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Objective:To observe the repair effect and possible mechanism of Dipsacus saponins Ⅵ on tibial fracture model rats.Methods:Thirty Sprague Dawley (SD) rats were randomly divided into model group, intervention group, and combination group, with 10 rats in each group, to establish a tibial fracture rat model using the sawing method. On the second day after surgery, the intervention group was intraperitoneally injected with 10 mg/kg of Chuanduduan saponin Ⅵ; The combination group received intraperitoneal injection of Dipsacus saponins Ⅵ 10 mg/kg and XAV939 1 mg/animal; The model group was intraperitoneally injected with 0.2 ml of physiological saline solution and 0.2 ml of dimethylsulfoxide (DMSO) solution; Once a day, continuous intervention for 14 days. After 2 to 4 weeks of intervention, Micro CT scan and X-ray scan were used to observe the fracture healing status; After 4 weeks of intervention, the wet weight of the tibia was detected; Hematoxylin eosin (HE) staining was used to observe the pathological changes of callus tissue; The Western blot method was used to detect the expression level of callus tissue β- catenin (β-catenin), p-β-catenin, glycogen synthase kinase 3β (GSK-3 β) and Runt related transcription factor 2 (Runx2) protein.Results:After 2 and 4 weeks of intervention, the bone volume fraction (BV/TV), number of trabeculae (Tb.N), Lane Sandhu score, and callus volume in the intervention group were higher than those in the model group (all P<0.05); After 2 and 4 weeks of intervention, the BV/TV, Tb.N, Lane Sandhu score, and callus volume in the combined group were lower than those in the intervention group (all P<0.05). The wet weight of the tibia in the intervention group was higher than that in the model group at 4 weeks after intervention ( P<0.05); The wet weight of the tibia in the combined group was lower than that in the intervention group ( P<0.05). The HE staining results showed that the model group had fibrous tissue hyperplasia and more bone trabeculae, but the maturity was not high and the thickening was not significant; The intervention group formed more bony callus, with orderly arrangement of bone trabeculae, partially mature, and obvious mineralization, consistent with the direction of stress; The combined group formed more cartilaginous and fibrous callus, with more mineralization at the edge of the cartilaginous callus and the formation of bone trabeculae. Abundant capillaries can be observed in the gaps. The expression level of Runx2 and p-β-catenin/β-catenin protein in callus tissue of the intervention group was higher than that of the model group, the protein expression GSK-3 β level was lower than that of the model group (all P<0.05); The expression level of Runx2 and p-β-catenin/β-catenin protein in the callus tissue of the combined group was lower than that of the intervention group; the protein expression level of GSK-3β was higher than that of the intervention group (all P<0.05). Conclusions:Dipsacus saponins Ⅵ can effectively promote fracture repair in tibial fracture model rats; It is possible to plays a role by activating the Wnt/β-catenin signaling pathway.
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Antecedentes: Las células madres intestinales generan las distintas estirpes celulares a dicho nivel. Estas se regulan por interacciones entre el epitelio y las células del nicho celular anexo. Estas se pueden ver dañadas en tratamientos con radiación, generando el síndrome gastrointestinal inducido por radiación. Se ha visto que células madre mesenquimales (MSC) y macrófagos de médula ósea (BMM) tienen propiedades de regeneración tisular. Objetivos: Evaluar la expresión génica de IL-4, Wnt6, VEGF y bFGF, a partir de cultivos celulares primarios independientes de MSC derivadas de tejido adiposo y BMM de ratones C57BL/6, por medio de PCR en tiempo real (qRT-PCR). Diseño experimental: A partir de un análisis in silico, se confeccionaron primers para evaluar la expresión génica de las moléculas propuestas, en los cultivos primarios por medio de qRT-PCR y electroforesis. Resultados y proyecciones: IL-4 y Wnt6 no son expresadas en las muestras de BMM y MSC. VEGF y bFGF son expresadas por diferentes células, dando expresión diferenciada. A futuro, se deben evaluar las mismas estirpes celulares en un ambiente inflamatorio y su efecto en la expresión génica, en especial VEGF y bFGF. Limitaciones: El número de moléculas en estudio es limitado y la expresión se evalúo solo a nivel genético.
Background: Intestinal stem cell generates diferents cellular types in their niche. They're regulated by interactions between epithelium and niche's cells, and can be damaged by medical radiation treatments causing radiation-induced gastrointestinal syndrome. It has seen that mesenchymal stem cells (MSC) d and bone marrow-derived macrophages (BMM) have propierties of tissular regeneration. Objectives: Determinated genetic expression of IL-4, Wnt6, VEGF and bFGF, in primary cellular cultures of MSC derivated of adipose tissue and BMM of C57BL/6 mice, through real time PCR (qRT-PCR). Methods: By an in silico analysis, we created primers to evaluate the proposed molecules in the primary cellular cultives, with qRT-PCR and electrophoresis. Results and projections: IL-4 and Wnt6 were not expressed in the MSC and BMM samples. VEGF and bFGF were expressed by different cells, giving differential expression. In the future, the same samples should be analyzed in an inflammatory environment, especially VEGF and bFGF. Limitations: The number of molecules are limited and the expression of them is only in a genetic level.
