Inhibitory effect of Astragalus polysaccharide on osteoporosis in ovariectomized rats by regulating FoxO3a /Wnt signaling pathway

Abstract Purpose: To investigate inhibitory effect of Astragalus polysaccharide (APS) on osteoporosis in ovariectomized rats by regulating FoxO3a/Wnt2 signaling pathway. Methods: Postmenopausal osteoporosis (PMOP) animal model was developed by excising the bilateral ovaries of rats. The model rats were administered with APS (200 mg/kg, 400 mg/kg, 800 mg/kg) by intragastric administration once daily for 12 weeks. Bone density, bone metabolism index and oxidative stress index were measured in all groups. Furthermore, the regulation of APS of FoxO3a / Wnt2 signaling pathway was observed. Results: APS has an estrogen-like effect, which can increase bone mass, lower serum ALP and BGP values, increase blood calcium content, and increase bone density of the femur and vertebrae in rats. At the same time, APS can increase the bone mineral content of the femur, increase the maximum stress, maximum load and elastic modulus of the ovariectomized rats, improve oxidative stress in rats by increasing the gene expression of β-catenin and Wnt2 mRNA and inhibiting the gene expression of FoxO3a mRNA. Conclusion: Astragalus polysaccharide can effectively alleviate oxidative stress-mediated osteoporosis in ovariectomized rats, which may be related to its regulation of FoxO3a/Wnt2/β-catenin pathway.

Effects of bone morphogenetic protein-2 (BMP-2) and vascular endothelial growth factor (VEGF) release from polylactide-poly (ethylene glycol)-polylactide (PELA) microcapsule-based scaffolds on bone

Multiple growth factors can be administered to mimic the natural process of bone healing in bone tissue engineering. We investigated the effects of sequential release of bone morphogenetic protein-2 (BMP-2) and vascular endothelial growth factor (VEGF) from polylactide-poly (ethylene glycol)-polylactide (PELA) microcapsule-based scaffolds on bone regeneration. To improve the double emulsion/solvent evaporation technique, VEGF was encapsulated in PELA microcapsules, to which BMP-2 was attached. The scaffold (BMP-2/PELA/VEGF) was then fused to these microcapsules using the dichloromethane vapor method. The bioactivity of the released BMP-2 and VEGF was then quantified in rat mesenchymal stem cells (rMSCs). Immunoblotting analysis showed that BMP-2/PELA/VEG promoted the differentiation of rMSCs into osteoblasts via the MAPK and Wnt pathways. Osteoblast differentiation was assessed through alkaline phosphatase expression. When compared with simple BMP-2 plus VEGF group and pure PELA group, osteoblast differentiation in BMP-2/PELA/VEGF group significantly increased. An MTT assay indicated that BMP-2-loaded PELA scaffolds had no adverse effects on cell activity. BMP-2/PELA/VEG promoted the differentiation of rMSCs into osteoblast via the ERK1/2 and Wnt pathways. Our findings indicate that the sequential release of BMP-2 and VEGF from PELA microcapsule-based scaffolds is a promising approach for the treatment of bone defects.

Mest Attenuates CCl4-Induced Liver Fibrosis in Rats by Inhibiting the Wnt/beta-Catenin Signaling Pathway

BACKGROUND/AIMS: The Wnt/beta-catenin signaling pathway has been reported to play an important role in liver fibrosis. This study was designed to investigate whether mesoderm-specific transcript homologue (Mest), a strong negative regulator of Wnt/beta-catenin signaling, could inhibit liver fibrosis. METHODS: pcDNA-Mest was transfected into hepatic stellate cells (HSCs) and rats. Rats were randomly divided into four groups: normal group (normal saline), treatment group (pcDNA-Mest+CCl4), control group (pcDNA-neo+CCl4), and model group (normal saline+CCl4). Changes in liver pathology were evaluated by hematoxylin and eosin and Masson's trichrome staining. The levels of alanine transaminase, aspartate transaminase, lactic dehygrogenase, hyaluronic acid, and laminin in the serum and hydroxyproline in the liver were detected by biochemical examination and radioimmunoassay, respectively. The expression and distribution of beta-catenin, alpha-smooth muscle actin (alpha-SMA), Smad3, and tissue inhibitor of metalloproteinase type I were determined, and the viability of the HSCs was tested. RESULTS: Our data demonstrate that Mest alleviated CCl4-induced collagen deposition in liver tissue and improved the condition of the liver in rats. Mest also significantly reduced the expression and distribution of beta-catenin, alpha-SMA and Smad3 both in vivo and in vitro, in addition to the viability of HSCs in vitro. CONCLUSIONS: We found that Mest attenuates liver fibrosis by repressing beta-catenin expression, which provides a new therapeutic approach for treating liver fibrosis.

Rspo1 promotes osteoblast differentiation via Wnt signaling pathway.

