DANCR Regulates hESC Differentiation Towards Definitive Endoderm / 中山大学学报(医学科学版)

ObjectiveTo explore the function of DANCR during the differentiation of human embryonic stem cells （hESC） toward definitive endoderm （DE）. MethodsThe in vitro DE differentiation system was established and its efficiency was verified. The correlation between the expression level of DANCR and DE differentiation process was detected. Using lentivirus system， we stably knocked down DANCR in hESC. The shDANCR hESC line was applied to DE differentiation， using qPCR and Western blot to detect the expression of DE marker genes SOX17 and FOXA2， and that of primitive streak marker genes Brachyury （T）， EOMES， MIXL1 and GSC. Dual luciferase reporter assay and qPCR were used to confirm the interaction between DANCR and the WNT pathway during DE differentiation. ResultsThe in vitro differentiation system mimicked DE differentiation efficiently. And the expression of DANCR was gradually downregulated during differentiation. DANCR was efficiently knocked down in the shDANCR hESC line （P < 0.001）. Compared with those in the control group， the expression levels of primitive markers Brachyury （T）， EOMES， MIXL1 and GSC， as well as DE markers SOX17 and FOXA2， were significantly decreased in shDANCR groups （P < 0.05）. Furthermore， the transcriptional activity of the WNT pathway in shDANCR groups was lower than that in the control group （P < 0.05）. And RNA levels of downstream genes of the WNT pathway， FZD5， FZD8， SFRP1， FRZB and ANKRD6， were significantly decreased in shDANCR groups （P < 0.05）. However， differences in protein levels of the TGFβ pathway effectors SMAD2/3 and p-SMAD2 were statistically insignificant in shDANCR and control groups （P > 0.05）. Forced activation of β-CATENIN rescued DANCR knock down-induced deficiency in DE differentiation. ConclusionsThe expression of DANCR decreases during DE differentiation. DANCR may promote DE differentiation through modulating the activity of the WNT pathway.

Correlation Between Wnt5a Expression and Vasculogenic Mimicry in Prostate Cancer Tissues / 肿瘤防治研究

Objective To investigate the correlation of Wnt5a expression and vasculogenic mimicry (VM) in prostate cancer tissues, and analyze their relationships with cancer stem cells (CSCs) characteristics and epithelial–mesenchymal transition (EMT). Methods Immunohistochemistry was conducted to detect the expression of Wnt5a in 50 prostate cancer tissues and 50 benign prostatic hyperplasia tissues. The expression levels of CD133, vimentin, and E-cadherin were detected in the prostate cancer tissues, and CD34/PAS double staining was used to detect VM structures. We analyzed the difference in Wnt5a level between prostate cancer and benign prostatic hyperplasia tissues, the clinical significance of Wnt5a and VM, the relationship of Wnt5a expression and VM, and the relationships of Wnt5a expression and VM with CD133, Vimentin, E-cadherin. Results The expression of Wnt5a was significantly higher in prostate cancer tissues than in benign prostatic hyperplasia (P < 0.05). A positive correlation was observed between Wnt5a expression and VM (P < 0.05). The expression levels of Wnt5a and VM were positively correlated with those of CD133 and vimentin (P < 0.05). Wnt5a expression and VM were positively correlated with Gleason score, vas deferens invasion and lymphatic metastasis (P < 0.05) of prostate cancer, and VM was also positively correlated with T stage of prostate cancer (P < 0.05). Conclusion The expression level of Wnt5a in prostate cancer tissues is elevated and positively related with VM formation. Wnt5a expression and VM are correlated with cancer stem cells characteristics and the expression of epithelial–mesenchymal transition marker proteins.

Sphingomyelin synthase 2 regulates the malignant biological behaviors of ovarian cancer TOV-21G cells through Wnt/β-catenin pathway / 中国肿瘤生物治疗杂志

@#[摘 要] 目的：探讨鞘磷脂合成酶2（SMS2）是否通过Wnt/β-catenin信号通路调控卵巢癌（OC）TOV-21G细胞增殖、迁移、侵袭、凋亡及其机制。方法：收集武汉市第三医院2022年7月至2023年5月间确诊的21例OC患者的癌及癌旁组织标本，免疫组化法检测OC组织SMS2表达水平。体外培养TOV-21G细胞，将细胞分为对照组、shRNA慢病毒阴性对照组（sh-NC组）、SMS2 shRNA慢病毒组（sh-SMS2组）、Wnt/β-catenin通路激活剂组LiCl（LiCl组）和sh-SMS2+LiCl组。Edu染色法、Transwell法、流式细胞术分别检测各组细胞的增殖、迁移和侵袭能力及凋亡水平，WB法检测细胞中SMS2、Ki67、cyclin D1、BAX、c-caspase3、Bcl-2及Wnt/β-catenin通路蛋白（β-catenin、c-Myc、MMP-9）的表达。构建TOV-21G细胞裸鼠移植瘤模型，观察敲低SMS2对移植瘤生长和SMS2、β-catenin表达的影响。结果：与癌旁组织比较，OC组织中SMS2呈高表达（P<0.01）。转染sh-SMS2后，TOV-21G细胞中SMS2表达水平显著降低（P<0.05），细胞的增殖、迁移和侵袭能力及Bcl-2、β-catenin、c-Myc、MMP-9蛋白表达均显著降低（均P<0.05），细胞凋亡率、BAX、c-caspase3蛋白表达均显著升高（均P<0.05）；LiCl处理能逆转敲低SMS2对TOV-21G细胞增殖、迁移和侵袭及Wnt/β-catenin通路的抑制作用（均P<0.05）。体内成瘤实验显示，敲低SMS2抑制裸鼠移植瘤的生长及SMS2、β-catenin蛋白的表达（均P<0.05）。结论：敲低SMS2表达通过Wnt/β-catenin信号通路抑制OC TOV-21G细胞的增殖、迁移、侵袭并促进细胞凋亡，同时LiCl处理则能逆转敲低SMS2对TOV-21G细胞增殖、迁移和侵袭的抑制作用。

