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.

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 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.

GLUL stabilizes N-Cadherin by antagonizing β-Catenin to inhibit the progresses of gastric cancer

Glutamate-ammonia ligase (GLUL, also known as glutamine synthetase) is a crucial enzyme that catalyzes ammonium and glutamate into glutamine in the ATP-dependent condensation. Although GLUL plays a critical role in multiple cancers, the expression and function of GLUL in gastric cancer remain unclear. In the present study, we have found that the expression level of GLUL was significantly lower in gastric cancer tissues compared with adjacent normal tissues, and correlated with N stage and TNM stage, and low GLUL expression predicted poor survival for gastric cancer patients. Knockdown of GLUL promoted the growth, migration, invasion and metastasis of gastric cancer cells in vitro and in vivo, and vice versa, which was independent of its enzyme activity. Mechanistically, GLUL competed with β-Catenin to bind to N-Cadherin, increased the stability of N-Cadherin and decreased the stability of β-Catenin by alerting their ubiquitination. Furthermore, there were lower N-Cadherin and higher β-Catenin expression levels in gastric cancer tissues compared with adjacent normal tissues. GLUL protein expression was correlated with that of N-Cadherin, and could be the independent prognostic factor in gastric cancer. Our findings reveal that GLUL stabilizes N-Cadherin by antagonizing β-Catenin to inhibit the progress of gastric cancer.

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.

Relationship of E-cadherin, Beta-catenin, N-cadherin, ZEB1 and ?SMA as Epithelial Mesenchymal Transition markers with prognostic factors in early and advanced stage laryngeal squamous cell carcinomas

Aims: To investigate the relationship between E-cadherin, beta-catenin, N-cadherin, ZEB1, and ?SMA as epithelial-mesenchymal transformation markers with tumor stage, lymph node metastasis (LNM), and overall survival (OS) in laryngeal squamous cell carcinomas (LSCC). Materials and Methods: A total of 100 cases diagnosed with LSCC were included in the study. Data about the lymphovascular invasion (LVI), perineural invasion (PNI), necrosis, and LNM were recorded by evaluating hematoxylin-eosin–stained slides. Markers of E-cadherin, beta-catenin, N-cadherin, ZEB1, and ?SMA were applied to the sections prepared from paraffin blocks of tumor samples. Results: Ninety-five male and five female patients were included in the study, and 38 of them exited. A significant relationship was observed between OS with advanced tumor stage, presence of LNM and PNI. A significant relationship was found between increased tumor Zeb1 expression and advanced tumor stage. In univariate and multivariate analyses, a significant negative relationship with OS, and increased Zeb1 expression in tumor and tumor stroma was seen. Any relationship was not observed between E-cadherin, beta-catenin, N-cadherin, and ?SMA and OS. Conclusion: Among the EMT markers, we evaluated in our study, it was seen that Zeb1, which is an EMT transcription factor, is associated with tumor stage, LNM, and OS. Remarkably, Zeb1 expression observed in tumor stroma was also significant for OS. Any similar data reported for LSCCs have not been encountered in the literature, and it was thought that it would be appropriate to support our findings with further studies to be performed on this subject.

Epidemiología de los factores ambientales relacionados con la vía β-catenina y su impacto en el desarrollo y progresión del cáncer colorrectal / Epidemiology of environmental factors related to beta-catenin pathway and its impact on the development and progression of colorectal cancer

Resumen El cáncer colorrectal (CCR) es una enfermedad compleja determinada por factores de riesgo genéticos, ambientales y de estilo de vida. Los factores genéticos (hereditarios) tienen gran influencia en su desarrollo, sin embargo, la mayoría de los casos de CCR son esporádicos y se desarrollan gradualmente a lo largo de varios años. Los principales factores ambientales de riesgo están asociados a la vía de señalización de β-catenina, entre ellos obesidad, falta de actividad física, consumo de carnes rojas y procesadas, alcoholismo y tabaquismo. La vía está relacionada con la regulación de la homeostasis celular, autorrenovación celular durante la embriogénesis y edad adulta. La principal recomendación para evitar el desarrollo del CCR es reducir los factores de riesgo, aumentar el consumo de frutas, verduras y granos, hacer ejercicio de manera rutinaria y limitar el consumo tanto de alcohol como de tabaco. Dado que los antecedentes familiares y la presencia de un síndrome hereditario aumentan el riesgo, se sugiere hacer exámenes periódicos para detectar CCR y emplear predictores del desarrollo como los marcadores bioquímicos y moleculares, los cuales se presentan en este trabajo.

