OBJECTIVETo explore the effects of down-regulated tryptase expression in mast cells on the synthesis and release of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) of vascular endothelial cells.

METHODSTryptase-siRNA (small-interfering RNA) vector was constructed to inhibit tryptase expression in P815 cells. The medium of P815 cells treated by the tryptase-siRNA (RNAi-P815 group) or pure vector (P815 group) was collected and used to culture bEnd.3 cells. The messenger RNAs (mRNAs) of IL-6 and TNF-alpha in bEnd.3 cells and their protein levels in the medium were measured by reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively.

RESULTSIL-6 and TNF-alpha mRNAs in bEnd.3 cells cultured in RNAi-P815-conditioned medium decreased significantly compared to those in P815-conditioned medium. Consistently, IL-6 and TNF-alpha protein levels in the medium of bEnd.3 of RNAi-P815 group were lower than those of P815 group.

CONCLUSIONReduced tryptase expression significantly inhibited the synthesis and release of IL-6 and TNF-alpha in vascular endothelial cells. RNA interference targeting tryptase expression may be a new anti-inflammatory strategy for vascular diseases.

Animals ; Cell Line ; Culture Media, Conditioned ; Down-Regulation ; genetics ; Endothelial Cells ; enzymology ; secretion ; Gene Expression Regulation, Enzymologic ; Interleukin-6 ; genetics ; secretion ; Mice ; RNA, Messenger ; genetics ; RNA, Small Interfering ; genetics ; Transgenes ; Tryptases ; genetics ; metabolism ; Tumor Necrosis Factor-alpha ; genetics ; secretion

In order to explore the new methods of biological treatment of human gliomas, this project is to study the biological properties of gliomas from four different aspects, the results show that there is a IL-6 autocrine loop in human gliomas and the growth of gliomas will be inhibited when the autocrine loop is broken. There is a magnificent predominant expression of Th2 cytokines in human gliomas and human glioma cells, the switching of Th2 to Th1 can inhibit the proliferation of glioma cells. The dosage of 100 micrograms/ml of erythromycin is the best of therapeutic effect. Angiostatin can not only inhibit the vascular endothelial growth, but also have the inhibitory role on the growth of glioma cells in vivo. The above studies have provided some new ideas and will be very helpful for the treatment of glioma patients.
Angiostatins ; pharmacology ; Animals ; Biological Therapy ; Brain Neoplasms ; secretion ; therapy ; Drug Resistance, Neoplasm ; Glioma ; secretion ; therapy ; Humans ; Interleukin-6 ; genetics ; secretion ; RNA, Messenger ; genetics ; secretion ; Th1 Cells ; metabolism ; Th2 Cells ; metabolism

OBJECTIVETo study the effect of a new obesity-related gene NYGGF4 on the insulin sensitivity and secretory function of adipocytes.

METHODS3T3-L1 preadipocytes transfected with either an empty expression vector (pcDNA3.1; control group) or an NYGGF4 expression vector (NYGGF4-pcDNA3.1) were cultured in vitro and differentiated into the matured adipocytes with the standard insulin plus dexamethasone plus 3-isobutyl-methylxanthine (MDI) induction cocktail. 2-deoxy-D-[3H] glucose uptake was determined by liquid scintillation counting. Western blot was performed to detect the protein content and translocation of glucose transporter 4 (GLUT4). The supernatant concentrations of TNF-alpha, IL-6, adiponectin and resistin were measured using ELISA.

RESULTSNYGGF4 over-expression in 3T3-L1 adipocytes reduced insulin-stimulated glucose uptake. NYGGF4 over-expression impaired insulin-stimulated GLUT4 translocation without affecting the total protein content of GLUT4. The concentrations of TNF-alpha, IL-6, adiponectin and resistin in the culture medium of 3T3-L1 transfected with NYGGF4 were not significantly different from those in the control group.

CONCLUSIONSNYGGF4 over-expression impairs the insulin sensitivity of 3T3-L1 adipocytes through decreasing GLUT4 translocation and had no effects on the secretory function of adipocytes.

