New mechanistic understanding of osteoclast differentiation and bone resorption mediated by P2X7 receptors and PI3K-Akt-GSK3ß signaling.

OBJECTIVE: Osteoporosis is a global health issue characterized by decreased bone mass and microstructural degradation, leading to an increased risk of fractures. This study aims to explore the molecular mechanism by which P2X7 receptors influence osteoclast formation and bone resorption through the PI3K-Akt-GSK3ß signaling pathway. METHODS: An osteoporosis mouse model was generated through ovariectomy (OVX) in normal C57BL/6 and P2X7f/f; LysM-cre mice. Osteoclasts were isolated for transcriptomic analysis, and differentially expressed genes were selected for functional enrichment analysis. Metabolite analysis was performed using liquid chromatography-tandem mass spectrometry (LC-MS/MS), and multivariate statistical analysis and pattern recognition were used to identify differential lipid metabolism markers and their distribution. Bioinformatics analyses were conducted using the Encyclopedia of Genes and Genomes database and the MetaboAnalyst database to assess potential biomarkers and create a metabolic pathway map. Osteoclast precursor cells were used for in vitro cell experiments, evaluating cell viability and proliferation using the Cell Counting Kit 8 (CCK-8) assay. Osteoclast precursor cells were induced to differentiate into osteoclasts using macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappa-beta ligand (RANKL), and tartrate-resistant acid phosphatase (TRAP) staining was performed to compare differentiation morphology, size, and quantity between different groups. Western blot analysis was used to assess the expression of differentiation markers, fusion gene markers, and bone resorption ability markers in osteoclasts. Immunofluorescence staining was employed to examine the spatial distribution and quantity of osteoclast cell skeletons, P2X7 protein, and cell nuclei, while pit assay was used to evaluate osteoclast bone resorption ability. Finally, in vivo animal experiments, including micro computed tomography (micro-CT), hematoxylin and eosin (HE) staining, TRAP staining, and immunohistochemistry, were conducted to observe bone tissue morphology, osteoclast differentiation, and the phosphorylation level of the PI3K-Akt-GSK3ß signaling pathway. RESULTS: Transcriptomic and metabolomic data collectively reveal that the P2X7 receptor can impact the pathogenesis of osteoporosis through the PI3K-Akt-GSK3ß signaling pathway. Subsequent in vitro experiments showed that cells in the Sh-P2X7 + Recilisib group exhibited increased proliferative activity (1.15 versus 0.59), higher absorbance levels (0.68 versus 0.34), and a significant increase in resorption pit area (13.94 versus 3.50). Expression levels of osteoclast differentiation-related proteins MMP-9, CK, and NFATc1 were markedly elevated (MMP-9: 1.72 versus 0.96; CK: 2.54 versus 0.95; NFATc1: 3.05 versus 0.95), along with increased fluorescent intensity of F-actin rings. In contrast, the OE-P2X7 + LY294002 group showed decreased proliferative activity (0.64 versus 1.29), reduced absorbance (0.34 versus 0.82), and a significant decrease in resorption pit area (5.01 versus 14.96), accompanied by weakened expression of MMP-9, CK, and NFATc1 (MMP-9: 1.14 versus 1.79; CK: 1.26 versus 2.75; NFATc1: 1.17 versus 2.90) and decreased F-actin fluorescent intensity. Furthermore, in vivo animal experiments demonstrated that compared with the wild type (WT) + Sham group, mice in the WT + OVX group exhibited significantly increased levels of CTX and NTX in serum (CTX: 587.17 versus 129.33; NTX: 386.00 versus 98.83), a notable decrease in calcium deposition (19.67 versus 53.83), significant reduction in bone density, increased trabecular separation, and lowered bone mineral density (BMD). When compared with the KO + OVX group, mice in the KO + OVX + recilisib group showed a substantial increase in CTX and NTX levels in serum (CTX: 503.50 versus 209.83; NTX: 339.83 versus 127.00), further reduction in calcium deposition (29.67 versus 45.33), as well as decreased bone density, increased trabecular separation, and reduced BMD. CONCLUSION: P2X7 receptors positively regulate osteoclast formation and bone resorption by activating the PI3K-Akt-GSK3ß signaling pathway.

The Combination of Zhuli Decoction and N-butylphthalide Inhibits Cell Apoptosis Induced by CO Poisoning through the PI3K/AKT/GSK-3ß Signaling Pathway.

Carbon monoxide poisoning (COP) represents a significant global health burden, characterized by its morbidity and high mortality rates. The pathogenesis of COP-induced brain injury is complex, and effective treatment modalities are currently lacking. In this study, we employed network pharmacology to identify therapeutic targets and associated signaling pathways of Zhuli Decoction (ZLD) for COP. Subsequently, we conducted both in vitro and in vivo experiments to validate the therapeutic efficacy of ZLD in combination with N-butylphthalide (NBP) for acute COP-induced injury. Our network pharmacology analysis revealed that the primary components of ZLD exerted therapeutic effects through the modulation of multiple targets and pathways. The in vitro and in vivo experiments demonstrated that the combination of NBP and ZLD effectively inhibited apoptosis and up-regulated the activities of P-PI3K (Tyr458), P-AKT (Ser473), P-GSK-3ß (Ser9), and Bcl-2, thus leading to the protection of neuronal cells and improvement in cognitive function in rats following COP, which was better than the effects observed with NBP or ZLD alone. The rescue experiment further showed that LY294002, a PI3K inhibitor, significantly attenuated the therapeutic efficacy of NBP + ZLD. The neuroprotection effects of NBP and ZLD against COP-induced brain injury are closely linked to the activation of the PI3K/AKT/GSK-3ß signaling pathway.

