Expression Of Notch Signaling Pathway Research Articles

Computational analysis of natural compounds targeted for NICD of notch pathway in hepatocellular carcinoma

Therapeutic drugs are required to fight against Hepatocellular carcinoma (HCC), which is one of the prominent cancer types and is considered the world's second leading cause of death related to cancer. HCC is caused by chronic usage of alcohol, obesity, hepatitis virus etc. Currently, the chemotherapeutics drugs available possess severe side effects and face multi-drug resistance due to which the mortality rate is high. The normal liver shows lower expression of notch receptors in comparison to HCC patients where overexpression of notch receptors is observed in the patient’s liver. The present study is based on screening natural compound databases using structure-based virtual screening to find compounds with the capability to inhibit the expression of Notch signaling pathway. 7929 compounds from three natural compound databases (NANPDB, EANPDB, and SANCDB) were subjected to LibDock succeeded by ADME and toxicity prediction using Biovia Discovery Studio Suite. To observe the binding system, molecular docking was performed between the natural compounds and the Notch intracellular domain (target molecule). The computational analysis resulted in a novel natural compound i.e., 5-(2′',3′'-epoxy-3′'-methylbutyl)-3-(3′-hydroxy-3′-methyl-1′-acetyloxy-but-2′-yl)indole. In silico ADMET analysis showed that this compound had good absorption levels with minimal toxic effects. In future, this compound can be utilized as a lead molecule for the development of an anti-HCC medication.

Lumiflavin Reduces Cisplatin Resistance in Cancer Stem-Like Cells of OVCAR-3 Cell Line by Inducing Differentiation.

Ovarian cancer stem-like cells (CSCs) play a vital role in drug resistance and recurrence of ovarian cancer. Inducing phenotypic differentiation is an important strategy to enhance the effects of chemotherapy and reduce the drug resistance of CSCs. This study found that lumiflavin, a riboflavin decomposition product, reduced the development of CSC resistance and enhanced the chemotherapy effect of cisplatin (DDP) on CSCs in DDP-resistant ovarian cancer OVCAR-3 cell line (CSCs/DDP) and was related to the induction of CSC phenotypic differentiation. Results showed that the development of DDP-resistant OVCAR-3 cells was related to the increase in the proportion of CSCs/DDP, and the treatment with lumiflavin reduced the DDP-resistance levels of OVCAR-3 cells and proportion of CSCs/DDP. Further investigation found that lumiflavin synergistic with DDP increased apoptosis, decreased mitochondrial membrane potential, and inhibited the clonal formation of CSCs/DDP. Meanwhile, in vivo experiments showed that lumiflavin dose-dependently enhanced the chemotherapy effect of DDP on tumor-bearing nude mice inoculated by CSCs/DDP. Lumiflavin treatment also reduced the ratio of CD133+/CD177+ to CD44+/CD24 cells, which is the identification of CSCs, in CSCs/DDP. In addition, transcriptome sequencing results suggested that the role of lumiflavin was related to the notch and stem cell pathway, and Western blot analysis showed that lumiflavin inhibited the protein expression of notch signaling pathway in CSCs/DDP. In conclusion, lumiflavin reduces the development of the drug resistance of OVCAR-3 cell and increases the sensitivity of CSCs/DDP to DDP by inducing phenotypic differentiation, which may have a potential role in the chemotherapy treatment of ovarian cancer.

Study on the difference of osteogenesis and Notch signaling pathway expression in biphasic calcium-phosphorus ceramic granule materials with different microstructure.

Different microstructures including micropore diameter, micropore volume, and micropore area of biphasic calcium phosphate (BCP, hydroxyapatite: β-tricalcium phosphate=8:2) ceramics granules were obtained by varying their sintering temperatures. Sprague-Dawley rat bone marrow-derived stem cells (BMSCs) were co-cultured with BCPs in vitro study and the BMSCs showed different degrees of proliferative activity under the influence of three materials. Cell proliferation and vitality were assessed. Three kinds of BCPs were implanted in the dorsal muscle of beagle dogs. At 1, 2, and 3 months, histological analyses were conducted to estimate the rate of osteogenesis. Expression of Notch pathway genes and osteogenic-related genes were detected by quantitative real-time polymerase chain reaction (q-rtPCR). The proportion of osteogenesis area increased to:48.75 ± 4.20%, 29.48 ± 1.55%, and 26.58 ± 3.86% at 3 months after the implantation (1050, 1150, 1250). Significant differences were observed in the upregulation of Notch pathway genes among different BCPs. BCPs with different micropore diameters have different ectopic osteogenesis effects and led to up-regulation of the Notch signaling pathway genes to different extents.

Electroacupuncture in promoting neural repair after spinal cord injury: Inhibiting the Notch signaling pathway and regulating downstream proteins expression.