3T3-L1 Cells ; Adipocytes ; drug effects ; secretion ; Adiponectin ; secretion ; Animals ; Carrier Proteins ; genetics ; physiology ; Glucose ; metabolism ; Glucose Transporter Type 4 ; analysis ; metabolism ; Insulin ; pharmacology ; Interleukin-6 ; secretion ; Mice ; Resistin ; analysis ; Transfection ; Tumor Necrosis Factor-alpha ; secretion

Interleukin (IL)-6 is an autocrine growth factor for mesangial cells. It is not known whether high glucose influences IL-6 production in mesangial cells. Angiotensin II (AGII) is involved in the progression of renal diseases including diabetic nephropathy. Therefore, we evaluated the effects of high glucose in concert with AGII on IL-6 production in human mesangial cells and the modulation by blocking AGII. After 48 hr of culture, IL-6 mRNA expression was analyzed by reverse transcription and polymerase chain reaction (PCR). Quantitative determination of IL-6 concentrations in the culture supernatants of mesangial cells was performed using a sandwich enzyme immunoassay kit. Incubation of mesangial cells with high glucose (450 mg/dL) reduced the ratio of PCR products for IL-6 to beta-actin on densitometric results, while AGII (10(-7)M) increased it. The IL-6 secretion in the supernatant was also increased by AGII and decreased by high glucose. The IL-6 mRNA expression and IL-6 secretion in combination of high glucose and AGII were higher than those in high glucose and similar with those in control media. The addition of losartan (10(-6)M) or captopril (10(-6)M) to high glucose had no additional effects on IL-6 production. These results suggest that whereas AGII increases IL-6 production, high glucose decreases it. The IL-6 production of mesangial cells in diabetic milieu may be complicated and depend on the local effects of high glucose and/or AGII.
Angiotensin II/*pharmacology ; Captopril/pharmacology ; Cells, Cultured ; Gene Expression/drug effects ; Glomerular Mesangium/cytology/*metabolism ; Glucose/*pharmacology ; Humans ; Interleukin-6/*biosynthesis/genetics/secretion ; Losartan/pharmacology

Gaucher disease is caused by a deficiency of glucocerebrosidase. Patients with Gaucher disease are divided into three major phenotypes: chronic nonneuronopathic, acute neuronopathic, and chronic neuronopathic, based on symptoms of the nervous system, the severity of symptoms, and the age of disease onset. The characteristics of patients with acute neuronopathic- and chronic neuronopathic-type Gaucher disease include oculomotor abnormalities, bulbar signs, limb rigidity, seizures and occasional choreoathetoid movements, and neuronal loss. However, the mechanisms leading to the neurodegeneration of this disorder remain unknown. To investigate brain dysfunction in Gaucher disease, we studied the possible role of inflammation in neurodegeneration during development of Gaucher disease in a mouse model. Elevated levels of the proinflammatory cytokines, IL-1alpha, IL-1beta, IL-6, and TNF-alpha, were detected in the fetal brains of Gaucher mice. Moreover, the levels of secreted nitric oxide and reactive oxygen species in the brains of Gaucher mice were higher than in wild-type mice. Thus, accumulated glucocerebroside or glucosylsphingosine, caused by glucocerebrosidase deficiency, may mediate brain inflammation in the Gaucher mouse via the elevation of proinflammatory cytokines, nitric oxide, and reactive oxygen species.
Up-Regulation/genetics ; Tumor Necrosis Factor-alpha/genetics/secretion ; Reverse Transcriptase Polymerase Chain Reaction ; Reactive Oxygen Species/metabolism ; RNA, Messenger/genetics/metabolism ; Nitric Oxide/metabolism ; Microglia/cytology/metabolism ; Mice, Knockout ; Mice, Inbred ICR ; Mice, Inbred C57BL ; Mice ; Interleukin-6/genetics/secretion ; Interleukin-1/genetics/secretion ; Inflammation/immunology ; Glucosylceramidase/genetics ; Gaucher Disease/*genetics/metabolism/pathology ; Cytokines/*genetics/immunology/secretion ; Cells, Cultured ; Brain/embryology/*metabolism/pathology ; Animals

OBJECTIVETo investigate the exact mechanism of SARS-CoV pathogenesis at the protein level.

METHODUnder the condition of the establishment of dendrtic cells (DC) culture method, we used recombinant adenovirus to infect mature DC to make clear the development changes in mRNA levels and secreted protein levels of proinflammatory cytokines of IL-1beta, IL-6, IL-8 and TNF-alpha by using RT-PCR and ELISA.

RESULTWe found that mRNA levels and secreted protein levels of IL-6 and TNF-alpha in DC had increased gradually after rAd-N infection during first 24 h compared with the control DC infected by rAd-LacZ (P < 0.05 or P < 0.01).

