NF-κB P65 Protein Levels Research Articles

The sinomenine enteric-coated microspheres suppressed the TLR/NF-κB signaling in DSS-induced experimental colitis

Sinomenine is a pure alkaloid with immunosuppressive effects that is extracted from the Chinese medicinal plant Sinomenium acutum. We studied the therapeutic effects of sinomenine on inflammatory bowel disease. In this study, we randomly divided mice into the following ten groups: Control group; DSS-induced colitis group; Salicylazosulfapyridine (SASP)-treated group; Chitosan-treated group; low-, medium-, and high-dose sinomenine-treated and sinomenine enteric-coated microspheres-treated groups. We recorded changes in colon length, disease activity index (DAI), and colon pathology, measured TLR4, MyD88, SIGIRR, NF-κB p65 protein levels and inflammatory serum cytokine levels.Except for the Control group, the weight of mice in each group decreased, the DAI of the DSS-induced colitis group was significantly higher than the other groups, and the DAIs of the sinomenine- and sinomenine enteric-coated microspheres-treated groups were significantly lower than that of the SASP-treated group. TLR4, MyD88, NF-κB p65 and proinflammatory cytokine expressions decreased dose dependently in the sinomenine and sinomenine enteric-coated microspheres-treated groups and were generally lower in the sinomenine enteric-coated microspheres groups. However, SIGIRR and anti-inflammatory IL-10 expressions exhibited the opposite pattern. Based on the superior therapeutic effect, sinomenine enteric-coated microspheres might regulate TLR/NF-κB signaling and would be beneficial for an effective and safe therapy of inflammatory bowel disease.

Stimulation of autophagy prevents intestinal mucosal inflammation and ameliorates murine colitis.

Defective autophagy contributes to the pathogenesis of inflammatory disorders such as inflammatory bowel disease and there are interactions between autophagy and inflammation. Here we have analysed the effects of autophagy stimulators on murine colitis. Mice were treated with intrarectal administration of 2,4,6-trinitrobenzenesulfonic acid (TNBS) (3.5mg·20g-1 ) and body weight was measured daily. Histological damage was scored 2 or 4days after treatment. Some mice received trehalose (3% in drinking water 3weeks before TNBS administration) or a daily administration of rapamycin (1.25mg·kg-1 , i.p.), betanin (1g·kg-1 , i.p.) or betanin+3-methyladenine (3MA) (10mg·kg-1 , i.p.). Protein levels of p-mTOR, p62, LC3, BCL10, NFκB, IκBα and p-IκBα in mucosa were determined by Western blots and mRNA expression of TNFα, IL1β, IL6, IL10, COX2, CCR7, CD11c, inducible NOS and CD86 by qRT-PCR. Impaired autophagy associated with body weight loss and intestinal damage was detected in the mucosa of TNBS-treated mice. Administration of trehalose, rapamycin or betanin prevented the impaired autophagic flux induced by TNBS and decreased mucosal protein levels of BCL10, p-IκBα and NFκB-p65 and the expression of pro-inflammatory cytokines and M1 macrophage markers. Blockade of autophagosome formation by treatment with 3MA, prevented the reduction in protein levels of p62, BCL10, p-IκBα and NFκB-p65 induced by betanin in TNBS-treated mice and weakened the protective effects of betanin on murine colitis. Pharmacological stimulation of mucosal autophagy reduced intestinal inflammation and improved murine colitis.

Artificial Zinc-Finger Transcription Factor of A20 Suppresses Restenosis in Sprague Dawley Rats after Carotid Injury via the PPARα Pathway.

The inhibition of inflammation and vascular smooth muscle cell (VSMC) proliferation is an ideal strategy to suppress intimal hyperplasia after percutaneous transluminal angioplasty (PTA). Evidence has indicated that overexpression of A20 suppresses neointima formation, but its low transfection efficiency limits its application. Hence, we upregulated A20 expression via transfection of rAd.ATF (recombinant adenovirus vector of artificial transcription factor) and rAd.A20 in rat carotid arteries after balloon dilatation (in vivo) and isolated VSMCs (in vitro). In vivo, we found that after rAd.ATF and rAd.A20 transfection, A20 expression was markedly increased, whereas proliferating cell nuclear antigen (PCNA) and nuclear factor κB p65 (NF-κBp65) protein levels were significantly decreased, and intimal hyperplasia and secretion of proinflammatory factors were significantly reduced when compared with empty vector and saline control groups. Most importantly, the rAd.ATF-treated group showed more significant inhibition on intimal hyperplasia and expression of PCNA than the rAd.A20-treated group. In vitro, compared with the control group, transfection of rAd.ATF and rAd.A20 significantly increased A20 expression, which upregulated the proliferator-activated receptor (PPAR) level for both mRNA and protein, and reduced migration and proliferation of VSMCs and lipopolysaccharide (LPS)-induced inflammation. Furthermore, the PPARα agonist GW6471 could partially restore the effect of A20 on VSMCs. Our findings indicate that the ATF of A20 inhibits neointimal hyperplasia and, therefore, constitutes a novel potential alternative to prevent restenosis.