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Animals , Mice , Radiation Injuries , Biological Factors/genetics , Interleukin-4/genetics , Vascular Endothelial Growth Factor A/genetics , Wnt Proteins/genetics , Mesenchymal Stem Cells/radiation effects , Stem Cells/radiation effectsABSTRACT
Objective:To investigate the relationship between spinal long chain noncoding RNA (lncRNA) and kindlin-1/Wnt3a signaling pathway in a rat model of neuropathic pain (NP).Methods:The experiment was performed in two parts.Experiment Ⅰ Sprague-Dawley rats of both sexes, aged 7 days, weighing 15-20 g, were selected.Rats were sacrificed, the dorsal horn of spinal cord was removed, and the primary astrocytes were extracted and cultured.Lipopolysaccharide 1 μg/ml was added to induce the activation of astrocytes for 24 h. The lncRNA binding to kindlin-1 was identified using PCR immunoprecipitation method.The localization of lncRNA FOXF1-AS1 in astrocytes was observed by fluorescence in situ hybridization, and the binding between lncRNA FOXF1-AS1 and kindlin-1 was detected by biotin-labeled magnetic bead method.Experiment Ⅱ Thirty clean-grade healthy male Sprague-Dawley rats, aged 10-12 weeks, weighing 250-280 g, were divided into 5 groups ( n=6 each) using a random number table method: sham operation control group (group C), NP group, lncRNA FOXF1-AS1 overexpression group (group F), lncRNA FOXF1-AS1 overexpression plus kindlin-1 shRNA group (group FK) and lncRNA FOXF1-AS1 overexpression + Wnt inhibitor group (group FW). NP was induced by chronic constrictive injury in anesthetized animals.In group F, lncRNA FOXF1-AS1 overexpression lentivirus 10 μl was intrathecally injected at 28 days before operation, and vector virus 10 μl was intrathecally injected in the other groups.In FK group, kindlin-1 interfering shRNA interference adenovirus 10 μl, and vector virus 10 μl was intrathecally injected in the other groups.In group FW, Wnt inhibitor IWP-2 10 μl was intrathecally injected at 1-3 days after operation, artificial cerebrospinal fluid 10 μl was intrathecally injected at the same time point in the other groups.Mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) were measured at 1 day before operation, at 4 days and 7 days after operation.The animals were sacrificed at the end of measurement of pain threshold at 7 days after operation, and the spinal cord tissues were taken for determination of the expression of kindlin-1, Wnt3a and glial fibrillary acidic protein (GFAP) (by Western blot) and the contents of tumor necrosis factor (TNF)-α and interleukin (IL)-1β (IL-1β) (using enzyme-linked immunosorbent assay). Results:ExperimentⅠ lncRNA FOXF1-AS1, which was expressed in the cytoplasm of astrocytes, combined with kindlin-1.Experiment Ⅱ Compared with C group, MWT was significantly decreased, TWL was shortened at 4 and 7 days after operation, the expression of kindlin-1, Wnt3a and GFAP in spinal cord was up-regulated, and the contents of TNF-α and IL-1β were increased in group NP ( P<0.05). Compared with NP group, MWT was significantly decreased, TWL was shortened at 4 and 7 days after operation, the expression of kindlin-1, Wnt3a and GFAP in spinal cord was up-regulated, and the contents of TNF-α and IL-1β were increased in F group, MWT was increased, TWL was prolonged at 4 and 7 days after operation, and the contents of TNF-α and IL-1β were decreased in group FK and group FW, the expression of kindlin-1, Wnt3a and GFAP was down-regulated in group FK, and the expression of kindlin-1 was up-regulated, and expression of Wnt3a and GFAP was down-regulated in group FW ( P<0.05). Compared with group F, MWT was significantly increased, TWL was prolonged at 4 and 7 days after operation, and the contents of TNF-α and IL-1β were decreased in group FK and group FW, the expression of spinal kindlin-1, Wnt3a and GFAP was down-regulated in group FK, and expression of Wnt3a and GFAP was down-regulated in group FW ( P<0.05). Conclusion:lncRNA FOXF1-AS1 can up-regulate kindlin-1 expression, activate Wnt3a signaling pathway, promote astrocyte activation, and then regulate inflammatory responses and is involved in the process of neuropathic pain in rats.
