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.

Effects of lead exposure on development of heart and spine in zebrafish embryos / 环境与职业医学

Background Lead is widely distributed. Lead exposure interferes with early life development in zebrafish, but the mechanisms by which lead exposure affects skeletal development and cardiac development are not clear as yet. Objective To investigate the molecular mechanisms of bone development and cardiac development toxicity induced by lead acetate exposure. Methods Zebrafish embryos were exposed to different concentrations of lead acetate (0, 6, 12, 24, and 48 μmol·L−1) for 3 h post-fertilization (3 hpf) until 5 d post-fertilization (5 dpf). The malformation phenotypes of 5 dpf were counted, and the mRNA expressions of spinal development-related genes (bmp2b, bmp4, bmp9, runx2a, runx2b) and heart development-related genes (nkx2.5, myh6, myh7) were detected by quantitative PCR (qPCR). Expressions of genes of development-related regulatory pathways including Wnt/β-catenin pathway (wnt5a, wnt8a, wnt10a, β-catenin) and TGF-β pathway (tgf-β1, tgf-β2) as well as key molecule eph of Eph-Ephrin signaling were analyzed. Results At 5 dpf, the zebrafish in the lead acetate treated groups showed deformed phenotypes including spinal curvature and pericardial sac edema compared to the control group. In the lead acetate groups at 24 and 48 μmol·L−1, the spinal curvature deformity rates reached 26.47% and 71.52% (P<0.01) respectively. The qPCR results revealed that the expression levels of spinal development-related genes bmp2b, bmp4, bmp9, runx2a, and runx2b were downregulated in the 48 μmol·L−1 exposure group compared to the control group by 82.8%, 58.0%, 88.7%, 85.5%, and 69.2%, respectively (P<0.05 or P<0.01); the expression levels of heart development-related genes myh6, myh7, and nkx2.5 were down-regulated by 63.7%, 58.9%, and 55.2%, respectively (P<0.01); the expression levels of wnt8a and β-catenin in the Wnt/β-catenin pathway were down-regulated by 71.5% and 47.3% (P < 0.05 or P < 0.01), respectively; the expression level of tgf- β1 in the TGF-β pathway was down-regulated by 67.5% (P<0.01); the expression level of eph was down-regulated by 86.9% (P<0.01). Conclusion Lead acetate exerts developmental toxic effects on zebrafish heart and bone by down-regulating the expressions of genes related to spinal development and heart development, as well as inhibiting development-related Wnt/β-catenin and TGF-β pathways and Eph-Ephrin signaling, causing malformed phenotypes such as spinal curvature and pericardial sac edema.

Proliferation Inhibitory Activity of Quinones from Blaps rynchopetera Defense Secretion on Colorectal Tumor Cells / 中国结合医学杂志

OBJECTIVE@#To explore the proliferation inhibitory effect of quinones from Blaps rynchopetera defense secretion on colorectal tumor cell lines.@*METHODS@#Human colorectal cancer cell HT-29, human colorectal adenocarcinoma cell Caco-2 and normal human colon epithelial cell CCD841 were chosen for the evaluation of inhibitory activity of the main quinones of B. rynchopetera defense secretion, including methyl p-benzoquinone (MBQ), ethyl p-benzoquinone (EBQ), and methyl hydroquinone (MHQ), through methyl thiazolyl tetrazolium assay. The tumor-related factors, cell cycles, related gene expressions and protein levels were detected by enzyme-linked immunosorbent assy, flow cytometry, RT-polymerase chain reaction and Western blot, respectively.@*RESULTS@#MBQ, EBQ, and MHQ could significantly inhibit the proliferation of Caco-2, with half maximal inhibitory concentration (IC50) values of 7.04 ± 0.88, 10.92 ± 0.32, 9.35 ± 0.83, HT-29, with IC50 values of 14.90 ± 2.71, 20.50 ± 6.37, 13.90 ± 1.30, and CCD841, with IC50 values of 11.40 ± 0.68, 7.02 ± 0.44 and 7.83 ± 0.05 µg/mL, respectively. Tested quinones can reduce the expression of tumor-related factors tumor necrosis factor α, interleukin (IL)-10, and IL-6 in HT-29 cells, selectively promote apoptosis, and regulate the cell cycle which can reduce the proportion of cells in the G1 phase and increase the proportion of the S phase. Meanwhile, tested quinones could up-regulate mRNA and protein expression of GSK-3β and APC, while down-regulate that of β-catenin, Frizzled1, c-Myc, and CyclinD1 in the Wnt/β-catenin pathway of HT-29 cells.@*CONCLUSION@#Quinones from B. rynchopetera defense secretion could inhibit the proliferation of colorectal tumor cells and reduce the expression of related factors, which would be functioned by regulating cell cycle, selectively promoting apoptosis, and affecting Wnt/β-catenin pathway-related mRNA and protein expressions.

