N-methyl-D-aspartate receptor (NMDAR),an important ionic glutamate receptor and a ligand and voltage-gated ion channel characterized by complex composition and functions and wide distribution,plays a key role in the pathological and physiological process of diseases or stress states.NMDAR can mediate apoptosis through different pathways such as mitochondrial and endoplasmic reticulum damage,production of reactive oxygen species and peroxynitrite,and activation of mitogen-activated protein kinase and calpain.This paper reviews the structure,distribution,and biological characteristics of NMDAR and the mechanisms of NMDAR-mediated apoptosis.
Apoptosis ; Humans ; Mitogen-Activated Protein Kinases/metabolism* ; Reactive Oxygen Species/metabolism* ; Receptors, N-Methyl-D-Aspartate/metabolism* ; Signal Transduction

OBJECTIVETo observe the expressions of discoidin domain receptor 2 (DDR2) in different phases of alcoholic liver fibrosis (ALF) in a rat model and to study the possible association between DDR2 and collagen deposition in ALF.

METHODSAfter an ALF rat model was established by alcohol gastrogavage and an olive oil diet, the liver histopathology was observed in different phases of the development of fibrosis. The expressions of DDR2 mRNA and protein were also detected by RT-PCR and Western blot respectively to make a dependability analysis with the index of ALF.

RESULTS(1) The expressions of DDR2 mRNA and protein increased gradually along with ALF aggravation. In the normal control group, they were respectively 1.023+/-0.132 and 0.321+/-0.027; in the model 1 group (week 12) they were 3.644+/-1.686, 0.476+/-0.046; in the model 2 group (week 16) they were 8.337+/-2.387, 0.738+/-0.057; and in the model 3 group (week 20) they were 15.730+/-4.569, 0.997+/-0.049. The differences of DDR2 mRNA (F = 21.74, P less than 0.01) and protein (F = 10.38, P less than 0.01) among these four groups were significant. (2) The expressions of DDR2 had a positive correlation with collagen type I, III, IV contents and the serum index of ALF, especially with type III and IV collagen and serum hexadecenoic acid.

CONCLUSIONThe expression of DDR2 in this ALF model correlates closely with collagen deposition in the liver, suggesting that it may play an important role in ALF pathogenesis.

Animals ; Collagen ; metabolism ; Discoidin Domain Receptors ; Disease Models, Animal ; Liver Cirrhosis, Alcoholic ; metabolism ; pathology ; Male ; Rats ; Rats, Wistar ; Receptor Protein-Tyrosine Kinases ; metabolism ; Receptors, Mitogen ; metabolism

OBJECTIVETo explore the role of discoidin domain receptors (DDRs) in the formation of the keloid.

METHODSThe real-time quantitative PCR was used to compare the DDRs expression in the keloids and normal fibroblasts.

RESULTSThe level of DDR1 expression was significantly higher in keloid than in normal fibroblast (20.98 vs 4.2, P <0.01; 7.9 vs 4.23, P <0.05). The level of DDR1 expression in keloid was also higher significantly than that in hypertropic scar (20.98 vs 7.9, P < 0.01). However, the level of DDR2 expression was somewhat higher in keloid than in normal fibroblasts, the difference seemed not to be significantly in probability (358, 332 vs 278, P > 0.05).

CONCLUSIONSDDRs may exert effect on keloid cell behaviours.

Cell Proliferation ; Cells, Cultured ; Cicatrix ; metabolism ; pathology ; Discoidin Domain Receptors ; Female ; Fibroblasts ; metabolism ; Humans ; Male ; Receptor Protein-Tyrosine Kinases ; metabolism ; Receptors, Mitogen ; metabolism

Recent studies showed that Eph/Ephrin tyrosine kinase family plays an important role in the development and functional maintenance of the nervous system, but its function in the sympathetic nervous system is still obscure. In the present study, we examined the effect of Eph/Ephrin-B1 signaling on the whole-cell currents mediated by either alpha7 or alpha3-nicotinic acetylcholine receptors (nAChRs) in acutly dissociated ciliary ganglion (CG) neurons. Firstly, we detected the effect of Ephrin-B1 on nAChRs currents. The neurons were randomly divided into control group, Ephrin-B1Fc-treated group that was stimulated by recombinant Ephrin-B1Fc, IgG-treated group, and Ephrin-B1-treated group. Secondly, we studied the regulatory mechanism of Ephrin-B1Fc on nAChRs currents. The neurons were randomly divided into control group, Ephrin-B1Fc-treated group, PP2 (inhibitor of Src tyrosine kinase) or PD98095 (antagonist of mitogen-activated protein kinase)-treated group, Ephrin-B1Fc + PP2 or PD98095-treated group. The results showed that there was no significant difference between the currents in control group, IgG-treated group and Ephrin-B1-treated group, but Ephrin-B1Fc significantly suppressed both alpha3-nAChRs and alpha7-nAChRs-mediated currents (P=0.002, P=0.003). Pretreatment with PP2 or PD98095 could partially rescue the Ephrin-B1Fc-induced suppression of currents mediated by alpha3-nAChRs or alpha7-nAChRs respectively. These results suggest that the Eph/Ephrin-B1 signaling may inhibit alpha3-nAChRs and alpha7-nAChRs-mediated currents on CG neurons, involving Src tyrosine kinase and mitogen-activated protein kinase signaling in the regulation of sympathetic nervous system.
Ephrin-B1 ; metabolism ; Ganglia, Parasympathetic ; enzymology ; Mitogen-Activated Protein Kinases ; metabolism ; Neurons ; enzymology ; Receptors, Nicotinic ; metabolism ; Signal Transduction ; alpha7 Nicotinic Acetylcholine Receptor ; metabolism ; src-Family Kinases ; metabolism

