Cinobufotalin prevents bone loss induced by ovariectomy in mice through the BMPs/SMAD and Wnt/β-catenin signaling pathways

Background:Osteoporosis is a chronic bone disease characterized by bone loss and decreased bone strength.However,current anti-resorptive drugs carry a risk of various complications.The deep learning-based efficacy prediction system(DLEPS)is a forecasting tool that can effectively compete in drug screening and prediction based on gene expression changes.This study aimed to explore the protective effect and potential mechanisms of cinobufotalin(CB),a traditional Chinese medicine(TCM),on bone loss.Methods:DLEPS was employed for screening anti-osteoporotic agents according to gene profile changes in primary osteoporosis.Micro-CT,histological and morphological analysis were applied for the bone protective detection of CB,and the osteogenic differentiation/function in human bone marrow mesenchymal stem cells(hBMMSCs)were also investigated.The underlying mechanism was verified using qRT-PCR,Western blot(WB),immunofluorescence(IF),etc.Results:A safe concentration(0.25mg/kg in vivo,0.05μM in vitro)of CB could effectively preserve bone mass in estrogen deficiency-induced bone loss and promote osteogenic differentiation/function of hBMMSCs.Both BMPs/SMAD and Wnt/β-catenin signaling pathways participated in CB-induced osteogenic differentiation,further regulating the expression of osteogenesis-associated factors,and ultimately promoting osteogenesis.Conclusion:Our study demonstrated that CB could significantly reverse estrogen deficiency-induced bone loss,further promoting osteogenic differentiation/function of hBMMSCs,with BMPs/SMAD and Wnt/β-catenin signaling pathways involved.

Pachymic acid exerts antitumor activities by modulating the Wnt/β-catenin signaling pathway via targeting PTP1B

Objective:To determine the inhibitory effects of pachymic acid on lung adenocarcinoma(LUAD)cells and elucidate its underlying mechanism.Methods:CCK-8,wound healing,Transwell,Western blot,tube formation,and immunofluorescence assays were carried out to measure the effects of various concentrations of pachymic acid on LUAD cell proliferation,metastasis,angiogenesis as well as autophagy.Subsequently,molecular docking technology was used to detect the potential targeted binding association between pachymic acid and protein tyrosine phosphatase 1B(PTP1B).Moreover,PTP1B was overexpressed in A549 cells to detect the specific mechanisms of pachymic acid.Results:Pachymic acid suppressed LUAD cell viability,metastasis as well as angiogenesis while inducing cell autophagy.It also targeted PTP1B and lowered PTP1B expression.However,PTP1B overexpression reversed the effects of pachymic acid on metastasis,angiogenesis,and autophagy as well as the expression of Wnt3a andβ-catenin in LUAD cells.Conclusions:Pachymic acid inhibits metastasis and angiogenesis,and promotes autophagy in LUAD cells by modulating the Wnt/β-catenin signaling pathway via targeting PTP1B.

Effects of Helicobacter pylori and Moluodan on the Wnt/β-catenin signaling pathway in mice with precancerous gastric cancer lesions

BACKGROUND Helicobacter pylori(H.pylori)is the primary risk factor for gastric cancer(GC),the Wnt/β-Catenin signaling pathway is closely linked to tumourigenesis.GC has a high mortality rate and treatment cost,and there are no drugs to prevent the progression of gastric precancerous lesions to GC.Therefore,it is necessary to find a novel drug that is inexpensive and preventive to against GC.AIM To explore the effects of H.pylori and Moluodan on the Wnt/β-Catenin signaling pathway and precancerous lesions of GC(PLGC).METHODS Mice were divided into the control,N-methyl-N-nitrosourea(MNU),H.pylori+MNU,and Moluodan groups.We first created an H.pylori infection model in the H.pylori+MNU and Moluodan groups.A PLGC model was created in the remaining three groups except for the control group.Moluodan was fed to mice in the Moloudan group ad libitum.The general condition of mice were observed during the whole experiment period.Gastric tissues of mice were grossly and microscopically examined.Through quantitative real-time PCR(qRT-PCR)and Western blotting analysis,the expression of relevant genes were detected.RESULTS Mice in the H.pylori+MNU group showed the worst performance in general condition,gastric tissue visual and microscopic observation,followed by the MNU group,Moluodan group and the control group.QRT-PCR and Western blotting analysis were used to detect the expression of relevant genes,the results showed that the H.pylori+MNU group had the highest expression,followed by the MNU group,Moluodan group and the control group.CONCLUSION H.pylori can activate the Wnt/β-catenin signaling pathway,thereby facilitating the development and progression of PLGC.Moluodan suppressed the activation of the Wnt/β-catenin signaling pathway,thereby decreasing the progression of PLGC.

