OBJECTIVETo investigate the effects of triptolide on Notch receptor and ligand expressions in rats with adjuvant-induced arthritis (AA).

METHODSForty rats were randomly divided into normal control (NC) group, model (MC) group, methotrexate group and triptolide groups. Rat models of AA were established by an intradermal injection of 0.1 mL Freund's complete adjuvant into the right paw. Twelve days after the injection, the rats were treated with corresponding drugs for 30 days; the rats in NC group and MC group were given saline only. Paw edema volume (E), arthritis index (AI), pulmonary function, histomorphologies, and Notch receptor/ ligand expression in the lung tissue were analyzed after the treatments.

RESULTSCompared with the NC group, E, AI, Notch3, Notch4, and Delta1 expressions in the lung tissues significantly increased while pulmonary function and pulmonary expressions of Notch1, Jagged1, and Jagged2 significantly decreased the model rats (P<0.01). Compared with the MC group, triptolide-treated rats showed significantly improved pulmonary functions, increased expressions of Notch1, Jagged1, and Jagged2 and decreased expressions of Notch3, Notch4, and Delta1 in the lungs (P<0.05, P<0.01); the therapeutic effect of triptolide was better than that of methotrexate.

CONCLUSIONTriptolide can reduce inflammatory reaction and immune complex deposition to improve joint and pulmonary symptoms in rats with AA possibly by up-regulating the expressions of Notch3, Notch4, and Delta1 and down-regulating the expressions of Jagged1, Jagged2, and Notch1.

Animals ; Arthritis, Experimental ; drug therapy ; metabolism ; Calcium-Binding Proteins ; metabolism ; Diterpenes ; pharmacology ; Down-Regulation ; Drugs, Chinese Herbal ; Epoxy Compounds ; pharmacology ; Intercellular Signaling Peptides and Proteins ; metabolism ; Intracellular Signaling Peptides and Proteins ; metabolism ; Jagged-1 Protein ; Jagged-2 Protein ; Ligands ; Lung ; drug effects ; metabolism ; physiopathology ; Membrane Proteins ; metabolism ; Methotrexate ; pharmacology ; Phenanthrenes ; pharmacology ; Rats ; Receptor, Notch3 ; Receptor, Notch4 ; Receptors, Notch ; metabolism ; Respiratory Insufficiency ; drug therapy ; Serrate-Jagged Proteins

This study was aimed to investigated the mRNA expression levels of Notch ligands- Delta-like-1 and Jagged-1 in bone marrow mesenchymal stem cells of patients with myelodysplastic syndrome (MDS), and to explore their relation with onset of MDS. Bone marrow mesenchymal stem cells of 38 patients with MDS and 16 normal subjects as control were collected to detect mRNA expression of Delta-like-1 and Jagged-1 by using real-time quantitative polymerase chain reaction. The results showed that the expression levels of Delta-like-1 and Jagged-1 in mesenchymal stem cells of MDS patients were significantly higher than that in normal controls (P < 0.05). According to WHO criteria, the mRNA expression of Delta-like-1 in RA/RAS, RCMD and RAEB groups were significantly higher than that in normal controls (P < 0.05), the mRNA expression of Jagged-1 in RAEB group was also significantly higher than that in normal controls (P < 0.05). The mRNA expression of Delta-like-1 was significantly correlated with the proportion of blasts in the bone marrow of MDS patients (r = 0.502, P < 0.05). The expression levels of Delta-like-1 and Jagged-1 in MDS patients with abnormal karyotypes were significantly higher than those in MDS patients with normal karyotypes (P < 0.05). The mRNA expression of Delta-like-1 in higher risk group according to International Prognostic Scoring System was significantly higher than that in lower risk group (P < 0.05), there was no significant difference in Jagged-1 expression levels between higher risk group and lower risk group (P > 0.05). It is concluded that the changes of Delta-like-1 and Jagged-1 expression level in MSC may play a role in the pathogenesis of myelodysplastic syndrome.
Calcium-Binding Proteins ; genetics ; Gene Expression Regulation, Neoplastic ; Humans ; Intercellular Signaling Peptides and Proteins ; genetics ; Intracellular Signaling Peptides and Proteins ; genetics ; Jagged-1 Protein ; Membrane Proteins ; genetics ; Mesenchymal Stromal Cells ; metabolism ; Myelodysplastic Syndromes ; genetics ; RNA, Messenger ; biosynthesis ; Serrate-Jagged Proteins