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Objective:To evaluate the effect of ulinastatin on hyperoxia-induced acute lung injury (ALI) and its relationship with Wnt/β-catenin signaling pathway in infantile rats.Methods:A total of 36 clean-grade Sprague-Dawley rats, aged 14 days, weighing 40-50 g, were divided into 3 groups ( n=12 each): control group (C group), hyperoxia-induced ALI group (ALI group) and ulinastatin group (UTI group). Hyperoxia-induced ALI was induced by inhaling oxygen at concentration greater than 90% for 72 h. At 1 day after the model was established successfully, ulinastatin 50 000 U/kg was injected intraperitoneally daily at the same time for 3 consecutive days in group UTI, while the equal volume of normal saline was injected intraperitoneally at the same time point in C and ALI groups.The animals were sacrificed at 4 days after the model was established successfully, the lung tissues were taken for determination of the wet/dry weight ratio (W/D ratio), for microscopic examination of the pathological changes which were scored, for measurement of interleukin-6 (IL-6) IL-1β and tumor necrosis factor-alpha (TNF-α) (by enzyme-linked immunosorbent assay) and for detection of the expression of phosphorylated glycogen synthase kinase (p-GSK-3β), Wnt3a and β-catenin by Western blot, and ultrastructure was examined with with an electron microscope. Results:Compared with C group, W/D ratio and lung injury score were significantly increased, the contents of IL-6, IL-1β and TNF-α were increased, and the expression of p-GSK-3β, Wnt3a and β-catenin were up-regulated in lung tissues in group ALI ( P<0.05). Compared with group ALI, W/D ratio and lung injury score were significantly decreased, the contents of IL-6, IL-1β and TNF-α were decreased, and the expression of p-GSK-3β, Wnt3a and β-catenin were down-regulated in lung tissues in group UTI ( P<0.05). The ultrastructure injury in group UTI was reduced as compared with group ALI. Conclusion:The mechanism by which ulinastatin can alleviate hyperoxia-induced ALI is related to inhibiting the activation of Wnt/β-catenin signaling pathway and decreasing inflammatory response in infantile rats.
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Objective:To evaluate the relationship between spinal kindlin-1/Wnt3a signaling pathway and inflammatory response in a rat model of neuropathic pain (NP).Methods:Twenty-four clean-grade healthy male Sprague-Dawley rats, aged 10-12 weeks, weighing 250-280 g, were divided into 4 groups ( n=6 each) using a random number table method: sham operation group (SH group), NP group, kindlin-1 shRNA group (K group) and Wnt3a inhibition group (W group). NP was induced by chronic constrictive injury in anesthetized animals.At 21 days before operation, kindlin-1 shRNA adenovirus vector 10 μl was intrathecally injected in group K, and empty viral vector 10 μl was intrathecally injected in SH, NP and W groups.Wnt inhibitor IWP-2 10 μl was intrathecally injected in group W, and artificial cerebrospinal fluid 10 μl was intrathecally injected in SH, NP and K groups at 1-3 days after operation.The mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) were measured at 1 day before operation and 4 and 7 days after operation, respectively.At the end of pain threshold measurement at 7 days after operation, the animals were sacrificed and the lumbar segments (L 4-6) of the spinal cord were obtained for determination of the contents of tumor necrosis factor (TNF)-α and interleukin (IL)-1β (IL-1β) (using enzyme-linked immunosorbent assay) and the expression of kindlin-1 and Wnt3a (by Western blot). Results:Compared with group SH, MWT was significantly decreased, TWL was shortened at 4 and 7 days after operation, and the contents of TNF-α and IL-1β in spinal cord were increased in NP, K, and W groups, the expression of kindlin-1 and Wnt3a was up-regulated in NP and W groups, and expression of Wnt3a was up-regulated in group K ( P<0.05). Compared with group NP, MWT was significantly increased and TWL was prolonged at 4 and 7 days after operation in K and W groups, the contents of TNF-α and IL-1β in spinal cord were decreased, and the expression of kindlin-1 and Wnt3a was down-regulated in group K, the contents of TNF-α and IL-1β in spinal cord were decreased, and the expression of Wnt3a was down-regulated in group W ( P<0.05), and no significant change was found in kindlin-1 expression ( P>0.05). Conclusion:Spinal kindlin-1 regulates the inflammatory response by up-regulating the expression of Wnt3a, and it is involved in the maintenance of NP in rats.
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The Wnt signaling pathway also plays a role in fibrotic diseases of various tissues and organs, such as pulmonary fibrosis, liver fibrosis, renal fibrosis and skin fibrosis.Activation of the Wnt signaling pathway is related to the fibrosis of the endometrium and ectopic endometrial tissue, and it is a key component of the potential mechanisms for the development intrauterine adhesions.It could also be a target for treatment via promoting endometrial proliferation and angiogenesis and enhancing colony formation and self-renewal ability of endometrial stem cells.Wnt signaling pathways may regulate the complicated cross-talking networks and balance the dynamic processes of fibrogenesis and repair in intrauterine adhesions, thus potentially providing a new route for studying the pathological mechanisms of intrauterine adhesions and finding therapeutic targets.
