OBJECTIVETo study the possible induction effect of exposure to 50 Hz magnetic field (MF) on clustering of cell membrane surface receptors for epidermal growth factor (EGF) and tumor necrosis factor (TNF), the starting site of signals of biological effects, and its possible intervention effect.

METHODSLung fibroblasts of Chinese hamster (CHL) were exposed to EGF, TNF, 0.4 mT 50 Hz MF, 0.4 mT noise MF, and 0.4 mT 50 Hz MF combined with 0.4 mT noise MF. Respectively, for different durations, following the treatment, EGF and TNF receptors on the cell membrane were marked by corresponding antibodies with immunohistochemical method, then observed under a confocal microscope.

RESULTSClustering of cell membrane receptors could be induced 5 min after treatment with EGF and TNF, as well as with 50 Hz MF at 0.4 mT, which reached the peak in 15 min. While noise MF with the same intensity did not induce clustering of cell membrane receptors. Superposition of noise MF with the same intensity could inhibit clustering of cell membrane receptors induced by 50 Hz MF.

CONCLUSIONClustering of EGF and TNF receptors on the cell membrane could be induced by 50 Hz MF, suggesting that membrane receptors would be one of the sites where MF signals coupled, and noise MF with the same intensity could inhibit these effects.

Animals ; Cell Line ; Cricetinae ; Electromagnetic Fields ; adverse effects ; Epidermal Growth Factor ; pharmacology ; Fibroblasts ; drug effects ; metabolism ; radiation effects ; Noise ; adverse effects ; Receptor, Epidermal Growth Factor ; metabolism ; Receptors, Cell Surface ; metabolism ; Receptors, Tumor Necrosis Factor ; metabolism ; Tumor Necrosis Factor-alpha ; pharmacology

To study the expression of the bFGF and its receptor in the mouse bone marrow by treatment with acute radioactive injury and Ligustrazine, 56 mice were divided into 3 groups: normal group, radiation-injured group and Ligustrazine group. After irradiation by 6.0 Gy 60Co gamma-ray, each mouse was orally given 0.1 ml Ligustrazine twice a day for 13 days in Ligustrazine group, and each mouse in radiation injured group was orally given equal amount of saline. On the 3rd, 7th, 14th day after irradiation, bone marrow mono-nuclear cells (BMMNC) were counted, and the expression levels of bPGF and bFGFR in bone marrow were evaluated by immunohistochemistry and flow cytometry analysis respectively. On the 3rd, 7th, 14th day after irradiation, expression of bFGF in bone marrow were significantly lower than in normal group (P<0.05 or P<0.01). Expressions of bFGF and bFGFR were much higher in Ligustrazine treated group than that in the control group (P<0.05 or P<0.01). Ligustrazine potentiate the expression of bFGF and bFGFR in bone marrow MNC to recover the bone marrow hematopoiesis inductive microenvironment, which is one of the mechanisms by which Ligustrazine rebuild the bone marrow hematopoiesis after acute radioactive injury.
Bone Marrow Cells/metabolism ; Fibroblast Growth Factor 2/*biosynthesis ; Hematopoiesis/drug effects ; Pyrazines/*pharmacology ; Radiation Injuries, Experimental/*metabolism ; Radiation-Protective Agents/pharmacology ; Receptors, Fibroblast Growth Factor/*biosynthesis