Spinal cord injury (SCI) is one of the serious central nervous system injuries and the incidence of SCI continues to increase. Previous studies have indicated that electroacupuncture (EA) is beneficial for promoting recovery after SCI. In the present study, we attempted to evaluate how EA can promote the neural repair in SCI model rats by observing changes in the Notch signaling pathway. Experimental rats were randomly divided into four groups. Each group had its own intervention period: 1 day, 7 days, 14 days, and 28 days, and five randomized subgroups: blank control (B) group, blank electroacupuncture (BE) group, sham operation (S) group, model control (M) group and EA group. Animals in the EA group and the BE group were treated with EA at Dazhui (GV14) and Mingmen (GV4) acupoints for 20 min. After the intervention period, the Basso-Beattie-Bresnahan (BBB) score was used to evaluate the neurological function. We found that BBB score increased in EA-treated groups. Hematoxylin and eosin staining was used to observe pathological changes in the injured spinal cord and the results showed that EA therapy could promote the repair of injured spinal cord tissue. Immunohistochemistry and Western blot methods were used to detect the expression of proteins Delta1, Presenilin1, Hes1, and Hes5 in the injured spinal cord. The results showed that the expression levels of Delta1, Presenilin1, Hes1, and Hes5 increased significantly after SCI and decreased after EA treatment. Our study suggested that the possible mechanism by which EA could benefit the recovery after SCI in rats may include inhibiting the Notch signaling pathway and regulating the downstream proteins expression. In addition, our study can provide reference for selecting acupoints and treatment cycle in the treatment of SCI.

Down-regulation of EGFL8 regulates migration, invasion and apoptosis of hepatocellular carcinoma through activating Notch signaling pathway

BackgroundOur previous studies have reported the down-regulation of EGFL8 correlates to the development and prognosis of colorectal and gastric cancer. The present study is carried out to explore the expression pattern and role of EGFL8 in hepatocellular carcinoma (HCC).Methods and materialsEGFL8 expression in 102 cases of HCC tissues matched with adjacent non-tumorous liver tissues, a normal liver cell line and three liver cancer cell lines with different metastatic capacity was detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blot. Moreover, the clinicopathological features and prognosis of HCC patients were correlated with expression of EGFL8. Subsequently, the gain-and loss-of-function experiments were carried out to investigate the biological function of EGFL8 in HCC. We also used N-[N-(3,5-Difluorophenacetyl-L-alanyl)]-(S)- phenylglycine t-butyl ester (DAPT), an inhibitor for Notch signaling pathway, in these experiments to verify the involvement of Notch signaling pathway in the effects of EGFL8. Additionally, a mouse model was established to investigate the effect of EGFL8 on metastasis of HCC cells. The expression of Notch signaling pathway in HCC cells and xenograft mouse tumors were detected by Western blot and immunohistochemistory.ResultsThe expression of EGFL8 was significantly decreased in HCC tissues and cell lines and EGFL8 down-regulation correlated to multiple nodules, vein invasion, high TNM stage and poor prognosis of HCC. Interestingly, the expression levels of EGFL8 in three liver cancer cell lines were negatively associated with their metastatic capacity. In vitro and in vivo experiments indicated that EGFL8 obviously suppressed metastasis and invasion of HCC cells but slightly promoted apoptosis. Meanwhile, the expression of Notch signaling pathway was obviously suppressed in EGFL8 overexpressed HCCLM3 cells and xenograft mouse tumors generated from these cells but markedly elevated in EGFL8 depleted Hep3B cells. Furthermore, the up-regulated expression of Notch signaling pathway and effects induced by EGFL8 knockdown in Hep3B cells could be counteracted by DAPT treatment.ConclusionThe down-regulation of EGFL8 was correlated to progression and poor prognosis of HCC and regulates HCC cell migration, invasion and apoptosis through activating the Notch signaling pathway, suggesting EGFL8 as a novel therapeutic target and a potential prognostic marker for HCC.

MiR-145 targeting BNIP3 reduces apoptosis of chondrocytes in osteoarthritis through Notch signaling pathway.

The purpose of this study was to explore the effect of micro ribonucleic acid (miR)-145 on the apoptosis of chondrocytes in osteoarthritis (OA), and to research the association between its targeting on B-cell lymphoma-2 (Bcl-2)/adenovirus E1B 19 kDa interacting protein 3 (BNIP3) and Notch signaling pathway and chondrocyte apoptosis. The mouse model of OA was established via surgery, and chondrocytes were isolated and cultured in vitro. Then, the chondrocytes were transfected with miR-145 inhibitor, miR-145 mimics, miR-negative control (NC), BNIP3-siRNA and BNIP3-vector, respectively, with those normally cultured as the control. After that, the expression levels of miR-145 and BNIP3 in cells were detected via quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR), the apoptosis rate was detected via flow cytometry, and the apoptosis level was detected using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. Moreover, the target gene sequences were predicted and compared using the software, and the BNIP3 Luciferase reporter vectors containing predicted target sites for miR-145 were constructed. Finally, the protein expressions of BNIP3, Notch1, and P21 were determined through Western blotting. The results of qRT-PCR showed that in OA chondrocytes, the expression of miR-145 was lower than that in normal chondrocytes (p<0.05), while the mRNA and protein expressions of BNIP3 were higher than those in normal chondrocytes (p<0.05). According to flow cytometry, the apoptosis rate was (4.4±0.6)% in normal cartilage tissues and (29.2±2.1)% in OA cartilage tissues. Overexpression of miR-145 significantly reduced chondrocyte apoptosis (p<0.05), while overexpression of BNIP3 markedly increased chondrocyte apoptosis (p<0.05). In addition, the Luciferase reporter system showed that miR-145 mimics evidently inhibited BNIP3 (p<0.05) and suppressed the Notch signaling pathway (p<0.05), while BNIP3 enhanced the expression of Notch signaling pathway (p<0.05). MiR-145 can reduce OA-induced chondrocyte apoptosis through targeted inhibition on BNIP3 and regulation on Notch signaling pathway.