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

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

Complement factor Ⅰ knockdown inhibits colon cancer development by affecting Wnt/β-catenin/c-Myc signaling pathway and glycolysis

BACKGROUND Colon cancer(CC)occurrence and progression are considerably influenced by the tumor microenvironment.However,the exact underlying regulatory mechanisms remain unclear.AIM To investigate immune infiltration-related differentially expressed genes(DEGs)in CC and specifically explored the role and potential molecular mechanisms of complement factor I(CFI).METHODS Immune infiltration-associated DEGs were screened for CC using bioinformatics.Quantitative reverse transcription polymerase chain reaction was used to examine hub DEGs expression in the CC cell lines.Stable CFI-knockdown HT29 and HCT116 cell lines were constructed,and the diverse roles of CFI in vitro were assessed using CCK-8,5-ethynyl-2’-deoxyuridine,wound healing,and transwell assays.Hematoxylin and eosin staining and immunohistochemistry staining were employed to evaluate the influence of CFI on the tumorigenesis of CC xenograft models constructed using BALB/c male nude mice.Key proteins associated with glycolysis and the Wnt pathway were measured using western blotting.RESULTS Six key immune infiltration-related DEGs were screened,among which the expression of CFI,complement factor B,lymphoid enhancer binding factor 1,and SRY-related high-mobility-group box 4 was upregulated,whereas that of fatty acid-binding protein 1,and bone morphogenic protein-2 was downregulated.Furthermore,CFI could be used as a diagnostic biomarker for CC.Functionally,CFI silencing inhibited CC cell proliferation,migration,invasion,and tumor growth.Mechanistically,CFI knockdown downregulated the expression of key glycolysis-related proteins(glucose transporter type 1,hexokinase 2,lactate dehydrogenase A,and pyruvate kinase M2)and the Wnt pathway-related proteins(β-catenin and c-Myc).Further investigation indicated that CFI knockdown inhibited glycolysis in CC by blocking the Wnt/β-catenin/c-Myc pathway.CONCLUSION The findings of the present study demonstrate that CFI plays a crucial role in CC development by influencing glycolysis and the Wnt/β-catenin/c-Myc pathway,indicating that it could serve as a promising target for therapeutic intervention in CC.

Activation of the wnt/β-catenin/CYP1B1 pathway alleviates oxidative stress and protects the blood-brain barrier under cerebral ischemia/reperfusion conditions

Accumulating evidence suggests that oxidative stress and the Wnt/β-catenin pathway participate in stroke-induced disruption of the blood-brain barrier.However,the potential links between them following ischemic stroke remain largely unknown.The present study found that cerebral ischemia leads to oxidative stress and repression of the Wnt/β-catenin pathway.Meanwhile,Wnt/β-catenin pathway activation by the pharmacological inhibito r,TWS119,relieved oxidative stress,increased the levels of cytochrome P4501B1(CYP1B1)and tight junction-associated proteins(zonula occludens-1[ZO-1],occludin and claudin-5),as well as brain microvascular density in cerebral ischemia rats.Moreove r,rat brain microvascular endothelial cells that underwent oxygen glucose deprivation/reoxygenation displayed intense oxidative stress,suppression of the Wnt/β-catenin pathway,aggravated cell apoptosis,downregulated CYP1B1and tight junction protein levels,and inhibited cell prolife ration and migration.Overexpression ofβ-catenin or knockdown ofβ-catenin and CYP1B1 genes in rat brain mic rovascular endothelial cells at least partly ameliorated or exacerbated these effects,respectively.In addition,small interfering RNA-mediatedβ-catenin silencing decreased CYP1B1 expression,whereas CYP1B1 knoc kdown did not change the levels of glycogen synthase kinase 3β,Wnt-3a,andβ-catenin proteins in rat brain microvascular endothelial cells after oxygen glucose deprivatio n/reoxygenation.Thus,the data suggest that CYP1B1 can be regulated by Wnt/β-catenin signaling,and activation of the Wnt/β-catenin/CYP1B1 pathway contributes to alleviation of oxidative stress,increased tight junction levels,and protection of the blood-brain barrier against ischemia/hypoxia-induced injury.

