OBJECTIVE: To investigate the effect of Arsenic trioxide (ATO) combined with Norcantharidin (NCTD) on the proliferation and apoptosis of Jurkat cells, and to evaluate its effect on the proliferation and apoptosis of acute T-lymphoblastic leukemia (T-ALL) based on the Wnt/β-catenin signaling pathway. METHODS: Jurkat cell lines were used as the study subjects and treated with different concentrations of ATO (0, 2, 4, 8, 16 μmol/L) and NCTD (0, 10, 25, 50, 100 μmol/L) for 72 hours, and the cell proliferation was detected by CCK-8. Meanwhile, flow cytometry was used to detect the apoptosis rate, EdU staining to detect cell proliferation viability, cell clone formation assay to assess cell cloning ability, Transwell assay to assess cell invasion ability, and Western blot to detect apoptosis and the expression of Wnt/β-catenin signaling pathway-related proteins. RESULTS: Compared with the control group, both ATO and NCTD effectively inhibited Jurkat cell proliferation when used alone, and the inhibition effect was more significant when used in combination ( P < 0.05). The combination significantly increased the apoptosis rate of Jurkat cells ( P < 0.05). Meanwhile, the combination significantly decreased the proliferation vitality and clone formation ability of the cells ( P < 0.05), and inhibited the invasion ability of Jurkat cells ( P < 0.05). Western blot analysis showed that the combination of ATO and NCTD significantly up-regulated the expression of pro-apoptotic proteins Bax and E-cadherin, and down-regulated the expression of anti-apoptotic proteins Bcl-2, c-myc and Cyclin D1 ( P < 0.05). CONCLUSION The combination of ATO and NCTD had a synergistic effect in inhibiting proliferation and promoting apoptosis in Jurkat cells, which may be related to the inhibition of Wnt/β-catenin signaling pathway.
Humans ; Apoptosis/drug effects* ; Jurkat Cells ; Cell Proliferation/drug effects* ; Arsenic Trioxide ; Wnt Signaling Pathway/drug effects* ; Bridged Bicyclo Compounds, Heterocyclic/pharmacology* ; beta Catenin/metabolism* ; Arsenicals/pharmacology* ; Oxides/pharmacology*

Excessive generation of reactive oxygen species (ROS) can lead to oxidative-antioxidative imbalance in the organism, resulting in oxidative stress. Hematopoietic stem/progenitor cells (HSPCs) exhibit high sensitivity to changes in ROS levels, and high levels of ROS can impair self-renewal capacity of HSPCs, leading to oxidative damage and even death. Wnt/β-catenin signaling pathway regulates hematopoiesis and plays an important role in determining the fate of stem cells, such as self-renewal, proliferation and differentiation of HSPCs. Studies have shown that Wnt/β-catenin signaling pathway is also closely related to oxidative stress. This article summarizes the relevant literature, and reviews the role of Wnt/β-catenin signaling pathway in oxidative stress, its impact on hematopoietic system, and the current research status of related mechanisms.