Bone marrow-derived c-kit positive stem cell administration protects against diabetes-induced nephropathy in a rat model by reversing PI3K/AKT/GSK-3ß pathway and inhibiting cell apoptosis.

Stem cell-based therapy has been proposed as a novel therapeutic strategy for diabetic nephropathy. This study was designed to evaluate the effect of systemic administration of rat bone marrow-derived c-kit positive (c-kit+) cells on diabetic nephropathy in male rats, focusing on PI3K/AKT/GSK-3ß pathway and apoptosis as a possible therapeutic mechanism. Twenty-eight animals were randomly classified into four groups: Control group (C), diabetic group (D), diabetic group, intravenously received 50 µl phosphate-buffered saline (PBS) containing 3 × 105 c-kit- cells (D + ckit-); and diabetic group, intravenously received 50 µl PBS containing 3 × 105 c-Kit positive cells (D + ckit+). Control and diabetic groups intravenously received 50 µl PBS. C-kit+ cell therapy could reduce renal fibrosis, which was associated with attenuation of inflammation as indicated by decreased TNF-α and IL-6 levels in the kidney tissue. In addition, c-kit+ cells restored the expression levels of PI3K, pAKT, and GSK-3ß proteins. Furthermore, renal apoptosis was decreased following c-kit+ cell therapy, evidenced by the lower apoptotic index in parallel with the increased Bcl-2 and decreased Bax and Caspase-3 levels. Our results showed that in contrast to c-kit- cells, the administration of c-kit+ cells ameliorate diabetic nephropathy and suggested that c-kit+ cells could be an alternative cell source for attenuating diabetic nephropathy.

PCMT1 regulates the migration, invasion, and apoptosis of prostate cancer through modulating the PI3K/AKT/GSK-3ß pathway.

Protein L-isoaspartate (D-aspartate) O-methyltransferase (PCMT1) is a repair enzyme that catalyzes the conversion of isomerized aspartic acid (iso-Asp) residues into their normal structure, thereby restoring the configuration and function of proteins. Studies have shown that PCMT1 is overexpressed in several tumors and affects patients' prognosis. However, there are few reports on the role of PCMT1 in prostate cancer (PCa). In the present research, with the assistance of The Cancer Genome Atlas Program (TCGA) database, we found that PCMT1 was overexpressed in PCa tissues. The results of quantitative reverse transcription-polymerase chain reaction (qRT-PCR), western blot and immunohistochemistry staining also showed that PCMT1 expression was significantly increased in PCa tissues and cell lines. In PCa clinical samples, PCMT1 expression was closely related to Gleason score, clinical stage, lymph node metastasis and bone metastasis. The experiments of overexpression and knockdown of PCMT1 in vitro or in vivo showed that PCMT1 can significantly promote the proliferation, migration and invasion of PCa cells, inhibit cell apoptosis, and promote the growth of PCa. We furthermore confirmed that PCMT1 regulated the migration, invasion and apoptosis of PCa cells by modulating the phosphatidylinositol 3-kinase/AKT kinase/glycogen-synthase kinase-3ß (PI3K/AKT/GSK-3ß) signaling pathway. Collectively, PCMT1 plays a cancer-facilitative role in PCa by promoting the proliferation, migration and invasion of PCa cells, and inhibiting apoptosis. Therefore, PCMT1 is considered to represent a novel target for treating PCa.

The Potent and Selective Histamine H3 Receptor Antagonist E169 Counteracts Cognitive Deficits and Mitigates Disturbances in the PI3K/AKT/GSK-3ß Signaling Pathway in MK801-Induced Amnesia in Mice.

The role of histamine H3 receptors (H3Rs) in memory and the prospective of H3R antagonists in pharmacological control of neurodegenerative disorders, e.g., Alzheimer's disease (AD), is well-accepted. Therefore, the procognitive effects of acute systemic administration of H3R antagonist E169 (2.5-10 mg/kg, i.p.) on MK801-induced amnesia in C57BL/6J mice using the novel object recognition test (NORT) were evaluated. E169 (5 mg) provided a significant memory-improving effect on MK801-induced short- and long-term memory impairments in NORT. The E169 (5 mg)-provided effects were comparable to those observed with the reference phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 and were abrogated with the H3R agonist (R)-α-methylhistamine (RAMH). Additionally, our results demonstrate that E169 ameliorated MK801-induced memory deficits by antagonism of H3Rs and by modulation of the level of disturbance in the expression of PI3K, Akt, and GSK-3ß proteins, signifying that E169 mitigated the Akt-mTOR signaling pathway in the hippocampus of tested mice. Moreover, the results observed revealed that E169 (2.5-10 mg/kg, i.p.) did not alter anxiety levels and locomotor activity of animals in open field tests, demonstrating that performances improved following acute systemic administration with E169 in NORT are unrelated to changes in emotional response or in spontaneous locomotor activity. In summary, these obtained results suggest the potential of H3R antagonists such as E169, with good in silico physicochemical properties and stable retained key interactions in docking studies at H3R, in simultaneously modulating disturbed brain neurotransmitters and the imbalanced Akt-mTOR signaling pathway related to neurodegenerative disorders, e.g., AD.