Kidney-tonifying and Blood-activating Prescription Regulates Wnt/β-catenin Signaling Pathway to Improve Uterine Spiral Artery Remodeling in Mouse Model of Recurrent Miscarriage / 中国实验方剂学杂志

ObjectiveTo observe the effects of the kidney-tonifying and blood-activating prescription on the Wnt/β-catenin signaling pathway and uterine spiral artery remodeling in a mouse model of recurrent miscarriage and to explore its underlying mechanism. MethodA mouse model of normal pregnancy was established by mating CBA/J mice with BALB/c mice. A mouse model of recurrent miscarriage was established by mating CBA/J mice with DBA/2 mice. The modeled mice of recurrent miscarriage were randomized into model， dydrogesterone， and low- and high-dose Chinese medicine groups. The mice in normal pregnancy were used as the control group. Each group consisted of 10 mice， and the drug administration lasted for 14 days. After the treatment， the embryo absorption rate of each group was recorded. Hematoxylin-eosin （HE） staining was employed to observe the pathological morphology of the uterine decidua， and the physiological transformation rate of spiral arteries （SPA） was evaluated. Real-time polymerase chain reaction （Real-time PCR） and Western blot were performed to determine the mRNA and protein levels， respectively， of matrix metalloproteinases （MMP）-2， MMP-9， vascular endothelial growth factor （VEGF）， and Wnt/β-catenin signaling pathway. ResultCompared with the control group， the model group presented increased embryo absorption rate （P<0.05）， decreased physiological transformation rate of uterine SPA （P<0.05）， cellular swelling， degeneration， and disordered arrangement in the uterine decidua tissue， and down-regulated mRNA and protein levels of key factors involved in SPA remodeling （MMP-2， MMP-9， VEGF） and the Wnt/β-catenin signaling pathway （Wnt2， β-catenin， Cyclin D1， c-Myc） （P<0.05）. Compared with the model group， both the low- and high-dose Chinese medicine reduced embryo absorption rate （P<0.05）， increased SPA physiological transformation rate （P<0.05）， improved uterine decidua tissue morphology， and increased decidua vessel count. Furthermore， they up-regulated the mRNA and protein levels of MMP-2， MMP-9， VEGF， and proteins in the Wnt/β-catenin signaling pathway （P<0.05）. ConclusionRecurrent miscarriage is associated with impaired uterine spiral artery remodeling. The kidney-tonifying and blood-activating prescription can promote uterine spiral artery remodeling by activating the Wnt/β-catenin signaling pathway and promoting the expression of VEGF， MMP-2， and MMP-9， thus treating recurrent miscarriage.

Down-regulation of METTL5 inhibits proliferation, migration and invasion of triple-negative breast cancer cells through Wnt/6-catenin signaling pathway / 中国药理学通报

Aim To investigate the role and potential mechanism of methyltransferase-like 5 (METTL5) in triple-negative breast cancer (TNBC) . Methods The expression of METTL5 in TNBC tumor tissues and cell lines was detected by immunohistochemistry and Western blot. After shRNA targeting METTL5 (shRNAMETTL5) was transfected into TNBC cells, cell proliferation, migration and invasion were detected by CCK-8, colony formation, wound healing and Transwell assays, respectively. Western blot was used to detect the expression of Wnt/p-catenin signaling-related key proteins. A xenograft tumor model was constructed to verify the effect of METTL5 knockdown on the growth of TNBC cells and Wnt/p-catenin signaling activity in vivo. Results The expression of METTL5 was up-regulated in TNBC tumor tissues and cell lines (P < 0. 01) . Knockdown of METTL5 significantly inhibited the proliferation, migration and invasion of TNBC cells and reduced the expression of Wnt/p-catenin signaling molecules (3-catenin, cyclin Dl, matrix metalloproteinase (MMP) -2 and MMP-7 (all P < 0. 01) . Knockdown of METTL5 reduced tumor growth and Wnt/pcatenin signaling activity in vivo. Conclusions Knockdown of METTL5 can inhibit the proliferation, migration and invasion of TNBC cells, which may be related to the inhibition of Wnt/p-catenin signaling pathway.

Research progress on bone metabolism and molecular mechanisms in accelerated aging SAMP6 mice / 中国药理学通报

Senile osteoporosis (SOP) is a systemic bone disease characterized by increased susceptibility to fractures. The pathogenesis of SOP is complex and not well understood. Currently, the rapid aging model mouse, senescence accelerated mouse prone 6 (SAMP6), is an ideal model for studying the mechanisms of SOP development and exploring its prevention and treatment. This model exhibits characteristics including increased bone fragility, degradation of bone microstructure, loss of bone matrix, and abnormal metabolism and dysfunction of bone cells, faithfully replicating the process of SOP occurrence and progression at both macroscopic and microscopic levels.