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Objective@#To explore the mechanism of llinc00052 regulating Wnt pathway affecting invasion and metastasis of gastric cancer.@*Methods@#SGC-7901 cells were selected from gastric cancer cell lines, and siRNAs related to INC0005 and had invasion and metastasis effects in gastric cancer cells were screened by binding of IncRNA expression profiles to qRT-PCR.Ilinc00052 and miRNA expression changes were studied by in vitro cell transfection experiments.Through molecular experiments, the expression of llinc00052 and the effect on Wnt/β-catenin expression were investigated to explore whether llinc00052 could affect the invasion and metastasis of gastric cancer cells by regulating miRNAs affecting Wnt/β-catenin signaling pathway.@*Results@#Transwell chamber test showed that the number of transmembrane cells in the untransfected plasmid group was (134.10±4.29), and the number of transmembrane cells in the overexpressed llinc000522 plasmid group was (169.24±6.99)(t=8.956, P=0.001). The scratch test showed that the migration distance in the llinc000522 overexpression transfection plasmid group was significantly higher than that in the no-load plasmid transfection group(r=0.907, P<0.01). The clone formation rate of llinc000522 overexpressed transfected plasmid group was significantly higher than that of the empty plasmid group[(92.75±6.32)% vs.(73.34±9.14)%](t=5.998, P<0.05). Compared with the transfection of blank plasmid, the expressions of Wnt1, Wnt3a, Wnt2 and β-catenin mRNA in the llinc000522 overexpression transfection group were significantly up-regulated(P<0.05), while the miRNA transfection group had no significant effect on the expression.The expressions of Wnt1, Wnt3a, Wnt2, and β-catenin mRNA were significantly increased [(1.82±0.11), (1.52±0.15), (1.42±0.21), (1.71±0.19)](P<0.05), but their expressions were still lower than those of the genes transfected with llinc000522 alone.Compared with the blank plasmid transfection, the expressions of Wnt1, Wnt3a, Wnt2 and β-catenin protein in the llinc000522 overexpression transfection group were significantly up-regulated(all P<0.05), while the miRNA transfection group had no significant effect on its expression.The protein expressions of Wnt1, Wnt3a, Wnt2 and β-catenin were also significantly increased[(1.53±0.09), (1.4±0.21), (1.33±0.07), (1.47±0.19)](P<0.05), but their expressions were still lower than those of the gene transfected with llinc000522 alone.@*Conclusion@#In gastric cancer cells, llinc00052 can affect the invasion and metastasis of gastric cancer by regulating the level of miRNA and affecting the Wnt/β-catenin pathway.
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Objective To explore the mechanism of llinc00052 regulating Wnt pathway affecting invasion and metastasis of gastric cancer.Methods SGC-7901 cells were selected from gastric cancer cell lines ,and siRNAs related to INC0005 and had invasion and metastasis effects in gastric cancer cells were screened by binding of IncRNA expression profiles to qRT -PCR.Ilinc00052 and miRNA expression changes were studied by in vitro cell transfection experiments.Through molecular experiments ,the expression of llinc00052 and the effect on Wnt／β-catenin expression were investigated to explore whether llinc 00052 could affect the invasion and metastasis of gastric cancer cells by regulating miRNAs affecting Wnt／β-catenin signaling pathway.Results Transwell chamber test showed that the number of transmembrane cells in the untransfected plasmid group was (134.10 ±4.29),and the number of transmembrane cells in the overexpressed llinc 000522 plasmid group was (169.24 ±6.99)(t＝8.956,P＝0.001). The scratch test showed that the migration distance in the llinc 000522 overexpression transfection plasmid group was significantly higher than that in the no -load plasmid transfection group (r＝0.907,P＜0.01).The clone formation rate of llinc000522 overexpressed transfected plasmid group was significantly higher than that of the empty plasmid group[(92.75 ±6.32)% vs.(73.34 ±9.14)%] (t＝5.998,P＜0.05).Compared with the transfection of blank plasmid,the expressions of Wnt1,Wnt3a,Wnt2 and β-catenin mRNA in the llinc000522 overexpression transfection group were significantly up -regulated(P＜0.05),while the miRNA transfection group had no significant effect on the expression.The expressions of Wnt1,Wnt3a,Wnt2,and β-catenin mRNA were significantly increased [(1.82 ± 0.11),(1.52 ±0.15),(1.42 ±0.21),(1.71 ±0.19)] ( P＜0.05),but their expressions were still lower than those of the genes transfected with llinc000522 alone.Compared with the blank plasmid transfection ,the expressions of Wnt1,Wnt3a,Wnt2 and β-catenin protein in the llinc000522 overexpression transfection group were significantly up-regulated(all P＜0.05),while the miRNA transfection group had no significant effect on its expression.The protein expressions of Wnt1,Wnt3a,Wnt2 and β-catenin were also significantly increased [(1.53 ±0.09),(1.4 ±0.21), (1.33 ±0.07),(1.47 ±0.19)](P＜0.05),but their expressions were still lower than those of the gene transfected with llinc000522 alone.Conclusion In gastric cancer cells, llinc00052 can affect the invasion and metastasis of gastric cancer by regulating the level of miRNA and affecting the Wnt ／β-catenin pathway.
