Effects of tumor necrosis factor alpha on proliferation and apoptosis of HSCs

AIM:To explore the role of tumor necrosis factor alpha(TNF-α) in the pathogenesis of liver fibrosis.METHODS:The proliferation and apoptosis of hepatic stellate cells (HSCs) in vitro were detected with flow cytometry, electron microscopy and TUNEL.RESULTS:The flow cytometry analysis showed that the cell proliferation index (PI) in the TNF-α(0.5 μg/L, 2.0 μg/L, 8.0 μg/L) groups was evidently lower than that in the control group (P＜0.05). In the cell cycle distribution, the portion of G₀/G₁ phase in the TNF-α groups was significantly higher than that in the control group(P＜0.05), but the portion of S phase in the TNF-α groups was evidently lower than that in the control group(P＜0.05). These indicated that TNF-α interfered with HSCs entrance into S phase from G₀/G₁ phase whereupon the proliferation of HSCs was inhibited. The apoptotic rate in the TNF-α groups was evidently higher than that in the control group(P＜0.05). The gene expression of bcl-2 and bax was also detected with flow cytometry. The expression of bcl-2 in the TNF-α groups was evidently lower than that in the control group(P＜0.05), but the expression of bax in the TNF-α groups was significantly higher than that in the control group(P＜0.05). TUNEL analysis showed the apoptotic rate of HSCs in the TNF-α(2.0 μg/L) group was 18.7%±2.5% compared with 5.3%±1.2% in the control group(P＜0.05).CONCLUSIONS:TNF-α interfered with HSCs entrance into S phase from G₀/G₁ phase whereupon the proliferation of HSCs was inhibited. TNF-α down-regulated bcl-2 gene expression and up-regulated bax gene expression whereupon the apoptosis of HSCs was induced.     

Lipopolysaccharide stimulates TNF-α and endothelin-1secretion from cultured rat Kupffer cells

AIM: To investigate lipopolysaccharide (LPS) stimulated cytokine secretion from normal rat Kupffer cells in vitro. METHODS: Kupffer cells were isolated from wistar rats liver and cultured. Tumor necrosis factor -α (TNF-α) and endothelin-1 (ET-1) secreted by LPS stimulated Kupffer cells were detected. RESULTS: LPS had an stimulative effect on Kupffer cell activity. LPS in definite concentrations promoted Kupffer cell secretion. CONCLUSION: LPS promotes Kupffer cell secretion, which may be associated with liver injury induced by LPS.     

Changes of DNA methylation at CpG sites in promoter region of TNF-α gene in DENV2-infected peripheral blood mononuclear cells

AIM：To examine DNA methylation at CpG sites in the promoter region of tumor necrosis factor-alpha (TNF-α) gene in dengue virus type 2 (DENV2)-infected peripheral blood mononuclear cells (PBMC).
METHODS：DNA methylation in the promoter region of TNF-α gene was measured by bisulfite sequencing PCR.
RESULTS：The promoter region of TNF-α gene was from -294 bp to +58 bp, including 11 CpG sites. The PCR products identified by aga-rose gel electrophoresis were consistent with the theoretical size. Two sites were methylated at 0 h and 6 h and 6 sites were methylated at 12 h in TNF-α gene promoter region in DENV2-infected PBMC. The average methylation rates were 103%, 121% and 255% at 0 h, 6 h and 12 h, respectively. Significant differences between 0 h and 12 h and between 6 h and 12 h were observed.
CONCLUSION：The DNA methylation in the promoter region of TNF-α gene is increased in DENV2-infected PBMCs.     

Synergic antitumor effect of verapamil and tumor necrosis factor alpha

AIM: To study the synergic antitumor effect of verapamil (VPL) and tumor necrosis factor alpha (TNFα). METHODS: In the presence of verapamil or verapami plus tumor necrosis factor, the proliferation, the expressions of c-myc and PCNA, the mean vessel density(MVD)of human hepatoma cell line(HepG₂) were studied in vivo and in vitro. RESULTS: It was demonstrated that the growth of HepG₂ was inhibited remarkably, under the presence of verapamil and TNFα simutaneously, the expressions of c-myc, PCNA and the MVD were also decreased.CONCLUSION:The calcium channel blocker can inhibit the proliferation of tumor and has a remarkable synergy with TNF.     