Sex determining region Y-box 9 induced microtubule formation and epithelial⁃mesenchymal transition in human oral squamous cell carcinoma CAL27 cells / 华西口腔医学杂志

OBJECTIVES@#This study aimed to explore the effect of sex determining region Y-box 9 (SOX9) on the microtubule formation and epithelial-mesenchymal transition (EMT) of human oral squamous cell carcinoma (OSCC) CAL27 and the underlying mechanism.@*METHODS@#SOX9-shRNA1 and SOX9-shRNA2 were designed and synthesized and then transfected into CAL27 cells. The expression of SOX9 was detected by quantitative real-time polymerase chain reaction. Microtubule formation assay was used to detect the change in the number of microtubule nodules after interfering with SOX9. Immunofluorescence was used to detect the Vimentin content. Western blot was used to detect the protein expression of EMT marker molecules and Wnt/β-catenin pathway-related proteins, such as E-cadherin, N-cadherin, Fibronectin, Wnt, β-catenin, T-cell factor-4 (TCF-4).@*RESULTS@#The expression level of SOX9 significantly decreased after transfection with SOX9-shRNA1 and SOX9-shRNA2 in CAL27 cells (@*CONCLUSIONS@#Interference with SOX9 decreased Vimentin content and inhibited the microtubule formation and protein expression of EMT marker molecules, as well as the expression of proteins related to the Wnt/β-catenin pathway. Thus, SOX9 can induce microtubule formation and EMT in CAL27, which was related to the inhibition of the Wnt/β-catenin pathway activation.

Intervention Mechanism of Yishen Huayu Prescription on Glomerular Podocyte Injury in Diabetic Nephropathy Rats Based on EMT Regulated by Wnt/β-catenin Pathway / 中国实验方剂学杂志

Objective:To investigate the intervention mechanism of Yishen Huayu prescription on glomerular podocyte injury in diabetic nephropathy （DN） rats based on epithelialmesenchymal transition （EMT） regulated by Wnt/<italic>β</italic>-catenin pathway. Method:The 60 SD rats were divided into control group， model group， Wnt-C59 group （0.03 g·kg^-1 Wnt/<italic>β</italic>-catenin pathway inhibitor）， low-dose group （8 g·kg^-1）， medium-dose group （16 g·kg^-1） and high-dose group （32 g·kg^-1）. After 12 weeks， various indexes ， including general signs， serum creatinine （SCr）， blood urea nitrogen （BUN）， renal index， urinary protein， blood glucose， renal pathological changes， podocyte and expressions of glomerular basement membrane injury and podocyte injury related proteins ［nephrin， synaptopodin］， Wnt/<italic>β</italic>-catenin pathway related proteins （Wnt1， <italic>β</italic>-catenin）， podocyte EMT related protein ［<italic>α</italic>-smooth muscle actin （<italic>α</italic>-SMA）， E-cadherin］， were compared between groups. Result:Compared with the control group， the renal tissue in the model group showed significant pathological changes， including diffuse thickening of glomerular mesangial matrix and severe foot process fusion， and a significant increase in SCr， BUN， renal indexes， urinary protein， blood glucose， Wnt1， <italic>β</italic>-catenin， and <italic>α</italic>-SMA expression levels （<italic>P</italic><0.05） as well as a significant decrease in nephrin， synaptopodin and E-cadherin expression levels（<italic>P</italic><0.05）. Compared with model group， SCr， BUN， renal index， urinary protein， blood glucose， Wnt1， <italic>β</italic>-catenin， and <italic>α</italic>-SMA expression levels in each intervention group significantly decreased （<italic>P</italic><0.05）， while the expression levels of nephrin， synaptopodin and E-cadherin significantly increased （<italic>P</italic><0.05）. Among intervention groups， the improvement of above indexes in high-dose Yishen Huayu prescription group was the most obvious （<italic>P</italic><0.05）， which was similar to the effect in Wnt-C59 group. Conclusion:Yishen Huayu prescription prevents podocyte EMT by inhibiting Wnt/<italic>β</italic>-catenin pathway， thereby repairing glomerular podocyte injury in rats with diabetic nephropathy.

Correlation between miR-206 expression and Wnt/β-catenin pathway in peripheral blood mononuclear cells of patients with hyperthyroidism / 中华地方病学杂志