Oxidative Stress ; Humans ; Wnt Signaling Pathway ; Hematopoietic Stem Cells ; Reactive Oxygen Species/metabolism* ; Hematopoietic System/metabolism* ; beta Catenin/metabolism* ; Hematopoiesis

OBJECTIVE: To investigate the Wnt signaling pathway and miRNAs mechanism of extracts of Plastrum Testudinis (PT) in the treatment of osteoporosis (OP). METHODS: Thirty female Sprague Dawley rats were randomly divided into 5 groups by random number table method, including sham group, ovariectomized group (OVX), ovariectomized groups treated with high-, medium-, and low-dose PT (160, 80, 40 mg/kg per day, respectively), with 6 rats in each group. Except for the sham group, the other rats underwent bilateral ovariectomy to simulate OP and received PT by oral gavage for 10 consecutive weeks. After treatment, bone mineral density was measured by dual-energy X-ray absorptiometry; bone microstructure was analyzed by micro-computed tomography and hematoxylin and eosin staining; and the expressions of osteogenic differentiation-related factors were detected by immunochemistry, Western blot, and quantitative polymerase chain reaction. In addition, Dickkopf-1 (Dkk-1) was used to inhibit the Wnt signaling pathway in bone marrow mesenchymal stem cells (BMSCs) and miRNA overexpression was used to evaluate the effect of miR-214 on the osteogenic differentiation of BMSCs. Subsequently, PT extract was used to rescue the effects of Dkk-1 and miR-214, and its impacts on the osteogenic differentiation-related factors of BMSCs were evaluated. RESULTS: PT-M and PT-L significantly reduced the weight gain in OVX rats (P<0.05). PT also regulated the bone mass and bone microarchitecture of the femur in OVX rats, and increased the expressions of bone formation-related factors including alkaline phosphatase, bone morphogenetic protein type 2, collagen type I alpha 1, and runt-related transcription factor 2 when compared with the OVX group (P<0.05 or P<0.01). Meanwhile, different doses of PT significantly rescued the inhibition of Wnt signaling pathway-related factors in OVX rats, and increased the mRNA or protein expressions of Wnt3a, β-catenin, glycogen synthase kinase-3β, and low-density lipoprotein receptor-related protein 5 (P<0.05 or P<0.01). PT stimulated the osteogenic differentiation of BMSCs inhibited by Dkk-1 and activated the Wnt signaling pathway. In addition, the expression of miR-214 was decreased in OVX rats (P<0.01), and it was negatively correlated with the osteogenic differentiation of BMSCs (P<0.01). MiR-214 mimic inhibited Wnt signaling pathway in BMSCs (P<0.05 or P<0.01). Conversely, PT effectively counteracted the effect of miR-214 mimic, thereby activating the Wnt signaling pathway and stimulating osteogenic differentiation in BMSCs (P<0.05 or P<0.01). CONCLUSION PT stimulates bone formation in OVX rats through β-catenin-mediated Wnt signaling pathway, which may be related to inhibiting miR-214 in BMSCs.
Animals ; MicroRNAs/genetics* ; Female ; Rats, Sprague-Dawley ; Wnt Signaling Pathway/genetics* ; Osteogenesis/genetics* ; Mesenchymal Stem Cells/cytology* ; Cell Differentiation/drug effects* ; Bone Density/drug effects* ; Ovariectomy ; Osteoporosis/drug therapy* ; beta Catenin/metabolism* ; Rats ; Intercellular Signaling Peptides and Proteins/metabolism* ; Drugs, Chinese Herbal/pharmacology*

OBJECTIVE: To explore the main components and potential mechanisms of Sangqi Qingxuan Liquid in the treatment of arterial vascular endothelial cells (AVECs) injury in hypertension through network pharmacology. METHODS: Traditional Chinese Medicine Systems Pharmacology and Analysis Platform (TCMSP) and Traditional Chinese Medicine Integrated Database (TCMID) were used to screen the active components of Sangqi Qingxuan Liquid (SQQX), which met the oral utilization rate and drug similarity criteria. An active component-target network was constructed using Cytoscape 3.6 software. A protein-protein interaction (PPI) network of targets associated with SQQX treatment for hypertension was constructed using the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database. The Metascape