Effect of electroacupuncture at Wnt/β-catenin signaling pathway on inhibiting cartilage degeneration in rats with knee osteoarthritis / 中国针灸

OBJECTIVE@#To explore the action mechanism of electroacupuncture (EA) for knee osteoarthritis (KOA) based on Wnt/beta-catenin (Wnt/β-catenin) signaling pathway.@*METHODS@#Ten rats were randomly selected into a sham-operation group among 50 male 2-month-old SD rats, and the KOA model was established in the remaining 40 rats by modified Hulth method. Four weeks after the model establishment, the rats were randomly divided into a model group, an experimental A group, an experimental B group and an experimental C group, 10 rats in each group. The rats in the sham-operation group and model group did not receive any intervention. The rats in the experimental A group were treated with EA at "Neixiyan" (EX-LE 4) and "Dubi"(ST 35) for 15 min. The rats in the experimental B group were treated with EA at "Neixiyan" (EX-LE 4) and "Dubi"(ST 35) for 30 min. The rats in the experimental C group were treated with EA at non-acupoint for 15 min. EA intervention was given once a day, five times a week, and totally 12-week treatment was given. After 12 weeks, the knee cartilage tissues were stained and the morphological changes were observed under light microscopy; the severity of cartilage degeneration was evaluated by modified Mankin's score; the content of interleukin-1β (IL-1β) in synovium tissues was detected by ELISA method; the content of Wnt-4, β-catenin and matrix metalloprotein-13 (MMP-13) in cartilage tissues was detected by Western blot method.@*RESULTS@#Compared with the sham-operation group, in the model group the morphology and structure of cartilage were disordered, the number of cells was significantly reduced, the matrix was decontaminated and tidal line was incomplete; the Mankin's score was significantly increased (0.05).@*CONCLUSION@#EA at "Neixiyan" (EX-LE 4) and "Dubi"(ST 35) may reduce the expression of MMP-13 and the production of inflammatory factor IL-1β through Wnt/β-catenin signaling pathway, thus inhibit the degradation of cartilage matrix and the apoptosis of chondrocyte, and improve the morphology and structure of cartilage.

Expression of WNT4 and its inhibitory factor SFRP1 in renal tissue of diabetic nephropathy rats / 基础医学与临床

Objective To observe the change of expression of WNT4/β-catenin signaling pathway and its inhibitory factor secreted frizzled-related protein 1 (SFRP1) in renal tissue in diabetic nephropathy(DN) rats, and to explore its possible role in the development of renal fibrosis. Methods Rats were randomly divided into normal control(NC) group and DN group, and equipped with 8 in each group. The IDDM model was prepared by tail vein injection of STZ 55 mg/kg. Hemotoxyin and eosin、Periodic Acid-Schiff and Masson stain were used to observe the morphological structure and fibrotic lesions in renal tissue;Immunohistochemical analysis was used to observe the protein expression of WNT4 and β-catenin in renal tissue;Western blot was used to detect the protein expression changes of WNT4, SFRP1, β-catenin, p-GSK-3β, GSK-3β, Collagenl, a-SMA, E-cadherin in renal tissue in each group;The mRNA expression of WNT4 and SFRPl in renal tissues of rat was detected by realtime PCR. Results Compared with NC group, renal tissue fibrosis was obvious in DN group. Compared with NC group, the protein and mRNA expressions of WNT4 significantly increased (P＜0.05), the protein expressions of β-catenin, p-GSK-3β, α-SMA and collagen I significantly increased (P < 0.05), the protein expressions of Ecadherin significantly decreased (P＜0. 05), the protein and mRNA expression of SFRPl significantly decreased (P＜0.05). Conclusions In the case of DN, the signal pathway of WNT4/β-catenin is abnormal activation. The expression of SFRPl is decreased, and that may inhibit this pathway and promote the development of renal fibrosis in DN.

Effects of WNT4 gene silencing on migration and invasion of renal tubular epithelial cells of rats stably transfected with PAX2 gene / 吉林大学学报(医学版)

