Selective NF-κB Inhibitor Research Articles

A Selective Nuclear Factor-κB Inhibitor, JSH-23, Exhibits Antidepressant-like Effects and Reduces Brain Inflammation in Rats.

Accumulating evidence suggests that nuclear factor (NF)-κB is involved in the pathophysiology of mood disorders. We conducted two experimental protocols in rats to investigate the effects of a selective NF-κB inhibitor (JSH-23) on (i) lipopolysaccharide (LPS)-induced inflammation and (ii) on behavioral phenotypes in rat models of depression (sucrose consumption test and forced swim test) and mania (amphetamine-induced hyperactivity test). Additionally, we tested the effects of JSH-23 on levels of inflammatory components (interleukin-6, prostaglandin E2, nuclear phospho-p65, and tumor necrosis factor-α) in the brain. Acute treatment with JSH-23 (10 mg/kg, intraperitoneally [ip]) led to potent anti-inflammatory effects in LPS-treated rats, including a diminished hypothermic response to LPS and a reduction in pro-inflammatory mediators' levels in the brain. Chronic treatment with JSH-23 (3 mg/kg, ip, once daily, for 14 days) resulted in robust antidepressant-like effects (increased sucrose consumption and decreased immobility time). The antidepressant-like effects of JSH-23 were mostly accompanied by a reduction in levels of pro-inflammatory mediators in the brain. On the other hand, JSH-23 did not reduce amphetamine-induced hyperactivity. Altogether, these data suggest that NF-κB may be a potential therapeutic target for pharmacological interventions for depression.

Astrocyte-specific activation of sigma-1 receptors in mPFC mediates the faster onset antidepressant effect by inhibiting NF-κB-induced neuroinflammation

Major depressive disorder (MDD) is a global health burden characterized by persistent low mood, deprivation of pleasure, recurrent thoughts of death, and physical and cognitive deficits. The current understanding of the pathophysiology of MDD is lacking, resulting in few rapid and effective antidepressant therapies. Recent studies have pointed to the sigma-1 (σ-1) receptor as a potential rapid antidepressant target; σ-1 agonists have shown promise in a variety of preclinical depression models. Hypidone hydrochloride (YL-0919), an independently developed antidepressant by our institute with faster onset of action and low rate of side effects, has recently emerged as a highly selective σ-1 receptor agonist; however, its underlying astrocyte-specific mechanism is unknown. In this study, we investigated the effect of YL-0919 treatment on gene expression in the prefrontal cortex of depressive-like mice by single-cell RNA sequencing. Furthermore, we knocked down σ-1 receptors on astrocytes in the medial prefrontal cortex of mice to explore the effects of YL-0919 on depressive-like behavior and neuroinflammation in mice. Our results demonstrated that astrocyte-specific knockdown of σ-1 receptor resulted in depressive-like behavior in mice, which was reversed by YL-0919 administration. In addition, astrocytic σ-1 receptor deficiency led to activation of the NF-κB inflammatory pathway, and crosstalk between reactive astrocytes and activated microglia amplified neuroinflammation, exacerbating stress-induced neuronal apoptosis. Furthermore, the depressive-like behavior induced by astrocyte-specific knockdown of the σ-1 receptor was improved by a selective NF-κB inhibitor, JSH-23, in mice. Our study not only reaffirms the σ-1 receptor as a key target of the faster antidepressant effect of YL-0919, but also contributes to the development of astrocytic σ-1 receptor-based novel drugs.

Accumulation of γδT cells in the decidua is promoted by RANKL which upregulates ICAM-1 via NF-κB.

The mechanism underlying the accumulation of γδT cells in the decidua, which helps maintain maternal-fetal immunotolerance in early pregnancy, is unknown. This study reveals that DSC-derived RANKL upregulates ICAM-1 expression via the NF-κB pathway to enable γδT cell accumulation in the early decidua. Decidual γδT (dγδT) cells help maintain maternal-fetal immunotolerance in early pregnancy. However, the mechanism underlying the accumulation of γδT cells in the decidua is unknown. Previous work showed that RANKL upregulated intercellular adhesion molecule 1 (ICAM-1) in decidual stromal cells (DSCs), and Rankl knockout mice had limited dγδT cell populations. In this study, we measured the expression levels of RANKL/RANK and ICAM-1 in DSCs, in addition to the integrins of ICAM-1 on dγδT cells, and the number of dγδT cells from patients with recurrent spontaneous abortion (RSA) and normal pregnant women in the first trimester. RSA patients showed significantly decreased RANKL/RANK and ICAM-1/CD11a signaling in decidua, and a decreased percentage of dγδT cells, which was positively correlated with DSC-derived RANKL and ICAM-1. Next, an in vitro adhesion experiment showed that the enhanced attraction of human DSCs to dγδT cells after RANKL overexpression was almost completely aborted by anti-ICAM-1. Furthermore, Rankl knockout mice showed a significant reduction in NF-κB activity compared with wild-type controls. Finally, we applied a selective NF-κB inhibitor named PDTC to validate the role of NF-κB in RANKL-mediated ICAM-1 upregulation. Taken together, our data show that DSC-derived RANKL upregulates ICAM-1 expression via the NF-κB pathway to enable γδT cell accumulation in the early decidua. A reduction in RANKL/ICAM-1 signaling in DSCs may result in insufficient accumulation of γδT cells in decidua and, in turn, RSA.

