NF-kappaB P65 Protein Research Articles

Role of nuclear transcription factor kappa B (NF-kappaB) for MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahyropyridine)-induced apoptosis in nigral neurons of mice

The biochemical and cellular changes that occur following treatment with MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahyropyridine) are remarkably similar to that seen in idiopathic Parkinson's disease. In this study, we investigated the time course changes of NF-kappaB (Nuclear factor kappa B) p65 protein and apoptosis in the substantia nigra after MPTP treatment in mice. Four administrations of MPTP at 2 h intervals showed a significant and severe decrease of the number of TH (tyrosine hydroxylase) immunopositive neurons in the substantia nigra of mice from 5 h up to 21 days posttreatment. Densities of DAT (dopamine transporter) immunoreactivity were also significantly decreased in nigral neurons of mice from 1 up to 21 days after MPTP treatment. GFAP (glial fibrillary acidic protein) immunopositive cells were increased significantly in the substantia nigra from 5 h up to 21 days after MPTP treatment. In contrast, isolectin B 4 positive microglia were increased markedly in the substantia nigra only 3 and 7 days after MPTP treatment. On the other hand, a significant increase of NF-kappaB p65 immunoreactivity was observed mainly in glial cells of the substantia nigra from 5 h to 3 days after MPTP treatment. A significant increase of ssDNA (single stranded DNA) immunopositive apoptotic neurons was also observed in the substantia nigra from 5 h to 3 days after MPTP treatment. These results demonstrate that dopaminergic neuronal loss may be caused by apoptosis due to increased cytokines and apoptosis-related proteins via the activation of NF-kappaB in reactive astrocytes of the substantia nigra after MPTP treatment in mice. Thus our findings suggest that the inhibition of NF-kappaB activation in astrocytes may be useful intervention in Parkinson's disease and other neurogenerative disorders where apoptosis or inflammation plays a key role in disease pathogenesis.

Upregulation of NF-κB upon differentiation of mouse embryonic stem cells

NF-kappaB is a transcriptional regulator involved in many biological processes including proliferation, survival, and differentiation. Recently, we reported that expression and activity of NF-kappaB is comparatively low in undifferentiated human embryonic stem (ES) cells, but increases during differentiation. Here, we found a lower expression of NF-kappaB p65 protein in mouse ES cells when compared with mouse embryonic fibroblast cells. Protein levels of NF-kappaB p65 and relB were clearly enhanced during retinoic acid-induced differentiation. Furthermore, increased DNA binding activity of NF-kappaB in response to TNF-alpha, an agonist of NF-kappaB signaling, was seen in differentiated but not undifferentiated mouse ES cells. Taken together with our previous data in human ES cells, it is likely that NF-kappaB expression and activity of the NF-kappaB signaling pathway is comparatively low in undifferentiated ES cells, but increases during differentiation of ES cells in general.

Synergistic inhibitory effect of sulforaphane and 5‐fluorouracil in high and low metastasis cell lines of salivary gland adenoid cystic carcinoma

The present study aimed to evaluate the growth-inhibitory effect of sulforaphane (SFN) and a traditional chemotherapy agent, 5-fluorouracil (5-Fu), against the proliferation of salivary gland adenoid cystic carcinoma high metastatic cell line (ACC-M) and low metastasis cell line (ACC-2). Furthermore, the expression of nuclear factor kappa B (NF-kappaB) which induces resistance to anticancer chemotherapeutic agents was also detected. The combination effect of SFN and 5-Fu was quantitatively determined using the method of median effect principle and the combination index. The nuclear NF-kappaB p65 expression after treatment with the SFN-5-Fu combination was also evaluated by western blot analysis. The ACC-M and ACC-2 cells exhibited relative resistant to 5-Fu. Treatment ACCs cells with SFN and 5-Fu in combination, led to synergistic inhibition on cell growth and a decreased expression in nuclear NF-kappaB p65 protein. This synergistic inhibitory effect was more significant in ACC-M cells, which is associated with the greatly decreased expression of NF-kappaB p65 (almost 5-fold) after the combination treatment. Our results demonstrate synergism between SFN and 5-Fu at higher doses against the ACC-M and ACC-2 cells, which was associated with the decreased expression of nuclear NF-kappaB p65 protein.