Isoliquiritigenin alleviates early brain injury after experimental intracerebral hemorrhage via suppressing ROS- and/or NF-\u03baB-mediated NLRP3 inflammasome activation by promoting Nrf2 antioxidant pathway

BackgroundIntracerebral hemorrhage (ICH) induces potently oxidative stress responses and inflammatory processes. Isoliquiritigenin (ILG) is a flavonoid with a chalcone structure and can activate nuclear factor erythroid-2 related factor 2 (Nrf2)-mediated antioxidant system, negatively regulate nuclear factor-κB (NF-κB) and nod-like receptor family, pyrin domain-containing 3 (NLRP3) inflammasome pathways, but its role and potential molecular mechanisms in the pathology following ICH remain unclear. The present study aimed to explore the effects of ILG after ICH and underlying mechanisms.MethodsICH model was induced by collagenase IV (0.2 U in 1 μl sterile normal saline) in male Sprague-Dawley rats weighing 280–320 g. Different doses of ILG (10, 20, or 40 mg/kg) was administrated intraperitoneally at 30 min, 12 h, 24 h, and 48 h after modeling, respectively. Rats were intracerebroventricularly administrated with control scramble small interfering RNA (siRNA) or Nrf2 siRNA at 24 h before ICH induction, and after 24 h, ICH model was established with or without ILG (20 mg/kg) treatment. All rats were dedicated at 24 or 72 h after ICH. Neurological deficits, histological damages, brain water content (BWC), blood-brain barrier (BBB) disruption, and neuronal degeneration were evaluated; quantitative real-time RT-PCR (qRT-PCR), immunohistochemistry/immunofluorescence, western blot, and enzyme-linked immunosorbent assay (ELISA) were carried out; catalase, superoxide dismutase activities and reactive oxygen species (ROS), and glutathione/oxidized glutathione contents were measured.ResultsILG (20 and 40 mg/kg) markedly alleviated neurological deficits, histological damages, BBB disruption, brain edema, and neuronal degeneration, but there was no significant difference between two dosages. ILG (20 mg/kg) significantly suppressed the NF-κB and NLRP3 inflammasome pathways and activated Nrf2-mediated antioxidant system. Gene silencing of Nrf2 aggravated the neurological deficits, brain edema, and neuronal degeneration and increased the protein levels of NF-κB p65, NLRP3 inflammasome components, and IL-1β. ILG delivery significantly attenuated the effects of Nrf2 siRNA interference mentioned above.ConclusionsIntraperitoneal administration of ILG after ICH reduced early brain impairments and neurological deficits, and the mechanisms were involved in the regulation of ROS and/or NF-κB on the activation of NLRP3 inflammasome pathway by the triggering of Nrf2 activity and Nrf2-induced antioxidant system. In addition, our experimental results may make ILG a potential candidate for a novel therapeutical strategy for ICH.

Association of NF-κB and its downstream pathway with acute radiation-induced myocardial fibrosis in rats

Objective To examine the pathological changes in the myocardial tissues such as inflammatory response and fibrosis in a rat model of acute radiation-induced heart damage (RIHD), and to explore whether NF-κB and its downstream pathway are associated with acute radiation-induced myocardial fibrosis. Methods Fourteen male adult Sprague-Dawley rats were randomly divided into control group and radiation group. Local heart irradiation was delivered to the precordial region of rats to establish an RIHD model in a single fraction with a dose of 20 Gy generated by a 6 MV linear accelerator. At 14 days after irradiation, the histopathological changes in myocardial and interstitial tissues were examined by HE staining; the distribution of collagen fibers was observed by Masson staining, and collagen volume fraction (CVF) was used as a semi-quantitative evaluation for myocardial collagen deposition, which was defined as the percentage of collagen area occupied in total area, and was compared using the independent-samples t test. The protein and mRNA expression levels of the NF-κB members p50 and p65 and the downstream pathway members hypoxia-inducible factor 1α(HIF-1α), connective tissue growth factor (CTGF), and type I (COL-1) were quantitatively analyzed by Western blot and qPCR, respectively. Results At 14 days after local heart irradiation, the radiation group showed significant myocardial edema and derangement, rupture of some myocardial cells, mild nuclear pyknosis, darkened nuclear staining, a small number of irregular nuclei, and myocardial interstitial inflammatory cell infiltration accompanied by increased fibroblast, as compared with the control group. The Masson staining showed that the collagen fibers in radiation group were widely distributed at the interstitial tissue and increased significantly compared with those in the control group; normal myocardial cells were in disordered array and had a tendency to be replaced by collagen fibers. The semi-quantitative analysis showed that radiation induced a significant increase in CVF (22.05% vs. 3.76%, P=0.003). Western blot and qPCR revealed that the protein and mRNA expression of p50, p65, HIF-1α, CTGF, and COL-1 was significantly higher in the radiation group than in the control group (all P <0.05). Conclusions The pathological features of acute RIHD include significant myocardial edema and myocardial interstitial inflammatory cell infiltration accompanied by increased fibroblasts and collagen fibers. Radiation exposure can activate NF-κB and cause the upregulation of HIF-1α and CTGF at both protein and mRNA levels, which may play an important role in the progression of radiation-induced myocardial inflammation to fibrosis. Key words: Radiation induced heart disease; Histopathology; Fibrosis; Gene expression; Protein expression