Role of Wnt signaling Pathway in Diabetic Osteoporosis and Traditional Chinese Medicine Intervention： A Review / 中国实验方剂学杂志

Diabetic osteoporosis （DOP） is a kind of bone complication caused by diabetes， which is characterized by the decrease of bone mineral density， the change of bone microstructure and the increase of bone fragility. The process of DOP is closely related to high glucose， insulin resistance， oxidative stress and other mechanisms. The Wnt/β-catenin signaling pathway plays an important role in mediating insulin resistance and bone metabolic balance in diabetes. Regulation of Wnt signal transduction promotes the expression of glycogen synthase kinase-3β（GSK-3β）phosphorylation and improves glucose and lipid metabolism. The Wnt/β-catenin signaling pathway is also an important way regulating osteocyte-driven bone remodeling， which not only plays an important regulatory role in the balance between osteoblasts and osteoclasts and improve bone metabolic homeostasis， but also promotes the expression of osteopontin， osteocalcin and type Ⅰ collagen， and improves bone proliferation and osteogenic differentiation by regulating the Wnt pathway. In recent years， the research of traditional Chinese medicine （TCM） in the prevention and treatment of DOP has gradually increased， and the exploration of TCM to interfere with the Wnt pathway to improve DOP has made some progress. This paper collects and summarizes the studies on the Wnt signaling pathway in glucose metabolism， bone metabolism and DOP worldwide in the past decade， as well as the related literature on the intervention of DOP by TCM compounds （classical and other compounds）， single Chinese medicine and TCM monomers based on the Wnt pathway， in order to provide a reference and direction for the development of new drugs for clinical prevention and treatment of DOP.

Prevention and Treatment of Lung Cancer by Traditional Chinese Medicine Targeting Wnt/β-catenin Signaling Pathway： A Review / 中国实验方剂学杂志

Lung cancer is one of the most common malignant tumors in the world， with its morbidity and mortality ranking at the top. The early symptoms are not obvious， and the biological structure is complex， so many patients have missed the optimal treatment time. At present， the treatment of lung cancer in modern medicine is dominated by first-line chemotherapy and surgical treatment with platinum-containing regimen， which has relatively large side effects， poor prognosis， and a high risk of metastasis and recurrence. With the gradual rise of targeted therapy and immunotherapy for lung cancer， the overall recovery of patients with lung cancer is still poor and the survival rate is low， despite more abundant treatment methods. From the perspective of holistic concept and syndrome differentiation， traditional Chinese medicine （TCM） plays an important role in the prognosis of tumor patients， with many targets， a wide range and light toxic and side effect. Modern studies have shown that the occurrence and development of lung cancer are closely related to the abnormality of multiple signaling pathways， and the Wnt/β-catenin signaling pathway， as one of the most important pathways in cancer， is involved in the whole process of lung cancer development by regulating the expression of related signaling proteins and genes. In recent years， many studies have confirmed that TCM monomers and TCM compounds can inhibit the epithelial-mesenchymal transition （EMT） process of lung cancer and the activity of lung cancer stem cells （LCSCs） by regulating the Wnt/β-catenin signaling pathway， induce lung cancer cell apoptosis， inhibit the proliferation， invasion and migration of lung cancer cells， and thus play an anti-lung cancer role. In recent years， research in this field has made breakthroughs， but there is a lack of systematic reviews and summaries. Thus， this paper reviewed relevant literature worldwide to analyze and interpret the mechanism of TCM intervention in the Wnt/β-catenin signaling pathway against lung cancer. The TCM monomers targeted to regulate this signaling pathway were summarized in four categories： promoting blood circulation for removing blood stasis， clearing heat and removing dampness， clearing heat and removing toxicity， and awakening the spirit. TCM compounds included Buzhong Yiqitang， Xuefu Zhuyutang， et al. This study aims to provide new ideas for clinical research and drug development for lung cancer.

Mechanism of Qingre Huayu Jianpi Prescription Inhibiting Development of Colitis-associated Colorectal Cancer in Mice / 中国实验方剂学杂志