Silencing of peroxiredoxin 2 suppresses proliferation and Wnt/β-catenin pathway,and induces senescence in hepatocellular carcinoma

Hepatocellular carcinoma(HCC),a common malignancy worldwide,still lacks effective clinical treatment.The study aimed to investigate the oncogenes that affect the progression of HCC and their possible mechanisms.In our study,we initially confirmed a higher level of PRDX2 in the bile of HCC patients compared to those with choledocholithiasis by 2-DE,LC-MS,and ELISA.Subsequently,we demonstrated the high expression of peroxiredoxin 2(PRDX2)in HCC based on the TCGA database and clinical sample analysis.Furthermore,PRDX2 overexpression enhanced the viability of HCC cells.And PRDX2 silencing induced senescence of HCC cells.In vivo,knockdown of PRDX2 significantly reduced the weight of xenograft tumors.PRDX2 also was found to activate the Wnt/β-catenin pathway by inducingβ-catenin nuclear translocation.Consequently,we proved that silencing PRDX2 could inhibit proliferation and Wnt/β-catenin pathway while promoting senescence in HCC cells.

Calculus bovis inhibits M2 tumor-associated macrophage polarization via Wnt/β-catenin pathway modulation to suppress liver cancer

BACKGROUND Calculus bovis(CB),used in traditional Chinese medicine,exhibits anti-tumor effects in various cancer models.It also constitutes an integral component of a compound formulation known as Pien Tze Huang,which is indicated for the treatment of liver cancer.However,its impact on the liver cancer tumor microenvironment,particularly on tumor-associated macrophages(TAMs),is not well understood.AIM To elucidate the anti-liver cancer effect of CB by inhibiting M2-TAM polarization via Wnt/β-catenin pathway modulation.METHODS This study identified the active components of CB using UPLC-Q-TOF-MS,evaluated its anti-neoplastic effects in a nude mouse model,and elucidated the underlying mechanisms via network pharmacology,transcriptomics,and molecular docking.In vitro assays were used to investigate the effects of CB-containing serum on HepG2 cells and M2-TAMs,and Wnt pathway modulation was validated by real-time reverse transcriptase-polymerase chain reaction and Western blot analysis.RESULTS This study identified 22 active components in CB,11 of which were detected in the bloodstream.Preclinical investigations have demonstrated the ability of CB to effectively inhibit liver tumor growth.An integrated approach employing network pharmacology,transcriptomics,and molecular docking implicated the Wnt signaling pathway as a target of the antineoplastic activity of CB by suppressing M2-TAM polarization.In vitro and in vivo experiments further confirmed that CB significantly hinders M2-TAM polarization and suppresses Wnt/β-catenin pathway activation.The inhibitory effect of CB on M2-TAMs was reversed when treated with the Wnt agonist SKL2001,confirming its pathway specificity.CONCLUSION This study demonstrated that CB mediates inhibition of M2-TAM polarization through the Wnt/β-catenin pathway,contributing to the suppression of liver cancer growth.

MicroRNA-329-3p inhibits the Wnt/β-catenin pathway and proliferation of osteosarcoma cells by targeting transcription factor 7-like 1

