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 Morindae Officinalis Radix Processed with Different Proportions of Glycyrrhizae Radix et Rhizoma on Improvement of Renal Function and HPG Axis of Kidney Yang Deficiency Model Rats Induced by Adenine / 中国实验方剂学杂志

ObjectiveTo explain the scientific connotation of Morindae Officinalis Radix （MOR） processed by Glycyrrhizae Radix et Rhizoma （Gly） by comparing the effect of raw products of MOR and processed products of MOR with different proportions of Gly （GMOs） on the improvement of renal function and hypothalamic-pituitary-gonadal （HPG） axis， the protein expression of Wnt/β-catenin and transforming growth factor-β1 （TGF-β1）/Smad signal pathways in kidney Yang deficiency model rats induced by adenine. MethodGMOs were prepared according to method under MOR in 2020 edition of Chinese Pharmacopoeia. Rat model of kidney Yang deficiency was established by intragastrical administration of adenine， levels of follicle stimulating hormone （FSH）， luteinizing hormone （LH）， estradiol （E2） and testosterone （T） were measured by enzyme-linked immunosorbent assay （ELISA）. Levels of urea nitrogen （BUN） and serum creatinine （SCr） were measured by spectrophotometry， hematoxylin-eosin （HE） staining was used to evaluate the pathological changes of kidney， testis and epididymis. Immunohistochemistry （IHC） was used to analyze the protein expression of E-cadherin， α-smooth muscle actin （α-SMA）， Wnt2b， β-catenin， Smad1 and Smad4. ResultMOR processed with 100∶6 and 100∶12 proportions of Gly （short for GMO/100∶6 and GMO/100∶12） had the most obvious improvement on the body posture of kidney Yang deficiency model rats. GMO/100∶12 had the best effect on reducing the levels of BUN， SCr， FSH， LH and the ratio of E2/T. GMO/100∶6 and GMO/100∶12 had the best effect on regulating the protein expression of E-cadherin， α-SMA， Wnt2b， β-catenin， Smad1 and Smad4. ConclusionGMO/100∶6 and GMO/100∶12 have the a good effect on the improvement of renal function and HPG axis in kidney Yang deficiency model rats induced by adenine， which is related with the fact that they can regulate Wnt/β-catenin pathway in renal and testicular tissue and TGF-β1/Smads pathway in testicular tissue.

Effect of Morindae Officinalis Radix Processed with Different Proportions of Glycyrrhizae Radix et Rhizoma on Improvement of Renal Function and HPG Axis of Kidney Yang Deficiency Model Rats Induced by Adenine / 中国实验方剂学杂志

ObjectiveTo explain the scientific connotation of Morindae Officinalis Radix （MOR） processed by Glycyrrhizae Radix et Rhizoma （Gly） by comparing the effect of raw products of MOR and processed products of MOR with different proportions of Gly （GMOs） on the improvement of renal function and hypothalamic-pituitary-gonadal （HPG） axis， the protein expression of Wnt/β-catenin and transforming growth factor-β1 （TGF-β1）/Smad signal pathways in kidney Yang deficiency model rats induced by adenine. MethodGMOs were prepared according to method under MOR in 2020 edition of Chinese Pharmacopoeia. Rat model of kidney Yang deficiency was established by intragastrical administration of adenine， levels of follicle stimulating hormone （FSH）， luteinizing hormone （LH）， estradiol （E2） and testosterone （T） were measured by enzyme-linked immunosorbent assay （ELISA）. Levels of urea nitrogen （BUN） and serum creatinine （SCr） were measured by spectrophotometry， hematoxylin-eosin （HE） staining was used to evaluate the pathological changes of kidney， testis and epididymis. Immunohistochemistry （IHC） was used to analyze the protein expression of E-cadherin， α-smooth muscle actin （α-SMA）， Wnt2b， β-catenin， Smad1 and Smad4. ResultMOR processed with 100∶6 and 100∶12 proportions of Gly （short for GMO/100∶6 and GMO/100∶12） had the most obvious improvement on the body posture of kidney Yang deficiency model rats. GMO/100∶12 had the best effect on reducing the levels of BUN， SCr， FSH， LH and the ratio of E2/T. GMO/100∶6 and GMO/100∶12 had the best effect on regulating the protein expression of E-cadherin， α-SMA， Wnt2b， β-catenin， Smad1 and Smad4. ConclusionGMO/100∶6 and GMO/100∶12 have the a good effect on the improvement of renal function and HPG axis in kidney Yang deficiency model rats induced by adenine， which is related with the fact that they can regulate Wnt/β-catenin pathway in renal and testicular tissue and TGF-β1/Smads pathway in testicular tissue.

