Intermittent fasting along with hydroalcoholic extract of Centella-asiatica ameliorates sub-acute hypoxia-induced ischemic stroke in adult zebrafish.

Reduced blood flow (hypoxia) to the brain is thought to be the main cause of strokes because it deprives the brain of oxygen and nutrients. An increasing amount of evidence indicates that the Centella-Asiatica (HA-CA) hydroalcoholic extract has a variety of pharmacological benefits, such as antioxidant activity, neuroprotection, anti-inflammatory qualities, and angiogenesis promotion. Intermittent fasting (IF) has neurological benefits such as anti-inflammatory properties, neuroprotective effects, and the ability to enhance neuroplasticity. The current study evaluates the combined effect of IF (for 1, 6, and 12 days) along with HA-CA (daily up to 12 days) in adult zebrafish subjected to hypoxia every 5 min for 12 days followed by behavioral (novel tank and open-field tank test), biochemical (SOD, GSH-Px, and LPO), inflammatory (IL-10, IL-1ß, and TNF-α), mitochondrial enzyme activities (Complex-I, II, and IV), signaling molecules (AMPK, MAPK, GSK-3ß, Nrf2), and imaging/staining (H&E, TTC, and TEM) analysis. Results show that sub-acute hypoxia promotes the behavioral alterations, and production of radical species and alters the oxidative stress status in brain tissues of zebrafish, along with mitochondrial dysfunction, neuroinflammation, and alteration of signaling molecules. Nevertheless, HA-CA along with IF significantly ameliorates these defects in adult zebrafish as compared to their effects alone. Further, imaging analysis significantly provided evidence of infarct damage along with neuronal and mitochondrial damage which was significantly ameliorated by IF and HA-CA. The use of IF and HA-CA has been proven to enhance the physiological effects of hypoxia in all dimensions.

Jaceosidin induces apoptosis and inhibits migration in AGS gastric cancer cells by regulating ROS-mediated signaling pathways.

Jaceosidin (JAC) is a natural flavonoid with anti-oxidant and other pharmacological activities; however, its anti-cancer mechanism remains unclear. We investigated the mechanism of action of JAC in gastric cancer cells. Cytotoxicity and apoptosis assays showed that JAC effectively killed multiple gastric cancer cells and induced apoptosis in human gastric adenocarcinoma AGS cells via the mitochondrial pathway. Network pharmacological analysis suggested that its activity was linked to reactive oxygen species (ROS), AKT, and MAPK signaling pathways. Furthermore, JAC accumulated ROS to up-regulate p-JNK, p-p38, and IκB-α protein expressions and down-regulate the p-ERK, p-STAT3, and NF-κB protein expressions. Cell cycle assay results showed that JAC accumulated ROS to up-regulate p21 and p27 protein expressions and down-regulate p-AKT, CDK2, CDK4, CDK6, Cyclin D1, and Cyclin E protein expressions to induce G0/G1 phase arrest. Cell migration assay results showed JAC accumulated ROS to down-regulate Wnt-3a, p-GSK-3ß, N-cadherin, and ß-catenin protein expressions and up-regulate E-cadherin protein expression to inhibit migration. Furthermore, N-acetyl cysteine pre-treatment prevented the change of these protein expressions. In summary, JAC induced apoptosis and G0/G1 phase arrest and inhibited migration through ROS-mediated signaling pathways in AGS cells.

TiO2 nanotube topography enhances osteogenesis through filamentous actin and XB130-protein-mediated mechanotransduction.

TiO2 nanotube topography, as nanomechanical stimulation, can significantly promote osteogenesis and improve the osteointegration on the interface of implants and bone tissue. However, the underlying mechanism has not been fully elucidated. XB130 is a member of the actin filament-associated protein family and is involved in the regulation of cytoskeleton and tyrosine kinase-mediated signalling as an adaptor protein. Whether XB130 is involved in TiO2 nanotubes-induced osteogenic differentiation and how it functions in mechano-biochemical signalling transduction remain to be elucidated. In this study, the role of XB130 on TiO2 nanotube-induced osteogenesis and mechanotransduction was systematically investigated. TiO2 nanotube topography was fabricated via anodic oxidation and characterized. The osteogenic effect was significantly accelerated by the TiO2 nanotube surface in vitro and vivo. XB130 was significantly upregulated during this process. Moreover, XB130 overexpression significantly promoted osteogenic differentiation, whereas its knockdown inhibited it. Filamentous actin depolymerization could change the expression and distribution of XB130, thus affecting osteogenic differentiation. Mechanistically, XB130 could interact with Src and result in the activation of the downstream PI3K/Akt/GSK-3ß/ß-catenin pathway, which accounts for the regulation of osteogenesis. This study for the first time showed that the enhanced osteogenic effect of TiO2 nanotubes could be partly due to the filamentous actin and XB130 mediated mechano-biochemical signalling transduction, which might provide a reference for guiding the design and modification of prostheses to promote bone regeneration and osseointegration. STATEMENT OF SIGNIFICANCE: TiO2 nanotubes topography can regulate cytoskeletal rearrangement and thus promote osteogenic differentiation of BMSCs. However, how filamentous actin converts mechanical stimulus into biochemical activity remains unclear. XB130 is a member of actin filament-associated protein family and involves in the regulation of tyrosine kinase-mediated signalling. Therefore, we hypothesised that XB130 might bridge the mechano-biochemical signalling transduction during TiO2 nanotubes-induced osteogenic differentiation. For the first time, this study shows that TiO2 nanotubes enhance osteogenesis through filamentous actin and XB130 mediated mechanotransduction, which provides new theoretical basis for guiding the design and modification of prostheses to promote bone regeneration and osseointegration.