Lappaconitine sulfate inhibits proliferation and induces mitochondrial-mediated apoptosis via regulating PI3K/AKT/GSK3ß signaling pathway in HeLa cells.

Lappaconitine (LA), a diterpenoid alkaloid extracted from the root of Aconitum sinomontanum Nakai, exhibits broad pharmacological effects, including anti-tumor activity. The inhibitory effect of lappaconitine hydrochloride (LH) on HepG2 and HCT-116 cells and the toxicity of lappaconitine sulfate (LS) on HT-29, A549, and HepG2 cells have been described. But the mechanisms of LA against human cervical cancer HeLa cells still need to be clarified. This study was designed to investigate the effects and molecular mechanisms of lappaconitine sulfate (LS) on the growth inhibition and apoptosis in HeLa cells. The cell viability and proliferation were evaluated using the Cell Counting Kit-8 (CCK-8) and 5-ethynyl-2´-deoxyuridine (EdU) assay, respectively. The cell cycle distribution and apoptosis were detected by flow cytometry analysis and 4', 6-diamidino-2-phenylindole (DAPI) staining. The mitochondrial membrane potential (MMP) was determined through the 5, 5', 6, 6'-tetrachloro-1, 1', 3, 3'-tetraethylbenzimi-dazolyl carbocyanine iodide (JC-1) staining. The cell cycle arrest-, apoptosis-, and the phosphatidylinositol-3-kinase/protein kinase B/glycogen synthase kinase 3ß (PI3K/AKT/GSK3ß) pathway-related proteins were estimated by western blot analysis. LS markedly reduced the viability and suppressed the proliferation of HeLa cells. LS induced G0/G1 cell cycle arrest through the inhibition of Cyclin D1, p-Rb, and induction of p21 and p53. Furthermore, LS triggered apoptosis through the activation of mitochondrial-mediated pathway based on decrease of Bcl-2/Bax ratio and MMP and activation of caspase-9/7/3. Additionally, LS led to constitutive downregulation of the PI3K/AKT/GSK3ß signaling pathway. Collectively, LS inhibited cell proliferation and induced apoptosis through mitochondrial-mediated pathway by suppression of the PI3K/AKT/GSK3ß signaling pathway in HeLa cells.

Study on Undercarboxylated Osteocalcin in Improving Cognitive Function of Rats with Type 2 Diabetes Mellitus by Regulating PI3K-AKT-GSK/3ß Signaling Pathwaythrough medical images.

This paper aims to clarify the effect of Undercarboxylated osteocalcin (ucOC) on cognitive function in rats with type 2 diabetes mellitus (T2DM). This research reviewed the cognitive function of 35 diabetic patients, 33 non-diabetic patients and the serum levels of Undercarboxylated osteocalcin (ucOC) in patients. What's more, we analyzed the correlation between serum ucOC levels and cognitive function. Diabetic rats were treated with high (30 µg·kg-1·d-1) and low (10 µg·kg-1·d-1) doses of ucOC to investigate its effects in regulating ucOC on blood lipid, blood glucose and cognitive function. We systematically detected the phosphorylation levels of cognitive level-related proteins (PI3K, AKT, and GSK/3ß) in the hippocampus by Western Blot. Finally, PI3K-Akt pathway involved in regulating cognitive function in diabetic rats by ucOC was verified with AKT pathway inhibitor LY294002. MoCA score and serum ucOC levels were significantly reduced in patients with diabetes mellitus. ucOC could concentration-dose-dependently decrease the blood glucose and lipid levels, and improve glucose metabolism and weaken insulin resistance in diabetic rats (P < 0.001). In addition, escape latency in diabetic rats was significantly higher than that of normal rats in the Morris maze test, and ucOC dose-dependently shortened the escape latency in diabetic rats (all with P < 0.05). After using AKT pathway inhibitor, ucOC failed to shorten the escape latency in diabetic rats. In conclusion, this study explored the relevant mechanisms in inducing cognitive dysfunction of T2DM, suggesting the potential value of ucOC as a drug to improve cognitive dysfunction in patients with T2DM in clinical.

NIGT1 represses plant growth and mitigates phosphate starvation signaling to balance the growth response tradeoff in rice.

