scholarly journals Expression pattern of Wif1 and β-catenin during development of anorectum in fetal rats with anorectal malformations

PeerJ ◽  
2018 ◽  
Vol 6 ◽  
pp. e4445 ◽  
Author(s):  
Xiao Bing Tang ◽  
Huan Li ◽  
Jin Zhang ◽  
Wei Lin Wang ◽  
Zheng Wei Yuan ◽  
...  
Keyword(s):  
Western Blot ◽  
Real Time ◽  
Expression Pattern ◽  
Immunohistochemical Staining ◽  
Anorectal Malformations ◽  
Rt Pcr ◽  
Rat Embryos ◽  
Fetal Rats ◽  
Normal Rat ◽  
Wnt Inhibitory Factor 1

Purpose This study was performed to investigate the expression pattern of Wnt inhibitory factor 1 (Wif1) and β-catenin during anorectal development in normal and anorectal malformation (ARM) embryos and the possible role of Wif1 and β-catenin in the pathogenesis of ARM. Methods ARM was induced with ethylenethiourea on the 10th gestational day in rat embryos. Cesarean deliveries were performed to harvest the embryos. The expression pattern of Wif1 and β-catenin protein and mRNA was evaluated in normal rat embryos (n = 288) and ARM rat embryos (n = 306) from GD13 to GD16 using immunohistochemical staining, Western blot, and real time RT-PCR. Results Immunohistochemical staining revealed that in normal embryos Wif1 was constantly expressed in the cloaca from GD13 to GD16. On GD13 and GD14, Wif1-immunopositive cells were extensively expressed in the cloaca. On GD15, the expression of Wif1 were mainly detected on the very thin anal membrane. In ARM embryos, the epithelium of the hindgut and urorectal septum demonstrated faint immunostaining for Wif1 from GD14 to GD16. Western blot and real time RT-PCR revealed that Wif1 and β-catenin protein and mRNA expression level was significantly decreased in the ARM groups compared with the normal group on GD14 and GD15 (p < 0.05). Conclusions This study demonstrated that the expression pattern of Wif1 and β-catenin was disrupted in ARM embryos during anorectal morphogenesis, which demonstrated that downregulation of Wif1 and β-catenin at the time of cloacal separation into the primitive rectum and urogenital septum might related to the development of ARM.

scholarly journals Expression pattern of Wif 1 during development of anorectum in fetal rats with anorectal malformations

2017 ◽  
Author(s):  
Xiao Bing Tang ◽  
Huan Li ◽  
Jin Zhang ◽  
Wei Lin Wang ◽  
Zheng Wei Yuan ◽  
...  
Keyword(s):  
Western Blot ◽  
Real Time ◽  
Expression Pattern ◽  
Immunohistochemical Staining ◽  
Anorectal Malformations ◽  
Rt Pcr ◽  
Rat Embryos ◽  
Fetal Rats ◽  
Normal Rat ◽  
Wnt Inhibitory Factor 1

Purpose: This study was performed to investigate the expression pattern of Wnt inhibitory factor 1 (Wif1) during anorectal development in normal and anorectal malformation (ARM) embryos and the possible role of Wif1 in the pathogenesis of ARM. Methods: ARM was induced with ethylenethiourea on the 10th gestational day in rat embryos. Cesarean deliveries were performed to harvest the embryos. The expression pattern of Wif1 protein and mRNA was evaluated in normal rat embryos (n=288) and ARM rat embryos (n=306) from GD13 to GD16 using immunohistochemical staining, Western blot, and real time RT-PCR. Results: Immunohistochemical staining revealed that in normal embryos Wif1 was constantly expressed in the cloaca from GD13 to GD16. On GD13 and GD14, Wif1-immunopositive cells were extensively expressed in the cloaca. On GD15, the expression of Wif1 were mainly detected on the very thin anal membrane. In ARM embryos, the epithelium of the hindgut and urorectal septum demonstrated faint immunostaining for Wif1 from GD14 to GD16. Western blot and real time RT-PCR revealed that Wif1 protein and mRNA expression level was significantly decreased in the ARM groups compared with the normal group on GD14 and GD15 (p<0.05).Conclusions: This study demonstrated that the expression pattern of Wif1 was disrupted in ARM embryos during anorectal morphogenesis, which demonstrated that downregulation of Wif1 at the time of cloacal separation into the primitive rectum and urogenital septum might related to the development of ARM.

