn group, CAC group [azoxymethane(AOM)/dextran sulfate sodium salt(DSS)](AOM/DSS), model group, and BXD group. Except for the control and AOM/DSS groups, the mice in the other groups were orally administered with Fn suspension twice a week. The AOM/DSS group, model group, and BXD group were also injected with a single dose of 10 mg·kg~(-1) AOM combined with three cycles of 2.5% DSS taken intragastrically. The BXD group received oral administration of BXD starting from the second cycle until the end of the experiment. The general condition and weight changes of the mice were monitored during the experiment, and the disease activity index(DAI) was calculated. At the end of the experiment, the colon length and weight of the mice in each group were compared. Hematoxylin-eosin(HE) staining was used to observe the pathological changes in the colon tissue. Enzyme-linked immunosorbent assay(ELISA) was used to detect the levels of interleukin(IL)-2, IL-4, and IL-6 inflammatory factors in the serum. Immunohistochemistry(IHC) was used to detect the expression of Ki67, E-cadherin, and ß-catenin in the colon tissue. Western blot was used to detect the protein content of Wnt3a, ß-catenin, E-cadherin, annexin A1, cyclin D1, and glycogen synthase kinase-3ß(GSK-3ß) in the colon tissue. The results showed that compared with the control group, the Fn group had no significant lesions. The mice in the AOM/DSS group and model group had decreased body weight, increased DAI scores, significantly increased colon weight, and significantly shortened colon length, with more significant lesions in the model group. At the same time, the colon histology of the model group showed more severe adenomas, inflammatory infiltration, and cellular dysplasia. The levels of IL-4 and IL-6 in the serum were significantly increased, while the IL-2 content was significantly decreased. The IHC results showed low expression of E-cadherin and high expression of Ki67 and ß-catenin in the model group, with a decreased protein content of E-cadherin and GSK-3ß and an increased protein content of Wnt3a, ß-catenin, annexin A1, and cyclin D1. After intervention with BXD, the body weight of the mice increased; the DAI score decreased; the colon length increased, and the tumor decreased. The histopathology showed reduced tumor proliferation and reduced inflammatory infiltration. The levels of IL-6 and IL-4 in the serum were significantly decreased, while the IL-2 content was increased. Meanwhile, the expression of E-cadherin was upregulated, and that of Ki67 and ß-catenin was downregulated. The protein content of E-cadherin and GSK-3ß increased, while that of Wnt3a, ß-catenin, annexin A1, and cyclin D1 decreased. In conclusion, BXD can inhibit CAC infected with Fn, and its potential mechanism may be related to the inhibition of Fn binding to E-cadherin, the decrease in annexin A1 protein level, and the regulation of the Wnt/ß-catenin pathway.

Exploring the effect of 6-BIO and sulindac in modulation of Wnt/ß-catenin signaling pathway in chronic phase of temporal lobe epilepsy.

The prospective involvement of the Wnt/ß-catenin signaling pathway in epilepsy, with the proposed therapeutic uses of its modulators, has been suggested; however, comprehensive knowledge in this regard is currently limited. Despite postulations about the pathway's significance and treatment potential, a systematic investigation is required to better understand its implications in chronic epilepsy. We investigated the role of key proteins like ß-catenin, GSK-3ß, and their modulators sulindac and 6-BIO, in Wnt/ß-catenin pathway during chronic phase of temporal lobe epilepsy. We also evaluated the role of modulators in seizure score, seizure frequency and neurobehavioral parameters in temporal lobe epilepsy. We developed status epilepticus model using lithium-pilocarpine. The assessment of neurobehavioral parameters was done followed by histopathological examination and immunohistochemistry staining of hippocampus as well as RT-qPCR and western blotting to analyse gene and protein expression. In SE rats, seizure score and frequency were significantly high compared to control rats, with notable changes in neurobehavioral parameters and neuronal damage observed in hippocampus. Our study also revealed a substantial upregulation of the Wnt/ß-catenin pathway in chronic epilepsy, as evidenced by gene and protein expression studies. Sulindac emerged as a potent modulator, reducing seizure score, frequency, neuronal damage, apoptosis, and downregulating the Wnt/ß-catenin pathway when compared to 6-BIO. Our findings emphasize the potential of GSK-3ß and ß-catenin as promising drug targets for chronic temporal lobe epilepsy, offering valuable treatment options for chronic epilepsy. The promising outcomes with sulindac encourages further exploration in clinical trials to assess its therapeutic potential.

