IκBα Protein Levels Research Articles

Astragaloside IV ameliorates necrotizing enterocolitis by attenuating oxidative stress and suppressing inflammation via the vitamin D3-upregulated protein 1/NF-κB signaling pathway.

Astragaloside IV (AS-IV) is a flavonoid from the plant Astragalus membranaceus (Fisch) Bge that has a wide range of therapeutic effects. The aim of the present study was to examine the effect of AS-IV on rats with necrotizing enterocolitis (NEC) under oxidative stress and inflammation. Newborn Sprague-Dawley rats were induced with NEC by asphyxia and hypothermia applied on 3 consecutive days. The rats were orally administered AS-IV at 25, 50 and 75 mg/kg for 4 days. The results revealed that AS-IV administration prevented NEC-induced decrease in the concentration of malondialdehyde and myeloperoxidase, and increase in the activity of glutathione (GSH) and superoxide dismutase in murine models. AS-IV also inhibited NEC-induced elevation in the levels of interleukin (IL)-6, IL-1β, tumor necrosis factor-α and nuclear factor (NF)-κB. The effects of AS-IV were achieved under inflammation and oxidative stress. Western blotting demonstrated that AS-IV substantially inhibited the phosphorylated (p)-IκBα, NF-κBp65, p-NF-κBp65 protein levels and increased vitamin D3 upregulated protein 1 (VDUP1) and IκBα protein levels. These data indicate that AS-IV may be effective in the protection of NEC-induced ileum degeneration by inhibiting the levels of inflammatory markers and oxidative stress via the regulation of the VDUP1/NF-κB signaling pathway.

Resveratrol ameliorates renal injury in spontaneously hypertensive rats by inhibiting renal micro-inflammation.

Micro-inflammation plays an important role in the pathogenesis of spontaneously hypertensive rat (SHR). In the present study, we investigated the therapeutic potential of resveratrol (RSV), a polyphenol with anti-fibrosis activity in hypertensive renal damage model. In SHR renal damage model, RSV treatment blunted the increase in urine albumin excretion, urinary β2-microglobulin (β2-MG), attenuated the decrease in creatinine clearance rate (CCR). The glomerular sclerosis index (1.54±0.33 compared with 0.36±0.07) and tubulointerstitial fibrosis (1.57±0.31 compared with 0.19±0.04) were significantly higher in SHRs compared with Wistar Kyoto rats (WKYs), which were significantly lower by RSV treatment. The increases in mesangium accumulation and the expression of renal collagen type I (Col I), fibronectin (Fn), plasminogen activator inhibitor-1 (PAI-1) and transforming growth factor-β1 (TGF-β1) in SHR were also reduced by RSV treatment. Nuclear factor κB (NF-κB) expression was increased in the cytoplasm and nuclei of the SHR kidneys, which was significantly decreased by RSV treatment. Furthermore, the protein level of IκB-α significantly decreased in the kidneys of the SHR when compared with the WKYs. RSV treatment partially restored the decreased IκB-α level. In SHR kidney, increased expression of interleukin-6 (IL-6), intercellular adhesion molecule-1 (ICAM-1) and monocyte chemoattractant protein 1 (MCP-1) were observed. These changes were attenuated by RSV treatment. No changes in blood pressure were detected between SHR group and SHR + RSV group. Taken together, the present study demonstrated that RSV treatment may significantly attenuate renal damage in the SHR model of chronic kidney disease (CKD). The renal protective effect is associated with inhibition of IL-6, ICAM-1 and MCP-1 expression via the regulation of the nuclear translocation of NF-κB, which suggesting that micro-inflammation may be a potential therapeutic target of hypertensive renal damage.

Protein degradation in a LAMP-2-deficient B-lymphoblastoid cell line from a patient with Danon disease

Danon disease, a condition characterized by cardiomyopathy, myopathy, and intellectual disability, is caused by mutations in the LAMP-2 gene. Lamp-2A protein, generated by alternative splicing from the Lamp-2 pre-mRNA, is reported to be the lysosomal membrane receptor essential for the chaperone-mediated autophagic pathway (CMA) aimed to selective protein targeting and translocation into the lysosomal lumen for degradation. To study the relevance of Lamp-2 in protein degradation, a lymphoblastoid cell line was obtained by EBV transformation of B-cells from a Danon patient. The derived cell line showed no significant expression of Lamp-2 protein. The steady-state mRNA and protein levels of alpha-synuclein, IΚBα, Rcan1, and glyceraldehyde-3-phosphate dehydrogenase, four proteins reported to be selective substrates of the CMA pathway, were similar in control and Lamp-2-deficient cells. Inhibition of protein synthesis showed that the half-life of alpha-synuclein, IΚBα, and Rcan1 was similar in control and Lamp-2-deficient cells, and its degradation prevented by proteasome inhibitors. Both in control and Lamp-2-deficient cells, induction of CMA and macroautophagy by serum and aminoacid starvation of cells for 8h produced a similar decrease in IΚBα and Rcan1 protein levels and was prevented by the addition of lysosome and autophagy inhibitors. In conclusion, the results presented here showed that Lamp-2 deficiency in human lymphoblastoid cells did not modify the steady-state levels or the degradation of several protein substrates reported as selective substrates of the CMA pathway.

