Influence of bifidobacterium on NF-κB and I κBα of experimental large bowel carcinoma

AIM:To explore the antitumor mechanisms of bifidobacteria adolescence in vivo. METHODS:The activity of NF-κB and its inhibiting protein I κBα of large bowel carcinoma tissues was detected by using laser scanning confocal microscope and immunohistochemistry.RESULTS:The positive cell density of NF-κB of large bowel carcinoma transplantation tumors in bifidobacterium injection group was markedly lower than that in tumor control group(P＜0.01).The expression of I κBα was contary in the two group. The average fluorescent strength of I κBα of large bowel carcinoma in bifidobacterium injection group was significantly higher than that in tumor control group(P＜0.01).CONCLUSION:Bifidobacteria adolescence could inhibit the degrade of I κBα and the activition of NF-κB in large bowel carcinoma in vivo.     

Effects of eicosapentaenoic acid on expression of nuclear factor κB and release of cytokines in human umbilical vein endothelial cells stimulated by lipopolysaccharide

AIM: To investigate the effects of eicosapentaenoic acid(EPA) on the expression of nuclear factor kappa B(NF-κB) and release of vascular endothelial growth factor(VEGF), IL-1α and IL-6 in cultured human umbilical vein endothelial cells(HUVECs) stimulated by lipopolysaccharide(LPS).METHODS: HUVECs were obtained from cell strain and cultured in vitro. HUVECs were divided into 4 groups: control group, LPS group, 0.030 g/L EPA treatment group and 0.050 g/L EPA treatment group. The cells were cultured with LPS alone in LPS group and incubated with EPA for 1 h in the EPA pretreatment groups at the concentrations of 0.030 g/L and 0.050 g/L before LPS stimulation. Twenty-four hours after stimulated by LPS, the protein expression of NF-κB p65 in HUVECs were assessed by Western blotting analysis at different time points. The production of VEGF, IL-1α and IL-6 in cultured HUVECs was evaluated by ELISA. The effects of EPA on the protein expression of NF-κB p65 and the production of VEGF, IL-1α and IL-6 in HUVECs challenged by LPS were also determined.RESULTS: Compared with control group, the protein expression of NF-κB p65 was significantly increased in HUVECs induced by LPS and was inhibited by EPA. Compared with control group, the protein expression of VEGF, IL-1α and IL-6 was dramatically increased in HUVECs induced by LPS and most of the increase was inhibited by EPA.CONCLUSION: LPS enhances the protein expression of NF-κB and the release of VEGF, IL-1α and IL-6. EPA inhibits the protein expression of NF-κB, and the production of VEGF and the inflammatory cytokines in cultured HUVECs stimulated by LPS, indicating that EPA may be useful for preventing and treating neovascular and inflammatory diseases.     

Effects of PDS on rat brain cortical nuclear factor kappa B in LPS shock

AIM:To explore the molecular mechanism of brain tissue injury induced by lipopolysaccharide (LPS),the effects of panaxadiol (PDS) on the expression of nuclear factor kappa B (NF-κB) in cerebral cortex of rat with LPS shock were studied. METHODS:Rats were randomly divided into LPS roup,LPS+dexamethasone group,LPS+PDS group and control group. The DNA binding activity and protein expression of NF-κB were observed. These indices were assayed at 1 h and 4 h after intravenous injection of LPS (4 mg·kg^-1). RESULTS:EMSA showed that PDS inhibited NF-κB DNA-binding activity in nuclear extracts at both 1 h and 4 h after LPS injection,compared with the LPS group (P<0.01). Western blotting showed that PDS down-regulated the expression of p65 and p50 protein in the nuclear extracts compared with the LPS group. However,the expression of p65 and p50 protein from cytosolic extracts did not show any significant change. CONCLUSION:PDS may alleviate brain injury by inhibiting NF-κB activation.     

Effects of burn sera on IκBα degradation and NF-κB activation in monocytes in vitro

AIM: To investigate the effects of burn sera on IκBα degradation, NF-κB activation in peripheral blood monocytes (PBMCs) in order to explore the role of burn sera on activation of monocytes. METHODS: PBMCs isolated from healthy volunteers were stimulated by sera from healthy volunteers and burn patients and by burn sera together with PDTC (pyrrolidine dithioncarbamate). Activation of monocytic NF-κB was tested by electrophoretic mobility shift assay (EMSA) and the degradation of monocytic IκBα was determined by Western blotting. RESULTS: When compared to that in control group, cytosolic IκBα degradation occurred within 30 min after PBMCs stimulated by burn sera, and peaked at 60 min. But IκBα gradually recovered in the cytoplasm after 2 h of stimulation. Meanwhile, activity of monocytic NF-κB was markedly increased, reached the peak at 30 min to 60 min after stimulation, and gradually decreased after 2 h of stimulation. PDTC (an antioxidants) effectively inhibited the monocytic IκBα degradation and activation of NF-κB induced by burn sera. CONCLUSION: Burn sera might induce the degradation of IκBα, then activate NF-κB, which ultimately lead to the secretion of cytokines from the monocytes.     