In the kidney, pericyte is the major source of myofibroblast (MyoF) in renal interstitium. It is reported that pericyte-myofibroblast transition（PMT）is one of the important pathomechanisms of renal interstitial fibrosis（RIF）. Among them, the main reasons for promoting RIF formation include pericyte recruitment, activation and isolation, as well as the lack of pericyte-derived erythropoietin. During the PMT startup process, pericyte activation and its separation from microvessels are controlled by multiple signal transduction pathways, such as transforming growth factor-β（TGF-β）pathway, vascular endothelial growth factor receptor (VEGFR) pathway and platelet derived growth factor receptor (PDGFR) pathway;Blocking of these signaling pathways can not only inhibit PMT, but also suppress renal capillaries reduction and further alleviate RIF. In clinic, many traditional Chinese medicine compound prescriptions, single traditional Chinese herbal medicine (CHM) and their extracts have the clear effects in alleviating RIF, and some of their intervention actions may be related to pericyte and its PMT. Therefore, the studies on PMT and its drug intervention will become the main development direction in the research field of anti-organ fibrosis by CHM.
Drugs, Chinese Herbal ; pharmacology ; Fibrosis ; Humans ; Kidney ; cytology ; drug effects ; pathology ; Myofibroblasts ; cytology ; Pericytes ; cytology ; Receptors, Platelet-Derived Growth Factor ; metabolism ; Signal Transduction ; Vascular Endothelial Growth Factor A ; metabolism

OBJECTIVETo study the effect of MNNG on inducement of non-targeted mutation and activation of several cellular signal transduction pathways, and to determine whether the activation of these signaling pathways was dependent on the DNA-damage.

METHODSVero cells were enucleated by discontinuous density centrifugation. The PKA activities were measured by enzyme-linked immunosorbent assay. The status of cell membrane receptors was studied with immunofluorescent staining and confocal microscopy.

RESULTIn enucleated cytoplasts, MNNG-treatment increased PKA activity for about 2.3-fold in accordance with the 2.7-fold up-regulation of PKA activity in whole vero cells exposed to MNNG. The clustering of cell surface receptors of epidermal growth factor and tumor necrosis factor alpha was also observed in cells exposed to MNNG; this phenomenon was also found in enucleated cells.

CONCLUSIONThe results indicate that the initiation of signal cascades induced by low concentration of MNNG might be associated with its interaction with cell surface receptors and/or direct activation of related signal proteins but not its DNA damage.

Animals ; Cell Nucleus ; physiology ; Cercopithecus aethiops ; Cyclic AMP-Dependent Protein Kinases ; metabolism ; DNA Damage ; Enzyme Activation ; drug effects ; Methylnitronitrosoguanidine ; toxicity ; Receptor, Epidermal Growth Factor ; drug effects ; metabolism ; Receptors, Tumor Necrosis Factor ; drug effects ; metabolism ; Signal Transduction ; drug effects ; Vero Cells

OBJECTIVEThis study was carried out to investigate the effects of insulin-like growth factor-II (IGF-II) and basic fibroblast growth factor (bFGF) on osteoprotegerin (OPG) secretion of periodontal ligament cells (PDLCs).

METHODSHealthy human premolars extracted for orthodontic reasons from 12-14 years old donators were obtained, and periodontal tissues were collected and cultured to obtain PDL cells. Primary or first passage PDLCs were cloned by means of limited dilutions. PDLCs with osteoblastic phenotypes were characterized as follows: Alkaline phosphatase activity, collagen III production and bone-like nodules formation. IGF-II and bFGF were added into culture media and their effects on PDLCs proliferation and OPG secretion were observed. The OPG concentrations in cell culture supernatants were detected by sandwich ELISA. Living cell numbers were demonstrated by MTT test. The average levels of OPG secretion by a single cell were calculated by dividing OPG concentration with MTT-test result.

RESULTSBoth IGF-II and bFGF upregulated the mtt values (P < 0.05), but ICF-II downregulated the opg/mtt values (P < 0.05), whereas bFGF had no significant effect on opg/mtt values (P > 0.05).

CONCLUSIONIGF-II enhances the proliferation of PDL cells but prohibits OPG secretion. Although bFGF has the same effect on the proliferation of PDL cells, it has no effect on OPG secretion. Before cytokines were used to enhance periodontal regeneration, their effects on local bone balance should also be studied.