Irbesartan ameliorates cardiac inflammation in type 2 diabetic db/db mice

To investigate the protective effects of irbesartan against cardiac inflammation associated with diabetes and obesity in the db/db mouse model of type 2 diabetes and explore the underlying mechanisms. Twenty- four 10-week-old diabetic db/db mice were equally randomized into irbesartan treatment (50 mg/kg per day) group and model group, using 12 nondiabetic littermates (db/+) as the controls, The mice were treated with irbesartan or saline vehicle for 16 consecutive weeks, after which the heart pathology was observed and the heart weight, body weight, and serum levels of fasting blood glucose (FBG), total cholesterol(TC), and triglycerides(TG) were measured. The expression of nuclear factor-kappaB (NF-κB) p65 in the myocardium was assessed with immunohistochemistry, the protein levels of P-IκBα ,IκBα and β-actin were analyzed with Western blotting, and the pro-inflammatory cytokines IL-6 and TNF-α mRNA were detected using quantitative real-time PCR (qPCR). Compared with db/+ mice, the saline-treated db/db mice developed obesity, hyperglycemia and hyperlipidemia (P<0.01). Histopathological examination of the heart tissue revealed inflammatory cell infiltration, increased myocardial interstitium and disorders of myocardial fiber arrangement. The diabetic mice showed increased P-IαBα and decreased IκBα protein levels, enhanced activity and expression of NF-κB in the hearts, and increased mRNA expression of IL-6 and TNF-α in the myocardium. These abnormalities were all associated with increased inflammatory response. Treatment with irbesartan improved the heart architecture and attenuated high glucose-induced inflammation in the diabetic mice. Treatment with irbesartan attenuates cardiac inflammation in type 2 diabetic db/db mice, and this effect was probably associated with the suppression of cardiac angiotensin II and NF-κB signaling pathway.

Protective effects of leucine against lipopolysaccharide-induced inflammatory response in Labeo rohita fingerlings.

The present study investigated the protective effects of leucine against lipopolysaccharide (LPS)-induced inflammatory responses in Labeo rohita (rohu) in vivo and in vitro. Primary hepatocytes, isolated from the hepatopancreas, were exposed to different concentrations of LPS for 24 h to induce an inflammatory response, and the protective effects of leucine against LPS-induced inflammation were studied. Finally, we investigated the efficiency of dietary leucine supplementation in attenuating an immune challenge induced by LPS in vivo. Exposure of cells to 10-25 μg mL(-1) of LPS for 24 h resulted in a significant production of nitric oxide and release of lactate dehydrogenase to the medium, whereas cell viability and protein content were reduced (p < 0.05). LPS exposure (10 μg mL(-1)) increased mRNA levels of the pro-inflammatory cytokines TNF-α, IL-1β and IL-8 in vitro (p < 0.05). However, pretreatment with leucine prevented the LPS-induced upregulation of TNF-α, IL-1β and IL-8 mRNAs by downregulating TLR4, MyD88, NF-κBp65, and MAPKp38 mRNA expression. Interestingly, mRNA expression of the anti-inflammatory cytokine, IL-10, which was increased by LPS treatment, was further enhanced (p < 0.05) by leucine pretreatment. The enhanced expression of IL-10 might inhibit the production of other pro-inflammatory cytokines. It was found that leucine pretreatment attenuated the excessive activation of LPS-induced TLR4-MyD88 signaling as manifested by lower level of TLR4, MyD88, MAPKp38, NF-κBp65 and increased level of IκB-α protein in leucine pre-treatment group. In vivo experiments demonstrated that leucine pre-supplementation could protect fish against LPS-induced inflammation through an attenuation of TLR4-MyD88 signaling pathway. Taken together, we propose that leucine pre-supplementation decreases LPS-induced immune damage in rohu by enhancing the expression of IL-10 and by regulating the TLR4-MyD88 signaling pathways.

Constitutive renal Rel/nuclear factor-κB expression in Lewis polycystic kidney disease rats.