miR-146a-5p-mediated suppression on trophoblast cell progression and epithelial-mesenchymal transition in preeclampsia

OBJECTIVE: This study aims to identify the effect of miR-146a-5p on trophoblast cell invasion as well as the mechanism in preeclampsia (PE). METHODS: Expression levels of miR-146a-5p and Wnt2 in preeclamptic and normal placentae were quantified. Trophoblast cells (HTR-8) were separately transfected with miR-146a-5p mimic, miR-146a-5p inhibitor, pcDNA3.1-Wnt2 or sh-Wnt2, and then the expression levels of miR-146a-5p, Wnt2, and epithelial-mesenchymal transition (EMT)-related proteins (Vimentin, N-cadherin and E-cadherin) were measured. Moreover, the proliferative, migratory and invasive capacities of trophoblast cells were detected, respectively. Dual luciferase reporter assay determined the binding of miR-146a-5p and Wnt2. RESULTS: Compared with normal placental tissues, the placentae from PE patients showed higher miR-146a-5p expression and lower Wnt2 expression. Transfection of miR-146a-5p inhibitor or pcDNA3.1-Wnt2 exerted pro-migratory and pro-invasive effects on HTR-8 cells and encouraged EMT in HTR-8 cells; transfection with miR-146a-5p mimic or sh-Wnt2 weakened the proliferative, migratory and invasive capacities as well as reduced EMT process of HTR-8 cells. Moreover, Wnt2 overexpression could partially counteract the suppressive effects of miR-146a-5p overexpression on the progression and EMT of HTR-8 cells. CONCLUSION: miR-146a-5p mediates trophoblast cell proliferation and invasion through regulating Wnt2 expression.

Effect of targeted regulation of the Wnt2 gene by microRNA(microRNA-21) on the proliferation and migration of HepG2 hepatoma cells / 临床肝胆病杂志

ObjectiveTo investigate the effect of targeted regulation of the Wnt2 gene by microRNA(miR-21) on the proliferation and migration of HepG2 hepatoma cells. MethodsQuantitative real-time PCR was used to measure the mRNA expression of miR-21 in HepG2 hepatoma cells and normal liver cell line LO2. HepG2 cells were transfected with miR-21 inhibitor, and then the expression of miR-21 and cell proliferation, migration, and apoptosis were analyzed for the inhibitor group and the control group. The protein expression of Wnt2 was measured for the two groups, and dual-luciferase reporter assay was used to verify the association between miR-21 and the Wnt2 gene. The t-test was used for comparison of continuous data between groups. ResultsThe relative expression of miR-21 in HepG2 cells was significantly higher than that in LO2 cells (1.978±0.035 vs 1.586±0.022, t=16.424, P＜0.05). After the transfection of miR-21 inhibitor, the inhibitor group had significantly lower expression of miR-21 than the control group (0.857±0.017 vs 1.684±0.039, t=33669, P＜0.05). Compared with the control group after the transfection of miR-21 inhibitor, the inhibitor group had a significant reduction in the proliferation ability of HepG2 cells (P＜0.05), a significantly lower number of cells passing through the Transwell chamber (83.72±15.06 vs 147.85±20.64, t=4.347, P＜0.05), and a significantly higher cell apoptosis rate (25.67%±3.95% vs 10.27%±2.14%, t=5937, P＜0.05). The inhibitor group had significantly lower relative expression of Wnt2 in HepG2 cells than the control group (0.862±0.127 vs 1.306±0.218, t=3.048, P＜0.05). TargetScan software showed that miR-21 inhibitor significantly inhibited the luciferase activity of the cells transfected with wild-type Wnt2-3′UTR plasmid (0.972±0.102 vs 0.612±0.092, t=4.219, P＜005), while there was no effect on the luciferase activity of the cells transfected with mutant Wnt2-3′UTR plasmid (0.982±0.093 vs 0911±0.128, t=0.972, P＞0.05). ConclusionInhibition of miR-21 expression can effectively inhibit the proliferation and migration of HepG2 cells, promote the apoptosis of HepG2 cells, and inhibit the over-activation of the Wnt signaling pathway, and therefore, it may become one of the potential target genes for liver cancer treatment.

