OBJECTIVE: To study the pathologic diagnosis and the injury time estimation in light closed encephalon injury. METHODS: Mice were hurt by fluid percussion, and were killed at 15, 30 min, 1, 3 , 6, 12 h, 1, 4, 7, 14 d respectively after injury. The expression of Fas-L in the cerebral cortex, thalamus, and hippocampi was detected by immunohistochemistry and the results were assessed by image analysis system. RESULTS: It is showed that the expression of Fas-L could be detected in 1 h after injury, and increased significantly in three hours, and it reached apex 12 h after injury, and decreased gradually four days after injury, and returned normal 14 days after injury. CONCLUSION This research demonstrated that Fas-L mediated apoptosis appeared not only around brain trauma but also in the brain tissue far away from the traumatic area. It indicted that the expression of Fas-L is a useful target for diagnosis of early brain injury; the regularity of Fas-L expression could be used as one of indication to date the time of brain injury.
Animals ; Apoptosis ; Brain/metabolism* ; Brain Injuries/pathology* ; Fas Ligand Protein ; Image Processing, Computer-Assisted ; Immunohistochemistry ; Male ; Membrane Glycoproteins/biosynthesis* ; Rats ; Rats, Wistar ; Time Factors ; Tumor Necrosis Factors/biosynthesis*

GITRL (Glucocorticoid-induced tumor necrosis factor receptor ligand) has been recently identified as a novel inhibitor of osteoclastogenesis and hence called Osteostat. In this study, we expressed recombinant extracellular domain of GITRL protein in Escherichia coli and analyzed its bioactivity. Using an Eco31I enzyme-based restriction and ligation method, we obtained an E. coli-preferred DNA sequence coding for the extracellular domain of human GITRL. The DNA was cloned into expression vector pQE-30Xa that encodes a fusion tag of 6xHis before the insert. The resultant recombinant expression vector pQE/GITRL was subsequently transformed into E. coli strain M15[pREP4]. After induction with Isopropyl beta-D-Thiogalactoside (IPTG), the cells produced the fusion protein mainly in the form of inclusion bodies as identified by SDS-PAGE. The recombinant protein was purified by affinity chromatography through Ni-NTA column and recognized by anti-His polyclonal antibody using Western blotting analysis. Moreover, we established a simple, efficient and sensitive reporter gene-based method to detect the activity of the recombinant protein. The results showed that the target protein was biologically active.
Base Sequence ; Escherichia coli ; genetics ; metabolism ; Extracellular Space ; metabolism ; Humans ; Molecular Sequence Data ; Recombinant Proteins ; analysis ; biosynthesis ; genetics ; Tumor Necrosis Factors ; biosynthesis ; genetics

OBJECTIVETo compare the expressions of transforming growth factor alpha (TGFalpha), tumor necrosis factor alpha (TNFalpha), and vascular endothelial growth factor (VEGF) between pheochromocytoma (PHEO) tissues and normal adrenal medulla tissues.

METHODSThe mRNA expressions of TGFalpha, TNFalpha, and VEGF detected by RT-PCR, were compared between 22 PHEO tissues and 18 normal adrenal medulla tissues (according with the principle of medical ethnics). Immunohistochemistry staining was performed on 27 PHEO tissues and 14 normal adrenal medulla tissues. The comparisons of the protein expression of TGFalpha, TNFalpha, and VEGF were analyzed in both of PHEO tissues and normal adrenal medulla tissues.

RESULTSCompared with normal adrenal medulla tissues, the expressions of TGFalpha and TNFalpha mRNA and protein were higher in PHEO tissues, and VEGF145 mRNA expression was also higher in PHEO tissues, while there was no significant difference of the mRNA expression of VEGF121 and VEGF165 between these two tissues. Positive staining rates for VEGF of endothelial cells and tumor cells were higher in PHEO tissues than in normal adrenal medulla tissues. Expressions of the TGFalpha, TNFalpha, and VEGF protein were higher in extra-adrenal PHEO than in adrenal PHEO. The TNFalpha immunohistochemistry staining rate was higher in the malignant or multiple PHEO than in the benign or single PHEO.