Study of the role of nuclear factor-kappa B in preterm birth with subclinical chorioamnionitis

To investigate the role of nuclear factor-kappa B (NF-kappaB) in preterm birth with subclinical chorioamnionitis. From October 2005 to October 2006, 111 cases including 36 cases of preterm birth in labor, 37 cases of full term gravida with spontaneous labor and 38 cases of full term gravida without threatened labor in the Hunan Province People's Hospital, third Xiangya Hospital of Central South University and Changsha Maternal and Child Care Service Center were enrolled in the study. After delivery, by pathology results of fetal membrane they were divided into two groups: subclinical chorioamnionitis group (subclinical infectious group) and non-infectious group. Immunohistochemical staining and RT-PCR were used to observe the change of the p65 subunit of NF-kappaB family in maternal blood and fetal membrane in subclinical infectious group and non-infectious group. (1) The incidence of subclinical chorioamnionitis: there were 24 cases of subclinical chorioamnionitis in the 36 cases of preterm birth in labor (67%), 7 cases in the 37 cases of full term gravida with spontaneous labor group (19%) and 3 cases in the 38 cases of full term gravida without threatened labor group (8%). There was a significant difference among the three groups (P < 0.01). In the totally 111 cases, 34 cases were classified as subclinical infectious group and 77 cases as non-infectious group. (2) In fetal membrane, the median of the average staining intensity of NF-kappaB p65 protein was higher in the subclinical chorioamnionitis group (8.0) than those in non-infectious group (4.0). Similarly, the average staining intensity of NF-kappaB p65 mRNA was higher in the subclinical infectious group (47.5 +/- 17.2) than those in non-infectious group (31.3 +/- 13.6). There was a significant difference between the two groups (P < 0.01). (3) In maternal blood, the expression of NF-kappaB p65 protein and mRNA was higher in subclinical chorioamnionitis group(8.0 and 42.6)than those in non-infectious group(4.0 and 23.6).There was a significant difference between the two groups (P < 0.01). (4) The concentration of NF-kappaB p65 protein in fetal membrane was positively correlated with that of maternal blood (r = 0.581, P < 0.01) and the concentration of NF-kappaB p65 mRNA in fetal membrane was positively correlated with that of maternal blood (r = 0.571, P < 0.01). The expression of NF-kappaB in maternal blood and fetal membrane in preterm birth with subclinical chorioamnionitis is higher and the two are correlated with each other. NF-kappaB p65 could be an accurate biochemical marker in predicting subclinical chorioamnionitis in preterm birth. NF-kappaB p65 plays an important role in the pathogenesis of subclinical chorioamnionitis in preterm birth.

Losartan reduced connexin43 expression in left ventricular myocardium of spontaneously hypertensive rats

To assess the effect of angiotensin II type 1 (AT(1)) receptor antagonist losartan on myocardium connexin43 (Cx43) gap junction (GJ) expression in spontaneously hypertensive rats (SHRs) and investigate possible mechanisms. Sixteen 9-week-old male SHRs and 8 age-matched male Wistar-Kyoto (WKY) rats were included in this study. SHRs were randomly divided into two groups to receive losartan at 30 mg/(kg x d) by oral gavage once daily for 8 weeks (SHR-L) or vehicle (0.9% saline) to act as controls (SHR-V); WKY rats receiving vehicle for 8 weeks served as normotensive controls. At the end of the experiment, rats were sacrificed and the hearts were removed. Expressions of Cx43 and nuclear factor-kappaB p65 (NF-kappaB p65) proteins in all three groups were observed and further investigations on the effect of angiotensin II type 1 receptor antagonist losartan (30 mg/(kg x d), 8 weeks) on Cx43 expression were conducted with Western blot and immunohistochemistry. NF-kappaB p65 protein in nuclear extracts was determined by Western blot. Left ventricular (LV) hypertrophy was prominent in SHRs, Cx43 and NF-kappaB p65 protein expressions were obviously upregulated and Cx43 distribution was dispersed over the cell surface. Treatment with losarton reduced the over-expressions of Cx43 and NF-kappaB p65 in LV myocardium. The distribution of Cx43 gap junction also became much regular and confined to intercalated disk after losartan treatment. Cx43 level was upregulated in LV myocardium of SHR during early stage of hypertrophy. Angiotensin II type 1 receptor antagonist losartan prevented Cx43 gap junction remodeling in hypertrophied left ventricles, possibly through the NF-kappaB pathway.