Abstract Colorectal cancer (CRC) is a complex disease, determined by genetic, environmental and lifestyle-associated risk factors. Genetic (inherited) factors have great influence on its development; however, most cases of CRC are sporadic and gradually develop over several years. The main environmental risk factors are associated with β-catenin signaling pathway, including obesity, lack of physical activity, consumption of red and processed meats, alcoholism, and smoking. The pathway is related to cell homeostasis regulation and cell self-renewal during embryogenesis and adulthood. The main recommendation for preventing the development of CRC is to reduce the risk factors, increase the consumption of fruits, vegetables and grains, exercise regularly and limit the consumption of both alcohol and tobacco. However, family history and the presence of a hereditary syndrome increase the risk, which is why carrying out periodic examinations to detect CRC is suggested, using development predictors such as biochemical and molecular markers, which are discussed in this work.

Immunohistochemical appraisal of epithelial mesenchymal transition type III in gall bladder cancer

Background: Epithelial-mesenchymal transition (EMT) is the heart of invasion. EMT associated with cancer progression and metastasis is known as type III EMT. Beta-catenin, E-cadherin, and MMP9 markers of EMT are routinely employed for diagnostic purposes. Aims: We employed these markers to study EMT by immunohistochemistry (IHC) in gall bladder cancer (GBC) with respect to depth of tumor invasion, clinical outcome, and disease-free survival. Settings and Design: This was a prospective case-control study. Material and Methods: Seventy gall bladders were included (50 GBC and 20 CC). After detailed histology, immunoexpression was studied in terms of percentage and strength of expression. Statistics Analysis Used: Expression was compared between CC and GBC by Student t test and analysis of variance. Kaplan–Meier was used for survival analysis, and the extent of agreement (“Kappa”) was calculated. Results and Conclusions: The age of incidence of GBC was 49.40 (+11.6) years with female predominance (F:M = 4:1). In 88% (44/50) of GBC, the fundus was involved. Moderately differentiated adenocarcinoma was most frequent [54%; 27/50]. Significant downregulation of E-cadherin (P = 0.022) and beta-catenin (P < 0.001) and upregulation in MMP9 (P < 0.001) were seen in GBC with respect to CC with significant association among them. MMP9 expression was significantly associated with higher tumor stage but with chemotherapeutic response. Our results display that epithelial-mesenchymal transition type III plays a role in GBC invasion. MMP9 overexpression and loss of membranous beta-catenin may be considered a marker for poor clinical outcomes and advanced disease.

β-catenin ameliorates myocardial infarction by preventing YAP-associated apoptosis

Abstract Objective: To explore whether the effect of β-catenin on MI and MI-induced cardiomyocyte apoptosis is YAP-dependent. Methods: The authors established an MI rat model by ligating the anterior descending branch of the left coronary artery, and an MI cell model by treating cardiomyocytes with H2O2. Results: β-catenin downregulation was observed in MI cardiac tissues and in H2O2-treated cardiomyocytes. Lentiviral-CTNNB1 was administered to MI rats to upregulate β-catenin expression in MI cardiac tissue. β-catenin recovery reduced the myocardial infarct area, fibrosis, and apoptotic cell death in MI rats. H2O2 treatment attenuated cell viability and induced cell death in cardiomyocytes, whereas β-catenin overexpression partially reversed these changes. Moreover, H2O2 treatment caused the deactivation of Yes-Associated Protein (YAP), as detected by increased YAP phosphorylation and reduced the nuclear localization of YAP. Upregulation of β-catenin expression reactivated YAP in H2O2-treated cardiomyocytes. Reactivation of YAP was achieved by administration of Mitochonic Acid-5 (MA-5) to H2O2-treated cardiomyocytes, and deactivation of YAP by CIL56 treatment in β-catenin-overexpressing H2O2-treated cardiomyocytes. MA-5 administration increased cell viability and repressed apoptosis in H2O2-treated cardiomyocytes, whereas CIL56 treatment counteracted the effects of β-catenin overexpression on cell survival and apoptosis. Conclusions: The present data indicate that β-catenin and YAP are effective treatment targets for MI, blocking the apoptotic death of cardiomyocytes.