ObjectiveTo explore the effect of Qingre Huayu Jianpi prescription （QHJ） on colitis-associated colorectal cancer （CAC） in mice， and its related mechanism. MethodC57BL/6 mice were randomly divided into four groups including the normal， model， QHJ low-dose （QHJ-L， 10 g·kg-1）， and QHJ high-dose （QHJ-H， 40 g·kg-1） groups. Azoxymethane （AOM） and dextran sodium sulfate （DSS） were combined to chemically build a CAC mouse model for 14 weeks. Each drug group was given intragastrically from the 5th week to the 14th week， once per day. An equal volume of water was fed to the normal and model groups. The mouse survival rate， colon length， weight， and pathological alterations were assessed. The protein expressions of Wnt-3a protein signaling （Wnt3a）， β-catenin， Non-phosphor-β-catenin （Non-p-β-catenin）， and cholesterol-binding glycoproteins 133 （CD133） were detected by Western blot. The localization and expression of the cluster of differentiation （CD） 80 and CD11 antigen-like family member B （CD11b） were detected by immunohistochemistry （IHC）. The colon organoids derived from CAC mice were isolated and cultured to detect the expression of Wnt signaling pathway-related proteins. ResultThe survival rate of the CAC mice was improved by QHJ treatment and the number of colon tumors was inhibited significantly. Compared with those in the normal group， the expression levels of Wnt3a， β-catenin， Non-p-β-catenin， and CD133 in colon tissues in the model group were significantly increased （P<0.05， P<0.01）. Compared with those in the model group， the levels of Wnt3a and β-catenin in the QHJ-L group were significantly decreased （P<0.01）， and the protein levels of Wnt3a， β-catenin， Non-p-β-catenin， and CD133 in the QHJ-H group were significantly decreased （P<0.05， P<0.01）. Meanwhile， the expression level of CD11b in the model group was significantly increased compared with that in the normal group while the CD80 level was significantly decreased （P<0.05， P<0.01）. Compared with those in the model group， CD11b in QHJ-L and QHJ-H groups was significantly decreased， and CD80 was significantly increased（P<0.05， P<0.01）. The expressions of Non-p-β-catenin and CD133 in colonic organoids of CAC model mice were significantly increased， while QHJ treatment could inhibit the expressions of Non-p-β-catenin and CD133 in colonic organoids （P<0.01）. ConclusionQHJ could inhibit the inflammation-cancer development in CAC mice， the mechanism of which might be related to regulating the microenvironment and inhibiting the over-activation of Wnt signaling.

Research progress of traditional Chinese medicine monomer and compound intervention on the Wnt signaling pathway in regulating bone metabolism / 中国药房

Bone metabolism refers to the decomposition and anabolism occurring during bone remodeling， and its balance is regulated by bone resorption and bone formation. A slight deviation of this balance causes various skeletal diseases， such as osteoporosis and renal osteodystrophy. Traditional Chinese medicine （TCM） monomers and compounds have certain advantages in treating bone metabolism diseases. The Wnt signaling pathway includes the canonical Wnt signaling pathway， dependent on β-catenin， and the non-canonical Wnt signaling pathway， independent of β-catenin. Both types of pathways can maintain bone metabolism balance by regulating bone formation and bone resorption and are essential for bone development， bone mass maintenance， and bone remodeling. A variety of TCM monomers （albiflorin， catalpol and icariin） and formulas （Zuogui pill， Yishen gugu prescription， Duzhong jiangu prescription， etc.） have been confirmed to promote differentiation of bone marrow mesenchymal stem cells， proliferation and differentiation of osteoblasts， bone injury repair， and osteoporosis improvement by activating the Wnt signaling pathway in recent years. Here， this article summarizes the research progress in the Wnt signaling pathway regulation of bone metabolism by TCM monomers and compounds to provide ideas for the clinical application of TCM and the research and development of new drugs for the prevention and treatment of bone metabolism diseases.

Mechanism of Medicated Serum of Buyang Huanwutang in Inhibiting Endothelial-to-mesenchymal Transition of Human Pulmonary Artery Endothelial Cells Based on Wnt1/β-catenin Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo investigate the role of the Wnt1/β-catenin signaling pathway in the intervention of medicated serum of Buyang Huanwutang （BYHWT） in endothelial-to-mesenchymal transition （EndMT） of human pulmonary artery endothelial cells （HPAECs） as well as its related mechanisms. MethodMedicated serum of BYHWT was prepared by gavage to New Zealand rabbits with a dosage of 53.36 g·kg-1·d-1 after decocting the medicine as usual. In addition， the same volume of normal saline was used to prepare blank serum. The HPAECs were cultured in vitro， and then induced by the transforming growth factor-β1 （TGF-β1） to establish the EndMT model. Five groups were established： blank group （10% blank serum）， model group （TGF-β1+10% blank serum）， low-dose BYHWT group （TGF-β1+2.5% medicated serum+7.5% blank serum）， medium-dose BYHWT group （TGF-β1+5% medicated serum+5% blank serum） and high-dose BYHWT group （TGF-β1+10% medicated serum）. Through Western blot， the expressions of Wnt1， β-catenin， and glycogen synthase kinase-3β （GSK-3β） were detected. In order to further clarify the mechanism of the Wnt1/β-catenin signaling pathway in the intervention of the medicated serum of BYHWT in inhibiting EndMT， the overexpression of β-catenin was confirmed by polymerase chain reaction after plasmid of overexpression β-catenin was constructed and transfected into the HPAECs. The HPAECs were intervened by 10% medicated serum with the optimal effect in previous studies. Then， they were divided into another five groups： the blank group （10% blank serum）， the model group （TGF-β1+10% blank serum）， the BYHWT group （TGF-β1+10% medicated serum）， the BYHWT+overexpression plasmid control group （TGF-β1+10% medicated serum+blank plasmid） and the BYHWT+β-catenin overexpression plasmid group （TGF-β1+10% medicated serum+β-catenin）. Apart from that， cell proliferation ability was detected by the methyl thiazolyl tetrazolium （MTT） method and cell migration ability by scratch assay and Transwell assay together. Immunofluorescence was adopted to detect the expressions of platelet endothelial cell adhesion molecule （PECAM-1/CD31）， vascular endothelial cadherin （VE-cadherin）， fibroblast-specific protein 1 （FSP1）， and α-smooth muscle actin （α-SMA）. ResultIn comparison to the blank group， the expressions of Wnt1 and β-catenin were significantly increased （P<0.01） while the expression of GSK-3β significantly decreased （P<0.01） in the model group. In comparison to the model group， the expressions of Wnt1 and β-catenin were significantly decreased （P<0.01） while the expression of GSK-3β was significantly increased （P<0.01） in the high-dose BYHWT group. The expression of β-catenin was significantly decreased （P<0.01） while the expression of GSK-3β was significantly increased （P<0.01） in the medium-dose BYHWT group. There was no significant difference in these indexes of the low-dose BYHWT group. In comparison to the blank group， proliferation and migration abilities were remarkably increased （P<0.01） and the immunofluorescence intensities of CD31 and VE-cadherin were decreased， while those of FSP1 and α-SMA were increased in the model group. In comparison to the model group， proliferation and migration abilities were significantly decreased （P<0.01） and the immunofluorescence intensities of CD31 and VE-cadherin were increased， while those of FSP1 and α-SMA diminished in the BYHWT group. Beyond that， the change trend of those indexes in the BYHWT+β-catenin overexpression plasmid group was consistent with that in the model group. In comparison to the BYHWT+overexpression plasmid control group， proliferation and migration abilities were significantly increased （P<0.01） and the immunofluorescence intensities of CD31 and VE-cadherin were decreased， while those of FSP1 and α-SMA were increased in the BYHWT+β-catenin overexpression plasmid group. ConclusionMedicated serum of BYHWT can inhibit EndMT of HPAECs by the Wnt1/β-catenin signaling pathway.