Berberine reverses free-fatty-acid-induced insulin resistance in 3T3-L1 adipocytes through targeting IKKβ

To investigate the effects and molecular mechanisms of berberine on improving insulin resistance induced by free fatty acids (FFAs) in 3T3-L1 adipocytes. The model of insulin resistance in 3T3-L1 adipocytes was established by adding palmic acid (0.5 mmol/L) to the culture medium. Berberine treatment was performed at the same time. Glucose uptake rate was determined by the 2-deoxy-[(3)H]-D-glucose method. The levels of IkB kinase beta (IKKbeta) Ser(181) phosphorylation, insulin receptor substrate-1(IRS-1) Ser(307) phosphorylation, expression of IKKbeta, IRS-1, nuclear transcription factor kappaB p65 (NF-kappaB p65), phosphatidylinositol-3-kinase p85 (PI-3K p85) and glucose transporter 4 (GLUT4) proteins were detected by Western blotting. The distribution of NF-kappaB p65 proteins inside the adipocytes was observed through confocal laser scanning microscopy (CLSM). After the intervention of palmic acid for 24 h, the insulin-stimulated glucose transport in 3T3-L1 adipocytes was inhibited by 67%. Meanwhile, the expression of IRS-1 and PI-3K p85 protein was reduced, while the levels of IKKbeta Ser(181) and IRS-1 Ser(307) phosphorylation, and nuclear translocation of NF-kappaB p65 protein were increased. However, the above indexes, which indicated the existence of insulin resistance, were reversed by berberine although the expression of GLUT4, IKKbeta and total NF-kappaB p65 protein were not changed during this study. Insulin resistance induced by FFAs in 3T3-L1 adipocytes can be improved by berberine. Berberine reversed free-fatty-acid-induced insulin resistance in 3T3-L1 adipocytes through targeting IKKbeta.

Glutamine decreases intestinal nuclear factor kappa B activity and pro-inflammatory cytokine expression after traumatic brain injury in rats

To investigate whether glutamine supplementation modulates intestinal nuclear factor kappa B (NF-kappaB) activity and pro-inflammatory cytokine expression after traumatic brain injury (TBI) in rats. Right parietal cortical contusion in male rats was made by the weight-dropping method. After trauma, the rats were randomly given chow alone or glutamine mixed chow for 5 d. Gut samples were extracted at 5 d postinjury. We measured NF-kappaB binding activity by electrophoretic mobility shift assay; NF-kappaB subunits p50 and p65 expression by immunohistochemistry; the concentrations of interleukin-1beta, tumor necrosis factor-alpha and interleukin-6 by enzyme-linked immunosorbent assay; intestinal mucosal morphological changes by histopathological study and electron microscopy; and apoptosis by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling staining. Administration of glutamine following TBI could decrease NF-kappaB binding activity, NF-kappaB p65 protein expression and concentrations of pro-inflammatory cytokines in the gut. TBI-induced damage of gut structure was ameliorated after glutamine supplementation. The results of the present study suggest that the therapeutic benefit of post-TBI glutamine supplementation might be due to its inhibitory effects on intestinal NF-kappaB activation and pro-inflammatory cytokine expression.

Memory and learning impairment induced by dexamethasone in senescent but not young mice

In this study, the memory and learning impairment induced by dexamethasone in young mice and senescent mice were evaluated by step-down inhibitory avoidance task and passive avoidance test. Colorimetric MTT(tetrazole 3-(4,5-dimethylthiazol-2-yl-)-2,5-diphenyltetrazolium bromide) assay and TUNEL staining were used to investigate the influence of dexamethasone on hippocampal neuronal cell death with amyloid beta-protein. It was determined the effect of dexamethasone on intracellular calcium ([Ca(2+)](i)) with amyloid beta-protein 25-35 by fluorescence imaging with a confocal laser microscope using fluo-3 acetoxymethylester (AM) as a fluorescent dye. The effect of dexamethasone on amyloid beta-protein 25-35-induced nuclear factor kappaB (NF-kappaB) was analyzed by western blot. The results showed that twenty one days dexamethasone exposure resulted in an impairment of memory and learning in senescent but not young mice. Pretreatment of isolated hippocampal neurons with dexamethasone increased the vulnerability of the hippocampal neurons to amyloid beta-protein 25-35, enhanced [Ca(2+)](i) and down-regulated the increased level of nuclear NF-kappaB p65 proteins induced by amyloid beta-protein 25-35. These results demonstrated that glucocorticoids could potentiate the neurotoxic action of amyloid beta-protein by further increasing the level of [Ca(2+)](i) and down-regulating the level of nuclear NF-kappaB protein. Since amyloid beta-protein increases in the brain with aging, glucocorticoids potentiation of the neurotoxic action of amyloid beta-protein maybe one of the mechanisms responsible for glucocorticoids-induced memory and learning impairment in senescent but not young mice, which maybe relevance to the etiology of Alzheimer's disease.