Effects of Cornu Bubali (CB) on the Adhesion of Leukocyte and Endothelial Cells

Background: The interaction between leukocytes and vascular endothelial cells is ubiquitous in the occurrence and development of many diseases, especially in the body's defense response. The purpose of the present study was to investigate the effect of cornu bubali (CB) on the adhesion of leukocytes to endothelial cells. Materials and methods: Human leukemic cell line (HL-60) and human umbilical vein endothelial cells (HUVECs) were used to simulate the adhesion effect between cells. After HUVECs were treated with TNF- α(15 ng/mL) combined with different dose of CB (15, 30 and 60 μmol/L) and dexamethasone (DEX, 2 μg/ml) for 24 h, HL-60 cells were added into the coculture system for another 1 h. CCK8 assay was performed to investigate cell viability of HUVECs. HL-60 cells adhesion to HUVECs was quantified using Hoechst 33342 staining. Subsequently, the levels of adhesion molecules were detected by the reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis and ELISA, respectively. RT-qPCR and western blot were performed to assess the levels of inflammatory cytokines, chemokines and the expression of Notch signaling pathway. Results: Treatment with CB could reduce the adherence of HL-60 to HUVECs induced by TNF- in a dose-dependent manner. CB inhibited the expression of ICAM-1, VCAM-1, CD44, IL-1β, COX-2 and CCL4 in HUVECs. Western blot and RT-qPCR analysis confirmed that CB prevented TNF-α -induced over-expression of Notch receptors (Notch1 and Notch2), Notch ligands (DLL1 and Jagged1), signaling effectors (Hes1) and adhesion related proteins (NF-κB/p65, p-I B and IκBκ) in HUVECs. Conclusion: CB induces interactions between leukocytes and endothelial cells through the activation of Notch signaling pathway. These data contribute to further explain the protective effect of CB against development of inflammatory process of hemorrhage in acute leukemia.

Notch Signaling Pathway Expression in the Skin of Leprosy Patients: Association With Skin and Neural Damage.

Introduction: Leprosy is an infectious disease caused by Mycobacterium leprae, a debilitating disease that affects the skin and peripheral nerves. It is possible that tissue changes during infection with leprosy are related to alterations in the activity of the Notch signaling pathway, an innate signaling pathway in the physiology of the skin and peripheral nerves.Methods: This is a descriptive observational study. Thirty skin biopsies from leprosy patients and 15 from individuals with no history of this disease were evaluated. In these samples, gene expressions of cellular components associated with the Notch signaling pathway, Hes-1, Hey-1, Runx-1 Jagged-1, Notch-1, and Numb, were evaluated using q-PCR, and protein expression was evaluated using immunohistochemistry of Runx-1 and Hes-1.Results: Changes were observed in the transcription of Notch signaling pathway components; Hes-1 was downregulated and Runx-1 upregulated in the skin of infected patients. These results were confirmed by immunohistochemistry, where reduction of Hes-1 expression was found in the epidermis, eccrine glands, and hair follicles. Increased expression of Runx-1 was found in inflammatory cells in the dermis of infected patients; however, it is not related to tissue changes. With these results, a multivariate analysis was performed to determine the causes of transcription factor Hes-1 reduction. It was concluded that tissue inflammation was the main cause.Conclusions: The tissue changes found in the skin of infected patients could be associated with a reduction in the expression of Hes-1, a situation that would promote the survival and proliferation of M. leprae in this tissue.

Long noncoding RNA NALT1-induced gastric cancer invasion and metastasis via NOTCH signaling pathway.