Role of nuclear factor κB on the expression of interleukin-6 in mouse mesangial cells induced by interleukin-1β

AIM:To investigate the regulatory role of nuclear factor κB (NF-κB) in the expression of interleukin-6 in mesangial cells (MC) induced by interleukin-1β.METHODS:Activation of NF-κB was measured by electrophoresis mobility shift assay (EMSA). RT/PCR and ELISA were used to detect IL-6 mRNA expression and IL-6 production, respectively.RESULTS:rhIL-1β could rapidly stimulate the activation of NF-κB in MC, and increase the expression of IL-6 mRNA and protein. PDTC, one of the inhibitor of NF-κB, could inhibit the expression of IL-6 in mRNA and protein in MC stimulated by rhIL-1β.CONCLUSION:IL-6 expression induced by IL-1β may be regulated by NF-κB in MC, NF-κB may modulate the immune-inflammatory reaction in glomerular disease.     

Effects of fractalkine on nuclear factor-κB activation and endogenous fractalkine mRNA expression in fibroblast-like synoviocytes from patients with rheumatoid arthritis

AIM: To investigate the effects of fractalkine(FKN) on nuclear factor kappa B (NF-κB) activation and endogenous FKN mRNA expression in fibroblast-like synoviocytes (FLS) from the patients with rheumatoid arthritis (RA). METHODS: RA-FLS were gained through tissue culture. Fractalkine at 100 μg/L was used to stimulate RA-FLS for 0 h, 1 h and 2 h. The expression of NF-κB p65 protein in cytoplasm and nucleus was detected by Western blotting, representing the activation of NF-κB in RA-FLS. RA-FLS was stimulated with fractalkine at concentration of 100 μg/L for 0 h, 12 h or 18 h, and the mRNA expression of FKN in RA-FLS was detected by RT-PCR.RESULTS: After stimulated with recombinant human FKN for 1 h, the expression of NF-κB p65 protein in the cytoplasm of RA-FLS was obviously lower than that in RA-FLS without FKN treatment in control group (P<0.05). After stimulated with FKN for 2 h, the expression of NF-κBp65 protein in nucleus was obviously higher than that in RA-FLS of control group (P<0.05). Recombinant human FKN at concentration of 100 μg/L induced endogenous FKN mRNA expression in RA-FLS in a time-dependent manner. The mRNA expression of FKN in RA-FLS obviously increased after stimulated with FKN for 18 h (P<0.05). CONCLUSION: FKN up-regulates the expression of endogenous FKN mRNA, suggesting a positive feedback. FKN can activate the NF-κB and may play an important role in the beginning of joint inflammation, angiogenesis and bone destruction.     

Expression of nuclear factor-κB in asthmatic guinea pigs and the effect of erigeron breviscapus on it

AIM:To explore the expression of nuclear factor-κB(NF-κB)in asthmatic guinea pigs,and the effect of erigeron breviscapus,a protein kinase C(PKC)inhibitor,on the expression of nuclear factor-κB(NF-κB).METHODS:48 guinea pigs were randomly divided into 6 groups(n=8).Airway resistance and eosinophilic inflammation of airway wall were examined,the expression of NF-κB in the lung tissue was detected by immunohistochemical staining.RESULTS:The expression of NF-κB was mainly found in airway epithelium,all the asthmatic animals showed significantly higher optical densities than that of the normal control group(P＜0.01),and the rats subjected therapeutic treatment for two weeks showed significantly lower NF-κB expression than those of the asthmatic groups(P＜0.01).Positive correlation exist between the airway resistance and the percentage of cells expressing NF-κB in epithelium,and between the amount of eosinophil in airway wall and the percentage of cells expressing NF-κB in epithelium(P＜0101).CONCLUSION:The increased expression of NF-κB in airway epithelium of the asthmatic guinea pigs suggested that NF-κB may be involved in asthma.And result that the increased expression of NF-κB was inhibited significantly by the treatment of the erigeron breviscapus suggested that PKC may play a significant role in the pathogenesis of asthma through NF-κB.     