scholarly journals Expression pattern of Wif 1 during development of anorectum in fetal rats with anorectal malformations

2017 ◽  
Author(s):  
Xiao Bing Tang ◽  
Huan Li ◽  
Jin Zhang ◽  
Wei Lin Wang ◽  
Zheng Wei Yuan ◽  
...  
Keyword(s):  
Western Blot ◽  
Real Time ◽  
Expression Pattern ◽  
Immunohistochemical Staining ◽  
Anorectal Malformations ◽  
Rt Pcr ◽  
Rat Embryos ◽  
Fetal Rats ◽  
Normal Rat ◽  
Wnt Inhibitory Factor 1

Purpose: This study was performed to investigate the expression pattern of Wnt inhibitory factor 1 (Wif1) during anorectal development in normal and anorectal malformation (ARM) embryos and the possible role of Wif1 in the pathogenesis of ARM. Methods: ARM was induced with ethylenethiourea on the 10th gestational day in rat embryos. Cesarean deliveries were performed to harvest the embryos. The expression pattern of Wif1 protein and mRNA was evaluated in normal rat embryos (n=288) and ARM rat embryos (n=306) from GD13 to GD16 using immunohistochemical staining, Western blot, and real time RT-PCR. Results: Immunohistochemical staining revealed that in normal embryos Wif1 was constantly expressed in the cloaca from GD13 to GD16. On GD13 and GD14, Wif1-immunopositive cells were extensively expressed in the cloaca. On GD15, the expression of Wif1 were mainly detected on the very thin anal membrane. In ARM embryos, the epithelium of the hindgut and urorectal septum demonstrated faint immunostaining for Wif1 from GD14 to GD16. Western blot and real time RT-PCR revealed that Wif1 protein and mRNA expression level was significantly decreased in the ARM groups compared with the normal group on GD14 and GD15 (p<0.05).Conclusions: This study demonstrated that the expression pattern of Wif1 was disrupted in ARM embryos during anorectal morphogenesis, which demonstrated that downregulation of Wif1 at the time of cloacal separation into the primitive rectum and urogenital septum might related to the development of ARM.

scholarly journals Pioglitazone Attenuates Vascular Fibrosis in Spontaneously Hypertensive Rats

PPAR Research ◽  
2012 ◽  
Vol 2012 ◽  
pp. 1-7 ◽  
Author(s):  
Dengfeng Gao ◽  
Ning Ning ◽  
Guanghua Hao ◽  
Xiaolin Niu
Keyword(s):  
Real Time ◽  
Immunohistochemical Staining ◽  
Spontaneously Hypertensive Rats ◽  
Pcr Analysis ◽  
Rt Pcr ◽  
Hypertensive Rats ◽  
Spontaneously Hypertensive ◽  
Ppar Γ ◽  
Collagen Iii ◽  
Wistar Kyoto

Objective. We sought to investigate whether the peroxisome proliferator-activated receptor-γ (PPAR-γ) ligand pioglitazone can attenuate vascular fibrosis in spontaneously hypertensive rats (SHRs) and explore the possible molecular mechanisms.Methods. SHRs (8-week-old males) were randomly divided into 3 groups (n=8each) for treatment: pioglitazone (10 mg/kg/day), hydralazine (25 mg/kg/day), or saline. Normal male Wistar Kyoto (WKY) rats (n=8) served as normal controls. Twelve weeks later, we evaluated the effect of pioglitazone on vascular fibrosis by Masson’s trichrome and immunohistochemical staining of collagen III and real-time RT-PCR analysis of collagen I, III and fibronectin mRNA.Vascular expression of PPAR-γ and connective tissue growth factor (CTGF) and transforming growth factor-β (TGF-β) expression were evaluated by immunohistochemical staining, western blot analysis, and real-time RT-PCR.Results. Pioglitazone and hydralazine treatment significantly decreased systolic blood pressure in SHRs. Masson’s trichrome staining for collagen III and real-time RT-PCR analysis of collagen I, III and fibronectin mRNA indicated that pioglitazone significantly inhibited extracellular matrix production in the aorta. Compared with Wistar Kyoto rats, SHRs showed significantly increased vascular CTGF expression. Pioglitazone treatment significantly increased PPAR-γ expression and inhibited CTGF expression but had no effect on TGF-β expression.Conclusions. The results indicate that pioglitazone attenuated vascular fibrosis in SHRs by inhibiting CTGF expression in a TGF-β-independent mechanism.