Monotropein Protects Mesenchymal Stem Cells from Lipopolysaccharide-Induced Impairments and Promotes Fracture Healing in an Ovariectomized Mouse Model.

Monotropein is one of the active ingredients in Morinda Officinalis, which has been used for the treatment in multiple bone and joint diseases. This study aimed to observe the in vitro effects of Monotropein on osteogenic differentiation of lipopolysaccharide treated bone marrow mesenchymal stem cells (bMSCs), and the in vivo effects of local application of Monotropein on bone fracture healing in ovariectomized mice. Lipopolysaccharide was used to set up the inflammatory model in bMSCs, which were treated by Monotropein. Molecular docking analysis was performed to evaluate the potential interaction between Monotropein and p65. Transverse fractures of middle tibias were established in ovariectomized mice, and Monotropein was locally applied to the fracture site using injectable hydrogel. Monotropein enhanced the ability of primary bMSCs in chondro-osteogenic differentiation. Furthermore, Monotropein rescued lipopolysaccharide-induced osteogenic differentiation impairment and inhibited lipopolysaccharide-induced p65 phosphorylation in primary bMSCs. Docking analysis showed that the binding activity of Monotropein and p65/14-3-3 complex is stronger than the selective inhibitor of NF-κB (p65), DP-005. Local application of Monotropein partially rescued the decreased bone mass and biomechanical properties of callus or healed tibias in ovariectomized mice. The expressions of Runx2, Osterix and Collagen I in the 2-week callus were partially restored in Monotropein-treated ovariectomized mice. Taking together, local application of Monotropein promoted fracture healing in ovariectomized mice. Inhibition of p65 phosphorylation and enhancement in osteogenesis of mesenchymal stem cells could be partial of the effective mechanisms.

Abstract TP222: Aging-associated Medin Induces Human Brain Microvascular Endothelial Cell Pro-inflammatory And Pro-thrombotic Activation Partly Via NFκB

Background: Vascular aging is characterized by endothelial dysfunction and proinflammatory & prothrombotic changes. It is responsible for aging-associated pathologies including stroke, vascular dementia (VaD) and Alzheimer’s disease (AD). The underlying mechanisms of vascular aging are poorly understood. Medin is an amyloidogenic protein that accumulates in aging vasculature, is associated with VaD and AD, and is a candidate mediator of vascular aging through unknown mechanisms. Our aim is to test the hypothesis that medin induces proinflammatory and prothrombotic changes in human brain microvascular endothelial cells (HBMVECs) through nuclear factor kappa B (NFκB). Methods: HBMVECs were exposed to vehicle, recombinant medin (5 μM, a physiologically relevant dose) without or with RO106-9920 (a small molecule selective NFκB inhibitor, 10 μM) or RO106-9920 alone for 20 hours (N=3-17/treatment). Cell lysates were measured for gene expression of inflammatory cytokines/chemokines (IL-8, IL-6, ICAM-1 and VCAM-1) and thrombotic factors (thrombomodulin (TM), a transmembrane glycoprotein and potent inhibitor of coagulation, plasminogen activator inhibitor-1 (PAI-1) which promotes thrombosis and tissue factor (TF) which initiates the coagulation cascade), using real time PCR. Results: See Figure. Medin increased gene expression of IL-8, IL-6, ICAM-1, VCAM-1 and PAI-1, reduced TM with no change in TF. Co-treatment of medin with RO106-9920 attenuated the increases in IL-8, IL-6, ICAM-1, VCAM-1 and the reduction in TM, with no change in PAI-1. Conclusions: Medin caused increased pro-inflammatory gene expression in HBMVECs. It also caused reduced TM and increased PAI-1 gene expression, signifying prothrombotic activation. Co-treatment with NFκB inhibitor prevented medin’s proinflammatory effect, and effect on TM, but not PAI-1. The effect of inducing endothelial cell activation supports medin as a potential novel target to treat vascular aging.

