Expression Of NF-κB P65 Subunit Research Articles

In vitro anti-inflammatory properties of Smilax campestris aqueous extract in human macrophages, and characterization of its flavonoid profile.

Extracts of Smilax campestris Griseb (Smilacaceae) have been employed in the treatment of several inflammatory diseases as a traditional herbal medicine. However, the cellular and molecular mechanisms involved in the observed effects remain elusive. Macrophages are known to play a central role in inflammatory responses. These cells are activated in response to a diversity of danger signals and produce several mediators of inflammation that eventually regulate the immune response. For all the above mentioned, scientific evidence is required to support the popular use of S. campestris. We aimed to investigate the anti-inflammatory effect of S. campestris aqueous extract (SME) in activated THP-1 human macrophages, on the production of some mediators of inflammation and oxidative stress in order to provide scientific support for its popular use. The characterization of SME was assessed by HPLC-MS/MS. The production of the pro-inflammatory cytokines and chemokines was evaluated by ELISA. The activity of metalloproteases was evaluated by zymography. The subcellular localization of the NF-κB transcription factor was analysed by Western blot. The superoxide anion and glutathione levels were assessed by flow cytometry. The cytotoxicity induced by SME in THP-1 macrophages was also investigated by the LDH release test. In the present study, we have identified catechin and glycosylated derivatives of quercetin (quercetin-3-O-glucoside, quercetin-3-O-galactoside, rutin and quercetin-3-rhamnoside) as major components of the aqueous SME. We found that SME significantly decreased the production of the pro-inflammatory cytokines tumour necrosis factor (TNF)- α, interleukin (IL)-1β, IL-6, IL-8 and monocyte chemoattractant protein (MCP)-1 and the activity of the metalloproteinase (MMP)-9, in lipopolysaccharide-activated macrophages derived from the monocytic cell line THP-1. Furthermore, SME diminished the expression of NF-κB p65 subunit in the nuclear fraction. In addition, SME decreased the production of superoxide anion in THP-1 macrophages, without altering the levels of reduced glutathione. These results suggest that SME exerts its anti-inflammatory effects in human activated macrophages by inhibiting the production of pro-inflammatory cytokines, matrix metalloproteinases and the NF-κB transcription factor pathway along with a reduction of oxidative stress mediators. Moreover, catechin and glycosylated derivatives of were identified by HPLC-MS/MS in SME. Our findings provide scientific support for the traditional use of the S. campestris extracts.

Ginsenoside Rg3 attenuates endometriosis by inhibiting the viability of human ectopic endometrial stromal cells through the nuclear factor-kappaB signaling pathway

ObjectiveTo investigate the effects of ginsenoside Rg3 on human ectopic endometriotic stromal cells in vitro. Materials and methodsEctopic endometrial tissue specimens were obtained from 6 female patients with ovarian endometriosis who underwent laparoscopic surgical procedures. Endometrial stromal cells derived from isolated ectopic endometriotic lesions were cultured, and the purity and homogeneity of cells were verified by Immunocytochemistry. The effect of Rg3 on cell proliferation was detected by Cell Counting Kit-8 (CCK8). After treatment with Rg3, the protein expression of NF-κB p65 subunit, VEGF, and caspases3 were measured by western blot analysis. Meanwhile, the mRNA expression of NF-κB p65 subunit was determined by Quantitative real-time polymerase chain reaction (RT-PCR). ResultsRg3 inhibited the proliferation of ectopic endometriotic cells in a time- and dose-dependent manner. The treatment with Rg3 significantly diminished the level of NF-κB p65 subunit as well as TNF-α induced nuclear translocation of NF-κB p65 subunit in ectopic endometriotic cells. Moreover, Rg3 upregulated the expression of caspases3 but suppressed the expression of VEGF. ConclusionOur results indicate that Ginsenoside Rg3 suppresses endometriosis by reducing the viability of human ectopic endometrial stromal cells involving the nuclear factor-kappaB signaling pathway in vitro.

