Shp-2／NF-κB Pathway Mediates the Inhibition of Lipoxin A4 onIL-1β-induced Synthesis of IL-6 in Glomerular Mesangial Cells

Objective: To examine whether lipoxin A4 (LXA4) has an antagonistic effect on IL-1β-induced synthesis of IL-6 in glomerular mesangial cells, and to explore the molecular mechanisms of signal pathway in LXA4 actions. Methods: The glomerular mesangial cells of rat were cultured and treated with IL-1β with or without preincubation with LXA4 at different concentrations. The amount of IL-6 in the supernatant of cells was analyzed by enzymelinked immunosorbent assay(ELISA). The expressions of mRNA of IL-6 were determined by RT-PCR. The expressions of Src homology 2( SH2 ) containing protein-tyrosine phosphatase 2(Shp-2) were assessed by immunoprecipitation and immunoblotting. Activities of DNA-binding of nuclear factor-kappa B(NF-κB) were measured by electrophoretic mobility shift assay(EMSA). Results:IL-1β- snulated secretion of protein and expression of mRNA of IL-6 in mesangial cells were inhibited by LXA4 in a dose-dependent manner. LXA4 antagonizes the phosphorylation of Shp-2 and activities of NF-κB induced by IL-1β Conclusion: LXA4 antagonists IL-1β-induced synthesis of IL-6 in glomerular mesangial cellsthrough the mechanism of Shp-2/NF-κB pathway-dependent signal transduction.     

Interleukin-13 inhibits cytokines synthesis by blocking nuclear factor-κB and c-Jun N-terminal kinase in human mesangial cells

Objective： Monocytes/macrophages, proinflammatory cytokines and chemokines are important in the pathogenesis of glomerulonephritis. Interleukin （IL） -13 has been shown to exert potent anti-inflammatory properties. This study was designed to investigate the effect of IL-13 on the expression of proinflammatory cytokines, chemokines and profibrogenic cytokines and the involved molecular mechanism in cultured human mesangial cells （HMCs）. Methods： The expressions of proinflammatory cytokines, chemokines and profibrogenic cytokines were determined by ribonuclease protection assay （RPA）. Activity of nuclear factor-kappa B （NF-κB） and activa- tor protein-1 （AP-1） was examined by electrophoretic mobility shift assay （EMSA）. NF-κB subunit p65 nuclear transportation and c-Jun N-terminal kinase （JNK） activity were assayed by immunoblot. Results： Recombinant IL-13 inhibited tumor necrosis factor-α （TNF-u）, IL-1α, IL-1β, monocyte chemoattractant protein-1 （MCP-1）, IL-8, and transforming growth factor-β1 （TGF-β1） mRNA expressions in a dose-dependent manner. Lipopoly- sacchorides （LPS） dramatically increased NF-κB DNA binding activity of HMCs, which was inhibited by IL-13 in a dose-dependent manner. LPS-activated NF-κB contained p50 and p65 dimers, but not c-Rel subunit. IL-13 blocked LPS-induced NF-κB subunit p65. LPS stimulated JNK/AP-1 activation, which was inhibited by IL-13 in a dose-dependent manner. Conclusion： IL-13 inhibits proinflammatory cytokines, chemokines, and profibrogenic cytokines synthesis by blocking NF-κB and JNK/AP-1 activation. These observations point to the importance of IL-13 in the modulation of inflammatory processes in the renal glomerulus.     

Triptolide-induced apoptosis by inactivating nuclear factor-kappa B apoptotic pathway in multiple myeloma <Emphasis Type="Italic">in vitro</Emphasis>

The effect of triptolide on proliferation and apoptosis of human multiple myeloma RPMI-8226 cells in vitro,as well as the roles of nuclear factor-kappa B(NF-κB) and IκBα was investigated.The effect of tritptolide on the growth of RPMI-8226 cells was studied by MTT assay.Apoptosis was detected by Hoechest 33258 staining and Annexin V/PI double staining assay.The expression of NF-κB and IκBα was observed by Western blot and confocal microscopy.The results showed that triptolide inactivated NF-κB apoptotic pathway in human multiple myeloma RPMI-8226 cells.Triptolide at nM range induced proliferation inhibition in a dose-and time-dependent manner and apoptosis in a dose-dependent fashion in RPMI-8226 cells.Besides,we observed the inhibition of NF-κB /p65 in the nuclear fraction was correlated with the increase in the protein expression of IκBα in the cytosol.These results suggested that triptolide might exhibit its strong anti-tumor effects via inactivation of NF-κB/p65 and IκBα.     