An important factor in the emergence and progre sion of osteosarcoma(OS)is the dysregulated expression of microRNAs(miRNAs).Transcription factor 7-like 1(TCF7LI),a member of the T cell factor/lymphoid enhancer factor(TCF/LEF)transcription factor family,interacts with the Wnt signaling pathway regulator β-catenin and acts as a DNA-specific binding protein.This study sought to elucidate the impact of the interaction between miR 3293p and TCF7L1 on.the growth and apoptosis of OS and analyze the regulatory expression relationship between miRNA and mRNA in osteosarcoma cells using a variety of approaches.MiR329-3p was significantly downregulated,while TCF7L1 was considerably up-regulated in all examined OS cell lines.Additionally,a clinical comparison study was performed using the TCGA database.Subsequently,the regulatory relationship between miR-329-3p and TCF7L1 on the proliferation and apoptosis of OS cells was verified through in vitro and in vivo experiments.When miR 329-3p was transfected into the OS cell line,the expression of TCF7L1 decreased,the proliferation of OS cells was inhibited,the cytoskeleton disintegrated,and the nucleus condensed to fom apoptotic bodies.The expression of proteins that indicate apoptosis increased simultaneously.The cell cycle was arrested in the G0/G1 phase,and the G1/S transition was blocked.The introduction of miR 3293p also inhibited downstream Cyclin D1 of the Wnt pathway.Xenograf experiments indicated that the overexpression of miR-329-3p signi ficanly inhibited the growth of OS xenografts in nude mice,and the expression of TCF7L1 and C-Myc in tumor tssues decreased.MiR 329-3p was significantly reduced in OS cells and played a suppressive role in tumorigenesis and proliferation by targeting TCF7L1 both in vitro and in vivo.Osteosarcoma cell cycle arrest and pathway inhibition were observed upon the regulation of TCF7LI by miR 3293p.Summarizing these results,it can be inferred that miR.3293p exerts anticancer efects in osteosarcoma by inhibiting TCF7L1.

Effects of Qigongwan on Wnt/β-catenin Signaling Pathway in Rats with Polycystic Ovary syndrome

[Objectives] To explore the therapeutic effect and mechanism of Qigongwan on PCOS model rats by detecting the changes in sex hormone levels in rats with polycystic ovary syndrome (PCOS), and observing the effects of ovarian pathological morphological changes, apoptosis and expression of Wnt/β-β catenin signaling pathway protein. [Methods] Ten of 40 female SD rats were randomly selected as the normal group, and the other 30 rats were treated with letrozole combined with high-fat diet to establish the PCOS rat model. After successful modeling, the model group was randomly divided into Qigongwan group, positive Daying-35 (Ethinylestradiol and Cyproterone Acetate Tablets) group and model group, with 10 rats in each group. Qigongwan group was given 14.7 g/(kg·d) by gavage, Daying-35 group was given 0.21 mg/(kg·d) by oral gavage, and normal group and model group were given the same amount of distilled water, and the intervention lasted for 21 d. ELISA method was used to detect the levels of hormones such as follicle-stimulating hormone (FSH), luteinizing hormone (LH), testosterone (T), estradiol (E 2) and progesterone (P) in serum. HE staining was used to observe the pathological morphological changes of ovarian tissues;TUNEL staining was used to observe apoptosis of ovarian tissue granule cells;the expression of Wnt, β-catenin protein in rat ovarian tissue was detected by immunohistochemistry. [Results] (i) Compared with the model group, Qigongwan group and Daying-35 group could significantly increase serum E 2 and P levels, significantly reduce serum T levels ( P <0.01), significantly reduce serum LH levels and LH/FSH ratio ( P <0.01), and increase serum FSH levels ( P <0.05) in different degrees. (ii)The results of HE staining showed that compared with the model group, Qigongwan and Daying-35 groups could improve follicular development and reduce atretic follicles in different degrees. Compared with Daying-35 group, the number of GC layers in Qigongwan group was significantly increased. (iii) The results of TUNEL staining showed that compared with the model group, the rate of TUNEL-positive cells in the Qigongwan group and Daying-35 group decreased significantly ( P <0.01). (iv) The immunohistochemical results showed that compared with the model group, the expression levels of wnt and β-catenin in the Qigongwan group and the Daying-35 group increased in different degrees ( P <0.05), and the expression range increased. [Conclusions] Qigongwan can regulate the secretion level of sex hormones such as FSH and LH, improve the pathological damage of ovarian tissue, and inhibit apoptosis of ovarian granule cells, and its mechanism may be related to the activation of Wnt/β-catenin signaling pathway.

TCM Intervention on Wnt/β-Catenin signaling pathway in the treatment of diabetic nephropathy

Diabetic nephropathy(DN)is the most serious microvascular complication of diabetes mellitus,which is highly prevalent worldwide.Abnormal activation of Wnt/β-catenin signaling pathway is an important mechanism of renal damage induced by hyperglycemia.Many studies have shown that TCM has the advantages of high efficiency and safety in the prevention and treatment of DN.Some TCM monomers and compounds repair podocyte function and inhibit transdifferentiation process by inhibiting the activation of Wnt/β-catenin signaling pathway,thus playing a protective role in kidney.Based on this,this paper will review the existing research results and related mechanisms of TCM intervention in Wnt/β-catenin signaling pathway in the treatment of DN,in order to promote the more effective and reasonable application of TCM in clinical practice.