database was used to perform enrichment analysis of gene ontology biological functions and MSigDB pathway enrichment analysis of proteins in the PPI network. Further analysis of the main components of SQQX was performed using UPLC-MS. Based on the results of network pharmacology, the mechanism of SQQX to improve the injury of AVECs in hypertension was verified through lentiviral transfection by Wnt/ β -catenin signaling pathway. AVECs induced by angiotensin II (Ang II ) was used to establish a model of endothelial function injury in hypertension. Cell viability, intracellular nitric oxide content, malonaldehyde content, and superoxide dismutase activity were measured to determine the optimal induction conditions. The optimal intervention conditions for SQQX were determined based on cell viability, cellular DNA activity, and the gradient method. The cells were further divided into blank, model, overexpression lentivirus negative control, overexpression lentivirus, overexpression lentivirus + SQQX intervention (2.47 mg/mL, 12 h), inhibition lentivirus negative control, inhibition lentivirus, and inhibition lentivirus + SQQX intervention (2.47 mg/mL, 12 h) groups. Finally, quantitative real-time PCR and Western blotting were performed to analyze the molecular mechanisms of SQQX in the Wnt/ β -catenin signaling pathway. RESULTS: The main SQQX components were betaine, buddleoside, and chlorogenic acid, in descending order. Network pharmacology analysis screened 12 pathways associated with the hypertensive vascular endothelium. The results showed that 1 µ mol/L for 12 h was the optimal condition for Ang II to induce AVECs injury, and 2.47 mg/mL SQQX intervention for 12 h was the optimal condition for treating AVECs injury. In the experimental validation based on the interaction network of the Wnt/ β -catenin signaling pathway, SQQX significantly decreased the expressions of β -catenin, Smad2, peroxisome proliferator-activated receptors (PPARs), endothelial nitric oxide synthase (eNOS), and endothelin-1 (ET-1) caused by the β -catenin overexpression lentivirus (P<0.05 or P<0.01). The function of vascular endothelial cells can be improved by the β -catenin inhibition lentivirus, and no obvious changes were observed after further intervention with SQQX. CONCLUSION SQQX may protect against AVECs injury by regulating the Wnt/β -catenin signaling pathway.
Drugs, Chinese Herbal/therapeutic use* ; beta Catenin/metabolism* ; Hypertension/metabolism* ; Endothelial Cells/metabolism* ; Protein Interaction Maps/drug effects* ; Humans ; Wnt Signaling Pathway/drug effects* ; Network Pharmacology ; Endothelium, Vascular/injuries* ; Cell Survival/drug effects* ; Angiotensin II/pharmacology* ; Nitric Oxide/metabolism*

Metastasis remains the primary cause of cancer-related mortality worldwide. Circulating tumor cells (CTCs) represent critical targets for metastasis prevention and treatment. Traditional Chinese medicine may prevent lung cancer metastasis through long-term intervention in CTC activity. Tiao-Shen-Zhi-Ai Formular (TSZAF) represents a Chinese medicine compound prescription utilized clinically for lung cancer treatment. This study combined three principal active ingredients from TSZAF into a novel TSZAF monomer combination (TSZAF mc) to investigate its anti-metastatic effects and mechanisms. TSZAF mc demonstrated significant inhibition of proliferation, migration, and invasion in CTC-TJH-01 and LLC cells, while inducing cellular apoptosis in vitro. Moreover, TSZAF mc substantially inhibited LLC cell growth and metastasis in vivo. Mechanistically, TAZSF mc significantly suppressed the Wnt/β-catenin signaling pathway and CXCL5 expression in lung cancer cells and tissues. Additionally, TAZSF mc notably reduced neutrophil infiltration in metastatic lesions. These findings indicate that TSZAF mc inhibits lung cancer growth and metastasis by suppressing the Wnt/β-catenin signaling pathway and reducing CXCL5 secretion, thereby decreasing neutrophil recruitment and infiltration. TSZAF mc demonstrates potential as an effective therapeutic agent for lung cancer metastasis.