Adolescent ; Cells, Cultured ; Child ; Fibroblast Growth Factor 2 ; pharmacology ; Glycoproteins ; biosynthesis ; Humans ; Insulin-Like Growth Factor II ; pharmacology ; Osteoprotegerin ; Periodontal Ligament ; cytology ; drug effects ; metabolism ; Receptors, Cytoplasmic and Nuclear ; biosynthesis ; Receptors, Tumor Necrosis Factor ; biosynthesis

The vascular endothelial growth factor (VEGF) family of soluble protein growth factors consists of key mediators of angiogenesis and lymphangiogenesis in the context of tumor biology. The members of the family, VEGF-A (also known as VEGF), VEGF-B, VEGF-C, VEGF-D, and placenta growth factor (PlGF), play important roles in vascular biology in both normal physiology and pathology. The generation of a humanized neutralizing antibody to VEGF-A (bevacizumab, also known as Avastin) and the demonstration of its benefit in numerous human cancers have confirmed the merit of an anti-angiogenesis approach to cancer treatment and have validated the VEGF-A signaling pathway as a therapeutic target. Other members of the VEGF family are now being targeted, and their relevance to human cancer and the development of resistance to anti-VEGF-A treatment are being evaluated in the clinic. Here, we discuss the potential of targeting VEGF family members in the diagnosis and treatment of cancer.
Angiogenesis Inhibitors ; therapeutic use ; Animals ; Antibodies, Monoclonal, Humanized ; therapeutic use ; Bevacizumab ; Humans ; Lymphangiogenesis ; Neoplasms ; drug therapy ; metabolism ; Neovascularization, Pathologic ; metabolism ; Placenta Growth Factor ; Pregnancy Proteins ; metabolism ; Receptors, Vascular Endothelial Growth Factor ; metabolism ; Signal Transduction ; drug effects ; Vascular Endothelial Growth Factor A ; classification ; metabolism ; Vascular Endothelial Growth Factor B ; metabolism ; Vascular Endothelial Growth Factor C ; metabolism ; Vascular Endothelial Growth Factor D ; metabolism

Impaired tumor necrosis factor receptor-1 (TNFR-1) signaling has been found in some malignant tumors with poor prognosis. However, the exact role of TNFR-1 signaling in fibrosarcoma remains unclear. Here, we explored the question by comparing the growth of TNFR-1 deficient (Tnfr1 (-)) and TNFR-1 competent (Tnfr1 (+)) fibrosarcoma FB61 cells (FB61-m and FB61-R1) in mice. TNFR-1 expression on fibrosarcoma cells delayed their growth in vivo but not in vitro. Moreover, reduced FB61-R1 tumor growth was also obtained in TNFR-1 knockout mice. The mechanism relies mainly on the TNFR-1-mediated downregulation of vascular endothelial growth factor (VEGF) production by tumor cells. Importantly, treatment of FB61-m tumors with melphalan resulted in a short delay of tumor growth, followed by a quick remission. However, when FB61-R1 tumors were treated with melphalan, tumor growth was similarly delayed at first and then completely rejected. Our results reveal evidence for TNFR-1 on tumor cells as a prerequisite in chemotherapy for fibrosarcoma, and provide novel insight into the therapeutic approach against some types of tumors using TNFR-1 angonist.
Animals ; Down-Regulation ; drug effects ; Fibrosarcoma ; drug therapy ; genetics ; pathology ; Gene Expression Regulation, Neoplastic ; drug effects ; Humans ; Melphalan ; administration & dosage ; Mice ; Molecular Targeted Therapy ; Receptors, Tumor Necrosis Factor, Type I ; genetics ; Signal Transduction ; drug effects ; Vascular Endothelial Growth Factor A ; biosynthesis

OBJECTIVETo study the mechanism underlying the inhibitory effects of peroxisome proliferator-activated receptor γ (PPARγ) agonists on transforming growth factor β1 (TGF-β(1))-induced scarring of skin.