This study was purposed to construct prokaryotic expression vector and to investigate the expression of Notch ligand Jagged1 in E.coli. An expression vector pET-hJagged1 was constructed, which can be inserted in Jagged1 with different lengths, but the DSL domain of human Jagged1 should be contained. Then the recombinant plasmids were transformed into the competent cell of E.coli BL21, and the expression of the fusion protein was induced by IPTG. Fusion protein was purified from the supernatant of cell lysates via the Nickel affinity chromatography. The results showed that prokaryotic expression vectors pET-hJagged1 (Bgl II), pET-hJagged1 (Hind I) and pET-hJagged1 (Stu I) were successfully constructed, but only pET-hJagged1 (Stu I) could express the soluble TRX-hJagged1. The purified TRX-Jagged1 protein could be obtained via the Nickel affinity chromatography, and then confirmed by Western Blot. It is concluded that prokaryotic expression vector pET-hJagged1 is successfully constructed, but only pET-hJagged1 (Stu I) can express the soluble TRX-hJagged1 and the TRX-Jagged1 fusion protein is obtained through the prokaryotic expression system, which laid a solid foundation for further to explore the effects of Jagged1 in hematopoietic and lymphoid system.
Calcium-Binding Proteins ; genetics ; metabolism ; Cloning, Molecular ; Escherichia coli ; metabolism ; Genetic Vectors ; Intercellular Signaling Peptides and Proteins ; genetics ; metabolism ; Jagged-1 Protein ; Membrane Proteins ; genetics ; metabolism ; Plasmids ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Serrate-Jagged Proteins

OBJECTIVE: To observe the changes of pulmonary function and Notch signaling pathway of lung tissues in adjuvant-induced arthritis rats, and to investigate the mechanism of reduced lung function. METHODS: A total of 30 rats were randomly divided into a normal group and a model group. Rats in the model group were induced to establish the adjuvant arthritis AA model by intradermally injecting 0.1 mL Freund's complete adjuvant into the right paw. After 30 days, we observed the paw edema volume, arthritis index, pulmonary function, histomorphology, and Notch receptor/ligand of the lung tissue. RESULTS: Compared with the normal group, the paw edema volume, arthritis index, average expiratory flow within 0.3 s (FEV0.3/FVC), and the level of Notch3, Notch4 and Jagged2 of the lung tissue in the model group was significantly increased, while maximum expiratory flow at 50% of vital capacity (FEF50), maximum expiratory flow at 75% of vital capacity (FEF75), forced expiratory flow (PEF) and the expression of Notch1 of Jagged1 and Delta1 in the lung were significantly decreased (P<0.05, P<0.01). There were significant positive correlations between FEV0.3/FVC and Notch4. FEV0.3/FVC, FEF25, FEF50 and Notch3, Delta1 were negatively correlated, respectively (P<0.05, P<0.01). CONCLUSION While arthritis occurs in AA rats, pulmonary function declines and significantly correlates with the expression of Notch receptor/ligand. The deposition of immune complex in the lung after the injection of CFA activates the Notch signaling pathway, and results in further decline of pulmonary function by signaling cascades.
Animals ; Arthritis, Experimental ; metabolism ; physiopathology ; Calcium-Binding Proteins ; metabolism ; Freund's Adjuvant ; Intercellular Signaling Peptides and Proteins ; metabolism ; Intracellular Signaling Peptides and Proteins ; metabolism ; Jagged-1 Protein ; Lung ; metabolism ; pathology ; Membrane Proteins ; metabolism ; Rats ; Receptors, Notch ; metabolism ; Respiratory Insufficiency ; metabolism ; physiopathology ; Serrate-Jagged Proteins ; Signal Transduction ; Vital Capacity

OBJECTIVETo investigate effects of paraquat on the mRNA expression of key elements of Notch signaling (Notch1, Jagged1 and DTX1) during differentiation process of human neural stem cells (hNSCs).