Objective:To explore the correlation between the expression level of microRNA (miR)-206 and the expression levels of Wnt-1 and β-catenin in Wnt/β-catenin pathway in peripheral blood mononuclear cells of patients with hyperthyroidism.Methods:Using a prospective design, 79 patients with hyperthyroidism admitted to Wuwei City People's Hospital from January 2018 to June 2020 were selected as observation group, and 70 cases of healthy physical examination in Wuwei City People's Hospital from January 2019 to September 2020 were selected as control group. The observation group was treated with anti-thyroid drugs, and tambazole was orally taken 10-20 mg/d during the treatment period; after the serum thyroid hormone was normal, the dosage was reduced and maintained at 5-10 mg/d for 12 months. Quantitative real-time PCR (qRT-PCR) was used to detect the expression levels of miR-206, Wnt-1 and β-catenin mRNA in peripheral blood mononuclear cells in observation group before and after treatment and control group.Results:The expression level of miR-206 in peripheral blood mononuclear cells of observation group before treatment was lower than that of control group (0.28 ± 0.07 vs 0.79 ± 0.15), and the expression levels of Wnt-1 and β-catenin mRNA were higher than those of control group (6.75 ± 1.39 vs 2.23 ± 0.65, 5.83 ± 1.24 vs 3.21 ± 0.86, P < 0.05). The expression level of miR-206 (0.54 ± 0.13) in peripheral blood mononuclear cells of observation group after treatment was higher than that before treatment, and the expression levels of Wnt-1 and β-catenin mRNA (3.62 ± 1.08, 4.34 ± 1.16) were lower than those before treatment ( P < 0.05). The expression level of miR-206 in peripheral blood mononuclear cells of patients with hyperthyroidism was negatively correlated with the expression levels of Wnt-1 and β-catenin mRNA ( r = - 0.763, - 0.798, P < 0.05). Conclusions:The expression level of miR-206 in peripheral blood mononuclear cells of patients with hyperthyroidism is low, while the expression levels of Wnt-1 and β-catenin mRNA are high. The expression level of miR-206 is negatively correlated with the expression levels of Wnt-1 and β-catenin mRNA.

Improvement Effects of Polygonatum sibiricum Polysaccharides on Myocardial Injury of Acute Myocardial Infarction Model Rats / 中国药房

OBJECTIVE：To study the imp rovement effects of Polygonatum si biricum polysaccharides（PSP）on the myocardial injury of acute myocardial infarction （AMI）model rats. METHODS ：The rats were randomly divided into blank control group ， model group ，aspirin group （positive control ，25 mg/kg），PSP low-dose ，medium-dose and high-dose groups （0.5，1，2 g/kg）， with 10 rats in each group. Except for blank control group ，AMI model was established by ligation of the anterior descending branch of the left coronary artery of rats in other groups. After modeling ，blank control group and model group were given normal saline intragastrically ，and administration groups were given relevant medicine intragastrically ，once a day ，for consecutive 28 days. After last medication ，left ventricular ejection fraction （LVEF）and left ventricular short axis shorting rate （LVFS）of rats were detected. The morphological changes of myocardial tissue were observed. The levels of oxidative stress indexes （SOD，MDA， ROS）in myocardial tissue of rats were detected by ELISA. The expression of apoptosis-related proteins （Bcl-2，Bax，caspase-3， caspase-8，caspase-9）and the Wnt/ β-catenin pathway-related proteins （Wnt1，β-catenin）in left ventricular anterior wall tissue of rats were detected by Western blot assay. RESULTS ：Compared with model group ，the levels of LVEF and LVFS ，the levels of SOD in myocardial tissue and protein expression of Bcl- 2 in left ventricular anterior wall tissue were increased significantly in PSP medium-dose，high-dose groups and aspirin group （P＜0.05）；the levels of MDA and ROS in myocardial tissue ，the protein expression of Bax ，caspase-3，caspase-8，caspase-9，Wnt1 and β-catenin in left ventricular anterior wall tissue were decreased significantly（P＜0.05）；myocardial tissue structure disorder and inflammatory cell infiltration were reduced. CONCLUSIONS ： PSP can relieve myocardial injury in AMI model rats ；its mechanism may be related to increasing SOD level in myocardial tissue ，decreasing MDA and ROS level ，regulating apoptosis-related proteins and Wnt/ β-catenin pathway-related proteins.

Mechanism of Biejiajian Wan in Reversing EMT in Rat Hetapic Oval Cells via Wnt/ β-catenin Pathway / 中国实验方剂学杂志