scholarly journals FOXD3/FOXD4 is required for the development of hindgut in the rat model of anorectal malformation

2018 ◽  
Vol 243 (4) ◽  
pp. 327-333 ◽  
Author(s):  
Luo-Jia Wang ◽  
Wei-Lin Wang ◽  
Hong Gao ◽  
Yu-Zuo Bai ◽  
Shu-Cheng Zhang
Keyword(s):  
Animal Models ◽  
Western Blot ◽  
Real Time ◽  
Digestive Tract ◽  
Anorectal Malformation ◽  
Aberrant Expression ◽  
Strong Basis ◽  
Rat Embryos ◽  
Ethylene Thiourea

Congenital anorectal malformation is the most common digestive tract malformation in newborns. It has been reported that FOXD3/FOXD4, a forkhead transcription factor, regulates the generation, migration, and differentiation of neural crest cells. However, whether FOXD3/FOXD4 takes part in anorectal malformation remains unclear. In the present study, we used ethylene thiourea to induce the animal models of anorectal malformation in rat embryos and to interrogate the role of FOXD3/FOXD4 in anorectal malformation pathogenesis. Hindgut samples of the animal models were collected at E15, E17, E19, and E21 days of age. The expression of FOXD3/FOXD4 was detected by immunohistochemistry, western blot, and quantitative real-time fluorescence PCR. By immunohistochemical staining, FOXD3/FOXD4 was observed in epithelial cells of the rectum and the anus both in normal and rat embryos with anorectal malformation. Expression level analysis by western blot indicated that FOXD3/FOXD4 expression increased in ethylene thiourea-induced anorectal malformation groups. mRNA expression as determined by quantitative real-time fluorescence PCR analysis was consistent with the western blot results. Tentative conclusions were drawn that FOXD3/FOXD4 is expressed in the hindgut in rat embryos and is upregulated in anorectal malformation. FOXD3/FOXD4 is required for the development of the hindgut, and its aberrant expression may be an important factor leading to the incidence of anorectal malformation. Impact statement Congenital anorectal malformation (ARM) is the most common digestive tract malformation in newborns. The pathophysiological ground remains unclear. In this study, we used animal models of ARM for the first time to interrogate the role of FOXD3/FOXD4 in ARM pathogenesis. The animal models of ARM were successfully induced by ethylene thiourea (ETU) in rat embryos providing a strong basis for pathogenesis study of this disease. Expression analysis of FOXD3/FOXD4 was carried out in these models, and the results shape a deeper understanding of FOXD3/FOXD4 being required for the normal development of the hindgut. The aberrant expression of FOXD3/FOXD4 may be an important factor leading to ARM incidence.

scholarly journals Investigation into imbalance of Th1/Th2 cells in cirrhotic, hypersplenic rats

2019 ◽  
Vol 48 (3) ◽  
pp. 030006051988944 ◽  
Author(s):  
Yunfu Lv ◽  
Yejuan Li ◽  
Ning Liu ◽  
Yonghong Dong ◽  
Jie Deng
Keyword(s):  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Chemokine Receptors ◽  
Immunohistochemical Staining ◽  
Th2 Cells ◽  
Intragastric Administration ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Chemokine Receptor Ccr3