METHODShNSCs exposed to PQ at the concentrations 0.10, 1.00, 10.00 M. Cell proliferation ability was assessed using MTT assay and mRNA expressions of Notch1, Jagged1 and DTX1 were detected by Real-time RT-PCR at 2, 4, 8, 12 d of differentiation.

RESULTSCompared with control group, NOTCH1, JAG1 mRNA expression levels exposed to PQ at the concentration of 0.10 M significantly reduced at 2, 4, 8 d and significantly went up at 12d (P < 0.01). Compared with control group, NOTCH1, JAG1 and DTX1 mRNA expression levels exposed to PQ at the concentration of 10.00 M significantly reduced at 2, 8, 12 d (P < 0.01). PQ could down-regulate Notch1, Jagged1 and DTX1 mRNA expressions at the early stage of differentiation, then up-regulate Notch1 mRNA expression, and down-regulate Notch1, Jagged1 and DTX1 mRNA expressions at the end of differentiation.

CONCLUSIONNotch signaling pathway may be involved in differentiation of neural stem cell exposed to PQ.

Calcium-Binding Proteins ; metabolism ; Cell Differentiation ; drug effects ; Cells, Cultured ; Embryonic Stem Cells ; cytology ; drug effects ; metabolism ; Humans ; Intercellular Signaling Peptides and Proteins ; metabolism ; Jagged-1 Protein ; Membrane Proteins ; metabolism ; Neural Stem Cells ; cytology ; drug effects ; metabolism ; Paraquat ; pharmacology ; Receptor, Notch1 ; metabolism ; Serrate-Jagged Proteins ; Signal Transduction ; drug effects ; Ubiquitin-Protein Ligases ; metabolism

OBJECTIVETo investigate the biological behavior including survival and proliferation of CD34 + CD38--Lin--cells when they are cultured at single cell level.

METHODSPurified umbilical cord blood CD34 + CD38--Lin--cells were separated at single cell level in 96-well plates using flow cytometry for four groups: control group (CD34 + CD38--Lin--cell plus stem cell medium) , Shh group (CD34 + CD38--Lin--cell plus stem cell medium and Shh), BMP-4 group (CD34 + CD38--Lin--cell plus stem cell medium and BMP-4), Jagged-1 group (CD34 + CD38--Lin--cell plus stem cell medium and Jagged-1). Methylcellulose medium was used in the colony-forming experiment which was also in four groups as previously. The number of cells and colony-forming units in each well for the four groups was evaluated at different time points (day 1, 3, 7) with fluorescence microscopy counting method.

RESULTSDivision of single cell was observed to be amplified in all of these groups from day 3. And meanwhile, after 1-week culture, the survival rates for the treated groups were all higher than the control group (Jagged-1 group > BMP-4 group > Shh group > control), while the cell number in each well was also highest in the Jagged-1 group (Jagged-1 group > BMP-4 group > control). The number of wells with a cell number of zero was significantly fewer in all treated groups (especially the Jagged-1 group) than in the control group; meanwhile, the number of wells with a cell number higher than 17 was evidently higher in all the treated groups (especially the BMP-4 group) more than controls. Colony-forming units for erythroid (BFU-E), granulocyte (CFU-G), macrophage (CFU-M), and granulocyte macrophage (CFU-GM) were observed for all of these experimental groups, and there was no significant difference between the four experimental groups.

CONCLUSIONSCD34 + CD38 - Lin - cell can achieve the survival, self-renewal and proliferation when cultured at single cell level, and the adding of Shh, BMP-4, and Jagged-1 can enhance such capabilities. However, CD34 + CD38 - Lin - cell can only maintain cell totipotency in its niche.