METHODSFibroblasts isolated from healthy adult skin were cultured in vitro and divided into blank control group (serum-free DMEM culture), TGF-β(1) group (with stimulation of 10 ng/mL TGF-β(1) for 48 hours), troglitazone group (with the same treatment as in TGF-β(1) group after stimulation of 10 µmol/L troglitazone for 2 hours), and 15-dioxygen prostaglandin J2 (15d-PGJ2) group (with the same treatment as in TGF-β(1) group after stimulation of 10 µmol/L 15d-PGJ2 for 2 hours) according to the stimulation added into DMEM. The expression of connective tissue growth factor (CTGF) was determined with Western blot. The mRNA levels of CTGF, matrix metalloproteinase-1 (MMP-1) and platelet-derived growth factor (PDGF) were determined with real-time fluorescence RT-PCR. Data were processed with one-way analysis of variance.

RESULTSThe expression of CTGF at mRNA and protein levels in skin fibroblasts were significantly increased in TGF-β(1) group as compared with control group; while expression of CTGF at mRNA and protein levels in 15d-PGJ2 and troglitazone groups were significantly decreased as compared with that in TGF-β(1) group. The mRNA level of MMP-1 in TGF-β(1) group (0.193 ± 0.051) was obviously lower than that in blank control group (1.281 ± 0.195, F = 12.811, P < 0.01), while the mRNA levels of MMP-1 in troglitazone group (0.417 ± 0.043) and 15d-PGJ2 group (0.485 ± 0.027) were significantly increased as compared with that in TGF-β(1) group (F = 12.811, P values all below 0.01). The mRNA level of PDGF in TGF-β(1) group (1.044 ± 0.237) was obviously higher than that in control group (0.349 ± 0.057, F = 16.848, P < 0.01), while the levels in troglitazone group (0.677 ± 0.055) and 15d-PGJ2 group (0.511 ± 0.017) were significantly decreased as compared with that in TGF-β(1) group (F = 16.848, P values all below 0.01).

CONCLUSIONSThe inhibitory effect of activated PPARγ on the expression of CTGF induced by TGF-β(1) may be the main mechanism of its inhibitory effect on TGF-β(1)-induced scarring on skin, and its influence on MMP-1 and PDGF may also be one of the underlying mechanisms.

Cell Line ; Connective Tissue Growth Factor ; metabolism ; Fibroblasts ; drug effects ; metabolism ; Humans ; Matrix Metalloproteinase 1 ; metabolism ; PPAR gamma ; agonists ; Receptors, Platelet-Derived Growth Factor ; metabolism ; Signal Transduction ; Transforming Growth Factor beta1 ; metabolism

OBJECTIVELipoxin A(4) is formed by the metabolism of arachidonic acid. Anti-inflammatory and anti-proliferative effect of lipoxin A(4) has been shown in many human diseases. Recently, as a novel high affinity receptor for ligand lipoxin A(4), Lipoxin A(4) receptor-like protein (LRLP) has been identified. Currently close attention is paid to the important contribution of connective tissue growth factor (CTGF) in lung fibrosis. The purpose of the study was to transfect LRLP gene into human lung fibroblasts and investigate the mechanism of its enhancing antagonistic effect of Lipoxin A(4) on human lung fibroblasts proliferation induced by connective tissue growth factor.

METHODSEukaryocytic expression vector pEGFP/LRLP which contained LRLP and green fluorescence protein fusion gene (GFP) was constructed and transfected into human lung fibroblasts (HLF). After selecting with G418, HLF/LRLP cell clone which stably expressed LRLP/GFP fusion protein was isolated and characterized by the laser scanning confocal microscope. Cultured HLF and HLF/LRLP were stimulated for 24 h with CTGF (1 microg/ml) in the presence and absence of pretreatment of Lipoxin A(4) (10.0 nmol/L) for 30 min. Inhibition of cell proliferation was determined by MTT assay. Cell cycle analysis was performed by flow cytometry. Western blot was used to detect the expression of cyclin D(1) protein. Electrophoretic mobility shift assay (EMSA) was employed to detect the DNA binding activity of STAT(3).