Objectives To evaluate the Th1/Th2 cell profile in spleens of cirrhotic and hypersplenic rats by investigating the expression of Th1-associated chemokine receptors CXCR3, CCR5 and Th2-associated chemokine receptor CCR3. Methods Experimental liver cirrhosis and hypersplenism were induced in rats by the intragastric administration of carbon tetrachloride (CCl4; 40% solution [0.3 ml/100g, twice/week for 8 weeks]) and confirmed by pathology and hemogram. Presence of the three chemokine receptors was investigated by real-time polymerase chain reaction (RT-PCR), immunohistochemical staining, and western blot analysis. Results By comparison with control animals (n=10), RT-PCR demonstrated that CXCR3 and CCR5-mRNA levels were significantly elevated in the hypersplenic rats (n=26) and CCR3-mRNA levels were lower. Immunohistochemical staining showed that by comparison with controls, the mean density of the Th1-associated CXCR3 and CCR5 receptors was significantly increased but there was no difference between groups in Th2-associated CCR3 receptors. Western blot analysis showed that by comparison with controls, hypersplenic rats had higher levels of CXCR3 and CCR5 protein but lower levels of CCR3 protein. Conclusions The abnormal expression of Th1-associated chemokine receptors in spleens of rats with cirrhosis and hypersplenism induced by CCL4 suggests that a functional imbalance between Th1/Th2 cells may play a role in the pathogenesis of hypersplenism.

scholarly journals NF-κB and pSTAT3 synergistically drive G6PD overexpression and facilitate sensitivity to G6PD inhibition in ccRCC

2020 ◽  
Author(s):  
Qiao Zhang ◽  
Zhe Yang ◽  
Yueli Ni ◽  
Honggang Bai ◽  
Qiaoqiao Han ◽  
...  
Keyword(s):  
Western Blot ◽  
Real Time ◽  
Signaling Pathway ◽  
Protein Interaction ◽  
Xenograft Model ◽  
Metabolic Reprogramming ◽  
Regulatory Function ◽  
Rt Pcr ◽  
Repressive Effect ◽  
The Impact

Abstract Background: Glucose 6-phosphate dehydrogenase (G6PD) serves key roles in cancer cell metabolic reprogramming, and has been reported to be involved in certain carcinogenesis. Previous results from our laboratory demonstrated that overexpressed G6PD was a potential prognostic biomarker in clear cell renal cell carcinoma (ccRCC), the most common subtype of kidney cancer. G6PD could stimulate ccRCC growth and invasion through facilitating reactive oxygen species (ROS)-phosphorylated signal transducer and activator of transcription 3 (pSTAT3) activation and ROS-MAPK-MMP2 axis pathway, respectively. However, the reasons for ectopic G6PD overexpression and the proliferation repressive effect of G6PD inhibition in ccRCC are still unclear. Methods: The impact of ROS accumulation on NF-κB signaling pathway and G6PD expression was determined by real-time RT-PCR and Western blot in ccRCC cells following treatment with ROS stimulator or scavenger. The regulatory function of NF-κB signaling pathway in G6PD transcription was analyzed by real-time RT-PCR, Western blot, luciferase and ChIP assay in ccRCC cells following treatment with NF-κB signaling activator/inhibitor or lentivirus infection. ChIP and Co-IP assay was performed to demonstrate protein-DNA and protein-protein interaction of NF-κB and pSTAT3, respectively. MTS assay, human tissue detection and xenograft model were conducted to characterize the association between NF-κB, pSTAT3, G6PD expression level and proliferation functions. Results: ROS-stimulated NF-κB and pSTAT3 signaling over-activation could activate each other, and exhibit cross-talks in G6PD aberrant transcriptional regulation. The underlying mechanism was that NF-κB signaling pathway facilitated G6PD transcription via direct DNA–protein interaction with p65 instead of p50. p65 and pSTAT3 formed a p65/pSTAT3 complex, occupied the pSTAT3-binding site on G6PD promoter, and contributed to ccRCC proliferation following facilitated G6PD overexpression. G6PD, pSTAT3, and p65 were highly expressed and positively correlated with each other in ccRCC tissues, confirming that NF-κB and pSTAT3 synergistically promote G6PD overexpression. Moreover, G6PD inhibitor exhibited tumor-suppressor activities in ccRCC and attenuated the growth of ccRCC cells both in vitro and in vivo . Conclusion: ROS-stimulated aberrations of NF-κB and pSTAT3 signaling pathway synergistically drive G6PD transcription through forming a p65/pSTAT3 complex. Moreover, G6PD activity inhibition may be a promising therapeutic strategy for ccRCC treatment.

