Anti-Toll-like receptor 2 antibody inhibits nuclear factor kappa B activation and attenuates cardiac damage in high-fat-feeding rats

Long-time con sumption of high-fat food is a direct cause of cardiovascular diseases, and high-fatrelated inflammation plays an important role in it. Toll-like receptors (TLRs), especially TLR2 and TLR4, play important roles in high-fat-related inflammation. However, the impact of TLR2 on high-fatassociated cardiovascular complications is still unknown. In this study, we try to investigate the relationship between TLR2 and high-fat-related cardiac injury. SD rats were allocated to either a control group which were fed with normal diet or a high-fat group which were fed with high-fat diet for 5 months. At the last mon th, rats fed with high-fat diet were intraperitoneally injected with control normal mouse IgG or anti-TLR2 antibody. Heart tissues were collected for further analysis. RT-qPCR and western blot analysis results revealed that TLR2 expression was increased in the heart tissues from rats fed with high-fat diet and anti-TLR2 antibody had no effect on TLR2 expression. However, anti- TLR2 antibody alleviated masson staining area, levels of TGF-pi and Collagen I mRNA, and decreased TUNEL-positive myocardial cells and caspase-3 activity, suggesting that anti-TLR2 antibody protected cardiac cells against high-fat-induced cardiac fibrosis and cell apoptosis. By using immunohistochemistry, RT-qPCR and ELISA, we found that anti-TLR2 antibody blocked NF-kB activation, inhibited the expression of inflammatory factors such as TNF-a, IL-1,IL-6 and IL-18 in the heart tissues from rats fed with high-fat diet. These results hinted that anti-TLR2 antibody might exert its protective effect via inhibition of the TLR2/NF-KB/inflammation pathway. Our findings suggest that anti-TLR2 antibody has a preventive function against high-fat-induced deleterious effects in the heart, and anti-TLR2 antibody may be used as an attractive therapeutic option for high-fat-induced cardiac injury.     

TNP-470 inhibits oxidative stress, nitric oxide production and nuclear factor kappa B activation in a rat model of hepatocellular carcinoma

The objective of this study is to determine if treatment with the angiogenesis inhibitor TNP-470 results in impairment of oxidative stress, inhibition of nuclear factor kappa B (NF-κB) activation and decrease of nitric oxide production in an experimental model of rat hepatocarcinogenesis. Tumour was induced by diethylnitrosamine and promoted by two-thirds hepatectomy plus acetaminofluorene administration. Experiments were carried out at 28 weeks after initiating the treatment. TNP-470 was administered at 30 mg/kg, three times per week from 20 to 28 weeks. Carcinomatous tissue growing outside dysplastic nodules and a marked expression of placental glutathione S-transferase were detected in rats with induced carcinogenesis. Liver concentrations of thiobarbituric acid reactive substances, reduced glutathione (GSH) and glutathione disulfide (GSSG) were significantly higher than those of controls and there was a significant increase in the GSSG/GSH ratio. Tumour growth was accompanied by augmented expression of inducible nitric oxide synthase, activation of (NF-κB) and proteolysis of IkappaB. All these effects were absent in animals receiving TNP-470. Our results indicate that TNP-470 inhibits oxidative stress, nitric oxide production and NF-κB activation induced by experimental hepatocarcinogenesis. These changes would contribute to the beneficial effects of TNP-470 in cancer treatment.     

Calpain inhibitor I reduces the activation of nuclear factor-kappaB and organ injury/dysfunction in hemorrhagic shock.