MiR-338-3p Enhances Ovarian Cancer Cell Sensitivity to Cisplatin by Downregulating WNT2B

PURPOSE: Chemoresistance is a concern in ovarian cancer patients, in whom survival remains. MicroRNA, a novel class of small RNAs, have frequently been found to be dysregulated in human malignancies and to act as negative regulators of gene expression. This study aimed to explore the function of miR-338-3p in cisplatin resistance in ovarian cancer and potential molecular mechanisms thereof. MATERIALS AND METHODS: The expression levels of miR-338-3p and WNT2B in ovarian cancer tissues and cells were estimated by real-time quantitative polymerase chain reaction (RT-qPCR). In addition, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazol-3-ium bromide (MTT), transwell, and flow cytometry assays were used to assess biological role of miR-338-3p in vitro. Western blot assay was conducted to measure protein expression of WNT2B, epithelial-mesenchymal transition (EMT)-related proteins, and apoptosis-related proteins. The relationship between miR-338-3p and WNT2B was confirmed by dual-luciferase reporter. Finally, a xenograft tumor model was developed to explore the effects of overexpression of miR-338-3p on tumor growth in ovarian cancer in vivo. RESULTS: MiR-338-3p was downregulated in cisplatin resistant ovarian cancer tissues and cells. Mechanistically, high expression of miR-338-3p enhanced cell sensitivity to cisplatin by inhibiting proliferation, motility, and EMT and by promoting apoptosis via targeting WNT2B expression in vitro. Furthermore, overexpression of miR-338-3p increased cisplatin sensitivity among ovarian cancer in an in vivo xenograft tumor model. CONCLUSION: MiR-338-3p enhances the sensitivity of ovarian cancer cells to cisplatin by downregulating WNT2B.

Wnt2 overexpression protects PINK1B9 transgenic flies by improving mitochondrial function / 医学研究生学报