scholarly journals High Glucose Enhances the Odonto/Osteogenic Differentiation of Stem Cells from Apical Papilla via NF-KappaB Signaling Pathway

2019 ◽  
Vol 2019 ◽  
pp. 1-10 ◽  
Author(s):  
Yanping Wang ◽  
Yanqiu Wang ◽  
Yadie Lu ◽  
Jinhua Yu
Keyword(s):  
Stem Cells ◽  
Western Blot ◽  
Osteogenic Differentiation ◽  
Real Time ◽  
Signaling Pathway ◽  
Mtt Assay ◽  
High Glucose ◽  
Rt Pcr ◽  
Alizarin Red ◽  
Apical Papilla

Objective. The transport and metabolism of glucose are important during mammalian development. High glucose can mediate the biological characteristics of mesenchymal stem cells (MSCs). However, the role of high glucose in the odonto/osteogenic differentiation of stem cells from apical papilla (SCAPs) is unclear. Materials and Methods. SCAPs were isolated and identified in vitro. Then, SCAPs were cultured in normal α-MEM and high glucose α-MEM separately. MTT assay was applied to observe the proliferation of SCAPs. ALP activity, alizarin red staining, real-time RT-PCR, and western blot were used to detect the odonto/osteogenic capacity of SCAPs as well as the participation of NF-κB pathway. Results. SCAPs in 25mmol/L glucose group expressed the maximum proteins of RUNX2 and ALP as compared with those in 5, 10, and 15 mmol/L groups. MTT assay showed that 25 mmol/L glucose suppressed the proliferation of SCAPs. ALP assay, alizarin red staining, real-time RT-PCR, and western blot showed 25 mmol/L high glucose can obviously enhance the odonto/osteogenic capacity of SCAPs. Moreover, the NF-κB pathway was activated in 25mmol/L glucose-treated SCAPs and the odonto/osteogenic differentiation was inhibited following the inhibition of NF-κB signaling pathway. Conclusions. High glucose can enhance the odonto/osteogenic capacity of SCAPs via NF-κB pathway.

Overexpression of the Anti-Apoptotic Genes mcl-1, bcl-w, bcl-xL and a1 Is Correlated with Chronic Myelogenous Leukemia Progression and Resistance to Gleevec.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2880-2880
Author(s):  
F. A. Castro ◽  
J. F. Jacysyn ◽  
A. G. Ulbrich ◽  
P. R. Tobo ◽  
L. R. Lopes ◽  
...  
Keyword(s):  
Gene Expression ◽  
Western Blot ◽  
Real Time ◽  
Chronic Phase ◽  
Rt Pcr ◽  
Blastic Crisis ◽  
Gapdh Gene ◽  
Blastic Phase ◽  
Cytogenetic Remission ◽  
Apoptotic Genes