Effect of nuclear factor kappa B inhibitor on the responsiveness of pulmonary artery rings to protein kinase C in rats with hypoxia-induced pulmonary hypertension

AIM: To investigate the role of nuclear factor kappa B (NF-κB) inhibitor in the responsiveness of isolated pulmonary artery rings to protein kinase C (PKC) in rats with hypoxia-induced pulmonary hypertension. METHODS: The pulmonary artery rings removed endothelium were prepared from model rats with hypoxia-induced pulmonary hypertension and control rats. The effects of PKC activator PMA (0.5 μmol/L) time-response cures and NF-κB inhibitor PDTC (0-1 000 μmol/L) concentration-response cures on pulmonary artery rings were observed. The responsiveness of each ring was tested by applying a maximally effective concentration of phenylephrine (10 μmol/L). Data were calculated as relative ratio by the maximally responseness ( P₀ ) setting at 100%, and the relative responseness tensions to PMA and PDTC were derived by dividing by the counts in P₀. t_1/2 and T show the time achieving half-maximal response and lasting maxima response to 0.5 μmol/L PMA, respectively. RESULTS: mPAP and RV/(LV+S)in hypoxia group were greater than those in control group(P<0.05).For the responseness of the artery rings to PMA of 0.5 mol/L,the relat ive tensions of hypoxia group were significantly higher(P<0.05)as compared with respective controls;mean t_1/2 in hypoxia group was shorter than that in control group(P<0.05).Mean T in hypoxia group was longer than that in control group(P<0.05).For the relative tensions of the artery rings to PDTC and PMA,hypoxia group were higher than those of controls in the range of PDTC 0-100 mol/L(P<0.05);the relative tensions of two group significantly decreased beyond PDTC of 500 mol/L(P<0.05). CONCLUSIONS: The responsiveness of pulmonary artery rings to PMA was increased during hypoxia and decreased to PDTC in concentration-dependent manner. These results further suggest that changes of PKC－NF-κB signaling pathway of pulmonary artery smooth muscle cells may be involved in vasoconstriction of hypoxia-induced pulmonary hypertension.     

Changes of nuclear factor-κB and inducible nitric oxide synthase in hypoxic pulmonary hypertension

AIM: To observe the changes of nuclear factor-κB (NF-κB) activity and inducible nitric oxide synthase (iNOS) expression in hypoxic pulmonary hypertension (HPH). METHODS :The rat model of HPH was used. The NF-κB activity and iNOS expression in lung tissue were determined by immunohistochemistry (IHC), in situ hybridization (ISH), RT-PCR and Western blot. RESULTS: ISH showed that iNOS mRNA expression in intraacinar pulmonary arteriole (IAPA) in H28d group (hypoxic treatment for 28 days) was stronger than that in normal group, H5d group and H14d group. RT-PCR showed that the iNOS mRNA in H28 group was 2.1 times, 1.9 times and 1.8 times higher than that in normal group, H5d group and H14d group, respectively. The nucleic anti-NF-κB stain was observed in H28d group, which was significantly stronger, but the I-κB amount was 2.8 times, 2.7 times and 2.5 times lower than that in normal group, H5d group and H14d group, respectively. CONCLUSION: The activity of NF-κB was correlated with the hypoxic pulmonary vessel structural remodeling and iNOS expression.     

Roles of nuclear factor-κB in the development of rat pancreatic fibrosis mediated by angiotensin II

AIM: To determine the effects of NF-κB on the development of rat pancreatic fibrosis mediated by angiotensin II. METHODS: Spraque-Dawley rats (200-300g) were randomly divided into normal group, control group and losartan-treatment group. Pancreatic fibrosis was induced by injection of 2% TNBS into biliopancreatic duct. Rats in losartan-treatment group and control group were respectively treated with losartan (10 mg·kg^-1·d^-1) by gavage and the same volume of saline vehicle. The expression, distribution, and activation of NF-κB were studied by Western blot, immunohistochemistry and TransAM^TM. Toluidine blue staining and transmission electron microscopy were also used to observe the number, distribution and degranulation of mast cells. In addition, RT-PCR was performed to detect the intrapancreatic ICAM-1 mRNA expression. RESULTS: The expression and activity of intrapancreatic NF-κB p65 protein were significantly increased on day 3 after operation, reaching peak on day 7 [(0.406±0.086)mg/g total protein].. Mast cell activation was observed and ICAM-1 mRNA levels on day 3 and 7 were up-regulated in control group. Losartan treatment inhibited NF-κB expression and activation, reduced mast cell infiltration and degranulation and decreased ICAM-1 mRNA expression compared with control rats. CONCLUSION: It might be associated with the expression and activation of NF-κB that angiotensin II mediates inflammation and fibrosis in the early stage of pancreatic fibrosis.     