Effects of curcumin on uterine leiomyoma in a rat model by inhibiting β-catenin/Wnt signaling pathway

Background:Women with uterine leiomyomas may suffer severe symptoms.To avoid risks of side effects,it is necessary to develop an optimal agent to shrink leiomyomas with fewer side effects and a lower recurrence rate.Curcumin may have a lower side effect in uterine leiomyoma treatment.Methods:We established the estrogen-and-progesterone-induced murine model of uterine leiomyoma.Next,we determined the expression of related genes of the β-catenin/Wnt signaling pathway by western blot,reverse transcription-polymerase chain reaction,and immunohistochemistry.We also noticed the morphological changes in uterine tissues by hematoxylin-eosin staining.Results:Curcumin plays an important role in Wnt/β-catenin signaling pathway-related expression including β-catenin,adenomatous polyposis coli,glycogen synthase kinase-3β,Wnt-11,and serum hormone concentrations.Conclusions:Curcumin could the down-regulation of serum hormone concentrations and inhibition of the β-catenin/Wnt signaling pathway in the treatment of uterine leiomyoma.

The Role and Mechanism of LncRNA-p21 in Regulating Gastric Cancer Metastasis by Mediating Wnt/β-Catenin Signaling Pathway

Objective:To study the mechanism of lncRNA p21 inhibiting the growth and metastasis of human gastric cancer SGC7901/GES-1 cells by mediating the Wnt/β-catenin signaling pathway.Methods:Lentiviral overexpression of lncRNA-p21 in human gastric cancer SGC7901/GES-1 cell transfections was observed and analyzed for in vitro migration,invasion,cell morphology and proliferation.Besides.Wnt/β-catenin signaling pathway was tested for direct involvement in lncRNA-p21-mediated inhibition of gastric cancer cell growth and proliferation.Wnt/β-catenin signaling pathway was validated using Li-C1.Results:Gastric cancer SGC7901/GES-1 cells in the overexpression of lncRNA-p21 showed changes in stellate morphology,low invasion,or spindle-shaped morphology.LncRNA-p21 inhibited the growth and proliferation of gastric cancer SGC7901/GES-1 cells both in vivo and in vitro,and Wnt/β-catenin signaling pathway mediated the proliferation,invasion,and migration of lncRNA-p21 on gastric cancer SGC7901/GES-1 cells.Conclusion:LncRNA-p21 can inhibit the growth and metastasis of gastric cancer SGC7901/GES-1 cells in vitro and in vivo,and the inhibition of lncRNA p21 is mainly mediated by inhibiting the Wnt/β-catenin signaling pathway.

Epimedium-Derived Flavonoids Modulate the Balance between Osteogenic Differentiation and Adipogenic Differentiation in Bone Marrow Stromal Cells of Ovariectomized Rats via Wnt/β-Catenin Signal Pathway Activation

Objective： To observe the function of wnt/β-catenin signal pathway on the process that epimedium-derived flavonoids （EFs） regulate the balance between osteogenic differentiation and adipogenic differentiation in bone marrow stromal ceils of ovariectomized rats, and to provide an experimental evidence for the mechanism of EFs on treating postmenopausal osteoporosis. Methods： Bone marrow stromal cells from ovariectomized rats were separated and cultivated in the condition of osteoinductive medium or liquid medium for 15 days. Low- （1 μg/mL）, medium- （10 μg/mL） and high- （100 μg/mL） dose EFs were administrated correspondingly. Alkaline phosphatase （ALP） staining, ALP activity determination, oil red O staining and real- time polymerese chain reaction （RT-PCR） were used to determine the effect of EFs on osteogenic differentiation and adipogenic differentiation in bone marrow stromal cells of ovariectomized rats. Moreover, in order to explore the mechanism of EFs on osteogenic differentiation and adipogenic differentiation in bone marrow stromal cells of ovariectomized rats, Dickkopf-related protein 1 （DKK1） was used in the medium group. Enzyme linked immunosorbent assay （ELISA） and RT-PCR were used to determine mRNA levels of 13-catenin, low density lipoprotein receptor-related protein 5 （LRP5） and T cell factor （TCF） protein, known as wnt/β-catenin signal pathway related factors. Results： EFs increased mRNA expression levels of ALP and early osteoblast differentiation factors, such as runt-related transcription factor 2 （Runx2）, osteocaicin and collagen I, and decreased mRNA expression levels of fat generation factors, such as peroxisome proliferator activated receptor gamma 2 （PPAR r/-2） and CCAAT enhancer-binding protein-α （C/EBP α） in a dose-dependent manner. While osteobiast differentiation factors were down-regulated, fat generation factors were up-regulated when DKK1 was applied. Also EFs up-regulated mRNA expression levels of β-catenin, LRP5 and TCF protein which could be blocked by DKK1. Conclusion： EFs regulate the balance between osteogenic differentiation and adipogenic differentiation in bone marrow stromal cells of ovariectomized rats by activating wnt/13-catenin signal pathway, which may be an important molecular mechanism of EFs on treatinq DostmenoDausal osteoDorosis.