ZNF554 Inhibits Endometrial Cancer Progression via Regulating RBM5 and Inactivating WNT/ß-Catenin Signaling Pathway.

OBJECTIVE: Uterine corpus endometrial carcinoma (UCEC), a kind of gynecologic malignancy, poses a significant risk to women's health. The precise mechanism underlying the development of UCEC remains elusive. Zinc finger protein 554 (ZNF554), a member of the Krüppel-associated box domain zinc finger protein superfamily, was reported to be dysregulated in various illnesses, including malignant tumors. This study aimed to examine the involvement of ZNF554 in the development of UCEC. METHODS: The expression of ZNF554 in UCEC tissues and cell lines were examined by qRT-PCR and Western blot assay. Cells with stably overexpressed or knocked-down ZNF554 were established through lentivirus infection. CCK-8, wound healing, and Transwell invasion assays were employed to assess cell proliferation, migration, and invasion. Propidium iodide (PI) staining combined with fluorescence-activated cell sorting (FACS) flow cytometer was utilized to detect cell cycle distribution. qRT-PCR and Western blotting were conducted to examine relative mRNA and protein levels. Chromatin immunoprecipitation assay and luciferase reporter assay were used to explore the regulatory role of ZNF554 in RNA binding motif 5 (RBM5). RESULTS: The expression of ZNF554 was found to be reduced in both UCEC samples and cell lines. Decreased expression of ZNF554 was associated with higher tumor stage, decreased overall survival, and reduced disease-free survival in UCEC. ZNF554 overexpression suppressed cell proliferation, migration, and invasion, while also inducing cell cycle arrest. In contrast, a decrease in ZNF554 expression resulted in the opposite effect. Mechanistically, ZNF554 transcriptionally regulated RBM5, leading to the deactivation of the Wingless (WNT)/ß-catenin signaling pathway. Moreover, the findings from rescue studies demonstrated that the inhibition of RBM5 negated the impact of ZNF554 overexpression on ß-catenin and p-glycogen synthase kinase-3ß (p-GSK-3ß). Similarly, the deliberate activation of RBM5 reduced the increase in ß-catenin and p-GSK-3ß caused by the suppression of ZNF554. In vitro experiments showed that ZNF554 overexpression-induced decreases in cell proliferation and migration were counteracted by RBM5 knockdown. Additionally, when RBM5 was overexpressed, it hindered the improvements in cell proliferation and migration caused by reducing the ZNF554 levels. CONCLUSION: ZNF554 functions as a tumor suppressor in UCEC. Furthermore, ZNF554 regulates UCEC progression through the RBM5/WNT/ß-catenin signaling pathway. ZNF554 shows a promise as both a prognostic biomarker and a therapeutic target for UCEC.

RSPO3 induced by Helicobacter pylori extracts promotes gastric cancer stem cell properties through the GNG7/ß-catenin signaling pathway.

BACKGROUND: Helicobacter pylori (H. pylori) accounts for the majority of gastric cancer (GC) cases globally. The present study found that H. pylori promoted GC stem cell (CSC)-like properties, therefore, the regulatory mechanism of how H. pylori promotes GC stemness was explored. METHODS: Spheroid-formation experiments were performed to explore the self-renewal capacity of GC cells. The expression of R-spondin 3 (RSPO3), Nanog homeobox, organic cation/carnitine transporter-4 (OCT-4), SRY-box transcription factor 2 (SOX-2), CD44, Akt, glycogen synthase kinase-3ß (GSK-3ß), p-Akt, p-GSK-3ß, ß-catenin, and G protein subunit gamma 7 (GNG7) were detected by RT-qPCR, western blotting, immunohistochemistry (IHC), and immunofluorescence. Co-immunoprecipitation (CoIP) and liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) were performed to identify proteins interacting with RSPO3. Lentivirus-based RNA interference constructed short hairpin (sh)-RSPO3 GC cells. Small interfering RNA transfection was performed to inhibit GNG7. The in vivo mechanism was verified using a tumor peritoneal seeding model in nude mice. RESULTS: H. pylori extracts promoted a CSC-like phenotype in GC cells and elevated the expression of RSPO3. RSPO3 knockdown significantly reduced the CSC-like properties induced by H. pylori. Previous studies have demonstrated that RSPO3 potentiates the Wnt/ß-catenin signaling pathway, but the inhibitor of Wnt cannot diminish the RSPO3-induced activation of ß-catenin. CoIP and LC-MS/MS revealed that GNG7 is one of the transmembrane proteins interacting with RSPO3, and it was confirmed that RSPO3 directly interacted with GNG7. Recombinant RSPO3 protein increased the phosphorylation level of Akt and GSK-3ß, and the expression of ß-catenin in GC cells, but this regulatory effect of RSPO3 could be blocked by GNG7 knockdown. Of note, GNG7 suppression could diminish the promoting effect of RSPO3 to CSC-like properties. In addition, RSPO3 suppression inhibited MKN45 tumor peritoneal seeding in vivo. IHC staining also showed that RSPO3, CD44, OCT-4, and SOX-2 were elevated in H. pylori GC tissues. CONCLUSION: RSPO3 enhanced the stemness of H. pylori extracts-infected GC cells through the GNG7/ß-catenin signaling pathway.