To determine the temporal expression and pattern of Rel/nuclear factor (NF)-κB proteins in renal tissue in polycystic kidney disease (PKD). The renal expression of Rel/NF-κB proteins was determined by immunohistochemistry, immunofluorescence and immunoblot analysis in Lewis polycystic kidney rats (LPK, a genetic ortholog of human nephronopthsis-9) from postnatal weeks 3 to 20. At each timepoint, renal disease progression and the mRNA expression of NF-κB-dependent genes (TNFα and CCL2) were determined. NF-κB was also histologically assessed in human PKD tissue. Progressive kidney enlargement in LPK rats was accompanied by increased renal cell proliferation and interstitial monocyte accumulation (peaking at weeks 3 and 10 respectively), and progressive interstitial fibrosis (with α smooth muscle actin and Sirius Red deposition significantly increased compared to Lewis kidneys from weeks 3 to 6 onwards). Rel/NF-κB proteins (phosphorylated-p105, p65, p50, c-Rel and RelB) were expressed in cystic epithelial cells (CECs) of LPK kidneys as early as postnatal week 3 and sustained until late-stage disease at week 20. From weeks 10 to 20, nuclear p65, p50, RelB and cytoplasmic IκBα protein levels, and TNFα and CCL2 expression, were upregulated in LPK compared to Lewis kidneys. NF-κB proteins were consistently expressed in CECs of human PKD. The DNA damage marker γ-H2AX was also identified in the CECs of LPK and human polycystic kidneys. Several NF-κB proteins are consistently expressed in CECs in human and experimental PKD. These data suggest that the upregulation of both the canonical and non-canonical pathways of NF-κB signaling may be a constitutive and early pathological feature of cystic renal diseases.

Andrographolide Inhibits Ovariectomy-Induced Bone Loss via the Suppression of RANKL Signaling Pathways.

Osteoporosis is a debilitating skeletal disorder with an increased risk of low-energy fracture, which commonly occurs among postmenopausal women. Andrographolide (AP), a natural product isolated from Andrographis paniculata, has been found to have anti-inflammatory, anti-cancer, anti-asthmatic, and neuro-protective properties. However, its therapeutic effect on osteoporosis is unknown. In this study, an ovariectomy (OVX) mouse model was used to evaluate the therapeutic effects of AP on post-menopausal osteoporosis by using micro-computed tomography (micro-CT). Bone marrow-derived osteoclast culture was used to examine the inhibitory effect of AP on osteoclastogenesis. Real time PCR was employed to examine the effect of AP on the expression of osteoclast marker genes. The activities of transcriptional factors NF-κB and NFATc1 were evaluated using a luciferase reporter assay, and the IκBα protein level was analyzed by Western blot. We found that OVX mice treated with AP have greater bone volume (BV/TV), trabecular thickness (Tb.Th), and trabecular number (Tb.N) compared to vehicle-treated OVX mice. AP inhibited RANKL-induced osteoclastogenesis, the expression of osteoclast marker genes including cathepsin K (Ctsk), TRACP (Acp5), and NFATc1, as well as the transcriptional activities of NF-κB and NFATc1. In conclusion, our results suggest that AP inhibits estrogen deficiency-induced bone loss in mice via the suppression of RANKL-induced osteoclastogensis and NF-κB and NFATc1 activities and, thus, might have therapeutic potential for osteoporosis.

Hint1 Up-Regulates IκBα by Targeting the β-TrCP Subunit of SCF E3 Ligase in Human Hepatocellular Carcinoma Cells.

There is increasing evidence that histidine triad nucleotide-binding protein 1 (HINT1) is a novel tumor suppressor. In the present study, we investigated the mechanism by which HINT1 promotes the stability of inhibitor of NF-κB α (IκBα) in the cytoplasm of hepatocellular carcinoma (HCC) cells, which was observed in our previous study (Wang et al. in Int J Cancer 124:1526-1534, 2009). We examined HINT1 and IκBα expression in HCC cell lines and determined the effect of HINT1 overexpression and knockdown on IκBα protein and mRNA expression in these cell lines. Then, ubiquitination assays were performed to investigate the effects of HINT1 expression plasmid transfection on IκBα ubiquitination. Next, the interaction between HINT1 and β-TrCP was investigated in immunoprecipitation and immunofluorescence assays. Our data showed that increased HINT1 expression in HepG2 and SMMC7702 cells markedly increased IκBα protein levels, while decreased HINT1 expression markedly decreased them. Overexpression or knockdown of HINT1 did not alter the transcription of IκBα, but HINT1 inhibited proteasomal IκBα degradation and reduced its ubiquitination levels. This inhibition might occur because HINT1 is a component of the SCF(β-TrCP) E3 ligase, which is responsible for IκBα ubiquitination and degradation. This study provides new evidence that HINT1 is a regulator of IκBα through SCF(β-TrCP) E3 ligase. These findings help to clarify the mechanism underlying the anticancer effects of HINT1.

