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Objective To evaluate the effect of electroacupuncture ( EA) preconditioning on hipp-ocampal I-kappa B-α ( IκB-α)∕nuclear factor κB ( NF-κB)∕intercellular adhesion molecule-1 ( ICAM-1) signaling pathway during cerebral ischemia-reperfusion ( I∕R) in mice. Methods A total of 120 healthy male C57BL∕6 mice, aged 10-12 weeks, weighing 20-25 g, were divided into 4 groups ( n=30 each) u-sing a random number table method: control group ( group C) , cerebral I∕R group ( group I∕R) , precondi-tioning with EA at non-acupoint+cerebral I∕R group ( group S+I∕R) and preconditioning with EA at Baihui acupoint + cerebral I∕R group ( group E+I∕R) . The cerebral I∕R injury model was established by occlusion of bilateral common carotid arteries followed by reperfusion for 72 h in mice anesthetized with halothane or chloral hydrate in group I∕R. Group S+I∕R received EA at the points 2 mm lateral to the acupoints of Baihui for 5 consecutive days, and then the cerebral I∕R injury model was established. Group E+I∕R received EA at Baihui acupoints with a sparse-dense wave at an intensity of 1 mA and a frequency of 2 Hz∕15 Hz for 30 min once a day for 5 consecutive days, and then the cerebral I∕R injury model was established. Neurobe-havioral score was assessed at 24 and 48 h of reperfusion. Then 5 mice in each group were sacrificed, and the hippocampal tissues were obtained and stained with haematoxylin and eosin for examination of the patho-logical changes in hippocampal CA1 region and for determination of the expression of IκB-α, NF-κB, ICAM-1, interleukin-6 ( IL-6) , IL-1β protein and mRNA by Western blot and real-time polymerase chain reaction, respectively. Results Compared with group C, neurobehavioral score was significantly in-creased, and the expression of hippocampal IκB-α, NF-κB, ICAM-1, IL-6 and IL-1βprotein and mRNA was up-regulated in I∕R, S+I∕R and E+I∕R groups ( P<0. 05) . Compared with group I∕R, neurobehavioral score was significantly decreased, and the expression of hippocampal IκB-α, NF-κB, ICAM-1, IL-6 and IL-1β protein and mRNA was down-regulated in group E+I∕R (P<0. 05), and no significant change was found in the parameters mentioned above in group S+I∕R (P>0. 05). Compared with group S+I∕R, neu-robehavioral score was significantly decreased, and the expression of hippocampal IκB-α, NF-κB, ICAM-1, IL-6 and IL-1β protein and mRNA was down-regulated in group E+I∕R ( P<0. 05) . Conclusion The mechanism by which EA preconditioning attenuates cerebral I∕R injury may be related to inhibiting activation of hippocampal IκB-α∕NF-κB∕ICAM-1 signaling pathway in mice.