Lung Neoplasms/genetics* ; Wnt Signaling Pathway/drug effects* ; Animals ; Humans ; Drugs, Chinese Herbal/pharmacology* ; Mice ; Neoplasm Metastasis/prevention & control* ; Cell Proliferation/drug effects* ; Cell Line, Tumor ; Neutrophil Infiltration/drug effects* ; Down-Regulation/drug effects* ; Cell Movement/drug effects* ; beta Catenin/genetics* ; Apoptosis/drug effects* ; Mice, Inbred C57BL ; Male ; Neoplastic Cells, Circulating/drug effects*

OBJECTIVE: To observe the effects of ultrasound-guided foraminal electroacupuncture on neuronal apoptosis and motor function in rats with spinal cord injury (SCI), and to explore the potential underlying mechanisms. METHODS: Thirty-six SPF-grade Sprague-Dawley rats were randomly assigned to a sham operation group, a model group, and an ultrasound-guilded electroacupuncture group (electroacupuncture group), with 12 rats in each group. In the sham operation group, the spinal cord was exposed and then the incision was sutured without contusion. In the other two groups, SCI models were established using a modified Allen's impact method. On days 1, 3, 7, and 14 after modeling, the electroacupuncture group received electroacupuncture intervention at the T₉/T₁₀ and T₁₀/T₁₁ intervertebral foramen under ultrasound guidance, avoiding spinal cord injury. Stimulation parameters were dense-disperse wave at 2 Hz/100 Hz and 1-2 mA for each session. Following interventions on days 1, 3, 7, and 14, the Basso-Beattie-Bresnahan (BBB) score was assessed; the inclined plane test was used to assess hindlimb grip strength in rats. After the intervention, HE staining was used to observe spinal cord morphology; TUNEL staining was used to detect neuronal apoptosis; ELISA was used to measure the serum levels of interleukin (IL)-6, IL-1β, and tumor necrosis factor-alpha (TNF-α); Western blot was used to analyze the protein expression of Wnt-4, β-catenin, c-Myc, Bax, Bcl-2, and NeuN in spinal tissue; quantitative real-time PCR was used to detect the mRNA expression of Wnt-4, β-catenin, c-Myc, Bax, Bcl-2, and NeuN. RESULTS: Compared with the sham operation group, the model group showed significantly reduced BBB scores (P<0.05), and reduced inclined plane angles (P<0.05) at all time points. Compared with the model group, the electroacupuncture group exhibited increased BBB scores on days 3, 7, and 14 (P<0.05), and higher inclined plane angles on days 1, 3, 7, and 14 (P<0.05). Compared with the sham operation group, the model group showed disorganized spinal cord structure with increased inflammatory cells and necrotic neurons, higher number of apoptotic neurons in spinal tissue (P<0.05), elevated serum IL-6, IL-1β, and TNF-α levels (P<0.05), increased protein and mRNA expression of Wnt-4, β-catenin, c-Myc, and Bax (P<0.05), and decreased protein and mRNA expression of Bcl-2 and NeuN in spinal tissue (P<0.05). Compared with the model group, the electroacupuncture group had fewer inflammatory cells and apoptotic neurons in spinal tissue (P<0.05), reduced serum IL-6, IL-1β, and TNF-α levels (P<0.05), increased protein and mRNA expression of Wnt-4, β-catenin, Bcl-2, and NeuN (P<0.05), and decreased protein and mRNA expression of c-Myc and Bax in spinal tissue (P<0.05). CONCLUSION Ultrasound-guided foraminal electroacupuncture could improve motor function in rats with SCI, potentially by regulating the expression of molecules related to the Wnt-4/β-catenin signaling pathway to inhibit neuronal apoptosis and inflammatory responses.
Animals ; Electroacupuncture/methods* ; Spinal Cord Injuries/physiopathology* ; Rats, Sprague-Dawley ; Rats ; Wnt Signaling Pathway ; Male ; Humans ; Female ; beta Catenin/metabolism* ; Apoptosis ; Ultrasonography ; Tumor Necrosis Factor-alpha/genetics* ; Spinal Cord/metabolism*

Human cytomegalovirus (HCMV) poses a significant risk of neural damage during pregnancy. As the most prevalent intrauterine infectious agent in low- and middle-income countries, HCMV disrupts the development of neural stem cells, leading to fetal malformations and abnormal structural and physiological functions in the fetal brain. This review summarizes the current understanding of how HCMV infection dysregulates the Wnt signaling pathway to induce fetal malformations and discusses current management strategies.
