Inhibitory Effect of Oxymatrine on Quartz-induced Secretion of TNF-α by the Pulmonary Alveolar Macrophages in the Fibroblast Proliferation

The massive proliferation of pulmonary fibroblasts is the common consequence shared by lung diseases. The over-expression of TNF-α plays an important role in the proliferation of pulmonary fibroblasts[1, 2]. Under patho-logical conditions, TNF-α is predominantly secreted by activated alveolar macrophages of lung. The high level of TNF-α acts on the receptors on the cell membrane of fibroblasts, mediates the conduction of intracellular sig-nals and eventually activates the nuclear factor…     

Effect of IL-Iβ and TNF-α on the expression of monocyte chemotactic protein-1 in endometriotic cells

Summary To investigate the clinical significance of monocyte chemotactic protein-1 (MCP-1) produced by endometriotic tissues, the endometriotic tissues were taken from 15 patients with endometriosis. MCP-1 mRNA and MCP-1 protein were determined by dot blot analysis and enzyme linked immunosorbent assay (ELISA) in endometriotic cells cultured with or without interleukin-1β (IL-lβ, 2 μg/L), tumor necrosis factor-α (TNF-α, 20 g/L). After exposure to IL-1β or TNF-α, the expression of MCP-1 mRNA in the endometriotic cells (8.635 ±0.826, 7.031 ±0.970, respectively) were significantly higher than that in the control group (4.482±0.435, P<0.05); The expression of MCP-1 protein in IL-lβ and TNF-α group was 4.52±0.09 μg/L,2.87±0.27 μg/L, respectively, which were significantly higher than 1.74±0.16 μg/L in control (P<0.01). The results suggested that IL-l\ and TNF-α could up-regulate the expression of MCP-1 in endometriotic cells, which might be related to the development of endometriosis.     

Antagonistic effects of trasilast on proliferation and collagen synthesis induced by TGF-\sB<Subscript>2</Subscript> in cultured human trabecular meshwork cellsin cultured human trabecular meshwork cells

Summary Whether tranilast had antagonistic effect on proliferation inhibition and collagen synthesis promotion induced by TGF-\sB₂ in cultured human trabecular meshwork cells was investigated. Suspension of 1×10⁴ cultured human trabecular meshwork cells of 3–5 passage was distributed in each well of a 96-well disk and divided into control group and experimental group. After 24 h, 0 μg/ml (control), 12.5 μg/ml, 25 μg/ml, 50μg/ml tranilast with 3.2 ng/ml TGF-\sB₂ were added into the incubation medium. Another 24 h later, proliferation and collagen synthesis in cultured human trabecular meshwork cells were examined respectively by using tetrazolium-based semiautomated colormetric (MTT) assay and³H-proline incorporation with liquid scintillation technique. The results showed absorbance (A) values of the experimental groups were 0.9036±0.3017, 1.1361±0.1352, 1.2457±0.1524 according to the different concentrations of tranilast, and 0.8956±0.1903 of the control group. In comparison with the control group, 25 μg/ml (q=3.23,P<0.05), 50 μg/ml (q′=4.70,P<0.01) tranilast significantly antagonized the decrease of theA values induced by TGF-\sB₂ in the cultured human trabecular meshowrk cells. In comparison with the control group [817.37±124.21 cpm/10⁴ cells], 12.5 μg/ml (620.33±80.46 cpm/10⁴ cells,q′=4.26,P<0.05), 25 μg/ml (59.4.58±88.13 cpm/10⁴ cells,q′=4.81,P<0.01), 50 μg/ml (418.64±67.90 cpm/10⁴ cells,q′=8.62,P<0.01) tranilast significantly inhibited the incorporation of³H-proline into the cultured human trabecular meshwork cells promoted by TGF-\sB₂ in a dose-dependent manner. It was concluded that tranilast had the antagonistic effect on the proliferation inhibition and collagen synthesis promotion induced by TGF-\sB₂ in the cultured human trabecular meshwork cells. Da Banghong, female, born in 1973, Resident. This project was supported by a grant from the National Natural Sciences Foundation of China (No. 38970758).     