Cytokine effects and cellular signaling pathways of grass carp HSP70 in head kidney leukocytes.

In mammals, the cytokine effects of heat shock protein 70 (HSP70) have been well documented. However, the precise role and molecular mechanism of fish HSP70 in cytokine production are poorly understood. In the present study, recombinant grass carp HSP70 (rgcHSP70) was prepared by using the E. coli expression system and its effect on interleukin (IL)-1β (gcil1β) and tumor necrosis factor (TNF)-α (gctnfα) mRNA expression was examined in grass carp head kidney leukocytes (HKLs). Compared with its heated-inactivated protein, rgcHSP70 enhanced the mRNA levels of these two cytokines in a dose- and time-dependent manner. More importantly, the results of enzyme-linked immunoabsorbent assay (ELISA) showed that rgcHSP70 markedly stimulated gcIL-1β and gcTNF-α secretion from the HKLs, for the first time indicating the ability of HSP70 to regulate cytokine release in fish. Moreover, rgcHSP70 displayed more potential to increase the cytokines' release instead of their transcription in the same cell model. Subsequently, the signaling mechanisms governing the cytokine effect of rgcHSP70 were investigated, showing that rgcHSP70-induced gcil1β mRNA expression was suppressed by inhibiting p38 MAPK and NF-κB pathways, while the stimulation of rgcHSP70 on gctnfα mRNA expression were blocked by inhibiting JNK MAPK and NF-κB pathways. In parallel, rgcHSP70 was also shown to activate NF-κB pathway through regulating grass carp IκBα protein levels in a time-dependent oscillation. These findings provide further understanding of cytokine effect and regulatory mechanism of HSP70 in fish and new insights into the roles of HSP70 in teleost immunity.

Cardioprotective effects and pharmacokinetic properties of a controlled release formulation of a novel hydrogen sulfide donor in rats with acute myocardial infarction.

We previously reported that S-propargyl-cysteine (SPRC) exerts cardioprotective effects by elevating H2S levels via the CSE/H2S pathway. In the present study, we investigated the cardioprotective effects and pharmacokinetic properties of a controlled release formulation of SPRC (CR-SPRC) in an in vivo rat model of myocardial infarction (MI). Rats were randomly assigned to seven groups that were pre-treated with CR-SPRC daily for 7 days prior to ligation of the left anterior descending coronary artery to induce MI. Cardiac function and infarct size were determined after MI, and we examined the activity of antioxidant enzymes, expression of anti-inflammation proteins and hydrogen sulfide levels. Mixed-mode, reversed-phase and cation-exchange HPLC-MS/MS were used to compare the pharmacokinetic properties of CR-SPRC and SPRC. CR-SPRC significantly reduced infarct size and creatine kinase (CK) and lactate dehydrogenase (LDH) leakage and it preserved cardiac function during MI. CR-SPRC displayed antioxidant properties, preserving glutathione (GSH), catalase (CAT) and superoxide dismutase (SOD) levels whereas reducing malondialdehyde (MDA) levels. Moreover, CR-SPRC significantly reduced the protein levels of inflammatory biomarkers (phospho-NF-κB p65/NF-κB p65, TNF-α) and increased cystathionine-γ-lyase (CSE) and Iκ-Bα protein levels. CR-SPRC had better pharmacokinetic properties than SPRC, with a reduced concentration peak (Cmax), prolonged time to reach peak concentration (Tmax), prolonged mean residence time (MRTinf) and increased AUC0-t. CR-SPRC showed protective effects against MI via the CSE/H2S pathway and demonstrated better cardioprotective effects than SPRC by prolonging the release of endogenous H2S.

Abstract 487: Saturated FFA Induced NF-κB Activation Is Independent of IKK/IκBα in Aortic Endothelial Cells

Accumulating evidence has suggested a linkage between free fatty acids (FFAs) and chronic inflammation. It has been shown that saturated FFAs inhibits insulin signal in adipocytes though the activation of IKK. In this study, we examined the effect of FFAs on NF-κB activation in bovine aortic endothelial cells. We found that the saturated FFAs, but not the unsaturated FFAs, caused the activation of NF-κB at a low but sustained level. As a result, ICAM-1 expression was also increased. Interestingly, inconsistent with the previous reports on insulin signal, no changes in IKK phosphorylation and IκBα protein level were observed. IKK16, an IKK inhibitor, failed to block saturated FFA induced NF-κB nuclear translocation. These results imply that saturated FFA induced NF-κB activation is independent of IKK activation and IκBα degradation in aortic endothelial cells, suggesting a novel mechanism for NF-κB activation.

