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.

Improvement effect of crocin on cognitive impairment of Alzheimer's disease rats through DKK3 regulation of GSK-3 β/β-Catenin pathway / 中国临床药理学与治疗学

AIM: To explore the improvement effect and mechanism of crocin on cognitive impairmrnt of Alzheimer's disease (AD) rats. METHODS: The hippocampus of SD rats were injected with Aβ 25-35 to establish AD model, then rats were randomly divided into AD group, AD + low, medium, high dose of crocin groups (10, 20, 40 mg/kg) and AD + donepezil group (1 mg/kg), intraperitoneal injection treatment for 4 weeks, set sham group. Dark avoidance test and water maze test were used to evaluate the learning and memory abilities of rats, ELISA was used to detect serum Aβ content, HE staining and Tunel staining were used to determine pathological changes and neuronal apoptosis of hippocampus of rats, immunohistochemistry was used to detect the expression of Brdu, Dcx and NeuN in hippocampus of rats, and Western blot was used to detect the protein expression of Aβ, DKK3, β-catenin, p-GSK-3β/GSK-3β, Caspase-3, Bax, Bcl-2 in hippocampus of rats. RESULTS: Compared to sham group, the learning and memory abilities of AD group rats were decreased, serum Aβ content increased, the pathological change in hippocampus was serious, neuronal apoptosis was increased, the expression of Brdu, Dcx, NeuN were decreased, the protein expression of Aβ, DKK3, p-GSK-3β/GSK-3β, Caspase-3, Bax were increased, protein expression of β-catenin, Bcl-2 were decreased (P<0.01). Compared to AD group, after the treatment of doses of crocin and donepezil, the learning and memory abilities of AD rats were improved, serum Aβ content were increased, and the pathological change in hippocampus were alleviated, neuronal apoptosis were reduced, the expression of Brdu, Dcx, NeuN were decreased, the protein expression of Aβ, DKK3, p-GSK-3β/ GSK-3β, Caspase-3, Bax were decreased, the protein expression of β-catenin, Bcl-2 were increased, notely, dose-dependent effect of crocin was significant. CONCLUSION: Crocin reduced neuronal apoptosis and mediated DKK3 to regulate GSK-3β/ β-catenin pathway to improve the cognitive impairment of AD rats.

Mechanism of Pingxiao capsule promoting breast cancer apoptosis through AKT1/β-catenin signal based on network pharmacology / 中国临床药理学与治疗学

AIM: To explore the potential mechanism of action of Pingxiao capsule in the treatment of breast cancer. METHODS: TCMSP, TCM-ID, GeneCards and other databases were used to screen the related targets of Pingxiao capsule and breast can-

Role and mechanism of NR1D1 in proliferation and migration of vascular adventitial fibroblasts / 中国药理学通报

Aim To explore the role and mechanism of nuclear receptor subfamily 1,group D,member 1(NR1D1)in the proliferation and migration of mouse adventitial fibroblasts(AFs). Methods Primary AFs isolated from C57BL/6J mice were cultured. Adenovirus carrying Nr1d1 gene was used to overexpress NR1D1 in AFs. The expression of β-catenin was restored by SKL2001. Proliferating cell nuclear antigen(Ki-67)immunofluorescence staining and CCK-8 staining were used to determine cell proliferation,and scratch test was used to determine cell migration. qPCR was used to determine the mRNA level of Nr1d1. Western blot was used to determine the protein levels of NR1D1 and β-catenin. To investigate the role of NR1D1 in intimal hyperplasia,20 male wild type C57BL/6J mice were randomly divided into sham group,carotid artery endothelial injury,sham+SR9009(NR1D1 agonist)group and carotid artery endothelial injury+SR9009(n=5 in each group). They were treated with DMSO or SR9009(100 mg·kg-1·d-1)via intraperitoneal injection for 14 days after operation,respectively. The degree of carotid intimal hyperplasia was measured by HE staining 28 days after operation. Results NR1D1 overexpression significantly reduced the percentage of Ki-67-positive cells(P<0.01),total cell number(P<0.01)and slowed down the rate of wound-healing(P<0.01). NR1D1 overexpression significantly inhibited the expression of β-catenin(P<0.05). After the expression of β-catenin was restored by SKL2001,the inhibitory effects of NR1D1 overexpression on the proliferation and migration of AFs were abolished(P<0.01). Enhanced activity of NR1D1 significantly ameliorated intimal hyperplasia after carotid endothelial injury(P<0.01). Conclusion NR1D1 may inhibit the proliferation and migration of AFs via suppressing the expression of β-catenin.