BACKGROUNDLong noncoding RNAs (lncRNAs) are aberrant and play critical roles in gastric cancer (GC) progression and metastasis. Searching for coexpressed lncRNA clusters or representative biomarkers related to malignant phenotypes of GC may help to elucidate the mechanism of tumor development and predict the prognosis of GC.AIMTo investigate the prognostic value of NOTCH1 associated with lncRNA in T cell acute lymphoblastic leukemia 1 (NALT1) in GC and the mechanism of its involvement in GC invasion and metastasis.METHODSRNA sequencing and corresponding clinical data were downloaded from The Cancer Genome Atlas database. The significance module was studied by weighted gene coexpression network analysis. A total of 336 clinical samples were included in the study. Gene silencing, reverse transcription polymerase chain reaction, western blotting, scrape motility assay, and Transwell migration assay were used to assess the function of hub-lncRNAs.RESULTSAt the transcriptome level, 3339 differentially expressed lncRNAs were obtained. weighted gene coexpression network analysis was used to obtain 15 lncRNA clusters and observe their coexpression. Pearson’s correlation showed that blue module was correlated with tumor grade and survival. NALT1 was the hub-lncRNA of blue module and was an independent risk factor for GC prognosis. NALT1 was overexpressed in GC and its expression was closely related to invasion and metastasis. The mechanism may involve NALT1 regulation of NOTCH1, which is associated with lncRNA in T cell acute lymphoblastic leukemia, through cis regulation, thereby affecting the expression of the NOTCH signaling pathway.CONCLUSIONNALT1 is overexpressed and promotes invasion and metastasis of GC. The mechanism may be related to regulation of NOTCH1 by NALT1 and its effect on NOTCH signaling pathway expression.

Abstract 408: Mir148a Family Regulates Cardiac Differentiation of Human Embryonic Stem Cells by Inhibiting The Dll1 -mediated Notch Signaling Pathway

Human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) can differentiate into spontaneously beating cardiomyocytes in vitro, and hold great promise for cardiovascular disease modeling, therapy and drug discovery. However, these applications were hampered by low yield and purity of the in vitro differentiated cardiomyocytes. Although miRNAs represent a type of potential candidates to promote cardiac differentiation, most of cardiomyocyte-enriched miRNAs have not been functionally investigated until now. This study investigated the roles of MIR148A family in cardiac differentiation from hESCs. The MIR148A family is composed of MIR148A , MIR148B and MIR152 , three highly conserved miRNAs sharing same seed sequences. The expression levels of all MIR148A family members were progressively increased during cardiac differentiation in vitro. By using CRISPR-Cas9-mediated knockout, we demonstrated that triple knockout of MIR148A family ( MIR148A -TKO), rather than individual knockout of each members, could grossly inhibited TNNT2 + cardiomyocyte generation. Ectopic expression of MIR152 significantly restored the cardiac differentiation efficiency of MIR148A -TKO hESCs. The transcriptome analysis identified a total of 1071 upregulated genes and 766 down-regulated genes in the MIR148A -TKO hESC-derived cells compared to wild-type hESC-derived cells at day 4 of cardiac differentiation. Gene Ontology analysis revealed that most genes down-regulated in MIR148A -TKO hESC-derived cells were involved in the events of lateral/cardiac mesoderm development, while the up-regulated genes were mainly involved in the events of paraxial/somitic development. Furthermore, the NOTCH ligand Delta-like1 ( DLL1 ) was validated as the target gene of MIR148A family, and the knockdown of DLL1 could inhibit target gene expression of Notch signaling pathway, and promote cardiac differentiation of MIR148A -TKO hESCs. Our findings demonstrate a new function of MIR148A family during cardiac differentiation. Synergistic inhibition of DLL1 -mediated Notch signaling represents a major mechanism for the MIR148A family to inhibit undesired lineage formation and promote hESC differentiation into cardiomyocytes.

Effect of tripterine on Notch signaling pathway in IgA nephropathy rats

Abstract: Objective: To investigate the effect of tripterine on Notch signaling pathway in renal tissue of IgA nephropathy rats. Methods: SD male rats were divided into the control group, IgAN nephropathy model group, benazepril group, 1mg/kg/d tripterine intervention group and 10mg/kg/d tripterine intervention group according to the random number table method, with 10 rats in each group. The urinary sediment and 24-hour urinary protein quantity were detected by conventional methods. The expressions of Notch1, Jagged1, Hes1 and Hey1 in renal tissue of rats were detected by real-time fluorescent quantitative PCR. Results: IgA nephropathy model was successfully established. The hematuria and proteinuria in model group were higher than those of control group (P&lt;0.05). The expressions of Notch1, Jagged1, Hes1 and hey1 in kidney tissue of IgA nephropathy rats were significantly increased (P&lt;0.05). Compared with the model group, hematuria and proteinuria in tripterine intervention group were alleviated. The expressions of Notch1, Jagged1, Hes1 and Hey1 in rat renal tissue were decreased (P&lt;0.05). Moreover, the expressions of Notch1, Jagged1, Hes1 and Hey1 in renal tissue of rats in 10mg/kg/d tripterine intervention group were decreased (P&lt;0.05). Conclusion: Tripterine can decrease the levels of hematuria and proteinuria in IgA nephropathy rats. The expression of Notch signaling pathway in IgA nephropathy rats is increased by the down-regulation of tripterine, suggesting that tripterine has a certain therapeutic effect on IgA nephropathy rat. And its role may be realized through this signal pathway so as to provide the new mentality for the diagnosis and treatment of IgA nephropathy.