PHF6 Maintains Acute Myeloid Leukemia Via Regulating NF-Κappab Signaling Pathway

Acute myeloid leukemia (AML) is a major hematopoietic malignancy characterized by the uncontrolled expansion of the immature myeloid cells in the hematopoietic system. Current chemotherapy regimens remain ineffective as assessed by low remission and 5-year-survival rate. Many efforts have been dedicated to decipher the molecular events underlying AML transformation. PHF6 is a conserved epigenetic regulator and has been found to be frequently altered in hematologic malignancies. Previous studies demonstrated that PHF6 plays a suppressive role in T-ALL leukemogenesis. However, the functional role(s) of PHF6 in AML initiation and progression remains unknown. Here in this study, we analyzed the mRNA expression of PHF6 in blood cells from healthy donors and leukemia cells from AML patients, and found that the PHF6 expression was significantly increased in AML cells when compared with that of normal PMNs (www.Bloodspot.com) (p < 0.05). Significantly, the average survival time of AML patients with high PHF6 expression was much shorter than that of AML patients with low PHF6 expression (p = 0.022). Overexpressing of PHF6 (PHF6 OE) in Kasumi-1 or K562 leukemia cell lines showed increased growth of Kasumi-1 and K562 cells, decreased apoptosis of Kasumi-1 cells. Additionally, we knocked down (KD) PHF6 expression in AML cells and CD34+ cord blood cells, and found that PHF6 KD significantly decreased the growth of Kasumi-1 and promoted cell apoptosis, while had little effects on apoptosis of CD34+ cord blood cells. These results suggested that PHF6 is required for the growth of myeloid leukemia cells but is dispensable for normal blood cells. To further evaluate the functional role of Phf6 in leukemogenesis in vivo, we generated Phf6 KO+RUNX1-ETO9a (RE9a) and Phf6 KO+MLL-AF9 (MA9) AML mice. We found the survival time of Phf6 KO+RE9a mice was significantly longer than that of WT Phf6+RE9a mice. The Phf6 KO+RE9a mice showed much milder disease phenotypes than WT Phf6+RE9a mice, including lower counts of GFP+ leukemia cells in the PB and BM, higher body weights, lower spleen and liver weights, and lower degree of extramedullary infiltration in spleen, liver, lung and brain. Extreme limiting dilution transplantation assays demonstrated a marked decrease in leukemia stem cell (LSC) activity in Phf6 KO+RE9a cells when compared with Phf6 WT+RE9a cells. Consistent with RE9a-induced AML mouse model, PHF6 deficiency impaired the self-renewal of LSCs and delayed the progression of MA9-induced AML, supporting a general oncogenic role of Phf6 in sustaining myeloid leukemia. To investigate the underlying molecular mechanisms of Phf6 in regulating AML development, we performed RNAseq to analyze the transcriptome programing changes associated with Phf6 deficiency in isogenic Phf6 WT+RE9a or Phf6 KO+RE9aAML cells. Gene set enrichment analysis (GSEA) showed up-regulation of apoptosis and down-regulation of NF-κB-regulated gene expressions in Phf6 KO+RE9a cells, suggesting that Phf6 depletion might promote AML cell apoptosis by blocking the NF-κB signaling. Immunocytochemistry studies showed that PHF6 depletion partially inhibited the nuclear translocation of p50. Co-immunoprecipitation experiments revealed that PHF6 directly bound to p50 in myeloid leukemia cells, suggesting that PHF6 might activate NF-κB signaling pathways by binding with p50 and maintaining the nuclear translocation of p50. To confirm this, we treated PHF6 OE AML cells with BAY11-7082 (a selective NF-κB inhibitor), and found BAY11-7082 significantly inhibited the growth of PHF6 OE Kasumi-1 cells in vitro. In summary, we revealed that PHF6 promoted RE9a- or MA9-induced AML progression in mice by activating NF-κB signaling pathway, and promoting P50 nuclear translocation. Our study thus defined a unique role for Phf6 in regulating NF-κB signaling pathway in AMLs, and potentially can be used as a therapeutic target for treating myeloid leukemia patients.

Macrophage Polarization Modulated by NF-κB in Polylactide Membranes-Treated Peritendinous Adhesion.

