Effect of NF‐κB Decoy Oligodeoxynucleotide on LPS/High‐Fat Diet‐Induced Atherosclerosis in an Animal Model

Abstract: Atherosclerosis is a chronic inflammatory process occurring in the walls of arteries, in large part due to the accumulation of inflammatory cells. This study was conducted to determine the effect of nuclear factor (NF)‐κB decoy oligodeoxynucleotide (ODN) in an atherosclerosis animal model. The mice received i.p. injections of lipopolysaccharide (LPS, 2 mg/kg) three times a week to induce atherosclerotic change, and fed an atherogenic diet for 12 weeks. NF‐κB decoy ODN (0.4 mg/kg) was injected into the tail vein. Treatment with NF‐κB decoy ODN decreased pro‐inflammatory cytokines, tumour necrosis factor (TNF)‐α and interleukin (IL)‐1β and inflammatory markers, vascular adhesion molecule (VCAM)‐1 and intercellular adhesion molecule (ICAM)‐1, in the LPS/Fat‐induced mice. In addition, the expression of proteins related to fibrosis, transforming growth factor (TGF)‐β1 and fibronectin were markedly decreased in the mice treated with NF‐κB decoy ODN compared with the LPS/Fat‐induced mice without decoy ODN treatment. These data suggest that NF‐κB decoy ODN may exert an inhibitory effect on the expression levels of pro‐inflammatory cytokines and cell adhesion molecules in atherosclerotic mice.     

NF‐κB/p53‐activated inflammatory response involves in diquat‐induced mitochondrial dysfunction and apoptosis

Inflammation generated by environmental toxicants including pesticides could be one of the factors underlying neuronal cell damage in neurodegenerative diseases. In this study, we investigated the mechanisms by which inflammatory responses contribute to apoptosis in PC12 cells treated with diquat. We found that diquat induced apoptosis, as demonstrated by the activation of caspases and nuclear condensation, inhibition of mitochondrial complex I activity, and decreased ATP level in PC12 cells. Diquat also reduced the dopamine level, indicating that cell death induced by diquat is due to cytotoxicity of dopaminergic neuronal components in these cells. Exposure of PC12 cells to diquat led to the production of reactive oxygen species (ROS), and the antioxidant N‐acetyl‐cystein attenuated the cytotoxicity of caspase‐3 pathways. These results demonstrate that diquat‐induced apoptosis is involved in mitochondrial dysfunction through production of ROS. Furthermore, diquat increased expression of cyclooxygenase‐2 (COX‐2) and tumor necrosis factor‐α (TNF‐α) via inflammatory stimulation. Diquat induced nuclear accumulation of NF‐κB and p53 proteins. Importantly, an inhibitor of NF‐κB nuclear translocation blocked the increase of p53. Both NF‐κB and p53 inhibitors also blocked the diquat‐induced inflammatory response. Pretreatment of cells with meloxicam, a COX‐2 inhibitor, also blocked apoptosis and mitochondrial dysfunction. These results represent a unique molecular characterization of diquat‐induced cytotoxicity in PC12 cells. Our results demonstrate that diquat induces cell damage in part through inflammatory responses via NF‐κB‐mediated p53 signaling. This suggests the potential to generate mitochondrial damage via inflammatory responses and inflammatory stimulation‐related neurodegenerative disease.     