A novel TNKS/USP25 inhibitor blocks the Wnt pathway to overcome multi-drug resistance in TNKS-overexpressing colorectal cancer

Modulating Tankyrases (TNKS), interactions with USP25 to promote TNKS degradation, rather than inhibiting their enzymatic activities, is emerging as an alternative/specific approach to inhibit the Wnt/β-catenin pathway. Here, we identified UAT-B, a novel neoantimycin analog isolated from Streptomyces conglobatus, as a small-molecule inhibitor of TNKS-USP25 protein-protein interaction (PPI) to overcome multi-drug resistance in colorectal cancer (CRC). The disruption of TNKS-USP25 complex formation by UAT-B led to a significant decrease in TNKS levels, triggering cell apoptosis through modulation of the Wnt/β-catenin pathway. Importantly, UAT-B successfully inhibited the CRC cells growth that harbored high TNKS levels, as demonstrated in various in vitro and in vivo studies utilizing cell line-based and patient-derived xenografts, as well as APCmin/+ spontaneous CRC models. Collectively, these findings suggest that targeting the TNKS-USP25 PPI using a small-molecule inhibitor represents a compelling therapeutic strategy for CRC treatment, and UAT-B emerges as a promising candidate for further preclinical and clinical investigations.

Eucommia granules activate Wnt/β-catenin pathway, and improve oxidative stress, inflammation, and endothelial injury in preeclampsia rats

ABSTRACT Purpose: Pre-eclampsia (PE) is a pregnancy-related complication. Eucommia is effective in the treatment of hypertensive disorders in pregnancy, but the specific effects and possible mechanisms of Eucommia granules (EG) in PE remain unknown. The aim of this study was to investigate the effects and possible mechanisms of EG in PE rats. Methods: Pregnant Sprague Dawley rats were divided into five groups (n = 6): the control group, the model group, the low-dose group, the medium-dose group, and the high-dose group of EG. The PE model was established by subcutaneous injection of levonitroarginine methyl ester. Saline was given to the blank and model groups, and the Eucommia granules were given by gavage to the remaining groups. Blood pressure and urinary protein were detected. The body length and weight of the pups and the weight of the placenta were recorded. Superoxide dismutase (SOD) activity and levels of malondialdehyde (MDA), placental growth factor (PIGF), and soluble vascular endothelial growth factor receptor-1 (sFIt-1) were measured in the placenta. Pathological changes were observed by hematoxylin-eosin staining. Wnt/β-catenin pathway-related protein expression was detected using Western blot. Results: Compared with the model group, the PE rats treated with EG had lower blood pressure and urinary protein. The length and weight of the pups and placental weight were increased. Inflammation and necrosis in the placental tissue was improved. SOD level increased, MDA content and sFIt-1/PIGF ratio decreased, and Wnt/β-catenin pathway-related protein expression level increased. Moreover, the results of EG on PE rats increased with higher doses of EG. Conclusions: EG may activate the Wnt/β-catenin pathway and inhibit oxidative stress, inflammation, and vascular endothelial injury in PE rats, thereby improving the perinatal prognosis of preeclamptic rats. EG may inhibit oxidative stress, inflammation, and vascular endothelial injury through activation of the Wnt/β-catenin pathway in preeclampsia rats, thereby improving perinatal outcomes in PE rats.