Inorganic phosphate (Pi) availability is an important factor which affects the growth and yield of crops, thus an appropriate and effective response to Pi fluctuation is critical. However, how crops orchestrate Pi signaling and growth under Pi starvation conditions to optimize the growth defense tradeoff remains unclear. Here we show that a Pi starvation-induced transcription factor NIGT1 (NITRATE-INDUCIBLE GARP-TYPE TRANSCRIPTIONAL REPRESSOR 1) controls plant growth and prevents a hyper-response to Pi starvation by directly repressing the expression of growth-related and Pi-signaling genes to achieve a balance between growth and response under a varying Pi environment. NIGT1 directly binds to the promoters of Pi starvation signaling marker genes, like IPS1, miR827, and SPX2, under Pi-deficient conditions to mitigate the Pi-starvation responsive (PSR). It also directly represses the expression of vacuolar Pi efflux transporter genes VPE1/2 to regulate plant Pi homeostasis. We further demonstrate that NIGT1 constrains shoot growth by repressing the expression of growth-related regulatory genes, including brassinolide signal transduction master regulator BZR1, cell division regulator CYCB1;1, and DNA replication regulator PSF3. Our findings reveal the function of NIGT1 in orchestrating plant growth and Pi starvation signaling, and also provide evidence that NIGT1 acts as a safeguard to avoid hyper-response during Pi starvation stress in rice.

[Electroacupuncture regulates glucose metabolism disorder through PI3K/Akt/GSK3ß signaling pathway in rats with depression].

OBJECTIVE: To explore the mechanism of electroacupuncture (EA) at "Zusanli" (ST36) on improving glucose metabolism disorder in chronic restraint induced depressed rats. METHODS: A total of 30 male SD rats were randomly divided into control, model and EA groups, with 10 rats in each group. The depression model was established by chronic restraint 2.5 h each day for 4 weeks. For rats in the EA group, EA stimulation (1 mA, 2 Hz, 30 min) was applied to bilateral ST36 during the modeling period, once a day for 4 weeks. The body weight of the rats was recorded before and after modeling. The behavior of rats was observed by sugar-water preference and forced swimming after modeling. The contents of glucose and glycosylated albumin in serum were determined by biochemical method. The histopathological morphology and liver glycogen content were observed by HE and PAS staining. The expression levels of phosphatidylinositol 3-kinase (PI3K), phosphorylated (p)-PI3K (p-PI3K), protein kinase B (Akt), p-Akt, glycogen synthase kinase-3ß (GSK3ß) and p-GSK3ß proteins in liver were determined by Western blot. RESULTS: Compared with the control group, the weight increment and sugar-water preference index decreased (P<0.01), the immobile swimming time was prolonged (P<0.01), the glucose and glycosylated albumin contents in serum increased (P<0.05), the expression of p-Akt protein and the ratio of p-Akt/Akt in liver tissues decreased (P<0.001), the expression of p-GSK3ß protein and the ratio of p-GSK3ß/GSK3ß in liver tissues increased (P<0.01,P<0.001) in the model group. Compared with the model group, the weight increment and sugar-water preference index increased (P<0.05), the immobile swimming time was shortened (P<0.05), the glucose and glycosylated albumin contents in serum decreased (P<0.05), the expressions of p-PI3K and p-Akt proteins and the ratio of p-PI3K/PI3K and p-Akt/Akt in liver tissues increased (P<0.05), the expression of p-GSK3ß protein and the ratio of p-GSK3ß/GSK3ß in liver tissues decreased (P<0.01) in the EA group. HE staining showed that the structure of the hepatic lobule was intact, no obvious inflammatory cell infiltration or fibrosis was observed in the lobule and interstitium, and no abnormalities were observed in the small bile duct, portal vein and artery in the portal area. PAS staining showed that the intensity of staining from the center of the hepatic lobule to the periphery of the hepatic lobule was gradually enhanced in the blank group, that is, the glycogen-rich granules in the hepatic cells were gradually increased; most of the hepatocytes were light colored and glycogen was lost significantly in the model group; while the intensity of hepatocyte staining increased, the staining intensity of the perilobular zone was weaker than that in the blank group, and the glycogen particles partially recovered in the EA group. CONCLUSION: EA intervention can regulate glucose metabolism disorder in chronic restraint induced depressed rats through PI3K/Akt/GSK3ß signaling pathway.

Allantoin ameliorates amyloid ß-peptide-induced memory impairment by regulating the PI3K/Akt/GSK-3ß signaling pathway in rats.

Alzheimer's disease (AD) is a brain disease that causes problems in memory, thinking, and behavior. Allantoin has been shown to have antioxidant, anti-inflammatory, and neuroprotective effects. In this study, we aimed to investigate the effect and mechanism of action of allantoin on AD-related memory impairment. We investigated the effect of allantoin on an amyloid ß1-42 peptide (Aß1-42)-induced AD model in rats and evaluated its memory-enhancing effect using the Morris water maze test. Pathological changes in the hippocampus and cortex were examined by hematoxylin-eosin staining. The expression of the phosphorylated Tau protein and PI3K/Akt/GSK-3ß signaling pathway was analyzed by western blotting. The results of the water maze test showed that after treatment with allantoin, the rats could reduce their swimming time and travel distances to find the platform. Allantoin treatment also increased the time spent in the quadrant in which the platform was located. Histological assessment showed that Aß1-42 could cause morphological alterations in nerve cells in the hippocampal CA1 region, and that allantoin could repair the damage to these cells. Western blotting revealed that allantoin treatment increased the expression of p-PI3K, p-Akt, and p-GSK-3ß and decreased p-Tau in the hippocampus and cortex of rats. These effects were inhibited by LY294002. These findings showed that allantoin could improve cognitive impairment in Aß1-42-induced rats by activating the PI3K/Akt/GSK-3ß signaling pathway to reduce abnormal hyperphosphorylation of Tau. Thus, allantoin may be a potential therapeutic agent for neurodegenerative diseases.

