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.     

Effects of Propofol on The mRNA Expression of Toll-like Receptor 4 in BV-2 Cells During Mimic Ischemia-Reperfusion Injury In Vitro

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.     

Effects of High Concentration Glucose on the Expression of NF-κB, Bax and Cytochrome C and Apoptosis of Islet Cells in Mice

The roles of NF-kappaB （NF-κB） expression, Bax activity and cytochrome C （Cyt C） release, apoptosis of islet cells induced by high concentration glucose were explored in vitro. Pancreatic islet cells, which were isolated from Kunming mice, were cultured with different concentrations of glucose in DMEM, and divided into the following groups： G1, G2, G3, G4, G5, and G6 groups, corresponding to the glucose concentrations of 5.6, 7.8, 11.1, 16.7, 22.5, and 27.6 mmol/L, respectively. After culture for 120 h, insulin secretion was evaluated by radioimmunoassay, and the NF-rd3 expression was detected by immunocytochemistry. Bax activity and Cyt C release were measured by immunofluorescence, and apoptosis was examined by Hoechst33342 assay. The results showed that in GI, G2 and G3 groups, insulin secretion was enhanced with the increase of glucose concentration, and the NF-κB expression was also increased （P〈0.05）, but Bax activity, Cyt C release and apoptosis rate showed no significant difference among them. However, in G4, G5, and G6 groups, apoptosis rate of islet cells, NF-rd3 expression, Bax activity, and Cyt C release were all significantly increased, and insulin secretion was impaired as compared with G1, G2, and G3 groups （P〈0.05）. It was concluded that the exposure of islet cells to high glucose could induce islet cells apoptosis as well as impaired insulin secretion. The NF-κB signaling pathway and mitochondria pathway in islet cells might play some roles in the progressive loss of islet cells in diabetes. The inhibition of the NF-κB expression could be an effective strategy for protecting pancreatic islet cells.     

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.     

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.     

Metformin inhibits nuclear factor-κB activation and inflammatory cytokines expression induced by high glucose via adenosine monophosphate-activated protein kinase activation in rat glomerular mesangial cells in vitro

Background The renoprotective mechanisms of adenosine monophosphate （AMP）-activated protein kinase （AMPK） agonist-metformin have not been stated clearly.We hypothesized that metformin may ameliorate inflammation via AMPK interaction with critical inflammatory cytokines The aim of this study was to observe the effects of metformin on expression of nuclear factor-κB （NF-κB）,monocyte chemoattractant protein-1 （MCP-1）,intercellular adhesion molecule-1 （ICAM-1） and transforming growth factor-beta 1 （TGF-β1） induced by high glucose （HG） in cultured rat glomerular mesangial cells （MCs）.Methods MCs were cultured in the medium with normal concentration glucose （group NG,5.6 mmol/L）,high concentration glucose （group HG,25 mmol/L） and different concentrations of metformin （group M1,M2,M3）.After 48-hour exposure,the supernatants and MCs were collected.The expression of NF-κB,MCP-1,ICAM-1,and TGF-β1 mRNA was analyzed by real time polymerase chain reaction.Westem blotting was used to detect the expression of AMPK,phospho-Thr-172 AMPK （p-AMPK）,NF-κB p65,MCP-1,ICAM-1,and TGF-β1 protein.Results After stimulated by HG,the expression of NF-κB,MCP-1,ICAM-1,TGF-β1 mRNA and protein of MCs in group HG increased significantly compared with group NG （P ＜0.05）.Both genes and protein expression of NF-κB,MCP-1,ICAM-1,TGF-β1 of MCs induced by high glucose were markedly reduced after metformin treatment in a dose-dependent manner （P ＜0.05）.The expression of p-AMPK increased with the rising of metformin concentration,presenting the opposite trend,while the level of total-AMPK protein was unchanged with exposure to HG or metformin.Conlusion Metformin can suppress the expression of NF-κB,MCP-1,ICAM-1 and TGF-β1 of glomerular MCs induced by high glucose via AMPK activation,which may partlv contribute to its reno-protection.     

Effect of N-tosyl-L-phenylalanylchloromethyl Ketone on Tumor Necrosis Factor-alpha -induced NF-κB Activation and Apoptosis in U937 Cell Line