CHIR-98014, a GSK 3ß Inhibitor, Protects Against Triptolide/Lipopolysaccharide-Induced Hepatotoxicity by Mitochondria-Dependent Apoptosis Inhibition.

Triptolide (TP) is a remarkable anti-inflammatory and immunosuppressive component separated from Tripterygium wilfordii Hook. F. However, its hepatotoxicity limits its application in the clinical. Our group has proposed a new perspective on TP-induced hepatotoxicity, in which TP enhances liver hypersensitivity upon lipopolysaccharide (LPS) stimulation. Because the cause of the disease is unknown, there is currently no uniform treatment available. In this study, we attempted to determine whether the GSK-3ß-JNK pathway affects liver damage and its regulatory mechanism in response to TP/LPS costimulation. In addition, we investigated the effect of CsA or the GSK 3ß inhibitor CHIR-98014 on TP/LPS-induced hepatotoxicity. The results showed that the TP/LPS cotreatment mice exhibited obvious hepatotoxicity, as indicated by a remarkable increase in the serum ALT and AST levels, glycogen depletion, GSK 3ß-JNK upregulation, and increased apoptosis. Instead of the specific knockdown of JNK1, the specific knockdown of JNK2 had a protective effect. Additionally, 40 mg/kg of CsA and 30 mg/kg of CHIR-98014 might provide protection. In summary, CHIR-98014 could protect against TP/LPS- or TP/TNF-α-induced activation of the GSK 3ß-JNK pathway and mitochondria-dependent apoptosis, improving the indirect hepatotoxicity induced by TP.

The effect and mechanism of selenium supplementation on the proliferation capacity of bovine endometrial epithelial cells exposed to lipopolysaccharide in vitro under high cortisol background.

Bovine endometritis severely inhibits uterine repair and causes considerable economic loss. Besides, parturition-induced high cortisol levels inhibit immune function, reduce cell proliferation, and further inhibit tissue repair. Selenium (Se) is an essential trace element for animals to maintain normal physiological function and has powerful antioxidant functions. This study investigated whether Se supplementation reduces endometrial damage and promotes tissue repair in cows with endometritis under stress and explored the underlying mechanism. Primary bovine endometrial epithelial cells were isolated and purified from healthy cows. The cells were treated with different combinations of lipopolysaccharide (LPS), cortisol, and various concentrations of Se. Data showed that LPS stimulation inhibited cell proliferation and increased cell apoptosis. High levels of cortisol further exacerbated these effects. Flow cytometry, scratch wound healing tests, and 5-ethynyl-2'-deoxyuridine (EdU) proliferation assays showed that Se supplementation promoted cell cycle progression, cell migration, and cell proliferation in the presence of LPS and cortisol. The quantitative PCR results showed that the expression of related growth factors was increased after Se supplementation. After administering various inhibitors, we further demonstrated that Se supplementation decreased the activity of glycogen synthetase kinase 3ß (GSK-3ß) through the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway to reduce the degradation of ß-catenin except the Wnt signal to promote cell proliferation. In conclusion, Se supplementation attenuated the cell damage induced by LPS at high cortisol levels and increased cell proliferation to promote uterine repair by elevating the mRNA expression of TGFB3 and VEGFA and activating the PI3K/AKT/GSK-3ß/ß-catenin signaling pathway.

After parturition, endometritis is a common bovine disease, which hinders endometrial repair and reduces bovine economic value. Besides, parturition-induced high cortisol levels cause immunosuppression, aggravate infection, and further inhibit cell proliferation and tissue repair. As an essential trace element, adding selenium to feed helps to maintain the normal physiological function of animals. This study developed a cellular model using lipopolysaccharide (LPS) and cortisol to simulate cows with endometritis in stress conditions. The results showed that Se supplementation attenuated bovine endometrial epithelial cell damage and promoted their proliferation in the presence of LPS and high cortisol levels, which are positively correlated with the concentration of Se. Besides, this study proved another molecular mechanism for Se to regulate ß-catenin except for the Wnt signal by affecting the ß-catenin degradation pathway.