ADP-ribosyl Cyclase 1 ; metabolism ; Antigens, CD34 ; metabolism ; Bone Morphogenetic Protein 4 ; chemistry ; Calcium-Binding Proteins ; chemistry ; Cell Culture Techniques ; Cell Proliferation ; Cell Survival ; Cells, Cultured ; Colony-Forming Units Assay ; Culture Media ; Fetal Blood ; cytology ; Hedgehog Proteins ; chemistry ; Hematopoietic Stem Cells ; cytology ; Humans ; Intercellular Signaling Peptides and Proteins ; chemistry ; Jagged-1 Protein ; Membrane Proteins ; chemistry ; Serrate-Jagged Proteins

OBJECTIVETo study the gene expression of Notch1 and Jagged1 in children with acute leukemia (AL) and their possible roles in the pathogenesis of AL.

METHODSMononuclear cells from bone marrow or peripheral blood of 47 children with AL and 20 controls (normal children or children with nonmalignant hematologic disease) were collected from February 2009 to July 2011. A two-step method to semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the gene expression of Notch1 and Jagged1. Of the 47 children with AL, there were 26 cases of B-ALL, 6 cases of T-ALL and 15 cases of AML.

RESULTSThe positive expression rate of Notch1 in the ALL and AML groups was higher than in the control group (P<0.05). The expression level of Notch1 in T-ALL children was higher than in B-ALL children (P<0.01). The positive expression rate of Jagged1 in the ALL and AML groups was not significantly different from the control group, however, the expression level of Jagged1 in the ALL and AML groups was higher than in the control group (P<0.05).

CONCLUSIONSThere are significant differences in the gene expression of Notch1 between children with different types of ALL, and a higher expression of Notch1 relates to T-ALL. The activation of Notch1 signal is common in children with AL. The abnormal gene expression of Notch1 in children with AML shows the role of Notch1 in AML. The gene expression of Jagged1 in children with ALL or AML is abnormal, and this needs to be confirmed by further research.

Acute Disease ; Adolescent ; Calcium-Binding Proteins ; genetics ; Child ; Child, Preschool ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Infant ; Intercellular Signaling Peptides and Proteins ; genetics ; Jagged-1 Protein ; Leukemia ; metabolism ; Leukemia, Myeloid, Acute ; metabolism ; Leukemia-Lymphoma, Adult T-Cell ; metabolism ; Male ; Membrane Proteins ; genetics ; Receptor, Notch1 ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Serrate-Jagged Proteins ; Signal Transduction

OBJECTIVETo study the expression of JAG1 and DLL1 in colorectal cancer and its clinical significance.

METHODSPatients with colorectal cancer were treated in the Center of Colorectal Surgery of the Third Affiliated Hospital of Nanjing University of TCM were collected prospectively and followed up. A tissue microarray was made and expressions of JAG1 and DLL1 were detected by immunohistochemical staining.

RESULTSA total of 146 cases with colorectal cancer were included. The differences in JAG1 expression were significant among different tumor differentiation types and the differences in DLL1 expression were significant among different tumor locations(all P<0.05). There were no significant differences in the expression of the two genes and microsatellite instability(MSI)(P>0.05). One hundred and thirty-four (91.8%) cases were followed up and the mean follow-up time was (42.3±13.3) months. Tumor-free survival was noticed in 86 patients. The overall survival was 93% at 1 year, 74% in 3 years, and 67% in 5 years. Multivariate analysis showed that long-term survival rate was related to TMN stage, pathology types, MSI status and expression of JAG1. The prognosis of patients with high expression of JAG1 was better than those with low and negative expression(P<0.05).

CONCLUSIONSThe expressions of JAG1 and DLL1 are related to tumor differentiation and tumor location. The expression of JAG1 gene is associated with long-term survival.

Adult ; Aged ; Aged, 80 and over ; Calcium-Binding Proteins ; genetics ; metabolism ; Colorectal Neoplasms ; genetics ; metabolism ; pathology ; Female ; Follow-Up Studies ; Humans ; Intercellular Signaling Peptides and Proteins ; genetics ; metabolism ; Jagged-1 Protein ; Male ; Membrane Proteins ; genetics ; metabolism ; Microsatellite Instability ; Middle Aged ; Prognosis ; Retrospective Studies ; Serrate-Jagged Proteins ; Survival Analysis ; Young Adult

OBJECTIVETo investigate whether Notch signaling is activated in hepatic stellate cells (HSCs), and to determine whether manipulation of the Notch signaling pathway can effect the activation of HSCs.