Effect of transfection of tumor necrosis factor α gene and its mutants on tumorigenicity of H22 tumor cells in vivo

AIM: To compare the tumorigenecity of H22 cells transfected with TNF-α gene and its mutants(secreted TNF-α mutant, S-TNF^m, transmembrane TNF-α mutant, TM-TNF^m and wild type of TNF-α, Wt-TNF) in vivo. METHODS: Three kinds of mouse liver cancer cell line H22 expressing TNF-α and its two mutants were mixed with untransfected H22 at different effector/target ratio separately. The growth of tumor was examined after injection of 2.5×10⁵(100 μL) mixed H22 tumor cells into mice. The lymphocyte infiltration in the site of tumor and the expression of Fas on tumor cells were detected by immunohistochemistry. RESULTS: The tumorigenecity of H22 cells transfected with TNF-α gene and its mutants was significantly weakened( P <0.01). Besides the cytotoxicity of the both forms of TNF, TM-TNF was found to induce the expression of death receptor Fas by tumor cells and S-TNF was shown to promote frank lymphocyte infiltration in the site of tumor. Furthermore, a transient decrease in body weight was found in mice inoculated with H22/S-TNF^m. CONCLUSION: The tumorigenecity of tumor cells was reduced by transfection with TM-TNF or S-TNF gene. It results from the cytotoxicity of TNF and their activation of tumorcidal mechanisms in vivo. TM-TNF may induce tumor cell apoptosis via the Fas pathway while S-TNF may exert its antitumor effect by recruiting and activating lymphocytes in the site of tumor.     

Proteomic analysis of the effects of tumor necrosis factor-α on endothelial cells

AIM: To investigate the affected proteins by tumor necrosis factor (TNF)-α in endothelial cells, and further explore the potential molecular mechanism of TNF-α on endothelial cells. METHODS: Nitric oxide (NO) production in the cultured human umbilical vein endothelial cells (HUVECs) was measured by a NO assay kit. Proteomic alterations were analyzed using two-dimensional electrophoresis, and peptide mass fingerprinting with matrix-assisted laser desorption/ionization-time of flight mass spectrometry. RESULTS: NO production in HUVECs decreased significantly after TNF-α treatement. Proteomics analysis showed 21 protein spots were changed including 9 spots that were increased and 11 spots that were decreased after TNF-α stimulation, and 1 spot was only detected in TNF-α activated cell gels. CONCLUSIONS: Decreased the expression of ecNOS by TNF-α might result in decreased NO production. Up-regulated MAP/ERK kinase 3 expression might imply that TNF-α activates the expression of adhesion molecules. Cytoskeletal protein actin is also involved in TNF-α injuried HUVECs. Proteomic analysis can find some clues for identifying new potential target of TNF-α.     

A novel mechanism of RhoA activation induced by tumor necrosis factor-α in mouse cerebral microvascular endothelial cells

AIM: To investigate the effects of tumor necrosis factor α (TNF-α) on RhoA activity in mouse cerebral microvascular endothelial cells.METHODS: The bEnd.3 cells, a mouse brain microvascular endothelial cell line, were cultured. RhoA activity was analyzed by pull-down assay 10 min, 30 min and 60 min after TNF-α treatment. Expression of RhoA protein was determined by Western blotting 1 h, 3 h, 6 h, 12 h and 24 h after TNF-α treatment. Small interfering RNA (siRNA) targeting to p115RhoGEF or control nsRNA was transfected into bEnd.3 cells. The expression of p115RhoGEF was determined by Western blotting, and RhoA activity was detected by pull-down assay 30 min after TNF-α treatment.RESULTS: RhoA activity peaked at 30 min after TNF-α treatment(P<0.01) . TNF-α significantly increased the protein expression of RhoA at 12 h and 24 h (P<0.05). Knock-down of p115RhoGEF by siRNA in bEnd.3 cells attenuated TNF-α-induced RhoA activation (P<0.05).CONCLUSION: TNF-α up-regulates RhoA activity and expression. p115RhoGEF may play a role in TNF-α-induced activation of RhoA.     