Suppression of oxidative stress and improvement of liver functions in mice by ursolic acid via LKB1-AMP-activated protein kinase signaling.

Hepatic cirrhosis is the final stage of liver dysfunction, characterized by diffuse fibrosis, which is the main response to the liver injury. This study is to investigate the effects of ursolic acid (UA) on liver functions and fibrosis in bile duct ligation (BDL) mice and to determine the underlying mechanisms. Cultured hepatocytes were treated with lipopolysaccharide (LPS) in the presence or absence of UA. The reactive oxygen species (ROS) level, protein levels of IκBα, iNOS and Cox-2, and NF-κB activation were detected, respectively. C57/BL6 and AMP-activated protein kinase (AMPK)α2(-/-) mice were subjected to BDL for 14 days. UA was administered by gavage. The markers of liver function and oxidative stress, and liver histopathology were analyzed after treatment. Treatment of hepatocytes with UA dose-dependently activates AMPK, which is abolished by silence of liver kinase B1 (LKB1). LPS significantly increased ROS productions, apoptosis, NF-κB activation, and expressions of iNOS and Cox-2 in cultured hepatocytes. All these effects were blocked by co-incubation with UA. Importantly, silence of LKB1, AMPK, or iNOS/Cox-2 by small interference RNA transfection reversed UA-induced effects in cultured cells. In an animal study, 14-day BDL induced liver fibrosis and liver injury, accompanied with increased oxidative stress and protein expressions of iNOS and Cox-2 in liver. Treatment of UA significantly attenuated the BDL-induced detrimental effects in wild-type mice but not in AMPKα2(-/-) mice. UA via LKB1-AMPK signaling offers protective effects on BDL-induced liver injury in mice, which may be related to inhibition of oxidative stress.

Ulinastatin suppresses lipopolysaccharide induced neuro-inflammation through the downregulation of nuclear factor-κB in SD rat hippocampal astrocyte

Astrocyte activation plays a pivotal role in neuroinflammation, which contributes to neuronal damage, so the inhibition of astrocyte activation may alleviate the progression of neurodegeneration. Recent studies have proved that urinary trypsin inhibitor ulinastatin could inhibit NF-kB activation. In our study, the inhibitory effects of ulinastatin on the production of pro-inflammatory mediators were investigated in lipopolysaccharide (LPS)-reduced primary astrocyte. Our results showed that ulinastatin significantly inhibited LPS-induced astrogliosis, which is measured by MTT and BrdU. Ulinastatin decreased the production of pro-inflammatory cytokines, such as TNF-α, IL-6, IL-1β, it significantly decreased both the mRNA and the protein levels of these pro-inflammatory cytokines and also increased the protein levels of IκB-α binded to NF-κB, which blocked NF-κB translocation to the nucleus and prevented its activity. Our results suggest that ulinastatin is able to inhibit neuroinflammation by interfering with NF-κB signaling. The study provides direct evidence of potential therapy methods of ulinastatin for the treatment of neuroinflammatory diseases.

Antiasthmatic Effects of Eugenol in a Mouse Model of Allergic Asthma by Regulation of Vitamin D3 Upregulated Protein 1/NF-κB Pathway

The aim of the study was to investigate the antiasthmatic effects of eugenol (EUG) and the possible mechanisms. Asthma model was established by ovalbumin induction. A total of 50 mice were randomly assigned to five experimental groups: control, OVA, OVA + dexamethasone (2 mg/kg), OVA + EUG (10 mg/kg), and OVA + EUG (20 mg/kg). Airway resistance (Raw) were measured, histological studies were evaluated by the hematoxylin and eosin (HE) staining, interleukin-4 (IL-4) and interleukin-5 (IL-5) were evaluated by enzyme-linked immunosorbent assay (ELISA), Vitamin D3 upregulated protein 1 (VDUP1), IκBα, P-IκBα, NF-κBP65, and p-NF-κBP65 were measured by Western blotting. Our study demonstrated that EUG inhibited OVA-induced increases in Raw and eosinophil count; IL-4 and IL-5 were recovered. Histological studies demonstrated that EUG substantially inhibited OVA-induced eosinophilia in the lung tissue. Western blotting studies demonstrated that EUG substantially inhibited P-IκBα, NF-κBP65, and p-NF-κBP65 protein levels and increased VDUP1 and IκBα protein levels. These findings suggest that EUG may effectively ameliorate the progression of asthma and could be used as a therapy for patients with allergic asthma.