RORα antagonist T0901317 promotes EMT in human gastric cancer MGC803 cells by RORα/β-catenin signal / 中国药理学通报

Aim To investigate the effect of RORα antagonist T0901317 promoting EMT (epithelial-mesenchymal transition) of human gastric cancer MGC803 cells by RORα/β-catenin signal. Methods Cell proliferation was detected by MTT. Cell migration and invasion were detected by cell scratch and Transwell assay respectively. RORα/β-catenin signaling molecules were detected by Western blot and immunofluorescence. RORα binding to β-catenin protein was detected by immunoprecipitation. Results MTT assay showed that the proliferation ability of T0901317 cells increased in a time-dependent manner compared with MGC803 cells (P < 0. 05). Cell scratches and Transwell experiments showed that the migration and invasion ability of T0901317 cells were significantly enhanced compared with MGC803 cells (P < 0. 05). Western blot analysis showed that RORα protein was significantly down-regulated after T0901317 compared with untreated group (P < 0. 05), and total β-catenin protein and nuclear β-catenin in MGC803 cells were up-regulated after T0901317 (P < 0. 05). Compared with the control group, RORα protein binding to β-catenin protein significantly decreased after T0901317 treatment (P < 0. 05). Compared with MGC803 cells treated with T0901317, the long spindle cells increased and the heteromorphism was more obvious. T0901317 significantly up-regulated the expression of Rac1, TGFβ1 and Vimentin in MGC803 cells (P < 0. 05), and inhibited the expression of E-cadherin (P < 0. 05). Conclusion T0901317 can promote the proliferation, migration, invasion and EMT in human gastric cancer MGC803 cells by RORα/β-catenin signal.

Mechanosensitive ion channel Piezo1 induces senescence of atrial fibroblasts by activating β-catenin / 中国药理学通报

Aim To observe whether the mechanosensitive ion channel Piezo1 was involved in the senescence of atrial fibroblasts by activating β-catenin based on our previous study which found marked increase of Piezo1 mRNA in senescent atrial fibroblasts. Methods Primary mouse atrial fibroblasts (MAFs) were isolated from male C57BL/6 mice (3-4 weeks) by enzyme digestion, and tert-butyl hydroperoxide (TBHP) was used to induce the senescence of cells. The ratio of senescent cells was detected by senescence-associated β-galactosidase (SA-β-Gal) staining. The protein levels of Piezo1, β-catenin/p-β-catenin, senescence-associated proteins p53 and p21 in the cells treated with TBHP (100 μmol · L

Effect of Liuwei Dihuang decoction on β-catenin and EMT in kidney tissue of UUO rats / 中国药理学通报

Aim To explore the effect of Liuwei Dihuang decoction ( LWDHD) on the expression of β-catenin, E-cadherin,α-SMA, the pathological changes of renal tissue, and the changes of an epithelial-mesen-chymal transformation ( EMT) in renal tissue of rats with unilateral ureteral obstruction ( UUO ) . Methods Forty-eight SPF grade SD rats were randomly divided into sham group ( Sham), model group ( UUO), Liuwei Dihuang decoction low, medium, and high groups ( LWDHD 3. 375, 6. 75, 13. 5 g · kg

Jiedu Recipe, a compound Chinese herbal medicine, inhibits cancer stemness in hepatocellular carcinoma via Wnt/β-catenin pathway under hypoxia / 中西医结合学报