Chronic unpredictable stress abrogates the endotoxin tolerance induced by repeated peripheral LPS challenge via the TLR4 signaling pathway

Endotoxin tolerance is induced by exposure to a repeated LPS challenge. Stress can exacerbate LPS-induced inflammation in the mouse hippocampus. However, the effects of stress on endotoxin tolerance have not been elucidated. The present experiments were designed to assess whether tolerance could be induced in the hippocampus by intraperitoneal injection of LPS at a dose of 250μg/kg for 7days, to investigate the effects of chronic unpredictable stress (CUS) on LPS-induced tolerance, and to assess underlying mechanisms in male Kunming mice. The levels of IL-1β in the hippocampus increased to a plateau after 4days of LPS injection. When the CUS protocol was performed with concurrent LPS injections for 7days, body weight decreased, and IL-1β expression in the hippocampus increased simultaneously. The expression of TLR4-MyD88-NF-κB signaling molecules and their negative regulators (PI3K, Akt) were also analyzed on day 7 after the last injection. The mRNA expression of TLR4 and MyD88 and the protein levels of TLR4 and NF-κBp65 were significantly increased, but the protein levels of PI3K and p-Akt were significantly decreased in the hippocampus of the mice. In conclusion, our findings show that endotoxin tolerance in the hippocampus was induced after four days of peripheral LPS challenge at a dose of 250μg/kg per day, and CUS abrogated this effect. CUS abolished endotoxin tolerance by reducing PI3K-mediated inhibition of TLR4-MyD88-NF-κB signaling pathways.

P009 Autophagy stimulation reduces mucosal NF-κB protein levels and ameliorates murine colitis

Background: A defective autophagy is involved in the pathogenesis of inflammatory disorders such as IBD. Cross talk interactions between autophagy and inflammation have been reported and we analyse the effects of autophagy stimulators on murine colitis. Methods: Mice were treated with intrarectal administration of TNBS (3.5 mg/20 mg mice) and body weight was measured every day (and expressed as a percentage of starting weight), and histological damage score analysed two or four days after treatment. Some mice received trehalose (3% in drinking water three weeks before TNBS administration) or a daily administration of rapamycin (1.25 mg/kg, i.p.), betanin (1g/kg, i.p.) or betanin + 3MA (10mg/kg, i.p.). Mucosal protein levels of p-mTOR, p62, LC3, BCL10, NF-κB, IκBα and p-IκBα were determined by WB and mRNA expression of TNFα, IL1β, IL6, IL10, COX2, CCR7, CD11c, iNOS and CD86 by qRT-PCR. Results: An impaired autophagy associated with body weight loss and intestinal damage was detected in the mucosa of TNBS-treated mice. Administration of trehalose, rapamycin or betanin prevented the impaired autophagic flux induced by TNBS and decreased the expression of pro-inflammatory cytokines and M1 macrophage markers (Fig. 1A) and mucosal protein levels of BCL10, p-IκBα and NF-κBp65. Blockade of the autophagosome formation by treatment of mice with 3MA prevented the reduction in both body weight loss (Fig. 1B) and protein levels of p62, BCL10, p-IκBα and NF-κBp65 (Fig 1C) induced by betanin in TNBS-treated mice and weakened the protective effects of betanin on murine colitis. Figure 1 Conclusions: Our results demonstrate that pharmacological stimulation of mucosal autophagy reduces intestinal inflammation and ameliorates murine colitis.

Protective effects of LBP on cerebral ischemia reperfusion injury in mice and mechanism of inhibiting NF-κB, TNF-α, IL-6 and IL-1β

To observe the protective effects of Lycium barbarum polysaccharides (LBP) on cerebral ischemia reperfusion injury in mice and explore its mechanism. Common carotid artery thread was used to cause middle cerebral artery ischemia, and the thread was taken out after 2 h ischemia to achieve cerebral ischemia reperfusion injury in mice. Therefore, the transient middle cerebral artery occlusion (tMCAO) models were established to observe the effects of LBP (25,50, 100 mg•kg⁻¹) on neurological outcome, infarct size and water contents. HE staining was used to observe its effects on neurocytes of cerebral tissues in mice. Western blotting was used to evaluate the protein expression levels of NF-κB p65. ELISA was used to evaluate the levels of TNF-α, IL-6 and IL-1β in the serum. According to the results, LBP markedly improved neurologic deficits, and decreased infarct size and water contents at 24 h after reperfusion in mice. Pathological section of brain tissues also proved its protective effects on neurocytes. Western blot analysis indicated that LBP markedly down-regulated the protein level of NF-κB p65. ELISA indicated that LBP decreased the levels of TNF-α, IL-6 and IL-1β in the serum 24 h after reperfusion.In conclusion, LBP has protective effects on cerebral ischemia reperfusion injury in mice, and this effect may be associated with inhibiting NF-κB and inflammatory reactions.