Traditional Chinese Medicine in Treatment of Diabetic Kidney Disease Based on Wnt/β-catenin Signaling Pathway： A Review / 中国实验方剂学杂志

Diabetic kidney disease （DKD） is a common complication of diabetes and a leading cause of end-stage kidney disease. Its pathogenesis is complex， and it presents a significant challenge in treatment， gradually becoming a major global public health issue. One of the main pathological changes in DKD is tubulointerstitial fibrosis， clinically characterized by proteinuria and declining kidney function， which severely impacts the daily life of patients. Currently， western medicine commonly uses methods such as controlling blood sugar and blood pressure， and reducing proteinuria to treat DKD， but the efficacy is unsatisfactory， and there are many side effects. As reported， traditional Chinese medicine （TCM） treatment for DKD has many advantages， such as low cost， significant efficacy， and minimal adverse reactions. More researchers focusing on DKD are turning their attention to TCM， and progress has been made in related studies both in China and abroad. The Wnt/β-catenin signaling pathway is relatively evolutionarily conserved and plays a crucial role in normal biological development and the entire life process. Studies have demonstrated that abnormal activation of the Wnt/β-catenin signaling pathway is related to renal fibrosis， which coincides with TCM theory of "collateral diseases". By reviewing relevant literature， this article reviewed the Wnt/β-catenin signaling pathway and its role in DKD and summarized the research status of TCM monomers， single drug extracts， and TCM formulas in improving renal fibrosis and treating DKD through the improvement of glomerular mesangial cells， renal tubular epithelial cells， and podocyte injury， aiming to provide new ideas and directions for TCM treatment of DKD.

Effect of Dickkopf-1 regulated Wnt pathway on transdifferentiation of hypoxia-induced lens epithelial cells / 国际眼科杂志(Guoji Yanke Zazhi)

AIM: To scrutinize the role of the Wnt/β-catenin signaling pathway in the epithelial-mesenchymal transition(EMT)of lens epithelial cells under hypoxic conditions, and to further analyze the effect of Dickkopf-1(DKK-1)expression on EMT of lens epithelial cells.METHODS: Human lens epithelial cells(HLEB3 cells)were propagated in vitro and then separated into two groups: one exposed to standard oxygen levels, added DMEM culture solution containing 10% FBS(normoxic group)and another subjected to low oxygen levels(hypoxic group). The hypoxic condition was emulated by applying a concentration of 100 μmol/L cobalt chloride(CoCl2)for 6, 12, 24, and 48h. The utilization of immunofluorescence staining enabled the detection of Wnt3a and DKK-1 expressions, along with the expression and localization of β-catenin protein in these groups. The expression of DKK-1 mRNA was discerned by quantitative real-time polymerase chain reaction(qRT-PCR).RESULTS: Immunofluorescence assays indicated an escalating trend in the Wnt3a and DKK-1 protein expression, which corresponded with the increasing duration of hypoxia. Likewise, an intensified nuclear accumulation of β-catenin protein was observed to be directly proportional to the length of hypoxia treatment. The qRT-PCR demonstrated that the difference in DKK-1 mRNA expression between the normoxic group and the group exposed to hypoxia for 6h was not statistically significant(P&#x003E;0.05), whereas the DKK-1 mRNA expression of the 12, 24, and 48h hypoxia groups were significantly increased(P&#x003C;0.001).CONCLUSION: Hypoxia can activate Wnt/β-catenin pathway in lens epithelial cells and induce the expression of DKK-1, thus regulating the Wnt/β-catenin pathway and affecting the EMT process.

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.