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Objective To examine the expressions of IKKε protein in the specimens and cells of epithelial ovarian cancer and investigate the effect of IKKε inhibitor on cell proliferation and apoptosis. Methods (1) A total of 118 cases of patients with the median age of 59 who have accepted surgical treatment due to ovarian cancer in the First Affiliated Hospital of Dalian Medical University from January 2006 to April 2013 were selected. Twenty cases of patients with the median age of 55 who have accepted hysterectomy and salpingo-oophorectomy due to uterine leiomyoma during the same period were selected as the control. The expressions of IKKε protein were detected by immunohistochemistry in normal ovarian tissues and epithelial ovarian cancer specimens,and the relationship between the expressions of IKKε and the clinical features of patients was analyzed. IKKε protein was determined by western blot in various ovarian cancer cells, including SKOV3, OV2008, C13, A2780S, A2780CP, OV4, OV5, OV8, and CAOV3 treated with or without IKKε inhibitor. The cellular proliferation and apoptosis of ovarian cancer cells after 48 hours treatment of IKKε inhibitor were analyzed by methyl thiazolyl tetrazolium (MTT) assay and flow cytometry, respectively. Results (1) The immunohistochemical results showed that IKKε was highly expressed in epithelial ovarian cancer specimens with the expression rate 66.1% (78/118), compared with normal ovarian tissue with the expression rate 35.0% (7/20), which exhibited statistically significant difference (χ2=6.993, P=0.008). The expression of IKKε protein was correlated with International Federation of Gynecology and Obstetrics (FIGO) stage, histological grade, the level of CA125 in preoperative serum and distribution of the tumor (P0.05). (2) IKKε was widely overexpressed in different levels in SKOV3, OV2008, C13, A2780S, A2780CP, OV4, OV5, OV8, and CAOV3 cells, and the expression of IKKε decreased as the increase of the concentration of IKKε inhibitor (0.1 and 0.5 μmol/L) in OV2008, C13, A2780S, and A2780CP cells after 48 hours treatment. Different concentrations of IKKε inhibitor (0.05, 0.1, 0.5, 1, 5, 10, and 25 μmol/L) significantly inhibited the proliferation of OV2008, C13, A2780S, A2780CP, and SKOV3 cells in a concentration-dependent manner (P<0.05), and the half maximal inhibitory concentration (IC50) was 0.43, 0.86, 0.10, 0.19, and 0.24 μmol/L, respectively. The cell apoptotic rate of OV2008, C13, A2780S, A2780CP, and SKOV3 cells was significantly increased after 48 hours treatment of IKKεinhibitor with the concentration of 0.1 and 0.5 μmol/L (P<0.05). Conclusions The IKKε protein in epithelial ovarian cancer specimens and cells is overexpressed. IKKε inhibitor could inhibit cellular proliferation and induce apoptosis in a concentration-dependent manner. Together, the result indicated that IKKε may be a candidate target for the treatment of ovarian cancer in future.
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Objective To investigate the effecof Iκkinases(IKK) inhibitoIKK16 on the proliferation and apoptosiof hu-man glioblastomcell line U87 and itpossible mechanism .MethodHuman glioblastomcell line U87 wacultured in vitro and divided into 4 group:blank group(withouintervention treatmen) ,control group(1% dimethyl sulfoxide) ,low dose IKK group (70 nmol/L IKK16) and high dose IKK group(200 nmol/L IKK16) .The Mtassay waused to detecthe effecof IKK16 on the proliferation of U87 cellathe low concentration and the high concentration .Western blowaused to investigate the effecof IKK16 on the expression of p65 ,cyclinD1 ,caspase-3 and bcl-2 .ResultThe U87 proliferation rate had no statistical difference be-tween the blank group and the control group(P＞0 .05) .Compared with the control group ,IKK16 could significantly suppresthe proliferation of U87 cell(P＜0 .05) .Athe same time ,IKK16 significantly reduced the expression of p65 ,CyclinD1 and Bcl-2 ,and increased the expression of caspase-3(P＜0 .05) with dose-dependenmanne.Conclusion IKK16 could suppresthe proliferation of human glioblastomcell line U87 and promote the apoptosi.