The expression of CD14 in rat Kupffer cells

AIM: To explore the expression of CD14 in rat Kupffer cells (KCs). METHODS: In rat KCs induced by LPS or the mediators from KCs induced by LPS, the changes of CD14 expression were measured by RT-PCR and immunohistochemistry.The expressions of TNFα mRNA、IL-6 mRNA or the concentrations of TNFα、IL-6 were estimated by in situ hybridization and radioimmunoassay, respectively. RESULTS: LPS increased the expression of CD14 in KCs in a dose-dependent fashion (LPS, 1 μg/L-10 mg/L) and in a time-dependent fashion(0.5 h-24 h, peaked at 3-6 hours). While the expression of CD14 in KCs stimulated by the active mediators from KCs which had been exposed to LPS 1 hour were obviously increased. CONCLUSIONS: There was a close relationship between LPS or the active mediators from KCs induced by LPS and the expressions of CD14. It is implied that the increase in CD14 expression may be induced by LPS and the cytokines produced by KCs, it also reveals that there is a auto-regulated loop in CD14 expression.     

uclear factor κB in LPS stimulated rat alveolar macrophages promotes TNF-α secretion

AIM: To investigate the activation of nuclear factor κB (NF-κB) induced by lipopolysaccharides (LPS) in rat alveolar macrophages (AMs) and its regulatory role in tumor necrosis factor (TNF-α) secretion. METHODS: The dynamic activity changes of NF-κB induced by LPS were determined with electrophoretic mobility shift assay (EMSA). Antisense oligonucleotides of NF-κB subunit (p65) were transfected into AMs prior to LPS stimulation. The effect of antisense oligonucleotide transfection on expressions of p65 and TNF-α in supernatant were measured with Western blotting and enzyme linked immunosorbent assay (ELISA), respectively.RESULTS: NF-κB activity increased markedly and reached its peak level at 4 h after LPS stimulation. After transfected with antisense oligonucleotides of NF-κB subunit (p65), expression of p65 and TNF-α in supernatant decreased markedly.CONCLUSION: NF-κB activity has a positive effect on regulating secretion of TNF-α in AMs induced by LPS.     

Mechanical stretch induces translocation of high-mobility group box 1 protein in THP-1 cells

AIM: To investigate the translocation of high-mobility group box 1 protein(HMGB1) in THP-1 cells induced by mechanical stretch. METHODS: The vector that expressed the fusion protein of HMGB1 and enhanced green fluorescent protein(EGFP) was constructed. The cDNA of HMGB1 and EGFP was subcloned into hemagglutinin(HA)-tagged vector pcDNA3-HA by two-step method. THP-1 cells were transfected with pcDNA3-HMGB1-EGFP and exposed to cyclic mechanical stretch at 20 % elongation using Flexercell 4000T cell stretching unit. The translocation of HMGB1 in THP-1 cells was observed under fluorescence microscope. RESULTS: The recombinant plasmid was verified by enzyme digestion. The green fluorescence accumulated in the nuclei of the cells, indicating that the fusion protein was highly expressed in THP-1 cells and localized in the nuclei. Eighteen hours after mechanical stretch, the green fluorescence was observed in the cytoplasm. At the same time, the green fluorescence was still localized in the nuclei of the control cells treated without mechanical stretch. CONCLUSION: The HMGB1-EGFP fusion protein is successfully and effectively expressed in THP-1 cells. Mechanical stretch induces the translocation of HMGB1 protein from nucleus to cytoplasm.     

Effects of aflatoxin G1 on proliferation and TNF-αsecretion of human peripheral blood mononuclear cells in vitro