There is limited evidence that inhibition of the activity of the cytosolic cysteine protease calpain reduces ischemia/reperfusion injury. The multiple organ injury associated with hemorrhagic shock is due at least in part to ischemia (during hemorrhage) and reperfusion (during resuscitation) of target organs. Here we investigate the effects of calpain inhibitor I on the organ injury (kidney, liver, pancreas, lung, intestine) and dysfunction (kidney) associated with hemorrhagic shock in the anesthetized rat. Hemorrhage and resuscitation with shed blood resulted in an increase in calpain activity (heart), activation of NF-kappaB (kidney), expression of iNOS and COX-2 (kidney), and the development of multiple organ injury and dysfunction, all of which were attenuated by calpain inhibitor I (10 mg/kg i.p.), administered 30 min prior to hemorrhage. Chymostatin, a serine protease inhibitor that does not prevent the activation of NF-kappaB, had no effect on the organ injury/failure caused by hemorrhagic shock. Pretreatment (for 1 h) of murine macrophages or rat aortic smooth muscle cells (activated with endotoxin) with calpain inhibitor I attenuated the binding of activated NF-kappaB to DNA and the degradation of IkappaBalpha, IkappaBbeta, and IkappaBvarepsilon. Selective inhibition of iNOS activity with L-NIL reduced the circulatory failure and liver injury, while selective inhibition of COX-2 activity with SC58635 reduced the renal dysfunction and liver injury caused by hemorrhagic shock. Thus, we provide evidence that the mechanisms by which calpain inhibitor I reduces the circulatory failure as well as the organ injury and dysfunction in hemorrhagic shock include 1) inhibition of calpain activity, 2) inhibition of the activation of NF-kappaB and thus prevention of the expression of NFkappaB-dependent genes, 3) prevention of the expression of iNOS, and 4) prevention of the expression of COX-2. Inhibition of calpain activity may represent a novel therapeutic approach for the therapy of hemorrhagic shock.     

Fluvastatin inhibits angiotensin II-induced nuclear factor kappa B activation in renal tubular epithelial cells through the p38 MAPK pathway

3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors has been shown to reduce the progression of renal disease independent of cholesterol-lowering effect, but the mechanism of potential protective effect remains unclear. Here, we investigate the effect of fluvastatin on activation of nuclear factor-κB (NF-κB) induced by angiotensin II (AngII) in rat kidney tubule epithelial cells (NRK-52E). Electrophoretic mobility shift assays (EMSA) was used to detect NF-κB activation. Phosphorylation of cellular p38 mitogen-activated protein kinase (p38MAPK) was determined by western blot analysis. AngII stimulated the DNA-binding activity of NF-κB and phosphorylation of p38MAPK in cultured NRK-52E cells in a dose-dependent (10⁻⁹–10⁻⁶ mol/l) manner (P < 0.01). AngII (10⁻⁶ mol/l) induced a rapid (5 min) increase of the p38MAPK phosphorylation. NF-κB DNA-binding activity was increased at as early as 30 min, peaked at 2 h after AngII treatment. This stimulatory effect of AngII on NF-κB was blocked by SB203580 (a specific inhibitor of p38MAPK). Incubation of cells with fluvastatin significantly inhibited the AngII-induced NF-κB activation in a dose-dependent (10⁻⁷–10⁻⁵ mol/l) manner (P < 0.05). Exogenous mevalonate (10⁻⁴mol/l) prevented the effect of fluvastatin on NF-κB activation. These results suggest the fluvastatin reduced AngII-induced NF-κB activation via the p38MAPK pathway in NRK-52E cells. The effect is at least partly due to blocking the biosynthesis of mevalonate.     

Salicylates inhibit flavivirus replication independently of blocking nuclear factor kappa B activation

Flaviviruses comprise a positive-sense RNA genome that replicates exclusively in the cytoplasm of infected cells. Whether flaviviruses require an activated nuclear factor(s) to complete their life cycle and trigger apoptosis in infected cells remains elusive. Flavivirus infections quickly activate nuclear factor kappa B (NF-kappaB), and salicylates have been shown to inhibit NF-kappaB activation. In this study, we investigated whether salicylates suppress flavivirus replication and virus-induced apoptosis in cultured cells. In a dose-dependent inhibition, we found salicylates within a range of 1 to 5 mM not only restricted flavivirus replication but also abrogated flavivirus-triggered apoptosis. However, flavivirus replication was not affected by a specific NF-kappaB peptide inhibitor, SN50, and a proteosome inhibitor, lactacystin. Flaviviruses also replicated and triggered apoptosis in cells stably expressing IkappaBalpha-DeltaN, a dominant-negative mutant that antagonizes NF-kappaB activation, as readily as in wild-type BHK-21 cells, suggesting that NF-kappaB activation is not essential for either flavivirus replication or flavivirus-induced apoptosis. Salicylates still diminished flavivirus replication and blocked apoptosis in the same IkappaBalpha-DeltaN cells. This inhibition of flaviviruses by salicylates could be partially reversed by a specific p38 mitogen-activated protein (MAP) kinase inhibitor, SB203580. Together, these results show that the mechanism by which salicylates suppress flavivirus infection may involve p38 MAP kinase activity but is independent of blocking the NF-kappaB pathway.     