R-spondin (Rspo)s proteins are a new group of Wnt/beta-catenin signaling agonists. These signaling molecules are known to be involved in the developmental stages of skeletal system. Recent studies in various murine osteoblast models have proposed that Rspo1 may interact with Wnt signaling pathway to induce differentiation in osteoblasts. Though findings in murine osteoblasts implicate a synergestic role of Rspo1 with Wnt signaling, still no study has addressed the similar role in more clinically applicable osteoblast models i.e., human cell lines or primary cells. Therefore, in the present study, we investigated the possible role of Rspo1 during differentiation process of human in vitro osteoblast cell models like primary osteoblasts or human osteoprogenitor cell line hFOB1.19 along with murine preosteoblast cell line MC3T3 E-1. Our results showed increase in Rspo1 at transcript level during differentiating phase of human primary osteoblasts and human FOB1.19 cells. We also found that Rspo1 (100 ng/mL) acts additively with Wnt3a to activate Wnt signaling, as confirmed by luciferase activity after transfection of TOPFLASH construct to hFOB1.19 cells. Similar additive role of Rspo1 and Wnt3a was apparent in alkaline phosphatase (ALP) activity analysis of human primary cells. Moreover, a reduction in ALP activity was observed with knock-down of Rspo1 by transfected shRNA in hFOB1.19 cells. These results suggested the possibility of autocrine regulation by Rspo1 on the osteogenic activities in human in vitro osteoblast models. Furthermore, these results were corroborated in MC3T3-E1, murine osteoblast cell model. Osteoblastic differentiation was induced by transfection of Rspo1 which was confirmed by increased ALP staining and qRT-PCR analysis of osteogenic markers, such as Runx2 and osteocalcin. In conclusion, present study highlights the role of Rspo1 in bone remodeling where it activates Wnt signaling to induce differentiation, as shown in human as well murine in vitro osteoblast cell models.

Canonical and noncanonical Wnt pathways: a comparison between endometrial cancer type I and atrophic endometrium in Brazil / Vias Wnt canônica e não canônica: uma comparação entre o câncer endometrial tipo I e endométrio atrófico no Brasil

CONTEXT AND OBJECTIVE: The Wnt pathway is involved in tumorigenesis of several tissues. For this reason, we proposed to evaluate Wnt gene expression in endometrial cancer type I. DESIGN AND SETTING: Cross-sectional study on materials gathered from the tissue bank of the Department of Pathology, Universidade Federal de São Paulo. METHODS: Endometrial specimens were obtained from surgeries performed between 1995 and 2005 at São Paulo Hospital, Universidade Federal de São Paulo. The material was divided into two groups according to tissue type: Group A, atrophic endometrium (n = 15); and Group B, endometrial adenocarcinoma (n = 45). We compared the immunohistochemical expression of Wnt1, Frizzled-1 (FZD1), Wnt5a, Frizzled-5 (FZD5) and beta-catenin between endometrial cancer type I and atrophic endometrium. RESULTS: Regarding Wnt1, FZD1 and Wnt5a expression, no significant association was observed between the groups. A significant association was observed between the groups in relation to FZD5 expression (P = 0.001). The proportion of FZD5-positive samples was significantly higher in group A (80.0 percent) than in group B (31.1 percent). Regarding the survival curve for FZD5 in group B, we did not find any significant association between atrophic endometrium and endometrial adenocarcinoma. We also did not find any significant association regarding beta-catenin expression (P = 1.000). CONCLUSION: FZD5 is downregulated in endometrial adenocarcinoma, in comparison with atrophic endometrium.

CONTEXTO E OBJETIVO: A via Wnt está envolvida na tumorigênese de diversos tipos de tecidos. Por essa razão, propusemo-nos a avaliar a expressão de genes da família Wnt no câncer endometrial tipo I. TIPO DE ESTUDO E LOCAL: Estudo transversal com coleta de materiais do banco de tecidos do Departamento de Patologia da Universidade Federal de São Paulo. MÉTODOS: Amostras endometriais foram obtidas de cirurgias que ocorreram entre 1995 e 2005 no Hospital São Paulo, Universidade Federal de São Paulo. Foram separados dois grupos segundo o tipo de tecido obtido: grupo A, com endométrio atrófico (n = 15); e grupo B, com adenocarcinoma endometrial (n = 45). Comparamos a expressão imunoistoquímica de Wnt 1, Frizzled-1 (FZD1), Wnt 5a, Frizzled-5 (FZD 5) e beta-catenina entre câncer endometrial tipo I e endométrio atrófico. RESULTADOS: Na expressão do Wnt1, FZD1 e Wnt5a, não observamos associação significante entre os grupos. Na expressão do FZD5, encontramos associação significante entre os grupos (P = 0,001). A proporção de positividade do FZD5 foi significantemente maior no grupo A comparado ao grupo B (31,1 por cento). Em relação à curva de sobrevida para o FZD5 no grupo B, não tivemos associação significante entre endométrio atrófico e adenocarcinoma do endométrio. Também não observamos associação significante na expressão da beta-catenina (P = 1,000). CONCLUSÃO: FZD5 é downregulated no adenocarcinoma endometrial quando comparado ao endométrio atrófico.