Foreign body reactions (FBR) to implants seriously impair tissue-implant integration and postoperative adhesion. The macrophage, owing to its phenotypic plasticity, is a major regulator in the formation of the inflammatory microenvironment; NF-κB signaling also plays a vital role in the process. It is hypothesized that NF-κB phosphorylation exerts a proinflammatory regulator in FBR to polylactide membranes (PLA-M) and adhesion. First, in vitro and in vivo experiments show that PLA-M induces NF-κB phosphorylation in macrophages, leading to M1 polarization and release of inflammatory factors. The inflammatory microenvironment formed due to PLA-M accelerates myofibroblast differentiation and release of collagen III and MMP2, jointly resulting in peritendinous adhesion. Therefore, JSH-23 (a selective NF-κB inhibitor)-loaded PLA membrane (JSH-23/PLA-M) is fabricated by blend electrospinning to regulate the associated M1 polarization for peritendinous anti-adhesion. JSH-23/PLA-M specifically inhibits NF-κB phosphorylation in macrophages and exhibits anti-inflammatory and anti-adhesion properties. The findings demonstrate that NF-κB phosphorylation has a critical role in PLA-induced M1 polarization and aggravating FBR to PLA-M. Additionally, JSH-23/PLA-M precisely targets modulation of NF-κB phosphorylation in FBR to break the vicious cycle in peritendinous adhesion therapy.

Cadmium exposure induces osteoporosis through cellular senescence, associated with activation of NF-κB pathway and mitochondrial dysfunction

Cadmium (Cd) is a heavy metal toxicant as a common pollutant derived from many agricultural and industrial sources. The absorption of Cd takes place primarily through Cd-contaminated food and water and, to a significant extent, via inhalation of Cd-contaminated air and cigarette smoking. Epidemiological data suggest that occupational or environmental exposure to Cd increases the health risk for osteoporosis and spontaneous fracture such as itai-itai disease. However, the direct effects and underlying mechanism(s) of Cd exposure on bone damage are largely unknown. We used primary bone marrow-derived mesenchymal stromal cells (BMMSCs) and found that Cd significantly induced BMMSC cellular senescence through over-activation of NF-κB signaling pathway. Increased cell senescence was determined by production of senescence-associated secretory phenotype (SASP), cell cycle arrest and upregulation of p21/p53/p16INK4a protein expression. Additionally, Cd impaired osteogenic differentiation and increased adipogenesis of BMMSCs, and significantly induced cellular senescence-associated defects such as mitochondrial dysfunction and DNA damage. Sprague-Dawley (SD) rats were chronically exposed to Cd to verify that Cd significantly increased adipocyte number, and decreased mineralization tissues of bone marrow in vivo. Interestingly, we observed that Cd exposure remarkably retarded bone repair and regeneration after operation of skull defect. Notably, pretreatment of melatonin is able to partially prevent Cd-induced some senescence-associated defects of BMMSCs including mitochondrial dysfunction and DNA damage. Although Cd activated mammalian target of rapamycin (mTOR) pathway, rapamycin only partially ameliorated Cd-induced cell apoptosis rather than cellular senescence phenotypes of BMMSCs. In addition, a selective NF-κB inhibitor moderately alleviated Cd-caused the senescence-related defects of the BMMSCs. The study shed light on the action and mechanism of Cd on osteoporosis and bone ageing, and may provide a novel option to ameliorate the harmful effects of Cd exposure.

Senescence-associated hyper-activation to inflammatory stimuli in vitro.

Aging is associated with an increased susceptibility to adverse inflammatory conditions such as sepsis and cytokine storm. We hypothesized that senescent cells (SnCs) play a central role in this age-associated pathology in part due to their expression of the senescence-associated secretory phenotype (SASP), which may prime SnCs to inflammatory stimulation. To test this hypothesis, we examined the expression of various inflammatory cytokines and chemokines at the levels of gene transcription and protein production in various SnCs in vitro in response to lipopolysaccharide (LPS), interleukin-1β (IL1β), and tumor necrosis factor α (TNFα) stimulation. We found that SnCs not only expressed higher basal levels of various inflammatory cytokines and chemokines as a manifestation of the SASP, but more importantly exhibited hyper-activation of the induction of a variety of inflammatory mediators in response to LPS, IL1β and TNFα stimulation as compared with non-SnCs. This senescence-associated hyper-activation is likely mediated in part via the p38MAPK (p38) and NFκB pathways because LPS stimulation elicited significantly higher levels of p38 phosphorylation and NFκB p65 nuclear translation in SnCs when compared to their non-senescent counterparts and inhibition of these pathways with losmapimod (a p38 specific inhibitor) and BMS-345541 (a selective NFκB inhibitor) attenuated LPS-induced expression of IL6, TNFα, CCL5, and IL1β mRNA in SnCs. These findings suggest that SnCs may play an important role in the age-related increases in the susceptibility to developing an exacerbated inflammatory response and highlight the potential to use senotherapeutics to ameliorate the severity of various devastating inflammatory conditions in the elderly.