A SPX domain vacuolar transporter links phosphate sensing to homeostasis in Arabidopsis.

Excess phosphate (Pi) is stored into the vacuole through Pi transporters so that cytoplasmic Pi levels remain stable in plant cells. We hypothesized that the vacuolar Pi transporters may harbor a Pi-sensing mechanism so that they are activated to deliver Pi into the vacuole only when cytosolic Pi reaches a threshold high level. We tested this hypothesis using Vacuolar Phosphate Transporter 1 (VPT1), a SPX domain-containing vacuolar Pi transporter, as a model. Recent studies have defined SPX as a Pi-sensing module that binds inositol polyphosphate signaling molecules (InsPs) produced at high cellular Pi status. We showed here that Pi-deficient conditions or mutation of the SPX domain severely impaired the transport activity of VPT1. We further identified an auto-inhibitory domain in VPT1 that suppresses its transport activity. Taking together the results from detailed structure-function analyses, our study suggests that VPT1 is in the auto-inhibitory state when Pi status is low, whereas at high cellular Pi status InsPs are produced and bind SPX domain to switch on VPT1 activity to deliver Pi into the vacuole. This thus provides an auto-regulatory mechanism for VPT1-mediated Pi sensing and homeostasis in plant cells.

The effects of the ethanol extract of Cordia myxa leaves on the cognitive function in mice.

BACKGROUND: Cordia myxa L. (Boraginaceae) is widely distributed in tropical regions and it's fruits, leaves and stem bark have been utilized in folk medicine for treating trypanosomiasis caused by Trypanosoma cruzi. A population-based study showed that T. cruzi infection is associated with cognitive impairments. Therefore, if C. myxa has ameliorating activities on cognitive function, it would be useful for both T. cruzi infection and cognitive impairments. METHODS: In this study, we evaluated the effects of an ethanol extract of leaves of C. myxa (ELCM) on memory impairments and sensorimotor gating deficits in mice. The phosphorylation level of protein was observed by the Western blot analysis. RESULTS: The administration of ELCM significantly attenuated scopolamine-induced cognitive dysfunction in mice, as measured by passive avoidance test and novel object recognition test. Additionally, in the acoustic startle response test, we observed that the administration of ELCM ameliorated MK-801-induced prepulse inhibition deficits. We found that these behavioral outcomes were related with increased levels of phosphorylation phosphatidylinositol 3-kinase (PI3K), protein kinase B (Akt) and glycogen synthase kinase 3 beta (GSK-3ß) in the cortex and extracellular signal-regulated kinase (ERK) and cAMP response element-binding protein (CREB) in the hippocampus by western blot analysis. CONCLUSIONS: These results suggest that ELCM would be a potential candidate for treating cognitive dysfunction and sensorimotor gating deficits observed in individuals with neurodegenerative diseases.

Schisandra chinensis (Turcz.) Baill. essential oil exhibits antidepressant-like effects and against brain oxidative stress through Nrf2/HO-1 pathway activation.

The present study aimed to evaluate the antidepressant-like effect of essential oils from Schisandra chinensis (Turcz.) Baill. (SEO) and its possible mechanisms of SEO. The behavioral despair mouse model in vivo and H2O2-induced PC12 cells model in vitro were employed. And the potential effective components were identified by the spectrum-effect relationships analysis. SEO significantly decreased the immobility time in the forced swimming test and tail suspension test, which indicated a promising antidepressant-like effect of SEO in depressed mice. The decreased levels of SOD, GSH, and CAT, and increased levels of MDA were significantly reversed by SEO treatment, which showed good antioxidant activities both in vitro and in vivo. Besides, SEO significantly promoted the nuclear translocation of Nrf2 and the expression of HO-1 in depressed mice and H2O2-induced PC12 cells. The histopathological examination results showed a potential neuronal protective effect of SEO in the hippocampus and cortex. Furthermore, the upregulation of PI3K/AKT/GSK3ß signaling was observed after SEO treatment in the H2O2-induced PC12 cells. Additionally, based on the spectrum-effect relationship analysis, 9 peaks were identified as positively correlated with the antioxidant activity of SEO. These results suggested that SEO promoted Nrf2/HO-1 pathway to improve the oxidative stress status and exerted the antidepressant-like effects.

Extracellular Matrix Protein 1 Regulates Colorectal Cancer Cell Proliferative, Migratory, Invasive and Epithelial-Mesenchymal Transition Activities Through the PI3K/AKT/GSK3ß/Snail Signaling Axis.