Summary: To investigate the effect of N-tosyl-L-phenylalanylchloromethyl ketone (TPCK) on tumor necrosis factor-alpha-induced NF-κB activation and apoptosis in U937 cell line, changes and subcellular localization of NF-κB/p65 and IκB-α were observed by fluorescencemicroscopy and expression and degradation of IκB-α by flow cytometry. The apoptosis of U937 cells was measured by flow cytometry and electrophoresis of DNA. Immunolfluorescence assay showed that NF-κB/p65,IκB-α only localized in cytoplasm. After TNF-α stimulation, p65 was localized only in nuclei, and IκB-α was only localized in cytoplasm and decreased. The changes of TNF-α stimulation were specifically inhibited by TPCK. Flow cytometry also revealed the downregulation of IκB-α protein during TNF-α-induced apoptosis and the down-regulation was specifically inhibited by TPCK. Flow cytometry also showed the apoptosis of U937 cells after TNF-α induction. DNA ladder can be detected in cells treated by TNF-α. It is concluded that degradation of IκB-α protein and NF-κB/p65 translocation occur during TNF-α-induced apoptosis of U937 cells, suggesting the activation of NF-κB.TPCK-sensitive protease plays an important role in the degradation of IκB-α protein induced by TNF-α in U937 cells. TPCK sensitive protease also plays an important role in the apoptosis of U937 cells induced by TNF-α.     

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.     

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.     

Huangqin Decoction Attenuates DSS-Induced Mucosal Damage and Promotes Epithelial Repair via Inhibiting TNF-α-Induced NF-κB Activation

Objective:To investigate the protective effect of Chinese herbal formula Huangqin Decoction(HQD)on ulcerative colitis mouse model induced by dextran sulphate sodium(DSS)and human intestinal epithelial cell injury induced by tumour necrosis factor-α(TNF-α).Methods:In vivo,30 male C57BL/6 mice were divided into 5 groups using a random number table(n=6 per group),including control,DSS,5-aminosalicylic acid(5-ASA),HQD low-(HQD-L)and high-dose(HQD-H)groups.The colitis mouse model was established by 3%(w/v)DSS water for 5 days.Meanwhile,mice in the HQD-L,HQD-H and 5-ASA groups were administrated with 100,200 mg/kg HQD or 100 mg/kg 5-ASA,respectively,once daily by gavage.After 9 days of administration,the body weight,disease activity index(DAI)score and colon length of mice were measured,the pathological changes of colons were analyzed by hematoxylin-eosin staining(HE)staining,and the levels of serum interleukin(IL)-6,IL-1βand TNF-αwere measured by enzyme linked immunosorbent assay.In vitro,the human colon epithelial normal cells(FHC cells)were exposed to HQD(0.6 mg/mL)for 12 h and then treated with TNF-α(10 ng/mL)for 24 h.The tight junction(TJ)protein expression levels of Claudin-4 and Occludin,and the protein phosphorylation levels of p65 and inhibitor of nuclear factor kappaB(NF-κB)-α(IκBα)were measured by Western blot.Results:In vivo,compared with the DSS group,HQD-H treatment attenuated the weight loss and reduced DAI score of mice on the 8th day(P<0.05).Moreover,HQD-H treatment ameliorated the colon shortening in the DSS-induced colitis mice(P<0.05).HE staining showed HQD attenuated the pathological changes of colitis mice,and the histological scores of HQD-H and 5-ASA groups were significantly decreased compared with the DSS group(P<0.05).Meanwhile,HQD-H and 5-ASA significantly decreased the serum IL-1β,IL-6 and TNF-αlevels of mice(P<0.05).In vitro experiments showed that HQD up-regulated Occludin and Claudin-4 protein expressions and inhibited p-p65 and p-IκBαlevels in FHC cells compared with the TNF-αgroup(P<0.05).Conclusion:HQD significantly relieved the symptoms in DSS-induced colitis mice by inhibiting pro-inflammatory cytokines expression and maintained the homeostasis of TJ protein in FHC cells by suppressing TNF-α-induced NF-κB activation.     

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.     

Role of Nuclear Transcription Factor-κB in Endotoxin-induced Shock in Rats

To investigate the role of NF-κB in endotoxic shock in rats. the model of endotoxinshock rats was induced by intravenous infusion of lipopolysaccharidc (LPS). 1 h. 2 h. 4 h and 6 h after LPS injection, the activation of NF-κB in blood mononuclear cells and the content of TNF-α and IL-6 in plasma was detected by enzyme-linked immunoadsordent assay (ELISA). The level of mean arterial pressure (MAP) and the histopathological changes of lung and liver were also observed. The activation of NF-κB in mononuclear cells increased 1 h after LPS injection and reached its peak 2 h after the injection, and its level was higher than that of normal group. The level of TNF-α was increased 1 h after the infusion and peaked 2 h after the injection, and its level was higher than that of normal group after LPS infusion. The content of IL-6 increased gradually with time. the IL-6 level was higher than that of normal group after LPS injection. MAP was decreased gradually with time and its level was lower than that of normal group after LPS injection. Pathological examination showed that endotoxic shock could cause pulmonary alveolar hemorrhage, edema and infiltration of inflammatory cell in lung tissue and congestion, edema, capillary dilation and inflammatory cell infiltration in liver tissue. It is concluded that NF-κB can up-regulate the expression of TNF-α and IL-6 in plasma and play an important role in endotoxin induced shock in rats.     