Humans ; Cytomegalovirus Infections/virology* ; Wnt Signaling Pathway ; Pregnancy ; Female ; Cytomegalovirus/physiology* ; Pregnancy Complications, Infectious/virology* ; Congenital Abnormalities/virology* ; Animals

Objective To investigate the effects of folate deficiency on changes in histone H3 lysine 4 (H3K4) mono-methylation (me1)-marked enhancers and the molecular mechanism underpinning the folate deficiency-induced neural tube defects (NTD). Methods Mouse embryonic stem cells (mESCs) were cultured in the folate-free DMEM medium (folate-deficient group) and the DMEM medium containing 4 mg/L folate (normal control group),respectively.Chromatin immunoprecipitation sequencing (ChIP-seq) was performed for H3K4me1. The mouse model of folate-induced NTD was established,and transcriptome sequencing (RNA-seq) was performed for the brain tissue of fetal mice to reveal the differential expression profiles.The results were validated through real-time quantitative polymerase chain reaction (RT-qPCR).The activity of the differential peak regions of H3K4me1 was verified through the luciferase reporter assay. Results The folate content in the mESCs cultured in the folate-free medium reduced compared with that in the normal control group (P=0.008).The H3K4me1-maked enhancers in the mESCs cultured in the folate-free medium induced significant changes in intronic regions,and these changes were concentrated in metabolic and energy metabolism processes (q=9.56×10^-48,P=1.28×10^-47).The differentially expressed genes harboring H3K4me1-marked enhancers in mESCs were mainly enriched in the Wnt signaling pathway (q=0.004,P=0.004 7).ChIP-qPCR results confirmed that H3K4me1 binding decreased in the differential peak regions of the Ldlrap1 gene (P=0.008),Camta1 gene (P=0.002),and Apc2 gene (P=0.012).The H3K4 demethylase inhibitor T-448 effectively reversed the H3K4me1 binding in the differential peak regions of the aforementioned genes (P=0.01).The results of RNA-seq for the brain tissue of NTD fetal mice showed significant enrichment of the differentially expressed genes in the Wnt signaling pathway (P=1.52×10^-5).The enrichment of differential peak regions of H3K4me1-marked enhancers in Apc2,Ldlrap1,and Camta1 genes in the brain tissue also showed significant changes.The differential peak region in Apc2 exhibited transcription factor activity (P=0.020). Conclusion Folate deficiency may affect changes in H3K4me1-marked enhancers to participate in the regulation of neural tube closure genes,thereby inducing the occurrence of NTD.
Neural Tube Defects/genetics* ; Animals ; Mice ; Folic Acid Deficiency/complications* ; Histones/metabolism* ; Folic Acid/metabolism* ; Methylation ; Mouse Embryonic Stem Cells/metabolism* ; Wnt Signaling Pathway ; Lysine/metabolism* ; Chromatin Immunoprecipitation Sequencing

BACKGROUND: Enzalutamide, a second-generation androgen receptor (AR) pathway inhibitor, is widely used in the treatment of castration-resistant prostate cancer. However, after a period of enzalutamide treatment, patients inevitably develop drug resistance. In this study, we characterized leucine-rich repeated G-protein-coupled receptor 5 (LGR5) and explored its potential therapeutic value in prostate cancer. METHODS: A total of 142 pairs of tumor and adjacent formalin-fixed paraf-fin-embedded tissue samples from patients with prostate cancer were collected from the Pathology Department at Sun Yat-sen Memorial Hos-pital. LGR5 was screened by sequencing data of enzalutamide-resistant cell lines combined with sequencing data of lesions with different Gleason scores from the same patients. The biological function of LGR5 and its effect on enzalutamide resistance were investigated in vitro and in vivo . Glutathione-S-transferase (GST) pull-down, coimmunoprecipitation, Western blotting, and immunofluorescence assays were used to explore the specific binding mechanism of LGR5 and related pathway changes. RESULTS: LGR5 was significantly upregulated in prostate cancer and negatively correlated with poor patient prognosis. Overexpression of LGR5 promoted the malignant progression of prostate cancer and reduced sensitivity to enzalutamide in vitro and in vivo . LGR5 promoted the phosphorylation of glycogen synthase kinase-3β (GSK-3β) by binding heat shock protein 90,000 alpha B1 (HSP90AB1) and mediated the activation of the Wingless/integrated (WNT)/β-catenin signaling pathway. The increased β-catenin in the cytoplasm entered the nucleus and bound to the nuclear AR, promoting the transcription level of AR, which led to the enhanced tolerance of prostate cancer to enzalutamide. Reducing HSP90AB1 binding to LGR5 significantly enhanced sensitivity to enzalutamide. CONCLUSIONS LGR5 directly binds to HSP90AB1 and mediates GSK-3β phosphorylation, promoting AR expression by regulating the WNT/β-catenin signaling pathway, thereby conferring resistance to enzalutamide treatment in prostate cancer.