Abstract Chronic myeloid leukemia (CML) is a stem cell disease where t(9;22) translocation is considered the primary molecular event leading to the appearance of the bcr-abl fusion gene and consequent cellular transformation. Bcr-Abl tyrosine-kinase inhibitors have been developed and are fairly successful in the treatment of CML. Despite their outstanding clinical activity in CML, they are not a definitive cure: the efficacy of imatinib mesylate (Gleevec®), for instance, in CML-blastic phase is reduced and reports of resistance and intolerance to it have been published. Since Bcr-abl initiates cellular modifications leading to an extreme resistance to apoptosis, we decided to investigate possible secondary targets for CML therapy. We evaluate the expression of known anti and pro-apoptotic genes in terms of CML progression and response to Gleevec. We studied 10 health controls and 71 CML patients in different phases (20 chronic phase, 20 accelerated phase, 10 blastic phase, 15 cytogenetic remission post-Gleevec® and 6 Gleevec® refractory patients). CML group was constituted by 26 men and 35 women, median age of 51.7 years (range 23–73 years), 5 men and 5 women, median age of 49.3 (range 25–72 years), were healthy controls. Peripheral blood mononuclear cells were isolated and expression of bax, bcl-w, mcl-1, bcl-2, a1 and bcl-xL was analyzed by real-time RT-PCR. Protein expression of Bcl-2 and Bcl-xL were analyzed by western-blot. The results of real-time RT-PCR and western-blot are expressed by relative expression, e.g. ratio of investigated genes or protein to the reference GAPDH gene and protein, respectively. We observed an increase of bcl-w (p<0.001), mcl-1 (p< 0.001), a1 (p<0.01) and bcl-xL (p<0.001) gene expression and a remarkable reduction of bcl-2 (p<0.001) in CML-BP patients (table 1). Patients in remission post-Gleevec® presented an anti-apoptotic gene expression profile similar to controls (p>0.05) and refractory patients profile seem to be analogous to blastic crisis (p>0.05). bax levels did not show significant changes in CML patients in different phases (p>0.05). Bcl-2 and Bcl-xL protein data support real-time RT-PCR findings. Taken together these results suggest that mcl-1, bcl-w, bcl-xL and a1 contribute to disease progression and resistance to treatment in CML patients. Further investigations on the state of the apototic machinery in CML patients should provide new approaches for drug design and consequently new efficient treatment for AP, BC and refractory CML patients. Table 1. Ratio of amplicons of the investigated genes to housekeeping (GAPDH). Gene C CP-CML AP-CML BP-CML CCR-CML R-CML C: control; CP: chronic phase; AP: accelerated phase; BP: blastic crisis; CCR: complete cytogenetic remission; R: refractory patients. Results expressed by mean /SD. mcl-1 3.6/0.9 4.4/1.0 8.5/5.0 12.6/2.7 2.4/0.7 13.7/3.6 a1 329.9/153.3 564.1/349.1 1,7000/564.4 972.4/564.0 434.2/98.1 968.8/2.4 bcl-w 4.2/1.1 12.8/3.2 3.4/0.6 17.2/8.6 3.5/0.8 19.8/3.2 bcl-xL 2.9/1.3 5.4/1.1 10.6/3.5 39.4/6.2 3.0/2.1 42.0/3.5 bcl-2 21.1/5.2 13.8/7.0 6.1/3.1 3.1/1.3 21.7/6.4 3.6/2.2 bax 3.2/1.3 4.2/0.8 3.7/1.3 5.2/2.5 4.5/2.7 4.2/2.7

scholarly journals Canonical, Non-Canonical and Atypical Pathways of Nuclear Factor кb Activation in Preeclampsia

2020 ◽  
Vol 21 (15) ◽  
pp. 5574
Author(s):  
Agata Sakowicz ◽  
Michalina Bralewska ◽  
Tadeusz Pietrucha ◽  
Dominika E Habrowska-Górczyńska ◽  
Agnieszka W Piastowska-Ciesielska ◽  
...  
Keyword(s):  
Western Blot ◽  
Real Time ◽  
Protein Level ◽  
Nuclear Factor Κb ◽  
Nuclear Factor ◽  
Rt Pcr ◽  
Protein Levels ◽  
Activation Pathways ◽  
Expression And Activity

Although higher nuclear factor κB (NFκB) expression and activity is observed in preeclamptic placentas, its mechanism of activation is unknown. This is the first study to investigate whether the canonical, non-canonical, or atypical NFκB activation pathways may be responsible for the higher activation of NFκB observed in preeclamptic placentas. The study included 268 cases (130 preeclamptic women and 138 controls). We studied the expression of the genes coding for NFκB activators (NIK, IKKα, IKKβ, and CK2α) and inhibitors (IκBα and IκBβ) using RT-PCR in real time. The RT-PCR results were verified on the protein level using ELISA and Western blot. To determine the efficiency of the pathways, the ratios of activator(s) to one of the inhibitors (IκBα or IκBβ) were calculated for each studied pathway. The preeclamptic placentas demonstrated significantly lower IKKα and CK2α but higher IκBα and IκBβ protein levels. In addition, the calculated activator(s) to inhibitor (IκBα or IκBβ) ratios suggested that all studied pathways might be downregulated in preeclamptic placentas. Our results indicate that preeclamptic placentas may demonstrate mechanisms of NFκB activation other than the canonical, non-canonical, and atypical forms. In these mechanisms, inhibitors of NFκB may play a key role. These observations broaden the existing knowledge regarding the molecular background of preeclampsia development.

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