CONCLUSIONSThe mRNA and protein expressions of TGFalpha, TNFalpha, and VEGF are higher in PHEO tissues than those in normal adrenal medulla tissues. Expressions of these cytokines vary in PHEO with different characteristic.

Adrenal Gland Neoplasms ; metabolism ; Adrenal Medulla ; metabolism ; Humans ; Pheochromocytoma ; metabolism ; RNA, Messenger ; biosynthesis ; genetics ; Transforming Growth Factor alpha ; biosynthesis ; genetics ; Tumor Necrosis Factor-alpha ; biosynthesis ; genetics ; Vascular Endothelial Growth Factors ; biosynthesis ; genetics

OBJECTIVETo investigate the effect of rosiglitazone on the expression of nuclear factor-kappaB (NF-kappaB) and coupling factor 6 (CF6) induced by tumor necrosis factor-alpha (TNF-alpha) in cultured human umbilical vein endothelial cells (HUVEC).

METHODSCultured HUVEC of passage 3-5 were stimulated with TNF-alpha and then cultured in the presence of rosiglitazone. The expression of CF6 and NF-kappaB subunit p65 were evaluated by immunocytochemistical method.

RESULTSPretreatment of HUVECs with rosiglitazone inhibited TNF-alpha-induced expression of CF6 in a dose-dependent manner. The activation of CF6 stimulated by TNF-alpha was suppressed by ROS in a dose-dependent manner.

CONCLUSIONTNF-alpha-induced enhancement of the gene expression and release of CF6 is mediated by activation of NF-kappaB signaling pathway. ROS can inhibit the activation of IKK, block NF-kappaB signaling pathway and inhibit the expression of CF6, which may be the mechanism underlying the action of TZDs on hypertension.

Cells, Cultured ; Endothelial Cells ; cytology ; drug effects ; metabolism ; Humans ; Hypoglycemic Agents ; pharmacology ; Immunohistochemistry ; Mitochondrial Proton-Translocating ATPases ; biosynthesis ; NF-kappa B ; biosynthesis ; Oxidative Phosphorylation Coupling Factors ; biosynthesis ; Thiazolidinediones ; pharmacology ; Tumor Necrosis Factor-alpha ; pharmacology ; Umbilical Veins ; cytology

This study was designed to evaluate the effect of Bazhen decoction on bone marrow depression induced by cyclophosphamide (CY) in mice. An experimental model of mouse bone marrow injury was established through cyclophosphamide induced and the following phenomena were observed. The techniques of culture of hematopoietic progenitor cell and hematopoietic growth factor assay were used. Bazhen decoction could obviously promote the proliferation of bone marrow cells of anaemic mice. The culture media of spleen cell, macrophage, lung and skeletal muscle treated with Bazhen decoction had much stronger stimulating effects on hematopoietic cells. The bone marrow cells of the anaemic mice could yield TNF through Bazhen decoction treatment. It was suggested that Bazhen decoction is clinically a hopeful drug used to cure bone marrow depression and attenuate the side effects of CY.
Anemia ; chemically induced ; drug therapy ; Animals ; Cyclophosphamide ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Hematopoiesis ; drug effects ; Hematopoietic System ; drug effects ; Mice ; Phytotherapy ; Tumor Necrosis Factors ; biosynthesis

OBJECTIVETo investigate the probabilities of core-biding factor a1 (Cbfa1) expression by human skin fibroblasts induced in vitro.

METHODSThe fibroblasts were isolated, purified from human skin, and were grown in incubation in the media of TNF-alpha, BMP-2, and combined TNF-alpha and BMP-2 at certain concentrations, respectively. The changes in biological features of these fibroblasts correlated with osteogenesis were detected by immunohistochemistry and RT-PCR assay.

RESULTSTNF-alpha could switch phenotype of collagen in fibroblasts from Type I and III to Type I and induce fibroblasts to express Ras and BMP type I receptor (BMPR-IA). TNF-alpha in combination with BMP-2 could induce fibroblasts to express Cbfa1 and osteocalcin mRNA.