Lithium Chloride Promotes Endogenous Synthesis of CLA in Bovine Mammary Epithelial Cells.

Although conjugated linoleic acid (CLA) can promote human health, its content in milk is insufficient to have a significant impact. The majority of the CLA in milk is produced endogenously by the mammary gland. However, research on improving its content through nutrient-induced endogenous synthesis is relatively scarce. Previous research found that the key enzyme, stearoyl-CoA desaturase (SCD) for the synthesis of CLA, can be expressed more actively in bovine mammary epithelial cells (MAC-T) when lithium chloride (LiCl) is present. This study investigated whether LiCl can encourage CLA synthesis in MAC-T cells. The results showed that LiCl effectively increased SCD and proteasome α5 subunit (PSMA5) protein expression in MAC-T cells as well as the content of CLA and its endogenous synthesis index. LiCl enhanced the expression of proliferator-activated receptor-γ (PPARγ), sterol regulatory element-binding protein 1 (SREBP1), and its downstream enzymes acetyl CoA carboxylase (ACC), fatty acid synthase (FASN), lipoprotein lipase (LPL), and Perilipin 2 (PLIN2). The addition of LiCl significantly enhanced p-GSK-3ß, ß-catenin, p-ß-catenin protein expression, hypoxia-inducible factor-1α (HIF-1α), and downregulation factor genes for mRNA expression (P < 0.05). These findings highlight that LiCl can increase the expression of SCD and PSMA5 by activating the transcription of HIF-1α, Wnt/ß-catenin, and the SREBP1 signaling pathways to promote the conversion of trans-vaccenic acid (TVA) to the endogenous synthesis of CLA. This data suggests that the exogenous addition of nutrients can increase CLA content in milk through pertinent signaling pathways.

Gallic acid induces osteoblast differentiation and alleviates inflammatory response through GPR35/GSK3ß/ß-catenin signaling pathway in human periodontal ligament cells.

BACKGROUND AND OBJECTIVE: Gallic acid (GA) possesses various beneficial functions including antioxidant, anticancer, anti-inflammatory as well as inhibiting osteoclastogeneis. However, effects on osteogenic differentiation, especially in human ligament periodontal (hPDL) cells, remain unclear. Thus, the aim of this study was to evaluate the function of GA on osteogenesis and anti-inflammation in hPDL cells and to explore the involved underlying mechanism. METHODS: Porphyromonas gingivalis lipopolysaccharide (Pg-LPS) treatment was used as a model for periodontitis. ROS production was determined by H2DCFDA staining. Trans-well and wound healing assays were performed for checking the migration effect of GA. Alizarin red and alkaline phosphatase activity (ALP) assays were performed to evaluate osteogenic differentiation. Osteogenesis and inflammatory-related genes and proteins were measured by real-time PCR and western blot. RESULTS: Our results showed that GA-treated hPDL cells had higher proliferation and migration effect. GA inhibited ROS production-induced by Pg-LPS. Besides, GA abolished Pg-LPS-induced inflammation cytokines (il-6, il-1ß) and inflammasome targets (Caspase-1, NLRP3). In addition, GA promoted ALP activity and mineralization in hPDL cells, lead to enhance osteoblast differentiation process. The effect of GA is related to G-protein-coupled receptor 35 (GPR35)/GSK3ß/ß-catenin signaling pathway. CONCLUSION: GA attenuated Pg-LPS-induced inflammatory responses and periodontitis in hPDL cells. Taken together, GA may be targeted for therapeutic interventions in periodontal diseases.

Crocin alleviates neurotoxicity induced by bupivacaine in SH-SY5Y cells with inhibition of PI3K/AKT signaling.

BACKGROUND: Bupivacaine, a common local anesthetic, can cause neurotoxicity and permanent neurological disorders. Crocin has been widely reported as a potential neuroprotective agent in neural injury models. OBJECTIVE: The aim of this study was to investigate the role and regulatory mechanism of crocin underlying bupivacaine-induced neurotoxicity. METHOD: Human neuroblastoma SH-SY5Y cells were treated with bupivacaine and/or crocin for 24 h, followed by detecting cell viability using 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay. The effect of crocin or bupivacaine on SH-SY5Y cell proliferation was measured by Ki67 immunofluorescence assay. The levels of apoptosis-related proteins and the markers in the PI3K/Akt signaling pathway were examined using western blot analysis. The activities of caspase 3, catalase (CAT), superoxide dismutase (SOD), malondialdehyde (MDA) and glutathione peroxidase (GSH-Px) were tested using respective commercial assay kits. Flow cytometry analysis was executed for detecting SH-SY5Y cell apoptosis. RESULT: Crocin attenuated bupivacaine-induced neurotoxicity in SH-SY5Y cells. Meanwhile, crocin inhibited SH-SY5Y cell apoptosis induced by bupivacaine via repressing the activity of caspase-3, reducing Bax expression, and elevating Bcl-2 expression. Moreover, crocin mitigated oxidative stress in SH-SY5Y cells by increasing the content of CAT, SOD, GSH-Px and reducing the content of MDA. Additionally, crocin protected against bupivacaine-induced dephosphorylation of Akt and GSK-3ß. The protective effects of crocin against bupivacaine-induced neurotoxicity in SH-SY5Y cells were counteracted by the Akt inhibitor. CONCLUSION: These results suggested that crocin may exert a neuroprotective function by promoting cell proliferation and suppressing apoptosis and oxidative stress in SH-SY5Y cells. Thus, crocin might become a promising drug for the treatment of bupivacaine-induced neurotoxicity.