LPS-induced NF-κB activation requires Ca2+ as a mediator in isolated pancreatic acinar cells of rat

Objective To investigate the effect of Ca2+ on lipopolysaccharide (LPS)-induced NF-κB activation in pancreatic acinar cells and the role of NF-κB in LPS-induced acinar cell injury. Methods Male rat pancreatic acinar cells were isolated by collagenase digestion, then exposed to varying concentrations of LPS (from 1 to 20 mg/L) in the presence or absence of EGTA. At various time points (30 minutes, 1 hour, 2 hours, 4 hours and 10 hours) after treatment with the agents, cell viability was determined by MTT. Nuclear translocation of NF-κB’s subunit p65 was visualized by immunofluorescence staining and nuclei protein was extracted to perform EMSA which was used to assay the activity of NF-κB binding to the DNA sequence containing the recognition site of NF-κB. Results LPS induced cell damage in a time- and concentration-dependent manner while EGTA attenuated LPS-induced cell damage (P&lt;0.05). NF-κB p65 immunofluorescence staining had increased intensity in the cytoplasm and indicated that nuclear translocation occurred within 30 minutes and its zenith was reached at 1 hour after LPS (10 mg/L) treatment. Testing of NF-κB DNA binding activity showed the same alteration phase as p65 immunofluorescence staining. NF-κB activation preceded the pathological alteration of pancreatic acinar cells. The Ca2+ chelator EGTA inhibited LPS-induced NF-κB activation. Conclusions NF-κB activation is an important early event in LPS-induced injury to pancreatic acinar cells. Ca2+ is an important mediator in the process of LPS-induced NF-κB activation.     

IgE PRODUCTION IS INVOLVED IN BUTYRATE-ENHANCED NK CELL ACTIVITY IN VIVO

It has been demonstrated that patients with asthma have a large number of NK cells and show a stronger NK activity. These results indicate that NK cell activity may be related to total IgE level in serum in healthy subjects.Previously, we have found that sodium butyrate (NaBu) markedly enhanced the IL-4-induced IgE production in the LPS-stimulated murine splenocytes in vitro, and inductive rat IgE production in vivo, and enhanced the NK cell activity ex vivo. We hypothesized that the IgE production might be involved in butyrate-enhanced NK cell activity in vivo. Mice were intraperitoneally treated/immunized with NaBu or/and Ascaris suum extract (ASC), and thespleen NK cell activity was evaluated. Furthermore, the effect of serum (NAS) on IL-2- or IFN-γ-induced spleenNK cell activity was determined. The spleen NK cell activity and IL-2- or IFN-γ-induced spleen NK cell activity of mice treated/immunizedwith NaBu or/and ASC were stronger than those of untreated/unimmunized mice. Although IL-4 blocked IL-2(100 U/ml)- or IFN-γ,( 100 U/ml)-induced increase in NK cell activity, these NK cell activities in mice treated/immunized with NaBu/ASC were not inhibited. IgE production showed a tendency to rise in NaBu-treatedmice serum, and a synergistic effect was observed with treatment of NaBu and ASC. Moreover, the NAS significantly increased IL-2(25 U/ml)-or IFN-γ-(25 U/ml)-induced NK cell activity, and its effect was inhibited by an-ti-mouse IgE mAb. These data show that IgE plays an important role in NAS-enhanced IL-2/IFN-γ-induced NK cell activity, andIL-4 does not inhibit IgE and IL-2/IFN-γ-induced NK cell activity in mice.     