The Mechanism of miR-1294 Targeting SOX15 to Regulate Wnt/β-catenin Signaling Pathway and Promote the Proliferation of Acute Lymphoblastic Leukemia Cells in Children / 中国实验血液学杂志

OBJECTIVE@#To explore the effect of abnormal miRNA expression on the proliferation of pediatric acute lymphoblastic leukemia (ALL) cells and its related mechanism.@*METHODS@#15 children with ALL and 15 healthy subjects were collected from the Second Affiliated Hospital of Hainan Medical University from July 2018 to March 2021. MiRNA sequencing was performed on their bone marrow cells, and validated using qRT-PCR. MiR-1294 and miR-1294-inhibitory molecule (miR-1294-inhibitor) were transfected into Nalm-6 cells, and the proliferation of Nalm-6 cells was detected by CCK-8 and colony formation assays. Western blot and ELISA were used to detect apoptosis of Nalm-6 cells. Biological prediction of miR-1294 was performed to find the target gene, which was verified by luciferase reporter assay. Si-SOX15 was transfected into Nalm-6 cells, Western blot was used to detect the expression of Wnt signaling pathway-related proteins and to verify the effect of si-SOX15 on the proliferation and apoptosis of Nalm-6 cells.@*RESULTS@#Compared with healthy subjects, 22 miRNAs were significantly upregulated in bone marrow cells of ALL patients, of which miR-1294 was the most significantly upregulated. In addition, the expression level of SOX15 gene was significantly reduced in bone marrow cells of ALL patients. Compared with the NC group, the miR-1294 group showed increased protein expression levels of Wnt3a and β-catenin, faster cell proliferation, and more colony-forming units, while caspase-3 protein expression level and cell apoptosis were reduced. Compared with the NC group, the miR-1294-inhibitor group showed reduced protein expression levels of Wnt3a and β-catenin, slower cell proliferation, and fewer colony-forming units, while caspase-3 protein expression level was increased and apoptosis rate was elevated. miR-1294 had a complementary base-pair with the 3'UTR region of SOX15 , and miR-1294 directly targeted SOX15 . The expression of miR-1294 was negatively correlated with SOX15 in ALL cells. Compared with the si-NC group, the si-SOX15 group showed increased protein expression levels of Wnt3a and β-catenin, accelerated cell proliferation, and decreased caspase-3 protein expression level and cell apoptosis rate.@*CONCLUSION@#MiR-1294 can target and inhibit SOX15 expression, thus activating the Wnt/β-Catenin signaling pathway to promote the proliferation of ALL cells, inhibit cell apoptosis, and ultimately affect the disease progression.

Traditional Chinese Medicine Intervenes in Signaling Pathways Associated with CKD-MBD： A Review / 中国实验方剂学杂志

Traditional Chinese medicine （TCM） has certain advantages in the treatment of chronic kidney disease-mineral and bone disorder （CKD-MBD）. In recent years， there have been many studies on the treatment of CKD-MBD by Chinese medicinal compounds and monomers. As revealed by literature retrieval， the research on the mechanism of Chinese medicine in intervening in signaling pathways related to CKD-MBD was mainly based on self-made Chinese medicinal compounds， and the action pathways involved fibroblast growth factor 23/Klotho （FGF23/Klotho） signaling pathway， Wnt/β-catenin signaling pathway， receptor activator of nuclear factor-κB/receptor activator of nuclear factor-κB ligand/osteoprotegerin （RANK/RANKL/OPG） system， and other signaling pathways. TCM can improve calcium and phosphorus metabolism and bone metabolism disorder， and regulate inflammatory reaction， oxidative stress， apoptosis， and autophagy by regulating this series of signaling pathways for the treatment of CKD-MBD. This paper introduced the research results of these signaling pathways and the mechanism of TCM in the treatment of CKD-MBD in order to provide ideas and references for the related research of Chinese medicine in the treatment of CKD-MBD.

Effects of rotenone exposure during pregnancy on GSK-3β and β-catenin protein expression in rat placenta / 环境与职业医学