Artificial Protein Crosstalk with a Molecule that Exchanges Binding Partners.

Drawing inspiration from allosteric signaling enzymes, whose catalytic and regulatory units are non-covalently linked, we have devised a method to establish unnatural, effector-mediated enzyme activation within native cells. The feasibility of this approach is demonstrated by introducing a synthetic regulatory unit (sRU) onto glycogen synthase kinase 3 (GSK-3) through non-covalent means. Our study reveals that this synthetic regulator mediates an unnatural crosstalk between GSK-3 and lactate dehydrogenase A (LDHA), whose expression is regulated by cellular oxygen levels. Specifically, with this approach, the constitutively active GSK-3 is transformed into an activable enzyme, whereas LDHA is repurposed as an unnatural effector protein that controls the activity of the kinase, making it unnaturally dependent on the cell's hypoxic response. These findings demonstrate a step toward imitating the function of effector-regulated cell-signaling enzymes, which play a key biological role in mediating the response of cells to changes in their environment. In addition, at the proof-of-principle level, our results indicate the potential to develop a new class of protein inhibitors whose inhibitory effect in cells is dictated by the cell's environment and consequent protein expression profile.

RCOR1 improves neurobehaviors and neuron injury in rat cerebral palsy by Endothelin-1 targeting-induced Akt/GSK-3ß pathway upregulation.

BACKGROUND: RE1 Silencing Transcription factor (REST) corepressor 1 (RCOR1) has been reported to orchestrate neurogenesis, while its role in cerebral palsy (CP) remains elusive. Besides, RCOR1 can interact with Endothelin-1 (EDN1), and EDN1 expression is related to brain damage. Therefore, this study aimed to explore the effects of RCOR1/EDN1 on brain damage during the progression of CP. METHODS: CP rats were established via hypoxia-ischemia insult, and injected with lentivirus-RCOR1, followed by examination of brain pathological conditions. The RCOR1 and EDN1 interaction was recognized using hTFtarget. Healthy rat cortical neuron cells received interference of RCOR1/EDN1 expression, and underwent oxygen-glucose deprivation/reoxygenation (OGD/R) treatment, after which phenotypic and molecular assays were conducted through the biochemical method, qRT-PCR and/or western blot. RESULTS: RCOR1 was low-expressed but EDN1 was high-expressed in CP model rats and OGD/R-treated neurons. RCOR1 overexpression ameliorated rat neurobehaviors, alleviated brain pathological conditions, reduced TUNEL-positive cells, decreased the levels of reactive oxygen species (ROS) and malondialdehyde (MDA), increased superoxide dismutase (SOD) level and repressed EDN1 expression in the brains of CP model rats. In neurons, RCOR1 overexpression counteracted OGD/R-induced viability decrease, reduction of the levels of RCOR1, SOD, Bcl-2, caspase-3, p-Akt/Akt and p-GSK-3ß/GSK-3ß, and elevation of the levels of EDN1, ROS, Bax, and cleaved caspase-3, while EDN1 overexpression did contrarily on these events. Moreover, there was a negative interplay between RCOR1 overexpression and EDN1 overexpression in OGD/R-induced neurons. CONCLUSION: RCOR1 ameliorates neurobehaviors and suppresses neuronal apoptosis and oxidative stress in CP through EDN1 targeting-mediated upregulation of Akt/GSK-3ß.

Nanoparticle and Nanotopography-Induced Activation of the Wnt Pathway in Bone Regeneration.