CONCLUSIONHuman skin fibroblast could be induced into pro-osteoblast expressing Cbfa1, an osteoblast-specific transcription factor and a regulation of osteoblast differentiation, and combined use of TNF-alpha and BMP-2 was one of the regulating factors.

Bone Morphogenetic Protein 2 ; Bone Morphogenetic Protein Receptors, Type I ; Bone Morphogenetic Proteins ; pharmacology ; Cells, Cultured ; Collagen ; biosynthesis ; Core Binding Factor Alpha 1 Subunit ; Core Binding Factors ; Fibroblasts ; metabolism ; Humans ; Neoplasm Proteins ; Osteocalcin ; biosynthesis ; Protein-Serine-Threonine Kinases ; biosynthesis ; RNA, Messenger ; analysis ; Receptors, Growth Factor ; biosynthesis ; Skin ; cytology ; Transcription Factors ; biosynthesis ; genetics ; Transforming Growth Factor beta ; Tumor Necrosis Factor-alpha ; pharmacology

Cytokines and growth factors are important regulatory proteins controlling the growth and differentiation of normal and malignant glial cells. In this study, we investigated the expression and origin of tumor necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta 1 (TGF-beta 1) in the subacute brain injury after a single high-dose irradiation using 60 Sprague-Dawley rats. The right cerebral hemispheres of rats were exposed to a single 10 Gy dose of gamma rays using Ir-192. The radiation effect was assessed at 1 week, 2 weeks, 4 weeks, 6 weeks, and 8 weeks after irradiation, and the results were compared with those in sham operation group. Histological changes characteristic of radiation injury were correlated with the duration after the single dose irradiation. The loss of cortical thickness also increased with the lapse of time after irradiation. The TNF-alpha expression in the irradiated cerebral hemispheres was significantly increased compared with that in the sham operation group. TGF-beta 1 expression was also increased in the irradiated hemispheres. Immunohistochemical study revealed that TGF-beta 1 was expressed predominantly by infiltrating macrophages and astrocytes around the necrotic areas. These findings indicate that TNF-alpha and TGF-beta 1 may play prominent roles in the radiation injuries after a single high-dose irradiation.
Animals ; Brain/immunology/pathology/*radiation effects ; Dose-Response Relationship, Radiation ; Immunohistochemistry/methods ; Rats ; Rats, Sprague-Dawley ; Time Factors ; Transforming Growth Factor beta/*biosynthesis ; Transforming Growth Factor beta1 ; Tumor Necrosis Factor-alpha/*biosynthesis

AIMTo explore the relations between anti-apoptotic role of dipfluzine (DIP) and the death signaling transduction pathway initiated by CD95 molecules, and the transcription factor involved in the transcription regulation of CD95 molecules in the hippocampal CA1 region after transient forebrain ischemia.

METHODSThe rat forebrain transient ischemia model was established through 15 min ischemia followed by 3 days reperfusion by using the four-vessel method. The rats were divided randomly into five groups: sham control group, ischemia/reperfusion (I/R) group, DIP treated groups (20, 40 and 80 mg x kg(-1) body weight, ig, separately). Western blotting and RT-PCR were performed to detect the expression changes of Fas, FasL, caspase 10 p20, caspase 8, I-kappaB-alpha, and p-I-kappaB-alpha molecules in protein and mRNA levels, separately, and immunohistochemistry for molecular localization of Fas and FasL in rat hippocampus.

RESULTSThe expression of Fas, FasL, and caspase 10 p20 in protein and mRNA levels increased after I/R, which was inhibited significantly after treatment with 20 and 40 mg x kg(-1) of DIP (P < 0.01). In 80 mg x kg(-1) of DIP group, the expression of Fas and FasL protein was not significantly different from that of I/R group (P > 0.05). The expression of caspase 8 and I-kappaB-alpha showed no significant differences in all groups (P > 0.05), and no gene expression was observed for p-I-kappaB-alpha protein in the study. DIP significantly affected molecular distribution of Fas and FasL protein in CA1 subregion of hippocampus.