Protective effects of SOD2 overexpression in human umbilical cord mesenchymal stem cells on lung injury induced by acute paraquat poisoning in rats

AimsTo study the protective effects and mechanisms of human umbilical cord mesenchymal stem cells (hUCMSCs) and overexpression of antioxidant gene SOD2 on lung injury by establishing a rat model of paraquat (PQ)-induced lung injury. Main methodsThe hUCMSCs cell line overexpressed SOD2 was established. After intraperitoneal injection of PQ solution (24 mg/kg) 3 h later, the different groups of hUCMSCs cell lines were injected through the tail veins of rats. Bronchoalveolar lavage fluid (BALF) was obtained to determine the protein level of inflammatory cytokines. Lung tissues were collected to test the wet/dry weight ratios (W/D), oxidative stress index and lung injury scores. Western blotting was used to detect SOD1, SOD2, HO-1, Nrf2, NF-κBp65 subunit, and cleaved caspase-3. Key findingsAfter treatment with cells built on the basis of hUCMSCs, the protein levels of TNF-α, IL-8, and ICAM-1 in BALF decreased, and meanwhile in lung tissues, MDA content was reduced, GSH-Px activity was elevated, and lung W/D ratio decreased. Additionally, protein expression of NF-κB p65 subunit and activated caspase-3 in lung tissues was down-regulated, whereas expression of SOD1, SOD2, HO-1, and Nrf-2 were up-regulated. The results of HE staining showed that lung injury was significantly alleviated in the hUCMSC treated group. It is noticeable that hUCMSCs and SOD2-overexpressed hUCMSCs effectively reduced PQ-induced lung injury in rats, and moreover, hUCMSCs overexpressed SOD2 were more effective compared with hUCMSCs only. SignificanceEvaluation of the efficacy and analysis of mechanism in the treatment of PQ induced ALI by appliance of SOD2-overexpressed hUCMSCs will provide the proof from bench to bedside.

DCLK1 promotes epithelial-mesenchymal transition via the PI3K/Akt/NF-κB pathway in colorectal cancer.

Double cortin-like kinase 1 (DCLK1) plays important roles during the epithelial-mesenchymal transition (EMT) process in human colorectal cancer (CRC). However, the role of DCLK1 in regulating the EMT of CRC is still poorly understood. In this study, we report evidence that DCLK1 acts as a potent oncogene to drive its extremely malignant character of EMT in an NF-κB-dependent manner in CRC cells. Mechanistic investigations showed that DCLK1 induced the NF-κBp65 subunit expression through the PI3K/Akt/Sp1 axis and activated NF-κBp65 through the PI3K/Akt/IκBα pathway during the EMT of CRC cells. Moreover, we found that silencing the expression of DCLK1 inhibited the invasion and metastasis of CRC cells in vivo. Collectively, our findings identify DCLK1 as a pivotal regulator of an EMT axis in CRC, thus implicating DCLK1 as a potential therapeutic target for CRC metastasis.

Potential malignant transformation in the gastric mucosa of immunodeficient mice with persistent Mycoplasma penetrans infection.

Mycoplasma infection has been reported in immunocompromised cancer patients; nevertheless, it is not clear if persistent Mycoplasma infection could facilitate the proliferation of cancer cells in immunocompromised organisms. The aim of this study was to examine the relationship between persistent Mycoplasma infection and malignant transformation in an immunodeficient host model. Immunodeficient mouse model was established using cyclophosphamide and mice gastric mucosal cells were infected with Mycoplasma penetrans (Mpe). After 18 weeks, mice were sacrificed and gastric mucosal Mpe infected cells were identified by fluorescence in situ hybridization (FISH). Moreover, pathological and ultrastructural changes in mice gastric mucosa were evaluated and the expression of multiple proto-oncogenes was examined by Western blot. Our data show that Mpe infection was detected in the blood of immunodeficient mice and Mpe persistent infection in mice gastric mucosa was confirmed by FISH. There were pathological and ultrastructural malignant transformation occurred in the gastric mucosa of infected mice compared to control mice. Mpe infected mice showed lower expression of p53 and p21 and higher H-ras expression compared to the control group. Moreover, expression of NF-κB p65 subunit increased in Mpe infected mice, similar to the TNF-α expression. Bax expression in gastric mucosa of Mpe infected mice was lower while Bcl-2 expression was higher than in the uninfected control group. Collectively these data demonstrate that persistent Mpe infection is associated with aberrant expression of multiple proto-oncogenes in gastric mucosa of immunodeficient mice which potentially facilitate the malignant transformation.

Curcumin-ER Prolonged Subcutaneous Delivery for the Treatment of Non-Small Cell Lung Cancer.