Synthesis and anti-hepatitis B virus activity of C4 amide-substituted isosteviol derivatives

A series of novel isosteviol derivatives having C4-amide substituents were synthesized in order to test for antiviral effects against the hepatitis B virus (HBV) in vitro. Among them, IN-4 [N-(propylcarbonyl)-4α-amino-19-nor-ent-16-ketobeyeran] (5) exhibited inhibitory activity against secretion of HBsAg and HBeAg as well as inhibition of HBV DNA replication. Therefore, the mechanism of its antiviral activity was further analyzed using HBV-transfected Huh7 cells. Exposure to IN-4 produced minimal inhibitory effects on viral precore/pregenomic RNA expression. However, expression levels of the 2.4/2.1-kb preS/major S RNA of the viral surface gene significantly decreased, along with intracellular levels of HBV DNA. A promoter activity analysis demonstrated that IN-4 significantly inhibited viral X, S, and preS expression levels but not viral core promoter activities. In particular, IN-4 was observed to significantly inhibit HBV gene regulation by disrupting nuclear factor (NF)-κB-associated promoter activity. In addition, the nuclear expression of p65/p50 NF-κB member proteins was attenuated following IN-4 treatment, while cytoplasmic IκBα protein levels were enhanced. Meanwhile, IN-4 was observed to inhibit the binding activity of NF-κB to putative DNA elements. Furthermore, transfection of a p65 expression plasmid into Huh7 cells significantly reversed the inhibitory effect of IN-4 on HBV DNA levels, providing further evidence of the central role of NF-κB in its antiviral mechanism. It is therefore suggested that IN-4 inhibits HBV by interfering with the NF-κB signaling pathway, resulting in downregulation of viral gene expression and DNA replication.

Sesquiterpene lactones inhibit advanced oxidation protein product-induced MCP-1 expression in podocytes via an IKK/NF-κB-dependent mechanism.

Inflammation is a relevant factor in the pathogenesis of diabetes nephropathy (DN). Sesquiterpene lactones (SLs), originally isolated from Tanacetum parthenium, have been reported to exhibit anti-inflammatory effects but few studies have examined their effects on DN. To determine whether advanced oxidation protein products (AOPPs) can induce the expression of chemokine monocyte chemoattractant protein- (MCP-) 1 in cultured mouse podocytes and to explore the mechanisms of the potential renoprotection of SLs, we treated podocytes with AOPPs and SLs (parthenolide and its derivatives micheliolide, compound 1, and compound 2). MCP-1 mRNA and protein expression were tested using quantitative real-time PCR and ELISA, respectively, and the protein levels of IKKβ, phospho-IKKβ, IκBα, NF-κB p65, phospho-NF-κB p65, and tubulin were analyzed by Western blotting. AOPPs activated the expression of MCP-1 mRNA and protein in a dose- and time-dependent manner, activated IKKβ and NF-κB p65, and promoted IκBα degradation. The IKK/NF-κB inhibitor parthenolide decreased AOPP-induced MCP-1 expression. Pretreatment with SLs inhibited MCP-1 mRNA and protein expression and suppressed IKKβ and NF-κB p65 phosphorylation and IκBα degradation. Taken together, these findings provide a novel explanation for the anti-inflammatory effects of SLs that will ultimately benefit DN and potentially other inflammatory and immune renal diseases.

Protective effect ofSalvia miltiorrhizaandCarthamus tinctoriusextract against lipopolysaccharide-induced liver injury