Objective:To study the effect of Biejiajian Wan on the epithelial-mesenchymal transition （EMT） of rat hepatic oval cells induced by transforming growth factor- β1（TGF-β1）， in order to explore its mechanism in reversing EMT. Method:WB-F344 cells were divided into five groups： blank group， TGF-β1 model group （10 μg·L-1TGF-β1）， low-dose group （10 μg·L-1TGF-β1+0.55 g·kg-1Biejiajian Wan）， medium-dose group （10 μg·L-1TGF-β1+1.1 g·kg-1Biejiajian Wan）， high-dose group （10 μg·L-1TGF-β1+2.2 g·kg-1Biejiajian Wan）. Except blank group， TGF-β1 was used to induce WB-F344 cells in all of the remaining groups to construct an EMT model. After the cells were treated with low， medium and high doses of Biejiajian Wan serum， the changes of migration ability of WB-F344 cells were detected by cell scratching test. The expressions of E-cadherin， N-cadherin and Vimentin were detected by Western blot. Real-time PCR was used to detect the changes in the expression of β-catenin mRNA. The expression of β-catenin was detected by cell immunofluorescence assay. Result:Compared with normal WB-F344 cells， the intercellular space of WB-F344 cells became loose from tight， and the morphology changed from cobblestone to fibroblast after TGF-β1 induced WB-F344 cells for 4 days， and the expression of E-cadherin protein decreased， while the expression of N-cadherin protein increased （P<0.01）， indicating that the EMT model of WB-F344 cells was successfully built. Compared with the blank group， the migration ability of WB-F344 cells in TGF-β1 model group was enhanced （P<0.01）， compared with TGF-β1 model group， Biejiajian Wan could significantly inhibit the migration ability of WB-F344 cells； specifically， the low-dose group had no statistical significance， and the medium and high-dose groups had statistical significance （P<0.05）. Western blot results showed that compared with the blank group， the expression of E-cadherin decreased， whereas those of N-cadherin and Vimentin increased in the TGF-β1 model group （P<0.01）， compared with TGF-β1 model group， E-cadherin protein expression was increased in the low， medium and high-dose groups， while the expressions of N-cadherin and Vimentin was decreased； specifically， the low-dose groups had no statistical significance， and the medium and high dose groups had statistical significance （P<0.05，P<0.01）. Real-time PCR results showed that compared with the blank group， the mRNA expression of β-catenin in the TGF-β1 model group was increased （P<0.05）， whereas compared with TGF-β1 model group， the mRNA expression of β-catenin in the low， medium and high-dose groups of Biejiajian Wan was reduced （P<0.01）. The results of cellular immunofluorescence showed that compared with the blank group， the fluorescence expression of β-catenin in the cell nucleus was enhanced in the TGF-β1 model group； and compared with the TGF-β1 model group， the expression of β -catenin in the cell nucleus of the low， medium and high-dose groups of Biejiajian Wan decreased， and the inhibitory effect of Biejiajian Wan on β-catenin in the cell nucleus was positively correlated with its concentration. Conclusion:Biejiajian Wan may reverse the EMT process that TGF-β1 induced WB-F344 cells， and inhibit the migration of WB-F344 cells by inhibiting Wnt/β-catenin signaling pathway.

Effect of ginkgo biloba extract on behaviors in rat with adolescent sedentariness / 解剖学报

Objective To investigate the effect of Ginkgo biloba extract on behaviors in rat with adolescent sedentariness and its possible mechanism. Methods Twenty-four male SD rats (4-week-old) were randomly divided into normal control (NC), adolescent sedentarines (AS), and Ginkgo biloba extract (GBE) groups. After 4 weeks intervention with GBE, open-field test and elevated plus-maze test were performed to detect the behavioral changes. The content of malondialdehyde (MDA) and activity of superoxide dismutase (SOD) in brain were determined by colorimetric method. Protein levels of glycogen synthase kinase 3β(GSK-3β) and β-catenin in cerebral white matter were determined by Western blotting. Results In open-field test, it was shown that the autonomic activity of rats in AS group increased, while the central regional travel time was reduced. Duration and number of residence in the open arm of elevated plus-maze test decreased in AS group. These anxiety-like behaviors were ameliorated by GBE intervention. Compared with the NC group, GSK-3β/ β-catenin ratio and the content of MDA was upregulated in AS group while downregulated in GBE group. And activity of SOD were downregulated in AS group while significantly upregulated in GBE group (P<0. 05). Conclusion Ginkgo biloba extract ameliorate anxiety-like behavior in rat with adolescent sedentariness. Wnt/ β-catenin pathway and antioxidant regulation may play a cerebral protective role.

Mechanism of electroacupuncture for regulation of lipid production and improvement in obesity by mediating Wnt/ β-catenin pathway through activating SIRT1 / 中国针灸

OBJECTIVE@#To explore the mechanism of electroacupuncture (EA) for the regulation of lipid production and improvement in obesity by mediating Wnt/β-catenin pathway through activating silent information regulator 1 (SIRT1).@*METHODS@#Of 75 Wistar male rats, 10 rats were selected randomly as the normal group and fed with standard diet. The rest rats were fed with high-fat diet for 8 weeks to establish the obesity model. Forty rats of successful modeling were randomized into a model group, an EA group, an EA plus inhibitor group (EA+I group) and an agonist group, 10 rats in each one. In the EA group, EA was applied at "Guanyuan" (CV 4), "Zhongwan" (CV 12), "Zusanli" (ST 36) and "Fenglong" (ST 40), with continuous wave, 2 Hz in frequency and around 1 mA in intensity. The needles were retained for 20 min. In the EA+I group, sirtinol solution was injected from caudal vein and EA was exerted simultaneously. In the agonist group, resveratrol solution was given by intragastric administration. The intervention of the above three groups was given once every two days, 3 times a week, consecutively for 8 weeks. Before and after intervention, body mass and Lee's index were recorded in the rats of each group. After intervention, the levels of serum total cholesterol (TC), triglyceride (TG) and free fatty acid (FFA) were detected in the rats of each group. After intervention, the mass of white adipose tissue (WAT) and the area of adipocytes were compared in the rats among the 5 groups. Using Western blot method, the protein expressions of SIRT1, glycogen synthase kinase-3β (GSK3β), β-catenin, cyclin D1 and peroxisome proliferators-activated receptor γ (PPARγ) were detected in WAT in the rats of each group.@*RESULTS@#After intervention, compared with the model group, the body mass and Lee's index were reduced in the rats of the EA group and the agonist group (@*CONCLUSION@#Electroacupuncture remarkably improves the body mass, Lee's index and blood lipid metabolism and reduces WAT mass and adipocyte size in obesity model rats, which is probably related to up-regulating the protein expression of SIRT1 in WAT, activating Wnt/β-catenin pathway and inhibiting the expression of PPARγ of downstream lipogenic gene so as to affect lipid production.