Effects of curcumin on peroxisome proliferator-activated receptor γ expression and nuclear translocation/redistribution in culture-activated rat hepatic stellate cells

The function of peroxisome proliferator-activated receptor gamma (PPARgamma) in hepatic fibrogenesis remains largely unknown. Curcumin is a natural substance extracted form Curcuma Longa Linn and has a variety of pharmacological effects. In this study, the effects of curcumin on the proliferation, activation and apoptosis of rat hepatic stellate cells (HSCs) through PPARgamma signaling were investigated. HSCs were isolated from the normal Sprague Dawley rats through in situ perfusion of the liver with Pronase E and density-gradient centrifugation with Nycodenz. Cells were treated with curcumin, troglitazone, salvianolic acid B or GW9662. The effect on HSCs proliferation was determined by MTT colorimetry. Total RNA was extracted by TRizol reagent and gene levels were determined by semi-quantitative RT-PCR. Total cellular and nuclear protein were isolated and separated by 10% sodium dodecy lsulfate polyacrylamide gel electrophoresis. Protein levels were determined by Western blot. Cell apoptosis was detected by Hoechst 33258 staining. PPARgamma subcellular distribution was detected by immunofluorescent staining. The activities of MMP-2 and 9 were measured by Gelatin zymograph assay. Curcumin suppressed HSCs proliferation in a dose-dependent manner. As HSCs underwent gradual activation with culture prolongation the PPARgamma nuclear expression level decreased. Curcumin up-regulated PPARgamma expression and significantly inhibited the production of alpha-SMA and collagen I. PPARgamma is expressed in the cytoplasm and nucleus and is evenly distributed in HSCs, but accumulated in the nucleus of HSCs and disappeared from cytoplasm after curcumin treatment. Hoechst 33258 staining showed that curcumin induced the apoptosis of culture-activated HSCs and significantly increased pro-apoptotic Bax expression and reduced anti-apoptotic Bcl-2 expression. Cyclin D1 gene, activated NFkappaB p65 protein and TGFbetaR-I protein expression were down-regulated significantly by curcumin. The activities of MMP-2 and MMP-9 were enhanced significantly by curcumin. Curcumin can inhibit the proliferation and activation of HSCs, induce the apoptosis of activated HSCs and enhance the activities of MMP-2 and MMP-9. The effects of curcumin are mediated through activating the PPARgamma signal transduction pathway and associated with PPARgamma nuclear translocation/redistribution.

Nuclear factor kappa‐B p50 and p65 subunits expression in dementia with Lewy bodies

Dementia with Lewy bodies (DLB) is the second most common cause of neurodegenerative dementia after Alzheimer's disease (AD). Parkinsonism in DLB is mainly caused by neuronal loss with Lewy bodies (LBs) in the substantia nigra, thereby inducing degeneration of the nigrostriatal dopaminergic pathway similar to that in Parkinson's disease (PD). To clarify the pathogenesis of DLB, it is important to investigate the mechanisms involved in the degenerative process of LB-bearing neurones. Several reports suggest a role for nuclear factor kappa-B (NFkappaB) in the manifestation of neurodegenerative conditions such as AD and PD. The aim of the present study was to investigate whether NFkappaB subunits are involved in the pathogenesis of neurodegeneration in DLB by measuring tyrosine hydroxylase (TH), NFkappaB p65 and p50 protein expression in frontal cortex and substantia nigra pars compacta of DLB and control human brains. An increase, although not statistically significant, in nigral TH expression in DLB cases was observed. There were no differences in the cortical and nigral expression levels of NFkappaB p65 subunit between control and DLB cases. Western blots of the frontal cortex showed no differences in the expression levels of NFkappaB p50 subunit. However, NFkappaB p50 levels were significantly decreased (P < 0.05) in the pars compacta of the substantia nigra in the DLB cases in comparison with controls. The decrease in the expression of the p50 subunit in the substantia nigra of DLB cases achieved in the present study may increase the vulnerability of the dopaminergic neurones to a possible neurotoxic effect of p65 subunit. Thus, normal levels of NFkappaB p65 might be toxic in neurones with a low expression of the NFkappaB p50 subunit.