Molecular mechanism of miR-203a targeting Runx2 to regulate thiram induced-chondrocyte development.

Thiram is a kind of organic compound, which is commonly used for sterilization, insecticidal and deodorization in daily life. Its toxicology has been broadly studied. Recently, more and more microRNAs have been shown to participate in the regulation of cartilage development. However, the potential mechanism by which microRNA regulates chondrocyte growth is still unclear. Our experiments have demonstrated that thiram can hamper chondrocytes development and cause a significant increase in miR-203a content in vitro and in vivo trials. miR-203a mimic significantly decrease in mRNA and protein expression of Wnt4, Runx2, COL2A1, ß-catenin and ALP, and significantly enhance the mRNA and protein levels of GSK-3ß. It has been observed that overexpression of miR-203a hindered chondrocytes development. In addition, Runx2 was confirmed to be a direct target of miR-203a by dual luciferase report gene assay. Transfection of si-Runx2 into chondrocytes reveals that significant downregulation of genes is associated with cartilage development. Overall, these results suggest that overexpression of miR-203a inhibits the expression of Runx2. These findings are conducive to elucidate the mechanism of chondrocytes dysplasia induced by thiram and provide new research ideas for the toxicology of thiram.

Prenylated Flavanone Isolated from Dalea Species as a Potential Multitarget-Neuroprotector in an In Vitro Alzheimer's Disease Mice Model.

Alzheimer's disease (AD) involves a neurodegenerative process that has not yet been prevented, reversed, or stopped. Continuing with the search for natural pharmacological treatments, flavonoids are a family of compounds with proven neuroprotective effects and multi-targeting behavior. The American genus Dalea L. (Fabaceae) is an important source of bioactive flavonoids. In this opportunity, we tested the neuroprotective potential of three prenylated flavanones isolated from Dalea species in a new in vitro pre-clinical AD model previously developed by us. Our approach consisted in exposing neural cells to conditioned media (3xTg-AD ACM) from neurotoxic astrocytes derived from hippocampi and cortices of old 3xTg-AD mice, mimicking a local neurodegenerative microenvironment. Flavanone 1 and 3 showed a neuroprotective effect against 3xTg-AD ACM, being 1 more active than 3. The structural requirements to afford neuroprotective activity in this model are a 5'-dimethylallyl and 4'-hydroxy at the B ring. In order to search the mechanistic performance of the most active flavanone, we focus on the flavonoid-mediated regulation of GSK-3ß-mediated tau phosphorylation previously reported. Flavanone 1 treatment decreased the rise of hyperphosphorylated tau protein neuronal levels induced after 3xTg-AD ACM exposure and inhibited the activity of GSK-3ß. Finally, direct exposure of these neurotoxic 3xTg-AD astrocytes to flavanone 1 resulted in toxicity to these cells and reduced the neurotoxicity of 3xTg-AD ACM as well. Our results allow us to present compound 1 as a natural prenylated flavanone that could be used as a precursor to development and design of future drug therapies for AD.

Association of glycogen synthase kinase-3ß with cognitive impairment in type 2 diabetes patients: a six-year follow-up study.