Objective PINK1 and Parkin are directly invoveled in the regulation and maintenance of mitochondrial functional morphology. We aim to explore the effect of Wnt2 overexpression on PINK1B9 Mutant Drosophila and its mechanism in this study. Methods The GAL4-UAS system was used to construct the normal control flies（W1118/ + ；MHC-GAL4/+）, PINK1B9 transgenic Drosophila model flies（UAS-PINK1B9 /y；MHC-GAL4 / +；Parkinson's disease model of Drosophila melanogaster), the Wnt2 overexpression flies（UAS-PINK1B9 /y；MHC-GAL4 / Wnt2 OE） and the Wnt2 RNAi flies（UAS-PINK1B9 /y；MHC-GAL4 /Wnt2）. On the 5th day, the abnormal wings phenotype rate and flying rate of flies were observed. The contents of Ndufs3 proteins were detected by Western blot. The mRNA expression levels of PGC-1α, Nrf1 and TFAM related to mitochondrial metabolism and synthesis were detected by real-time fluorescence quantitative PCR. The morphology of mitochondria was observed by electron microscopy. Complex I and Complex II function was detected by high-resolution mitochondrial respiratory system. Results Compared with the normal control flies, PINK1B9 transgenic Drosophila model flies showed increased abnormal wings phenotype rate([1.87±0.06]% vs [68.79±0.70]%), decreased flying rate([ 97.51±0.52)% vs(3.95±0.53)%], and the differences were statistically significant(P<0.05); Compared with PINK1B9 transgenic Drosophila model flies, the Wnt2 RNAi flies showed decreased abnormal wings phenotype rate[(10.14±1.72)%], increased flying rate([41.83±2.57]%)(P<0.05). Compared with the normal control flies, PINK1B9 transgenic Drosophila model flies showed decreased expression levels of PGC-1α and Nrf1,Ndufs3 proteins, Complex I and Complex II(P<0.05);On the contrary, the Wnt2 RNAi flies showed increased trends compared with PINK1B9 transgenic Drosophila model flies(P<0.05). Conclusion Overexpression of Wnt2 protects PINK1B9 transgenic Drosophila models, which is related to the improvement of mitochondrial function.

Repair effect of meisoindigo on myocardial damage in type 1 diabetes mellitus in rats / 中国药理学通报

Aim To observe the influence of meisoindi-go on the alteration of Wnt/β-catenin signaling in type 1 diabetic rats ' myocardium and clarify its role in the development of diabetic cardiomyopathy .Methods The type 1 diabetes rat model was established by injec-tion of streptozocin after one-week adaptive feeding . The successful modeling rats were randomly divided in-to DM model group of 4 weeks and 8 weeks, meisoindi-go group of 4 weeks and 8 weeks.Fasting blood glu-cose(FBG) levels were tested.HE staining was used to observe the pathological changes of myocardial struc-tures.The alteration of GSK-3β, p-GSK-3β, Wnt2,β-catenin, NF-κB-p65, p-NF-κB-p65 in myocardium was determined by Western blot and immunohistochem-istry.Results Compared with control group , FBG levels of type 1 diabetic rats significantly increased ( P<0.01 ) , while body weight levels significantly de-creased ( P<0.01 ); compared with DM group , FBG levels of 8 weeks meisoindigo group significantly de-creased ( P <0.01 ) .Myocardial histological analysis revealed that DM induced myocardial focal myocyte hypertrophy , solubility , necrosis , fiber tissue hyperplasi-a; compared with DM group , these symptoms were eased in meisoindigo group of 4 weeks and 8 weeks. Compared with control group , the expression of p-GSK-3β, Wnt2, β-catenin, p-NF-κB-p65 level increased, especially with DM group of 8 weeks(P<0.01).The expression of p-GSK-3β, Wnt2,β-catenin, p-NF-κB-p65 level in meisoindigo group of 4 weeks and 8 weeks decreased significantly(P<0.01).Conclusions The repair effect of meisoindigo on myocardial damage in type 1 diabetic rats may be caused by lowering the ex-pression of proteins in Wnt/β-catenin signaling and in-hibiting the activation of Wnt/β-catenin signaling path-way, participating in the repair of myocardial damage and inflammatory in diabetic rats .Further researches on its mechanism in repairing diabetic myocardial dam-age may find new therapeutic targets for diabetic car-diomyopathy .