In prior reports, extracellular matrix protein 1 (ECM1) upregulation has been reported in colorectal cancer (CRC) patient tumor tissues, and has been suggested to be related to the metastatic progression of CRC, although the underlying mechanisms have yet to be clarified. In this study, we found that ECM1 was overexpressed in both CRC tissues and cell lines. Upregulation of ECM1 was correlated with tumor size, lymph node status and TNM stage in CRC patients. Knocking down ECM1 suppressed CRC cell growth, migration and invasion, in addition to reducing the expression of Vimentin and increasing E-cadherin expression. The overexpression of ECM1, in contrast, yielded the opposite phenotypic outcomes while also promoting the expression of p-AKT, p-GSK3ß, and Snail, which were downregulated when ECM1 was knocked down. Treatment with LY294002 and 740 Y-P reversed the impact upregulation and downregulation of ECM1 on CRC cell metastasis and associated EMT induction. In vivo analyses confirmed that ECM1 overexpression was able to enhance EMT induction and CRC tumor progression. In conclusion, ECM1 influences CRC development and progression in an oncogenic manner, and regulates CRC metastasis and EMT processes via the PI3K/AKT/GSK3ß/Snail signaling axis.

A diarylheptanoid compound from Alpinia officinarum Hance ameliorates high glucose-induced insulin resistance by regulating PI3K/AKT-Nrf2-GSK3ß signaling pathways in HepG2 cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Alpinia officinarum Hance, a perennial natural medicine-food herb, has been traditionally used to treat colds, stomachache, and diabetes for thousands of years. 1,7-Diphenyl-4E-en-3-heptanone (DPH5), a diarylheptanoid isolated from the rhizome of A. officinarum has been reported to be safe and to have antioxidant and hypoglycemic effects, suggesting its potential in the treatment of insulin resistance (IR). AIM OF THE STUDY: Aim of to investigate the protective effect of DPH5 on IR and elucidate its underlying mechanism of action. MATERIALS AND METHODS: HepG2 cells were used as the research objects. Glucose uptake and reactive oxygen species (ROS) levels in high glucose-induced insulin-resistant HepG2 cells were assessed using flow cytometry. Glucose consumption and the levels of malondialdehyde (MDA) and superoxide dismutase (SOD) were analyzed using the corresponding assay kits. The expression of mRNA and proteins related to insulin signaling, glucose metabolism, and antioxidant factor, including insulin receptor substrate-1 (IRS1), phosphatidylinositol 3-kinase (PI3K), protein kinase B (AKT), translocation of glucose transporter-4, glycogen synthase kinase-3ß (GSK3ß), glucokinase (GCK), pyruvate kinase (PK), phosphoenolpyruvate carboxykinase (PEPCK), glucose-6-phosphatase (G6Pase), nuclear factor-erythroid 2 related factor 2 (Nrf2), heme oxygenase-1 (HO-1), NADPH quinoneoxidoreductase (NQO1), and glutathione peroxidase (GSH-Px) was determined using real-time quantitative polymerase chain reaction and western blotting. Furthermore, molecular docking was performed to determine the spatial mechanism of DPH5 on the key targets PI3K, AKT, Nrf2, and GSK3ß. RESULTS: DPH5 could improve IR that manifested as increased glucose uptake and glucose consumption in insulin-resistant HepG2 cells. Moreover, DPH5 could enhance antioxidant capacity by activating Nrf2/HO-1 elements, including increasing Nrf2, HO-1, SOD, NQO1, and GSH-Px expression and reducing MDA, ROS, and JNK levels, thereby improving oxidative stress and ultimately alleviating IR. Additionally, DPH5 could promote the expression of IRS1, PI3K, AKT, GSK3ß, GCK, and PK, and downregulate the expression of PEPCK and G6pase, thereby accelerating glucose utilization and enhancing insulin sensitivity. The mechanism underlying the effect of DPH5 in alleviating IR was related to the PI3K/AKT- and Nrf2/HO-1-mediated regulation of the GSK3ß signaling pathway, and the results were further confirmed using the specific inhibitors LY294002 and ML385. Results from molecular docking indicated that there were different regulatory sites and interacting forces between DPH5 and PI3K, AKT, Nrf2, and GSK3ß; however, the binding force was relatively strong. CONCLUSIONS: DPH5 improved oxidative stress and glucose metabolism via modulating the PI3K/AKT-Nrf2-GSK3ß pathway, thereby ameliorating IR. Overall, our findings suggest the potential of DPH5 as a natural medicine to treat type-2 diabetes mellitus.

Lignans from Eucommia ulmoides Oliver leaves exhibit neuroprotective effects via activation of the PI3K/Akt/GSK-3ß/Nrf2 signaling pathways in H2O2-treated PC-12 cells.