Non-small-cell lung cancer therapy is a challenge due to poor prognosis and low survival rate. There is an acute need for advanced therapies having higher drug efficacy, low immunogenicity and fewer side effects which will markedly improve patient compliance and quality of life of cancer patients. The purpose of this study was to develop a novel hybrid curcumin nanoformulation (Curcumin-ER) and evaluate the therapeutic efficacy of this formulation on a non-small cell lung cancer xenograft model. Use of curcumin, a natural anticancer agent, is majorly limited due to its poor aqueous solubility and hence it's low systemic bioavailability. In this paper, we carried out the nanoformulation of Curcumin-ER, optimized the formulation process and determined the anticancer effects of Curcumin-ER against human A549 non-small cell lung cancer using in vitro and in vivo studies. Xenograft tumors in nude mice were treated with 20 mg/kg subcutaneous injection of Curcumin-ER and liposomal curcumin (Lipocurc) twice a week for seven weeks. Results showed that tumor growth was suppressed by 52.1% by Curcumin-ER treatment and only 32.2% by Lipocurc compared to controls. Tumor sections were isolated from murine xenografts and histology and immunohistochemistry was performed. A decrease in expression of NFκB-p65 subunit and proliferation marker, Ki-67 was observed in treated tumors. In addition, a potent anti-angiogenic effect, characterized by reduced expression of annexin A2 protein, was observed in treated tumors. These results establish the effectiveness of Curcumin-ER in regressing human non-small cell lung cancer growth in the xenograft model using subcutaneous route of administration. The therapeutic efficacy of Curcumin-ER highlights the potential of this hybrid nanoformulation in treating patients with non-small cell lung cancer.

Increased expression of nuclear factor kappa-B p65 subunit in adenomyosis.

ObjectiveNuclear factor kappa-B (NF-κB) is a critical proinflammatory regulator that has been suggested to play a pivotal role in the pathogenesis and pathophysiology of endometriosis. In the present study, we aimed to evaluate whether the expression of NF-κB p65 subunit is increased in the eutopic endometrium and/or in the adenomyosis nodule of women with adenomyosis.MethodsThirty-three women with histologically confirmed adenomyosis after laparoscopic or transabdominal hysterectomy were recruited. Women with carcinoma in situ of uterine cervix without evidence of adenomyosis or endometriosis (n=32) served as controls. Formalin-fixed, paraffin-embedded archival tissues were sectioned and immunostained utilizing a monoclonal anti-human NF-κB p65 subunit antibody, and the immunoreactivity of NF-κB p65 subunit was compared between women with and without adenomyosis.ResultsThe immunoreactivities of both the nuclear and the cytoplasmic NF-κB p65 subunit were significantly increased in the stromal cells in the eutopic endometrium as well as in the adenomyosis nodule of women with adenomyosis compared with controls, respectively. The nuclear expression of NF-κB p65 subunit was significantly higher in the glandular cells in the eutopic endometrium as well as the adenomyosis nodule of women with adenomyosis compared with controls, respectively.ConclusionThe expression of NF-κB p65 is increased in the eutopic endometrium and adenomyosis nodule of women with adenomyosis, which strongly suggest that NF-κB plays a critical role in the pathogenesis and/or pathophysiology of adenomyosis.

Knockdown of NF-κB p65 subunit expression suppresses growth of nude mouse lung tumour cell xenografts by inhibition of Bcl-2 apoptotic pathway.

Nuclear factor-kappaB (NF-κB) is an important transcriptional factor and regulates a variety of pathophysiologic process involved in cell survival and death. This study aimed to assess the effects of NF-κB p65 subunit knockdown in suppression of nude mouse lung tumour cell xenografts and understands the underlying molecular events. A nude mouse Lewis lung carcinoma cell xenograft model was established, and the mice were intraperitoneally injected with NF-κB p65 small interfering RNA (siRNA) and sacrificed after 2weeks of tumour cell injection. Tumour xenografts were harvested for terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, Western blot and quantitative reverse transcription-polymerase chain reaction analyses. Results found that compared to the phosphate-buffered saline (PBS)-treated or the negative control (NC) siRNA-treated mice tumour xenograft weight and volume were significantly decreased in the NF-κB p65 siRNA-treated mice. The TUNEL positive (apoptosis) cells in xenograft sections were 45 ± 5 in PBS and 38 ± 3 in NC siRNA but increased to 271 ± 11 in p65 siRNA-treated mice. Compared to the PBS or the NC mice, levels of Bcl-2 mRNA and protein in tumour xenografts were significantly downregulated in p65 siRNA-treated mice. Knockdown of NF-κB p65 subunit expression can significantly inhibit the growth of nude mouse Lewis tumour cell xenografts by inducing tumour cell apoptosis and downregulating pro-apoptotic protein Bcl-2 expression. Targeting NF-κB p65 subunit expression as a potential therapeutic strategy needs further evaluation in treating human lung cancer.