核因子—kB的研究进展

机体对损伤及微生物和侵进入防御反应时,活化的核因子－ｋＢ（ＮＦ－ｋＢ）可诱导细胞合成各种生物大分子。细胞处于静息状态时,ＮＦ－ｋＢ与ｋＢ抑制蛋白（ＩｋＢｓ）结合形成三聚体存在于细胞质内。当细胞受到外界因素刺激时,ＮＦ－ｋＢ与ＩｋＢｓ分离,ＮＦ－ｋＢ进入细胞核内,其亚基形成环状结构与ＤＮＡ接触,启动基因转录。随后,新合成的ＩｋＢｓ又与ＮＦ－ｋＢ结合返回细胞质,不同ＩｋＢｓ亚型发挥不同的生理功能,同时     

Translational repression mediates activation of nuclear factor kappa B by phosphorylated translation initiation factor 2

Numerous stressful conditions activate kinases that phosphorylate the alpha subunit of translation initiation factor 2 (eIF2alpha), thus attenuating mRNA translation and activating a gene expression program known as the integrated stress response. It has been noted that conditions associated with eIF2alpha phosphorylation, notably accumulation of unfolded proteins in the endoplasmic reticulum (ER), or ER stress, are also associated with activation of nuclear factor kappa B (NF-kappaB) and that eIF2alpha phosphorylation is required for NF-kappaB activation by ER stress. We have used a pharmacologically activable version of pancreatic ER kinase (PERK, an ER stress-responsive eIF2alpha kinase) to uncouple eIF2alpha phosphorylation from stress and found that phosphorylation of eIF2alpha is both necessary and sufficient to activate both NF-kappaB DNA binding and an NF-kappaB reporter gene. eIF2alpha phosphorylation-dependent NF-kappaB activation correlated with decreased levels of the inhibitor IkappaBalpha protein. Unlike canonical signaling pathways that promote IkappaBalpha phosphorylation and degradation, eIF2alpha phosphorylation did not increase phosphorylated IkappaBalpha levels or affect the stability of the protein. Pulse-chase labeling experiments indicate instead that repression of IkappaBalpha translation plays an important role in NF-kappaB activation in cells experiencing high levels of eIF2alpha phosphorylation. These studies suggest a direct role for eIF2alpha phosphorylation-dependent translational control in activating NF-kappaB during ER stress.     

Chronic inhibition of nuclear factor kappa B attenuates aldosterone/salt-induced renal injury

AimsRecent studies suggested that nuclear factor kappa B (NF-κB) plays a key role in the pathogenesis of renal injury. This study investigated whether NF-κB inhibition attenuates progressive renal damage in aldosterone/salt-induced renal injury and its mechanisms.Main methodsAdult male rats were uninephrectomized and treated with one of the following for 4 weeks: vehicle (0.5% ethanol, subcutaneously); vehicle/1% NaCl (1% NaCl in drinking solution); aldosterone/1% NaCl (1% NaCl in drinking solution and aldosterone, 0.75 μg/h, subcutaneously); or aldosterone/1%NaCl + pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-κB (100 mg/kg/day, by gavage). The activity of NF-κB was measured by EMSA and immunohistochemistry, CTGF and ICAM-1 were measured by Western blot and real-time PCR, and TGF-β and CTGF were measured by immunohistochemistry.Key findingsRats that received aldosterone/1% NaCl exhibited hypertension and severe renal injury. Renal cortical mRNA levels of CTGF, TGF-β, ICAM-1 and collagen IV, protein expression of CTGF and ICAM-1, and NF-κB–DNA binding activity were significantly upregulated in rats that received aldosterone/1% NaCl. Treatment with PDTC significantly decreased the percentage of cells positive for CTGF and TGF-β; mRNA levels of CTGF, TGF-β, ICAM-1 and collagen IV, and protein levels of CTGF and ICAM-1 were also inhibited by PDTC.SignificanceThese data suggest that the NF-κB signal pathway plays a role in the progression of aldosterone/salt-induced renal injury.     