Expression and clinical significance of Wnt2 and Dvl protein in esophageal aquamous carcinoma / 天津医药

Objective To detect the expressions of Wnt2 and dishevelled (Dvl) protein in esophageal squamous carci-noma, and analyze their relationship with the occurrence and development of esophageal squamous cell carcinoma. Meth-ods The expression levels of Wnt2 and Dvl protein were detected by Western blot assay in 60 samples of esophageal carci-noma and adjacent non-carcinomatous esophageal tissues, and their relationship with clinical pathological features were ana-lyzed. Results The relative expression levels of Wnt2 and Dvl protein were higher in esophageal squamous carcinoma tis-sue (0.512 ± 0.406, 1.218±1.082) than those of esophageal tissue adjacent to carcinoma (0.153 ± 0.189, 0.505±0.358). There were significant differences in the expression levels of Wnt2 and Dvl protein between different infiltration depth, different TNM stages, and lymph node metastasis (P<0.01). There was a positive correlation between Wnt2 and Dvl protein in esopha-geal squamous carcinoma (r=0.718, P<0.01). Conclusion The high expression levels of Wnt2 and Dvl protein promote the development and metastasis of esophageal squamous cell carcinomas collaboratively via Wnt2 signal transduction path-ways.

Alteration of Wnt/β-catenin signaling pathway in type2 diabetic rats’ aorta and regulation of SIRT1 / 中国药理学通报

Aim To investigate the alteration of Wnt/β-catenin signaling and sirtuins 1 in type 2 diabetic rats’ aorta and clarify its role in the development of di-abetes aortic disease. Methods The type 2 diabetes rat model was established by injection of streptozocin after five-week of high fat diet. The rats were randomly divided into control group, DM model group of 2 weeks, 4 weeks, 8 weeks and 12 weeks. Fasting blood glucose ( FBG ) , total cholesterol ( TC ) , triglyceride ( TG) , high density lipoprotein-cholesterol( HDL-C) , low density lipoprotein- cholesterol ( LDL-C ) and fast-ing insulin( FINS) levels were tested. HE staining was used to observe the pathological changes of aortal struc-tures. The alteration of Wnt2, β-catenin, TCF4, SIRT1 and sFRP2 in aortawas determined by Western blot and RT-PCR. Results Compared with control group, TC, TG, LDL-C levels of type 2 diabetic rats were significantly increased, HDL-C levels were signif-icantly reduced( P0. 05). But the expression of TCF4 and SIRT1 was enhanced continuously in DM compared with control group while sFRP2 decreased in the duration of DM development. Conclusions Wnt/β-catenin signa-ling pathway was activated in diabetic aortal injury by regulation of SIRT1 via sFRP2 . Further researches on its mechanism of actionin DM aorta injury may find a new therapeutic target for the disease.

Alteration of Wnt/β-catenin signaling pathway in early diabetic rat myocardium / 中国药理学通报

Aim To investigate the alteration of Wnt/β-catenin signaling pathway in early diabetic rat myo-cardium and clarify its role in development of diabetic cardiomyopathy. Methods The diabetes mellitus ( DM) model was prepared by intraperitoneal injection of streptozotocin ( STZ, 60 μg · g-1 ) . The alteration of Wnt/β-catenin signaling pathway was determined by Western blot and immunohistochemistry. HE staining was used to analyze the change of myocardial pathologi-cal structure. Results Cardiac histological analyses revealed that DM induced cardiomyocyte degeneration and necrosis. Myocardial Wnt2, β-catenin and c-Myc were enhanced in 2 wk DM compared with control group while DKK1 showed no significant alteration. Conclusion Wnt/β-catenin signaling pathway is acti-vated in early diabetic myocardial injury. Further re-searches on its role in DM myocardium may find a new therapeutic target for diabetic cardiomyopathy.

Expressions of Wnt2 and β-catenin in Doxorubicin-induced myocardial injury and their relationships with p53 / 中华实用儿科临床杂志