Metformin ameliorates the proinflammatory state in patients with carotid artery atherosclerosis through sirtuin 1 induction

Metformin is a widely used classic antidiabetic drug. However, its clinical pharmacologic mechanism remains poorly understood. In the present study, we investigated the anti-inflammatory effects of metformin on circulating peripheral blood mononuclear cells (MNCs) of patients with carotid artery atherosclerosis (AS). A total of 42 patients with carotid artery AS were randomly assigned to metformin (500 mg twice a day; Met; n = 21) or placebo control (Con; n = 21) groups. After 12 weeks of treatment, plasma concentrations of high-sensitivity C-reactive protein (hs-CRP), interleukin 6 (IL-6), and tumor necrosis factor α (TNF-α) significantly decreased in the Met group compared with the Con group. In addition, treatment with metformin significantly reduced the expression of IL-6 and TNF-α at the messenger RNA level and attenuated nuclear factor kappa B (NF-κB) DNA binding activity in MNCs. Intriguingly, metformin did not alter the expression of NF-κB p65 subunit, but markedly inhibited its acetylation. Furthermore, metformin significantly induced sirtuin 1 (SIRT1) expression in MNCs. Moreover, we found that metformin treatment dramatically induced SIRT1 expression, blocked p65 acetylation, and inhibited NF-κB activity and the expression of inflammatory factors in MNCs in vitro. We conclude that metformin has a novel direct protective role to ameliorate the proinflammatory response through SIRT1 induction, p65 acetylation reduction, NF-κB inactivation, and inflammatory inhibition in peripheral blood MNCs of patients with carotid artery AS.

(+)-Naltrexone is neuroprotective and promotes alternative activation in the mouse hippocampus after cardiac arrest/cardiopulmonary resuscitation.

Despite dramatic improvement in cardiopulmonary resuscitation (CPR) and other techniques for cardiac arrest (CA), the majority of survivors continue to show signs of decreased memory or executive cognitive function. Such memory impairment may be due to hippocampal CA1 neuronal death, which is delayed by several days after CA/CPR. Classical microgliosis in the CA1 region may contribute to neuronal death, yet the role of a key activation receptor Toll Like Receptor 4 (TLR4) has not been previously investigated for such neuronal death after CA/CPR. We show that (+)-naltrexone was neuroprotective after CA/CPR. TLR4 blockade was associated with decreased expression of markers for microglial/macrophage activation and T cell and B cell infiltration, as well as decreased pro-inflammatory cytokine levels. Notably, IL-10 expression was elevated in response to CA/CPR, but was not attenuated by (+)-naltrexone, suggesting that the local monocyte/microglial phenotype had shifted towards alternative activation. This was confirmed by elevated expression of Arginase-1, and decreased expression of NFκB p65 subunit. Thus, (+)-naltrexone and other TLR4 antagonists may represent a novel therapeutic strategy to alleviate the substantial burden of memory or executive cognitive function impairment after CA/CPR.

Knockdown of NF-κB p65 subunit expression suppresses growth of nude mouse lung tumor cell xenografts by activation of Bax apoptotic pathway.

Nuclear factor-kappaB (NF-κB) is an important transcriptional factor and regulates avariety of pathophysiologic process involved in cell survival and death. The present study assesses the effects of NF-κB p65 subunit knockdown in suppression of nude mouse lung tumor cell xenografts and understands the underlying molecular events.A nude mouse Lewis lung carcinoma cell xenograft model was established and the mice were intraperitoneally injected with NF-κB p65 siRNA and sacrificed after two weeks of tumor cell injection. Tumor xenografts were harvested for TUNEL, Western blot, and qRT-PCR analyses.Compared to the PBS-treated or the negative control (NC) siRNA-treated mice, tumor xenograft weight and volume was significantly decreased in the NF-κB p65 siRNA-treated mice. The TUNEL positive (apoptosis) cells in xenograft sections were 45 ± 5 in PBS and 38 ± 3 in NC siRNA, but increased to 271 ± 11 in p65 siRNA-treated mice. Compared to the PBS or the NC mice, levels of Bax mRNA and protein in tumor xenografts were significantly upregulated in p65 siRNA-treated mice. Knockdown of NF-κB p65 subunit expression significantly inhibited the growth of nude mouse Lewis tumor cell xenografts by induction of tumor cell apoptosis and significantly up-regulation of pro-apoptotic protein Bax expression. Future study will confirm the current data and targeting NF-κB p65 subunit expression as apotential therapeutic strategy in treating human lung cancer. NF-κB, lung cancer, apoptosis, small interfering RNA, xenografts.