Effect of BN52021 on NFκ-Bp65 expression in pancreatic tissues of rats with severe acute pancreatitis

To investigate dynamic changes and significance of expression of NF-kappaBp65 in pancreatic tissues of rats with severe acute pancreatitis (SAP), as well as BN52021 effects. Wistar male rats were randomly divided into negative control group (NC group, n=60), SAP-model group (SAP group, n=60), and BN52021-treated group (BN group, n=60), and each of the above groups was respectively divided into 6 subgroups at different time points after operation (1 h, 2 h, 3 h, 6 h, 12 h, and 24 h) (n=10). By RT-PCR and Western blot, NF-kappaBp65 mRNA and its protein expression in pancreatic tissues of rats were detected respectively. The expression of NF-kappaBp65 mRNA dynamically changed in both SAP groups and BN groups. The mRNA level was higher in SAP groups than NC groups at 2 h, 3 h, 12 h, and 24 h after operation (P<0.05), higher in BN groups than NC groups at all time points (P<0.05), and higher in BN groups than SAP group at 1 h (P<0.05). The NF-kappaBp65 protein level was higher in SAP groups than NC groups at 1 h, 3 h, and 6 h (P<0.01), and 2 h, 12 h, and 24 h (P<0.05), higher in BN groups than NC groups at all time points (P<0.05), and lower in BN groups than SAP groups at 1 h, 3 h, and 6 h (P<0.05). The expression of NF-kappaBp65 in pancreatic tissues is dynamically changed and the changes play an important role in pathogenesis of SAP. BN52021 exerts therapeutic effects through reducing the expression level of NF-kappaBp65 protein in the early stage of SAP.

Influence of dexamethasone on inflammatory mediators and NF-kappaB expression in multiple organs of rats with severe acute pancreatitis.

To observe the therapeutic effects of dexamethasone on rats with severe acute pancreatitis (SAP) and investigate the influences of dexamethasone on the inflammatory mediators and NF-kappaB expression in multiple organs of SAP rats as well as the mechanisms involved. Ninety Sprague-Dawley (SD) rats with SAP were randomly divided into the model group (n = 45) and dexamethasone treatment group (n = 45), and another 45 rats were selected for the sham operation group. All groups were randomly subdivided into the 3 h, 6 h and 12 h groups, each group containing 15 rats. The survival of all groups and pathological changes of multiple organs (liver, kidney and lung) were observed at different time points after the operation. The pathological score of multiple organs was carried out, followed by the determination of amylase, endotoxin and TNF-alpha contents in blood. The tissue microarray was used to detect the expression levels of NF-kappaB p65 protein in multiple organs. There was no marked difference between the model group and treatment group in the survival rate. The amylase content of the treatment group was significantly lower compared to the model group at 12 h (P < 0.01, 7791.00 vs 9195.00). Moreover, the endotoxin and TNF-alpha levels of the treatment group were significantly lower than that of the model group at 6 h and 12 h (P < 0.01, 0.040 vs 0.055, 0.042 vs 0.059 and P < 0.05, 58.30 vs 77.54, 38.70 vs 67.30, respectively). Regarding the changes in liver NF-kappaB expression, the model group significantly exceeded the sham operation group at 3 h (P < 0.01, 1.00 vs 0.00), and the treatment group significantly exceeded the sham operation group at 12 h (P < 0.01, 1.00 vs 0.00), whereas no marked difference was observed between the model group and treatment group at all time points. The kidney NF-kappaB expression level in the treatment group significantly exceeded the model group (P < 0.05, 2.00 vs 0.00) and the sham operation group (P < 0.01, 2.00 vs 0.00) at 12 h. No NF-kappaB expression in the lung was found in any group. Dexamethasone can lower the amylase, endotoxin and TNF-alpha levels as well as mortality of SAP rats. NF-kappaB plays an important role in multiple organ injury. Further studies should be conducted to determine whether dexamethasone can ameliorate the pathological changes of multiple organs by reducing the NF-kappaB expression in the liver and kidney. The advantages of tissue microarrays in pancreatitis pathological examination include time- and energy- saving, and are highly efficient and representative. The restriction of tissue microarrays on the representation of tissues to various extents due to small diameter may lead to the deviation of analysis.

RETRACTED: The anti-inflammatory activity of 1,25-dihydroxyvitamin D3 in macrophages