BACKGROUND: Neuronal apoptosis and oxidative stress have the most crucial influence on neurodegenerative diseases, including Parkinson's disease. Rat adrenal pheochromocytoma cells (PC-12) induced by H2O2 are one of the primary in vitro models of Parkinson's disease (PD) . Previous studies have found that E ulmoides leaf extract exerts good neuroprotective activity and has the potential to treat neurodegenerative diseases. However, the molecular pathways involved in the neuroprotective effects of its primary leaf component, lignans, have not yet been well elucidated yet. PURPOSE: This study aimed to evaluate the neuroprotective effects of lignans in E. ulmoides leaves and to explore the underlying mechanism. METHODS: Cell viability was measured using the CCK-8 assay. Apoptosis was assessed by calcein/PI staining. The release levels of ROS and LDH were assessed using a commercial assay kit. The enzyme activities of SOD and GPx were measured using kits. The establishment of the compound-target-pathway-disease network was performed using a database and computer software. Antioxidant proteins (HO-1, NQO-1, and Cat) and related regulatory proteins (Nrf2, GSK-3ß, p-GSK 3ß (Ser 9), Akt, p-Akt (Tyr326), PI3K) were detected by western blotting. Apoptosis in the zebrafish head was assessed using acridine orange (AO) staining. RESULTS: In the present study, 12 lignans were isolated and characterized from E. ulmoides leaves, including a new compound, (-)-7­epi-pinoresinol mr1 (1). Compounds 1-12 exerted neuroprotective effects in H2O2-treated PC-12 cells by increasing cell viability, improving the enzyme activity of SOD and GPx, and reducing levels of ROS and LDH. Compared to the positive control group (25 µM hesperetin), cell viability in response to 25 µM compound 1 (78.0 ± 0.8%) was highest, but its relative percent LDH release (20.1 ± 2.5%) was the lowest; 25 µM compound 4 resulted in the lowest ROS release levels (101.7 ± 2.6%) and highest SOD enzyme activity (35.9 ± 4.2 U/mg), and the GPx enzyme activity of 25 µM compound 1 was strongest (197.6 ± 0.6 U/mg). Next, the potential targets (PI3K, GSK-3ß) of the test compounds' antioxidant activity were identified using pharmacological network analysis. Using DAVID software for pharmacological network analysis, potential targets (PI3K, GSK-3ß, and SOD2) of 12 lignans were identified. Based on the initial screening results, biological experiments confirmed that diepoxylignans 1, 2, and 4 exerted significant neuroprotection by regulating the PI3K/AKT/GSK-3ß/Nrf2 signaling pathways, increasing protein expression of HO-1, NQO-1, and CAT, and enhancing the antioxidant enzyme activity of SOD and GPx. CONCLUSION: Our experiments first propose that the diepoxylignans from E. ulmoides leaves exert neuroprotective effects via activation of the PI3K/Akt/GSK-3ß/Nrf2 signaling pathway. These findings further indicate that lignans could be the primary components of E. ulmoides Oliver as agents for the prevention and treatment of neurodegenerative diseases. Collectively, Eucommia ulmoides leaves with important research value may be a potential candidate for traditional Chinese medicine for treating oxidative stress-related neurodegenerative diseases.

Deep Sea Water Alleviates Tau Phosphorylation and Cognitive Impairment via PI3K/Akt/GSK-3ß Pathway.

Deep sea water (DSW), as a noticeable natural resource, has been demonstrated to contain high levels of beneficial minerals and exert marked anti-diabetes effects. Epidemiological studies show that type 2 diabetes mellitus (T2DM) is closely related to high danger of Alzheimer's disease (AD); moreover, Akt/GSK-3ß signaling is the main underlying pathway that connects these two diseases. Besides, it has been demonstrated that minerals in DSW, such as Mg, Se, and Zn, could effectively treat cognitive deficits associated with AD. Herein, we first observed the protection of DSW against cognitive dysfunction in T2DM rats, then furtherly explored the neuroprotective mechanism in SH-SY5Y cell model. In T2DM rats, DSW obviously elevated the concentrations of elements Mg, V, Cr, Zn, and Se in brain and improved learning and memory dysfunction in behavior assays, including Morris water maze (MWM) and new object recognition (NOR). Western blot (WB) results demonstrated that DSW could stimulate PI3K/Akt/GSK-3ß signaling, arrest Tau hyperphosphorylation at serine (Ser) 396 and threonine (Thr)231, which was confirmed by immunohistochemistry (IHC). In order to further confirm the mechanism, we employed wortmannin to inhibit PI3K in SH-SY5Y cells; results showed that pretreatment with wortmannin almost abolished DSW-induced decreases in phosphorylated Tau. Taken together, these data elucidated that DSW could improve Tau hyperphosphorylation and cognitive impairment, which were closely related with the stimulation of Akt/GSK-3ß signaling, and the neuroprotective effects of DSW should be contributed to the synergistic effects of major and trace elements in it, such as Mg, V, Cr, Zn, and Se. These experimental evidence indicated that DSW may be explored as natural neuroprotective food for the prevention and treatment of AD.

PINK1 overexpression prevents forskolin-induced tau hyperphosphorylation and oxidative stress in a rat model of Alzheimer's disease.

PTEN-induced putative kinase 1 (PINK1)/parkin pathway mediates mitophagy, which is a specialized form of autophagy. Evidence shows that PINK1 can exert protective effects against stress-induced neuronal cell death. In the present study we investigated the effects of PINK1 overexpression on tau hyperphosphorylation, mitochondrial dysfunction and oxidative stress in a specific rat model of tau hyperphosphorylation. We showed that intracerebroventricular (ICV) microinjection of forskolin (FSK, 80 µmol) induced tau hyperphosphorylation in the rat brain and resulted in significant spatial working memory impairments in Y-maze test, accompanied by synaptic dysfunction (reduced expression of synaptic proteins synaptophysin and postsynaptic density protein 95), and neuronal loss in the hippocampus. Adeno-associated virus (AAV)-mediated overexpression of PINK1 prevented ICV-FSK-induced cognition defect and pathological alterations in the hippocampus, whereas PINK1-knockout significantly exacerbated ICV-FSK-induced deteriorated effects. Furthermore, we revealed that AAV-PINK1-mediated overexpression of PINK1 alleviated ICV-FSK-induced tau hyperphosphorylation by restoring the activity of PI3K/Akt/GSK3ß signaling. PINK1 overexpression reversed the abnormal changes in mitochondrial dynamics, defective mitophagy, and decreased ATP levels in the hippocampus. Moreover, PINK1 overexpression activated Nrf2 signaling, thereby increasing the expression of antioxidant proteins and reducing oxidative damage. These results suggest that PINK1 deficiency exacerbates FSK-induced tau pathology, synaptic damage, mitochondrial dysfunction, and antioxidant system defects, which were reversed by PINK1 overexpression. Our data support a critical role of PINK1-mediated mitophagy in controlling mitochondrial quality, tau hyperphosphorylation, and oxidative stress in a rat model of Alzheimer's disease.