Effect of resveratrol on platelet aggregation in vivo and in vitro

Objective Low or moderate consumption of red wine has a greater benefit than the consumpti on of other beverages in the prevention of atherosclerosis and coronary heart di sease and this is increasingly attributed to the polyphenol compounds in red win e, such as resveratrol. In the present study, we investigated the effect of res veratrol on platelet aggregation in vitro and in vivo. Methods Platelet aggregation in rabbits and normal subjects was measured using Born’s me thod. Results Resveratrol, at 10-1000 μmol/L, significantly inhibited platelet aggregation i n vitro induced by collagen, thrombin, and ADP in healthy subjects. The inhibit ory effect was concentration- dependent. Hypercholesterolemia induced by high- cholesterol diet enhanced ADP- induced platelet aggregation. Resveratrol 4 mg ·kg(-1)·d(-1)inhibited ADP- induced platelet aggregation in vivo de spite no changes in serum lipid levels. Conclusions Resveratrol inhibits platelet aggregation both in vitro and in vivo. This may b e one of the mechanisms by which resveratrol prevents atherosclerosis.     

Homocysteine-induced Enhanced Expression of Tissue Factor in Human Vascular Smooth Muscle Cells

The homocysteine (Hcy)-induced tissue factor (TF) expression in human vascular smooth muscle cells (VSMCs) and the effect of Hcy on the activity of nuclear factor-kappaB (NF-кB) and the expression of inducible nitric oxide synthase (iNOS) were investigated. Human umbilical artery VSMCs were cultured by tissue explanting method, identified by α-actin immunohistochemistry, and incubated with different concentrations of Hcy/PTDC (NF-кB inhibitor). Semi-quantitative RT-PCR was performed to detect the expression of TF mRNA in VSMCs. Flow cytometry was used to assay the expression of TF protein on the surface of VSMCs and the expression of iNOS in VSMCs. Western blot was carried out to detect the expression of NF-кB protein in nuclei. The results showed that Hcy could induce VSMCs expressing TF mRNA significantly after the VSMCs were incubated with Hcy at concentrations of 10, 100, 500 μmol/L respectively. There was low expression level of TF protein on the surface of the resting VSMCs and Hcy could also induce VSMCs expressing TF pro- tein on the cell surface in different concentrations. Additionally, Hcy could rapidly induce the activation of NF-кB and this effect could be significantly inhibited by PDTC. Hcy alone could not induce the expression of iNOS in VSMCs. It was concluded that Hcy could significantly induce the expression of TF in VSMCs and enhance the activation of NF-ΚB, subsequently mediate TF gene expression and protein synthesis. NF-кB-mediated expression of TF in VSMCs might be the important mechanism of atherosclerosis and thrombosis induced by Hcy.     

Dexamethasone Induces Apoptosis of Primary Cultured Osteoblasts

Objective: To investigate the effect of glucocorticoids on cell viability of osteoblasts and explore the cytological and molecular mechanisms of osteoblasts apoptosis induced by Glucocorticoids. Methods: Dexamethasone was used to induce apoptosis of primary cultured osteoblast-like cells from skulls of suckling mouse. Surviving rate of cells, apoptosis rate, activity of Caspase-3 and DNA binding activity of nuclear factor-κB were assessed respectively. Results: It was found that Dexamethasone increased apoptosis rate of osteoblast-like cells, activated Caspase-3 while inhibited the activity of NF-κB, in a concentration-dependent and time-dependent manner. Conclusion: These results suggested that Dexamethasone induced apoptosis of primary cultured and non-transformed osteoblasts, which was Caspase-3 dependent, while NF-κB might play a protective role in the process through inhibition of Caspase-3.     