NF‐κB activation mediates LPS‐or zymosan‐induced hypotension and inflammation reversed by BAY61‐3606, a selective Syk inhibitor,in rat models of septic and non‐septic shock

We have previously demonstrated that the activation of the spleen tyrosine kinase (Syk)/inhibitory‐κB (IκB)‐α/nuclear factor‐κB (NF‐κB) p65 signalling pathway contributes to hypotension and inflammatory response in a rat models of zymosan (ZYM)‐induced non‐septic shock. The purpose of this study was to further examine the possible mechanism underlying the effect of inhibition of Syk by BAY61‐3606 via NF‐κB activity at the level of nuclear translocation regarding the production of vasodilator and proinflammatory mediators in lipopolysaccharide (LPS) (septic)‐ and ZYM (non‐septic)‐induced shock. Administration of LPS (10 mg/kg, ip) or ZYM (500 mg/kg, ip) to male Wistar rats decreased mean arterial pressure and increased heart rate that was associated with an increase in the activities of cyclooxygenase and nitric oxide synthase, tumour necrosis factor‐α, and interleukin‐8 levels, and NF‐κB activation and nuclear translocation in sera and/or cardiovascular and renal tissues. BAY61‐3606 (3 mg/kg, ip), the selective Syk inhibitor, given 1 hour after LPS‐ or ZYM injection reversed all the above‐mentioned effects. These results suggest that Syk contributes to the LPS‐ or ZYM‐induced hypotension and inflammation associated with transactivation of NF‐κB in septic and non‐septic shock.     

Involvement of NF‐κB in the upregulation of cystathionine‐γ‐lyase,a hydrogen sulfide‐forming enzyme,and bladder pain accompanying cystitis in mice

Hydrogen sulfide (H₂S) is generated from l ‐cysteine by multiple enzymes including cystathionine‐γ‐lyase (CSE), and promotes nociception by targeting multiple molecules such as Ca_v3.2 T‐type Ca²⁺ channels. Bladder pain accompanying cyclophosphamide (CPA)‐induced cystitis in mice has been shown to involve the functional upregulation of the CSE/H₂S/Ca_v3.2 pathway. Therefore, we investigated whether NF‐κB, as an upstream signal of the CSE/H₂S system, contributes to bladder pain in mice with CPA‐induced cystitis. Bladder pain‐like nociceptive behaviour was observed in CPA‐treated mice, and referred hyperalgesia was evaluated by the von Frey test. Isolated bladder weights were assessed to estimate bladder swelling, and protein levels were measured by Western blotting. CPA, administered intraperitoneally, induced nociceptive behaviour, referred hyperalgesia and increased bladder weights in mice. β‐Cyano‐l ‐alanine, a reversible selective CSE inhibitor, prevented CPA‐induced nociceptive behaviour, referred hyperalgesia, and, in part, increases in bladder weight. CPA markedly increased phosphorylated NF‐κB p65 levels in the bladder, an effect that was prevented by pyrrolidine dithiocarbamate (PDTC), an NF‐κB inhibitor. PDTC and curcumin, which inhibits NF‐κB signals, abolished CPA‐induced nociceptive behaviour, referred hyperalgesia and, in part, increases in bladder weight. CPA caused the overexpression of CSE in the bladder, and this was prevented by PDTC or curcumin. The CPA‐induced activation of NF‐κB signals appeared to cause CSE overexpression in the bladder, contributing to bladder pain and in part swelling, possibly through H₂S/Ca_v3.2 signaling. Therefore, NF‐κB‐inhibiting compounds including curcumin may be useful for the treatment of cystitis‐related bladder pain.     

C‐phycocyanin protects against ethanol‐induced gastric ulcers in rats: Role of HMGB1/NLRP3/NF‐κB pathway