Sustained Inhibition of NF-κB Activity Mitigates Retinal Vasculopathy in Diabetes

This study investigated the effects of long-term NF-κB inhibition in mitigating retinal vasculopathy in a type 1 diabetic mouse model (Akita, Ins2Akita). Akita and wild-type (C57BL/6J) male mice, 24 to 26 weeks old, were treated with or without a selective inhibitor of NF-κB, 4-methyl-N1-(3-phenyl-propyl) benzene-1,2-diamine (JSH-23), for 4 weeks. Treatment was given when the mice were at least 24 weeks old. Metabolic parameters, key inflammatory mediators, blood-retinal barrier junction molecules, retinal structure, and function were measured. JSH-23 significantly lowered basal glucose levels and intraocular pressure in Akita. It also mitigated vascular remodeling and microaneurysms significantly. Optical coherence tomography of untreated Akita showed thinning of retinal layers; however, treatment with JSH-23 could prevent it. Electroretinogram demonstrated that A- and B-waves in Akita were significantly smaller than in wild type mice, indicating that JSH-23 intervention prevented loss of retinal function. Protein levels and gene expression of key inflammatory mediators, such as NOD-like receptor family pyrin domain-containing 3, intercellular adhesion molecule-1, inducible nitric oxide synthase, and cyclooxygenase-2, were decreased after JSH-23 treatment. At the same time, connexin-43 and occludin were maintained. Vision-guided behavior also improved significantly. The results show that reducing inflammation could protect the diabetic retina and its vasculature. Findings appear to have broader implications in treating not only ocular conditions but also other vasculopathies.

SARS-CoV-2 spike protein S1 subunit induces pro-inflammatory responses via toll-like receptor 4 signaling in murine and human macrophages

Coronavirus disease 2019 (COVID-19), an infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has now spread globally. Some patients develop severe complications including multiple organ failure. It has been suggested that excessive inflammation associated with the disease plays major role in the severity and mortality of COVID-19. To elucidate the inflammatory mechanisms involved in COVID-19, we examined the effects of SARS-CoV-2 spike protein S1 subunit (hereafter S1) on the pro-inflammatory responses in murine and human macrophages. Murine peritoneal exudate macrophages produced pro-inflammatory mediators in response to S1 exposure. Exposure to S1 also activated nuclear factor-κB (NF-κB) and c-Jun N-terminal kinase (JNK) signaling pathways. Pro-inflammatory cytokine induction by S1 was suppressed by selective inhibitors of NF-κB and JNK pathways. Treatment of murine peritoneal exudate macrophages and human THP-1 cell-derived macrophages with a toll-like receptor 4 (TLR4) antagonist attenuated pro-inflammatory cytokine induction and the activation of intracellular signaling by S1 and lipopolysaccharide. Similar results were obtained in experiments using TLR4 siRNA-transfected murine RAW264.7 macrophages. In contrast, TLR2 neutralizing antibodies could not abrogate the S1-induced pro-inflammatory cytokine induction in either RAW264.7 or THP-1 cell-derived macrophages. These results suggest that SARS-CoV-2 spike protein S1 subunit activates TLR4 signaling to induce pro-inflammatory responses in murine and human macrophages. Therefore, TLR4 signaling in macrophages may be a potential target for regulating excessive inflammation in COVID-19 patients.

Acetylcholine ameliorated TNF-α-induced primary trophoblast malfunction via muscarinic receptors†.

Oxidative stress and apoptosis of trophoblasts are involved in preeclampsia (PE). Numerous studies have shown that acetylcholine (ACh), the principal vagal neurotransmitter, plays a crucial role in attenuating oxidative stress, inflammation, and apoptosis in a variety of human diseases. However, the role of ACh in PE management remains unclear. Here, we aimed to determine the effects of ACh on TNF-α-treated human primary trophoblast cells. Western blotting, CCK-8, DHE, TUNEL immunofluorescence staining, transwell assays, and wound-healing assays were performed to evaluate the role of ACh in vitro. We found that both TNF-α expression and the apoptotic index were higher in placentas from preeclamptic women than in normal placentas. TNF-α enhanced oxidative stress and increased the number of TUNEL-positive nuclei, Bax/Bcl-2 ratio, and the cleaved caspase-3/caspase-3 ratio while decreasing cell viability in primary human trophoblast cells. TNF-α promoted cell migration and invasion. PDTC, a selective NF-κB inhibitor, significantly blunted TNF-α-induced effects. ACh treatment attenuated oxidative stress and apoptosis while further promoting migration and invasion of TNF-α-treated primary trophoblast cells. The effects of ACh could be reversed by the muscarinic receptor antagonist atropine. Overall, our findings indicate that ACh significantly ameliorates TNF-α-induced oxidative stress and apoptosis of human primary trophoblast cells via muscarinic receptors. This is the first time that the improvement of vagal activity served as a therapeutic strategy for PE-like trophoblasts, suggesting its potential value in clinical practice.