Social Isolation in Male Rats During Adolescence Inhibits the Wnt/β-Catenin Pathway in the Prefrontal Cortex and Enhances Anxiety and Cocaine-Induced Plasticity in Adulthood / 神经科学通报·英文版

In adult animals, it is well established that stress has a proactive effect on psychostimulant responses. However, whether only a short period of stress during adolescence can also affect cocaine responses later in life and what mechanisms are involved are unknown. Here, we showed that 5 days of social isolation during rat adolescence had a long-term impact on anxiety-like behaviors, cocaine-induced conditioned place preference, and the expression of sensitization during adulthood. At the molecular level, social isolation decreased the activity of the Wnt/β-catenin pathway in the prefrontal cortex (PFC). Furthermore, after the expression of cocaine sensitization, isolated rats showed an increase in this pathway in the nucleus accumbens. Together, these findings suggest that, adolescent social isolation by altering the Wnt/β-catenin pathway in the developing PFC might increase the cocaine responses during adulthood, introducing this pathway as a novel neuroadaptation in the cortical-accumbens connection that may mediate a stress-induced increase in vulnerability to drugs.

Epigenetic deregulations of Wnt/β-catenin and transforming growth factor beta-Smad pathways in esophageal cancer: Outcome of DNA methylation

Background: Promoter methylation of tumor suppressor genes (TSGs) is a well-reported portent in carcinogenesis; hence, it is worthy to investigate this in high-risk Northeast population of India. The study was designed to investigate methylation status of 94 TSGs in esophageal squamous cell carcinoma (ESCC). Further, the effect of OPCML promoter methylation on gene expression was analyzed by immunohistochemistry. Moreover, in silico protein–protein interactions were examined among 8 TSGs identified in the present study and 23 epigenetically regulated genes reported previously by our group in ESCC. Materials and Methods: Methylation profiling was carried out by polymerase chain reaction array and OPCML protein expression was examined by tissue microarray-based immunohistochemistry. Results: OPCML, NEUROG1, TERT, and WT1 genes were found hypermethylated and SCGB3A1, CDH1, THBS1, and VEGFA were hypomethylated in Grade 2 tumor. No significant change in OPCML expression was observed among control, Grade 1, and Grade 2 tumor. Conclusively, hypermethylation of the studied OPCML promoter in Grade 2 tumor produced no effect on expression. Unexpectedly, OPCML expression was downregulated in Grade 3 tumor in comparison to other groups signifying that downregulation of OPCML expression may lead to higher grade of tumor formation at the time of diagnosis of ESCC in patients. Significant interactions at protein level were found as VEGFA:PTK2, CTNNB1:CDH1, CTNNB1:VEGFA, CTNNB1:NEUROG1, CTNND2:CDH1, and CTNNB1:TERT. These interactions are pertinent to Wnt/β-catenin and TGF-β-Smad pathways. Conclusions: Deranged OPCML expression may lead to high-grade ESCC as well as epigenetically regulated genes, that is, CDH1, CTNNB1, CTNND2, THBS1, PTK2, WT1, OPCML, TGFB1, and SMAD4 may alter the Wnt/β-catenin and TGF-β-Smad pathways in ESCC. Further study of these genes could be useful to understand the molecular pathology of ESCC with respect to epithelial-mesenchymal transition (EMT) mediated by Wnt/β-catenin and TGF-β signaling pathways

Study on the Mechanism of Wound Healing Promotion of Panax notoginseng-Bletilla striata Gum Sponge on Diabetic Foot Ulcer Model Rats Based on Wnt/ β-catenin Signal Pathway / 中国药房