Nystose attenuates bone loss and promotes BMSCs differentiation to osteoblasts through BMP and Wnt/β-catenin pathway in ovariectomized mice

Increasing the osteogenic differentiation ability and decreasing the adipogenic differentiation ability of bone marrow mesenchymal stem cells(BMSCs)is a potential strategy for the treatment of osteoporosis(OP).Naturally derived oligosaccharides have shown significant anti-osteoporotic effects.Nystose(NST),an oligosaccharide,was isolated from the roots of Morinda officinalis How.(MO).The aim of the present study was to investigate the effects of NST on bone loss in ovariectomized mice,and explore the underlying mechanism of NST in promoting differentiation of BMSCs to osteoblasts.Administration of NST(40,80 and 160 mg/kg)and the positive control of estradiol valerate(0.2 mg/kg)for 8 weeks significantly prevented bone loss induced by ovariectomy(OVX),increased the bone mass density(BMD),improved the bone microarchitecture and reduced urine calcium and deoxypyridinoline(DPD)in ovariectomized mice,while inhibited the increase of body weight without significantly affecting the uterus weight.Furthermore,we found that NST increased osteogenic differentiation,inhibited adipogenic differentiation of BMSCs in vitro,and upregulated the expression of the key proteins of BMP and Wnt/β-catenin pathways.In addition,Noggin and Dickkopf-related protein-1(DKK-1)reversed the effect of NST on osteogenic differentiation and expression of the key proteins in BMP and Wnt/β-catenin pathway.The luciferase activities and the molecular docking analysis further supported the mechanism of NST.In conclusion,these results indicating that NST can be clinically used as a potential alternative medicine for the prevention and treatment of postmenopausal osteoporosis.

Heterogeneity beyond tumor heterogeneity—SULF2 involvement in Wnt/β-catenin signaling activation in a heterogeneous side population of liver cancer cells

Introduction:Sulfatase 2(SULF2),an endogenous extracellular sulfatase,can remove 6-O-sulfate groups of glucosamine residues from heparan sulfate(HS)chains to modulate the Wnt/β-catenin signaling pathway,which plays an important role in both liver carcinogenesis and embryogenesis.Side population(SP)cells are widely identified as stem-like cancer cells and are closely related to carcinoma metastasis,recurrence,and poor patient prognosis.However,the roles of SULF2 in SP cells of hepatomas are unclear,and the underlying mechanism is undefined.Objectives:This study aimed to compare the heterogeneity between SP cells and non-side population(NSP)cells derived from three different liver cancer cell lines and to elucidate the involvement of the SULF2-Wnt/β-catenin axis in liver cancer stem cells(CSCs)and its impact on the processes of carcinogenesis and invasiveness.Methods:In this work,three different liver cancer SP cells(HepG2,Huh7,and PRC/PRL/5)were sorted by flow cytometry.We also examined the migration and invasion behaviors of SP and NSP cells.To determine if this high tumorigenic potential of SP cells is correlated to SULF2,qPCR,western blotting,and immunofluorescence analysis were conducted.We also performed nude mouse xenograft experiments for in vivo analysis.Results:The results from the in vitro colony formation assay showed that SP cells exhibited a 2-fold higher colony formation efficiency compared to their NSP counterparts.The SP cells exhibited significantly higher potentials in terms of their migratory capacity and invasive ability compared to NSP cells.We found that higher expression of SULF2 in SP cells was associated with greater capabilities for clonogenicity,migration,and invasion.It was also linked to higher activation of the Wnt/β-catenin signaling pathway via stimulation of key downstream factors,particularlyβ-catenin,c-Myc,and cyclin D1.Further,a positive correlation between the upregulated SULF2 expression and tumorigenesis in the in vivo nude mouse xenograft models was demonstrated,highlighting that the potential underlying mechanism was Wnt/β-catenin signaling pathway activation.Conclusion:Our findings show that variable SULF2 expression was associated with differential activation of the Wnt/β-catenin signaling pathway,which could lead to behavioral differences between SP and NSP cells and also among the SP cells of the three liver cancer cell lines assessed.It was reasonably concluded that the SULF2-Wnt/β-catenin axis could play an important role in the tumorigenicity of liver cancer stem cells.