OBJECTIVEDifferent from primary membranous nephropathy, hepatitis B virus associated membranous nephropathy (HBV-MN) shows lower deposits of membrane attack complex (C5b-9) in glomerular subepithelium. The causes of relatively low complement activation in this disease remain unclear. The aim of this study was to investigate the influence of hepatitis B x protein (HBx) on the expression of CD59 and Crry in mouse podocytes.

METHODCultured mouse podocytes were divided into adenovirus vector hepatitis B virus X gene (Ad-HBx) transfected group (Ad-X group), blank podocytes group (B group) and adenovirus vector transfected group (Ad group). CD59 and Crry mRNA expression were assayed by semiquantitative RT-PCR. CD59 and Crry expression were tested by flow cytometry. The effect of HBx on complement activation was evaluated with MTT method. And then, the effects of P38MAPK, PI-3K and ERK1/2 pathway inhibitors (SB203580, LY294002, U0126) and DMSO on CD59 and Crry expression were respectively detected by flow cytometry.

RESULTProteins CD59 and Crry expression rates (%) in group B, Ad group and Ad-X group were 17.71 ± 3.81, 18.29 ± 3.36 and 45.7 ± 9.01; 18 ± 2.31, 21.78 ± 2.01 and 47.45 ± 9.95, respectively. Compared with group B, CD59 and Crry expression in group Ad was not significantly different (P values for both > 0.05), but CD59 and Crry protein expression in Ad-X group was significantly higher than that in groups B and Ad (P values for both < 0.005); CD59 and Crry gene expression in group Ad was not significantly different from that in group B (P values for both > 0.05). However, CD59 and Crry gene expression of Ad-X group was significantly higher than that in groups B and Ad (P values for both < 0.05). Flow cytometry detected CD59 protein expression rates (%) were 17.35 ± 1.24, 46.19 ± 9.77, 43.03 ± 6.83 and 40.04 ± 6.39 and Crry protein expression rates (%) were 18.14 ± 3.56, 31.95 ± 1.68, 31.95 ± 1.69 and 37.14 ± 3.92 after SB203580, LY294002, U0126 and DMSO were added to Ad-X group respectively. P38 pathway inhibition resulted in significantly lower CD59 and Crry expression than Ad-X group (P values for all < 0.005), but PI-3K, ERK1/2 pathway inhibitors and DMSO had no significant effect on the expression of CD59 and Crry (P values for all > 0.05). The inhibition rates of cell lysis were significantly higher in Ad-X group than in groups B and Ad at each serum dilution point (P values for all < 0.05), while groups B and Ad had no significant difference in cell viability.

CONCLUSIONHBx can up-regulate CD59 and Crry expression in podocytes through activating P38 pathway, resulting in decreased complement activation, which may facilitate latent HBV infection in podocytes and play a role in development of hepatitis B virus associated glomerulonephritis (HBV-GN).

Animals ; CD59 Antigens ; metabolism ; Cells, Cultured ; Mice ; Podocytes ; immunology ; metabolism ; Receptors, Complement ; metabolism ; Trans-Activators ; metabolism ; p38 Mitogen-Activated Protein Kinases ; metabolism