Extract of Fructus Schisandrae chinensis Inhibits Neuroinflammation Mediator Production from Microglia via NF-κB and MAPK Pathways

Objective: To investigate the anti-neuroinflammation effect of extract of Fructus Schisandrae chinensis(EFSC) on lipopolysaccharide(LPS)-induced BV-2 cells and the possible involved mechanisms. Methods: Primary cortical neurons were isolated from embryonic(E17-18) cortices of Institute of Cancer Research(ICR) mouse fetuses. Primary microglia and astroglia were isolated from the frontal cortices of newborn ICR mouse. Different cells were cultured in specific culture medium. Cells were divided into 5 groups: control group, LPS group(treated with 1 μg/mL LPS only) and EFSC groups(treated with 1 μg/mL LPS and 100, 200 or 400 mg/mL EFSC, respectively). The effect of EFSC on cells viability was tested by methylthiazolyldiphenyltetrazolium bromide(MTT) colorimetric assay. EFSC-mediated inhibition of LPS-induced production of pro-inflammatory mediators, such as nitrite oxide(NO) and interleukin-6(IL-6) were quantified and neuron-protection effect against microglia-mediated inflammation injury was tested by hoechst 33258 apoptosis assay and crystal violet staining assay. The expression of pro-inflammatory marker proteins was evaluated by Western blot analysis or immunofluorescence. Results: EFSC(200 and 400 mg/mL) reduced NO, IL-6, inducible nitric oxide synthase(iN OS) and cyclooxygenase 2(COX-2) expression in LPS-induced BV-2 cel s(P0.01 or P0.05). EFSC(200 and 400 mg/mL) reduced the expression of NO in LPS-induced primary microglia and astroglia(P0.01). In addition, EFSC al eviated cel apoptosis and inflammation injury in neurons exposed to microglia-conditioned medium(P0.01). The mechanistic studies indicated EFSC could suppress nuclear factor(NF)-κB phosphorylation and its nuclear translocation(P0.01). The anti-inflammatory effect of EFSC occurred through suppressed activation of mitogen-activated protein kinase(MAPK) pathway(P0.01 or P0.05). Conclusion: EFSC acted as an anti-inflammatory agent in LPS-induced glia cel s. These effects might be realized through blocking of NF-κB activity and inhibition of MAPK signaling pathways.     

The role of NF-κB in hepatocellular carcinoma cell

Objective To evaluate the role of nuclear factor-kappaB (NF-κB) and IκBα in hepatocellular cacinoma (HCC) SMMC7721 cells, the consequence of NF-κB inhibition in SMMC7721 cells transfected with mutated IκBα (mIκBα) plasmid and the effect of stable inhibition of NF-κB activity in combination with Doxorubicin.Methods Western blot was used to determine the expression of NF-κB and IκBα in SMMC7721 cells and normal liver cells. Nuclear protein was used to evaluate the binding of the 32P-labeled tandem κB sequence using electrophoretic mobility shift assay and the expression of NF-κB using Western blot between SMMC7721 cells transfected with mIκBα plasmid (SMMC7721-MT) and control cells. Furthermore, cell viability was plotted between SMMC7721-MT and control cells. The binding of κB sequence and cell viability between SMMC7721-MT and control cells at different concentrations of Doxorubicin were also investigated.Results Western blot analysis for nuclear extract showed more P50 (NF-κB1) and P65 (RelA) expression in SMMC7721 cells compared with normal liver cells. The expression of cytosolic IκBα protein in SMMC7721 cells was less than that in normal cells. SMMC7721-MT cells inhibited NF-κB nuclear translocation at 0, 24, 48 and 96 hours. Furthermore, NF-κB cannot be detected in the nuclear protein of SMMC7721-MT cells by Western blot. By calculating cell viability, the proliferation of SMMC7721-MT cells was shown to be suppressed more significantly than that of control cells. NF-κB in untransfected cells was activated by Doxorubicin in a dose-dependent manner, but that in SMMC7721-MT cells was not induced at low concentrations of Doxorubicin. Compared with untransfected cells, the viability of SMMC7721-MT cells was significantly suppressed at the same concentration of Doxorubicin (P&lt;0.01）.Conclusions The present study demonstrates that upregulation of NF-κB and downregulation of inhibitory kappaB (IκBα) in SMMC7721 cells are related with the growth of hepatocellular cacinoma cells. Stable expression of mIκBα in SMMC7721-MT cells can inhibit NF-κB nuclear translocation and suppress cell growth. Furthermore, stable inhibition of NF-κB activity in combination with Doxorubicin can significantly inhibit cell proliferation in SMMC7721-MT cells. Thus, modulation of NF-κB may represent an improvement in the efficacy of HCC therapies and be worthy of further research and investigation.