Gastric ulcer is a widespread inflammatory disease with high socio‐economic burden. C‐phycocyanin is one of the active constituents of Spirulina microalgae, and although it is well known for its antioxidant and anti‐inflammatory properties, its protective effects against gastric ulcer have not yet been identified. High‐mobility group box 1 (HMGB1) is a nuclear protein that, once secreted extracellularly, initiates several inflammatory reactions, and it is involved in the pathogenesis of gastric ulcer. The aim of the present study was to investigate the anti‐inflammatory and anti‐ulcerogenic effects of C‐phycocyanin against ethanol‐induced gastric ulcer targeting HMGB1/NLRP3/NF‐κB pathway. Ulcer induction showed increase in HMGB1 expression through activation of nucleotide‐binding domain and leucine‐rich repeat‐containing protein 3 (NLRP3) inflammasome and nuclear factor kappa p65 (NF‐κB p65). Moreover, oxidative stress and inflammatory markers were elevated in the ulcer‐treated group compared to the normal control group. However, pre‐treatment with C‐phycocyanin significantly reduced HMGB1 expression via suppression of NLRP3/NF‐κB, oxidative markers, IL‐1β, tumour necrosis factor‐α (TNF‐α) and ulcer index value. These results were consistent with histopathological and immunohistochemistry examination. Thus, C‐phycocyanin is a potential therapeutic strategy with anti‐inflammatory and anti‐ulcerogenic effects against ethanol‐induced gastric ulcer.     

MiR‐101a ameliorates AngII‐mediated hypertensive nephropathy by blockade of TGFβ/Smad3 and NF‐κB signalling in a mouse model of hypertension

Hypertensive nephropathy, clinically characterized by progressive renal fibrosis and inflammation, is a severe complication of hypertension. The objectives of this study were to investigate the roles of miR‐101a in relieving angiotensin II (Ang II)‐mediated hypertensive nephropathy and uncover the possible underlying mechanisms. A hypertensive mouse model was established via continuous 28‐day AngII infusion. Systolic blood pressure (SBP), ratio of urine albumin to creatinine, blood urea nitrogen (BUN), serum creatinine (Scr) and glomerular filtration rate (GFR) were evaluated. Dual luciferase reporter assay was used to explore the target of miR‐101a. mRNA levels of miR‐101a, TGFβRI, fibrotic markers (Collagen I and α‐SMA) and pro‐inflammatory cytokines (IL‐1β and TNF‐α) were determined by real‐time PCR. Protein levels of TGFβRI, Collagen I, α‐SMA, IL‐1β, TNF‐α, t‐p65, P‐p65, t‐Smad3, P‐Smad3, t‐IκBα and P‐IκBα were detected by western blot. MiR‐101a mimics significantly improved GFR and inhibited AngII‐induced increase in the ratio of urine albumin to creatinine, BUN and Scr. MiR‐101a mimics partially abolished AngII‐induced increase in the mRNA and protein level of fibrotic markers by targeting TGFβRI and inhibiting TGFβ/Smad3 pathway. Moreover, TGFβRI inhibitor galunisertib inhibited AngII‐mediated renal injury in mice with hypertensive nephropathy. Additionally, miR‐101a overexpression blocked AngII‐induced up‐regulation of pro‐inflammatory markers via suppressing NF‐κB pathway. MiR‐101a exhibited protective effects against hypertensive nephropathy via inhibiting TGFβ/Smad3 and NF‐κB signalling pathways.     

Inhibition of NF‐κB and metastasis in irinotecan (CPT‐11)‐resistant LoVo colon cancer cells by thymoquinone via JNK and p38

Clinically used chemotherapeutics can effectively eliminate most tumor cells. However, they cause unwanted side effects and result in chemoresistance. To overcome such problems, phytochemicals are now used to treat cancers by means of targeted therapy. Thymoquinone (TQ) is used to treat different cancers (including colon cancer) and is an NF‐κB inhibitor. Irinotecan resistant (CPT‐11‐R) LoVo colon cancer cell line was previous constructed by step‐wise CPT‐11 challenges to un‐treated parental LoVo cells and expresses EGFR/IKKα/β/NF‐κB pathway. TQ resulted in reduced total and phosphorylation of IKKα/β and NF‐κB and decreased metastasis in CPT‐11‐R cells. TQ not only reduced activity of ERK1/2 and PI3K but also activated JNK and p38. Furthermore, TQ was also found to suppress metastasis through activation of JNK and p38. Therefore, TQ suppressed metastasis through NF‐κB inhibition and activation of JNK and p38 in CPT‐11‐R LoVo colon cancer cells. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 669–678, 2017.     