Background: Our previous multicenter case-control study showed that aging, up-regulation of platelet glycogen synthase kinase-3ß (GSK-3ß), impaired olfactory function, and ApoE ϵ4 genotype were associated with cognitive decline in type 2 diabetes mellitus (T2DM) patients. However, the causal relationship between these biomarkers and the development of cognitive decline in T2DM patients remains unclear. Methods: To further investigate this potential relationship, we designed a 6-year follow-up study in 273 T2DM patients with normal cognitive in our previous study. Baseline characteristics of the study population were compared between T2DM patients with and without incident mild cognitive impairment (MCI). We utilized Cox proportional hazard regression models to assess the risk of cognitive impairment associated with various baseline biomarkers. Receiver operating characteristic curves (ROC) were performed to evaluate the diagnostic accuracy of these biomarkers in predicting cognitive impairment. Results: During a median follow-up time of 6 years (with a range of 4 to 9 years), 40 patients (16.13%) with T2DM developed MCI. Participants who developed incident MCI were more likely to be older, have a lower education level, have more diabetic complications, a higher percentage of ApoE ϵ4 allele and a higher level of platelet GSK-3ß activity (rGSK-3ß) at baseline (P<0.05). In the longitudinal follow-up, individuals with higher levels of rGSK-3ß were more likely to develop incident MCI, with an adjusted hazard ratio (HR) of 1.60 (95% confidence interval [CI] 1.05, 2.46), even after controlling for potential confounders. The AUC of the combination of age, rGSK-3ß and ApoEϵ4 allele predicted for incident MCI was 0.71. Conclusion: Platelet GSK-3ß activity could be a useful biomarker to predict cognitive decline, suggesting the feasibility of identifying vulnerable population and implementing early prevention for dementia.

Diverse effects of a Cyperus rotundus extract on glucose uptake in myotubes and adipocytes and its suppression on adipocyte maturation.

Cyperus rotundus rhizomes have been used in longevity remedies in Thailand for nourishing good health, which led us to investigate the effect on energy homeostasis, especially glucose utilization in myotubes and adipocytes, and on inhibition of lipogenesis in adipocytes. The results showed that an ethyl acetate extract of C. rotundus rhizomes (ECR) containing 1.61%w/w piceatannol, with a half-maximal concentration of 17.76 ± 0.03 µg/mL in 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay, caused upregulation and cell-membrane translocation of glucose transporters GLUT4 and 1 in L6 myotubes but downregulation and cytoplasmic localization of GLUT4 expression in 3T3-L1 adipocytes and was related to the p-Akt/Akt ratio in both cells, especially at 100 µg/mL. Moreover, ECR (25-100 µg/mL) significantly inhibited lipid accumulation via Adenosine Monophosphate-Activated Protein Kinase (AMPK), Acetyl CoA Carboxylase (ACC), and Glycogen Synthase Kinase (GSK) pathways. Its immunoblot showed increased expression of p-AMPKα/AMPKα and p-ACC/ACC but decreased expression of p-Akt/Akt and p-GSK3ß/GSK3ß in 3T3-L1 adipocytes. Moreover, the decreased expression of the adipogenic effectors, perilipin1 and lipoprotein lipase, in ECR-incubated adipocytes (50 and 100 µg/mL) indicated reduced de novo lipogenesis. Our study elucidated mechanisms of C. rotundus that help attenuate glucose tolerance in skeletal muscle and inhibit lipid droplet accumulation in adipose tissue.

The contribution of the WNT pathway to the therapeutic effects of montelukast in experimental murine airway inflammation induced by ovalbumin and lipopolysaccharide.