4-Methoxycinnamic acid attenuates schizophrenia-like behaviors induced by MK-801 in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Scrophularia buergeriana has been used for traditional medicine as an agent for reducing heat in the blood and for nourishing kidney 'Yin'. Therefore, S. buergeriana might be a potential treatment for mental illness, especially schizophrenia, which may be attenuated by supplying kidney Yin and reducing blood heat. In a pilot study, we found that S. buergeriana alleviated sensorimotor gating dysfunction induced by MK-801. AIM OF THE STUDY: In the present study, we attempted to reveal the active component(s) of S. buergeriana as a candidate for treating sensorimotor gating dysfunction, and we identified 4-methoxycinnamic acid. We explored whether 4-methoxycinnamic acid could affect schizophrenia-like behaviors induced by hypofunction of the glutamatergic neurotransmitter system. MATERIALS AND METHODS: Mice were treated with 4-methoxycinnamic acid (3, 10, or 30 mg/kg, i.g.) under MK-801-induced schizophrenia-like conditions. The effect of 4-methoxycinnamic acid on schizophrenia-like behaviors were explored using several behavioral tasks. We also used Western blotting to investigate which signaling pathway(s) is involved in the pharmacological activities of 4-methoxycinnamic acid. RESULTS: 4-Methoxycinnamic acid ameliorated MK-801-induced prepulse inhibition deficits, social interaction disorders and cognitive impairment by regulating the phosphorylation levels of PI3K, Akt and GSK-3ß signaling in the prefrontal cortex. And there were no adverse effects in terms of catalepsy and motor coordination impairments. CONCLUSION: Collectively, 4-methoxycinnamic acid would be a potential candidate for treating schizophrenia with fewer adverse effects, especially the negative symptoms and cognitive dysfunctions.

Protective effects of safranal on hypoxia/reoxygenation-induced injury in H9c2 cardiac myoblasts via the PI3K/AKT/GSK3ß signaling pathway.

Safranal (SFR), an active ingredient extracted from saffron, exhibits a protective effect on the cardiovascular system. However, the mechanism of SFR against hypoxia/reoxygenation (H/R)-induced cardiomyocyte injury has previously not been investigated in vitro. The aim of the present study was therefore to observe the protective effects of SFR on H/R-induced cardiomyocyte injury and to explore its mechanisms. A H/R injury model of H9c2 cardiac myoblasts was established by administering 800 µmol/l CoCl2 to H9c2 cells for 24 h and reoxygenating the cells for 4 h to induce hypoxia. H9c2 cardiac myoblasts were pretreated with SFR for 12 h to evaluate the associated protective effects. A Cell Counting Kit-8 assay was used for cell viability detection, and the expression levels of lactate dehydrogenase (LDH), creatine kinase-MB (CK-MB), glutathione peroxidase (GSH-px), catalase (CAT), superoxide dismutase (SOD), malondialdehyde (MDA) and caspase-3, and the intracellular Ca2+ concentration were measured using the corresponding commercial kits. Levels of reactive oxygen species (ROS) in the cells were detected using 2,7-dichlorodihydrofluorescein diacetate. Flow cytometry was used to determine the degree of apoptosis and the level of mitochondrial membrane potential (MMP). Moreover, the expression levels of phosphorylated (p-)PI3K, AKT, p-AKT, glycogen synthase kinase 3ß (GSK3ß), p-GSK3ß, Bcl-2, Bax, caspase-3 and cleaved caspase-3 were measured using western blot analysis. Results of the present study demonstrated that the H9c2 cardiac myoblasts treated with SFR exhibited significantly improved levels of viability and significantly reduced levels of ROS, compared with the H/R group. Furthermore, compared with the H/R group, SFR treatment significantly increased the MMP levels and antioxidant enzyme levels, including CAT, SOD and GSH-px; whereas the levels of CK-MB, LDH, MDA and intracellular Ca2+ concentration were significantly decreased. Moreover, the results of the present study demonstrated that SFR significantly reduced caspase-3, cleaved caspase-3 and Bax protein expression levels, but upregulated the Bcl-2 protein expression levels. SFR also increased the protein expressions of PI3K/AKT/GSK3ß. In summary, the results suggested that SFR may exert a protective effect against H/R-induced cardiomyocyte injury, which occurs in connection with the inhibition of oxidative stress and apoptosis via regulation of the PI3K/AKT/GSK3ß signaling pathway.