OBJECTIVE@#Jiedu Recipe (JR), a Chinese herbal remedy, has been shown to prolong overall survival time and decrease recurrence and metastasis rates in patients with hepatocellular carcinoma (HCC). This work investigated the mechanism of JR in HCC treatment.@*METHODS@#The chemical constituents of JR were detected using liquid chromatography-mass spectrometry. The potential anti-HCC mechanism of JR was screened using network pharmacology and messenger ribonucleic acid (mRNA) microarray chip assay, followed by experimental validation in human HCC cells (SMMC-7721 and Huh7) in vitro and a nude mouse subcutaneous transplantation model of HCC in vivo. HCC cell characteristics of proliferation, migration and invasion under hypoxic setting were investigated using thiazolyl blue tetrazolium bromide, wound healing and Transwell assays, respectively. Image-iT™ Hypoxia Reagent was added to reveal hypoxic conditions. Stem cell sphere formation assay was used to detect the stemness. Epithelial-mesenchymal transition (EMT) markers like E-cadherin, vimentin and α-smooth muscle actin, and pluripotent transcription factors including nanog homeobox, octamer-binding transcription factor 4, and sex-determining region Y box protein 2 were analyzed using Western blotting and real-time polymerase chain reaction. Western blot was performed to ascertain the anti-HCC effect of JR under hypoxia involving the Wnt/β-catenin pathway.@*RESULTS@#According to network pharmacology and mRNA microarray chip analysis, JR may potentially act on hypoxia and inhibit the Wnt/β-catenin pathway. In vitro and in vivo experiments showed that JR significantly decreased hypoxia, and suppressed HCC cell features of proliferation, migration and invasion; furthermore, the hypoxia-induced increases in EMT and stemness marker expression in HCC cells were inhibited by JR. Results based on the co-administration of JR and an agonist (LiCl) or inhibitor (IWR-1-endo) verified that JR suppressed HCC cancer stem-like properties under hypoxia by blocking the Wnt/β-catenin pathway.@*CONCLUSION@#JR exerts potent anti-HCC effects by inhibiting cancer stemness via abating the Wnt/β-catenin pathway under hypoxic conditions. Please cite this article as: Guo BJ, Ruan Y, Wang YJ, Xiao CL, Zhong ZP, Cheng BB, Du J, Li B, Gu W, Yin ZF. Jiedu Recipe, a compound Chinese herbal medicine, inhibits cancer stemness in hepatocellular carcinoma via Wnt/β-catenin pathway under hypoxia. J Integr Med. 2023; 21(5): 474-486.

Moxibustion as an adjunctive treatment for rheumatoid arthritis and its effects on the serum levels of SOST and β-catenin / 中国针灸

OBJECTIVES@#To observe the clinical efficacy of moxibustion as an adjunctive treatment for rheumatoid arthritis (RA) based on conventional medication and its effects on serum sclerostin (SOST) and β-catenin levels, exploring the potential mechanisms by which moxibustion may protect joint bones in RA patients.@*METHODS@#Seventy-six RA patients were randomly divided into an observation group (38 cases, 3 cases dropped out) and a control group (38 cases, 4 cases were eliminated, 2 cases dropped out). The patients in the control group were treated with conventional oral medication; based on the treatment of the control group, the patients in the observation group were treated with moxibustion. The direct moxibustion was applied at Zusanli (ST 36) on both sides and ashi points around small joints, and indirect moxibustion was applied at Shenshu (BL 23) on both sides and ashi points around large joints. The treatment was given three times a week for a total of 5 weeks. The count of pain and swollen joint, morning stiffness score, disease activity score of 28 joints (DAS28), visual analogue scale (VAS) score, health assessment questionnaire (HAQ) score, and serum levels of SOST, β-catenin, and tumor necrosis factor-α (TNF-α) were evaluated before and after treatment in the two groups.@*RESULTS@#Compared those before treatment, after treatment, both groups showed a reduction in pain and swollen joint count (P<0.01, P<0.05), morning stiffness, DAS28, VAS, and HAQ scores (P<0.01, P<0.05), with the observation group having lower scores than the control group (P<0.01). Serum levels of SOST, β-catenin, and TNF-α after treatment in the observation group were lower than those in both before treatment and the control group (P<0.01, P<0.05). There was a positive correlation between the difference in serum β-catenin levels before and after treatment and the difference in serum SOST (r=0.578, P<0.001) and TNF-α (r=0.403, P<0.05) levels in the observation group.@*CONCLUSIONS@#In addition to medication, moxibustion as an adjunctive treatment could significantly alleviate joint pain and reduce disease activity in RA patients, suggesting a potential role in joint protection. This mechanism may be related to the inhibition of the inflammatory factor TNF-α, regulation of β-catenin levels, and reduction in the production of the endogenous negative regulator protein SOST within the Wnt/β-catenin signaling pathway.

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.

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.