Involvement of the Toll-Like Receptor/Nitric Oxide Signaling Pathway in the Pathogenesis of Cervical Cancer Caused by High-Risk Human Papillomavirus Infection.

Human papillomavirus (HPV) can activate Toll-like receptor (TLR)/nitric oxide (NO) signaling pathways; however, whether the TLR/NO pathway is involved in cervical cancer caused by high-risk HPV (HR-HPV) remains unclear. In this study, 43 HR-HPV-positive patients with cervical cancer (CC group), 39 HR-HPV-positive patients with a healthy cervix (HR-HPV group), and 33 HR-HPV-negative controls were recruited. NO concentration in cervical canal and expression of inducible NO synthase (iNOS) in cervical tissues were detected. Expressions of key TLR/NO pathway genes (TLR3/4/7/8, NF-κB p65, and iNOS) in cervical epithelial cells were detected by quantitative reverse transcription PCR. Expressions of TLR4, NF-κB p65, and iNOS in CaSki, HeLa, and C33a cells were determined by Western blot. NO concentration in cervical canal of CC group was significantly higher than in other groups (P < 0.05). Positive rates of iNOS in cervical tissues were 72.1%, 28.2%, and 3.1% in the CC group, HR-HPV group, and controls, respectively (P < 0.05). Levels of TLR3, TLR4, TLR7, TLR8, NF-κB p65, and iNOS in cervical epithelial cells were higher in CC group than in other groups (P < 0.05). Both mRNA and protein levels of TLR4, NF-κB p65, and iNOS were higher in HPV-positive HeLa and CaSki cells than in HPV-negative C33a cells (P < 0.05). Together, these results suggest that TLR/NO signaling pathway may be involved in pathogenesis of cervical cancer caused by HR-HPV.

Role of GABAB receptors and p38MAPK/NF-κB pathway in paclitaxel-induced apoptosis of hippocampal neurons

Context: The effects of the anticancer drug paclitaxel on learning and memory are rarely studied. Objective: This study investigated changes in GABAB receptor expression during paclitaxel-induced apoptosis of hippocampal neurons and the role of the p38MAPK/NF-κB pathway in this process. Materials and methods: Hippocampal neurons isolated from neonatal Sprague–Dawley rats were divided into six groups: Control (C), SB (10 µL of 10-µmol/L SB203580), SN (53 µg/mL SN50), N (1 µmol/L paclitaxel), SB + N (10 µmol/L SB203580 + 1 µmol/L paclitaxel) and SN + N (53 µg/mL SN50 + 1 µmol/L paclitaxel). Cells in different groups were treated with corresponding agents for 24 h at 37 °C. The apoptosis rate and protein levels of GABAB1 receptors and NF-κB p65 were evaluated. Rat models of neuropathic pain was induced by paclitaxel and were divided into four groups such as N, B + N, SN + N and SN + B + N groups. Rats in the N group received intrathecal injections of normal saline solution. Rats in the B + N group received intrathecal injections of 10 μL baclofen (0.05 μg/μL). Rats in the SN + N and SN + B + N groups received intrathecal injections of SN50 and SN50 plus baclofen, respectively. Spatial learning and memory were evaluated in rat models based on the escape latency and the number of crossings over the platform and protein levels of GABAB1 receptors, NF-κB, IL-1β and TNFα were measured by immunohistochemistry assay and western blot. Results: The neuronal apoptosis rate was significantly increased in N (49.16 ± 3.12)%, SB + N (31.18 ± 3.02)% and SN + N (28.47 ± 3.75)% groups, accompanied by increased levels of GABAB1 receptors and NF-κB p65 (p < 0.05). The paclitaxel-treated rats demonstrated significantly increased latency (24.32 ± 2.94)s and decreased the crossings number (3.14 ± 0.63) after 15 d in the Morris water maze (p < 0.05). Immunohistochemistry assay showed that compared with the N group (GABAB1:9.0 ± 1.6, NF-κB p65:29.6 ± 2.4, IL-1β: 30.4 ± 3.4, TNFα: 31.0 ± 3.4), B + N, SN + N and SN + B + N groups evidently increased levels of GABAB1 receptor (B + N:SN + N:SN + B + N = 19.4 ± 2.1:20.8 ± 1.9:28.0 ± 1.9) but significantly decreased levels of NF-κB p65 (B + N:SN + N:SN + B + N = 21.2 ± 1.5:18.6 ± 2.1:12.6 ± 1.5), IL-1β (B + N:SN + N:SN + B + N = 22.0 ± 1.0:19.6 ± 1.8:14.6 ± 1.5) and TNF α (B + N:SN + N:SN + B + N = 23.0 ± 1.6:22.2 ± 0.8:16.6 ± 1.7). Similar findings were found in western blot analysis. Discussions and conclusions: Paclitaxel may reduce cognitive function in rats through the p38MAPK/NF-κB pathway and GABAB1 receptors.