AIM: To explore the effects of aflatoxin G₁(AFG₁ )on proliferation and TNF-α secretion of human peripheral blood mononuclear cells(HPBM) in vitro. METHODS: The effects of AFG₁ on proliferation of HPBM were analysed with flow cytometric (FCM) DNA analysis and MTT bioassay, while that on TNF-α secretion was detected with ELISA.RESULTS: FCM analysis revealed that 6 h after treatment, proliferation index(PI) of 1000 μg/L AFG₁ treated HPBM was significantly higher than that of control. 24 h after AFG₁ treatment, stimulating effects on proliferation was found in HPBM treated with AFG₁ at 200 μg/L and 1 000μg/L.Regression analysis showed that PI was postively correlated with the concentrations of AFG₁ in the concentration range from 0 to 1 000μg/L( r=0. 5122 and 0.5119 respectively,P＜0.05).MTT bioassay showed that the A value of the cells treated with AFG₁ at 2 000 μg/L was higher than that of the control. Double antibody sandwich enzyme linked immunosorbent assay (ELISA) results showed that AFG₁ at a dose of 100 μg/L could significantly inhibit lipopolysaccharide-induced TNF-α secretion.CONCLUSION: AFG₁ could stimulate the proliferation of HPBM and could decrease TNF-α secretion at certain concentration.     

Effects of interferon-γ, tumor necrosis factor-α and interleukin-1β on apoptosis of human airway smooth muscle cellsin vitro

AIM:To clarify if interferon-γ(IFN-γ), tumor necrosis factor-α(TNF-α)and interleukin-1β(IL-1β)can induce apoptosis of human airway smooth muscle cells (ASMCs) in vitro.METHODS:Human ASMCs were isolated and cultured in DMEM containing 10% fetal bovine serum. Passage 4-6 cell was used in the experiment. IFN-γ,TNF-α and IL-1β, were used separately or together in the treatment of human ASMCs. The effects of IFN-γ,TNF-α and IL-1β on the growth of the cells was detected by MTT method at the hour 0,24,48 and 72. Light microscopy and electron microscopy were used to examine the morphological change. DNA fragmentation was analyzed by agarose gel electrophoresis. SP immunohistological staing method was performed to detect the change of expressions of p 53, bcl- 2 and bax gene. The apoptosis cell percentage were detected by in situ end labeling technique (TUNEL)of fragmental DNA. RESULTS:(1)IFN-γ or IFN-γ together with TNF-α and IL-1β decreased the number of viable cells in a time dependent manner. (2) Light and electron microscopic examination showed cell shrinkage, membrane blebbing, nuclear contraction, chromatin condensation and nuclear fragmentation in human ASMCs. (3) Agarose gel electrophoresis showed a characteristic"ladder"of DNA bands representing integer multiples of the internucleosomal fragments (about 180-200 bp) in cytokine cotreated human ASMCs. (4)The expression of p 53 and bax gene in cytokine cotreated group was significantly higher than in control group, but the expression of bcl-2 gene was lower than in control group. (5)Stimultaneous treatment with IFN-γ(4×10⁵ U/L),TNF-α(4×10⁵ U/L)and /or IL-1β (10×10⁴ U/L) induced apoptosis of human ASMCs. Apoptotic index of human ASMCs in cytokine co-treated group was significantly higher than in control group (P＜0.01).CONCLUSION:Stimultaneous treatment with IFN-γ,TNF-α and /or IL-1β induced apoptosis of human ASMCs. These immune cytokines may play an important role in airway remodeling of asthma and of chronic obstructive pulmonary disease.     

Effects of lipopolysaccharide,IL-6 and TNFα on the tissue factor expression of astrocytes

AIM:To study the effects of lipopolysaccharide(LPS), interleukin-6(IL-6)and tumor necrosis factor α (TNFα) on tissue factor(TF) expression of astrocytes. METHODS:Astrocytes were identified with anti-glial fibrillary acidic protein antibody. The TF activity of cell lysate was measured with one stage clotting assay. RESULTS:TF activity of astrocytes of LPS,IL-6,TNFα groups were obviously higher than that of the control group(P <0.05); While LPS,IL-6 and TNFα were combined with trifluoperazine or H₇, their inductive effects were inhibited. CONCLUSION:LPS,IL-6 and TNFα promoted the TF expression of astrocytes and its mechanisms may connected with Calcium/Camodulin and protein kinase C pathway.     