OBJECTIVE： To study the mechanism of wound healing promotion of Panax notoginseng-Bletilla striata gum sponge on diabetic foot ulcer （DFU） model rats. METHODS： Healthy SD rats were selected and given high-lipid and high-glucose diet， intraperitoneal injection of streptozotocin once to establish diabetes model. Neodymium-iron-boron magnet was used to press the back of rats to make ulcer wound then established DFU model. Totally 60 DFU model rats were randomly divided into group A（blank group， i.e. normal saline gauze group）， group B（vaseline gauze group），group C （gelatin sponge group） and group D （P. notoginseng-B. striata gum sponge group）， with 15 rats in each group. The rats were given corresponding gauze/sponge to cover the wound for intervention treatment， changing dressing once every 1-2 days. On the 3rd and 7th day after intervention， the wound healing of rats in each group was observed with naked eyes， and the wound healing rate was calculated. The wound margin tissue was collected to obtain HE staining section， and histopathological observation was conducted under microscope. mRNA expression of β-catenin， GSK-3β and Rspo3 in wound tissue were determined by RT-PCR. RESULTS： On the 3rd and 7th day after intervention， compared with group A， B， C， healing rate of group D was increased significantly （P＜0.05）； inflammatory cell infiltration， collagen deposition， capillary and granulation tissue growth in wound tissue increased significantly. The mRNA expression levels of β-catenin and Rspo3 all increased， and those of GSK-3β all decreased； except for the difference of β-catenin at the 3rd day and GSK-3β at the 7th day after intervention between group D and group C were not significant， the difference of other indicators was statistically significant （P＜0.05）. CONCLUSIONS： P. notoginseng-B. striata gum sponge can effectively promote the wound healing in DFU model rats， the mechanism of which may be associated with up-regulating the expression of β-catenin and Rspo3 mRNA and down-regulating the expression of GSK-3β mRNA.

Renal Protective Effect of Tangshiping Capsule on Diabetic Nephropathy Kkay Mice and Its Effect on Wnt/β-catenin Signal Transduction Pathway / 中国实验方剂学杂志

Objective:To investigate the renal protective effect of Tangshenping capsule (Tangshenping) on diabetic nephropathy (DN) KKAy mice and its effect on Wnt/β-catenin signaling pathway. Method:Sixty female Sprague-Dawley KKAy mice aged 10 weeks old were induced with KKAy rat feed for 10 weeks. The DN animal model was successfully determined with blood glucose (>16.7 mmol·L-1) and 24 hour urine protein (>0.4 mg). The model mice were randomly divided into a model group, an irbesartan group, and low, medium and high-dose Tangshenping group, with 10 female C57BL/6J mice as a control group. The treatment groups were given the corresponding drugs by gavage. The normal group and the model group were given an equal volume of deionized water by gavage. The intragastric dose was 0.01 mL·g-1 body weight coefficient once a day. The general conditions of the mice were observed, the body mass was weighed every 4 weeks, and 24 h urine protein was quantified. At the 26th week, the blood was collected from eyeballs, and the mice were put to death. The quality of the kidneys, serum blood urea nitrogen (BUN), serum creatinine (SCr), triglyceride (TG), malondialdehyde (MDA), nitric oxide (NO) and superoxide dismutase (SOD) content were measured. In situ hybridization and immunohistochemistry were used to detect the expressions of Wnt4, glycogen synthase kinase 3β(GSK3β) and β-catenin in kidney tissues. Result:Compared with model group, body mass, kidney mass/body mass, and 24 h urine protein were significantly lower in high-dose Tangshenping group (PPPβ and β-catenin were decreased (PConclusion:Tangshenping may inhibit the activation of Wnt/β-catenin signaling pathway, reverse the transdifferentiation of renal tubular epithelial cells in DN KKAy mice, delay the progression of renal interstitial fibrosis, and then exert renal protection.

TNF-α promotes the proliferation of colon cancer cells by activating the Wnt/β-catenin pathway / 西安交通大学学报(医学版)

Objective To investigate the effect of treatment with TNF-α on growth ability of colon cancer cells and its possible mechanism.Methods Human colon cancer HT-29 cells were treated with TNF-α(10 nmol/L). The effect of TNF-αon the proliferative ability was examined by cell growth curve assay and MTT assay,respectively. Cell cycle was detected by flow cytometry.Protein levels ofβ-catenin,c-myc and cyclinD1 were detected by Western blot.We also observed the effect of the Wnt/β-catenin pathway blockage by XAV9 3 9 on TNF-α's promoting prolif-eration of colon cancer cells and proteins related to the Wnt/β-catenin pathway.Results Upon treatment with TNF-α,the proliferative ability of HT-29 cells was enhanced (all P＜0.05),G0/G1 phase cell ratio was decreased (P＜0.05),and S phase cell ratio was increased (P＜0.05).The protein levels ofβ-catenin,c-myc and cyclinD1 were increased in TNF-α-treated HT-29 cells (all P＜0.05).XAV939 treatment resulted in a significant inhibition of cell proliferation ability (all P＜0.05)as well as the protein levels ofβ-catenin,c-myc and cyclinD1 (all P＜0.05) in the TNF-α-treated HT29 cells.Conclusion TNF-αmay be involved in the occurrence and development of colon cancer by activating the Wnt/β-catenin pathway and promoting the proliferation of colon cancer cells.