Exploring the role of the WNT5A rs566926 polymorphism and its interactions in non-syndromic orofacial cleft: a multicenter study in Brazil

Abstract Associations between the WNT5A rs566926 variant and non-syndromic orofacial cleft (NSOC) have been reported in different populations. Objective This study aimed to investigate the role of the rs566926 single nucleotide polymorphism (SNP) in WNT5A and its interactions with SNPs in BMP4, FGFR1, GREM1, MMP2, and WNT3 in the occurrence of NSOC in a Brazilian population. Methodology A case-control genetic association study was carried out involving participants from four regions of Brazil, totaling 801 patients with non-syndromic cleft lip with or without cleft palate (NSCL±P), 273 patients with cleft palate only (NSCPO), and 881 health volunteers without any congenital condition (control). Applying TaqMan allelic discrimination assays, we evaluated WNT5A rs566926 in an ancestry-structured multiple logistic regression analysis, considering sex and genomic ancestry as covariates. Interactions between rs566926 and variants in genes involved in the WNT5A signaling pathway (BMP4, FGFR1, GREM1, MMP2, and WNT3) were also explored. Results WNT5A rs566926 was significantly associated with an increased risk of NSCL±P, particularly due to a strong association with non-syndromic cleft lip only (NSCLO), in which the C allele increased the risk by 32% (OR: 1.32, 95% CI: 1.04-1.67, p=0.01). According to the proportions of European and African genomic ancestry, the association of rs566926 reached significant levels only in patients with European ancestry. Multiple interactions were detected between WNT5A rs566926 and BMP4 rs2071047, GREM1 rs16969681 and rs16969862, and FGFR1 rs7829058. Conclusion The WNT5A rs566926 polymorphism was associated with NSCL±P, particularly in individuals with NSCLO and high European ancestry. Epistatic interactions involving WNT5A rs566926 and variants in BMP4, GREM1, and FGFR1 may contribute to the risk of NSCL±P in the Brazilian population.

Genetic polymorphism of WNT9A is functionally associated with thumb osteoarthritis in the Chinese population

Abstract Background In a recent genome-wide association study, novel genetic variations of WNT9A were reported to be involved in the etiopathogenesis of thumb osteoarthritis (TOA) in Caucasians. Our purposes were to replicate the association of WNT9A with the development of TOA in the Chinese population and to further unveil the functional role of the risk variants. Methods SNP rs11588850 of WNT9A were genotyped in 953 TOA patients and 1124 healthy controls. The differences of genotype and allele distributions between the patients and healthy controls were evaluated using the Chi-square test. Luciferase Reporter Assay was performed to investigate the influence of variant on the gene expression. Results There was significantly lower frequency of genotype AA in TOA patients than in the controls 74.9% vs. 81.9%, p < 0.001). The frequency of allele A was remarkably lower in the patients than in the controls (86.3% vs. 90.5%, p < 0.001), with an odds ratio of 0.66 (95% CI = 0.54-0.80). Luciferase Reporter Assay showed that the construct containing mutant allele G of rs11588850 displayed 29.1% higher enhancer activity than the wild allele A construct (p < 0.05). Conclusions Allele G of rs11588850 was associated with the increased risk of TOA possibly via up-regulation of WNT9A expression. Further functional analysis into the regulatory role of rs11588850 in WNT9A expression can shed new light on the genetic architecture of TOA. Key Points Genetic variants of WNT9A were associated with the incidence and severity of TOA. Allele G of rs11588850 was associated with an increased transcriptional activity of WNT9A promoter. Allele G of rs11588850 may add to the risk of TOA possibly via up-regulation of WNT9A expression. Further functional analysis into the regulatory role of rs11588850 in WNT9A expression can shed new light on the genetic architecture of TOA.

Recent advances in prostate cancer: WNT signaling, chromatin regulation, and transcriptional coregulators / 亚洲男科学杂志(英文版)

Prostate cancer is one of the most common diseases in men worldwide. Surgery, radiation therapy, and hormonal therapy are effective treatments for early-stage prostate cancer. However, the development of castration-resistant prostate cancer has increased the mortality rate of prostate cancer. To develop novel drugs for castration-resistant prostate cancer, the molecular mechanisms of prostate cancer progression must be elucidated. Among the signaling pathways regulating prostate cancer development, recent studies have revealed the importance of noncanonical wingless-type MMTV integration site family (WNT) signaling pathways, mainly that involving WNT5A, in prostate cancer progression and metastasis; however, its role remains controversial. Moreover, chromatin remodelers such as the switch/sucrose nonfermentable (SWI/SNF) complex and chromodomain helicase DNA-binding proteins 1 also play important roles in prostate cancer progression through genome-wide gene expression changes. Here, we review the roles of noncanonical WNT signaling pathways, chromatin remodelers, and epigenetic enzymes in the development and progression of prostate cancer.