Inhibition of the Hedgehog Pathway Decreases the Quiescent CD34+CD38- Population in Acute Myeloid Leukemia

IntroductionAcute myeloid leukemia (AML) is a clonal disease with a reduced life expectancy due to a high relapse rate. One explanation is that leukemic stem cells (LSC) evade the action of conventional chemotherapy due to their quiescent state. Several mechanisms have been proposed that regulate their quiescence, however, by analogy with normal hematopoietic stem cells, a key role may be carried out by the signaling pathways Notch and Hedgehog (Hh). The objectives of this study are to analyze the role of Notch and Hh pathways in the quiescence of LSC and to verify if the pharmacological inhibition of the Notch and Hh pathways decreases the percentage of quiescent LSC. In this way, LSCs would be sensitized to chemotherapy treatments and the high relapse rate of AML could be reduced.MethodsExpression of GLI1, a transcription factor of the Hh signaling pathway and NOTCH Internal Cleaved Domain (NICD) were analyzed in the hematopoietic stem and progenitor cells of four patients diagnosed with AML. The selection of quiescent fraction was performed by flow cytometry using anti-CD34-FITC, anti-CD117-PerCP, anti-CD45PE-Cy7, anti-CD38-APC-Cy7 and anti-KI67-BV510 antibodies. KI67 negative cells were considered quiescent. The activation of NOTCH and Hh pathways was studied using rabbit anti-GLI, anti-NICD primary antibodies and anti-rabbit BV421 secondary antibodies. Results were expressed in median (range) or mean ± standard deviation.Dose-response curves of inhibitors of the Notch pathway (BMS-906024, inhibitor of γ-secretase), Hh pathway (BMS-833923, SMO inhibitor and GANT61, GLI inhibitor), and cytarabine (AraC) were made to study drug potency in the OCI-AML3 cell line. We also analyzed its synergistic behavior in combination with Arac by calculating the combination index (CI) of each of them. These experiments were conducted in triplicate and values were expressed as the mean ± standard deviation.Finally, the effect of BMS-833923 and BMS-906024 on the quiescence of the CD34+CD38- cells of two patients diagnosed with AML was studied by flow cytometry.The paired samples t-test was used in the statistical analysis of GLI and NICD expression between G0 and proliferating cells and in the statistical analysis of the decrease of quiescent cells due to Notch and Hh inhibitors.ResultsFirst of all, hematopoietic and progenitor cells were quantified in four AML patients: the median of the percentage of CD34+CD38- cells with respect to total cells in the bone marrow of the AML patient studied was 1.1% (range: 0.12%-9.05%), within which 71.50% (range: 64.30%-88.43%) are quiescent. Interestingly, we found a trend for a higher expression of NOTCH signaling pathway in the proliferating CD34+CD38- cells (relative median fluorescent intensity (MFI) = 1.91 (range: 1.51-3.34)) compared to the quiescent fraction (relative MFI=1.55 (range: 1.18-1.94); p=0.105). But no differences were found in expression of GLI1.Before studying the effect on cellular quiescence of Notch and Hh inhibitors in monotherapy and in combination with AraC, we evaluated their effect on cell viability. The most potent drug studied was AraC (IC50 = 4.055 μM), followed by inhibitors of the Hh pathway (IC50 BMS-833923 = 5.041 μM; IC50 GANT61 = 7.042 μM) on the OCI-AML3 cell line. In contrast, the γ-secretase inhibitor (BMS-906024) showed no effect. Moreover, it was found that the combination 0.8 μM AraC plus 8 μM BMS-833923 was the most synergistic (CI = 0.53, 15% viability with respect to DMSO control).Subsequently, the effect of the SMO inhibitor on the quiescent CD34+CD38- cells of two patients diagnosed with CD34+ AML was analyzed: BMS-833923 decreased the percentage of quiescent CD34+CD38- cells by 88.5±16.3% in monotherapy (p=0.083) and in presence of AraC by 85.8 ± 21.2% (p=0.113) (figure 1).ConclusionThe use of SMO inhibitors for the treatment of AML is promising because it increases the sensitivity of leukemic cells to chemotherapy and facilitates their action by reducing the percentage of quiescent LSC. This could mean a decrease in the probability of relapse in patients with AML treated with Hh inhibitors. These results were derived from an ongoing project and more patients are being studied in order to confirm the explained results.This work is partially funded by the Madrid Association of Hematology and Hemotherapy. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Effects of tumor necrosis factor-α on osteogenic differentiation and Notch signaling pathway in human periodontal ligament stem cells