Role of Δ133p53 isoform in NF-κB inhibitor PDTC-mediated growth inhibition of MKN45 gastric cancer cells.

AIMTo investigate the role of Δ133p53 isoform in nuclear factor-κB (NF-κB) inhibitor pyrrolidine dithiocarbamate (PDTC)-mediated growth inhibition of MKN45 gastric cancer cells.METHODSThe growth rate of MKN45 cells after treatment with different concentrations of only PDTC or PTDC in combination with cisplatin was detected by the CCK-8 assay. mRNA expression levels of Δ133p53, p53β, and the NF-κB p65 subunit and p65 protein levels were detected by reverse transcription-polymerase chain reaction (RT-PCR) and immunofluorescence, respectively. Growth of MKN45 cells was significantly inhibited by PDTC alone in a dose-dependent manner (P < 0.01). Moreover, the inhibitory effect of cisplatin was remarkably enhanced in a dose-dependent manner by co-treatment with PDTC (P < 0.01).RESULTSRT-PCR analysis revealed that mRNA expression of p65 was curbed significantly in a dose-dependent manner by treatment with only PDTC (P < 0.01), and this suppressive effect was further enhanced when co-treated with cisplatin (P < 0.01). With respect to the other p53 isoforms, mRNA level of Δ133p53 was significantly reduced in a dose-dependent manner by treatment with only PDTC or PTDC in combination with cisplatin (P < 0.01), whereas p53β mRNA expression was not altered by PDTC treatment (P > 0.05). A similar tendency of change in p65 protein expression, as observed for the corresponding mRNA, was detected by immunofluorescence analysis (P < 0.01). Pearson correlation analysis demonstrated that Δ133p53 and p65 mRNA expression levels were positively related, while no significant relationship was observed between those of p65 and p53β (r = 0.076, P > 0.01).CONCLUSIONΔ133p53 isoform (not p53β) is required in PDTC-induced inhibition of MKN45 gastric cancer cells, indicating that disturbance in the cross-talk between p53 and NF-κB pathways is a promising target in pharmaceutical research for the development of treatment strategies for gastric cancer.

Sirtuin1 (SIRT1) Regulates Tumor Necrosis Factor-alpha (TNF-α-Induced) Aquaporin-2 (AQP2) Expression in Renal Medullary Collecting Duct Cells Through Inhibiting the NF-κB Pathway.

BackgroundAquaporin-2 (AQP2) plays a major role in water reabsorption in the renal collecting duct, and is involved in a variety of renal disease. Recent studies have indicate that sirtuin1 (SIRT1) exerts renoprotective properties against kidney diseases. This study aimed to determine the potential role of SIRT1 in AQP2 expression induced by tumor necrosis factor-alpha (TNF-α) and to disclose the underlying mechanism in renal inner medullary collecting duct (IMCD) cells.Material/MethodsQuantitative real-time PCR and Western blotting were respectively identified mRNA and protein expression. Immunofluorescence staining was used to detect the localization of AQP2. Small-interfering RNA (siRNA) was carried out for mechanism study.ResultsResults showed that AQP2 was clearly increased in the plasma membrane and decreased in the cytoplasm of IMCD cells treated with AVP. TNF-α treatment in IMCD cells significantly reduced SIRT1 and AQP2 expression, and increased acetylated NF-κBp65 protein level in time- and concentration-dependent manners. Moreover, SIRT1 overexpression or the activator SRT1720 augmented AQP2 expression and reduced the acetylation of NF-κBp65, which was reversed by SIRT1 siRNA or the inhibitors Ex527 and sirtinol in TNF-α-induced IMCD cells. Knockdown of NF-κBp65 or NF-κBp65 inhibition by pyrrolidine dithiocarbamate (PDTC) enhanced AQP2 expression in IMCD cells exposed to TNF-α. Importantly, knockdown of NF-κBp65 augmented the up-regulation of SIRT1 on AQP2 expression in IMCD cells induced by TNF-α.ConclusionsThese findings indicate that SIRT1 increases AQP2 expression in TNF-α-induced IMCD cells via the NF-κB-dependent signalling pathway, which might provide novel insight to understanding the renoprotective effects of SIRT1 in kidney diseases.