Dexmedetomidine preconditioning for myocardial protection in ischaemia‐reperfusion injury in rats by downregulation of the high mobility group box 1‐toll‐like receptor 4‐nuclear factor κB signalling pathway

Pharmacological preconditioning reduces myocardial infarct size in ischaemia‐reperfusion (I‐R) injury. Dexmedetomidine, a selective α₂‐adrenoceptor agonist, has a proven cardioprotective effect when administered prior to I‐R, although the underlying mechanisms for this effect are not fully understood. We evaluated whether dexmedetomidine preconditioning could induce a myocardio‐protective effect against I‐R injury by inhibiting associated inflammatory processes through downregulation of the high mobility group box 1 (HMGB1)‐toll‐like receptor 4 (TLR4)‐nuclear factor κB (NF‐κB) signalling pathway. Seventy rats were randomly assigned to seven groups: a control and six test groups, involving I‐R for 30 and 120 minutes, respectively, in isolated rat hearts and different pretreatment protocols with dexmedetomidine (10 nmol/L) as well as yohimbine (1 μmol/L) and recombinant HMGB1 peptide (rHMGB1; 20 μg/L), alone or in combination with dexmedetomidine. Cardiac function was recorded; myocardial HMGB1, TLR4, and NF‐κB activities and levels of tumour necrosis factor‐α (TNF‐α) and interleukin‐6 (IL‐6) were measured as were lactate dehydrogenase (LDH) and creatine kinase (CK) in coronary outflow. Dexmedetomidine preconditioning significantly improved cardiac function (P<.05), downregulated the expression of HMGB1‐TLR4‐NF‐κB, reduced levels of TNF‐α and IL‐6 in isolated ventricles during I‐R injury, and significantly reduced CK and LDH levels in coronary outflow (P<.05). All of these effects were partially reversed by yohimbine (P<.05) or rHMGB1 (P<.05). Dexmedetomidine preconditioning alleviated myocardial I‐R injury in rats through inhibition of inflammatory processes associated with downregulation of the HMGB1‐TLR4‐NF‐κB signalling pathway via activation at α₂‐adrenergic receptors.     

Nonylphenol regulates cyclooxygenase‐2 expression via Ros‐activated NF‐κB pathway in sertoli TM4 cells

The aim of this study was to investigate the signaling pathways involved in the cyclooxygenase (COX)‐2 regulation induced by nonylphenol (NP) in mouse testis Sertoli TM4 cells. Our results showed that treatment of TM4 cells with NP increased COX‐2 protein expression and interleukin‐6 (IL)‐6 and prostaglandin E2 (PGE2) secretion in a dose‐dependent manner. Pretreatment with reactive oxygen species (ROS) scavenger, N‐acetylcysteine (NAC), attenuated NP‐induced ROS production, COX‐2 expression, and IL‐6 and PGE2 release in TM4 cells. Exposure to NP stimulated activation of NF‐κB, whereas the NF‐κB inhibitor, pyrrolidine dithiocarbamate, attenuated NP‐enhanced COX‐2 expression and IL‐6 and PGE2 release in TM4 cells in a dose‐dependent manner. Furthermore, NAC blocked NP‐induced activation of NF‐κB. In addition, inhibition of COX‐2 mitigated NP‐induced IL‐6 release. In conclusion, NP induced ROS generation, activation of NF‐κB pathway, COX‐2 upregulation, and IL‐6 and PGE2 secretion in TM4 cells. NP may regulate COX‐2 expression via ROS‐activated NF‐κB pathway in Sertoli TM4 cells. © 2014 Wiley Periodicals, Inc. Environ Toxicol 30: 1144–1152, 2015.     