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Hepatic macrophages and hepatic progenitor cells play an important role in the development and repair of liver fibrosis. The polarized state of macrophages can affect the differentiation orientation of hepatic progenitor cells, and the Wnt signaling pathway may play a key role in the crosstalk between hepatic macrophages and hepatic progenitor cells. This article overviews the role of macrophages and hepatic progenitor cells and the mechanism of the crosstalk between them in the pathological state of liver fibrosis. It is pointed out that in-depth studies are needed to investigate the genome and phenotype of hepatic macrophages and clarify the mechanism of action of macrophages in regulating the differentiation of hepatic progenitor cells, in order to provide a basis for the treatment of liver fibrosis.
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In modern society,the spectrum of human diseases has changed significandy. Cerebrovascular diseases have become a huge threat to human health. Vascular smooth muscle cells,which constitute the main components of the cerebrovascular wall, play a key role in cerebrovascular diseases. Understanding the physiological metabolism of vascular smooth muscle cells is helpful to the research of cerebrovascular diseases. In recent years, researchers found that Wnt protein and Wnt signaling pathway were widely involved in cerebrovascular diseases. Wnt signaling pathway plays an important role in the proliferation,migration and apoptosis of smooth muscle cells. This review, based on Wnt signaling pathway, will explore the relationship between vascular smooth muscle cells and cerebrovascular lesions.
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The breast cancer is the most common malignant tumor of women, and its treatments were widely explored.A lot of studies have confirmed that the abnormal activation of WNT/ beta-catenin signaling pathway and the abnormal expression of miRNA are closely related to the occurrence and development of breast cancer.miRNA could target the regulation of WNT/beta-catenin signaling pathway and affect the occurrence and development of many human tumors as an endogenous non-coding small molecule RNA.Therefore, the mechanism of WNT/beta-catenin signaling pathway in breast cancer and its interaction with miRNA may provide new ideas for the treatment of breast cancer.The article reviewed the composition of the WNT/beta-catenin signaling pathway and the role of this signaling pathway and its interaction with miRNA in the pathogenesis and progression of breast cancer.
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Objective To observe the effect of hydroxysafflor yellow A (HSYA) on the protection of blood brain barrier of cerebral ischemia mice, and explore the mechaniam. Methods Seventy-two C57/BL mice were divided into 6 groups: the sham operation group, the cerebral ischemia mice group, the TLR4 blocking group, the TLR4 blocking+cerebral ischemia mice group, the HSYA intervention+cerebral ischemia mice group, HSYA intervention+TLR4 blocking+cerebral ischemia mice group. Cerebral ischemia mice group were subjected to the middle cerebral artery occlusion (MCAO) model, TLR4 blocking was used, while TLR4 blocking was injected TLR4 antibody via right common carotid artery, and HSYA intervention group was injected 2 mg/kg HSYA by tail vein 0.5 h before cerebral ischemia. RT-PCR and western blot were applied to detect the mRNA and protein expression change of Wnt3a and β-catenin in each group. Results Compared with the cerebral ischemia mice group,the expression of TLR4 mRNA(1.63 ± 0.05,1.53 ± 0.04,1.84 ± 0.03 vs. 1.97 ± 0.05) significantly decreased (P<0.05 or P<0.01), the Wnt3a mRNA (0.56 ± 0.01, 0.58 ± 0.01, 0.50 ± 0.04 vs.0.42 ± 0.03),β-catenin mRNA(0.61 ± 0.03,0.74 ± 0.02,0.58 ± 0.04 vs.0.50 ± 0.03),Claudin-5 mRNA (0.54 ± 0.02, 0.58 ± 0.01, 0.47 ± 0.01 vs. 0.46 ± 0.02) mRNA significantly increased(P<0.05 or P<0.01), the protein expression of TLR4 (1.73 ± 0.05, 1.57 ± 0.03, 1.79 ± 0.08 vs. 1.89 ± 0.02) significantly decreased (P<0.05 or P<0.01), the protein expression of Wnt3a (0.67 ± 0.03, 0.74 ± 0.03, 0.57 ± 0.01 vs. 0.46 ± 0.01), Occludin(0.66 ± 0.02,0.73 ± 0.02,0.67 ± 0.01 vs.0.53 ± 0.01),Claudin-5(0.71 ± 0.01,0.73 ± 0.01,0.66 ± 0.01 vs. 0.64 ± 0.03) significantly increased (P<0.05 or P<0.01) in the TLR4 blocking+cerebral ischemia mice group, the HSYA intervention+cerebral ischemia mice group, HSYA intervention+TLR4 blocking+cerebral ischemia mice group. Conclusions TLR4 plays a critical regulatory role on the activation of Wnt3a and β-catenin in cerebral ischemic mice model. HSYA could intervene on the tight junction of cerebral ischemic brain through the intervention of Wnt3a and β-catenin, thus exerting the protection for cerebral ischemic brain.