Sphk1 promotes breast epithelial cell proliferation via NF-κB-p65-mediated cyclin D1 expression.

Lipid metabolism is crucially involved with the promotion of malignant progression and metastasis in various cancers. Growing evidence suggests that many types of cancers express high levels of sphingosine kinase 1 (Sphk1), which is known to mediate cell proliferation We hypothesized that Sphk1/sphingosine-1-phosphate (S1P) signaling contributes to tumor progression. In MCF10A and MCF10A-Sphk1 breast epithelial cells, we used TNF-α to activate the Sphk1/S1P pathway and the measured expression levels of NF-κBp65 and cyclin D1 mRNA and protein in the presence and absence of an NF-κB-p65 inhibitor. Chromatin immunoprecipitation assays were performed to determine whether NF-κB-p65 binds to the cyclin D1 promoter. We found that overexpression of Sphk1 induced NF-κB-p65 activation, increased expression of cyclin D1, shortened the cell division cycle, and thus promoted proliferation of breast epithelial cells. These findings provide insight into the mechanism by which an Sphk1/NF-κB-p65/cyclin D1 signaling pathway mediates cell proliferation.

Artesunate suppresses RANKL-induced osteoclastogenesis through inhibition of PLCγ1-Ca2+–NFATc1 signaling pathway and prevents ovariectomy-induced bone loss

Bone lytic diseases including osteoporosis, rheumatoid arthritis, and bone metastatic tumors affect hundreds of millions people worldwide. Targeting over-activated osteoclasts as an anti-resorptive treatment becomes an important strategy to treat osteolytic diseases. Artesunate is a compound derived from artemisinin (qinghaosu) and has been used to treat malaria and rheumatoid arthritis clinically in China, but its role in osteolysis is unknown. Here, we found that artesunate could suppress RANKL-induced osteoclastogenesis and bone resorption from 1.56 to 12.5μM. Artesunate obviously reduced RANKL-induced NF-κB-luc activity at 50μM, but had no effects on RANKL-induced NF-κB activation (NF-κB luciferase activity, IκB-α degradation and nuclear NF-κB p65 protein level) from 3.125 to 12.5μM in pre-osteoclastic RAW264.7 cells. Interestingly, artesunate could significantly inhibit RANKL-induced NFATc1 activation measured by NFAT luciferase activity, NFATc1 mRNA and nuclear NFATc1 protein levels from 3.125 to 12.5μM. Further study revealed that artesunate inhibited RANKL up-regulated PLCγ1 activation, intracellular calcium, and calcineurin (PP2B-Aα) protein expression from 3.125 to 12.5μM. In addition, the NFATc1 targeted osteoclast-specific genes expression including cathepsin K, MMP-9, and TRAP was reduced by artesunate. Finally, we showed that artesunate was able to reverse the bone loss in an ovariectomized mouse model in vivo accompanied with reduced RANKL, RANKL/OPG, and TRAP-5b levels. This study indicates that artesunate inhibits RANKL-induced osteoclastogenesis and bone loss by inhibiting PLCγ1-Ca2+-calcineurin-NFATc1 pathway. Collectively, our data suggest that artesunate is a potential treatment option against RANKL-mediated osteolytic bone disease.

Effects of celecoxib on expression of PPARγ and NF-κB in type 2 diabetes rats with non-alcoholic steatohepatitis

Objective: To investigate the protective effect of celecoxib against non-alcoholicsteatohepatitis and its impact on the expression of peroxisome proliferator-activated receptor gamma (PPARγ)/NF-κB in type 2 diabetes rats. Methods: Thirty-six male Sprague-Dawley rats were randomly and equally divided into three groups: control group, model group, and model+celecoxib group. After the model was successfully established, all rats were sacrificed to isolate serum and liver tissues. Serum levels of alanine transaminase (ALT) and aspartate aminotransferase (AST) were measured, liver pathological changes were analyzed by HE and oil red O staining, and the protein expression of hepatic PPARγ, NF-κB p65, and COX-2 was determined by immunohistochemistry and Western blot. Results were statistically analyzed by t-test. Results: The levels of ALT and AST in the model group (301.8±5.40 and 345.4±9.63 U/L) were significantly higher than those in the control group (33±4.06 and 76.6±7.64 U/L) and those in the model+celecoxib group (88.8±13.07 and 99.8±20.51 U/L). HE and oil red O staining demonstrated hepatic steatosis and inflammation in liver tissues. Immunohistochemical results showed that the integral optical densities (IODs) of NF-κB p65 and COX-2 in the model group (4.04E5±2.42E4 and 6.62E5±2.64E4) were significantly higher than those in the control group (4.23E4±6.82E3 and 4.82E4±3.71E3) (t= 48.86 and 72.93, bothP< 0.01) and those in the model+celecoxib group (2.74E5±4.38E4 and 2.31E5±2.09E4) (t= 9.02 and 40.51, bothP< 0.01). There was no significant difference in the IOD of PPARγbetween the model group (4.50E4±2.38E3) and the control group (4.26E4±5.09E3). However, the IOD of PPARγin the model+celecoxib group (3.22E5±1.54E4) was significantly higher than that in the model group (t= 61.82,P< 0.01). Western blot analysis showed that the relative protein levels of hepatic NF-κB p65 and COX-2 in the model group (4.93±0.76 and 3.04±0.23) were significantly higher than those in the control group (1.00±0.13 and 1.00±0.15) (t= 11.44 and 16.64, bothP< 0.01) and those in the model+celecoxib group (2.44±0.32 and 1.26±0.11) (t= 6.80 and 15.81, bothP< 0.01). The relative protein level of hepatic PPARγin the model+celecoxib group (0.98±0.09) was significantly higher than that in the model group (0.37±0.03) (t= 15.08,P< 0.01). Conclusion: Celecoxib protects type 2 diabetes rats against non-alcoholic steatohepatitis probably via modulating the expression of PPARγand NF-κB.