Background Research on non-target organ damage of biological pesticides has attracted much attention. Rotenone exposure may be far beyond the occupational environment, and the exposureduring pregnancy may be increased through bioaccumulation, fruit or vegetable residues, and other forms of oral intake. At present, the effects of rotenone on placental development and its mechanism are still unknown. Objective To investigate the developmental damage of rat placenta and evaluate the expression levels of glycogen synthase kinase (GSK-3β) and beta catenin (β-catenin) followed by rotenone exposure through the placental barrier during pregnancy, as well as to propose possible associated mechanisms. Methods Eighteen sexually mature SD female infertile rats without specific pathogens were selected and divided into three groups: blank control group (0.9% saline), corn oil group, and rotenone group (corn oil + 2 mg·kg−1 rotenone) by random number method, six female animals in each group. Another six male rats were selected and mated to the female rats at night with a female to male ratio of 3:1 per cage. Pregnant rats were given 0.9% saline, corn oil, and 2 mg·kg−1 rotenone preparation by isovolumetric gavage once daily for the entire gestation period (19 d), and their conditions were observed after the last dose. The pregnant rats were anesthetized, and the size of the placenta and blood perfusion were detected by ultrasound the next day of the last dose of rotenone. Then, 3 pregnant rats in each group were sacrificed immediately and the placenta and umbilical cord tissues were dissected. The remaining 9 pregnant rats gave birth naturally, and the fetuses were observed for developmental evaluation and weighed. The histopathological changes of umbilical cord and placenta were observed by hematoxylin-eosin staining. The reactive oxygen species levels of placenta tissues were detected by flow cytometry. The Ca2+-ATPase activity of placenta tissues was detected by colorimetric method. The localization and levels of GSK-3β and β-catenin expression of placenta were detected by immunohistochemistry. The p-GSK-3β/GSK-3β and p-β-catenin/β-catenin protein expression in placental tissues were measured by Western blotting. Results No injury or death was recorded during the pregnant rats receiving rotennon administration. Adverse pregnancy outcomes such as fetal absorption and postpartum stillbirth were found in the rotenone group, and the weight of the fetal mice decreased (P<0.05). The B-ultrasound showed disc-shaped placenta with a thick middle and thin edge, smooth fetal surface, rough maternal surface, visible placental lobules, granular echotexture of the placenta with comma-like echogenic densities, and chorionic plate showing deep indentations, no calcification, degeneration, or necrosis in each group. Compared with the corn oil group, the fetal surface diameter of the placenta was reduced in the rotenone group (P<0.05). The Doppler color ultrasound showed that interplacental blood flow was reduced in the rotenone group, while interplacental blood flow was abundant in the blank control and the corn oil groups. The hematoxylin-eosin staining results showed that smooth muscle cells in the umbilical cord tissues of rats were loosely arranged, with fuzzy nuclei and inflammatory infiltration in the rotenone group. The placental trophoblast cells were small in size, disorderly arranged with nuclear fragmentation and cytoplasm turbidity. The tissue reactive oxygen species level in the rotenone group was higher than those in the other two groups (P<0.05). The Ca2+-ATPase activity of placental tissues was reduced in the rotenone group (P<0.05). The immunofluorescence low-magnification observation showed that GSK-3β and β-catenin were expressed in placental tissue, weak fluorescence expression in the decidua basalis, strong fluorescence expression in the labyrinthine layer structure. The labyrinthine layer under high magnification showed that compared with the blank control group and the corn oil group, the brightness of β-catenin fluorescence expression in the rotenone group decreased (P<0.05), and the brightness of GSK-3β expression increased (P<0.05). The Western blotting results showed that the expression of β-catenin and p-GSK-3β proteins decreased (P<0.01), and the expression of GSK-3β protein increased (P<0.01) in the rotenone group. No significant expression of p-β-catenin protein was detected in the placenta tissue of each group. Conclusion Rotenone exposure during pregnancy induces placental hypoperfusion, growth retardation, and oxidative stress in rats, as well as down-regulation of β-catenin and p-GSK-3β protein expression, and up-regulation of GSK-3β protein expression, which may further lead to abnormal pregnancy and fetal restricted growth.

Physical exercise suppresses hepatocellular carcinoma progression by alleviating hypoxia and attenuating cancer stemness through the Akt/GSK-3β/β-catenin pathway / 中西医结合学报