In a previous study we demonstrated a down-regulatory effect of vitamin D active metabolite (1,25(OH)(2)D(3)) and its vitamin D(2) analog (1,24(OH)(2)D(2)) on TNFalpha expression in macrophages. We also found an inhibitory effect in the physiological concentration (10(-10)M) of 1,25(OH)(2)D(3) which was dose-dependent. This down-regulation, caused by the decrease in NFkappaB activity by 1,25(OH)(2)D(3) and 1,24(OH)(2)D(2), was demonstrated in P388D1 cells transfected with NFkappaB reporter gene (p NFkappaB-Luc) and by EMSA. In our present study we investigated the processes leading to reduced NFkappaB activity on P388D1 cells. A decrease in nuclei NFkappaB-p65 and an increase in cytosolic NFkappaB-p65, were measured, while no changes in total NFkappaB-p65 mRNA and protein levels were observed. Simultaneously, a significant increase in both mRNA and protein levels of the NFkappaB-cytosolic inhibitor, IkappaBalpha, were determined. The half-life of IkappaBalpha-mRNA increased, with a parallel decrease in the phosphorylation of its protein, as the first step of ubiquitinization and degradation. The present results demonstrate that 1,25(OH)(2)D(3) and 1,24(OH)(2)D(2) inhibit TNFalpha expression in macrophages, by increasing IkappaBalpha and decreasing NFkappaB activity. Since NFkappaB is a major transcription factor for TNFalpha and other inflammatory mediators, these findings suggest that 1,25(OH)(2)D(3) and 1,24(OH)(2)D(2) may be used therapeutically as anti-inflammatory agents.

Reversal Effect of Thalidomide on Established Hepatic Cirrhosis in Rats via Inhibition of Nuclear Factor-κB/Inhibitor of Nuclear Factor-κB Pathway

Suppression of nuclear factor-kappaB (NF-kappaB)/inhibitor of nuclear factor-kappaB (IkappaB) signaling pathway is a potential property of thalidomide. This study was designed to investigate the effects of thalidomide on expressions of NF-kappaB, IkappaB and intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule (VCAM-1) in established rat liver cirrhosis. Rat liver cirrhosis was achieved by IP injection of carbon tetrachloride (CCl4) three times weekly for 8 weeks. CCl4 was then discontinued and thalidomide (100 mg/kg) or its vehicle was administered daily by gavage for 6 weeks. Hydroxyproline (HYP) content in liver was detected by biochemical assay. NF-kappaBp65, ICAM-1, VCAM-1 and alpha-smooth muscle actin (alpha-SMA) protein in the liver, IkappaBalpha protein in cytoplasm and NF-kappaBp65 protein in nucleus and ICAM-1, VCAM-1 mRNA levels in the liver were studied using immunohistochemistry, Western blot, and reverse transcriptase polymerase chain reaction, respectively. Compared with the spontaneous recovery of cirrhosis, the histopathology of liver of rats given thalidomide was significantly improved. HYP content in liver, the expressions of ICAM-1, VCAM-1 mRNA and protein, NF-kappaBp65 and alpha-SMA protein were decreased significantly and IkappaBalpha protein in liver was elevated significantly in this group. Thalidomide may exert its effect on downregulation of NF-kappaB-induced adhesion molecules and activation of hepatic stellate cell via inhibition of degradation of IkappaB to reverse established rat hepatic cirrhosis.

Alleviation of neuropathic pain by intrathecal injection of antisense oligonucleotides to p65 subunit of NF-κB

Treatment of neuropathic pain remains a challenge. The current study investigated the therapeutic effect of intrathecal administration of NF-kappaB antisense oligodeoxynucleotides (ODNs) on mechanical allodynia and thermal hyperalgesia in a chronic constriction injury (CCI) model of rats. Lumbar intrathecal catheters were implanted in male Sprague-Dawley rats and a CCI model was established. Thermal and mechanical nociceptive thresholds were assessed with paw withdrawal latency (PWL) to radiant heat and von Frey filaments. The phosphorothioate-modified antisense ODNs to p65 subunit of NF-kappaB were administered intrathecally on each of five consecutive days post-CCI. Nuclear NF-kappaB p65 expression was assessed by western blot. CCI induced mechanical allodynia and thermal hyperalgesia and significantly increased NF-kappaB p65 protein expression. Intrathecal injection of antisense ODN markedly suppressed the expression of NF-kappaB p65 protein and significantly attenuated CCI-induced mechanical allodynia and thermal hyperalgesia. The activation of NF-kappaB pathway may contribute to neuropathic pain in CCI rats. Suppression of NF-kappaB could be a potential new strategy for the treatment of neuropathic pain.