Objective: To explore the development of a CAR-T cells targeting CLL-1 and verify its function. Methods: The expression levels of CLL-1 targets in cell lines and primary cells were detected by flow cytometry. A CLL-1 CAR vector was constructed, and the corresponding lentivirus was prepared. After infection and activation of T cells, CAR-T cells targeting CLL-1 were produced and their function was verified in vitro and in vivo. Results: CLL-1 was expressed in acute myeloid leukemia (AML) cell lines and primary AML cells. The transduction rate of the prepared CAR T cells was 77.82%. In AML cell lines and AML primary cells, CLL-1-targeting CAR-T cells significantly and specifically killed CLL-1-expressing cells. Compared to untransduced T cells, CAR-T cells killed target cells and secreted inflammatory cytokines, such as interleukin-6 and interferon-γ, at significantly higher levels (P<0.001) . In an in vivo human xenograft mouse model of AML, CLL-1 CAR-T cells also exhibited potent antileukemic activity and induced prolonged mouse survival compared with untransduced T cells [not reached vs 22 days (95%CI 19-24 days) , P=0.002]. Conclusion: CAR-T cells targeting CLL-1 have been successfully produced and have excellent functions.
Animals ; Cell Line, Tumor ; Cytokines ; Humans ; Immunotherapy, Adoptive ; Lectins, C-Type ; Leukemia, Myeloid, Acute/metabolism* ; Mice ; Receptors, Mitogen ; T-Lymphocytes

OBJECTIVE: To investigate the effects of Toll like receptors on the osteogenesis of human pe-riodontal ligament stem cells (hPDLSCs) and probable molecular mechanism. METHODS: Real-time PCR and flow cytometry were applied to test the expression of TLRs in hPDLSCs and the positive cell percentage of TLR. hPDLSCs were cultured in osteogenic medium for 7 to 14 days with different TLR agonists at various concentrations . The effect of different TLR on osteogenic differentiation of hPDLSCs was evaluated by alizarin red S staining, alkaline phosphatase (ALP) staining and ALP activity assay. Western blotting was used to analyze the phosphorylation levels of extracellular regulated protein kinases (ERK), c-Jun N-terminal protein kinase (JNK), P38, AKT and expression of Runx2 an osteogenic related gene after treatment with TLR agonists, compared with the effect of inhibitors of mitogen activated protein kinase (MAPK) or protein kinase B (PKB or AKT) on Runx2 expression of hPDLSCs cultured in osteogenic medium. RESULTS: Higher expressions of TLR1,3,4,6 were found in hPDLSCs through real-time PCR. Positive cell percentage of TLR was determined by flow cytometry and described as TLR1: 2.82%±0.68%; TLR2: 1.26%±0.09%; TLR3: 13.23%±2.05%; TLR4: 3.64%±0.79%; TLR6: 3.21%±1.64%, whose tendency was comparable to their mRNA expression in hPDLSCs. Most TLR ligands had no effect on the ALP staining, activity and mineralization of hPDLSCs at lower concentration except for 0.1 mg/L PolyI:C could induce the osteogenic ability of hPDLSCs. On the contrary, Higher concentration of TLR ligands (PolyI:C: 10 mg/L, LPS: 10 mg/L , Pam3CSK4: 1 mg/L, FSL-1: 50 μg/L) had obviously inhibitory effect on osteogenic differentiation of hPDLSCs. Activation of TLR using higher concentration of TLR ligands could downregulate the phosphorylation levels of ERK, P38, JNK and AKT, and also reduced the expression of Runx2, compared with the untreated control. The inhibitors of MAPK (U0126, SP600125,SB203580) and inhibitor of AKT (perifosine) could also inhibit Runx2 expression. CONCLUSION Higher concentration of TLR ligands could inhibit osteogenic differentiation of hPDLSCs. This inhibitory effect seemed to be related to decreased phosphorylation of MAPK and AKT.
Cell Differentiation ; Cells, Cultured ; Humans ; Ligaments ; Mitogen-Activated Protein Kinases/metabolism* ; Osteogenesis ; Periodontal Ligament/metabolism* ; Phosphorylation ; Proto-Oncogene Proteins c-akt/metabolism* ; Stem Cells ; Toll-Like Receptors/metabolism*

To elucidate the intracellular signaling pathways for VLDL-induced VLDLR transcription, Western blot analysis was used to examine phosphorylated ERK1/2 protein. It was found that that VLDL induced an increase in ERK1/2 activity in a protein kinase C (PKC)-dependent manner in murine RAW264.7 macrophages. By using different protein kinases inhibitors or activators it was observed that the effect of VLDL-induced VLDL receptor transcription, which is monitored by RTPCR analysis of VLDL receptor mRNA, was not affected by the inhibitor of p38 kinase and cAMP analog, but completely abolished by pretreatment of the cells with PD 98059, an inhibitor of MEK and GF 109203X, an inhibitor of PKC. These results demonstrated that the PKC/ERK1/2 cascade is the essential signaling pathway by which VLDL activates VLDL receptor mRNA expression.
Animals ; CCAAT-Binding Factor ; metabolism ; Cattle ; Cells, Cultured ; Gene Expression Regulation ; Humans ; Lipoproteins, VLDL ; metabolism ; Macrophages ; cytology ; metabolism ; Mitogen-Activated Protein Kinase 1 ; metabolism ; Mitogen-Activated Protein Kinase 3 ; Mitogen-Activated Protein Kinases ; metabolism ; physiology ; Muscle, Smooth, Vascular ; metabolism ; Phosphorylation ; RNA, Messenger ; metabolism ; Rats ; Receptors, LDL ; metabolism ; Signal Transduction ; Transcription Factors ; metabolism ; Transcription, Genetic