CONCLUSIONDIP inhibits the death signaling transduction pathway initiated by CD95 molecules in rat hippocampal CA1 subregion, and NF-kappaB transcription factor may not be involved in the transcription regulation of CD95 molecules after transient forebrain ischemia.

Animals ; Apoptosis ; drug effects ; Brain Ischemia ; metabolism ; pathology ; Calcium Channel Blockers ; pharmacology ; Cinnarizine ; analogs & derivatives ; pharmacology ; Fas Ligand Protein ; Female ; Hippocampus ; metabolism ; Membrane Glycoproteins ; biosynthesis ; genetics ; NF-kappa B ; metabolism ; Neurons ; metabolism ; RNA, Messenger ; biosynthesis ; genetics ; Rats ; Rats, Sprague-Dawley ; Reperfusion Injury ; metabolism ; pathology ; Signal Transduction ; Tumor Necrosis Factors ; biosynthesis ; genetics ; fas Receptor ; biosynthesis ; genetics

Tumor necrosis factor receptor-related 2 (TR2, HVEM or TNFRSF-14) plays an important role in immune responses, however, the mechanisms regulating its expression are unclear. To understand the control of TR2 gene expression, we studied the upstream region of the gene. Gel supershift assays revealed inducible binding of nuclear factor of activated T cells (NFAT) to a putative NFAT site within the TR2 promoter. Furthermore, cotransfection of a dominant negative NFAT construct, or siRNA for NFAT, resulted in increased expression of a TR2 reporter gene. Our findings demonstrate that NFAT negatively regulates TR2 expression in activated T cells.
Animals ; Base Sequence ; CD4-Positive T-Lymphocytes/metabolism ; Cells, Cultured ; Down-Regulation ; Mice ; Mice, Inbred C57BL ; Molecular Sequence Data ; NFATC Transcription Factors/*physiology ; Receptors, Tumor Necrosis Factor, Member 14/*biosynthesis ; T-Lymphocytes/*metabolism

OBJECTIVETo study the correlation between the expression of Egr-1 and NF-kappaB and the up-regulation of TNF-alpha and TGF-beta1 in macrophages after stimulation by silica in-vitro.

METHODSMacrophages were treated with antibodies against Egr-1 and NF-kappaB and antisense oligonucleotides. The level of TNF-alpha protein in the cell supernatant was then measured using enzyme-linked immunoadsorbent assay (ELISA). The expression of TGF-beta1 protein was detected by immunocytochemistry. The expression of TNF-alpha and TGF-beta1 mRNAs was also monitored by reverse transcriptase-polymerase chain reaction (RT-PCR).

RESULTSCompared with silica-stimulated macrophages untreated with antibodies, the cells treated with 10 micro g/ml of Egr-1 or NF-kappaB antibodies were associated with reduced expression of TNF-alpha and TGF-beta1 proteins and mRNAs (P < 0.05). Compared with silica-stimulated untransfected group, the antisense group was associated with obvious reduction in the expression of TNF-alpha and TGF-beta1 proteins and mRNAs (P < 0.05).

CONCLUSIONThe expression of TNF-alpha and TGF-beta1 mRNAs and proteins are associated with activation of Egr-1 and NF-kappaB in macrophages, after stimulation by silica. It is possible that the corresponding antibodies and antisense oligonucleotides may become a potential therapeutic tool in the management of silicosis in the future.

Animals ; Antibodies ; immunology ; Cells, Cultured ; DNA-Binding Proteins ; genetics ; immunology ; Early Growth Response Protein 1 ; Immediate-Early Proteins ; genetics ; immunology ; Macrophages ; cytology ; metabolism ; Mice ; NF-kappa B ; genetics ; immunology ; Oligonucleotides, Antisense ; pharmacology ; RNA, Messenger ; biosynthesis ; genetics ; Silicon Dioxide ; pharmacology ; Silicosis ; etiology ; Transcription Factors ; genetics ; immunology ; Transforming Growth Factor beta ; biosynthesis ; genetics ; Transforming Growth Factor beta1 ; Tumor Necrosis Factor-alpha ; biosynthesis ; genetics