Ethanol Extract of Lycopodium serratum Thunb. Attenuates Lipopolysaccharide-Induced C6 Glioma Cells Migration via Matrix Metalloproteinase-9 Expression

Objectives: To elucidate how ethanol extract of L. serratum (ELS) could exert anti-migratory effects on glioma with the suppression of nuclear factor kappa B (NF-κB) downstream pathway. Methods: Cell viability of ELS on C6 glioma was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Nitric oxide (NO) assay and 2'',7''-dichlorofluorescin diacetate (DCFH-DA) assay were applied to measure NO production and reactive oxygen species (ROS) generation on lipopolysaccharide (LPS)-induced C6 glioma cells. NF-κB, mitogen-activated protein kinase (MAPK), inducible nictric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein were determined by Western blot. Wound healing assay was used to investigate the inhibitory effect of ELS on fetal bovine serum (FBS)-induced migration and matrix metalloproteinase (MMP)-9 and -2 activity was examined by zymography. Results: ELS suppressed LPS-induced phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 through inhibiting the expression of chemokine CCL2 (or monocyte chemoattractant protein-1, MCP-1). In addition, ELS inhibited the expression of iNOS, COX-2, and the production of NO by LPS in C6 glioma cells. ELS also significantly decreased serum-induced migration of C6 glioma cells in scratch wound healing in a dose-dependent manner (P<0.01). The activity of MMP-9 and -2 were also significantly attenuated by ELS with LPS treatment (P<0.01). Conclusion: Our results suggest that downregulation of MMP-9 gene expression might be involved in the anti-migration effect of ELS against LPS-induced C6 glioma cells.     

Expression of NF-κB in hRPE Cells Stimulated by PDTC and IL-1β

The constitutive expression of nuclear-factor-κB (NF-κB) in human pigment epithelial (hRPE) cells cultivated in vitro and the possible changes when incubated with PDTC and IL-I were investigated. The synchronized hRPE cells in vitro were divided into two groups. In nonPDTC group, hRPE cells were exposed respectively to IL-1β and NS (for detecting the constitutive expressions of NF-κB in hRPE cells) ; In PDTC group, PDTC-pretreated hRPE cells were exposed respectively to IL-1β?Aand NS. (for detecting the constitutive expression of NF-κB in PDTC-pretreated hRPE cells). The expression of NF-κB in hRPE cells in two groups was detected by immunofluorescence stain and flow cytometry. The results showed that the constitutive expression of NF-κB in hRPE cells in vitro was 8.05 %, and increased to 30.26 % by IL-1β. After PDTC pretreatment, the constitutive expression of NF-κB in hRPE cells was decreased to 3.74%, and 3.66 % by IL-l,respectively. It was concluded that the expressions of NF-κB in hRPE cells could be increased significantly by IL-1βand depressed effectively by PDTC. Also, PDTC could significantly inhibit the activation of NF-κB induced by IL-1β.     

Inhibition of NF-κB by mutant IκBα enhances TNF-α-induced apoptosis in HL-60 cells by controlling bcl-xL expression

Backgound The aim of this study was to explore whether the inhibition of nuclear factor-κB (NF-κB)activation by mutant IκBα (S32,36→A) can enhance TNF-α-induced apoptosis of leukemia cells and to investigate the possible mechanism. Methods The mutant IκBα gene was transfected into HL-60 cells by liposome-mediated techniques. G418 resistant clones stably expressing mutant IκBα were obtained by the limiting dilution method. TNF-α-induced NF-κB activation was measured by electrophoretic mobility shift assay (EMSA). The expression of bcl-xL was detected by RT-PCR and Western blot after 4 hours exposure of parental HL-60 and transfected HL-60 cells to a variety of concentrations of TNF-α. The percentage of apoptotic leukemia cells was evaluated by flow cytometry (FCM). Results Mutant IκBα protein was confirmed to exist by Western blot. The results of EMSA showed that NF-κB activation by TNF-α in HL-60 cells was induced in a dose-dependent manner, but was almost completely inhibited by mutant IκBα repressor in transfected cells. The levels of bcl-xL mRNA and protein in HL-60 cells increased after exposure to TNF-α, but changed very little in transfected HL-60 cells. The inhibition of NF-κB activation by mutant IκBα enhanced TNF-α-induced apoptosis. Thecytotoxic effects of TNF-α were amplified in a time- and dose-dependent manner. Conclusions NF-κB activation plays an important role in the resistance to TNF-α-induced apoptosis. The inhibition of NF-κB by mutant IκBα could provide a new approach that may enhance the antileukemia effects of TNF-α or even of other cytotoxic agents.