CONCLUSIONIt is suggested that N protein may be related to the excessive secretion of proinflammatory cytokines during SARS-CoV infection at the acute phase.

Adenoviridae ; genetics ; immunology ; Animals ; Cercopithecus aethiops ; Cytokines ; genetics ; secretion ; Dendritic Cells ; metabolism ; virology ; Enzyme-Linked Immunosorbent Assay ; Interleukin-6 ; genetics ; metabolism ; RNA, Messenger ; metabolism ; SARS Virus ; genetics ; immunology ; metabolism ; Severe Acute Respiratory Syndrome ; metabolism ; Tumor Necrosis Factor-alpha ; genetics ; metabolism

OBJECTIVETo explore the effect of LPS on the expression of CD14 and the activation of Kupffer cells (KCs).

METHODSRat KCs were isolated and cultured with LPS. Immunohistochemistry and RT-PCR methods were employed to determine the changes in the CD14 expression and the concentration of TNFalpha, IL-6 and NO in the supernatant of the cultured KCs with LPS.

RESULTS(1) The expression of CD14mRNA and the synthesis of CD14 protein in the KCs increased evidently when stimulated by various concentrations of LPS, and the CD14mRNA expression was correlated in dose-dependent manner with LPS levels. (2) The expression of CD14mRNA and the synthesis of CD14 protein in KCs induced by LPS (10 micro g/ml) increased significantly and peaked at 3 approximately 6 hours. (3) The expression of CD14mRNA and the synthesis of CD14 protein in freshly cultured KCs were obviously up-regulated by the active mediators produced by KCs after being stimulated by LPS. (4) The release of TNFalpha, IL-6 and NO from cultured KCs was evidently down-regulated by the addition of anti-CD14McAb in the presence of serum or by the addition of LPS in the absence of serum, but up-regulated by the concomitant addition of LPS and LBP.

CONCLUSION(1) The CD14mRNA expression and the protein synthesis in cultured KCs were closely related to LPS and the active mediators produced from the KCs.The increased CD14 expression was possibly caused by LPS, and the further increase of the expression might be closely correlated to the cytokines released from the KCs. (2) The KC activation by low concentration of LPS was CD14 dependent.

Animals ; Cells, Cultured ; Dose-Response Relationship, Drug ; Gene Expression Regulation ; drug effects ; Interleukin-6 ; secretion ; Kupffer Cells ; cytology ; drug effects ; metabolism ; Lipopolysaccharide Receptors ; genetics ; metabolism ; Lipopolysaccharides ; pharmacology ; Nitric Oxide ; secretion ; RNA, Messenger ; drug effects ; genetics ; metabolism ; Rats ; Reverse Transcriptase Polymerase Chain Reaction ; Tumor Necrosis Factor-alpha ; secretion

Senescence-associated secretory phenotype (SASP) is often a concomitant result of cell senescence, embodied by the enhanced function of secretion. The SASP factors secreted by senescent cells include cytokines, proteases and chemokines, etc, which can exert great influence on local as well as systemic environment and participate in the process of cell senescence, immunoregulation, angiogenesis, cell proliferation and tumor invasion, etc. Relative to the abundance of SASP models in human cells, the in vitro SASP model derived from mouse cells is scarce at present. Therefore, the study aimed to establish a mouse SASP model to facilitate the research in the field. With this objective, we treated the INK4a-deficient mouse NIH-3T3 cells and the wildtype mouse embryonic fibroblasts (MEF) respectively with mitomycin C (MMC), an anticarcinoma drug which could induce DNA damage. The occurring of cell senescence was evaluated by cell morphology, β-gal staining, integration ratio of EdU and Western blot. Quantitative RT-PCR and ELISA were used to detect the expression and secretion of SASP factors, respectively. The results showed that, 8 days after the treatment of NIH-3T3 cells with MMC (1 μg/mL) for 12 h or 24 h, the cells became enlarged and the ratios of β-gal-positive (blue-stained) cells significantly increased, up to 77.4% and 90.4%, respectively. Meanwhile, the expression of P21 protein increased and the integration ratios of EdU significantly decreased (P < 0.01). Quantitative RT-PCR detection showed that the mRNA levels of several SASP genes, including IL-6, TNF-α, IL-1α and IL-1β increased evidently. ELISA detection further observed an enhanced secretion of IL-6 (P < 0.01). On the contrary, although wildtype MEF could also be induced into senescence by MMC treatment for 12 h or 24 h, embodied by the enlarged cell volume, increased ratios of β-gal-positive cells (up to 71.7% and 80.2%, respectively) and enhanced expression of P21 protein, the secretion of IL-6 displayed no significant change. Our study indicated that, although MMC could induce senescence in both mouse NIH-3T3 cells and wildtype MEF, only senescent NIH-3T3 cells displayed the canonical SASP phenomena. Current study suggested that senescent NIH-3T3 cells might be an appropriate in vitro SASP model of mouse cells.
Animals ; Cell Proliferation ; Cellular Senescence ; drug effects ; Cyclin-Dependent Kinase Inhibitor p21 ; genetics ; metabolism ; Cytokines ; genetics ; metabolism ; DNA Damage ; Fibroblasts ; drug effects ; Interleukin-6 ; secretion ; Mice ; Mitomycin ; pharmacology ; NIH 3T3 Cells ; Phenotype