Effect of Wnt/β-catenin signaling pathway in neural differentiation of human bone marrow mesenchymal stem cells / 中国修复重建外科杂志

OBJECTIVE@#To explore the effect of basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), and the combination of bFGF and EGF in the neural differentiation of human bone marrow mesenchymal stem cells (hBMSCs), and the role of Wnt/β-catenin signaling pathway in this process.@*METHODS@#The identified 4th-generation hBMSCs were divided into five groups according to different induction conditions, namely control group (group A), EGF induction group (group B), bFGF induction group (group C), EGF and bFGF combined induction group (group D), and EGF, bFGF, and Dickkopf-related protein 1 (DKK-1) combined induction group (group E). After 7 days of continuous induction, the cell morphology was observed by inverted fluorescence phase contrast microscopy, levels of genes that were related to neural cells [Nestin, neuron-specific enolase (NSE), microtubule-associated protein 2 (MAP-2), and glial fibrillary acidic protein (GFAP)] and key components of the Wnt/β-catenin signaling pathway (β-catenin and Cyclin D1) were detected by RT-PCR, and the levels of proteins that were related to neural cells (Nestin and GFAP) as well as genes that were involved in Wnt/β-catenin signaling pathway [β-catenin, phosphorylation β-catenin (P-β-catenin), Cytoplasmic β-catenin, and Nuclear β-catenin] were explored by cellular immunofluorescence staining and Western blot.@*RESULTS@#When compared to groups A and B, the typical neuro-like cell changes were observed in groups C-E, and most obviously in group D. RT-PCR showed that the relative expressions of Nestin, NSE, and MAP-2 genes in groups C-E, the relative expressions of GFAP gene in groups D and E, the relative expression of NSE gene in group B, the relative expressions of β-catenin gene in groups C and D, and the relative expressions of Cyclin D1 gene in groups B-D significantly increased when compared with group A ( P<0.05). Compared with group E, the relative expressions of Nestin, NSE, MAP-2, GFAP, β-catenin, and CyclinD1 genes significantly increased in group D ( P<0.05); compared with group C, the relative expression of Nestin gene in group D significantly decreased ( P<0.05), while NSE, MAP-2, and GFAP genes significantly increased ( P<0.05). The cellular immunofluorescence staining showed that the ratio of NSE- and GFAP-positive cells significantly increased in groups C-E than in group A, in group D than in groups C and E ( P<0.05). Western blot assay showed that the relative expression of NSE protein was significantly higher in groups C and D than in group A and in group D than in groups C and E ( P<0.05). In addition, the relative expression of GFAP protein was significantly higher in groups C-E than in group A and in group D than in group E ( P<0.05). Besides, the relative expressions of β-catenin, Cytoplasmic β-catenin, Nuclear β-catenin, and the ratio of Nuclear β-catenin to Cytoplasmic β-catenin were significantly higher in groups C and D than in group A and in group D than in group E ( P<0.05), whereas the relative expression of P-β-catenin protein was significantly lower in groups C and D than in group A and in group D than in group E ( P<0.05).@*CONCLUSION@#Different from EGF, bFGF can induce neural differentiation of hBMSCs. In addition, EGF can enhance the hBMSCs neural differentiation of bFGF, while the Wnt/β-catenin signaling pathway may play a positive regulatory role in these processes.

Blaps rynchopetera combined with cyclophosphamide affects proliferation and apoptosis of lung cancer cells via Wnt/β-catenin signaling pathway / 中国中药杂志

This study aims to investigate the effects of Blaps rynchopetera Fairmaire and/or cyclophosphamide on the proliferation and apoptosis of lung cancer cells and decipher the underlying mechanism. B. rynchopetera and cyclophosphamide-containing serum and blank serum were prepared from SD rats. Cell counting kit-8(CCK-8) assay was employed to examine the proliferation of lung cancer cell lines A549 and Lewis treated with corresponding agents. The Jin's formula method was used to evaluate the combined effect of the two drugs. According to the evaluation results, appropriate drug concentrations and lung cancer cell line were selected for subsequent experiments, which included control, B. rynchopetera, cyclophosphamide, B. rynchopetera + cyclophosphamide, and B. rynchopetera + Wnt/β-catenin pathway agonist lithium chloride(LiCl) groups. Immunocytochemistry was employed to measure the expression of proliferation-related proteins in Lewis cells after drug interventions. Flow cytometry was employed to determine the cell cycle and apoptosis. The expression levels of proliferating cell nuclear antigen(PCNA), cyclinD1, B-cell lymphoma 2(Bcl-2), Bcl-2-assiocated X protein(Bax), Wnt1, and β-catenin were determined by Western blot. The results showed that B. rynchopetera and/or cyclophosphamide significantly inhibited the proliferation of A549 and Lewis cells. Compared with B. rynchopetera alone, the combination increased the inhibition rate on cell proliferation. The combination of B. rynchopetera and cyclophosphamide demonstrated a synergistic effect according to Jin's formula-based evaluation. Compared with the control group, the B. rynchopetera, cyclophosphamide, and B. rynchopetera + cyclophosphamide groups showed increased proportion of Lewis cells in G_0/G_1 phase, increased apoptosis rate, up-regulated expression of Bax, and down-regulated expression of PCNA, cyclinD1, Bcl-2, Wnt1, and β-catenin. Compared with the cyclophosphamide group, the combination group showed increased proportion of cells in G_0/G_1 phase, increased apoptosis rate, up-regulated expression of Bax, and down-regulated expression of PCNA, cyclinD1, Bcl-2, Wnt1, and β-catenin. Compared with the B. rynchopetera group, the B. rynchopetera + LiCl group had deceased proportion of cells in G_0/G_1 phase, decreased apoptosis rate, down-regulated expression of Bax, and up-regulated expression of PCNA, cyclinD1, Bcl-2, Wnt1, and β-catenin. The results indicated that B. rynchopetera could inhibit the proliferation, arrest the cell cycle, and induce the apoptosis of lung cancer cells by inhibiting the Wnt/β-catenin signaling pathway. Moreover, B. rynchopetera had a synergistic effect with cyclophosphamide.