The wingless/integrase-1 (WNT) pathway involved in the pathogenesis of inflammatory airway diseases has recently generated considerable research interest. Montelukast, a leukotriene receptor antagonist, provides therapeutic benefits in allergic asthma involving eosinophils. We aimed to investigate the role of the WNT pathway in the therapeutic actions of montelukast (MT) in a mixed type of allergic-acute airway inflammation model induced by ovalbumin (OVA) and lipopolysaccharide (LPS) in mice. Female mice were sensitized with intraperitoneal OVA-Al(OH)3 administration in the initiation phase and intranasal OVA followed by LPS administration in the challenge phase. The mice were divided into eight groups: control, asthmatic, and control/asthmatic treated with XAV939 (inhibitor of the canonical WNT pathway), LGK-974 (inhibitor of the secretion of WNT ligands), or MT at different doses. The inhibition of the WNT pathway prevented tracheal 5-HT and bradykinin hyperreactivity, while only the inhibition of the canonical WNT pathway partially reduced 5-HT and bradykinin contractions compared to the inflammation group. Therefore, MT treatment hindered 5-HT and bradykinin hyperreactivity associated with airway inflammation. Furthermore, MT prevented the increases in the phosphorylated GSK-3ß and WNT5A levels, which had been induced by airway inflammation, in a dose-dependent manner. Conversely, the MT application caused a further increase in the fibronectin levels, while there was no significant alteration in the phosphorylation of the Smad-2 levels in the isolated lungs of the mice. The MT treatment reversed the increase in the mRNA expression levels of interleukin-17A. An increase in eosinophil and neutrophil counts was observed in bronchoalveolar lavage fluid samples obtained from the mice in the inflammation group, which was hampered by the MT treatment. The inhibition of the WNT pathway did not alter inflammatory cytokine expression or cell infiltration. The WNT pathway mediated the therapeutic effects of MT due to the inhibition of GSK-3ß phosphorylation as well as the reduction of WNT5A levels in a murine airway inflammation model.

Scaffold Adhering to Peptide-Based Biomimetic Extracellular Matrix Composite Nanobioglass Promotes the Proliferation and Migration of Skin Fibroblasts Through the GSK-3ß/ß-Catenin Signaling Axis.

Introduction: Nano-mesoporous bioactive glass and RGD peptide-coated collagen membranes have great potential in wound healing. However, the application of their compound has not been further studied. Our purpose is to prepare a novel bioactive collagen scaffold containing both NMBG stent and adhesion peptides (BM), which then proves its promising prospect the assessment of physical properties, biocompatibility, GSK-3ß/ß-catenin signaling axis and toxicological effects. Methods: The structural and morphological changes of BM were analyzed using scanning electron microscopy (SEM) and Energy Dispersive Spectroscopy (EDS). In vivo, wound healing of BM was assessed in SD rats through dynamic monitoring and calculation of wound healing rate. Immunohistofluorescence (IHF), H&E, and Masson staining were utilized; in vitro, primary cell culture, and a variety of assays including CCK-8, Transwell, Scratch, Immunocytofluorescence (ICF), and Western blot (WB) were performed, both for morphology and molecular analysis. Results and Discussion: Preparation of BM involved attaching NMBG to RGD-exposed collagen while avoiding the use of toxic chemical reagents. BM exhibited a distinctive superficial morphology with increased Si content, indicating successful NMBG attachment. In vivo studies on SD rats demonstrated the superior wound healing capability of BM, as evidenced by accelerated wound closure, thicker epithelial layers, and enhanced collagen deposition compared to the NC group. Additionally, BM promoted skin fibroblast migration and proliferation, possibly through activation of the GSK-3ß/ß-catenin signaling axis, which was crucial for tissue regeneration. This study underscored the potential of BM as an effective wound-healing dressing. Conclusion: A new method for synthesizing ECM-like membranes has been developed using nano-mesoporous bioactive glass and collagen-derived peptides. This approach enhances the bioactivity of biomaterials through surface functionalization and growth factor-free therapy.

NLRP3 Inflammasome Priming in the Retina of Diabetic Mice Requires REDD1-Dependent Activation of GSK3ß.

Purpose: Inflammasome activation has been implicated in the development of retinal complications caused by diabetes. This study was designed to identify signaling events that promote retinal NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) inflammasome activation in response to diabetes. Methods: Diabetes was induced in mice by streptozotocin administration. Retinas were examined after 16 weeks of diabetes. Human MIO-M1 Müller cells were exposed to hyperglycemic culture conditions. Genetic and pharmacological interventions were used to interrogate signaling pathways. Visual function was assessed in mice using a virtual optomotor system. Results: In the retina of diabetic mice and in Müller cell cultures, NLRP3 and interleukin-1ß (IL-1ß) were increased in response to hyperglycemic conditions and the stress response protein Regulated in Development and DNA damage 1 (REDD1) was required for the effect. REDD1 deletion prevented caspase-1 activation in Müller cells exposed to hyperglycemic conditions and reduced IL-1ß release. REDD1 promoted nuclear factor κB signaling in cells exposed to hyperglycemic conditions, which was necessary for an increase in NLRP3. Expression of a constitutively active GSK3ß variant restored NLRP3 expression in REDD1-deficient cells exposed to hyperglycemic conditions. GSK3 activity was necessary for increased NLRP3 expression in the retina of diabetic mice and in cells exposed to hyperglycemic conditions. Müller glia-specific REDD1 deletion prevented increased retinal NLRP3 levels and deficits in contrast sensitivity in diabetic mice. Conclusions: The data support a role for REDD1-dependent activation of GSK3ß in NLRP3 inflammasome transcriptional priming and in the production of IL-1ß by Müller glia in response to diabetes.