Clinicopathological features and expression of regulatory mechanism of the Wnt signaling pathway in colorectal sessile serrated adenomas/polyps with different syndrome types

BACKGROUND Colorectal cancer(CRC)is the third most common cancer worldwide,with the fourth highest mortality among all cancers.Reportedly,in addition to adenomas,serrated polyps,which account for 15%-30%of CRCs,can also develop into CRCs through the serrated pathway.Sessile serrated adenomas/polyps(SSAs/Ps),a type of serrated polyps,are easily misdiagnosed during endoscopy.AIM To observe the difference in the Wnt signaling pathway expression in SSAs/Ps patients with different syndrome types.METHODS From January 2021 to December 2021,patients with SSAs/Ps were recruited from the Endoscopy Room of Shanghai Traditional Chinese Medicine-Integrated Hospital,affiliated with Shanghai University of Traditional Chinese Medicine.Thirty cases each of large intestine damp-heat(Da-Chang-Shi-Re,DCSR)syndrome and spleen-stomach weakness(Pi-Wei-Xu-Ruo)syndrome were reported.Baseline comparison of the general data,typical tongue coating,colonoscopy findings,and hematoxylin and eosin findings was performed in each group.The expression of the Wnt pathway-related proteins,namelyβ-catenin,adenomatous polyposis coli,and mutated in colorectal cancer,were analyzed using immunohistochemistry.RESULTS Significant differences were observed with respect to the SSAs/Ps size between the two groups of patients with different syndrome types(P=0.001).The other aspects did not differ between the two groups.The Wnt signaling pathway was activated in patients with SSAs/Ps belonging to both groups,which was manifested asβ-catenin protein translocation into the nucleus.However,SSAs/Ps patients with DCSR syndrome had more nucleation,higherβ-catenin expression,and negative regulatory factor(adenomatous polyposis coli and mutated in colorectal cancer)expression(P<0.0001)than SSA/P patients with Pi-Wei-Xu-Ruo syndrome.In addition,the SSA/P size was linearly correlated with the related protein expression.CONCLUSION Patients with DCSR syndrome had a more obvious Wnt signaling pathway activation and a higher risk of carcinogenesis.A high-quality colonoscopic diagnosis was essential.The thorough assessment of clinical diseases can be improved by combining the diseases of Western medicine with the syndromes of traditional Chinese medicine.

Actinidia chinensis Planch.root extract downregulates the Wnt/β-catenin pathway to treat gastric cancer:a mechanism study based on network pharmacology

Background:Actinidia chinensis Planch.roots(AcRoots)have been applied as an anti-inflammatory and antitumor drug in the treatment of gastric cancer(GC).However,their mechanisms against GC cells remain unclear.To investigate the anticancer effect of AcRoots in GC and the possible underlying mechanism by using network pharmacology.Methods:Differentially expressed genes between gastric precancerous lesions and cancer were analyzed in Gene Expression Omnibus datasets,and these genes were overlapped with potential targets of AcRoots.Potential targets and pathways for AcRoots treatment of GC predicted by network pharmacology.Furthermore,we used the GC cell line HGC27 to explore the molecular mechanisms in the context of hub genes in apoptosis,invasion,metastasis,and epithelial to mesenchymal transition-promoting factors.Molecular docking between hub targets and active drug components was also performed.Results:Network pharmacological analysis suggested that the potential mechanism was related to the Wnt pathway and predicted nine hub genes.In in vitro studies,AcRoots significantly decreased HGC27 cell viability and promoted apoptosis by upregulating caspase3 and downregulating Bcl2.Moreover,it suppressed invasion and metastasis as well as the expression of epithelial to mesenchymal transition-related factors.In addition,AcRoots affected the phosphorylation level of GSK3β(Ser9)in the Wnt pathway to promote the degradation ofβ-catenin,resulting in the downregulation of the downstream target genes c-myc,cyclin D1 and snail.All the experimental results were consistent with the network pharmacology results.Conclusion:This study combined network pharmacology with in vitro experiments to provide valid evidence for the clinical promotion of AcRoots.