Dehydroxymethylepoxyquinomicin,a novel nuclear factor‐κB inhibitor,prevents inflammatory injury induced by interferon‐γ and histamine in NCTC 2544 keratinocytes

1. The novel nuclear factor (NF)‐κB inhibitor dehydroxymethylepoxyquinomicin (DHMEQ) is a derivative of the antibiotic epoxyquinomicin C from Amycolatopsis sp. that has been found to inhibit tumour necrosis factor (TNF)‐α‐induced activation of NF‐κB by suppressing nuclear translocation of NF‐κB. The aim of the present study was to determine the effects of DHMEQ on interferon (IFN)‐γ‐ and histamine‐activated NCTC 2544 keratinocytes. 2. Keratinocytes were stimulated or not with 200 U/mL IFN‐γ and 10^?4 mol/L histamine in the absence or presence of different concentrations of DHMEQ (1, 5 and 10 μg/mL) or hydrocortisone (10^?5 mol/L), which was used as a reference anti‐inflammatory drug. After 48 h, each sample was tested for the presence of intercellular adhesion molecule (ICAM)‐1 by western blot analysis, as well as for the release of monocyte chemoattractant protein (MCP)‐1, RANTES and interleukin (IL)‐8 using specific sandwich ELISAs. To verify the effect of DHMEQ on cell viability of non‐stimulated NCTC 2544 keratinocytes, the 3‐(4,5‐dimethyl‐2 thiazoyl)‐2,5‐diphenyl‐2H‐tetrazolium bromide (MTT) assay was used. 3. The results showed that 10 μg/mL DHMEQ potently inhibited ICAM‐1 production (by 50%), as well as the release of MCP‐1 (to 25% of control), RANTES (to 5% of control) and IL‐8 (to 2% of control). The results of the MTT assay indicated that DHMEQ has no effect on cell viability. 4. In conclusion, DHMEQ inhibits the IFN‐γ‐ and histamine‐induced activation of the keratinocyte cell line NCTC 2544. The anti‐inflammatory effects of DHMEQ could be exploited by applying the drug topically alone or in combination with sub‐toxic concentrations of anti‐inflammatory drugs to producer a synergistic effect.     

Esculetin inhibits histamine‐induced expression of inflammatory cytokines and mucin in nasal epithelial cells

Allergic rhinitis (AR) is a type of respiratory disease closely associated with chronic inflammation. Esculetin is a natural coumarin derivative and has been reported to possess anti‐allergic and anti‐inflammatory effects. However, the roles of esculetin in AR have not been studied. In this study, we aimed to examine the effect of esculetin on AR using an in vitro model. The human nasal epithelial cells (HNEpC) were stimulated by histamine for 24 hours with or without the pretreatment of esculetin. The mRNA levels and production of inflammatory cytokines including IL‐6 and IL‐8, as well as mucin 5AC (MUC5AC) were measured using qRT‐PCR and ELISA, respectively. The results showed that esculetin suppressed histamine‐induced expression and secretion of IL‐6, IL‐8, and MUC5AC in HNEpCs. Furthermore, we examined the effect of esculetin on NF‐κB pathway by detecting the expression levels of NF‐κB p65, p‐p65 and IκBα using western blot analysis. Esculetin treatment suppressed the histamine‐induced p‐p65 expression and p‐IκBα degradation. Inhibiting NF‐κB pathway suppressed histamine‐induced production of IL‐6, IL‐8, and MUC5AC in HNEpCs. These findings suggested that esculetin suppressed histamine‐induced production of inflammatory cytokines and mucin in HNEpCs, which were partly mediated by the inhibition of NF‐κB pathway.     