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Objective To explore the effect of I kappa B kinase (IKK) on liver during hepatic ischemia reperfusion (HIR) in rats.Methods Wister rats were divided randomly into HIR group in which hepatic reperfusion was given after 60 minutes of ischemia by interruption of the arterial and portal venous blood supply to the left lobes and middle lobes of the liver; HIR + PDTC treatment group in which PDTC (120 mg/kg) were injected via the dorsum vein of penis before ischemia reperfusion; and sham control group in which midline laparotomy was performed without vascular occlusion and treatment.Expression levels of IKK were measured with In situ hybridization(ISH).The NF-κB activities were determined with EMSA.Expression levels of TNF-α were measured with immunohistochemistry (IH).Serum levels of ALT were measured.Results Expression level of IKK was increased markedly from 0 to 12h and peaked 6h after reperfusion in HIR group.NF-κB was activated 0 ～ 12h after reperfusion and activities of NF-κB were maximal 6h after reperfusion in HIR group rats compared with sham control group.Expression level of TNFα was increased markedly from 0 to 12h and peaked 6h after reperfusion in HIR group.Serum levels of ALT were increased significantly after reperfusion in H1R group.Expression level of IKK was lowered markedly in HIR + PDTC group from 0 to 12h after reperfusion.NF-κB activities were significantly lower in HIR +PDTC group than in HIR group from 0 to 12h after reperfusion.Expression level of TNF-α was lowered markedly in HIR + PDTC group as compared with HIR group from 0 to 12h after reperfusion.Serum level of ALT was decreased significantly after reperfusion in HIR + PDTC group as compared with HIR group.Conclusion HIR can activate IKK-β which promotes the activation of NF-κB,then NF-κB results in upregulation transcription of TNF-α gene which gives rise to the release of other inflammatory cytokines and triggers uncontrolled inflammatory response,and induces hepatic injury.Blocking IKK-NF-κB pathway may be an effective approach to checking the generation and development of ALI,PDTC plays important prophylaxis and treatment roles in hepatic injury after HIR.
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Objective To observe the effects of gypenosides (GP) on nuclear factor κB (NF-κB) and p38 mitogen activated protein kinase (p38MAPK) signaling pathways in photodamaged skin of mice,and to explore the mechanisms underlying the protective effects of GP against photodamage.Methods Eighty Balb/C female mice were randomly divided into 8 groups: blank control group receiving no treatment,ultraviolet B (UVB) model group receiving UVB irradiation for 60 seconds,GP group Ⅰ receiving topical GP treatment followed by UVB irradiation,GP group Ⅱ receiving UVB irradiation followed by topical GP treatment,VitE group Ⅰ receiving topical VitE treatment followed by UVB irradiation,VitE group Ⅱ receiving UVB irradiation followed by topical VitE treatment,matrix group Ⅰ receiving topical matrix treatment followed by UVB irradiation,matrix group Ⅱ receiving UVB irradiation followed by topical matrix treatment.UVB irradiation lasted 60 seconds at one time,and was given once every other day for 7 times to establish a skin model of photodamage.The interval between irradiation and topical treatment was 30 minutes in all the groups except the control group and UVB model group.After the last treatment,mice were sacrificed.Western blot was performed to measure the protein expressions of inhibitor of NF-κB(IκB),inhibitor of NF-κB kinase (IKK),p38MAPK as well as phosphorylated p38MAPK (pp38) in skin tissue from the mice.Results No expressions of IκB or IKK were observed in the blank control group.The expression level of IκB was 0.40 ± 0.07 in UVB model group,significantly lower than that in GP group Ⅰ (1.63 ± 0.85,P ＜ 0.05) and GP group Ⅱ (0.90 ± 0.40,P ＜ 0.05),whereas the level of IKK protein was higher in UVB model group than in the GP group Ⅰ and Ⅱ (2.01 ± 1.75vs.0.23 ± 0.12 and 0.45 ± 0.29,both P ＜ 0.05).No significant difference was observed in the expression of IκB or IKK proteins between the GP group Ⅰ,Ⅱ,VitE group Ⅰ and Ⅱ or in the expression of p38MAPK between the 8 groups.The phosphorylated p38MAPK expression in UVB model group was significantly higher than that in GP group Ⅰ and Ⅱ (0.835 ± 0.049 vs.0.425 ± 0.054 and 0.571 ± 0.090,both P＜ 0.05),but similar to that in VitE groups.Conclusions UVB can activate NF-κB and phosphorylated p38MAPK signaling pathways; GP 1.5％ cream can inhibit UVB-induced activation of NF-κB and p38MAPK pathways,which may be one of the mechanisms underlying its protective effects against inflammation and photodamage.