Objective; To investigate the effects of WNT4 gene silecing on the migration and invasion of renal tubular epithelial cells of the rats stably transfected with paired box gene 2 (PAX2) gene, and to clarify whether the PAX2 gene regulates the biological activities of renal tubular epithelial cells through WNT4 gene or not. Methods; The renal tubular epithelial cells transfected with PAX2 gene in the logarithmic phase were divided into stably transfected group and control group. The cells in stably transfected group were divided into stably transfected control group (without WNT4 siRNA silencing) and silencing 72 h group (with WNT4 siRNA silencing for 72 h). The relative expression amounts of PAX2 and WNT4 proteins in renal tubular epithelial cells of the rats in stably transfected group and control group were detected by Western blotting method. The relative expression amounts of WNT4 mRNA in renal tubular epithelial cells of the rats in stably transfected control group and silencing 72 h group were detected by Real time PCR method. The migration rates of renal tubular epithelial cells of the rats in stably transfected control group and silencing 72 h group were detected by cell wound scratch assay. The number of transmembrane renal tubular epithelial cells of the rats in stably transfected control group and silencing 72 h group was detected by Transwell experiment. Results: The Western blotting results showed that the relative expression amounts of PAX2 and WNT4 proteins in renal tubular epithelial cells of the rats in stably transfected group were higher than those in control group (P0. 05); the migration rate of renal tubular epithelial cells in silencing 72 h group after 18 h was lower than that in stably transfected control group (P<0. 05). The Transwell experiment results showed that the number of transmembrane renal tubular epithelial cells in silencing 72 h group was lower than that in stably transfected control group (P<0. 05). Conclusion: PAX2 gene can regulate the biological activities of renal tubular epithelial cells through WNT4 gene.

Identification and expression analysis of two Wnt4 genes in the spotted scat (Scatophagus argus)

Background: WNT4 is a protein that plays a crucial role in ovarian differentiation and development in mammals, with a relatively well understood function in mammalian gonadal differentiation. The role of WNT4 in teleost fish; however, remains unclear. In the present study, cDNAs of Wnt4a and Wnt4b were cloned and characterized in the spotted scat. The expression patterns of two Wnt4 genes in the gonads at different stages of development and in fish after treatment with 17a-methyltestosterone (MT) were investigated. Results: The tissue distribution showed that Wnt4a was expressed in various tissues, including the gonads, gills, spleen, brain, and fin. Interestingly, Wnt4b not only was expressed in the gills, brain, and spleen, but also was obviously expressed in the ovary. During gonad development, Wnt4a was highly expressed in the testis at stage I and Wnt4b was mainly expressed in the ovary at stages II-III. After MT treatment, the mRNA expression of Wnt4a increased significantly up to 40 d, and the transcript level of Wnt4b decreased at 20 d. Conclusions: These results suggest that Wnt4a may be involved in gonad development and plays a role in the process of spermatogonial proliferation. Our results also demonstrate that Wnt4b is not only expressed in the nervous system, but also in the ovary and it may be involved in ovary development of the spotted scat.

Sequence Analysis and Molecular Characterization of Wnt4 Gene in Metacestodes of Taenia solium

Wnt proteins are a family of secreted glycoproteins that are evolutionarily conserved and considered to be involved in extensive developmental processes in metazoan organisms. The characterization of wnt genes may improve understanding the parasite's development. In the present study, a wnt4 gene encoding 491amino acids was amplified from cDNA of metacestodes of Taenia solium using reverse transcription PCR (RT-PCR). Bioinformatics tools were used for sequence analysis. The conserved domain of the wnt gene family was predicted. The expression profile of Wnt4 was investigated using real-time PCR. Wnt4 expression was found to be dramatically increased in scolex evaginated cysticerci when compared to invaginated cysticerci. In situ hybridization showed that wnt4 gene was distributed in the posterior end of the worm along the primary body axis in evaginated cysticerci. These findings indicated that wnt4 may take part in the process of cysticerci evagination and play a role in scolex/bladder development of cysticerci of T. solium.

Modulation of Wnt/β-catenin signaling pathway by irbesartan in highglucose-induced tubular epithelial-mesenchymal transition / 中国药理学通报

Aim To investigate the effects of irbesartan on Wnt/β-catenin signaling pathway in tubular epithelial-mesenchymal transition(EMT)in HKCs induced by high-glucose.Methods Human kidney proximal tubular epithelial cell line(HKCs)cultured in vitro was divided into four groups:normal-glucose group,mannitol control group,high-glucose group and high-glucose plus irbesartan group.Immunocytochemistry staining was used to observe the expression of β-catenin;the protein expression of Wnt4,β-catenin,E-cadherin and α-SMA was assessed by Western blot;Wnt4 and β-catenin mRNA were detected by reverse transcription-polymerase chain reaction(RT-PCR).Results Compared with normal-glucose and mannitol control group,both the protein and the mRNA of Wnt4 were up-regulated in HKCs stimulated by high-glucose.α-SMA expression significantly increased but E-cadherin decreased in HG group.The cytoplastic and nuclear fraction of β-catenin enhanced with highglucose stimulation.But no difference of the total protein and mRNA of β-catenin was observed between highglucose-treatment and control groups.Highglucose induced Wnt4 and β-catenin expression in a time-dependent manner,both peaking at 24 h.Irbesartan reduced the promotional effect of HG on Wnt4 and α-SMA expression,and nuclear translocation of β-catenin.HG-mediated inhibition of E-cadherin was also restored by irbesartan.Conclusion These data supported a functional role for Wnt/β-catenin signaling pathway during epithelial-mesenchymal transition of HKCs induced by high glucose and suggested that irbesartan might reverse tubular EMT by regulating activity of Wnt/β-catenin pathway.