Background and Aims: Recent research has focused on developing nanoparticle and nanotopography-based technologies for bone regeneration. The Wingless-related integration site (Wnt) signaling pathway has been shown to play a vital role in this process, in particular in osteogenic differentiation and proliferation. The exact mechanisms by which nanoparticles and nanotopographies activate the Wnt signaling pathway, however, are not fully understood. This review aimed to elucidate the mechanisms by which nanoscale technologies activate the Wnt signaling pathway during bone regeneration. Methods: The terms "Wnt," "bone," and "nano*" were searched on PubMed and Ovid with no date limit. Only original research articles related to Wnt signaling and bone regeneration in the context of nanotopographies, nanoparticles, or scaffolds with nanotopographies/nanoparticles were reviewed. Results: The primary mechanism by which nanoparticles activated the Wnt pathway was by internalization through the endocytic pathway or diffusion through the cell membrane, leading to accumulation of nonphosphorylated ß-catenin in the cytoplasm and subsequently downstream osteogenic signaling (e.g., upregulation of runt-related transcription factor 2 [RUNX2]). The specific size of the nanoparticles and the process of endocytosis itself has been shown to modulate the Wnt-ß-catenin pathway. Nanotopographies were shown to directly activate frizzled receptors, initiating Wnt/ß-catenin signaling. Additional studies showed nanotopographies to activate the Wnt/calcium (Wnt/Ca2+)-dependent and Wnt/planar cell polarity pathways through nuclear factor of activated T cells, and α5ß1 integrin stimulation. Finally, scaffolds containing nanotopographies/nanoparticles were found to induce Wnt signaling through a combination of ion release (e.g., lithium, boron, lanthanum, and icariin), which inhibited glycogen synthase kinase 3 beta (GSK-3ß) activity, and through similar mechanisms to the nanotopographies. Conclusion: This review concludes that nanoparticles and nanotopographies cause Wnt activation through several different mechanisms, specific to the size, shape, and structure of the nanoparticles or nanotopographies. Endocytosis-related mechanisms, integrin signaling and ion release were the major mechanisms identified across nanoparticles, nanotopographies, and scaffolds, respectively. Knowledge of these mechanisms will help develop more effective targeted nanoscale technologies for bone regeneration. Impact statement Nanoparticles and nanotopographies can activate the Wingless-related integration site (Wnt) signaling pathway, which is essential for bone regeneration. This review has identified that activation is due to endocytosis, integrin signaling and ion release, depending on the size, shape, and structure of the nanoparticles or nanotopographies. By identifying and further understanding these mechanisms, more effective nanoscale technologies that target the Wnt signaling pathway can be developed. These technologies can be used for the treatment of nonunion bone fractures, a major clinical challenge, with the potential to improve the quality of life of millions of patients around the world.

Wireless Photoactivated Targeted Nanosystem for Oncotherapy Via Synergistic Effects of Hyperthermia/Redox Stress Amplification/GSK-3ß Activity Inhibition.

Highly soluble salts and gas mediated therapies are emerging antitumor strategies. However, the therapeutic efficacy remains restricted by difficulty in delivering them to the tumor site and poorly controlled release in deep tissues. Here, an intelligent wireless photoactivated targeted nanosystem is designed for delivering LiCl and H2 to tumors for therapy. LiCl causes cell death by inhibiting the activity of GSK-3ß. H2 selectively interacts with reactive oxygen species in the tumor, leading to redox stress, which induces apoptosis. The significant heat generated by the nanosystem not only kills tumor cells but also accelerates the dissolution of LiCl and the release of H2. The rapid dissolution of LiCl leads to a surge in intracellular osmotic pressure, which further intensifies the redox stress response and enhances the efficiency of therapy. The nanosystem shows efficient tumor therapeutic capability via synergistic effects of hyperthermia/redox stress amplification/GSK-3ß activity inhibition.

Total Astragalus saponins can reverse type 2 diabetes mellitus-related intestinal dysbiosis and hepatic insulin resistance in vivo.

Objective: Intestinal flora homeostasis in rats with type 2 diabetes mellitus (T2DM) was evaluated to explore the effects of total Astragalus saponins (TAS) on hepatic insulin resistance (IR). Methods: Six-week-old male Sprague-Dawley rats were fed high-fat and high-sugar diet for 4 weeks and intraperitoneally injected with streptozotocin to induce T2DM, and they were then randomly divided into control, model, metformin, and TAS groups. Stool, serum, colon, and liver samples were collected after 8 weeks of drug administration for relevant analyses. Results: TAS reduced fasting blood glucose, 2-hour postprandial blood glucose, area under the curve of oral glucose tolerance test, glycated serum protein, homeostasis model assessment of insulin resistance, total cholesterol, triglyceride, and low-density lipoprotein cholesterol levels in T2DM rats but increased insulin, C-peptide, and high-density lipoprotein cholesterol levels. Moreover, TAS improved the morphology and structure of liver and colon tissues and improved the composition of the intestinal microbiome and bacterial community structure at different taxonomic levels. In addition, TAS increased the protein expression of hepatic IRS-1, PI3K, PDK1, and p-AKT and decreased the protein expression of p-GSK-3ß. Meanwhile, TAS increased the mRNA expression of liver PDK1, PI3K, and GS and decreased the mRNA expression of GSK-3ß. Conclusion: TAS can ameliorate T2DM-related abnormal glucose and blood lipid metabolism, intestinal dysbiosis, and IR.