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The Wnt/β-catenin signaling pathway is crucial in bone development and homeostasis.Normally, it has been suggested to play a significant role in regulation of osteogenic differentiation of bone marrow mesenchymal stem cells, osteoclastogenesis and?偅ibone mineral density. Dysregulated Wnt/β-catenin signaling pathway is responsible for bone tumor, metabolic bone disease, degenerative bone disease and it related to development and invasion of osteosarcoma and Ewing′s sarcoma.Studies of the molecular mechanisms of Wnt/β-catenin signaling pathway provide theoretical basis of a potential target for bone diseases.
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Objective To evaluate the role of spinal kindlin-1 in neuropathic pain in rats and the relationship with Wnt3a.Methods Eighteen clean-grade healthy male Sprague-Dawley rats,weighing 250-280 g,aged 10-12 weeks,were divided into 3 groups (n =6 each) using a random number table:sham operation group (group S),neuropathic pain group (group NP) and kindlin-1 inhibitor group (group K).Neuropathic pain was induced by chronic compression of the sciatic nerve.The sciatic nerve was only exposed but not ligated in group S.In group K,shRNA was intrathecally injected at 21 days before operation to inhibit the expression of kindlin-1.Vector virus was intrathecally injected at 21 days before operation in S and NP groups.The mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) were measured at 1 day before operation and 1,4,7,10 and 13 days after operation.Rats were sacrificed at 13 days after measurement of pain threshold and the spinal cord was removed for determination of the expression of kindlin-1 and Wnt3a (by Western blot) and expression of Wnt3a mRNA (by real-time polymerase chain reaction).Results Compared with group S,the MWT was significantly decreased and the TWL was shortened at 4,7,10 and 13 days,and the expression of Wnt3a protein and mRNA and kindlin-1 was up-regulated in group NP (P＜0.05).Compared with group NP,the MWT was significantly increased and the TWL was prolonged at 4,7,10 and 13 days,and the expression of Wnt3a protein and mRNA and kindlin-1 was down-regulated in group K (P＜0.05).Conclusion Kindlin-1 is involved in the development of neuropathic pain by up-regulating the expression of Wnt3a in rats.
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Objective@#To investigate the effect of Wnt3a on osteogenic differentiation of human dental pulp stem cells (DPSC).@*Methods@#DPSCs were subjected to different concentrations of Wnt3a (0, 5, 20, 50 and 100 μg/L) and at seven days after culture the alkaline phosphatase (ALP) activity was tested. Mineralized nodule formation was examined by alizarin red staining. Osteogenic-related gene expression of bone sialoprotein (BSP), osteocalcin (OCN), collagen type Ⅰ (COL-Ⅰ), Runt-related transcription factor-2 (RUNX2) was examined by quantitative real-time PCR (qPCR).@*Results@#After seven days of induction by DPSC, Wnt3a protein could inhibit the ALP activity (concentration 0: 1.076±0.203, 5 μg/L: 0.828±0.118, 20 μg/L: 0.505±0.044, 50 μg/L: 0.499±0.038, 100 μg/L: 0.483±0.060). The expression of OCN in 5 μg/L Wnt3a group (0.092±0.005) was lower than that in culture medium (0.858±0.190)(P<0.05). Alizarin red staining showed that 5 μg/L Wnt3a had no mineralization induction effect on DPSC.@*Conclusions@#Wnt3a could inhibit osteogenic differentiation of dental pulp stem cells.
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Objective To investigate the effects and related mechanism of recombinant Wnt 3a protein on vascular smooth muscle cells(VSMCs) migration and adhesion .Methods Primary rat VSMCs were cultured .The experiment was divided into the Wnt3a group and control group .The VSMC migration ability was detected by Transwell experiment .And the ability of VSMC ad-hesion to extracelluar matrix was detected by the celluar matrix adhesion assay .The protein expression levels of β-catenin ,phos-phor-β-catenin (Ser675) ,GSK-3β,phosphor-GSK-3β(Ser9) and integrin-linked kinase(ILK) in VSMCs were detected by Western blot and analyzed .Results The number of VSMCs migration in the Wnt3a group was significantly higher than that in the control group (P<0 .05) .Compared with the control group ,the number of VSMCs adhesion to collagen Ⅰ and the optical density value in the Wnt3a group were significantly increased (P<0 .05) ,and the protein expression levels of β-catenin ,Ser675 ,GSK-3β,Ser9 and ILK in the Wnt3a group were significantly up-regulated (P<0 .05) .Conclusion Wnt3a can regulate VSMCs migration and adhesion by ILK .