METHODSThe expression of Notch signaling components in unactivated or TGF-b1-activated HSC-T6 cells was detected by Taqman Probe-based gene expression analysis. Differential expression of Notch3 and Jagged1 was detected by immunofluorescence analysis. Notch3-mediated expression of the myofibroblastic markers, a-SMA and collagen I, was detected in HSC-T6 cells transfected with pcDNA3.1-N3ICD or Notch3 siRNA by Western blotting.

RESULTSNotch signaling components were expressed in both unactivated and activated HSC-T6 cells, but the TGF-b1-treated cells showed significantly higher expression levels of Jagged1 (3.9-fold, F = 2543.482), Notch3 (4.2-fold, F = 287.982), and HES1 (3.2-fold, F = 1719.851). Transfection-mediated over-expression of Notch3 led to significantly increased expression of a-SMA (6.8-fold, t = 13.157) and collagen I (5.5-fold, t = 9.810) (both P less than 0.01). Transient knock-down of Notch3 expression by siRNA decreased expression of the myofibroblastic markers (a-SMA by approximately 90%, t = 19.863 and collagen I by 84%, t = 10.376; both, P less than 0.01). Moreover, knock-down of Notch3 antagonized the TGF-b1-induced expression of a-SMA and collagen I.

CONCLUSIONNotch signaling may participate in liver fibrogenesis by regulating HSC activation. Selective interruption of Notch3 may represent a new anti-fibrotic strategy to treat liver fibrosis.

Animals ; Calcium-Binding Proteins ; genetics ; metabolism ; Cell Line ; Hepatic Stellate Cells ; metabolism ; Intercellular Signaling Peptides and Proteins ; genetics ; metabolism ; Jagged-1 Protein ; Membrane Proteins ; genetics ; metabolism ; RNA, Messenger ; genetics ; RNA, Small Interfering ; Rats ; Receptor, Notch3 ; Receptors, Notch ; genetics ; metabolism ; Serrate-Jagged Proteins ; Signal Transduction

OBJECTIVETo observe the effect of C-reactive protein (CRP) on the expressions of Notch pathway components in human peripheral blood endothelial progenitor cells (EPC) in vitro.

METHODSMononuclear cells isolated by density gradient centrifugation of human peripheral blood mixed with 6% hydroxyethyl starch (Hes) were plated on fibronectin-coated 6-well culture dishes. After 7 days, the adherent cells were cultured in the presence of 10 and 20 mg/L CRP for 48 h, and the proliferation, migration, and adhesion abilities of the cells were observed. The mRNA expressions of Notch-1 and its ligand Jagged-1 in the EPCs were measured by RT-PCR, and their protein expressions by Western blotting.

RESULTSCRP at 10 and 20 mg/L caused a significant reduction in the number of viable EPCs (61∓3 and 54∓3, respectively) as compared with PBS (71∓4, P<0.05). CRP also resulted in a significant suppression of the proliferation, migration and adhesion capacities of the EPCs. The mRNA and protein expressions of Jagged-1 and Notch-1 in the EPCs significantly increased following CRP exposure in comparison with PBS treatment.

CONCLUSIONCRP can suppress the proliferation, migration and adhesion capacities of the EPCs probably by affecting the expressions of the Notch-1 pathway components.

C-Reactive Protein ; pharmacology ; Calcium-Binding Proteins ; genetics ; metabolism ; Cell Adhesion ; Cell Movement ; Cell Proliferation ; Cells, Cultured ; Endothelial Cells ; cytology ; metabolism ; Humans ; Intercellular Signaling Peptides and Proteins ; genetics ; metabolism ; Jagged-1 Protein ; Leukocytes, Mononuclear ; cytology ; metabolism ; Membrane Proteins ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Receptor, Notch1 ; genetics ; metabolism ; Serrate-Jagged Proteins ; Signal Transduction ; drug effects ; Stem Cells ; cytology ; metabolism