Long non-coding RNA-MIAT promotes proliferation of colorectal cancer by regulating Wnt/β-catenin pathway / 中华普通外科杂志

Objective To analyze the expression of MIAT in colorectal cancer and explore its molecular mechanism.Methods The expression of MIAT mRNA in colorectal cancer tissues and paracancerous tissues,colorectal cancer cell lines and human normal colorectal mucosa cells was detected by RT-qPCR.HCT-116 cells were transfected with MIAT overexpression vector and infected with knockdown lentivirus,cell proliferation and cell cycle were detected,Western blot was used to analyze the β3-catenin protein levels,luciferase assay was used to detect β-catenin promoter transcription level,C-myc,CCND1,CD44 and Oct3/4 mRNA levels was measured by RT-qPCR.Results The expression of MIAT in colorectal cancer tissues was significantly higher than that in adjacent tissues (t =4.093,P ＜ 0.05).Overexpression of MIAT could promote the proliferation of HCT-116 cells,decrease the rate of G0/G1 phase,activate Wnt/β-catenin pathway and increase its downstream target gene mRNA expression levels (all P ＜ 0.05).Knockdown MIAT shows the inhibition effect.Conclusions MIAT is a tumor-promoting gene of colorectal cancer and may play a role in tumor promotion by activating Wnt/β3-catenin pathway.

miR-34a Inhibitor May Effectively Protect against Sevoflurane-Induced Hippocampal Apoptosis through the Wnt/β-Catenin Pathway by Targeting Wnt1

PURPOSE: Research has shown that sevoflurane-induced toxicity causes neurodegeneration in the developing brain. miR-34a has been found to negatively regulate ketamine-induced hippocampal apoptosis and memory impairment. However, the role of miR-34a in sevoflurane-induced hippocampal neurodegeneration remains largely unclear. MATERIALS AND METHODS: C57/BL6 mice (7-day-old) inhaled 2.3% sevoflurane for 2 h/day over 3 consecutive days. miR-34a expression was reduced through intracerebroventricular injection with miR-34a interference lentivirus vector (LV-anti-miR-34a) into mouse hippocampus after anesthesia on the first day of exposure. Hippocampal apoptosis was detected by TUNEL assay and flow cytometry analysis. Spatial memory ability was evaluated by the Morris water maze test. The interaction between miR-34a and Wnt1 was confirmed by luciferase reporter assay, RNA immunoprecipitation, Western blot, and immunofluorescence staining. The effects of miR-34a on protein levels of B-cell lymphoma 2 (Bcl-2), bcl-2-like protein 4 (Bax), and Wnt/β-catenin pathway-related proteins were evaluated using Western blot analysis. RESULTS: Sevoflurane upregulated hippocampal miR-34a, and miR-34a inhibitor attenuated sevoflurane-induced hippocampal apoptosis and memory impairment. miR-34a negatively regulated Wnt1 expression by targeting miR-34a in hippocampal neurons. Moreover, forced expression of Wnt1 markedly undermined miR-34a-mediated enhancement of sevoflurane-induced apoptosis of hippocampal neurons, while Wnt1 silencing greatly restored anti-miR-34a-mediated repression of sevoflurane-induced apoptosis of hippocampal neurons. Increased expression of miR-34a inhibited the Wnt/β-catenin pathway in hippocampal neurons exposed to sevoflurane, while anti-miR-34a exerted the opposite effects. CONCLUSION: miR-34a inhibitor may effectively protect against sevoflurane-induced hippocampal apoptosis via activation of the Wnt/β-catenin pathway by targeting Wnt1.

Scutellaria barbata D. Don inhibits colorectal cancer growth via suppression of Wnt/β-catenin signaling pathway / 中国结合医学杂志

<p><b>OBJECTIVE</b>To investigate the effect of the ethanol extract of Scutellaria barbata D. Don (EESB) on colorectal cancer (CRC) growth and Wnt/β-catenin signaling pathway in vivo and in vitro.</p><p><b>METHODS</b>In vivo experiment, CRC xenograft mouse model was constructed with injection of HT-29 cells. Following xenograft implantation, twenty mice were randomly divided into EESB-treated group (n=10) and control group (n=10) by a random number table, and were given with intra-gastric administration of 2 g/kg EESB or saline, 5 days a week for 16 days, respectively. At the end of experiment, tumors were removed and weighed by electronic scales. The proliferation biomarker Ki-67 of tumor was evaluated by immunohistochemistry (IHC) assay. In vitro study, HT-29 cells were treated with 0, 0.5, 1.5, 2.5 mg/mL EESB for 24 h. At the end of the treatment, the viability and survival of HT-29 cells were determined by methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay and colony formation assay, respectively. The mRNA expression of c-Myc, Survivin and adenomatous polyposis coli (APC) was examined by reverse transcription-polymerase chain reaction (RT-PCR) both in tumor tissues of CRC xenograft mice and HT-29 cells. Protein expression of c-Myc, Survivin, APC, and β-catenin as well as β-catenin phosphorylation level were evaluated by IHC assay or Western blotting.</p><p><b>RESULTS</b>EESB significantly reduced tumor weight in CRC xenografts mice, compared with the control group (P<0.05). IHC assay showed that EESB significantly inhibited protein expression of Ki-67 in tumor tissues (P<0.05). MTT assay showed that EESB significantly reduced HT-29 cell viability in a dose-dependent manner (P<0.05). Colony formation assay showed that EESB dose-dependently decreased the survival of HT-29 cells (P<0.05). In addition, RT-PCR assay showed that EESB decreased the mRNA expression of c-Myc and Survivin and increased APC expression, both in tumor tissues of CRC xenograft mice and HT-29 cells (P<0.05). IHC assay or Western blotting showed that EESB decreased protein expression of β-catenin, c-Myc and Survivin, as well as increased APC expression and β-catenin phosphorylation in tumor tissues or HT-29 cells (P<0.05).</p><p><b>CONCLUSIONS</b>EESB significantly reduced tumor growth in CRC xenografts mice, and inhibited the viability and survival of HT-29 cells. EESB could suppress the activation of the Wnt/β-catenin pathway, which might be one of the mechanisms whereby Scutellaria barbata D. Don exerts its anticancer activity.</p>