Effects of propofol on the mRNA expression of toll-like receptor 4 in BV-2 cells during mimic ischemia-reperfusion injury <Emphasis Type="Italic">in vitro</Emphasis>

This study investigated the effects of propofol on the mRNA expression of Toll-like receptor-4 （TLR4） in BV-2 cells during mimic ischemia-reperfusion （I/R） injury in vitro. BV-2 cells, a mouse microglia line, were cultured and divided into 4 groups at random： control group （group C）, ischemia/reperfusion group （group I/R）, low-dose propofol （25 μmol/L） intervention group （group PF25） and high-dose propofol （100 μmol/L） intervention group （group PF100）. The mRNA expression of TLR4 and NF-κB was measured by means of RT-PCR. TNF-α levels in the supernatants of BV-2 cells were detected by ELISA. The results showed that the mRNA expression of TLR4 and NF-κB was significantly higher in groups I/R, PF25 and PF100 than in group C （P〈0.01）. And the TNF-α level in the supernatants was elevated in groups I/R, PF25 and PF100 as compared with that in group C （P〈0.01）. After pre-treatment with propofol, the mRNA expressions of TLR4 and NF-κB and the TNF-α level were significantly decreased in groups PF25 and PF100 in comparison to those in group I/R （P〈0.01）. And the decrease in those indicators was more significant in group PF100 than in group PF25 （P〈0.01）. It was concluded that propofol exerted brain-protecting effects during I/R injury by suppressing the mRNA expressions of TLR4 and NF-κB and deceasing the TNF-α level.     

Effect of Jianpi Huoxue decoction (健脾活血方)-containing serum on tumor necrosis factor-α secretion and gene Expression of endotoxin receptors in RAW264.7 cells induced by lipopolysaccharide

Objective:To evaluate the effect of Jianpi Huoxue decoction(健脾活血方,JHD)-containing serum on tumor necrosis factor-α(TNF-α) secretion and endotoxin receptor gene expression in RAW264.7 cells induced by lipopolysaccharide(LPS).Methods:The cytotoxicity of blank-control serum and JHD-containing serum at different concentrations were evaluated through the lactate dehydrogenase(LDH) assay in RAW264.7 cells.RAW264.7 cells were divided into six groups:5%blank-control serum group(C1,n=3),5%blank-control serum plus...     

Effect of Naotaifang extract on changes of coagulation in human umbilical cord vein endothelial cell and fibrinolysis function induced by recombinant human Tumor Necrosis Factor-α

Objective: To observe the changes of coagulation and fibrinolysis function in human umbilical cord vein endothelial cells (HUVEC) induced by recombinant human tumor necrosis factor-α (rhTNF-α) and the effect of Naotaifang extract (NTE) on it.Methods: Cultured HUVEC is randomly divided into six groups: Control group, NTE control group (only 2 g/L NTE), rhTNF-α group (100 μg/ L rhTNF-α), and low-dosage, middle-dosage and high-dosage NTE group (100 μg/L rhTNF-α and 0. 67 g/L, 2 g/L, 6 g/L NTE). The coagulation activity of frozen-dissolved HUVEC, von Willebrand factor (vWF) content in the conditioned medium and tissue-type plasminogen activator (tPA) and plasminogen activator inhibitor (PAD activity were to be detected after 24 hrs.Results: Compared with the control group, PAI activity were enhanced, vWF release markedly increased in conditioned medium of TNF-α group (P < 0.01) and the frozen-dissolved HUVEC markedly shortens the rabbit plasma prothrombin time, and the above changes could be significantly inhibited by the 3 dosages of NTE (P < 0.05,P < 0. 01).Conclusion: NTE is effective in inhibiting the coagulation activity of the HUVEC non-stimulated or stimulated by rhTNF-α to enhance the vWF release, and to adjust fibrinolytic function, and mainly to inhibit the PAI activity. The item is supported by Natural Science Foundation of Hunan Province (No. 98JJY 2027) and Young-middle Aged Science and Technological Fund of Hunan Province (NO. 00JZYZ145)     

Cytokine-induced cell surface expression of adhesion molecules in vascular endothelial cellsIn vitro

Summary  Regulation of the adhesion molecules expression by cytokine in vascular endothelial cells was investigated. Human umbilical vein endothelial cells (HUVEC) were stimulated with cytokines, TNF-α (1–250 U/ml) or IL-1? (0. 1–50 U/ml) for 24 h. HUVEC were also cultured with cytokines, TNF-α (100 U/ml) or IL-1? (10 U/ml), for 4–72 h, cell surface expression of adhesion molecules (ICAM-1 and VCAM-1) were detected and quantitated by immunocytochemical methods and computerized imaging analysis technique. Adhesion molecules expression were up-regulated by TNF-α, IL-1? in a concentration- and time-dependent manner. Some significant differences were observed between the effects of cytokines on the ICAM-1 and on VCAM-1 expression. Cytokines might directly induce the expression of ICAM-1 and VCAM-1 in vascular endothelial cells. Our observations indicate differential functions of the two adhesion molecules during the evolution of inflammatory responses in stroke.     