Wilforine inhibits rheumatoid arthritis pathology through the Wnt11/ß-catenin signaling pathway axis.

BACKGROUND: Wilforine (WFR) is a monomeric compound of the anti-RA plant Tripterygium wilfordii Hook. f. (TwHF). Whether WFR has anti-RA effect, its molecular mechanism has not been elucidated. AIM OF THE STUDY: Our study aims to clarify how WFR inhibits fibroblast-like synovial cells (FLS) activation and improves RA through Wnt11 action on the Wnt11/ß-catenin signaling pathway. METHODS: The therapeutic effect of WFR on collagen-induced arthritis (CIA) rats was evaluated using methods such as rat arthritis score. The inhibitory effects and signaling pathways of WFR on the proliferation and inflammatory response of CIA FLS and RA FLS were studied using ELISA, CCK-8, RT-qPCR, Western blot, and immunofluorescence methods. RESULTS: WFR could effectively alleviate the arthritis symptoms of CIA rats; reduce the levels of IL-6, IL-1ß, and TNF-α in the peripheral blood of CIA rats; and inhibit the expression of MMP3 and fibronectin. The data showed that WFR has a significant inhibitory effect on FLS proliferation. Furthermore, WFR inhibited the activation of Wnt/ß-catenin signaling pathway and decreased the expression of Wnt11, ß-catenin, CCND1, GSK-3ß, and c-Myc, while the effects of WFR were reversed after overexpression of Wnt11. CONCLUSIONS: WFR improves RA by inhibiting the Wnt11/ß-catenin signaling pathway, and Wnt11 is the direct target of WFR. This study provides a new molecular mechanism for WFR to improve RA and contributes to the clinical promotion of WFR.

Wheat germ peptide improves glucose metabolism and insulin resistance in HepG2 hepatocytes via regulating SOCS3/IRS1/Akt pathway.

Evidence has demonstrated that oxidative stress plays a crucial role in regulating cellular glucose metabolism. In previous studies, wheat germ peptide (WGP) was found to effectively mitigate oxidative stress induced by high glucose. Based on the information provided, we hypothesized that WGP could exhibit antihyperglycemic and anti-insulin-resistant effects in cells. The insulin-resistant cell model was established by insulin stimulation. The glucose consumption, glycogen content, and the activities of hexokinase and pyruvate kinase following WGP treatment were measured. The protein expression of SOCS3, phosphorylated insulin receptor substrate-1 (p-IRS1), IRS1, phosphorylated protein kinase B (p-Akt), Akt, glucose transporter 2 (GLUT2), phosphorylated GSK 3ß, GSK 3ß, FOXO1, G6P, and phosphoenolpyruvate carboxykinase were assessed by western blot analysis. Our results demonstrated that WGP treatment increased cellular glucose consumption and glycogen synthesis and enhanced hexokinase and pyruvate kinase activities. Additionally, WGP treatment was observed to cause a significant reduction in the expression of SOCS3, FOXO1, G6P, and phosphoenolpyruvate carboxykinase, as well as in the ratio of p-IRS1/IRS1. Conversely, the expression of GLUT2 and the ratios of p-Akt/Akt and p-GSK3ß/GSK3ß were upregulated by WGP. These findings suggested that WGP can activate the SOCS3/IRS1/Akt signaling pathway, thus promoting the phosphorylation of GSK-3ß and increasing the expression of FOXO1 and GLUT2, which contribute to enhancing glycogen synthesis, inhibiting gluconeogenesis, and promoting glucose transport in insulin-resistant HepG2 cells.

Different ratios of Schisandra chinensis and Evodia rutaecarpa combination for treating Alzheimer's disease.

Schisandra chinensis and Evodia rutaecarpa are traditional Chinese herbs that have been used for many years to treat neurodegenerative diseases. In Chinese medicine, multiple herbs are often used in combination to enhance their efficacy, and different combination ratios can produce different therapeutic effects, thus flexibly responding to the needs of various patients. This study aimed to investigate the effects of different ratios of Schisandra and Evodia herbs on learning and memory impairment in rats with Alzheimer's disease (AD) and their specific mechanisms of action. Morris water maze and hematoxylin and eosin (HE) staining experiments were performed to evaluate the effects of different ratios of Schisandra-Evodia on learning memory in AD model rats. Immunohistochemical experiments were performed to investigate the effects of Schisandra-Evodia on the Aß1-42 and P-Tau proteins, and protein immunoblotting (WB) was performed to determine the expression of key proteins in two pathways, BDNF/TrkB/CREB and GSK-3ß/Tau. Our experimental results show that all Schisandra-Evodia groups showed significant neuroprotective effects, improved learning memory impairment, and reduced levels of Aß1-42 and P-Tau proteins in AD model rats. Schisandra-Evodia upregulated BDNF, P-TrkB/TrkB, and P-CREB/CREB protein expression and downregulated GSK-3ß and P-Tau/Tau protein expression. Among the different Schisandra-Evodia ratio groups, the 2:1 group showed the strongest therapeutic effect on AD. Our research results indicate that Schisandra-Evodia can reduce Aß1-42 and P-Tau protein content by modulating the activity of two pathways, BDNF/TrkB/CREB and GSK-3ß/Tau, thus improving neuronal cell damage and cognitive deficits caused by AD. In addition, we found that a Schisandra-Evodia ratio of 2:1 had the most profound therapeutic effect on AD.