Various Wavelengths of Light-Emitting Diode Light Regulate the Proliferation of Human Dermal Papilla Cells and Hair Follicles via Wnt/β-Catenin and the Extracellular Signal-Regulated Kinase Pathways

BACKGROUND: The human dermal papilla cells (hDPCs) play an important role in regulation of hair cycling and growth. OBJECTIVE: The aim of this study was to investigate the effect of different wavelengths of light-emitting diode (LED) irradiation on the proliferation of cultured hDPCs and on the growth of human hair follicles (HFs) in vitro. METHODS: We examined the effect of LED irradiation on Wnt/β-catenin signaling and mitogen-activated protein kinase (MAPK) pathways in hDPCs. Anagen HFs were cultured with LED irradiation and elongation of each hair shaft was measured. RESULTS: The most potent wavelength in promoting the hDPC proliferation is 660 nm and 830 nm promoted hDPC proliferation to a lesser extent than 660 nm. Various wavelengths significantly increased β-catenin, Axin2, Wnt3a, Wnt5a and Wnt10b mRNA expression. LED irradiation significantly increased β-catenin and cyclin D expression, and the phosphorylation of MAPK and extracellular signal-regulated kinase (ERK). HFs irradiated with 415 nm and 660 nm grew longer than control. CONCLUSION: Our result suggests that LED has a potential to stimulate hDPC proliferation via the activation of Wnt/β-catenin signaling and ERK pathway. To our best knowledge, this is the first report which investigated that the effect of various wavelengths of LED on hDPC proliferation and the underlying mechanisms.

Regulation of allergic airway inflammation in asthmatic mice by Wnt/β-catenin pathway of dendritic cells / 西安交通大学学报(医学版)

Objective To investigate the role of Wnt/β-catenin pathway in regulating allergic airway inflammation in asthmatic mice.Methods We induced dendritic cells (DCs) from bone marrow of BALB/c mice,and then treated the cells with LiCl and PKF118-310,separately.We observed the morphological features of DCs under light microscope.Mixed lymphocyte reaction (MLR) was used to observe the functional changes of DCs.Western blot was used to detect the expressions of GSK-3β and β-catenin at the protein level.We established a mouse asthma model by using ovalbumin (OVA),and then treated these mice with LiCl and PKF118-310.The total number of cells and eosinophil percentage in BALF were determined.The lungs of mice were observed by HE staining to evaluate the degree of allergic inflammation.The cytokines in BALF and spleen cells supernatant were assayed by enzyme-linked immunoassay (ELISA),and the total IgE in the serum was also measured by ELISA.The protein expression levels of GSK-3β and β-catenin in lung tissue were assayed by Western blot.Results ① The DCs treated with LiCl promoted the proliferation of allogeneic T lymphocytes in MLR more weakly than those treated with PKF118-310 (P＜0.01).② The GSK-3β protein expression level of DCs treated with LiCl was significantly lower than DCs treated with PKF118-310.In contrast,the β-catenin protein expression of DCs treated with LiCl was higher than that of DCs treated with PKF118-310 (P ＜ 0.01).③ The total number of cells and eosinophil percentage in BALF were significantly increased in the experimental group compared with those in the control group (P＜0.01).There was also a significant difference between LiCl group and PKF118-310 group (P＜0.01).④ In the three experimental groups,the severity of inflammation in the lungs of LiCl group was weaker than that in PKF118-310 group (P＜0.05).⑤ Compared with that in the normal control group,IL-4 in BALF and spleen cell culture supernatant of the experimental group was significantly higher while IFN-γ was the opposite (P＜0.01).LiCl group had the lowest level of IL-4 and the highest level of IFN-γ;PKF groups was the opposite (P＜0.05).⑥ The total IgE in serum was significantly increased in the experimental group compared with the control group (P＜0.01).There was also a significant difference between LiCl group and PKF118-310 group (P＜0.05).⑦ GSK-3β protein expression was significantly lower in LiCl group than in PKF118-310 group (P＜0.05),while β-catenin protein expression was significantly higher in LiCl group than in PKF118-310 group (P＜0.05).Conclusion LiCl and PKF118-310 can affect the severity of asthma by regulating Wnt/β-catenin signal pathway and the expressions of GSK-3β andβ-catenin protein,which provides a new direction for asthma treatment.