Tumor Antigen-pulsed CD8α＋ Dendritic Cells Induce T Cell-mediated Graft-versus-tumor Effect In Vitro

The graft-versus-tumor (GVT) effect of T cells induced by tumor antigen-pulsed CD8α+dendritic cells (DCs) in vitro was investigated in this study.Immature CD8α+ DCs were prepared from C57BL/6 (H-2b) bo...     

The Change of Interleukin-6 and Tumor Necrosis Factor in Patients with Obstructive Sleep Apnea Syndrome

Obstructive sleep apnea syndrome( OSAS) is avery common disease,which is characterized by re-current apneas caused by occlusion of upper airwayduring sleep,leading to arterial oxygen desaturationand a decrease in tissue oxygen concentration.Pa-tients with OSAS exhibit dramatic fragmentation ofsleep,a decrease in the duration of stage3and 4sleep,decreased percentages of rapid eye movementsleep ( REMS) ,and excessive daytime somnolence.The cause and pathogenesis of OSAS are unclear.Recentl…     

The Role of IκBα in TNF-α-induced Apoptosis in Hepatic Stellate Cell Line HSC-T6

To investigate the role of NF-κB in TNF-α induced apoptosis in HSC-T6, a mutant IκBα was transfected into HSC-T6 cells by lipofectin transfection technique and its transient effect was examined 48 h after the transfection. The activation of NF-κB was detected by immune fluorescence cytochemistry and Western blotting with anti-p65 antibody. The apoptosis and the rate of inhibition by TNF-α in both transfected and untransfected HSC-T6 cells were measured respectively by FAC-Scan side scatter analysis and MTF methods. Our results showed that TNF-α could activate NF-κB in untransfected cells but not in transfected HSC-T6 cells. The percentage of apoptosis in transfected cells were significantly higher than that in the untransfected ones （P〈0.01） and it was also true of the inhibition rate （P〈0.01）. It is concluded that the resistance of HSC-T6 towards apoptosis induced by TNF-α can be mediated by NF-κB activation. The inhibition of NF-κB activation by mutant IκBα can attenuate the resistance of HSC-T6 cells and increase its sensitivity to TNF-α.     

Effect of ephrin-A1/EphA2 on invasion of trophoblastic cells

The effect of axon guidance factors ephrin-A 1/EphA2 on the invasion of trophoblastic cells and the possible mechanism were investigated in this study.The expression of EphA2 in vascular endothelial ce...     

Antitumor effects of the fibroblasts transfected TNF-α gene and its mutants

Summary To compare the anti-tumor effects of transmembrane TNF-α (TM-TNF) and secreted TNF-α (S-TNF)in vivo, mouse fibroblasts NIH3T3 were transfected separately with three types of retrovirus containing wild type TNF-α (Wt-TNF), TM-TNF mutant (TM-TNFm), S-TNF mutant (S-TNFm). Southern blot, RT-PCR, FACS and bioassay were used to investigate TNF-α gene integration, expression and its biological activity. It was found that both fixed cells and supernatant of NIH3T3/Wt-TNF, the fixed cells of NIH3T3/TM-TNFm and the supernatant of NIH3T3/S-TNFm could express high level of TNF-α or its mutants and effectively kill H22in vitro. The trans-fected NIH3T3 were separately injected into the mice at the sites of H22 tumor cell inoculation according to a ratio of 5∶1 or 1∶1 (effector/target cells, E/T) after the third day of H22 challenge, respectively. At the E/T=5∶1, the NIH3T3/TM-TNFm induced the highest tumor regression, while NIH3T3/S-TNFm exerted the strongest tumor depressing effect at the E/T=1∶1in vivo. No obvious side effects were noted throughout the course of treatment. The results suggest that both TM-TNF and S-TNF could cause tumor regression. The anti-tumor effect of TM-TNF would be more powerful and safe than that of S-TNF at the proper E/T ratio. LI Qingfen, female, born in 1965, Associate Professor     