Hydroalcoholic root extracts of Houttuynia cordata (Thunb.) standardized by UPLC-Q-TOF-MS/MS promotes apoptosis in human hepatocarcinoma cell HepG2 via GSK-3ß/ß-catenin/PDL-1 axis.

Houttuynia cordata (Thunb.), an important medicinal plant of Northeast India, Korea, and China, is used to treat various ailments and for anticancer research. Knowing its traditional practices, we are interested in the mode-of-action of HCT on HepG2 to co-relate the traditional practice with modern drug therapeutics. UPLC-Q-ToF-Ms analysis of HCT reveals identification of 14 metabolites. Network pharmacology analysis of the 14 compounds showed interaction with 232 different targets with their potential involvement in hepatocellular carcinoma. Whole extracts impart cytotoxicity on variety of cell lines including HepG2. There was a significant morphological alteration in treated HepG2 cells due to impairment of cytoskeletal components like ß and γ- tubulin. Arrest at G1-S checkpoint was clearly indicated downregulation of Cyclin D1. The root extracts actuated apoptosis in HepG2 as evident from altered mitochondrial membrane potential, Annexin V- FITC, BrdU-PI, AO/EtBr assays, and modulations of apoptotic protein expression but without ROS generation. Whole extracts caused abrogation of epithelial to mesenchymal transition with repression of Snail, N-Cadherin, Vimentin, MMP-9, and upregulation of Pan-Cadherin. Pathway analysis found GSK-3ß in Wnt/ß-Catenin signaling cascade to be involved through Hepatocellular carcinoma (hsa05225) pathway. The GSK-3ß/ß-Catenin/PDL-1 signaling was found to be inhibited with the downregulation of pathway components. This was further confirmed by application of EGF, an inducer of the GSK-3ß/ß-Catenin pathway that neutralized the effect of Houttuynia cordata (Thunb.) root extract on the said pathway. Network pharmacology analysis also confirms the synergy network with botanical-bioactive-target-disease which showed Kaempferol to have the highest degree of association with the said pathway.

Sevoflurane inhibits cholangiocarcinoma via Wnt/ß-catenin signaling pathway.

BACKGROUND: Cholangiocarcinoma (CCA) is a refractory malignancy derived from bile duct epithelial cells. This study aimed to explore the role and molecular mechanisms of action of sevoflurane in CCA. METHODS: CCK-8 assay was used to assess the proliferation of cholangiocarcinoma cells, and flow cytometry was used to detect cholangiocarcinoma cell apoptosis. The effects of sevoflurane on TFK1 and QBC939 cell migration and invasion were investigated using a Transwell assay. Western blotting and RT-qPCR were used to assess the expression of apoptosis-related proteins and genes, and gene expression of the Wnt/ß-catenin signaling pathway. RESULTS: Our study found that sevoflurane inhibited cholangiocarcinoma cell proliferation in a dose-dependent manner. In addition, sevoflurane induced cholangiocarcinoma cell apoptosis, inhibited cholangiocarcinoma cell migration and invasion, as well as the Wnt/ß-catenin signaling pathway evidenced by decreased Wnt3a, ß-catenin, c-Myc, and Cyclin D1 protein and mRNA expression, reduced p-GSK3ß protein expression and p-GSK3ß/GSK3ß ratio. Further mechanistic studies revealed that Wnt/ß-catenin pathway inducer SKL2001 reversed the inhibitory effect of sevoflurane on cholangiocarcinoma cells. CONCLUSIONS: Sevoflurane induces apoptosis and inhibits the growth, migration, and invasion of cholangiocarcinoma cells by inhibiting the Wnt/ß-catenin signaling pathway. This study not only revealed the role of sevoflurane in the development of CCA but also elucidated new therapeutic agents for CCA.

Protective effect of manganese treatment on insulin resistance in HepG2 hepatocytes.