To evaluate the effects of tumor necrosis factor-α (TNF-α) on osteogenic differentiation and Notch signaling pathway of periodontal ligament stem cells (PDLSCs) and to investigate the regulatory role of Notch signaling pathway on the osteogenic differentiation of PDLSCs under the influence of TNF-α. PDLSCs were obtained through enzyme digestion and tissue block method. The expression levels of stem cell surface markers CD105, CD90, CD146, CD45, and CD31 were detected by fluorescence activated cell sorter (FACS). PDLSCs were divided into experimental (10 ng·mL⁻¹ TNF-α) and control groups (0 ng·mL⁻¹ TNF-α). The proliferation ability of PDLSCs was detected using cell counting kit-8 (CCK-8). The effect of TNF-α on the osteogenic ability of PDLSCs were tested by measuring alkaline phosphatase (ALP) activity and conducting alizarin red staining and quantitative real-time polymerase chain reaction (PCR). We tested Notch signal pathway receptors Notch1, Notch2, ligand JAG1, JGA2, and downstream gene Hes-1. Changes in DLL1 expression were detected by quantitative real-time PCR. FACS profiling showed that PDLSCs were strongly positive for CD105, CD90, and CD146 but negative for CD45 and CD31. CCK-8 results showed that TNF-α could promote the proliferation of PDLSCs (P<0.05). ALP activity in the experimental group was lower than that in the control group (P<0.05). Alizarin red staining showed that the experimental group had decreased mineralized nodules as compared with the control group. Quantitative real-time PCR results showed that the mRNA expression of osteogenic marker genes cementum attachment protein (CAP), osteopontin (OPN), and Runt-related transcription factor 2 (Runx2) significantly decreased in the experimental group as compared with those in the control group (P<0.05). The expression levels of Notch1, Notch2, JAG1, JGA2 and Hes-1 were significantly decreased (P<0.05), whereas those of Notch3 and DLL1 were increased in Notch signaling pathway-related molecules (P<0.05). TNF-α can promote PDLSCs proliferation and inhibit bone differentiation and Notch signaling pathway expression, indicating that the Notch signaling pathway regulates PDLSCs osteogenic differentiation.

Inhibition of nuclear translocation of notch intracellular domain (NICD) by diosgenin prevented atherosclerotic disease progression

Notch signaling plays a pivotal role in homeostasis and cardiovascular development. The role of notch signaling in atherosclerosis cannot be complete without analysing the key role of notch in macrophages, which trigger the inflammatory response and subsequent plaque formation in atherosclerosis. Diosgenin showed its anti-atherosclerotic property by the unifying mechanism of suppressing the expression of notch signaling pathway, particularly the nuclear translocation of notch intracellular domain (NICD) in aorta and in differentiated macrophage cells. It is further confirmed by the inhibition of NICD by DAPT (N-[N-(3, 5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester), which also restricted the differentiation of macrophage. Hence, inhibition of nuclear translocation of NICD by diosgenin aids in preventing atherosclerosis.

Human placenta mesenchymal stem cells suppress airway inflammation in asthmatic rats by modulating Notch signaling.

Neurogenic locus notch homolog protein (Notch) signaling mediates intracellular communication and may regulate cell fate decisions, including cell proliferation, differentiation, and apoptosis. Mesenchymal stem cells (MSCs) possess immunomodulatory properties and the potential for use in stem cell replacement treatments. The aim of the present study was to evaluate the therapeutic effects of human placenta‑deviated MSCs (hPMSCs) in asthma and to investigate the mechanisms of Notch signaling mediated by transplanted MSCs. A Sprague‑Dawley rat ovalbumin (OVA)‑sensitized acute asthma model was established and challenged. MSCs derived from human placenta (hPMSCs) were transplanted into the asthmatic rats. Transplantation resulted in reduced Notch‑1, Notch‑2 and jagged‑1, and increased Notch‑3, Notch‑4 and delta‑like ligand (delta)‑4 expression in lung, blood, and lymph samples. Notch‑1, Notch‑2, and jagged‑1 expression in OVA‑treated rats was significantly decreased compared with controls and hPMSC‑treated rats; however, Notch‑3, Notch‑4 and delta‑4 expression was significantly increased. Serum interferon‑γ significantly increased after hPMSCs transplantation, whereas interleukin‑4 and immunoglobulin E decreased. In OVA‑treated rats, Notch‑1, Notch‑2 and jagged‑1 levels were increased in the lymph compared with the blood, although Notch‑4 and delta‑4 levels were decreased. Peribronchial infiltration of cells and goblet cell hyperplasia were markedly decreased in the OVA + hPMSCs group compared with those in the OVA‑treated and control groups. Alterations in Notch signaling pathway expression were accompanied by decreased inflammatory cell infiltration, goblet cell hyperplasia and mucus production in lung tissues. The results of the present study are consistent with hPMSC suppression of asthma symptoms and inflammation by regulating the Notch signaling pathway in the rat asthma model.