OBJECTIVE@#Physical exercise, a common non-drug intervention, is an important strategy in cancer treatment, including hepatocellular carcinoma (HCC). However, the mechanism remains largely unknown. Due to the importance of hypoxia and cancer stemness in the development of HCC, the present study investigated whether the anti-HCC effect of physical exercise is related to its suppression on hypoxia and cancer stemness.@*METHODS@#A physical exercise intervention of swimming (30 min/d, 5 d/week, for 4 weeks) was administered to BALB/c nude mice bearing subcutaneous human HCC tumor. The anti-HCC effect of swimming was assessed in vivo by tumor weight monitoring, hematoxylin and eosin (HE) staining, and immunohistochemistry (IHC) detection of proliferating cell nuclear antigen (PCNA) and Ki67. The expression of stemness transcription factors, including Nanog homeobox (NANOG), octamer-binding transcription factor 4 (OCT-4), v-Myc avian myelocytomatosis viral oncogene homolog (C-MYC) and hypoxia-inducible factor-1α (HIF-1α), was detected using real-time reverse transcription polymerase chain reaction. A hypoxia probe was used to explore the intratumoral hypoxia status. Western blot was used to detect the expression of HIF-1α and proteins related to protein kinase B (Akt)/glycogen synthase kinase-3β (GSK-3β)/β-catenin signaling pathway. The IHC analysis of platelet endothelial cell adhesion molecule-1 (CD31), and the immunofluorescence co-location of CD31 and desmin were used to analyze tumor blood perfusion. SMMC-7721 cells were treated with nude mice serum. The inhibition effect on cancer stemness in vitro was detected using suspension sphere experiments and the expression of stemness transcription factors. The hypoxia status was inferred by measuring the protein and mRNA levels of HIF-1α. Further, the expression of proteins related to Akt/GSK-3β/β-catenin signaling pathway was detected.@*RESULTS@#Swimming significantly reduced the body weight and tumor weight in nude mice bearing HCC tumor. HE staining and IHC results showed a lower necrotic area ratio as well as fewer PCNA or Ki67 positive cells in mice receiving the swimming intervention. Swimming potently alleviated the intratumoral hypoxia, attenuated the cancer stemness, and inhibited the Akt/GSK-3β/β-catenin signaling pathway. Additionally, the desmin+/CD31+ ratio, rather than the number of CD31+ vessels, was significantly increased in swimming-treated mice. In vitro experiments showed that treating cells with the serum from the swimming intervention mice significantly reduced the formation of SMMC-7721 cell suspension sphere, as well as the mRNA expression level of stemness transcription factors. Consistent with the in vivo results, HIF-1α and Akt/GSK-3β/β-catenin signaling pathway were also inhibited in cells treated with serum from swimming group.@*CONCLUSION@#Swimming alleviated hypoxia and attenuated cancer stemness in HCC, through suppression of the Akt/GSK-3β/β-catenin signaling pathway. The alleviation of intratumoral hypoxia was related to the increase in blood perfusion in the tumor. Please cite this article as: Xiao CL, Zhong ZP, Lü C, Guo BJ, Chen JJ, Zhao T, Yin ZF, Li B. Physical exercise suppresses hepatocellular carcinoma progression by alleviating hypoxia and attenuating cancer stemness through the Akt/GSK-3β/β-catenin pathway. J Integr Med. 2023; 21(2): 184-193.

Fibroblasts overpressing WNT2b cause impairment of intestinal mucosal barrier / 南方医科大学学报

OBJECTIVE@#To investigate the mechanism by which fibroblasts with high WNT2b expression causes intestinal mucosa barrier disruption and promote the progression of inflammatory bowel disease (IBD).@*METHODS@#Caco-2 cells were treated with 20% fibroblast conditioned medium or co-cultured with fibroblasts highly expressing WNT2b, with the cells without treatment with the conditioned medium and cells co-cultured with wild-type fibroblasts as the control groups. The changes in barrier permeability of Caco-2 cells were assessed by measuring transmembrane resistance and Lucifer Yellow permeability. In Caco-2 cells co-cultured with WNT2b-overexpressing or control intestinal fibroblasts, nuclear entry of β-catenin was detected with immunofluorescence assay, and the expressions of tight junction proteins ZO-1 and E-cadherin were detected with Western blotting. In a C57 mouse model of dextran sulfate sodium (DSS)-induced IBD-like enteritis, the therapeutic effect of intraperitoneal injection of salinomycin (5 mg/kg, an inhibitor of WNT/β-catenin signaling pathway) was evaluated by observing the changes in intestinal inflammation and detecting the expressions of tight junction proteins.@*RESULTS@#In the coculture system, WNT2b overexpression in the fibroblasts significantly promoted nuclear entry of β-catenin (P < 0.01) and decreased the expressions of tight junction proteins in Caco-2 cells; knockdown of FZD4 expression in Caco-2 cells obviously reversed this effect. In DSS-treated mice, salinomycin treatment significantly reduced intestinal inflammation and increased the expressions of tight junction proteins in the intestinal mucosa.@*CONCLUSION@#Intestinal fibroblasts overexpressing WNT2b causes impairment of intestinal mucosal barrier function and can be a potential target for treatment of IBD.