Antagonizing binding of cell cycle and apoptosis regulatory protein 1 (CARP-1) to the NEMO/IKKγ protein enhances the anticancer effect of chemotherapy

NF-κB is a pro-inflammatory transcription factor that critically regulates immune responses and other distinct cellular pathways. However, many NF-κB-mediated pathways for cell survival and apoptosis signaling in cancer remain to be elucidated. Cell cycle and apoptosis regulatory protein 1 (CARP-1 or CCAR1) is a perinuclear phosphoprotein that regulates signaling induced by anticancer chemotherapy and growth factors. Although previous studies have reported that CARP-1 is a part of the NF-κB proteome, regulation of NF-κB signaling by CARP-1 and the molecular mechanism(s) involved are unclear. Here, we report that CARP-1 directly binds the NF-κB-activating kinase IκB kinase subunit γ (NEMO or NF-κB essential modulator) and regulates the chemotherapy-activated canonical NF-κB pathway. Importantly, blockade of NEMO-CARP-1 binding diminished NF-κB activation, indicated by reduced phosphorylation of its subunit p65/RelA by the chemotherapeutic agent adriamycin (ADR), but not NF-κB activation induced by tumor necrosis factor α (TNFα), interleukin (IL)-1β, or epidermal growth factor. High-throughput screening of a chemical library yielded a small molecule inhibitor of NEMO-CARP-1 binding, termed selective NF-κB inhibitor 1 (SNI)-1). We noted that SNI-1 enhances chemotherapy-dependent growth inhibition of a variety of cancer cells, including human triple-negative breast cancer (TNBC) and patient-derived TNBC cells in vitro, and attenuates chemotherapy-induced secretion of the pro-inflammatory cytokines TNFα, IL-1β, and IL-8. SNI-1 also enhanced ADR or cisplatin inhibition of murine TNBC tumors in vivo and reduced systemic levels of pro-inflammatory cytokines. We conclude that inhibition of NEMO-CARP-1 binding enhances responses of cancer cells to chemotherapy.

Myeloid cell–targeted miR-146a mimic inhibits NF-κB–driven inflammation and leukemia progression in vivo

AbstractNF-κB is a key regulator of inflammation and cancer progression, with an important role in leukemogenesis. Despite its therapeutic potential, targeting NF-κB using pharmacologic inhibitors has proven challenging. Here, we describe a myeloid cell–selective NF-κB inhibitor using an miR-146a mimic oligonucleotide conjugated to a scavenger receptor/Toll-like receptor 9 agonist (C-miR146a). Unlike an unconjugated miR146a, C-miR146a was rapidly internalized and delivered to the cytoplasm of target myeloid cells and leukemic cells. C-miR146a reduced expression of classic miR-146a targets (IRAK1 and TRAF6), thereby blocking activation of NF-κB in target cells. IV injections of C-miR146a mimic to miR-146a–deficient mice prevented excessive NF-κB activation in myeloid cells, and thus alleviated myeloproliferation and mice hypersensitivity to bacterial challenge. Importantly, C-miR146a showed efficacy in dampening severe inflammation in clinically relevant models of chimeric antigen receptor (CAR) T-cell–induced cytokine release syndrome. Systemic administration of C-miR146a oligonucleotide alleviated human monocyte-dependent release of IL-1 and IL-6 in a xenotransplanted B-cell lymphoma model without affecting CD19-specific CAR T-cell antitumor activity. Beyond anti-inflammatory functions, miR-146a is a known tumor suppressor commonly deleted or expressed at reduced levels in human myeloid leukemia. Using The Cancer Genome Atlas acute myeloid leukemia data set, we found an inverse correlation of miR-146a levels with NF-κB–related genes and with patient survival. Correspondingly, C-miR146a induced cytotoxic effects in human MDSL, HL-60, and MV4-11 leukemia cells in vitro. The repeated IV administration of C-miR146a inhibited expression of NF-κB target genes and thereby thwarted progression of disseminated HL-60 leukemia. Our results show the potential of using myeloid cell–targeted miR-146a mimics for the treatment of inflammatory and myeloproliferative disorders.