Increased nuclear expression of nuclear factor kappa-B p65 subunit in the eutopic endometrium and ovarian endometrioma of women with advanced stage endometriosis

ObjectiveTo evaluate whether the nuclear expression of nuclear factor kappa-B (NF-κB) p65 subunit is increased in the eutopic endometrium and/or the ovarian endometrioma of women with advanced stage endometriosis.DesignCase-controlled laboratory study in a university hospital.Materials and MethodsWe recruited a total of 66 women who underwent hysterectomy due to carcinoma in situ (CIS) of the uterine cervix (n=32) or advanced stage endometriosis (n=34) as well as 30 nulliparous women who underwent ovarian cystectomy due to endometrioma. Immunohistochemistry was performed to compare the nuclear NF-κB p65 subunit immunoreactivity between women with and without advanced stage endometriosis. The nuclear NF-κB p65 subunit expression and DNA binding were analyzed utilizing Western blotting, enzyme-linked immunosorbent assay (ELISA), and electrophoretic mobility shift assay (EMSA) in endometrial cells treated with tumor necrosis factor-alpha (TNF-α) or interleukin-1beta (IL-1β).ResultsThe immunoreactivity of the nuclear NF-κB p65 subunit was significantly increased in the eutopic endometrium as well as in the ovarian endometrioma of women with advanced stage endometriosis compared to the CIS group. In vitro treatment of endometrial cells with TNF-α and IL-1β led to a significant increase of nuclear NF-κB p65 subunit expression and DNA binding.ConclusionThe nuclear expression of NF-κB p65 is increased in the eutopic endometrium and ovarian endometrioma of women with advanced stage endometriosis, which strongly suggest that NF-κB signaling plays a crucial role in the pathogenesis and/or pathophysiology of endometriosis. ObjectiveTo evaluate whether the nuclear expression of nuclear factor kappa-B (NF-κB) p65 subunit is increased in the eutopic endometrium and/or the ovarian endometrioma of women with advanced stage endometriosis. To evaluate whether the nuclear expression of nuclear factor kappa-B (NF-κB) p65 subunit is increased in the eutopic endometrium and/or the ovarian endometrioma of women with advanced stage endometriosis. DesignCase-controlled laboratory study in a university hospital. Case-controlled laboratory study in a university hospital. Materials and MethodsWe recruited a total of 66 women who underwent hysterectomy due to carcinoma in situ (CIS) of the uterine cervix (n=32) or advanced stage endometriosis (n=34) as well as 30 nulliparous women who underwent ovarian cystectomy due to endometrioma. Immunohistochemistry was performed to compare the nuclear NF-κB p65 subunit immunoreactivity between women with and without advanced stage endometriosis. The nuclear NF-κB p65 subunit expression and DNA binding were analyzed utilizing Western blotting, enzyme-linked immunosorbent assay (ELISA), and electrophoretic mobility shift assay (EMSA) in endometrial cells treated with tumor necrosis factor-alpha (TNF-α) or interleukin-1beta (IL-1β). We recruited a total of 66 women who underwent hysterectomy due to carcinoma in situ (CIS) of the uterine cervix (n=32) or advanced stage endometriosis (n=34) as well as 30 nulliparous women who underwent ovarian cystectomy due to endometrioma. Immunohistochemistry was performed to compare the nuclear NF-κB p65 subunit immunoreactivity between women with and without advanced stage endometriosis. The nuclear NF-κB p65 subunit expression and DNA binding were analyzed utilizing Western blotting, enzyme-linked immunosorbent assay (ELISA), and electrophoretic mobility shift assay (EMSA) in endometrial cells treated with tumor necrosis factor-alpha (TNF-α) or interleukin-1beta (IL-1β). ResultsThe immunoreactivity of the nuclear NF-κB p65 subunit was significantly increased in the eutopic endometrium as well as in the ovarian endometrioma of women with advanced stage endometriosis compared to the CIS group. In vitro treatment of endometrial cells with TNF-α and IL-1β led to a significant increase of nuclear NF-κB p65 subunit expression and DNA binding. The immunoreactivity of the nuclear NF-κB p65 subunit was significantly increased in the eutopic endometrium as well as in the ovarian endometrioma of women with advanced stage endometriosis compared to the CIS group. In vitro treatment of endometrial cells with TNF-α and IL-1β led to a significant increase of nuclear NF-κB p65 subunit expression and DNA binding. ConclusionThe nuclear expression of NF-κB p65 is increased in the eutopic endometrium and ovarian endometrioma of women with advanced stage endometriosis, which strongly suggest that NF-κB signaling plays a crucial role in the pathogenesis and/or pathophysiology of endometriosis. The nuclear expression of NF-κB p65 is increased in the eutopic endometrium and ovarian endometrioma of women with advanced stage endometriosis, which strongly suggest that NF-κB signaling plays a crucial role in the pathogenesis and/or pathophysiology of endometriosis.