Objective To investigate the expressions of Wnt2 and β-catenin in Doxorubicin (DOX)-induced myocardial injury and to explore their roles in myocardial cell apoptosis.Methods Cardiomyoblast cells were damaged by different concentrations of DOX(1 mg/L,2 mg/L,3 mg/L,4 mg/L) for 72 h.The effect of different concentrations of DOX on cardiomyocyte growth curve was detected according to the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-h-tetrazolium bromide(MTT) assay.DOX(1 mg/L) was used to induce the model of cardiomyoblast cell injury.Cardiomyocytes were divided into 4 groups:group A:DOX-injured cardiomyocytes for 12 h ;group B:DOX-injured cardiomyocytes for 24 h ; group C:DOX-injured cardiomyocytes for 48 h; group D:normal cardiomyocytes.The expressions of Wnt2,β-catenin and p53 were observed by Western blot and reverse transcription polymerase chain reaction(RT-PCR) at the time point of 12 h,24 h and 48 h.Results DOX significantly inhibited cardiomyocyte proliferation in a dose dependent fashion.The protein and mRNA expressions of Wnt2 increased in the DOX-induced myocardial injury group compared with the group D,with statistical significance (F =224.115,P ＜ 0.05) ;The expressions of β-catenin,p53 were significantly increased compared with the group D,and the higher expression appeared with the time extending(F =188.145,231.927,all P ＜ 0.05).Significantly positive correlation between Wnt2 and β-catenin expression was observed(r =0.940,P ＜ 0.05).Conclusions These findings suggest that Wnt2/β-catenin signaling pathway may play important roles in the cardiovascular disease and be useful for exploring the molecular mechanism of myocardial injury..

The expression of Wnt2 in human breast cancer tissues and its effect on the invasion of breast cancer cells / 肿瘤

Objective: To investiget the expression of Wnt2 in human breast cancer tissues and its effect on the invasion of breast cancer cells. Methods: The serum Wnt2 content of patients with breast cancer and the healthy volunteers was detected by ELISA. The expressions of Wnt2 in breast cancer tissues and their adjacent para-cancerous tissues were observed by immunohistochemistry. The primary cells from the breast cancer cells with high- and low-expression of Wnt2 were cultured. The capacities of invasion and adhesion of the breast cancer cells with high- and low-expression of Wnt2 were determined by Transwell assay and cell adhesion experiment, respectively. Results: The serum Wnt2 content of patients with breast cancer was higher than that of the healthy volunteers (P < 0.05). The positive expression rate of Wnt2 in breast cancer tissues was higher than that in the adjacent paracancerous tissues (P < 0.01). The capacities of invasion and adhesion of breast cancer cells with high-expression of Wnt2 were higher than those of the cells with low-expression of Wnt2 (all P < 0.05). Conclusion: The expression level of Wnt2 is higher in breast cancer. The invasion capacity of the breast cancer cells with high-expression of Wnt2 is relatively stronger.

Expression of Oct4 and Wnt2 in human glioma tissues and its clinical significance / 中国肿瘤生物治疗杂志

Objective:To investigate the expression of Oct4 and Wnt2 in human glioma tissues and its relationship with the clinicopathological features of glioma. Methods: Fifty-six paraffin blocks were obtained from glioma patients receiving surgery. The diagnosis of these patients were confirmed by pathology in our hospital from 2006-2009. Immunohistochemi-cal staining was used to examine Oct4 and Wnt2 expression in the brain tissues of 10 patients with acute brain injury and 56 glioma tissues (including 15 recurrent cases). Results: The normal brain tissues were negative of Oct4, with only one case showing weak Wnt2 expression. Thirty-four of the 56 glioma tissues showed positive expression of Oct4 (60.7%), and 40 showed positive expression of Wnt2 (71.4%). Positive expression rates of Oct4 and Wnt2 in low-grade and high-grade glioma tissues were 46.2 %, 73.3% and 57.7 %, 83.3%, respectively (P < 0.05). Oct4 positive rates in the recrudescence and newly diagnosed glioma tissues were 86.7% and 51.2%, respectively (P < 0.05). Oct4 expression in the glioma tissues was positively correlated with that of Wnt2 (r = 0.537, P < 0.01). Conclusion: Expression of Oct4 and Wnt2 is associated with the malignant degrees of glioma, and Oct4 expression is related to the recurrence of glioma. Oct4 might participate in the development and progression of brain glioma through Wnt signaling pathway.