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Objective To evaluate the role of Wnt/β-catenin signaling pathway in lithium chloride preconditioning-induced improvement in postoperative cognitive function of aged rats.Methods A total of 100 pathogen-free healthy male Sprague-Dawley rats,aged 18 months,weighing 540-650 g,were divided into 5 groups (n =20 each) using a random number table:control group (group C),surgery group (group S),lithium chloride preconditioning group (group L),secreted frizzled-related protein 1 (sFRP-1) group (group F) and lithium chloride preconditioning plus sFRP-1 group (group L+F).Lithium chloride 2 mmol/kg was intraperitoneally injected once a day for 5 consecutive days before operation in L and L+ F groups.In F and L + F groups,sFRP-1 10 μl (concentration 10 μg/ml) was injected into the ventricle at 1 day before operation.Ten rats in each group were randomly sacrificed at 1 day after operation,and the hippocampi were removed for determination of the expression of phosphorylated glycogen synthase kinase-3 beta (p-GSK-3β),3-catenin and Wnt in hippocampal tissues (by Western blot).The rest rats underwent Morris water maze test at day 3-7 after operation,and the concentrations of amyloid beta 42 (A3-42) and phosphorylated tau-181 protein (p-tau-181) in cerebrospinal fluid (CSF) were detected by enzyme-linked immunosorbent assay at day 7 after operation.Results Compared with group C,the escape latency was significantly prolonged at 3-7 days after operation,and the concentration of Aβ-42 in CSF was increased in the other four groups,and the expression of Wnt,p-GSK-3β and β-catenin in hippocampal tissues was down-regulated in group S,the expression of Wnt,p-GSK-3β and β-catenin in hippocampal tissues was significantly up-regulated in L and L+F groups,and the concentration of p-tau-181 in CSF was significantly increased in S and L+F groups (P＜0.05).Compared with group S,the escape latency was significantly shortened at 3-7 days after operation in group L and at 5-7 days after operation in group L+F,and the expression of Wnt,p-GSK-3β and β-catenin was significantly up-regulated,and the concentrations of Aβ-42 and p-tau-181 in CSF were decreased in L and L+F groups (P＜0.05).Compared with group L,the escape latency was significantly prolonged at 3-6 days after operation,the expression of Wnt,p-GSK-3β and β-catenin was down-regulated,and the concentration of p-tau-181 in CSF was increased in group L+F (P＜ 0.05).Compared with group F,the escape latency was significantly shortened at 3-7 days after operation,the expression of Wnt,p-GSK-3β and β-catenin was up-regulated,and the concentrations of Aβ-42 and ptau-181 in CSF were decreased in group L+F (P＜0.05).Conclusion The mechanism by which lithium chloride preconditioning improves in postoperative cognitive function is partially related to activating Wnt/β-catenin signaling pathway in aged rats.
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Background Studies showed that microRNA (miR)-375 suppresses the growth,apoptosis,migration and adhesion of tumor cells,and it plays a regulation to the changes of vascular endothelial growth factor (VEGF) in tumor tissue to arrest neovascularization.However,whether miR-375 intervenes the formation of new blood vessel in eyes is unelucidated.Objective This study was to explore the effects of miR-375 on human retinal capillary endothelial cells (HRCECs) function induced by hypoxia.Methods HRCECs were cultured using IMDM and divided into normal control group,CoCl2 model group,CoCl2 +miR-375 mimic group,CoCl2 +miR-375 mimic control group,CoCl2+miR-375 inhibitor group and CoCl2 +miR-375 inhibitor control group,and hypoxia cell models were created by adding 200 μmol/L CoCl2.MiR-375 and frizzled 4 (FZD4) small interfering RNA (siRNA) were transfected into the cells by 50 nmol/L miRNA lipidosome for 48 hours.The proliferation of the cells was detected by MTT assay;migrated number of the cells was examined by Transwell chamber assay;ELISA was employed to detect the concentrations of VEGF and VE-cadherin in the medium;the expression of β-catenin,cyclinD1,matrix metalloproteinase-2 (MMP2) and VEGF proteins were analyzed by Western blot;tube length of vessel formation was evaluated by Matrigel assay.Cultured cells were divided into normal control group,CoCl2 model group,CoCl2 +mock group and CoCl2 + FZD4 siRNA group,the relative expression of FZD4,a miR-375 targeted gene,was detected by luciferase reporter.Results The relative expression of miR-375 mRNA was significantly increased in the CoCl2 +miR-375 mimic group compared with the CoCl2 + miR-375 mimic control group and reduced in the CoCo2 + miR-375 inhibitor group compared with the CoCo2 + miR-375 inhibitor control group (t =-19.237,8.764,both at P＜0.01),with a higher transfected efficacy for miR-375.The cell proliferative fold,migrated cell number,VEGF and VE-Cadherin contents in the medium and the tube length were significantly different among the CoCl2 model group,CoCl2 +miR-375 mimic group,CoCl2 +miR-375 mimic control group,CoCl2 +miR-375 inhibitor group and CoCl2 +miR-375 inhibitor control group (F =24.324,26.776,14.113,19.225,15.040,all at P＜0.001),and those in the CoCl2 +miR-375 mimic group were evidently reduced in the CoCl2 +miR-375 mimic group compared with the CoCl2 +miR-375 mimic control group,while those in the CoCl2 +miR-375 inhibitor group were considerably elevated in comparison with the CoCl2 +miR-375 inhibitor control group (all at P＜0.01).The expressions of β3-catenin,cyclinD1,MMP2 and VEGF protein were significantly different among the normal control group,CoCl2 model group,CoCl2 +miR-375 mimic group,CoCl2+miR-375 mimic control group,CoCl2 +miR-375 inhibitor group and CoCl2 +miR-375 inhibitor control group (F=11.753,13.283,16.770,10.334,all at P＜0.001).In addition,the cell proliferative fold,migrated cell number and the tube length were significantly increased in the CoCl2 model group and CoCl2+mock group,and those in the CoCl2+FZD4 siRNA group were decreased in comparison with the CoCl2 +mock group (all at P＜0.05).Conclusions MiR-375 inhibits the growth,migration and tube formation ability of HRCECs in hypoxic status probably by regulating the activation of Wnt pathway via directly targeting FZD4.