Effect of tongbiling on the synoviocyte function in adjuvant arthritis rats

Objective: To study the effect of Tongbiling (TBL) on the proliferation of synovial fibroblast and interleukin-1 (IL-1), tumor necrosis factor-α (TNF-α) and prostaglandin E₂(PGE₂) secreted by synoviocytes in adjuvant arthritis (AA) rats.Methods: Synovial fibroblast was derived from culture of tissue piece. The effect of primary synoviocyte culture supernatants on the fibroblast proliferation were assayed and IL-1, TNF-α bioactivity and PGE₂ content of supernatants of cultured synoviocytes were measured.Results: TBL could significantly inhibit the synovial fibroblast proliferation (P < 0.001), and down-regulate IL-1, TNF-α and PGE₂ productions (P < 0. 001); indomethacin could obviously promote the synovial fibroblast proliferation (P < 0.001). It significantly inhibited PGE₂ production, but further up-regulated IL-1 and TNF-α secreted by synoviocytes (P<0.01).Conclusion: The therapeutical effect of TBL on AA might be associated with its down-regulating the secretory function of synoviocyte, then restoring the abnormal proliferation of fibroblast to normal levels. This program was supported by the Nature Science Foundation of Guangdong Province (No. 960550)     

Effects of Baicalin on Expression of Inducible Nitric Oxide Synthase in Cultured Fibroblasts Stimulated by Cytokines

Objective: To investigate the effects of baicalin on expression of inducible nitric oxide synthase (iNOS) in fibroblasts and its mechanisms in treating psoriasis. Methods: Fibroblasts cultured in vitro were stimulated with tumor necrosis factor-α(TNF-α), interferon-γ (IFN-γ), interleukin-8 (IL-S) in different groups. iNOS was detected by western blot and immunocytochemistry assay, and in addition, the effects of baicalin on its expression were investigated. Results: Fibroblasts did not express iNOS without cytokine stimulation. When treated for 24 h with 1. 0× 106 U/L TNF-α, 0.2× 106U/L IFN-γ, 0.2× 106 pg/L IL-8 alone or in combinations indicated, fibroblasts produced iNOS when stimulated by TNF-α alone while neither IFN-γ nor IL-8 could induce the production of iNOS. The combination of TNF-α and IL-8 induced a strong expression of iNOS, the combined exposure of three kinds of cytokines showed an even stronger effects. The strongly stained area was in the cytoplasm near the nuclei. Expression of iNOS induced by TNF-α and IL-8 was inhibited by 50 μg/ mi of baicalin. Conclusion: Fibroblasts might express iNOS when stimulated by certain cytokines. Baicalin decreased production of nitric oxide through inhibiting the expression of iNOS, furthermore it reduced inflammation, which might be part of its mechanisms in treating psoriasis.     

Expression of macrophage inflammatory protein 1α in the endothelial cells exposed to diamide

Summary In order to study whether the endothelial cells (ECs) with lipid peroxidation induced by diamide can express and secrete macrophage inflammatory protein 1α (MIP-1α), the expression of MIP-1α protein in the cells was detected by cell enzyme-linked immunosorbent assay (ELISA) and that of MIP-1α mRNA was determined by cell in situ hybridization and nuclease S1 protection assay after the ECs were exposed to different concentrations of diamide for 4 h. The chemotactic activity of MIP-1α was tested by micropore filter method using modified Boyden chambers. Cell ELISA showed that the expression of MIP-1α protein in endothelial cells exposed to 1 μmol/L, 5 μmol/L and 10 μmol/L diamide was 1.9-fold, 2.3-fold and 1.7-fold respectively as much as that in the control cells, which was statistically significant by analysis of variance. In situ hybridization revealed that the mRNA expression of ECs treated with 1 μmol/L, 5 μmol/L and 10 μmol/L diamide was 1.3-fold, 3.0-fold and 1.7-fold as much as that in the control group, which had statistical significance (F=188. 93,P<0.01). The mRNA expression in 5 μmol/L dimide treated ECs, measured by nuclease S1 protection assay, was 3.4-fold as much as that in the control group (t=8.70,P<0.05). Chemotactic response (99.50±4.31 μm) to the culture medium conditioned by 5 μmol/L diamide treated ECs, which was stronger than that (66.47±3.25 μm) conditioned by the ECs (F=404.31,P<0.05), was significantly decreased (F=192.25,P<0.05) after adding MIP-1α antibody. It suggests that diamide, a lipid peroxidation inducer, could stimulate ECs to produce high level of MIP-1α, and might play an important role in atherogenesis by promoting the migration of peripheral blood monocytes into arterial intima. YANG Limin, female, born in 1973, M. D., Ph.D. This project was supported by a grant from National Natural Sciences Foundation of China (No. 39730220).     