Effect of Sanhuang Tangshenkang on Wnt/β-catenin Signaling Pathway in Bone Tissue of Diabetic Rats / 中国实验方剂学杂志

ObjectiveTo observe the effect of Sanhuang Tangshenkang on the Wnt/β-catenin signaling pathway in the bone tissue of diabetic rats. MethodA high-sugar and high-fat diet was administered for 4 weeks， along with intraperitoneal injection of freshly prepared 2% streptozotocin （pH 4.5） at 30 mg·kg-1 body weight to induce a diabetes model in rats. The rats with diabetes were randomly divided into model group， low- and high-dose Sanhuang Tangshenkang groups （12.8， 38.4 g·kg-1）， and Gushukang group （1.8 g·kg-1） according to the blood glucose level. Rats of the same age were fed on a regular diet and assigned to the control group. After 12 weeks of respective treatments with drugs or physiological saline， the fasting blood glucose （FBG） levels of the rats were measured using an automated biochemical analyzer. Enzyme-linked immunosorbent assay （ELISA） was used to detect fasting serum insulin （FINS）， bone-specific alkaline phosphatase （BALP）， and tartrate-resistant acid phosphatase （TRAP） levels. Micro-computed tomography （Micro-CT） was used to scan the femurs of rats to observe bone tissue microstructure and measure bone mineral density （BMD）. Hematoxylin-eosin （HE） staining and safranin O/fast green staining were performed to observe pathological changes in the femoral bone tissue. Immunohistochemistry （IHC） and Western blot were used to detect the expression of Wnt3a， low-density lipoprotein receptor-related protein 5 （LRP-5）， and β-catenin proteins. ResultCompared with the control group， the model group showed a significant increase in FBG， FINS， and TRAP levels （P<0.01）， a significant decrease in BALP level （P<0.01）， a significant decrease in BMD （P<0.01）， and disorganized， elongated， and sparse bone trabecular structures with fractures and increased lipid droplets. Additionally， the expression of Wnt3a， LRP-5， and β-catenin proteins decreased （P<0.05， P<0.01）. Compared with the model group， the low- and high-dose Sanhuang Tangshenkang groups showed a reduction in FBG and an increase in BALP （P<0.05）. The low-dose Sanhuang Tangshenkang group also exhibited a decrease in FINS （P<0.05）. All treatment groups showed a significant decrease in TRAP （P<0.01）， varying degrees of improvement in BMD （P<0.05， P<0.01））， increased and denser bone trabeculae with more regular arrangements and reduced lipid droplets， and improved bone microstructure morphology. The average optical density values of Wnt3a， LRP-5， and β-catenin proteins were significantly increased in all drug-treated groups （P<0.05， P<0.01）， and the expression of Wnt3a， LRP-5， and β-catenin proteins was elevated （P<0.05， P<0.01）. ConclusionSanhuang Tangshenkang may regulate the imbalance of the Wnt/β-catenin signaling pathway by increasing the expression of Wnt3a， LRP-5， and β-catenin proteins in bone tissue， which may promote bone formation， reduce bone resorption， and lower blood glucose levels， thereby achieving the effect of preventing and treating diabetic osteoporosis.

Cross-regulation of Wnt/β-catenin and NF-κB signaling pathway and their involvement in the differentiation of mesenchymal stem cells / 口腔医学

@#Both Wnt/β-catenin signaling pathway and NF-κB signaling pathway are highly conservative pathways that regulate a variety of biological processes, and their cross-regulation have attracted attention in many biological and medical research fields. In this review, we summarize the cross-regulation between Wnt/β-catenin signaling pathway and NF-κB signaling pathway and discuss their involvement in the multi-directional differentiation of mesenchymal stem cells.