Expressão da proteína Wnt4 e seu possível papel como um antígeno associado ao câncer de próstata / [Expression of the Wnt4 protein and its possible role as an antigen associated with prostate cancer]

A ativação aberrante da via de sinalização de Wnt tem sido freqüentemente associada à tumorigênese do câncer de próstata (CaP). No entanto, pouco se sabe sobre o papel e perfil de expressão do membro Wnt4 no desenvolvimento deste tumor. Este trabalho apresenta dois principais objetivos: 1- Avaliar a expressão da proteína Wnt4 em amostras tumorais e não tumorais (hiperplasia prostática benigna - HPB) de próstata através de imunohistoquímica em microarranjos de tecido (TMA) e 2- Avaliar o potencial papel da proteína Wnt4 como antígeno associado ao CaP. A análise do perfil de expressão da proteína Wnt4 em amostras tumorais e de HPB apresentou marcação citoplasmática e de membrana, não havendo diferenças estatisticamente significativas no valor mediano da intensidade de marcação entre elas. Ressalta-se uma marcação mais intensa para Wnt4 na borda apical das células epiteliais voltadas para o lúmen do ácinos prostáticos. O perfil de expressão de Wnt4 não apresentou correlação com o Escore de Gleason, níveis de PSA, recidiva clínica e presença de um segundo tumor. No entanto, pacientes cujas amostras de tumor de próstata marcavam mais fortemente para a proteína Wnt4 apresentavam recorrência bioquímica da doença num tempo menor em relação a pacientes cujas amostras apresentavam marcação fraca ou ausente. A expressão das proteínas E-caderina e Topoisomerase II-α também foi analisada nas mesmas amostras do TMA e seus perfis de marcação também não apresentaram correlação com os parâmetros clínicopatológicos investigados. A expressão de Topoisomerase II-α foi maior em amostras de CaP quando comparadas com amostras de HPB. Neste estudo, mostramos haver correlação entre o perfil de marcação das proteínas Wnt4, Ecaderina e Topoisomerase II-α. Ensaios de imunoblot utilizando a proteína Wnt4 recombinante identificaram a presença de autoanticorpos contra esta proteína em uma mistura de soros de pacientes com CaP, mas não em amostras de soros de indivíduos controles sadios. Em conjunto nossos dados mostram que a proteína Wnt4 não é diferencialmente expressa entre tecidos de CaP e HPB e que quando superexpressa em amostras de tumores de CaP parece apresentar correlação com menor tempo de recorrência bioquímica. Este estudo também apresenta a primeira evidência de que a proteína Wnt4 é capaz de induzir resposta imune humoral em pacientes com CaP, podendo constituir, portanto, um potencial antígeno associado ao CaP.

Aberrant Wnt signaling pathway activation has been associated with prostate cancer (PCa) tumorigenesis. However, the role and expression profiling of Wnt4 member in PCa tumorigenesis is poorly understood. This work presents two main objectives: 1- Evaluate the expression of Wnt4 in tumor and non tumor prostate samples (benign prostate hyperplasia - BPH) through immunohistochemistry in tissue microarrays (TMA) and 2- Evaluate the potential role of Wnt4 as a tumor associated antigen in PCa. Wnt4 expression profiling in PCa and BPH presented a cytoplasmatic and membrane staining pattern, with no significant statistical difference at the median intensity staining values between these two samples. It is noteworthy the stronger Wnt4 staining at the apical border of epithelial cells toward the prostatic acini lumen. Wnt4 expression pattern did not present any correlation to Gleason score, PSA levels, clinical relapse and presence of a second tumor. However, PCa patients whose tumor samples were strongly stained for Wnt4 presented biochemical recurrence earlier than those whose tissues presented weak or absent Wnt4 staining. The expression of E-cadherin and Topoisomerase II-α were also analyzed in these same samples on this TMA and their expression profiling did not present any correlation to the clinical-pathological parameters analyzed as well. The expression of Topoisomerase II-α was higher in PCa tumor samples as compared to BPH tissues. In this study, we showed a correlation between the expression profiling of Wnt4, E-cadherin and Topoisomerase II-α. Immunoblot assays using the Wnt4 recombinant protein identified autoantibodies against this protein in a pool of serum samples from PCa patients, but not in a pool sample from healthy donor individuals. As a whole, our data show that Wnt4 protein is not differentially expressed between PCa e BPH tissues and that there is correlation between highly Wnt4 protein expression in PCa tumor tissues and an earlier biochemical recurrence. This study also provides the first clues that Wnt4 could trigger humoral immune response in PCa patients and hence corresponding to a potential PCa tumor associated antigen.