RESULTS(1) HLF/LRLP cell clone which stably expressed LRLP and GFP fusion protein was successfully obtained. (2) Proliferation of HLF and HLF/LRLP was induced by 1 microg/ml CTGF. Pretreatment with 10 nm Lipoxin A(4) inhibited the proliferation of HLF and HLF/LRLP. And the inhibitory rate of HLF/LRLP was significantly higher than that of HLF [(54.1 +/- 4.2)%, (21.2 +/- 3.7)%, P < 0.05]. (3) The flow cytometry analysis showed that compared with HLF, more HLF/LRLP were arrested at G(0)/G(1) phase in the presence of pretreatment of Lipoxin A(4). [(76.3 +/- 3.5)%, (60.8 +/- 2.0)%, P < 0.05]. (4) Ten nmol/L Lipoxin A(4) antagonized CTGF induced increase of cyclin D(1) protein expression in HLF and HLF/LRLP. And its antagonistic effect on HLR/LRLP was stronger than that on HLF (P < 0.05). (5) Ten nmol/L Lipoxin A(4) antagonized CTGF induced increase of STAT(3) DNA binding activity, and its antagonistic effect on HLF/LRLP was more powerful than that on HLF (P < 0.05).

CONCLUSIONSTransfection of Lipoxin A(4) receptor-like protein gene enhanced the inhibitory effect of Lipoxin A(4) on human lung fibroblasts proliferation induced by CTGF. Its mechanism might be related to regulation of cyclin D(1) protein expression and STAT(3) DNA binding activity.

Connective Tissue Growth Factor ; antagonists & inhibitors ; Cyclin D1 ; analysis ; DNA ; metabolism ; Fibroblasts ; cytology ; drug effects ; Humans ; Lipoxins ; pharmacology ; Lung ; cytology ; drug effects ; Receptors, Formyl Peptide ; genetics ; physiology ; Receptors, Lipoxin ; genetics ; physiology ; STAT3 Transcription Factor ; metabolism ; Transfection

Neurotrophins protect neurons against excitotoxicity; however the signaling mechanisms for this protection remain to be fully elucidated. Here we report that activation of the phosphatidyl inositol 3 kinase (PI3K)/Akt pathway is critical for protection of hippocampal cells from staurosporine (STS) induced apoptosis, characterized by nuclear condensation and activation of the caspase cascade. Both nerve growth factor (NGF) and brain-derived growth factor (BDNF) prevent STS-induced apoptotic morphology and caspase-3 activity by upregulating phosphorylation of the tropomyosin receptor kinase (Trk) receptor. Inhibition of Trk receptor by K252a altered the neuroprotective effect of both NGF and BDNF whereas inhibition of the p75 neurotrophin receptor (p75NTR) had no effect. Impairment of the PI3K/Akt pathway or overexpression of dominant negative (DN)-Akt abolished the protective effect of both neurotrophins, while active Akt prevented cell death. Moreover, knockdown of Akt by si-RNA was able to block the survival effect of both NGF and BDNF. Thus, the survival action of NGF and BDNF against STS-induced neurotoxicity was mediated by the activation of PI3K/Akt signaling through the Trk receptor.
Animals ; Apoptosis/*drug effects ; Brain-Derived Neurotrophic Factor/*metabolism ; Cell Line ; Cell Survival/drug effects ; Cytoprotection/*drug effects ; Gene Knockdown Techniques ; Hippocampus/*cytology ; Nerve Growth Factor/*metabolism ; Neurons/*cytology/drug effects/metabolism ; PC12 Cells ; Proto-Oncogene Proteins c-akt/metabolism ; Rats ; Receptors, Nerve Growth Factor/metabolism ; Signal Transduction/drug effects ; Staurosporine/*pharmacology