Characteristics in the secretion of liver TNF-α induced by infusion of LPS into veins in goats

AIM: To study the role of liver in immune regulation in experimental endotoxemia. METHODS: 17 castrated male goats were subjected to simultaneously installing catheters in jugular, hepatic and portal veins by surgery. Four days later, lipopolysaccharide (LPS) was infused in term of three groups as followings: In group ①, LPS of 20 EU (endotoxin unit, EU)·kg^-1 was infused into portal vein; In group ②, LPS of 20 EU·kg^-1 was infused into jugular vein and LPS of 1 500 EU·kg^-1 infused into jugular vein in group ③. Before and after infusion, blood samples were collected from the three veins through the catheters for 8 h.The plasma levels of TNF-α were measured by RIA. RESULTS: In group ①, the plasma TNF-α levels of hepatic and portal vein rose to peak value at 5 h, but that of the jugular vein did not changed. In group ②, the plasma TNF-α levels in hepatic vein rose to peak value at 3 h. The TNF-α levels of jugular vein rose to peak value at 1 h and the one in portal vein enhanced continuously between 0-8 h. In group ③, the plasma TNF-α levels in jugular, hepatic and portal vein rose to significant peaks at 1 h simultaneously. CONCLUSION: During experimental endotoxemia,liver showed different dynamic characteristics in TNF-α secretion according to the pathway and doses of LPS delivery.     

Effects of glycine on intracellular of free calcium and tumor necrosis factor-α of cardiomyocytes during hypoxia/reoxygenation

AIM: To observe the influence of glycine on intracellular free calcium, the concentration of tumor necrosis factor-α and the survival rate of myocardial cells during hypoxia/reoxygenation (H/R). METHODS: The simulated model of myocardial ischemia-reperfusion with the primary cultured cardiomyocytes of neonatal rats was established, and the cultured cardiomyocytes were divided into seven groups, control group, hypoxia/reoxygenation group, glycine (0.5 mmol/L) plus hypoxia/reoxygenation group, glycine (1.0 mmol/L) plus hypoxia/reoxygenation group, glycine (2.0 mmol/L) plus hypoxia/reoxygenation group, glycine (4.0 mmol/L) plus hypoxia/reoxygenation group, 4.0 mmol/L glycine group. RESULTS: Within certain concentration (0.5-2.0 mmol/L), the glycine could inhibit the calcium overload resulting from hypoxia/reoxygenation injury in cells in a dose-dependent manner with the optimal inhibitory effect at 2.0 mmol/L. Glycine inhibited the secretion of tumor necrosis factor-α from myocardial cells and increased the survival rate of myocardial cells. CONCLUSION: Glycine has a protective effect on hypoxia/reoxygenation myocardial cells, which may be related to inhibiting calcium overload and decreasing the production of tumor necrosis factor-α.     

Mast cells change cellular cholesterol content and inhibit cholesterol efflux from THP-1 macrophage-derived foam cells

AIM:Mast cells (MC) are present in the arterial intima,the site of atherogenesis. The present studies explore the effect of MC on cholesterol content,distribution and efflux in THP-1 macrophage-derived foam cells (THP-1FCs）. METHODS:THP-1FCs were incubated with high-density lipoproteins 3 (HDL₃) in the absence or presence of mast cell granules (MCGs) harvested from compound 48/80-stimulated rat peritoneal MC. The intracellular cholesterol level,cholesterol effluxing capacity,ATP-binding cassette transporter A1(ABCA1) mRNA and HDL₃ treated with MCGs were detected to characterize the role of MC on intracellular cholesterol. RESULTS:MCGs had high levels of cellular total cholesterol(TC),free cholesterol(FC) but not esterifed cholesterol(EC) compared to control group where the TC concentrations ranged from 527.3 mg/g to 917.9 mg/g cellular protein with EC accounting for 7.6% of the cholesterol. Cholesterol efflux was 14% less in MCGs group compared to control group. ABCA1 mRNA expression in MCG-treated THP-1FCs remained unchanged in 20 hours. In contrast,treatment of HDL₃ with MCGs resulted in rapid degradation of the main HDL₃ apoliproteins,apoA-Ⅰ. SDS-PAGE revealed that a minor polypeptide band with about 26 kD molecular mass appeared below the apoA-Ⅰband. Densitometric analysis of the gel demonstrated that ≈ 28% of apoA-Ⅰhad been degraded by the MCGs. CONCLUSION:These results indicate that MC decreases cholesterol efflux,increases cellular accumulation in TC and FC by depleting HDL₃ and apoA-Ⅰ,but not by inhibiting ABCA1 mRNA expression.