Schisandra B inhibits proliferation, migration and invasion of bladder cancer by regulating the Wnt/β‑catenin signaling pathway

Objective:To investigate the effects of Schisandra B on proliferation,migration,invasion of bladder cancer and to further investigate its molecular mechanism.Methods:Bladder cancer cells were subjected to different concentrations of Schisandra B solution(0,20,40,80μmol/L).CCK-8 assay was used to detect the effect of schisandra B on bladder cancer cell proliferation.Transwell migration assay and wound healing assay were used to detect the effect of Schisandra B on the migration of bladder cancer cells.Transwell invasion assay was used to detect the effect of schisandra B on invasion ability of bladder cancer cells.The expression levels of intracellularβ-catenin and c-myc protein were measured by western blot.Results:Schisandra B inhibited the proliferation of T24 and UM-UC-3 cells in a concentration and time dependent manner(P<0.05).The rate of wound healing and number of migration and invasion cells decreased with the increase of Schisandra B concentration(P<0.05).The expression ofβ-catenin and c-myc decreased after treatment with Schisandra B in bladder cancer cells(P<0.05).Conclusion:Schisandra B can inhibit the proliferation,migration and invasion of human bladder cancer T24 and UM-UC-3 cells,and the main mechanism for its inhibitory effect may be related to the inactivation of the Wnt/β-catenin signaling pathway.

Exosomal miR-320e through wnt2targeted inhibition of the Wnt/β-catenin pathway allevisate cerebral small vessel disease and cognitive impairment

BACKGROUND Exosomal miRNAs play crucial roles in many central nervous system diseases.Cerebral small vessel disease(CVSD)is a small vessel disease that is affected by various factors.This study aimed to investigate the role of exosomal miR-320e in the Wnt/β-catenin pathway stimulated by oxidative stress and assess its clinical correlation with psychiatric symptoms in patients with CVSD.AIM To explore whether exosomal miR-320e could suppress the Wnt/β-catenin pathway and play a protective role in CVSD progression,as well as examine its potential correlation with cognitive impairment and depression in patients with CVSD.METHODS Differentially expressed exosomal miRNAs were filtered by sequencing plasma exosomes from patients with CVSD and healthy controls.Bioinformatics and dual luciferase analyses were used to confirm the binding of miR-320e to Wnt2,and the mRNA and protein levels of downstream components in the Wnt/β-catenin pathway were evaluated when overexpressed or with knockdown of miR-320e under H2O2-induced oxidative stress.In addition,Wnt2-targeting siRNA was used to confirm the role of miR-320e in the Wnt2-mediated inhibition of the Wnt/β-catenin pathway.A retrospective analysis was conducted among patients with CVSD to confirm the correlation between miR-320e expression and the severity of cognitive impairment and depression,which were quantified using the Montreal Cognitive Assessment(MoCA)/Executive Function Assessment(EFA),and the Hamilton Depression Scale(HAMD)/Beck Depression Inventory(BDI),respectively.RESULTS High-throughput sequencing revealed that exosomal miR-320e was downregulated in patients with CVSD.Bioinformatics analysis and dual-luciferase reporter gene experiments showed that exosomal miR-320e inhibited the Wnt/β-catenin pathway in response to oxidative stress by targeting the 3'noncoding region of Wnt2.Uptake of exosomes carrying miR-320e into endothelial cells could also target Wnt2 and inhibit the Wnt2/β-catenin pathway.Elevated miR-320e expression may protect patients with CVSD from relatively severe cognitive impairment and depression,as it was found to have a positive correlation with the MoCA/EFA and HAMD/BDI scores.CONCLUSION Our results suggest that exosomal miR-320e suppresses the Wnt/β-catenin pathway and may play a protective role in CVSD progression.