OBJECTIVETo explore the effects of Bushen Shuji Granule (BSG) on inhibiting the interleukin 6 (IL-6) level in the synovial fluid sample of fibroblast cells from the ankylosing spondylitis (AS) patients.

METHODSUsing serum pharmacologic method, the IL-6 level in the culture fluid sample of fibroblast cells was observed by ELISA method with different concentrations of medicated serum containing BSG. The IL-6 level at the mRNA level was detected using reverse transcriptase-polymerase chain reaction (RT-PCR). The vehicle serum and sulfasalazine (SSZ) serum were taken as controls.

RESULTSResults of ELISA showed the IL-6 level in the AS group was more than that in the vehicle serum group with obvious statistical difference. BSG could obviously inhibited the IL-6 level, showing statistical difference when compared with the vehicle serum group. Besides, obvious dose-dependent correlation existed between BSG and its inhibition on fibroblast proliferation. And the IL-6 level at the mRNA level in the AS group was higher than that in the vehicle serum group, showing statistical difference by semi-quantitative analysis.

CONCLUSIONBSG could play its clinical role of anti-inflammation and anti-fibrosis through inhibiting the IL-6 level in the culture fluid sample of fibroblast cells.

Animals ; Cell Line ; Drugs, Chinese Herbal ; pharmacology ; Female ; Fibroblasts ; drug effects ; secretion ; Humans ; Interleukin-6 ; metabolism ; Male ; RNA, Messenger ; genetics ; Rats ; Rats, Sprague-Dawley ; Spondylitis, Ankylosing ; Synovial Fluid ; metabolism ; Synovial Membrane

This study is to explore the effect of ginkgolide B (BN52021) on the production of nitric oxide (NO), interleukin (IL)-6 and regulated upon activation normal T cell expressed and secreted (RANTES) from astrocytes induced by stimulators. Primary cultured rat astrocytes were stimulated with lipopolysaccharides (LPS), the production of NO was assayed using Griess reaction; U251 cells were stimulated with IL-1 beta, the contents of IL-6 and RANTES in the supernatant were measured using ELISA. The mRNA expressions of IL-6 and RANTES were detected using RT-PCR. LPS (10 ng mL(-1) to 10 microg mL(-1)) could stimulate rat astrocytes to produce NO in a dose-dependent manner. Ginkgolide B at the concentrations of 0.1-10 micromol L(-1) were shown to decrease NO production significantly. IL-1 beta could induce the mRNA expression and protein secretion of IL-6 from U251 cells, as well as RANTES. Ginkgolide B at concentrations of 0.1-10 micromol L(-1) were shown to inhibit RANTES secretion, and to inhibit mRNA expression of IL-6 and RANTES at concentration of 10 micromol L(-1). Ginkgolide B has inhibitory effect on the production of NO, IL-6 and RANTES from astrocytes treated with inflammatory stimulators.
Animals ; Astrocytes ; cytology ; metabolism ; Cell Line, Tumor ; Cells, Cultured ; Chemokine CCL5 ; genetics ; metabolism ; Dose-Response Relationship, Drug ; Ginkgolides ; administration & dosage ; pharmacology ; Glioblastoma ; metabolism ; pathology ; Humans ; Interleukin-1beta ; Interleukin-6 ; genetics ; secretion ; Lactones ; administration & dosage ; pharmacology ; Lipopolysaccharides ; Male ; Mice ; Mice, Inbred C57BL ; Nitric Oxide ; metabolism ; Platelet Activating Factor ; antagonists & inhibitors ; RNA, Messenger ; metabolism ; Rats ; Rats, Wistar