Expression of Notch signaling pathway during osteoarthritis in the temporomandibular joint

PurposeOur study aim was to characterize the expression of Notch molecules during temporomandibular joint osteoarthritis (TMJOA), thus exploring the mechanism and roles that Notch signaling possibly plays in the initiation and progression of TMJOA. Materials and methodsA total of 126 mice were divided randomly into experimental groups, a sham-surgery group and a normal control group. In the experimental group, total discectomy was performed in the right temporomandibular joint (TMJ) to induce TMJOA; the sham-operation group underwent the same procedure without disc removal, and the normal control group was left undisturbed. Fourteen mice in each group were sacrificed in batches respectively at 1, 2, and 4 weeks postoperatively. Histology was performed to examine TMJOA in eight condyles each group, and a modified Mankin scoring system was used for evaluation. Immunohistochemistry was carried out to characterize the expression of the Notch markers Notch1, Jagged1, Hes1, and Hes5. Real-time polymerase chain reaction (RT-PCR) was performed for further detection and analysis of Notch markers in six condyles in each group. ResultsNotch1, Jagged1, and Hes5 were activated in the experimental group, with expression levels that increased dramatically over time, whereas the control group showed no fluctuation. Hes1 expression was suppressed at the beginning but was up-regulated afterward. ConclusionsOur data suggest that Notch signaling is activated in TMJOA with a much more abundant expression in osteoarthritis cartilage.

Effects of VPA on the Expression of Notch Signaling Pathway in Multiple Myeloma RPMI 8226 Cell Line

To investigate the effects of valproic acid(VPA) on the expression of intracellular domain of Notch1 (ICN1) and Hes1 in multiple myeloma RPMI 8226 cell line. Experiments were divided into 4 group: blank control group and groups of cells treated with VPA of different concentration (2, 4, 8 mmol/L), the cell proliferation was detected by MTT method, RT-PCR was applied to detect the mRNA expression level of ICN1 and Hes1. Western blot was used to detect the protein expression of ICN1 and Hes1. The proliferation of the RPMI 8226 cell was obviously inhibited by different concentration of VPA (2, 4, 8 mmol/L) at the same time. The same concentration of VPA was used to treat RPMI8226 cell for different time (24, 48, 72 h), the cell proliferation was obviously inhibited. Compared with control group, the mRNA and protein expression of ICN1 was significantly depressed at different concentration of VPA(2, 4, 8 mmol/L) for 48 h (P<0.05). Compared with control group, the mRNA and protein expression of Hes1 was depressed at different concentration 2, 4, 8 mmol/L)of VPA for 48 h (P<0.05). VPA inhibits the proliferation of the RPMI 8226 cell in a time- and dose- dependent manner; VPA down-regulates the mRNA and protein expression level of ICN1 and Hes1 in RPMI8226 cell; thus VPA might inhibit cell proliferation possibly through the inhibition of Notch signaling pathway in multiple myeloma cells.

Mo1955 Notch Signaling Pathway Protects Intestine From Ischemia/Reperfusion Injury in Rats Through STAT3 Signaling

Background: Notch signaling has been shown to play an important role in the maintenance of intestinal homeostasis. The purpose of the present study was to investigate the role of Notch signaling in the regulation of intestinal injury in an ischemia reperfusion (I/R) rat model. Method: Male Sprague-Dawley rats were subjected to sham operation or I/R by occlusion of the superior mesenteric artery (SMA) for 30 min. DAPT (a γ-secretase inhibitor, 500μg/100g) or DMSO was given intraperitoneally 2 h before I/R. Intestinal tissue samples were collected at 12 h after reperfusion. The expression of Notch signaling pathway and phosphorylation of STAT were examined by Real-time PCR, Western blot, immunofluorescence and Co-Immunoprecipitation. Apoptosis of intestinal epithelial cells were examined by TUNEL staining. Neutrophil infiltration was assessed by examining myeloperoxidase (MPO) activity in the gut. Finally, an in vitro IEC-6 culture system was further applied to investigate the role of Notch signaling in the regulation of intestinal epithelal apoptosis after I/R injury. Results: In this study, Notch signaling was activated after intestinal I/R injury. The proteins expression of NICD-1 and Hes-5 were increased significantly (NICD-1: 0.35±0.12 vs 0.79±0.27, p<0.05; Hes-5:0.42±0.15 vs 0.93±0.31, p<0.05 ) and immunofluorescence results showed that the proteins were colocalized mainly in the intestinal epithelial cells after I/R injury. Blocking Notch signaling by DAPT led to aggravated intestinal I/R injury, as manifested by deteriorated intestinal barrier function (TER: 78±9.5V.cm2 vs 62±7.3V.cm2), increased inflammation and intestinal epithelial cells apoptosis (18±1.9 cells/100crypts vs 32±2.2 cells/ 100crypts). Notch signaling blockade also resulted in down-regulation of Hes5, leading to reduced formation of the Hes5-STAT3 complex and hypophosphorylation of STAT3 both in vivo and in vitro. Overexpression of a constitutively active STAT3 attenuated I/R injury by reducing apoptosis. Furthermore, IEC-6 cells were protected from I/R injury induced apoptosis after the activation of Notch signaling by ligand stimulation through increased activation of STAT3. Conclusion: Notch signaling was activated and played an important role in the protection of intestine from I/R injury through Hes-5 dependent activation of STAT3.