Berberine improves airway inflammation and inhibits NF-κB signaling pathway in an ovalbumin-induced rat model of asthma

Objectives: Berberine has been reported for its various activities including anti-inflammatory effects and has been used in treating many diseases. However, its effects on airway inflammation in asthma have not been investigated. This study mainly aimed to detect its effects on the airway inflammation and the nuclear factor-κB (NF-κB) signaling pathway activity in a rat model of asthma. Methods: Asthma was induced by ovalbumin (OVA) sensitization and challenge. The asthmatic rats were respectively treated with vehicle PBS or berberine (100 mg/kg or 200 mg/kg) for 28 days. The control rats were treated with PBS. Inflammatory cells in bronchoalveolar lavage fluid (BALF) were counted and the lung inflammation was scored. Levels of NF-κB p65 (mRNA and protein), phosphorylated NF-κB p65 (p-NF-κB p65), inhibitory κB alpha (IκBα) (mRNA and protein) and phosphorylated IκBα (p-IκBα), as well as NF-κB p65 DNA-binding activity, were measured to assess the activity of NF-κB signaling pathway. Levels of the downstream inflammatory mediators of NF-κB signaling, IL-1β, IL-4, IL-5, IL-6, IL-13 and IL-17 in BALF, were measured. Besides, the serum levels of OVA-specific immunoglobulin (Ig)E were measured. Results: Results showed that OVA increased the number of inflammatory cells in BALF, elevated lung inflammation scores, enhanced the NF-κB signaling activity and promoted the production of IgE in rats. Berberine dose-dependently reversed the alterations induced by OVA in the asthmatic rats. Conclusions: The findings suggested a therapeutic potential of berberine on OVA- induced airway inflammation. The ameliorative effects on the OVA-induced airway inflammation might be associated with the inhibition of the NF-κB signaling pathway.

Examination of CK2α and NF-κB p65 expression in human benign prostatic hyperplasia and prostate cancer tissues.

Protein kinase CK2 plays a critical role in cell growth, proliferation, and suppression of cell death. CK2 is overexpressed, especially in the nuclear compartment, in the majority of cancers, including prostate cancer (PCa). CK2-mediated activation of transcription factor nuclear factor kappa B (NF-κB) p65 is a key step in cellular proliferation, resulting in translocation of NF-κB p65 from the cytoplasm to the nucleus. As CK2 expression and activity are also elevated in benign prostatic hyperplasia (BPH), we sought to increase the knowledge of CK2 function in benign and malignant prostate by examination of the relationships between nuclear CK2 and nuclear NF-κB p65 protein expression. The expression level and localization of CK2α and NF-κB p65 proteins in PCa and BPH tissue specimens was determined. Nuclear CK2α and NF-κB p65 protein levels are significantly higher in PCa compared with BPH, and these proteins are positively correlated with each other in both diseases. Nuclear NF-κB p65 levels correlated with Ki-67 or with cytoplasmic NF-κB p65 expression in BPH, but not in PCa. The findings provide information that combined analysis of CK2α and NF-κB p65 expression in prostate specimens relates to the disease status. Increased nuclear NF-κB p65 expression levels in PCa specifically related to nuclear CK2α levels, indicating a possible CK2-dependent relationship in malignancy. In contrast, nuclear NF-κB p65 protein levels related to both Ki-67 and cytoplasmic NF-κB p65 levels exclusively in BPH, suggesting a potential separate impact for NF-κB p65 function in proliferation for benign disease as opposed to malignant disease.