A NAD(P)H oxidase mimic for catalytic tumor therapy via a deacetylase SIRT7-mediated AKT/GSK3ß pathway.

Nicotinamide adenine dinucleotide (NADH) and its phosphorylated form, NADPH, are essential cofactors that play critical roles in cell functions, influencing antioxidation, reductive biosynthesis, and cellular pathways involved in tumor cell apoptosis and tumorigenesis. However, the use of nanomaterials to consume NAD(P)H and thus bring an impact on signaling pathways in cancer treatment remains understudied. In this study, we employed a salt template method to synthesize a carbon-coated-cobalt composite (C@Co) nanozyme, which exhibited excellent NAD(P)H oxidase (NOX)-like activity and mimicked the reaction mechanism of natural NOX. The C@Co nanozyme efficiently consumed NAD(P)H within cancer cells, leading to increased production of reactive oxygen species (ROS) and a reduction in mitochondrial membrane potential. Meanwhile, the generation of the biologically active cofactor NAD(P)+ promoted the expression of the deacetylase SIRT7, which in turn inhibited the serine/threonine kinase AKT signaling pathway, ultimately promoting apoptosis. This work sheds light on the influence of nanozymes with NOX-like activity on cellular signaling pathways in tumor therapy and demonstrates their promising antitumor effects in a tumor xenograft mouse model. These findings contribute to a better understanding of NAD(P)H manipulation in cancer treatment and suggest the potential of nanozymes as a therapeutic strategy for cancer therapy.

Unraveling the mechanism in l-Caldesmon regulating the osteogenic differentiation of PDLSCs: An innovative perspective.

Maxillofacial bone defect is one of the common symptoms in maxillofacial, which affects the function and aesthetics of maxillofacial region. Periodontal ligament stem cells (PDLSCs) are extensively used in bone tissue engineering. The mechanism that regulates the osteogenic differentiation of PDLSCs remains not fully elucidated. Previous studies demonstrated that l-Caldesmon (l-CALD, or CALD1) might be involved in the osteogenic differentiation of PDLSCs. Here, the mechanism by which CALD1 regulates the osteogenic differentiation of PDLSCs is investigated. The osteogenic differentiation of PDLSCs is enhanced with Cald1 knockdown. Whole transcriptome sequencing (RNA-seq) analysis shows that bone morphogenetic proteins (BMP) signaling pathway and Wingless type (Wnt) pathway have significant change with Cald1 knockdown, and the expressions of Wnt-induced secreted protein 1 (WISP1), BMP2, Smad1/5/9, and p-Smad1/5/9 are significantly upregulated, while Glycogen synthase kinase 3ß (GSK3ß) and p-GSK3ß are downregulated. In addition, subcutaneous implantation in nude mice shows that knockdown of Cald1 enhances the osteogenic differentiation of PDLSCs in vivo. Taken together, this study demonstrates that knockdown of Cald1 enhances the osteogenic differentiation of PDLSCs by BMP and Wnt signaling pathways, and provides a novel approach for subsequent clinical treatment.

IL-6 Enhances the Activation of PI3K-AKT/mTOR-GSK-3ß by Upregulating GRPR in Hippocampal Neurons of Autistic Mice.