Mo1956 Isolated Right Colon Ischemic Colitis Has Different Features and Outcomes Compared to Non-Right Colon Ischemic Colitis: A Multicenter Community Study

Background: Notch signaling has been shown to play an important role in the maintenance of intestinal homeostasis. The purpose of the present study was to investigate the role of Notch signaling in the regulation of intestinal injury in an ischemia reperfusion (I/R) rat model. Method: Male Sprague-Dawley rats were subjected to sham operation or I/R by occlusion of the superior mesenteric artery (SMA) for 30 min. DAPT (a γ-secretase inhibitor, 500μg/100g) or DMSO was given intraperitoneally 2 h before I/R. Intestinal tissue samples were collected at 12 h after reperfusion. The expression of Notch signaling pathway and phosphorylation of STAT were examined by Real-time PCR, Western blot, immunofluorescence and Co-Immunoprecipitation. Apoptosis of intestinal epithelial cells were examined by TUNEL staining. Neutrophil infiltration was assessed by examining myeloperoxidase (MPO) activity in the gut. Finally, an in vitro IEC-6 culture system was further applied to investigate the role of Notch signaling in the regulation of intestinal epithelal apoptosis after I/R injury. Results: In this study, Notch signaling was activated after intestinal I/R injury. The proteins expression of NICD-1 and Hes-5 were increased significantly (NICD-1: 0.35±0.12 vs 0.79±0.27, p<0.05; Hes-5:0.42±0.15 vs 0.93±0.31, p<0.05 ) and immunofluorescence results showed that the proteins were colocalized mainly in the intestinal epithelial cells after I/R injury. Blocking Notch signaling by DAPT led to aggravated intestinal I/R injury, as manifested by deteriorated intestinal barrier function (TER: 78±9.5V.cm2 vs 62±7.3V.cm2), increased inflammation and intestinal epithelial cells apoptosis (18±1.9 cells/100crypts vs 32±2.2 cells/ 100crypts). Notch signaling blockade also resulted in down-regulation of Hes5, leading to reduced formation of the Hes5-STAT3 complex and hypophosphorylation of STAT3 both in vivo and in vitro. Overexpression of a constitutively active STAT3 attenuated I/R injury by reducing apoptosis. Furthermore, IEC-6 cells were protected from I/R injury induced apoptosis after the activation of Notch signaling by ligand stimulation through increased activation of STAT3. Conclusion: Notch signaling was activated and played an important role in the protection of intestine from I/R injury through Hes-5 dependent activation of STAT3.

Mo1957 Characteristics of Recurrent vs. Non-Recurrent Ischemic Colitis: A Multicenter Community Study

Background: Notch signaling has been shown to play an important role in the maintenance of intestinal homeostasis. The purpose of the present study was to investigate the role of Notch signaling in the regulation of intestinal injury in an ischemia reperfusion (I/R) rat model. Method: Male Sprague-Dawley rats were subjected to sham operation or I/R by occlusion of the superior mesenteric artery (SMA) for 30 min. DAPT (a γ-secretase inhibitor, 500μg/100g) or DMSO was given intraperitoneally 2 h before I/R. Intestinal tissue samples were collected at 12 h after reperfusion. The expression of Notch signaling pathway and phosphorylation of STAT were examined by Real-time PCR, Western blot, immunofluorescence and Co-Immunoprecipitation. Apoptosis of intestinal epithelial cells were examined by TUNEL staining. Neutrophil infiltration was assessed by examining myeloperoxidase (MPO) activity in the gut. Finally, an in vitro IEC-6 culture system was further applied to investigate the role of Notch signaling in the regulation of intestinal epithelal apoptosis after I/R injury. Results: In this study, Notch signaling was activated after intestinal I/R injury. The proteins expression of NICD-1 and Hes-5 were increased significantly (NICD-1: 0.35±0.12 vs 0.79±0.27, p<0.05; Hes-5:0.42±0.15 vs 0.93±0.31, p<0.05 ) and immunofluorescence results showed that the proteins were colocalized mainly in the intestinal epithelial cells after I/R injury. Blocking Notch signaling by DAPT led to aggravated intestinal I/R injury, as manifested by deteriorated intestinal barrier function (TER: 78±9.5V.cm2 vs 62±7.3V.cm2), increased inflammation and intestinal epithelial cells apoptosis (18±1.9 cells/100crypts vs 32±2.2 cells/ 100crypts). Notch signaling blockade also resulted in down-regulation of Hes5, leading to reduced formation of the Hes5-STAT3 complex and hypophosphorylation of STAT3 both in vivo and in vitro. Overexpression of a constitutively active STAT3 attenuated I/R injury by reducing apoptosis. Furthermore, IEC-6 cells were protected from I/R injury induced apoptosis after the activation of Notch signaling by ligand stimulation through increased activation of STAT3. Conclusion: Notch signaling was activated and played an important role in the protection of intestine from I/R injury through Hes-5 dependent activation of STAT3.