Effect of CYFIP2 overexpression on biological function and Wnt/β-catenin signaling pathway of bladder cancer cell line T24 / 国际生物医学工程杂志

Objective:To investigate the effects of cytoplasmic fragile X mental retardation protein 1 binding protein 2 (CYFIP2) overexpression on the biological functions and Wnt/β-catenin signaling pathways of bladder cancer T24 cells.Methods:The control group was T24 cells transfected with the empty pcDNA3 vector, and the overexpression group was T24 cells transfected with the CYFIP2 overexpression vector. The expression of CYFIP2 mRNA and protein was detected by reverse transcriptase, quantitative polymerase chain reaction, and Western Blot. The effect of CYFIP2 overexpression on T24 cell proliferation was detected by CCK-8. The effect of CYFIP2 overexpression on T24 cell migration and invasion was detected by Transwell. The effects of CYFIP2 overexpression on Wnt/β-catenin signaling pathway in T24 cells were detected by Western Blot.Results:Compared with the control group, the expression levels of CYFIP2 mRNA and protein were increased in the overexpression group (all P < 0.001), and the cell proliferation, migration, and invasion abilities were reduced (all P < 0.01). β-catenin, c-Myc, and Cyclin D1 protein expression were down-regulated in CYFIP2 overexpressed T24 cells (all P < 0.05), while the protein levels of p-β-catenin were increased ( P < 0.05). Conclusions:CYFIP2 overexpression can inhibit T24 cell proliferation, migration, and invasion, and its possible molecular mechanism is related to the inhibition of Wnt/β-catenin signaling pathway.

Traditional Chinese Medicine Regulates Wnt/β-catenin Signaling Pathway to Treat Steroid-induced Femoral Head Necrosis： A Review / 中国实验方剂学杂志

Steroid-induced necrosis of the femoral head （SNOFH） is a common orthopedic disease，which is difficult to cure and has poor clinical prognosis. The number of SNOFH patients in China is still increasing year by year，which seriously threatens human health. Long-term non-standard or short-term extensive use of hormone （GC） is an important reason for the occurrence of this disease. At present，SNOFH is mostly treated by surgical methods such as hip replacement，which has limitations of great harm to patients and high cost. In recent years，with the continuous deepening and innovation of traditional Chinese medicine（TCM） research，the use of TCM to treat SNOFH has been widely used in clinical practice. The main TCM pathogenesis of SNOFH is kidney deficiency and blood stasis. Therefore，TCM monomer and compound compound of tonifying kidney and promoting blood circulation are used to treat SNOFH. And TCM has obvious therapeutic effect，small side effects，less cost and other advantages. Glycoprotein/beta chain protein secretion （Wnt/beta- catenin） signaling pathway as a classic signaling pathway is closely related to the bone，between its by promoting bone marrow mesenchymal stem cell update，enhance the activity of osteoblast and suppress the apoptosis，which adjust the metabolic balance of bone tissue，increase bone density，will play an important role in the process of bone formation. In recent years，the use of TCM monomers and compounds to regulate Wnt/β-catenin signaling pathway to accelerate bone marrow mesenchymal stem cells，promote their transformation into osteoblasts，and maintain bone metabolic balance mechanism to treat SNOFH has become a new research hotspot. This article reviews the research progress of TCM in the prevention and treatment of SNOFH by regulating Wnt/β-catenin signaling pathway，in order to provide reference for the application of TCM in the treatment of SNOFH.

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.