Introduction: Objectives: manganese (Mn) is closely related to type 2 diabetes mellitus and insulin resistance (IR), but the exact mechanism is unclear. This study aimed to explore the regulatory effects and mechanism of Mn on IR using hepatocyte IR model induced by high palmitate (PA), high glucose (HG) or insulin. Methods: HepG2 cells were exposed to PA (200 µM), HG (25 mM) or insulin (100 nM) respectively, alone or with 5 µM Mn for 24 hours. The expression of key proteins in insulin signaling pathway, intracellular glycogen content and glucose accumulation, reactive oxygen species (ROS) level and Mn superoxide dismutase (MnSOD) activity were detected. Results: compared with control group, the expression of phosphorylated protein kinase B (Akt), glycogen synthase kinase-3ß (GSK-3ß) and forkhead box O1 (FOXO1) in the three IR groups was declined, and this decrease was reversed by Mn. The reduction of intracellular glycogen content and increase in glucose accumulation in IR groups were also inhibited by Mn. Additionally, the production of ROS was increased in IR models, compared with normal control group, while Mn reduced the excessive production of ROS induced by PA, HG or insulin. However, Mn did not alter the activity of MnSOD in the three IR models. Conclusion: this study demonstrated that Mn treatment can improve IR in hepatocytes. The mechanism is probably by reducing the level of intracellular oxidative stress, enhancing the activity of Akt/GSK-3ß/FOXO1 signal pathway, promoting glycogen synthesis, and inhibiting gluconeogenesis.

Introducción: Objetivos: el manganeso (Mn) está estrechamente relacionado con la diabetes mellitus tipo 2 y la resistencia a la insulina (RI), pero el mecanismo exacto aún no está claro. Este estudio tuvo como objetivo explorar los efectos reguladores y el mecanismo del Mn sobre la RI utilizando un modelo de RI en hepatocitos inducido por palmitato alto (PA), glucosa alta (HG) o insulina. Métodos: las células HepG2 se expusieron a PA (200 µM), HG (25 mM) o insulina (100 nM), solas o junto con 5 µM de Mn durante 24 horas. Se evaluó la expresión de proteínas clave en la vía de señalización de la insulina, el contenido intracelular de glucógeno y la acumulación de glucosa, el nivel de especies reactivas de oxígeno (ROS) y la actividad superóxido dismutasa del manganeso (MnSOD). Resultados: en comparación con el grupo de control, la expresión de proteína quinasa B fosforilada (Akt), la glucógeno sintasa quinasa-3ß (GSK-3ß) y la proteína forkhead box O1 (FOXO1) en los tres grupos de RI se redujo, y esta disminución fue revertida por el Mn. La reducción del contenido de glucógeno intracelular y el aumento de la acumulación de glucosa en los grupos de RI también fueron inhibidos por el Mn. Además, la producción de ROS aumentó en los modelos de RI en comparación con el grupo de control normal. Mientras que el Mn redujo la producción excesiva de ROS inducida por PA, HG o insulina. Sin embargo, el Mn no alteró la actividad de la MnSOD en los tres modelos de RI. Conclusión: este estudio demostró que el tratamiento con Mn puede mejorar la RI en hepatocitos. El mecanismo probablemente sea mediante la reducción del nivel de estrés oxidativo intracelular, mejorando la actividad de la vía de señalización Akt/GSK-3ß/FOXO1, promoviendo la síntesis de glucógeno e inhibiendo la gluconeogénesis.

NRF1 knockdown alleviates lipopolysaccharide-induced pulmonary inflammatory injury by upregulating DKK3 and inhibiting the GSK-3ß/ß-catenin pathway.

Excessive inflammatory injury is the main cause of the incidence of severe neonatal pneumonia (NP) and associated deaths. Although dickkopf-3 (DKK3) exhibits anti-inflammatory activity in numerous pathological processes, its role in NP is still unknown. In this study, human embryonic lung WI-38 and MRC-5 cells were treated with lipopolysaccharide (LPS) to induce inflammatory injury of NP in vitro. The expression of DKK3 was downregulated in LPS-stimulated WI-38 and MRC-5 cells. DKK3 overexpression decreased LPS-induced inhibition of cell viability, and reduced LPS-induced apoptosis of WI-38 and MRC-5 cells. DKK3 overexpression also reduced LPS-induced production of pro-inflammatory factors such as ROS, IL-6, MCP-1, and TNF-α. Nuclear respiratory factors 1 (NRF1) knockdown was found to upregulate DKK3 and inactivate the GSK-3ß/ß-catenin pathway in LPS-injured WI-38 and MRC-5 cells. NRF1 knockdown also suppressed LPS-induced inhibition on cell viability, repressed LPS-induced apoptosis, and inhibited the accumulation of ROS, IL-6, MCP-1, and TNF-α in LPS-injured WI-38 and MRC-5 cells. DKK3 knockdown or re-activation of the GSK-3ß/ß-catenin pathway reversed the inhibitory effects of NRF1 knockdown on LPS-induced inflammatory injury. In conclusion, NRF1 knockdown can alleviate LPS-triggered inflammatory injury by regulating DKK3 and the GSK-3ß/ß-catenin pathway.