Inhibitory effects of dexamethasone against polyinosinic:polycytidylic acid-induced chemotactic factor expression in bronchial epithelial cells and the underlying mechanism

Objective To investigate the effect of dexamethasone on polyinosinic: polycytidylic acid(PIC)-induced chemotactic factor expression in human bronchial epithelial(16hBE) cells and the underlying mechanism.Methods 16hBE cells were treated with different concentrations of PIC(0.001,0.01,0.1,and 1 μg/ml) and dexamethasone(0.1,1,and 10 μmol/L).IL 8 and IP-10 mRNA levels were detected by RT-PCR 6 h after stimulation.IL-8 and IP-10 protein levels in the culture supernatant were detected by ELISA 24 h after stimulation.NF-κB p65 subunit expression was detected by immunohistochemical staining.Results PIC concentration-dependently(0.001,0.01,and 0.1 μg/ml) increased the expression of IL-8 and IP-10 mRNA and protein compared with the control group,with significant differences found when PIC at 0.01 μg/ml and 0.1 μg/ml(P0.05,P0.01).When the concentration of PIC was 1 μg/ml,the expressions of IL-8 and IP-10 were decreased at both mRNA and protein levels.Pretreatment with dexamethasone(1 μmol/L and 10 μmol/L) for 0.5 h significantly inhibited the IL-8 and IP-10 expression at both mRNA and protein levels(P0.05,P0.01).Dexamethasone pretreatment(1 μmol/L) significantly inhibited PIC-induced NF-κB p65 subunit expression(P0.01).Conclusion Glucocorticoids can suppress PIC-induced IL-8 and IP-10 expression in human bronchial epithelial cells,probably through activation of NF-κB pathway.

Nicotine Decreases Beta-Amyloid Through Regulating BACE1 Transcription in SH-EP1-α4β2 nAChR-APP695 Cells

Alzheimer's disease (AD) is a neurodegenerative disorder that affects the elderly population. Deposition of beta-amyloid (Aβ) in the brain is a hallmark of AD pathology. In our previous study, we have constructed a cell line expressing human APP695 (hAPP695) in SH-EP1 cells stably transfected with human nicotinic receptor (nAChR) α4 subunit and β2 subunit gene. In present study, we found that activation of α4β2 nAChR by nicotine and epibatidine decreased secreted Aβ level in the cell line and hippocampal neurons, but had no effects on full-length APP695 and sAPP-α. Nicotine also decreases BACE1 and PSEN1 expression, as well as ERK1 and NFκB P65 subunit expression in the cell line. Furthermore, BACE1 promoter activity is, but PSEN1 not, decreased by nicotine in the cell line. All the results suggest that activation of α4β2 nAChR decreases Aβ through regulating BACE1 transcription by ERK1-NFκB pathway. Additionally, analysis of BACE1 promoter activity by dual-luciferase reporter assay may be useful for drug screening as a high throughput method.