Autism spectrum disorder (ASD) is a neurological disorder associated with brain inflammation. The underlying mechanisms could be attributed to the activation of PI3K signaling in the inflamed brain of ASD. Multiple studies highlight the role of GRPR in regulating ASD like abnormal behavior and enhancing the PI3K signaling. However, the molecular mechanism by which GRPR regulates PI3K signaling in neurons of individuals with ASD is still unclear. In this study, we utilized a maternal immune activation model to investigate the effects of GRPR on PI3K signaling in the inflamed brain of ASD mice. We used HT22 cells with and without GRPR to examine the impact of GRP-GRPR on the PI3K-AKT pathway with IL-6 treatment. We analyzed a dataset of hippocampus samples from ASD mice to identify hub genes. Our results demonstrated increased expression of IL-6, GRPR, and PI3K-AKT signaling in the hippocampus of ASD mice. Additionally, we observed increased GRPR expression and PI3K-AKT/mTOR activation in HT22 cells after IL-6 treatment, but decreased expression in HT22 cells with GRPR knockdown. NetworkAnalyst identified GSK-3ß as the most crucial gene in the PI3K-AKT/mTOR pathway in the hippocampus of ASD. Furthermore, we found that IL-6 upregulated the expression of GSK-3ß in HT22 cells by upregulating GRP-GRPR. Our findings suggest that IL-6 can enhance the activation of PI3K-AKT/mTOR-GSK-3ß in hippocampal neurons of ASD mice by upregulating GRPR.

Polyphyllin I enhances tumor necrosis factor-related apoptosis-inducing ligand-induced inhibition of human osteosarcoma cell growth downregulating the Wnt/ß-catenin pathway.

OBJECTIVE: To investigate the synergistic effects of polyphyllin I (PPI) combined with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) on the growth of osteosarcoma cells through downregulating the Wnt/ß-catenin signaling pathway. METHODS: Cell viability, apoptosis and cell cycle distribution were examined using cell counting kit-8 and flow cytometry assays. The morphology of cancer cells was observed with inverted phase contrast microscope. The migration and invasion abilities were examined by xCELLigence real time cell analysis DP system and transwell assays. The expressions of poly (adenosine diphosphate-ribose) polymerase, C-Myc, Cyclin B1, cyclin-dependent kinases 1, N-cadherin, Vimentin, Active-ß-catenin, ß-catenin, p-glycogen synthase kinase 3ß (GSK-3ß) and GSK-3ß were determined by Western blotting assay. RESULTS: PPI sensitized TRAIL-induced decrease of viability, migration and invasion, as well as increase of apoptosis and cell cycle arrest of MG-63 and U-2 OS osteosarcoma cells. The synergistic effect of PPI with TRAIL in inhibiting the growth of osteosarcoma cells was at least partially realized through the inactivation of Wnt/ß-catenin signaling pathway. CONCLUSION: The combination of PPI and TRAIL is potentially a novel treatment strategy of osteosarcoma.

PELI1: key players in the oncogenic characteristics of pancreatic Cancer.

BACKGROUND: Pancreatic cancer (PC) is a highly malignant gastrointestinal tumor, which is characterized by difficulties in early diagnosis, early metastasis, limited therapeutic response and a grim prognosis. Therefore, it is imperative to explore potential therapeutic targets for PC. Currently, although the involvement of the Pellino E3 Ubiquitin Protein Ligase 1 (PELI1) in the human growth of some malignant tumors has been demonstrated, its association with PC remains uncertain. METHODS: Bioinformatics, qRT-PCR, Western blot and IHC were used to detect the expression of PELI1 in pancreas or PC tissues and cells at mRNA and protein levels. The effects of PELI1 on the proliferation and metastatic ability of pancreatic cancer in vitro and in vivo were investigated using CCK8, cloning formation, EdU, flow cytometry, IHC, Transwell assay, wound healing, nude mice subcutaneous tumorigenesis and intrasplenic injection to construct a liver metastasis model. The interactions of PELI1 with proteins as well as the main functions and pathways were investigated by protein profiling, Co-IP, GST-pull down, Immunofluorescence techniques, immunohistochemical co-localization and enrichment analysis. The rescue experiment verified the above experimental results. RESULTS: The mRNA and protein expression levels of PELI1 in PC tissues were upregulated and were associated with poor prognosis of patients, in vitro and in vivo experiments confirmed that PELI1 can affect the proliferation and metastatic ability of PC cells. Co-IP, GST-pull down, and other experiments found that PELI1 interacted with Ribosomal Protein S3 (RPS3) through the FHA structural domain and promoted the polyubiquitination of RPS3 in the K48 chain, thereby activates the PI3K/Akt/GSK3ß signaling pathway. Moreover, ubiquitinated degradation of RPS3 further reduces Tumor Protein P53 (p53) protein stability and increases p53 degradation by MDM2 Proto-Oncogene (MDM2). CONCLUSION: PELI1 is overexpressed in PC, which increased ubiquitination of RPS3 proteins and activates the PI3K/Akt/GSK3ß signaling pathway, as well as reduces the protective effect of RPS3 on p53 and promotes the degradation of the p53 protein, which facilitates the progression of PC and leads to a poor prognosis for patients. Therefore, PELI1 is a potential target for the treatment of PC.