9-cis-retinoic acid (9CRA) plays an important role in the immune response; this includes cytokine production and cell migration. We have previously demonstrated that 9CRA increases expression of chemokine receptors CCR1 and CCR2 in human monocytes. To better understand how 9CRA induces CCR1 and CCR2 expression, we examined the contribution of signaling proteins in human monocytic THP-1 cells. The mRNA and surface protein up-regulation of CCR1 and CCR2 in 9CRA-stimulated cells were weakly blocked by the pretreatment of SB202190, a p38 MAPK inhibitor, and PD98059, an upstream ERK inhibitor. Activation of p38 MAPK and ERK1/2 was induced in both a time and dose-dependent manner after 9CRA stimulation. Both p38 MAPK and ERK1/2 phosphorylation peaked at 2 h after a 100 nM 9CRA treatment. 9CRA increased calcium influx and chemotactic activity in response to CCR1-dependent chemokines, Lkn-1/CCL15, MIP-1alpha/CCL3, and RANTES/CCL5, and the CCR2-specific chemokine, MCP-1/CCL2. Both SB202190 and PD98059 pretreatment diminished the increased calcium mobilization and chemotactic ability due to 9CRA. SB202190 inhibited the expression and functional activities of CCR1 and CCR2 more effectively than did PD98059. Therefore, our results demonstrate that 9CRA transduces the signal through p38 MAPK and ERK1/2 for CCR1 and CCR2 up-regulation, and may regulate the pro-inflammatory process through the p38 MAPK and ERK-dependent signaling pathways.
Calcium Signaling/drug effects ; Cell Line ; Chemokines/pharmacology ; Chemotaxis, Leukocyte/drug effects ; Enzyme Activation/drug effects ; Extracellular Signal-Regulated MAP Kinases/*metabolism ; Flavonoids/pharmacology ; Gene Expression Regulation/*drug effects ; Humans ; Imidazoles/pharmacology ; Mitogen-Activated Protein Kinase 1/metabolism ; Mitogen-Activated Protein Kinase 3/metabolism ; Monocytes/drug effects/*enzymology ; Pyridines/pharmacology ; RNA, Messenger/genetics/metabolism ; Receptors, CCR1 ; Receptors, CCR2 ; Receptors, Chemokine/*genetics/metabolism ; Tretinoin/*pharmacology ; p38 Mitogen-Activated Protein Kinases/*metabolism

OBJECTIVETo study the effects of dopamin receptors-2 (DR2) on myocardial ischemic postconditioning and explore its underlying mechanisms.

METHODSThe myocardial ischemic postconditioning (PC) model was established in cultured primary rat neonatal cardiomyocytes which were then randomly assigned in the following groups: Nomial control group, Isehemia/reperfusion (L'R) group, PC (ischemic postconditioning) group, PC + Bro (Bromocriptine, a DB2 antagonist) group, PC + Hal (Haloperidol, a DB2 repressor) and PC + Hal + Bro groups. The lactate dehydrogenase (LDH) and superoxide dismutase (SOD) activity and malondialdehyde (MDA) content in cell medium were analyzed by colorunetry. The cell ultrastructure changes were observed by transmission electron microscope. The cell apoptosis was analyzed using flowcytometiy. The protein expression level of D112 and activity of p-p38 and p-JNK were detected by Western blot.

RESULTSCompared with the nonnal control group, hR increased the protein expression level of DB2, enhanced LDH activity and MDA content, promoted cell injury and apoptosis, decreased SOD activity, up-regulated the activity of p-p38 and p-JNK. Compared with the hR group, although PC further increased the expression of DR2 protein, it decreased LDH activity and MDA content, cell injury and apoptosis, increased SOD activity, down-regulated activity of p-p38 and p-JNK. Bromocriptine treatment further enhanced PC-induced canlioprotective effect, yet Hal addition attenuated this enhancing effect exerted by bromocriptine.

CONCLUSIONThe activation of DB2 is involved in the protective effect of ischemic postconditioning on myocardial ischemia/reperfusion injury through down-regulating the activity of p-p38 and p-JNK.

Animals ; Apoptosis ; Cells, Cultured ; Ischemic Postconditioning ; JNK Mitogen-Activated Protein Kinases ; metabolism ; Myocardial Reperfusion Injury ; prevention & control ; Myocytes, Cardiac ; pathology ; Rats ; Rats, Wistar ; Receptors, Dopamine D2 ; physiology ; p38 Mitogen-Activated Protein Kinases ; metabolism