Protectin DX attenuates IL-1β-induced inflammation via the AMPK/NF-κB pathway in chondrocytes and ameliorates osteoarthritis progression in a rat model

Protectin DX (PDX) has been reported to have extensive anti-inflammatory effects. However, it is unknown whether PDX acts as an anti-inflammatory agent in the context of osteoarthritis (OA). This study aimed to evaluate the anti-inflammatory activity of PDX in vitro and in vivo in a model of OA. Primary rat chondrocytes were preincubated with PDX 1 h prior to IL-1β treatment for 24 h. We found that PDX was nontoxic, and pretreatment with PDX increased cell viability in IL-1β-induced chondrocytes. Preincubation with PDX also efficiently inhibited the degradation of type II collagen dose-dependently. Additionally, the expression of MMP-3, MMP-13, ADAMTS4, iNOS, COX-2, NO, and PGE2 decreased after IL-1β stimulation when cells were preincubated with PDX. Moreover, PDX inhibited the increase in phosphorylated NF-κB p65 and IκBα upon IL-1β stimulation, and the negative effects of IL-1β on chondrocytes were partially blocked by treatment with pyrrolidine dithiocarbamate (PDTC), a selective NF-κB inhibitor. In addition, we found that PDX increased AMPK phosphorylation in IL-1β-mediated chondrocytes. The phosphorylation of AMPK could be inhibited by compound C, a classic AMPK inhibitor. Compound C also remarkably reversed the decrease in p65 phosphorylation and MMP-13 expression caused by PDX. Furthermore, nuclear translocation of NF-κB was visible by immunofluorescence after PDX-induced AMPK activation. Additionally, we verified that PDX ameliorated cartilage degradation in monosodium iodoacetate (MIA)-induced OA rats through histological evaluation and ELISA of TNF-α in the serum and intra-articular lavage fluid. In conclusion, we have shown that PDX suppresses inflammation in chondrocytes in vitro and in vivo, likely through the AMPK/NF-κB signaling pathway. Our results suggest that PDX could be a useful novel therapeutic agent for OA treatment.

Role of NF-κB in cytochrome P450 epoxygenases down-regulation during an inflammatory process in astrocytes

Cytochrome P450 (CYP) epoxygenases and their metabolic products, epoxyeicosatrienoic acids (EETs), have been proposed as important therapeutic targets in the brain. However, CYP expression can be modified by the presence of diverse pro-inflammatory cytokines and the subsequent activation of the NF-κB pathway. It has been indicated that CYP epoxygenases are down-regulated by inflammation in the heart, kidney and liver. However, up to this point, there has been no evidence regarding regulation of CYP epoxygenases during inflammation in the brain. Therefore, in order to explore the effects of inflammation and NF-κB activation in CYP2J3 and CYP2C11 regulation, rat primary astrocytes cultures were treated with LPS with and without IMD-0354 (selective NF-κB inhibitor). Cyp2j3 and Cyp2c11 mRNA expression was determined by qRT-PCR; protein expression was determined by immunofluorescence and by Western Blot and total epoxygenase activity was determined by the quantification of EETs by ELISA. NF-κB binding sites in Cyp2j3 and Cyp2c11 promoter regions were bioinformatically predicted and Electrophoretic Mobility Shift Assays (EMSA) were performed to determine if each hypothetic response element was able to bind NF-κB complexes. Results shown that LPS treatment is able to down-regulate astrocyte CYP2J3 and CYP2C11 mRNA, protein and activity. Additionally, we have identified NK-κB as the transcription factor involved in this regulation.

Entamoeba histolytica Induce Signaling via Raf/MEK/ERK for Neutrophil Extracellular Trap (NET) Formation.

Amoebiasis, the disease caused by Entamoeba histolytica is the third leading cause of human deaths among parasite infections. E. histolytica was reported associated with around 100 million cases of amoebic dysentery, colitis and amoebic liver abscess that lead to almost 50,000 fatalities worldwide in 2010. E. histolytica infection is associated with the induction of inflammation characterized by a large number of infiltrating neutrophils. These neutrophils have been implicated in defense against this parasite, by mechanisms not completely described. The neutrophil antimicrobial mechanisms include phagocytosis, degranulation, and formation of neutrophil extracellular traps (NETs). Recently, our group reported that NETs are also produced in response to E. histolytica trophozoites. But, the mechanism for NETs induction remains unknown. In this report we explored the possibility that E. histolytica leads to NETs formation via a signaling pathway similar to the pathways activated by PMA or the Fc receptor FcγRIIIb. Neutrophils were stimulated by E. histolytica trophozoites and the effect of various pharmacological inhibitors on amoeba-induced NETs formation was assessed. Selective inhibitors of Raf, MEK, and NF-κB prevented E. histolytica-induced NET formation. In contrast, inhibitors of PKC, TAK1, and NADPH-oxidase did not block E. histolytica-induced NETs formation. E. histolytica induced phosphorylation of ERK in a Raf and MEK dependent manner. These data show that E. histolytica activates a signaling pathway to induce NETs formation, that involves Raf/MEK/ERK, but it is independent of PKC, TAK1, and reactive oxygen species (ROS). Thus, amoebas activate neutrophils via a different pathway from the pathways activated by PMA or the IgG receptor FcγRIIIb.