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Objective To explore the roles of IκBα and TGF-β1 on airway inflammation in rats with chronic bronchitis induced by smoking and study the effects and mechanism of anti-inflammation of pretreatment with ambroxol. Methods Sixty male wistar rats were randomly divided into four groups: Normal dosage group, model group, high dosage group and low dosage group. The rats with chronic bronchitis were established by smoking. For high and low dosage group, rats were pretreated respectively with ambroxol group, rats were pretreated with normal saline through peritoneal injection as much as the low dosage group.After 76 days, the histopathologic changes stained in hemotoxylin and eosin (H. E.) of bronchopulmonary tissues were observed under opticalmicroscope, white cell counts and differential analysis were performed in BALF, the expression of IκBα and TGF-β1 were detected by immunohistochemistry. Results The pathological changes of model group were in consistent with that of chronic bronchitis, but the degrees were significantly reduced in high and low dosage groups. Compared with those in normal group, the white cell count and the neutrophilic granulocyte count of BALF in model group were significantly increased and the macrophagocyte count decreased, and the expression of IκBαwas significantly decreased(t =3.24,3.31,3.29,3.48, P ＜0. 05) and the TGF-β1 significantly increased (P ＜0. 05). Compared with those in the model group, the white cell count and the neutrophilic granulocyte count of BALF in high and low dosage group were significantly decreased and the macrophagocyte count increased, the expression of IκBαwas significantly increased (t =2. 86,2. 97,2. 92,3.52,2.42,2. 88,2. 58,3.48, P ＜0. 05) and the TGF-β1 significantly decreased (P ＜0. 05) . Compared with those in low dosage group, the expression of TGF-β1 was decreased and the expression of IκBαincreased in high dosage group (t =2. 82,3.64, P ＜0. 05). Conclusions Down-regulating the expression of IκBα and up-regulating of TGF-β1 may be involved in the process of airway inflammation in rats with chronic bronchitis induced by smoking. Ambroxol might have better effects on ameliorating airway inflammation by up-regulating the expression of IκBα and down-regulating of TGF-β1.
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The expression of inducible nitric oxide synthase (iNOS) is a critical factor in both normal physiological functions and the pathogenesis of disease. This study was undertaken to determine the molecular mechanism by which nitric oxide (NO) exerts negative feedback regulation on iNOS gene expression. Isolated rat hepatocytes stimulated with cytokines exhibited a marked increase in NO production as well as iNOS mRNA and protein levels, which were significantly reduced by pretreatment of the NO donors S-nitroso-N-acetyl-D, L-penicillamine (SNAP) and V-PYRRO/NO. This effect of SNAP was inhibited when NO was scavenged using red blood cells. Pretreatment with oxidized SNAP, 8-Br-cGMP, NO2-, or NO3- did not suppress the cytokine-induced NO production. Moreover, LPS/ IFN-gamma-stimulated RAW264.7 cells, which produce endogenous NO, expressed lower levels of iNOS, IL-1beta, IL-6 and TNF-alpha mRNAs, without changes in their mRNA half-lives, than those in the presence of the iNOS inhibitor NG-monomethyl- L-arginine. The iNOS gene transcription rate exhibited an 18-fold increase after cytokine stimulation, which was significantly inhibited by SNAP pretreatment. SNAP also blocked cytokine- induced increase in NF-kappa B activation, iNOS promoter activity, nuclear translocation of cytosolic NF-kappa B p65 subunit, and I kappa B alpha degradation, which correlated with its inhibitory effect on phosphorylation and ubiquitination of I kappa B. These data indicate that NO down-regulates iNOS gene expression and NO production by inhibiting the post-translational processes of I kappa B alpha thereby preventing NF-kappa B activation. These results identify a novel negative feedback mechanism whereby NO down-regulates iNOS gene expression.