Effect of total flavonoids of buckwheat flower and leaf on myocardial cell apoptosis and Wnt/β-catenin/PPARγ pathway in arrhythmic rats / 中国中药杂志

This paper aimed to investigate the effect of total flavonoids of buckwheat flower and leaf on myocardial cell apoptosis and Wnt/β-catenin/peroxisome proliferator-activated receptor γ(PPARγ) pathway in arrhythmic rats. SD rats were randomly divided into a control group, a model group, a low-dose(20 mg·kg~(-1)) group of total flavonoids of buckwheat flower and leaf, a medium-dose(40 mg·kg~(-1)) group of total flavonoids of buckwheat flower and leaf, a high-dose(80 mg·kg~(-1)) group of total flavonoids of buckwheat flower and leaf, a propranolol hydrochloride(2 mg·kg~(-1)) group, with 12 rats in each group. Except the control group, rats in other groups were prepared as models of arrhythmia by sublingual injection of 1 mL·kg~(-1) of 0.002% aconitine. After grouping and intervention with drugs, the arrhythmia, myocardial cells apoptosis, myocardial tissue glutathione peroxidase(GSH-Px), catalase(CAT), malondialdehyde(MDA), serum interleukin-6(IL-6), prostaglandin E2(PGE2) levels, myocardial tissue apoptosis, and Wnt/β-catenin/PPARγ pathway-related protein expression of rats in each group were measured. As compared with the control group, the arrhythmia score, the number of ventricular premature beats, ventricular fibrillation duration, myocardial cell apoptosis rate, MDA levels in myocardial tissues, serum IL-6 and PGE2 levels, Bax in myocardial tissues, and Wnt1 and β-catenin protein expression levels increased significantly in the model group, whereas the GSH-Px and CAT levels, and Bcl-2 and PPARγ protein expression levels in myocardial tissues reduced significantly. As compared with the model group, the arrhythmia score, the number of ventricular premature beats, ventricular fibrillation duration, myocardial cell apoptosis rate, MDA leve in myocardial tissues, serum IL-6 and PGE2 levels, Bax in myocardial tissues, and Wnt1 and β-catenin protein expression levels reduced in the drug intervention groups, whereas the GSH-Px and CAT levels and Bcl-2 and PPARγ protein expression levels in myocardial tissues increased. The groups of total flavonoids of buckwheat flower and leaf were in a dose-dependent manner. There was no significant difference in the levels of each index in rats between the propranolol hydrochloride group and the high-dose group of total flavonoids of buckwheat flower and leaf. The total flavonoids of buckwheat flower and leaf inhibit the activation of Wnt/β-catenin pathway, up-regulate the expression of PPARγ, reduce oxidative stress and inflammatory damage in myocardial tissues of arrhythmic rats, reduce myocardial cell apoptosis, and improve the symptoms of arrhythmia in rats.

Analysis of Mechanism of Antidepressant Effect of Sophora flavescens Seed Extract / 中国实验方剂学杂志

ObjectiveTo explore the antidepressant effect of Sophora flavescens seed extract and its molecular mechanism. MethodA mouse depression model was established by intraperitoneal injection of lipopolysaccharide（LPS）， and normal group， model group， fluoxetine group（2.5 mg·kg-1）， and S. flavescens seed low， medium and high dose groups（200， 400， 800 mg·kg-1） were set up for 7 d of consecutive gavage. Then the antidepressant effect of S. flavescens seed extract was evaluated by using open field test， elevated plus maze test and forced swimming test. Pathological morphological changes in the hippocampal tissue was observed by hematoxylin-eosin（HE） staining. Protein expression levels of G1/S-specific cyclin D1（Cyclin D1）， Wnt1， β-catenin and phosphorylated glycogen synthase kinase-3β（p-GSK-3β） in mouse brain tissues were detected by Western blot. Hippocampal cell apoptosis was detected by terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate（dUTP） nick end labeling（TUNEL）. ResultThe results of mouse behavioral experiments showed that compared with the normal group， the speed of movement in the open field and the distance of movement in the central area of the open field， and the time spent on the open arms of the elevated plus maze were significantly reduced in the model group（P<0.01）， while immobility time in the forced swimming test was significantly increased（P<0.05）. Compared with the model group， the S. flavescens seed medium and high dose groups had increased speed of movement in the open field test and time spent on the open arms of the elevated plus maze test（P<0.05， P<0.01）， and decreased immobility time in the forced swimming test（P<0.05）， the distance of movement in the central area of the open field test increased in the high dose group（P<0.05）. HE staining results showed that compared with the normal group， the hippocampal neuron structure of mice in the model group was damaged. Compared with the model group， after treatment of S. flavescens seed extract， the pathological state of the mouse hippocampal neuron structure was alleviated， and the neurons increased， were neatly arranged， and the cytoplasm was clear. Western blot results showed that the protein expression levels of Wnt1 and β-catenin in mouse brain tissue were significantly decreased（P<0.01）， while the protein expression levels of Cyclin D1 and p-GSK-3β were significantly increased（P<0.01） after LPS injection. Compared with the model group， protein expression levels of Wnt1 and β-catenin in brain tissue of S. flavescens seed medium and high dose groups were significantly increased（P<0.01）， while the protein expression levels of Cyclin D1 and p-GSK-3β were significantly decreased（P<0.01）. TUNEL staining results showed that the hippocampal cell apoptosis rate in the model group was significantly increased compared with that of the normal group（P<0.01）， while the hippocampal cell apoptosis rate in the S. flavescens seed medium and high dose groups was significantly decreased compared with that of the model group（P<0.01）. ConclusionS. flavescens seed extract can effectively improve the severity of depression in LPS-induced depressed mice， and its molecular mechanism is related to the regulation of neuroinflammation and hippocampal neuronal apoptosis mediated by Wnt/β-catenin signaling pathway.