Suppression of human 8-oxoguanine DNA glycosylase (OGG1) augments ultrasound-induced apoptosis in cervical cancer cells

PurposeHuman 8-oxoguanine DNA glycosylase (OGG1) is a major base excision repair enzyme, and it was reported to suppress the activation of intrinsic apoptotic signaling pathway in response to oxidative stress. In this study, our aim was to investigate the effects of OGG1 downregulation on ultrasound-induced apoptosis in cervical cancer cells. MethodsOGG1 expression was silenced by shRNA in the cervical cancer SW756 and CaSki cells. Cell viability was evaluated by MTT assay after OGG1 knockdown following ultrasound treatment. Ultrasound-induced apoptosis was measured by Annexin V-FITC/propidium iodide. Intracellular reactive oxygen species (ROS) production and Ca2+ concentration were detected using a fluorescent probe, 2′,7′-dichlorofluorescin diacetate (DCFH-DA) and a green fluorescent dye fluo-4AM, respectively. Western blotting was used to analyze the expression of Bcl-2, Bax, cleaved caspase-3, and nuclear factor-κB p65 (NF-κB p65). ResultsThe results indicated that OGG1 knockdown did not suppress cell proliferation, but significantly augmented ultrasound-induced inhibitory effects on the cell viability, and increased ultrasound-induced early apoptosis and late apoptosis and necrosis in the SW756 and CaSki cells when exposure to ultrasound (1MHz) at 1.5W/cm2 for 30 and 60s. OGG1 knockdown significantly increased intracellular ROS production and Ca2+ concentration after incubation of 6, 24, and 48h post-ultrasound treatment. The downregulation of Bcl-2 protein and the upregulation of Bax, cleaved caspase-3, and NF-κB p65 protein levels were observed in the shRNA-OGG1 cells and mock-shRNA cells, but no significant change of these protein levels was found between of them. ConclusionsThese results indicate that downregulation of OGG1 expression can augment ultrasound-induced apoptosis in cervical cancer cells, which suggests that OGG1 suppression might provide a new insight for ultrasound-induced therapeutic effects on cervical cancer treatment.

Endothelial lipase is upregulated by interleukin-6 partly via the p38 MAPK and p65 NF-κB signaling pathways.

To investigate the effects of inflammatory factor interleukin (IL)-6 on the expression of endothelial lipase (EL) and its potential signaling pathways in atherosclerosis, a primary culture of human umbilical vein endothelial cells (HUVECs) was established and treated as follows: i) Control group without any treatment; ii) recombinant human (rh)IL-6 treatment (10 ng/ml) for 0, 4, 8, 12 and 24 h; iii) p38 mitogen-activated protein kinases (MAPKs) inhibitor (SB203580, 10 µmol/l) pretreatment for 1 h prior to rhIL-6 (10 ng/ml) treatment; iv) nuclear factor (NF)-κB activation inhibitor (pyrrolidine dithiocarbamate, 10 mmol/l) pretreatment for 1 h prior to rhIL-6 (10 ng/ml) treatment. EL levels were detected by immunocytochemical staining and western blot analysis. Proliferation of HUVECs was detected by immunostaining of proliferating cell nuclear antigen (PCNA) and an MTT assay. p38 MAPK and NF-κB p65 levels were detected by western blotting. The results showed that rhIL-6 treatment increased EL expression and proliferation of HUVECs. NF-κB p65 and MAPK p38 protein levels also increased in a time-dependent manner in HUVECs after rhIL-6 treatment. NF-κB inhibitor and MAPK p38 inhibitor prevented the effects of rhIL-6 on EL expression. In conclusion, inflammatory factor IL-6 may participate in the pathogenesis of atherosclerosis by increasing EL expression and the proliferation of endothelial cells via the p38 MAPK and NF-κB signaling pathways.

Evaluations of thyme extract effects in human normal bronchial and tracheal epithelial cell lines and in human lung cancer cell line

Thyme (Thymus vulgaris) is used traditionally to prepare herbal remedies possessing expectorant, mucolytic, antitussive and antispasmodic properties.The aim of the present study was to investigate the effects of a standardized hydroalcoholic extract of thyme on primary human airway (bronchial/tracheal) epithelial cell lines in a model of lung inflammation induced by LPS. In addition, the effects of thyme extract on human lung cancer cell line (H460) were analysed. Thyme extract showed significant anti-inflammatory properties by reducing the NF-κB p65 and NF-κB p52 transcription factors protein levels followed by the decrease of pro-inflammatory cytokines (IL-1 beta and IL-8), and Muc5ac secretion in human normal bronchial and tracheal epithelial cells. Moreover, the extract showed cytotoxic effects on H460 cancer cells, modulated the release of IL-1 beta, IL-8 and down-regulated NF-κB p65 and NF-κB p52 proteins.Taken together, these results substantiated the traditional uses of thyme in the treatment of respiratory diseases. Thyme extract might be an effective treatment of chronic diseases based on inflammatory processes when hypersecretion of mucus overwhelms the ciliary clearance and obstructs airways, causing morbidity and mortality. Moreover thyme extract, evaluated in H460 lung cancer cell line, demonstrated to induce cell cytotoxicity in addition to reduce inflammatory cell signals.