To investigate the hepatoprotective effects and mechanisms of an extract of Salvia miltiorrhiza and Carthamus tinctorius in vivo. C57BL/6J mice were randomly assigned to five groups and intraperitoneally administered 0.9% saline, Salvia miltiorrhiza and Carthamus tinctorius extract [Danhong injection (DHI), 0.75 and 3 g/kg mixed extract] or reduced glutathione for injection (RGI, 300 mg/kg) for 30 min before exposure to lipopolysaccharide (LPS, 16 mg/kg). After intraperitoneal LPS stimulation for 90 min or 6 h, the mice were sacrificed by ether anaesthesia, and serum and liver samples were collected. Histological analysis (H&E) and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling (TUNEL) staining were performed. Alanine transferase (ALT), aspartate transaminase (AST), total bilirubin (TBil), glutathione-S-transferase (GST), malondialdehyde (MDA), tumour necrosis factor (TNF)-α, interleukin (IL)-6, and caspase-3 levels were measured. Bax, Bcl-2, P-IκBα, IκBα, P-NF-κB p65, and NF-κB p65 protein levels were determined by Western blot. TNF-α, IL-6, caspase-3, Bax and Bcl-2 mRNA expression was measured by real-time reverse transcription-polymerase chain reaction (RT-PCR). Hematoxylin-eosin staining and TUNEL results suggested that DHI (3 g/kg) treatment alleviated inflammatory and apoptotic (P < 0.01) injury in the liver of mice. DHI treatment dose-dependently blunted the abnormal changes in biochemical parameters such as ALT (72.53 ± 2.83 for 3 g/kg, P < 0.01), AST (76.97 ± 5.00 for 3 g/kg, P < 0.01), TBil (1.17 ± 0.10 for 3 g/kg, P < 0.01), MDA (0.81 ± 0.36 for 3 g/kg, P < 0.01), and GST (358.86 ± 12.09 for 3 g/kg, P < 0.01). Moreover, DHI (3 g/kg) remarkably decreased LPS-induced protein expression of TNF-α (340.55 ± 10.18 for 3 g/kg, P < 0.01), IL-6 (261.34 ± 10.18 for 3 g/kg, P < 0.01), and enzyme activity of caspase-3 (0.93 ± 0.029 for 3 g/kg, P < 0.01). The LPS-induced mRNA expression of TNF-α, IL-6 and caspase-3 was also decreased by DHI. Western blot analysis revealed that DHI antagonised LPS-stimulated decrease of Bcl-2 and increase of Bax protein expression. Furthermore, DHI inhibited LPS-induced IκBα and NF-κB p65 phosphorylation. DHI may be a multi-function protectant against acute hepatic injury in mice through its anti-inflammatory, anti-oxidative and anti-apoptotic activities.

Effect of Candida albicans on the production of interleukin-6 by and activation of IκBα in human THP-1 monocytes

Objective To investigate the effect of Candida albicans on the production of interleukin-6 by and activation of IκBα in an acute monocytic leukemia cell line THP-1.Methods THP-1 cells were classified into three groups to be stimulated by heat-killed C.albicans cells in concentrations of 105 and 106 colony-forming units (CFU)/ml and lipopolysaccharide (100 μg/L) in vitro respectively.Those remaining untreated served as the blank control group.After additional culture for different durations,real time reverse transcription PCR and enzyme-linked immunosorbent assay were performed to measure the mRNA and protein expression levels of interleukin-6 (IL-6) respectively,and Western blot was conducted to determine the levels of total and phosphorylated IκBα.Statistical analysis was carried out by t test.Results A significant increase was observed in the mRNA expression level (2-△△α) of IL-6 in THP-1 cells at 3 and 6 hours after starting treatment with C.albicans at 105 CFU/ml (6.48 ± 0.30 vs.0.84 ± 0.16,125.34 ± 1.47 vs.1.22 ± 0.22,both P ＜ 0.01),and at 1,3 and 6 hours after starting treatment with C.albicans at 106 CFU/ml (2.96 ± 0.35 vs.1.03 ± 0.16,8.57 ± 1.27 vs.0.84 ± 0.16,588.10 ± 2.31 vs.1.22 ± 0.22,P＜ 0.05 or 0.01) compared with the blank control group,but no significant difference was noted between THP-1 cells at 1 hour after starting treatment with C.albicans at 105 CFU/ml and those remaining untreated (1.48 ± 0.06 vs.1.03 ± 0.16,P＞ 0.05).The protein expression level of IL-6 was (924.9 ± 30.13) ng/L in THP-1 cells at 24 hours after starting treatment with C.albicans at 106 CFU/ml,significantly different from that in the blank control group (P ＜ 0.001).There was a marked elevation in the level of phosphorylated IκBα protein,but an obvious reduction in the level of IκBα protein in THP-1 cells treated with C.albicans at 106 CFU/ml for 30 and 60 minutes.Conclusion Human THP-1 monocytes may play a role in innate immune responses against C.albicans through activation of nuclear factor-κB and production of IL-6. Key words: Candida albicans; Leukemia, monocytic, acute; Interleukin-6; I-kappa B proteins; NF-kappa B

Abstract 3551: Non-coding RNA HOTAIR connects DNA damage signaling to NF-κB activation in cisplatin resistant ovarian cancer