Expression of low-density lipoprotein receptor-associated protein 11 in colorectal cancer tissues and its effects on proliferation and apoptosis of SW480 cells / 中国肿瘤生物治疗杂志

@#[摘 要] 目的：探讨低密度脂蛋白受体相关蛋白11（LRP11）在结直肠癌（CRC）组织中的表达及其对结肠癌SW480细胞增殖与凋亡的影响。方法：利用生物信息学方法分析TCGA数据库中LRP11在CRC组织中的表达水平。用慢病毒感染技术分别将sh-LRP11及sh-NC质粒转染至SW480细胞，采用qPCR、WB法检测感染后各组细胞中LRP11的mRNA和蛋白的表达，CCK-8法、流式细胞术分别检测细胞的增殖活力、凋亡率及细胞周期分布情况，WB法检测SW480细胞中cyclin D1、BAX、Bcl-2、β-catenin、活化β-catenin等蛋白的表达水平。结果：TCGA数据库数据分析显示，LRP11 mRNA在CRC组织中的表达水平显著高于正常组织（P<0.05）。与sh-NC组比较，sh-LRP11组SW480细胞的增殖活力明显降低、细胞凋亡率显著升高（均P<0.01），细胞中BAX表达显著升高、Bcl-2表达显著降低（均P<0.01）；G0/G1期细胞增多、S期细胞明显减少（均P<0.01），cyclin D1的蛋白表达显著降低（P<0.01）；Wnt/β-catenin信号通路中β-catenin和活化β-catenin的蛋白表达均显著下降（均P<0.01）。结论：LRP11 mRNA在CRC组织中呈高表达，干扰LRP11表达可抑制结肠癌SW480细胞增殖并促进其凋亡，为CRC提供了一种潜在的治疗靶点。

Effects of Tuina on cartilage degradation and chondrocyte terminal differentiation in rats with knee osteoarthritis (KOA) via the Wnt/β-catenin signaling pathway / 针灸推拿医学（英文版）

Objective: To investigate the therapeutic effects of Tuina (Chinese therapeutic massage) in a knee osteoarthritis (KOA) rat model and its influence on proteins associated with the Wnt/β-catenin signaling pathway. Methods: A total of 32 specific-pathogen-free grade Sprague-Dawley rats were used. Eight rats were randomly selected as the control group (CG). The remaining 24 rats underwent intra-articular injections with 0.2 mL of 4％ papain to prepare the KOA rat models. After the model was established, the 24 rats were randomly and equally assigned to 3 groups, including a model group (MG), a Tuina group (TG), and a positive medicine group (PMG), with 8 rats in each group. The Lequesne score was applied to evaluate the success of model development. After the model was successfully established, the CG did not receive any intervention, and the TG was treated with local, clockwise annular Rou-Kneading around the knee joint with the thumbs. The pressure in the longitudinal direction was 3 N, and the frequency was designed to be 120-140 times/min for 15 min, followed by flexing the joint 10 times. The PMG was intragastrically administered with celecoxib [24 mg/(kg·bw)] every day. These interventions were performed once a day, 6 d per week, for a total of 4 weeks. After treatment, the Lequesne score was applied again to assess the severity of the KOA in the rats; hematoxylin-eosin (HE) staining and a mixture of equal volumes of aqueous solutions of safranin O-fast green were used to stain and observe the cartilage morphology and structure; the modified Mankin score was applied to evaluate the pathology; enzyme-linked immunosorbent assay method was used to quantify the C-telopeptide fragments of type Ⅱ collagen (CTX-Ⅱ) and cartilage oligomeric matrix protein (COMP); Western blotting was then applied to quantify Wnt4, β-catenin, matrix metalloproteinase 13 (MMP-13), and bone morphogenetic protein 2 (BMP-2) protein expression; immunohistochemistry was conducted to determine the percentage of collagen type X (ColX)-positive cells. Results: The Lequesne score of the TG and PMG was both lower than that of the MG (P<0.01); the HE staining, safranin O-fast green stained morphology and structure, and modified Mankin scores of the TG and the PMG were also better than those in the MG (P<0.01). Compared with the CG, the amounts of CTX-Ⅱ and COMP in the serum were significantly increased (P<0.01); the expression of Wnt4, β-catenin, MMP-13, and BMP-2 proteins in the cartilage tissue was significantly increased (P<0.01), and the percentage of ColX-positive chondrocytes was significantly increased (P<0.01) in the MG. In comparison with those in the MG, the amounts of CTX-Ⅱ and COMP were significantly decreased (P<0.01), the expression of Wnt4, β-catenin, MMP-13, and BMP-2 proteins was significantly decreased (P<0.01), and the percentage of ColX-positive chondrocytes was significantly decreased (P<0.01) in the TG and PMG. Compared with the PMG, the contents of CTX-Ⅱ and COMP and the expression of Wnt4, β-catenin, MMP-13, and BMP-2 proteins were decreased (P<0.05 or P<0.01); the percentage of ColX-positive chondrocytes was significantly decreased (P<0.01) in the TG. Conclusion: Tuina can relieve the degeneration of KOA, and the mechanism may be related to the down-regulation of the Wnt/β-catenin signaling pathway, the decrease in MMP-13 and BMP-2 protein expression, the reduction in chondrocyte extracellular matrix degradation, and slowing down the terminal cell differentiation.