Scaffold-Hopping Approach To Discover Potent, Selective, and Efficacious Inhibitors of NF-κB Inducing Kinase.

NF-κB-inducing kinase (NIK) is a protein kinase central to the noncanonical NF-κB pathway downstream from multiple TNF receptor family members, including BAFF, which has been associated with B cell survival and maturation, dendritic cell activation, secondary lymphoid organ development, and bone metabolism. We report herein the discovery of lead chemical series of NIK inhibitors that were identified through a scaffold-hopping strategy using structure-based design. Electronic and steric properties of lead compounds were modified to address glutathione conjugation and amide hydrolysis. These highly potent compounds exhibited selective inhibition of LTβR-dependent p52 translocation and transcription of NF-κB2 related genes. Compound 4f is shown to have a favorable pharmacokinetic profile across species and to inhibit BAFF-induced B cell survival in vitro and reduce splenic marginal zone B cells in vivo.

Punicalagin induces senescent growth arrest in human papillary thyroid carcinoma BCPAP cells via NF-κB signaling pathway

Papillary thyroid carcinoma (PTC) is the most common endocrine carcinoma. Our previous study revealed that punicalagin (PUN), an active component from pomegranate, triggered autophagic cell death and DNA damage response (DDR) in papillary thyroid carcinoma BCPAP cells. But the detailed anti-cancer mechanisms of punicalagin against PTC still remained to be further explored. DDR activation is a proven cause of cellular senescence, which mediates anti-tumor processes under certain circumstances. In this study, we reported that punicalagin treatment generated a senescent phenotype of BCPAP cells characterized as altered morphology, increased cell granularity and senescence-associated β-galactosidase (SA-β-Gal) staining. Senescence induced by punicalagin treatment was further confirmed by cell cycle arrest and upregulation of cyclin-dependent kinase inhibitor p21. Meanwhile, the senescence-associated secretory phenotype (SASP) included high levels of inflammatory cytokines, principally IL-6 and IL-1β. Furthermore, punicalagin exposure caused the phosphorylation and subsequent degradation of IκBα as well as the nuclear translocation of p65, suggesting the activation of NF-κB signaling pathway. Inhibition of NF-κB by pyrrolidine dithiocarbamate (PDTC), a selective inhibitor of NF-κB, partially reversed the cellular senescent phenotype induced by punicalagin in BCPAP cells as evidenced by the decreased fraction of SA-β-Gal staining positive cells and blockage of SASP generation. These results collectively showed that punicalagin treatment induced senescent growth arrest and SASP via triggering NF-κB activation. These observations elucidated novel anti-cancer mechanisms of punicalagin and might provide new potential prospects for PTC therapy.

Blockade of nuclear factor-κB (NF-κB) pathway inhibits growth and induces apoptosis in chemoresistant ovarian carcinoma cells.

Epithelial ovarian cancer (EOC) has exhibited marginal improvement in survival rate, despite advances in surgical debulking and chemotherapy regimens. Although the majority of EOC patients achieve a clinical remission after induction therapy, over 80% relapse and succumb to chemoresistant disease. In this regard, it is of paramount importance to elucidate molecular mechanisms and signaling pathways which promote therapy resistance in EOC in order to devise novel and more effective treatment strategies. In this study, we showed that activation of nuclear factor-κB (NF-κB) is significantly higher in therapy-resistant EOC cells compared to chemosensitive counterparts, which was positively associated with resistance to cisplatin, carboplatin, paclitaxel and erlotinib. Bay 11-7082, a highly selective NF-κB inhibitor, reduced cell proliferation, clonogenicity and anoikis resistance in the therapy-resistant EOC cells and induced apoptotic cell death. Moreover, Bay 11-7082 decreased the expression of pro-survival, inflammatory and metastatic genes and synergistically increased anti-proliferative efficacy of cisplatin, carboplatin, paclitaxel and erlotinib. Altogether, these findings suggest that NF-κB is an attractive therapeutic target in EOC to be exploited in translational oncology and Bay 11-7082 is a potential anti-cancer drug to overcome chemoresistance and inhibit proliferation of the EOC cells.