Free fatty acids induce endothelial dysfunction and activate protein kinase C and nuclear factor-κB pathway in rat aorta

BackgroundInsulin resistance is associated with an inappropriate elevation of plasma free fatty acids (FFAs) and endothelial dysfunction. In this study, we asked if elevated circulating FFA levels led to impaired insulin signaling and endothelial dysfunction in-vivo via activation of PKC-mediated inflammatory pathways. MethodsSprague–Dawley (S–D) rats were infused with 1) 20% intralipid+heparin (FFA group) or 2) saline alone (Control group) for 6h. The intact aorta thoracica and aorta abdominalis were then removed. Aortic rings were isolated and evaluated for endothelial-dependent and non-dependent relaxation in an organ bath. The activities of eNOS and PKC were measured in endothelial homogenates prepared from endothelial cells harvested from the aorta. The expression levels of insulin signaling molecules IRS-1, Akt, eNOS, ERK1/ERK2, PKC-α, NFκB-p65 subunit and IκB-α in rat aortic endothelium were determined by immunohistochemistry and Western blot. ResultsElevation of FFAs resulted in a 35.9% reduction in the response to acetylcholine (p<0.01), a 26% decline in plasma NOx levels (p<0.05), a 53% decrease in eNOS activity and a 34±9% inhibition in IRS-1 tyrosine phosphorylation (p<0.05). We also found a 46% decrease in Akt phosphorylation and a 36% decrease in eNOS phosphorylation. FFA-induced endothelial insulin resistance was associated with 82% increase in total membrane-associated PKC activity, a 1.7-fold increase in total PKC-α protein, 1.29-fold decrease in IκB-α expression levels and 1.47-fold increase in NF-κB p65 subunit expression in rat aortic endothelium. ConclusionThe molecular mechanisms underlying FFA-induced endothelial insulin resistance and eNOS inhibition may provide an important link implicating the PKC and IκB-α/NF-κB pathways in FFA-mediated inhibition of vascular insulin signaling.

Simvastatin induces derepression of PTEN expression via NFκB to inhibit breast cancer cell growth

Sustained activation of Akt kinase acts as a focal regulator to increase cell growth and survival, which causes tumorigenesis including breast cancer. Statins, potent inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase, display anticancer activity. The molecular mechanisms by which statins block cancer cell growth are poorly understood. We demonstrate that in the tumors derived from MDA-MB-231 human breast cancer cell xenografts, simvastatin significantly inhibited phosphorylation of Akt with concomitant attenuation of the expression of the anti-apoptotic protein Bcl XL. In many cancer cells, Bcl XL is a target of NFκB. Simvastatin inhibited the DNA binding and transcriptional activities of NFκB resulting in marked reduction in transcription of Bcl XL. Signals transmitted by anti-neoplastic mechanism implanted in the cancer cells serve to obstruct the initial outgrowth of tumors. One such mechanism represents the action of the tumor suppressor protein PTEN, which negatively regulates Akt kinase activity. We provide the first evidence for significantly increased levels of PTEN in the tumors of simvastatin-administered mice. Importantly, simvastatin markedly prevented binding of NFκB to the two canonical recognition elements, NFRE-1 and NFRE-2 present in the PTEN promoter. Contrary to the transcriptional suppression of Bcl XL, simvastatin significantly increased the transcription of PTEN. Furthermore, expression of NFκB p65 subunit inhibited transcription of PTEN, resulting in reduced protein expression, which leads to enhanced phosphorylation of Akt. Taken together, our data present a novel bifaceted mechanism where simvastatin acts on a nodal transcription factor NFκB, which attenuates the expression of anti-apoptotic Bcl XL and simultaneously derepresses the expression of anti-proliferative/proapoptotic tumor suppressor PTEN to prevent breast cancer cell growth.

Combined Glutamine and Arginine Decrease Proinflammatory Cytokine Production by Biopsies from Crohn’s Patients in Association with Changes in Nuclear Factor-κB and p38 Mitogen-Activated Protein Kinase Pathways 3