Differential Inhibitory Effects of the Polyphenol Ellagic Acid on Inflammatory Mediators NF‐κB,iNOS, COX‐2, TNF‐α, and IL‐6 in 1,2‐Dimethylhydrazine‐Induced Rat Colon Carcinogenesis

Abstract: Dietary polyphenols have been found to possess preventive and therapeutic potential against several types of cancers. We investigated the effect of ellagic acid on colon cancer induced by 1,2‐dimethylhydrazine in rats. Male Wistar albino rats were divided into four groups. Group 1 served as control, group 2 rats received ellagic acid 60 mg/kg bodyweight/every day p.o. throughout the experiment. Rats from groups 3 and 4 were given subcutaneous (s.c.) injections of 1,2‐dimethylhydrazine (20 mg/kg body weight) once a week for the first 15 weeks; rats in group 4 received ellagic acid as in group 2 after the last injection of 1,2‐dimethylhydrazine and continued till the end of the experimental period of 30 weeks. 1,2‐dimethylhydrazine‐induced rats exhibited alterations in cancer tumour markers [5′‐nucleotidase (5′‐ND), gamma glutamyl transpeptidase (γ‐GT), carcinoembryonic antigen (CEA), alphafetoprotein (AFP) and cathepsin‐D (CD)]; pathophysiological markers [alkaline phosphatase (ALP) and lactate dehydrogenase (LDH)] and oral administration of ellagic acid restored the levels of these marker enzymes. Nuclear factor‐kappa B (NF‐κB) actively involved in the regulation of both pro‐inflammatory proteins [inducible nitric oxide synthase (iNOS) and cyclooxygenase‐2 (COX‐2)] and pro‐inflammatory cytokines [tumour necrosis factor (TNF)‐α and interleukin (IL)‐6] and in our study 1,2‐dimethylhydrazine‐induced group exhibited elevated expressions of all these inflammatory proteins. Ellagic acid administration reduced the expressions of NF‐κB, COX‐2, iNOS, TNF‐α and IL‐6 as confirmed by immunohistochemical, immunoblot and immunofluorescence analysis during 1,2‐dimethylhydrazine‐induced colon carcinogenesis. In conclusion, ellagic acid demonstrates anti‐inflammatory property by iNOS, COX‐2, TNF‐α and IL‐6 down‐regulation due to inhibition of NF‐κB and exerts its chemopreventive effect on colon carcinogenesis.     

Design and optimize N‐substituted EF24 as effective and low toxicity NF‐κB inhibitor for lung cancer therapy via apoptosis‐to‐pyroptosis switch

As NF‐κB signaling pathway is constitutively activated in lung cancer, targeting NF‐κB has a potential for the treatment. EF24 has been proved to be a NF‐κB inhibitor with good antitumor activity, while whose toxicity possibly became one of the obstacles to enter into clinical application. In order to find high efficiency and low toxicity NF‐κB inhibitors, EF24 was modified and 13d was screened out. It was proved that 13d possessed an effective combination of inhibiting NF‐κB pathway and showing lower cytotoxicity on normal cells as well as less toxicity in acute toxicity experiment compared with the lead compound of EF24. In addition, 13d was found to inhibit cell vitality, arrest cell cycle in G2/M phase, promote cell apoptosis, and suppress the xenograft tumor growth. Furthermore, 13d was elucidated to induce pyroptosis developing from apoptosis, which was associated with the inhibition of NF‐κB. Taken together, it was suggested that 13d was a potent antitumor agent.     

Synthesis and SAR Studies of Dual AKT/NF‐κB Inhibitors Against Melanoma

The protein Kinase B alpha (AKT) and nuclear factor kappa‐light‐chain‐enhancer of activated B cells (NF‐κB) pathways are central regulators of cellular signaling events at the basis of tumor development and progression. Both pathways are often up‐regulated in different tumor types including melanoma. We recently reported the identification of compound 1 (BI‐69A11) as inhibitor of the AKT and the NF‐κB pathways. Here, we describe SAR studies that led to novel fluorinated derivatives with increased cellular potency, reflected in efficient inhibition of AKT and IKKs. Selected compounds demonstrated effective toxicity on melanoma, breast, and prostate cell lines. Finally, a representative derivative showed promising efficacy in an in vivo melanoma xenograft model.