Abstract The Polycomb Repressive Complex 2 (PRC2) has been implicated in cancer and a role for PRC2 during DNA damage response (DDR) has recently been reported. To examine changes in gene expression with DDR in cisplatin (cddp)-resistant ovarian cancer (OC), we performed whole transcriptome RNA-seq analysis of isogenic cddp-sensitive and -resistant OC cell lines (A2780 and A2780-cp). Differential expression of PRC components (SUZ12, EZH1, SIRT1, PHC1 &amp;2) (P&amp;lt;0.05), NF-κB pathway members (NCAM, ABCB9, RAGE, IL4R, IL6R, BCL2L11) (P&amp;lt;0.05), and long non-coding RNAs (lncRNAs) known to associate with PRC2 (P&amp;lt;0.05), including Hox transcript antisense intergenic RNA or HOTAIR, was observed. In tumors from patients with high grade serous OC at diagnosis, basal expression of HOTAIR was greater (P&amp;lt;0.01) compared to normal ovarian surface epithelium and marked overexpression of HOTAIR was observed in tumors obtained from patients who had developed platinum-resistant OC. Ablation of HOTAIR using dsiRNA resensitized A2780-cp to cddp, while ectopic over-expression of HOTAIR increased (P&amp;lt;0.05) A2780 cell survival after cddp treatment (3-fold vs. control). To further examine HOTAIR regulation, we conducted a promoter analysis using bioinformatics tools and luciferase assays. We identified a putative p65-NF-κB binding site (906-GGGACACCCC-915) 906 bp upstream of the HOTAIR transcription start site. Treatment of A2780 cells with the NF-κB activator TNF-α induced HOTAIR (16-fold vs. control) and NF-κB enrichment (3-fold assessed by ChIP assays) at the HOTAIR promoter. Furthermore, in A2780-cp compared to A2780, total Iκ-Bα levels were reduced (P&amp;lt;0.05) and nuclear p65 levels were increased, indicating that endogenous activation of NF-κB contributes to cddp resistance and DDR. Consistent with this observation, cddp treatment (20μM for 0-24hrs) of A2780 cells increased (P&amp;lt;0.05) HOTAIR expression by 5- and 16-fold at 8 and 24 hrs and decreased (P&amp;lt;0.05) Iκ-Bα protein levels at similar time points. Furthermore, inhibiting NF-κB by either gliotoxin (5μM) or Bay-11 (3μM) completely abolished cddp-induced HOTAIR expression in A2780 cells, demonstrating that NF-κB is a HOTAIR transcriptional activator during cddp-induced DDR. Importantly, EZH2 and histone H3 lysine-27 trimethylation (H3K27me3) levels were enriched (6- and 17-fold) in the Iκ-Bα promoter at 24 and 48 hours post cddp treatment, and HOTAIR depletion using dsiRNA reduced the observed EZH2-H3K27me3 enrichment at the Iκ-Bα promoter, demonstrating that HOTAIR recruits PRC2 complex to the Iκ-Bα promoter to prolong NF-κB activation during cddp-induced genotoxic stress. Mouse xenograft studies with A2780 cells overexpressing HOTAIR are ongoing. The results of this study support a role for HOTAIR as a positive regulator of the NF-κB pathway and PRC2 during cisplatin-induced DNA damage. We further suggest that HOTAIR may serve as a therapeutic target in cisplatin-resistant OC. Citation Format: Ali R. Ozes, Dave F. Miller, Yunlong Liu, Kenneth P. Nephew. Non-coding RNA HOTAIR connects DNA damage signaling to NF-κB activation in cisplatin resistant ovarian cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3551. doi:10.1158/1538-7445.AM2014-3551

Anti-viral effect of a compound isolated from Liriope platyphylla against hepatitis B virus in vitro

The compound LPRP-Et-97543 was isolated from Liriope platyphylla roots and was observed to have potential anti-viral effects in HepG2.2.15 cells against hepatitis B virus (HBV). The antiviral mode was further clarified, and the HBV-transfected Huh7 cells were used as the platform. During viral gene expression, LPRP-Et-97543 treatment had apparent effects on the viral precore/pregenomic and S/preS RNA. Promoter activity analysis demonstrated that LPRP-Et-97543 significantly reduced Core, S, and preS but not X promoter activities. Further examination showed that putative signaling pathways were involved in this inhibitory effect, indicating that NF-κB may serve a putative mediator of HBV gene regulation with LPRP-Et-97543. In addition, the nuclear expression of p65/p50 NF-κB member proteins was attenuated with LPRP-Et-97543 and augmented cytoplasmic IκBα protein levels but without affecting the expression of these proteins in HBV non-transfected cells during treatment. Moreover, LPRP-Et-97543 reduced the binding activity of NF-κB protein to CS1 element of HBV surface gene in a gel retardation analysis and inhibited CS1 containing promoter activity in HBV expressed cells. However, HBV transfection significantly enhances CS1 containing promoter activity without compound treatment in cells. Finally, transfection of the p65 expression plasmid significantly reversed the inhibitory effect of LPRP-Et-97543 on the replicated HBV DNA level in HBV positive cells. In conclusion, this study suggests that the mechanism of HBV inhibition by LPRP-Et-97543 may involve the feedback regulation of viral gene expression and viral DNA replication by HBV viral proteins, which interferes with the NF-κB signaling pathway.