Subject(s)
Animals , Rats , Cell Line , Cell Nucleus/metabolism , Cyclic GMP/analogs & derivatives , Cytokines/genetics , Down-Regulation , Hepatocytes/metabolism , I-kappa B Proteins/metabolism , Lipopolysaccharides/pharmacology , Nitric Oxide/biosynthesis , Nitric Oxide Synthase/biosynthesis , Penicillamine/analogs & derivatives , Phosphorylation , Promoter Regions, Genetic/genetics , Protein Processing, Post-Translational , Protein TransportABSTRACT
PURPOSE: The nuclear factor-kappa B (NF-kappa B) has been known to regulate the inflammatory and immune process by transcription of inflammatory intermediates. The purpose of the present study is to show the difference in activity of NF-kappa B and its inhibitory factor-I kappa B alpha in patients with rheumatoid arthritis, osteoarthritis and normal control subjects. MATERIALS AND METHODS: Synovial membrane samples were obtained at the time of orthopedic surgery from the knees of 7 patients with RA and 7 patients with OA. Two control samples were obtained from an amputee with no history of arthritis. We designed the primer of the subunit p65 of NF-kappa B and I kappa B alpha, measured the activity of them by RT-PCR, and analyzed the expression of NF-kappa B by immunohistochemical staining. RESULTS: From the results of RT-PCR, the expression levels of NF-kappa B was found to be higher in synovial tissues obtained from patients with RA than from synovial tissue obtained from patients with OA, and the least from the control group. The expression levels of I kappa B alpha were not different statistically among the three groups. Immunohistochemical staining for the NF-kappa B was dominant in synovial tissue from patients with RA. The result of immunohistochemical staining was similar to the results of RT-PCR for NF-kappa B. The localization of the staining was predominantly nuclear. CONCLUSION: In this study, activity of NF-kappa B of rheumatoid arthritis was higher than the other group, but expressions of I kappa B alpha were no different between the diseases. Further studies about specific inhibitors of NF-kappa B will benefit the development of rheumatoid arthritis regimens with greater efficacy.
Subject(s)
Humans , Amputees , Arthritis , Arthritis, Rheumatoid , I-kappa B Proteins , Knee , NF-kappa B , Orthopedics , Osteoarthritis , Synovial MembraneABSTRACT
BACKGROUND: Cyclosporin A(CsA) and tacrolimus(FK506) have been widely used as immunosuppressants. The effects of CsA, or FK506, on the IkappaB/NF-kappaB pathway have been shown to vary according to the cell type. However, their effects on the IkappaB/NF-kappaB pathway have not been reported in bronchial epithelial cells. In this study, the effects of CsA and FK506 on the IkappaB/NF-kappaB pathway in bronchial epithelial cells, monocytes, lymphocytes and alveolar macrophages were evaluated. The relationship between their effects on the IkappaB/NF-kappaB pathway and IkappaB kinase(IKK) activity was also investigated. METHODS: BEAS-2B and A549 cells, pulmonary alveolar macrophages, peripheral blood monocytes and lymphocytes were used. The cells were pre-treated with CsA, or FK506, for various time periods, followed by stimulation with TNF-alpha, LPS or IL-1beta. The I(kappa)B(alpha) expressions were assayed by Western blot analyses. The IKK activity was evaluated by an in vitro immune complex kinase assay, using GST-I(kappa)B(alpha) as the substrate. RESULTS: Neither CsA nor FK506 affected the level of I(kappa)B(alpha) expression in any of the cell types used in this study. CsA pre-treatment inhibited the TNFalpha-induced I(kappa)B(alpha) degradation in bronchial epithelial cells. In contrast, the TNFalpha-induced I(kappa)B(alpha) degradation was not affected by FK506 pre-treatment. However, FK506 suppressed the cytokine-induced I(kappa)B(alpha) degradation in the pulmonary alveolar macrophages, peripheral blood monocytes and lymphocytes. The inhibitory effect of CsA, or FK506, on I(kappa)B(alpha) degradation was not related to IKK. CONCLUSIONS: CsA and FK506 suppressed the I(kappa)B(alpha) degradation in bronchial epithelial cells, mono. cytes, lymphocytes and alveolar macrophages, so this may not be mediated through IKK.