FAK-related nonkinase attenuates hypertrophy induced by angiotensin-II in cultured neonatal rat cardiac myocytes

To examine the inhibitory effect of FAK-related nonkinase (FRNK) in cardiac hypertrophy in vitro and investigate the possible mechanisms. A functional fragment of FRNK cDNA was amplified by reverse transcription-polymerase chain reaction and cloned into the vector pcDNA3.1. Hypertrophy in neonatal rat cardiac myocytes was established with angiotensin-II stimulation. The pcDNA3.1-FRNK or pcDNA3.1 was respectively transfected into cardiomyocytes by Lipofectamine 2000. The surface area and mRNA expression of atrial natriuretic peptide (ANP) of myocytes were employed to detect cardiac hypertrophy. NF-kappaB p65 protein in nuclear extracts, phosphorylation levels of ERK1/2 (p-ERK1/2) and AKT (p-AKT), as well as total ERK1/2, and AKT in variant treated cardiomyocytes were determined by Western blot. Under the stimulation of angiotensin II, the surface area of myocytes and levels of ANP mRNA were significantly increased. But transient transfection with pcDNA3.1-FRNK in advance may reduce the surface area and expression of ANP mRNA of hypertrophic myocytes. The protein levels of NF-kappaB p65 in nuclear extracts and p-ERK1/2, p-AKT in FRNK treated cardiomyocytes were significantly decreased compared with that in angiotensin-II induced cardiomyocytes, while different treatments had little effect on total ERK1/2 and AKT. FRNK may inhibit angiotensin-II-induced cardiomyocyte hypertrophy via decreasing phosphorylation levels at ERK1/2 and AKT, consequently downregulating nuclear translocation of NF-kappaB p65.

The effects of resveratrol, a phytoalexin derived from red wines, on chronic inflammation induced in an experimentally induced colitis model

Neutrophil infiltration, proinflammatory cytokines, eicosanoid generation and oxidative stress have been implicated in colitis. Resveratrol is a polyphenolic compound found in grapes and wine, with multiple pharmacological actions, including anti-inflammatory, antioxidant, antitumour and immunomodulatory activities. In a previous report, we documented that resveratrol decreases the degree of inflammation associated with acute experimental colonic inflammation, but its effects on chronic experimental colitis remain undetermined. The aim of this research was to investigate the effects of resveratrol on the chronic colonic injury caused by intracolonic instillation of trinitrobenzenesulphonic acid (TNBS) in rats. The inflammatory response was assessed by histology and myeloperoxidase activity. Tumour necrosis factor alpha (TNF-alpha) production, histological and histochemical analysis of the lesions were also carried out. We determined the production of prostaglandin (PG) E2 and D2 in colon mucosa, as well as cyclooxygenase (COX)-1 and -2 and nuclear transcription factor NF-kappa B (NF-kappaB) p65 protein expression. Finally, since resveratrol has been found to modulate apoptosis, we intended to elucidate its effects on colonic mucosa under chronic inflammatory conditions. Resveratrol (10 mg kg(-1) day(-1)) significantly attenuated the damage score and corrected the disturbances in morphology associated to injury. In addition, the degree of neutrophil infiltration and the levels of TNF-alpha were significantly ameliorated. Resveratrol did not modify PGD2 levels but returned the decreased PGE2 values to basal levels and also reduced COX-2 and the NF-kappaB p65 protein expression. Furthermore, treatment of rats with resveratrol caused a significant increase of TNBS-induced apoptosis in colonic cells. In conclusion, resveratrol reduces the damage in chronic experimentally induced colitis, alleviates the oxidative events, returns PGE2 production to basal levels and stimulates apoptosis in colonic cells.

Vitamin D decreases NFκB activity by increasing IκBα levels

In a previous study we demonstrated the inhibitory effect of 1,25-dihydroxyvitamin D (1,25(OH)(2)D(3)) and its less calcaemic analog 1,24(OH)(2)D(2) on the production of tumour necrosis factor alpha (TNFalpha) by human peritoneal macrophages. The aim of the present study is to examine whether this vitamin D inhibition of TNFalpha is mediated by its major transcription factor, nuclear factor-kappaB (NFkappaB). Murine macrophage cells (P388D1) were incubated with 10(-7) M 1,25(OH)(2)D(3) or 1,24(OH)(2)D(2) and then stimulated with lipopolysaccharide. NFkappaB activity was assayed using a reporter gene and by electrophoretic mobility shift assay (EMSA). In addition, we evaluated mRNA and protein levels of NFkappaB-p65 and of IkappaBalpha, a potent NFkappaB inhibitor, and phosphorylated IkappaBalpha. Both 1,25(OH)(2)D(3) and 1,24(OH)(2)D(2) induced a 60% reduction of TNFalpha secretion. By using a reporter gene and EMSA we found that vitamin D markedly reduced NFkappaB activity. 1,25(OH)(2)D(3) or 1,24(OH)(2)D(2) decreased NFkappaB-p65 levels in the nucleus and increased NFkappaB-p65 levels in the cytosol; no changes were observed in the total levels of NFkappaB-p65 protein and mRNA. Concurrently, vitamin D induced a significant increase in mRNA and protein levels of IkappaBalpha (approximately 6.5- and 4.5-fold, respectively). Elevated levels of IkappaBalpha can be explained by the vitamin D-induced prolongation of IkappaBalpha-mRNA half-life from 110 to 190 min and by the decrease in IkappaBalpha phosphorylation. Vitamin D up-regulates IkappaBalpha levels by increasing mRNA stability and decreasing IkappaBalpha phosphorylation. The increase in IkappaBalpha levels reduces nuclear translocation of NFkappaB and thereby downgrades its activity. Since NFkappaB is a major transcription factor of inflammatory mediators, these findings suggest that the less-calcaemic analog, 1,24(OH)(2)D(2) may be effective as an anti-inflammatory therapeutic agent.