Male ; Humans ; Phenylthiohydantoin/pharmacology* ; Benzamides ; Receptors, G-Protein-Coupled/genetics* ; Nitriles ; Cell Line, Tumor ; HSP90 Heat-Shock Proteins/metabolism* ; Drug Resistance, Neoplasm/genetics* ; Prostatic Neoplasms/drug therapy* ; beta Catenin/metabolism* ; Receptors, Androgen/genetics* ; Animals ; Mice ; Wnt Signaling Pathway/physiology*

This study investigates the therapeutic effect and underlying mechanism of Compound Ziyin Granules(CZYG) on postmenopausal osteoporosis(PMOP) induced by bilateral ovariectomy in rats. Six-month-old female SD rats were randomly divided into sham-operated(sham) group, ovariectomy(OVX) model group, high-, medium-, and low-dose CZYG groups, and alendronate sodium(AS) group. After 30 days of model establishment, treatment was administered by gavage once daily for 8 weeks, followed by sample collection. Enzyme-linked immunosorbent assay(ELISA) was used to measure serum levels of calcium ions, alkaline phosphatase(AKP), estrogen(E_2), osteoprotegerin(OPG), osteocalcin(BGP), tartrate-resistant acid phosphatase(TRAP), and type Ⅰ procollagen N-terminal propeptide(PINP). Hematoxylin-eosin(HE) staining was used to observe the histopathological changes in the femurs of rats, while micro-computed tomography(micro-CT) was used to analyze the microstructure of the distal femur. Western blot analysis was performed to measure the expression levels of bone metabolism-related proteins, including wingless-type MMTV integration site family member 2(Wnt2), β-catenin, low-density lipoprotein receptor-related protein 5(LRP5), glycogen synthase kinase-3β(GSK-3β). The mRNA expression levels of Wnt2, β-catenin, LRP5, GSK-3β, p-GSK-3β were determined by quantitative real-time PCR(qRT-PCR). Thirty days after bilateral ovariectomy, compared to the sham group, the OVX group showed significant increases in body weight and significant decreases in uterine coefficient. After 8 weeks of treatment, compared to the OVX group, CZYG(medium and high doses) and AS reduced body weight, with high-dose CZYG and AS significantly increasing the uterine coefficient. Serum levels of AKP and TRAP were significantly elevated, while levels of calcium, E_2, BGP, and OPG were significantly decreased in the OVX group. Compared to the OVX group, CZYG and AS significantly reduced serum levels of AKP and TRAP, while high-dose CZYG and AS notably increased the levels of E_2, BGP, OPG, and PINP. Micro-CT and HE staining results indicated that CZYG(medium and high doses) and AS significantly increased bone tissue volume, trabecular number, bone mineral density, and improved the microstructure of the femur. Compared to the OVX group, high-dose CZYG and AS significantly upregulated the protein and mRNA expression levels of Wnt2, β-catenin, and LRP5, and downregulated the phosphorylation level of p-GSK-3β. These results suggest that CZYG can improve PMOP by promoting estrogen secretion, improving bone metabolism indicators, increasing trabecular number and bone mineral density. Its mechanism may be related to the regulation of the Wnt/β-catenin signaling pathway.
Animals ; Female ; Rats, Sprague-Dawley ; Osteoporosis, Postmenopausal/genetics* ; Rats ; Wnt Signaling Pathway/drug effects* ; Humans ; Drugs, Chinese Herbal/administration & dosage* ; beta Catenin/genetics* ; Osteoprotegerin/metabolism* ; Ovariectomy ; Calcium/blood* ; Bone Density/drug effects*