Subject(s)
Antigen-Antibody Complex , Blotting, Western , Cyclosporine , Epithelial Cells , Immunosuppressive Agents , Lymphocytes , Macrophages, Alveolar , Monocytes , NF-kappa B , Phosphotransferases , Tacrolimus , Tumor Necrosis Factor-alphaABSTRACT
BACKGROUND AND OBJECTIVES: K B is a transcription factor in immune and inflammatory reactions, and exerts its effect by expressing cytokines and chemokines, enzymes, receptors and adhesion molecules. Many of the inflammatory proteins that are expressed in respiratory airways are also regulated, at least in part, by NF-K B. The purpose of this study is to investigate the NF-K B and its inhibitory protein, I-K B expression in normal nasal mucosa and allergic rhinitis. MATERIALS AND METHODS: We have evaluated 20 allergic rhinitis mucosa and 7 normal inferior turbinate. Immunohistochemical study and RT-PCR were done for NF-K B and I-K B expression. RESULTS: NF-K B and I-K B were localized at the epithelium, and in the subepithelial inflammatory cells, vascular endothelial cells, and glandular endothelial cells in both normal nasal mucosa and allergic rhinitis. Compared to normal nasal mucosa, both activated and inactivated forms of NF-K B were significantly increased in the epithelial cell layer of allergic rhinitis. However, for the I-K R expression, no difference could be observed. RT-PCR revealed a significant difference in the expression level of NF-K B mRNA between nasal mucosa and allergic rhinitis, but I- K B expression showed no difference. CONCLUSIONS: This results show that NF-K B is usually activated in the nasal epithelial cell layer and NF-K B may play a role in the inflammatory reaction of allergic rhinitis. But further study is required for the role of I-K B.
Subject(s)
Chemokines , Cytokines , Endothelial Cells , Epithelial Cells , Epithelium , I-kappa B Proteins , Mucous Membrane , Nasal Mucosa , NF-kappa B , Rhinitis , RNA, Messenger , Transcription Factors , TurbinatesABSTRACT
Objective To detect the activation of NF－? B signaling pathway in peripheral blood mononuclear cells(PBMCs) of patients with systemic lupus erythematosus(SLE) and investigate its clinical significance. Methods Activation of NF－? B was detected by electrophoretic mobility shift assay( EMSA), the expression of I? B? protein and its phosphorylated products were detected by Western blot. Anti-dsDNA antibody, IgG and IgM in supernatant of PBMC culture were tested by ELISA. Results (1)Elevated activation of NF－? B in PBMC of SLE patients was found when compared with the controls(P
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The aim of the study was to explore the role of NF ?B activation in the pathogenesis of smoke inhalation injury and to evaluate the effects of dexamethasone on NF ?B activation. 130 Wistar rats were inflicted with smoke inhalation injury and randomized equally into the control group, smoke inhalation injury group and dexamethasone treatment group.The expressions of NF ?B P 65 ,I?B ?,TNF ?,and ICAM 1 proteins in the lung tissue were determined by immunohistochemistry at 2,6,12,24,48,72 hours after smoke inhalation injury. The results showed that,after smoke inhalation ,the expressions of NF ?BP 65 ,TNF ?,and ICAM 1 increased,whereas the expression of I?B ? decreased. In the dexamethasone treatment group,the expressions of NF ?BP 65 ,TNF ?,and ICAM 1 were down regulated, and I?B ? was up regulated.These results suggest that NF ?B activation may participate in the pathogenesis of smoke inhalation injury.Dexamethasone could suppress NF ?B activation, thus partially blocked the production of cytokines and adhesion molecules,and in turn reduced the damage of inflammatory response. Therefore NF ?B activation might be a key point in the development of smoke inhalation injury and modulation of activation of NF?B might be a potential therapeutic strategy to treat this injury at the transcription level.