Glutamine (Gln) and arginine (Arg) are conditionally essential amino acids with immunomodulatory properties. The aim of the study was to assess the effects of Gln and Arg alone or in combination on cytokine release by cultured colonic biopsies from patients with active Crohn’s disease (CD). Ten consecutive patients [mean (range) age 26 (18–39) y] with active colonic CD (mean CD activity index: 383.7 ± 129.8) were prospectively included in the study. Eight colonic biopsies were obtained via a colonoscopy and incubated during 18 h with low (physiological) or high (pharmacological) doses of Arg (0.1 or 2 mmol/L designated as Arglow or Arghigh, respectively) and Gln (0.6 or 10 mmol/L designated as Glnlow or Glnhigh, respectively). The concentrations of cytokines [interleukin (IL)-4, IL-10, IL-8, IL-6, tumor necrosis factor-α (TNFα), IL-1β, interferon-γ) were assessed by ELISA, and nitric oxide (NO) production was evaluated by Griess assay. Nuclear factor (NF)-κB p65 subunit, inhibitor of NFκB-α, and p38 mitogen-activated protein kinase (MAPK) were assessed by immunoblotting. Arghigh/Glnhigh decreased the production of TNFα, IL-1β, IL-8, and IL-6 (each P < 0.01). Arglow/Glnhigh decreased IL-6 and IL-8 production (both P < 0.01), whereas Arghigh/Glnlow did not affect cytokine and NO production. Arglow/Glnhigh and Arghigh/Glnhigh decreased NF-κB p65 subunit expression, whereas p38 MAPK was decreased only by Arghigh/Glnhigh. Combined pharmacological doses of Arg and Gln decreased TNFα and the main proinflammatory cytokines release in active colonic CD biopsies via NF-κB and p38 MAPK pathways. These results could be the basis of prospective studies evaluating the effects of enteral supply of combined Arg and Gln during active CD.

Angiotensin II-induced skeletal muscle insulin resistance mediated by NF-κB activation via NADPH oxidase

Reduced insulin sensitivity is a key factor in the pathogenesis of type 2 diabetes and hypertension. Skeletal muscle insulin resistance is particularly important for its major role in insulin-mediated glucose disposal. Angiotensin II (ANG II) is integral in regulating blood pressure and plays a role in the pathogenesis of hypertension. In addition, we have documented that ANG II-induced skeletal muscle insulin resistance is associated with generation of reactive oxygen species (ROS). However, the linkage between ROS and insulin resistance in skeletal muscle remains unclear. To explore potential mechanisms, we employed the transgenic TG(mRen2)27 (Ren-2) hypertensive rat, which harbors the mouse renin transgene and exhibits elevated tissue ANG II levels, and skeletal muscle cell culture. Compared with Sprague-Dawley normotensive control rats, Ren-2 skeletal muscle exhibited significantly increased oxidative stress, NF-kappaB activation, and TNF-alpha expression, which were attenuated by in vivo treatment with an angiotensin type 1 receptor blocker (valsartan) or SOD/catalase mimetic (tempol). Moreover, ANG II treatment of L6 myotubes induced NF-kappaB activation and TNF-alpha production and decreased insulin-stimulated Akt activation and GLUT-4 glucose transporter translocation to plasma membranes. These effects were markedly diminished by treatment of myotubes with valsartan, the antioxidant N-acetylcysteine, NADPH oxidase-inhibiting peptide (gp91 ds-tat), or NF-kappaB inhibitor (MG-132). Similarly, NF-kappaB p65 small interfering RNA reduced NF-kappaB p65 subunit expression and nuclear translocation and TNF-alpha production but improved insulin-stimulated phosphorylation (Ser(473)) of Akt and translocation of GLUT-4. These findings suggest that NF-kappaB plays an important role in ANG II/ROS-induced skeletal muscle insulin resistance.

Glutamine inhibits over-expression of pro-inflammatory genes and down-regulates the nuclear factor kappaB pathway in an experimental model of colitis in the rat

We investigated the effects of glutamine on markers of oxidative stress, nuclear factor kappaB (NF-κB) activation, and pro-inflammatory mediators in a rat model of experimental colitis induced by intracolonic administration of 7% acetic acid. Glutamine (25 mg/kg) was given by rectal route 48 and 24 h before acetic acid instillation. Glutamine significantly reduced gross damage and histopathological scores, and partially prevented the decrease of anal pressure observed in the animals receiving acetic acid. Increases in the cytosolic concentration of TBARS and hydroperoxide-initiated chemiluminescence were significantly prevented in glutamine-treated animals. Acetic acid instillation induced a marked increase of the NF-κB p65 subunit expression in the nucleus and resulted in significant changes in the cytosolic protein level of IκB kinases (IKKα and IKKβ) and the non-phosphorylated form of the inhibitor IκBα. Protein levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) were significantly increased. All these effects were partially prevented by administration of glutamine. It is concluded that the anti-inflammatory activity of glutamine in a rat model of acetic acid-induced colitis may be mediated, at least in part, by inhibition of the expression of certain pro-inflammatory mediators which are regulated by the oxidative stress-sensitive NF-κB signalling pathway.