Expression and activity of inducible nitric oxide synthase and endothelial nitric oxide synthase correlate with ethanol-induced liver injury

To study the expression and activity of inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) in rats with ethanol-induced liver injury and their relation with liver damage, activation of nuclear factor-kappaB (NF-kappaB) and tumor necrosis factor-alpha (TNF-alpha) expression in the liver. Female Sprague-Dawley rats were given fish oil (0.5 mL) along with ethanol or isocaloric dextrose daily via gastrogavage for 4 or 6 wk. Liver injury was assessed using serum alanine aminotransferase (ALT) activity and pathological analysis. Liver malondialdehyde (MDA), nitric oxide contents, iNOS and eNOS activity were determined. NF-kappaB p65ìiNOS, eNOS and TNF-alpha protein or mRNA expression in the liver were detected by immunohistochemistry or reverse transcriptase-polymerase chain reaction (RT-PCR). Chronic ethanol gavage for 4 wk caused steatosis, inflammation and necrosis in the liver, and elevated serum ALT activity. Prolonged ethanol administration (6 wk) enhanced the liver damage. These responses were accompanied with increased lipid peroxidation, NO contents, iNOS activity and reduced eNOS activity. NF-kappaB p65, iNOS and TNF-alpha protein or mRNA expression were markedly induced after chronic ethanol gavage, whereas eNOS mRNA expression remained unchanged. The enhanced iNOS activity and expression were positively correlated with the liver damage, especially the necro-inflammation, activation of NF-kappaB, and TNF-alpha mRNA expression. iNOS expression and activity are induced in the liver after chronic ethanol exposure in rats, which are correlated with the liver damage, especially the necro-inflammation, activation of NF-kappaB and TNF-alpha expression. eNOS activity is reduced, but its mRNA expression is not affected.

Alcohol suppresses IL-2-induced CC chemokine production by natural killer cells.

Natural killer (NK) cells are a critical component of the host innate immune system. We investigated whether alcohol impairs NK cell function, particularly production of CC chemokines induced by interleukin (IL)-2, the natural ligands for CCR5 receptor. Primary NK cells and NK cell line (YTS) were cultured with or without alcohol (10 to 80 mM) for three hours. The culture supernatants were then harvested and used to treat human peripheral blood monocyte-derived macrophages and a HeLa cell line, which expresses CD4, CCR5, and CXCR4 receptors (MAGI cells). CC chemokine expression by YTS and primary NK cells treated with or without alcohol was analyzed with the real-time RT-PCR and ELISA. [Ca(2)(+)]i and Western blot assays were used to determine calcium-mediated intracellular signaling pathway and NF-kappaB p65 expression. HIV strains (Bal and UG024) were used to infect macrophages and MAGI cells. In addition, ADA (macrophage-tropic strain) and murine leukemia virus (MLV) envelope-pseudotyped HIV infection was carried out in macrophages. HIV infectivity was determined by HIV reverse transcriptase (RT) and beta-galactosidase activity assays. Alcohol inhibited IL-2-induced CC chemokine (CCL3 and CCL4) expression by NK cells. Functional tests demonstrated that this reduced expression of CC chemokines was associated with diminished anti-HIV ability of NK cells. Alcohol also reduced the ability of NK cells to response to CCL3-mediated chemotaxis. Alcohol inhibited IL-2-induced NF-kappaB p65 protein expression and calcium mobilization by NK cells. Alcohol, through the inhibition of IL-2-induced NF-kappaB p65 protein expression and intracellular calcium mobilization, suppressed NK cell production of CC chemokines. This suppression of CC chemokine production was associated with diminished anti-HIV activity of NK cells. Thus, by inhibiting NK cell-mediated innate immunity against HIV, alcohol consumption may have a cofactor role in the immunopathogenesis of HIV disease.