ABSTRACT
PURPOSE: Although static magnetic fields (SMFs) have been used in dental prostheses and osseointegrated implants, their biological effects on osteoblastic and cementoblastic differentiation in cells involved in periodontal regeneration remain unknown. This study was undertaken to investigate the effects of SMFs (15 mT) on the osteoblastic and cementoblastic differentiation of human osteoblasts, periodontal ligament cells (PDLCs), and cementoblasts, and to explore the possible mechanisms underlying these effects. METHODS: Differentiation was evaluated by measuring alkaline phosphatase (ALP) activity, mineralized nodule formation based on Alizarin red staining, calcium content, and the expression of marker mRNAs assessed by reverse transcription polymerase chain reaction (RT-PCR). Signaling pathways were analyzed by western blotting and immunocytochemistry. RESULTS: The activities of the early marker ALP and the late markers matrix mineralization and calcium content, as well as osteoblast- and cementoblast-specific gene expression in osteoblasts, PDLCs, and cementoblasts were enhanced. SMFs upregulated the expression of Wnt proteins, and increased the phosphorylation of glycogen synthase kinase-3β (GSK-3β) and total β-catenin protein expression. Furthermore, p38 and c-Jun N-terminal kinase (JNK) mitogen-activated protein kinase (MAPK), and nuclear factor-κB (NF-κB) pathways were activated. CONCLUSIONS: SMF treatment enhanced osteoblastic and/or cementoblastic differentiation in osteoblasts, cementoblasts, and PDLCs. These findings provide a molecular basis for the beneficial osteogenic and/or cementogenic effect of SMFs, which could have potential in stimulating bone or cementum formation during bone regeneration and in patients with periodontal disease.
Subject(s)
Humans , Alkaline Phosphatase , Blotting, Western , Bone Regeneration , Calcium , Dental Cementum , Dental Prosthesis , Gene Expression , Glycogen Synthase , Guided Tissue Regeneration, Periodontal , Immunohistochemistry , JNK Mitogen-Activated Protein Kinases , Magnetic Fields , Miners , Osteoblasts , Periodontal Diseases , Periodontal Ligament , Phosphorylation , Polymerase Chain Reaction , Protein Kinases , Regeneration , Relative Biological Effectiveness , Reverse Transcription , RNA, Messenger , Signal Transduction , Wnt ProteinsABSTRACT
PURPOSE: Wnt7a is a glycoprotein involved in embryonic development and the progression of different types of malignant tumors. This study aimed to detect the level of Wnt7a expression in breast cancer and explore its role in the disease progression and prognosis. METHODS: A total of 258 patients diagnosed with invasive ductal carcinoma of the breast were included in this study. Using tissue microarray and immunohistochemical staining, we evaluated the association between Wnt7a expression and clinicopathological parameters, and the prognostic value of Wnt7a. RESULTS: Wnt7a expression was significantly correlated with estrogen receptor (ER) expression (odds ratio, 3.95; 95% confidence interval [CI], 1.99–7.80; p < 0.001). On univariate and multivariate analyses, loss of Wnt7a expression was associated with poor disease-free survival (DFS) (multivariate hazard ratio [HR], 9.12; 95% CI, 1.80–46.09; p=0.008), but not with poor overall survival (OS). In the ER-positive group (n=114), loss of Wnt7a expression was an independent prognostic factor for shorter DFS (multivariate HR, 13.54; 95% CI, 1.11–165.73; p=0.042) and OS (multivariate HR, 4.76; 95% CI, 1.29–17.61; p=0.019) on univariate and multivariate analyses. However, in the ER-negative group, there was no significant difference in DFS and OS according to Wnt7a expression. CONCLUSION: The loss of Wnt7a expression might be a meaningful factor in assessing DFS and OS, especially in ER-positive breast cancer.