Advances in IκB protein family research

IκB is an inhibitor of nuclear factor-kappa B (NF-κB) and presents in the majority of cells. Eight structurally related members of the mammalian IκB family have been described:IκBα, IκBβ, IκBε, Bcl-3, IκBγ, IκBδ, p100 and p105. The ankyrin repeat domain of IκB can interact with NF-κB, and sequester NF-κB in the cytoplasm as inactive complexes. Most recently, IκBα has been found to inhibit p53 tumor suppressor protein by binding p53 to form a cytoplasmic p53·IκBα complex and studies have shown that p100 profoundly sensitizes cells to death-receptor-mediated apoptosis. The current review is to describe the members of IκB family, their related signaling pathways, and their application in tumor therapy.     

Effect of nuclear factor-κB on proliferation of airway smooth muscle cells regulated by protein kinase C in asthmatic rats

AIM: To investigate the effect of protein kinase C (PKC)- nuclear factor-κB (NF-κB) signal pathway on proliferation of airway smooth muscle cells (ASMCs) in asthmatic rats.METHODS: (1) 16 Wistar rats were divided into asthmatic group (8 rats) and control group (8 rats).ASMCs from asthmatic group and control group were treated with PKC agonist PMA and NF-κB inhibitor PDTC.The proliferation of ASMCs was examined by cell cycle analysis,MTT colorimetric assay and proliferating cell nuclear antigen (PCNA) immunofluorescence staining,respectively.NF-κB activity was detected by NF-κB p65 immunofluorescence staining and electrophoretic mobility shift assay (EMSA),respectively.RESULTS: The percentage of S phase,A value,the positive expression rate of PCNA,the positive expression rate of NF-κB p65 and EMSA value in asthmatic ASMCs treated with PMA were higher than those in asthmatic ASMCs without treatment (P<0.05).After asthmatic ASMCs previously treated with PDTC,then with PMA,the above figures were lower than those in asthmatic ASMCs only treated with PMA and without treatment (P<0.05).The above figures in asthmatic ASMCs only treated with PDTC were lower than those in asthmatic ASMCs without treatment (P<0.05).CONCLUSION: NF-κB may contribute to the proliferation of ASMCs in asthmatic rat,in which PKC－NF-κB signal pathway is involved.     

Effects of diterpenoid C from ether extract of Radix Curcumae on nuclear factor-κB activity in LPS-induced human gastric adenocarcinoma SGC7901 cells

AIM: To observe the effects of diterpenoid C from ether extract of Radix Curcumae (RC) on the activity of nuclear factor-κB in human gastric adenocarcinoma SGC7901 cells stimulated with lipopolysaccharide (LPS). METHODS: SGC7901 cells were normally cultured, induced by LPS, or treated with LPS plus RC. The protein expression of IKKα, IKKβ, p65, phosphorylated p65 and phosphorylated IκBα was assayed by Western blotting. NF-κB DNA binding activity was determined by electrophoretic mobility shift assay. RESULTS: RC reduced the protein expression of IKKα, IKKβ, p65, phosphorylated p65 and phosphorylated IκBα induced by LPS. NF-κB DNA binding activity increased much greatly by LPS stimulation, while RC resisted the action of LPS. CONCLUSION: RC may attenuate the secretion of inflammatory cytokines by inhibiting the activation of NF-κB signaling pathway.     

Effect of GRK5 on activation of rat astrocytes

AIM: To study the effect of G-protein-coupled receptor kinase 5 (GRK5) on the activation of astrocytes in the brain cortex of newborn Wistar rats. METHODS: GRK5 gene was silenced in the model of rat brain cortex astrocytes in vitro for 24 h. N-acetylcysteine (NAC), which is a known inhibitor of NF-κB, was added into the culture medium according to gene silencing for 24 h. The expression levels of GFAP and caspase-3 were detected by the method of immunofluorescence, and the mRNA levels of NF-κB, TNF-α, IL-1β and iNOS were determined by real-time PCR. Moreover, the activity of SOD and concentrations of TNF-α and NO were measured. RESULTS: GRK5 gene silencing increased the expression of NF-κB at mRNA and protein levels obviously (P<0.01), and the mRNA levels of IL-1β and iNOS increased synchronously (P<0.01). Furthermore, caspase-3-positive cells in GRK5 siRNA group were increased compared with control siRNA group (P<0.01). Treatment with NAC obviously reduced the activity of NF-κB and weakened the effects induced by GRK5 siRNA (P<0.05). CONCLUSION: GRK5 siRNA increases NF-κB activity and induces the activation of astrocytes.