Inhibition of MST1 ameliorates neuronal apoptosis via GSK3ß/ß-TrCP/NRF2 pathway in spinal cord injury accompanied by diabetes.

AIMS: Spinal cord injury (SCI) is a devastating neurological disease that often results in tremendous loss of motor function. Increasing evidence demonstrates that diabetes worsens outcomes for patients with SCI due to the higher levels of neuronal oxidative stress. Mammalian sterile 20-like kinase (MST1) is a key mediator of oxidative stress in the central nervous system; however, the mechanism of its action in SCI is still not clear. Here, we investigated the role of MST1 activation in induced neuronal oxidative stress in patients with both SCI and diabetes. METHODS: Diabetes was established in mice by diet induction combined with intraperitoneal injection of streptozotocin (STZ). SCI was performed at T10 level through weight dropping. Advanced glycation end products (AGEs) were applied to mimic diabetic conditions in PC12 cell line in vitro. We employed HE, Nissl staining, footprint assessment and Basso mouse scale to evaluate functional recovery after SCI. Moreover, immunoblotting, qPCR, immunofluorescence and protein-protein docking analysis were used to detect the mechanism. RESULTS: Regarding in vivo experiments, diabetes resulted in up-regulation of MST1, excessive neuronal apoptosis and weakened motor function in SCI mice. Furthermore, diabetes impeded NRF2-mediated antioxidant defense of neurons in the damaged spinal cord. Treatment with AAV-siMST1 could restore antioxidant properties of neurons to facilitate reactive oxygen species (ROS) clearance, which subsequently promoted neuronal survival to improve locomotor function recovery. In vitro model found that AGEs worsened mitochondrial dysfunction and increased cellular oxidative stress. While MST1 inhibition through the chemical inhibitor XMU-MP-1 or MST1-shRNA infection restored NRF2 nuclear accumulation and its transcription of downstream antioxidant enzymes, therefore preventing ROS generation. However, these antioxidant effects were reversed by NRF2 knockdown. Our in-depth studies showed that over-activation of MST1 in diabetes directly hindered the neuroprotective AKT1, and subsequently fostered NRF2 ubiquitination and degradation via the GSK3ß/ß-TrCP pathway. CONCLUSION: MST1 inhibition significantly restores neurological function in SCI mice with preexisting diabetes, which is largely attributed to the activation of antioxidant properties via the GSK3ß(Ser 9)/ß-TrCP/NRF2 pathway. MST1 may be a promising pharmacological target for the effective treatment of spinal cord injury patients with diabetes.

Phosphorylation of Tau Protein by CDK2/cyclin A and GSK3ß Recombinant Kinases: Analysis of Phosphorylation Patterns by Nuclear Magnetic Resonance Spectroscopy.

Posttranslational modifications (PTMs) of proteins can be investigated by Nuclear Magnetic Resonance (NMR) spectroscopy as a powerful analytical tool to define modification sites, their relative stoichiometry, and crosstalk between modifications. As a Structural Biology method, NMR provides important additional information on changes in protein conformation and dynamics upon modification as well as a mapping of binding sites upon biomolecular interactions. Indeed, PTMs not only mediate functional modulation in protein-protein interactions, but can also induce diverse structural responses with different biological outcomes. Here we present protocols that have been developed for the production and phosphorylation of the neuronal tau protein. Under its aggregated form, tau is a hallmark of Alzheimer's disease and other neurodegenerative diseases named tauopathies involving tau dysfunction and/or mutations. As a common feature shared by various tauopathies, tau aggregates are found into a form displaying an increased, abnormal phosphorylation, also referred to hyperphosphorylation. We have used NMR to investigate the phosphorylation patterns of tau induced by several kinases or cell extracts, how phosphorylation affects the local and overall conformation of tau, its interactions with partners (proteins, DNA, small-molecules, etc.) including tubulin and microtubules, and its capacity to form insoluble fibrillar aggregates. We present here detailed protocols for in vitro phosphorylation of tau by the recombinant kinases CDK2/cyclin A and GSK3ß, the production of the recombinant kinases thereof, as well as the analytical characterization of phosphorylated tau by NMR spectroscopy.