Dicoumarol alters cellular redox state and inhibits nuclear factor kappa B to enhance arsenic trioxide-induced apoptosis

Cellular reduction/oxidation (redox) state was found toplay an important role in regulation of cell proliferation,differentiation and apoptosis [1,2]. Researches in the pastdecade showed that the chemotherapeutic effects ofa number of cytotoxic drugs are influenced by the cellularredox state. Arsenic trioxide (As2O3), as a novel chemo-therapeutic agent treating acute promyelocytic leukemia(APL), induces apoptosis of leukemia cells with de-pendence on the cellular redox state [3,4]. Redox sta…     

Attachment of Staphylococcus aureus is required for activation of nuclear factor kappa B in human osteoblasts

Nuclear factor kappa B (NF-κB) plays a prominent role in the pathogenesis of infectious diseases. Staphylococcus aureus (S. aureus), which can attach to and invade human osteoblasts, is the most common causative agent of osteomyelitis. To determine whether S. aureus can activate NF-κB in human osteoblasts and explore the possible factors of activation in response to infection, we used flow cytometry, enzyme-linked immunosorbent assay, immunoblots, and electrophoretic mobility shift assays to quantify the invasion of bacteria, to measure the interleukin-6 (IL-6) of culture supernatants, and to investigate the IκBα degradation and NF-κB activation in human osteoblasts. Moreover, we explored the possible factors responsible for the activation of NF-κB by preventing S. aureus from physically touching human osteoblasts or inhibiting the invasion of S. aureus into human osteoblasts under co-culture conditions, by incubating proteinase K-treated or ultraviolet-killed S. aureus with human osteoblasts and by treating human osteoblasts with peptidoglycan (PGN) or lipoteichoic acid (LTA). We found that S. aureus induced the IκBα degradation and NF-κB activation, which could regulate IL-6 secretion in the culture supernatants of human osteoblasts in response to infection. In addition, the maximal IκBα degradation and NF-κB activation in human osteoblasts occurred prior to the maximal invasion of S. aureus. It was the attachment not invasion or the secreted soluble factor(s), PGN, LTA of S. aureus, that could induce the IκBα degradation and NF-κB activation in human osteoblasts. These results indicated that S. aureus can activate NF-κB in human osteoblasts and that the attachment of S. aureus is required for this activation in response to infection.     

Neuropeptide-inducible upregulation of proteasome activity precedes nuclear factor kappa B activation in androgen-independent prostate cancer cells

Background

Upregulation of nuclear factor kappa B (NF??B) activity and neuroendocrine differentiation are two mechanisms known to be involved in prostate cancer (PC) progression to castration resistance. We have observed that major components of these pathways, including NF??B, proteasome, neutral endopeptidase (NEP) and endothelin 1 (ET-1), exhibit an inverse and mirror image pattern in androgen-dependent (AD) and -independent (AI) states in vitro.

Methods

We have now investigated for evidence of a direct mechanistic connection between these pathways with the use of immunocytochemistry (ICC), western blot analysis, electrophoretic mobility shift assay (EMSA) and proteasome activity assessment.

Results

Neuropeptide (NP) stimulation induced nuclear translocation of NF??B in a dose-dependent manner in AI cells, also evident as reduced total inhibitor ??B (I??B) levels and increased DNA binding in EMSA. These effects were preceded by increased 20?S proteasome activity at lower doses and at earlier times and were at least partially reversed under conditions of NP deprivation induced by specific NP receptor inhibitors, as well as NF??B, I??B kinase (IKK) and proteasome inhibitors. AD cells showed no appreciable nuclear translocation upon NP stimulation, with less intense DNA binding signal on EMSA.

Conclusions

Our results support evidence for a direct mechanistic connection between the NPs and NF??B/proteasome signaling pathways, with a distinct NP-induced profile in the more aggressive AI cancer state.