Specific inhibitory protein Dkk-1 blocking Wnt/β-catenin signaling pathway improve protectives effect on the extracellular matrix

The present study examined the role of Wnt/β-catenin signaling pathway in the degeneration of nucleus pulposus cells and the protective effect of DKK1 on nucleus pulposus cells.The model of nucleus pul...     

Interleukin-1β, tumor necrosis factor-α and lipopolysaccharide induce expression of monocyte chemoattractant protein-1 in calf aortic smooth muscle cells

Summary To investigate whether interleukin-1β(IL-1β), tumor necrosis factor-α (TNF-α) and lipopolysaccharide (LPS) induce expression of monocyte chemoattractant protein-1 (MCP-1) mRNA and protein in calf aortic smooth muscle cells (SMCs), calf aortic SMCs were cultured by a substrate-attached expiant method. The cultured SMCs were used between the third to the fifth passage. After the cells became confluent, the SMCs were exposed to 2 ng/ml IL- 1β, 20 ng/ml TNF-1α and 100 ng/ml LPS respectively, and the total RNA of SMCs which were incubated for 4 h at 37 C were extracted from the cells by using guanidinium isothiocyanate method. The expression of MCP-1 mRNA in SMCs was detected by using dot blotting analysis using a probe of γ-³²P-end-labelled 35-mer oligonucleotide. After a 24-h incubation, the media conditioned by the cultured SMCs were collected. The MCP-1 protein content in the conditioned media was determined by using sandwich ELISA. The results were as follows: Dot blotting analysis showed that the cultured SMCs could express MCP-1 mRNA. After a 4-h exposure to IL-1β, TNF-α and LPS, the MCP-1 mRNA expression in SMCs was increased (3.6-fold, 2.3-fold and 1.6-fold, respectively). ELISA showed that the levels of MCP-1 protein in the conditioned media were also increased (2. 9-fold, 1.7-fold and 1.1-fold, respectively). The results suggest that calf aortic SMCs could express MCP-1 mRNA and protein. IL-1β and TNF-α can induce strong expression of MCP- 1 mRNA and protein, and the former is more effective than the latter. This project was supported by a grant from National Natural Science Foundation of China (No. 39470289).     

Effect of salvia miltiorrhiza Bge on left ventricular hypertrophy and the expression of tumor necrosis factor-α in spontaneously hypertensive rats

Summary The effects of salvia miltiorrhiza Bge (SMB) on left ventricular hypertrophy (LVH) and the expression of tumor necrosis factor-α (TNF-α) in the left ventricle of spontaneously hypertensive rats and the action mechanism were investigated. Normal Wistar-kyoto (WKY) rats were used as negative control, and spontaneously hypertensive rats (SHR) were randomly assigned to receive placebo or SMB. SMB (1 g/kg·d) was injected intraperitoneally for 12 weeks. Systolic blood pressure (SBP) and left ventricular mass index (LVMI) were measured. HE, VG and immunohistochemical staining combined with computed morphometry were employed to evaluate the cardiomyocyte size, diameter, the collagen volume fraction (CVF), perivascular circumferential area (PVCA), and tumor necrosis factor-α (TNF-α) expression in the left ventricular tissue. The results showed, as compared with WKY rats, the SBP, LVMI, cardiomyocyte size, diameter, CVF, PCVA, and TNF-α expression were increased markedly in the 20-week-old spontaneously hypertensive rats. SMB decreased LVMI (P<0.01), size of cardiomyocytes (P<0.01), collagen volume fraction (P<0.01), perivascular circumferential area (P<0.01), and TNF-α expression (P<0.01), but had no effect on SBP (P>0.05). It was suggested that chronic administration of SMB could inhibit and reverse the development of LVH in spontaneously hypertensive rats independent of BP. TNF-α may be involved in the reversal mechanism of LVH by SMB. SUN Lianping, male, born in 1966, Associate Professor     

Immunoregulation of lupus-like NZB/W F1 mice by antimurine IL-1α, IL-6 antibodies

Summary Anti-murine IL-1α, IL-6 antibodies were intra-peritoneally injected to the lupus-like NZB/W F1 mice of 4 months with the dosage of 10 μg per day for three days and then per month for three months. The mice were killed at the age of 11 months. The results showed that the treatment of the dosage could not absolutely prevent lupus nephritis; it could alleviate proteinuria, obviously reduce the levels of serum IL-1α and inhibit the secretion of IL-1α by celiac macrophage. As to the level of IL-6 and TNF-α no significant change was observed. The project is surported by the National Science Foundation of China (No. 39270637)