The NF-κB Transcriptional Footprint Is Essential for SARS-CoV-2 Replication.

SARS-CoV-2, the etiological agent of COVID-19, is characterized by a delay in type I interferon (IFN-I)-mediated antiviral defenses alongside robust cytokine production. Here, we investigate the underlying molecular basis for this imbalance and implicate virus-mediated activation of NF-κB in the absence of other canonical IFN-I-related transcription factors. Epigenetic and single-cell transcriptomic analyses show a selective NF-κB signature that was most prominent in infected cells. Disruption of NF-κB signaling through the silencing of the NF-κB transcription factor p65 or p50 resulted in loss of virus replication that was rescued upon reconstitution. These findings could be further corroborated with the use of NF-κB inhibitors, which reduced SARS-CoV-2 replication in vitro. These data suggest that the robust cytokine production in response to SARS-CoV-2, despite a diminished IFN-I response, is the product of a dependency on NF-κB for viral replication. IMPORTANCE The COVID-19 pandemic has caused significant mortality and morbidity around the world. Although effective vaccines have been developed, large parts of the world remain unvaccinated while new SARS-CoV-2 variants keep emerging. Furthermore, despite extensive efforts and large-scale drug screenings, no fully effective antiviral treatment options have been discovered yet. Therefore, it is of the utmost importance to gain a better understanding of essential factors driving SARS-CoV-2 replication to be able to develop novel approaches to target SARS-CoV-2 biology.

Zinc finger protein A20 regulates the development and progression of osteoarthritis by affecting the activity of NF-κB p65.

OBJECTIVE: To investigate the role of Zinc finger protein A20 in osteoarthritis (OA) by regulating NF-κB p65. METHODS: A20, MMP1, MMP13 and IL-1ß expressions in human OA cartilage samples were detected by qRT-PCR. IL-1ß-induced chondrocyte was treated with A20 lentivirus activation particle, pyrrolidine dithiocarbamate (PDTC, a NF-κB inhibitor) with/without A20 siRNA. IL-6, TNF-α, and PGE2 levels were measured by ELISA, and NO production by Greiss reaction. Destabilization of the medial meniscus (DMM) surgery was used to construct the OA models, followed by injection of A20 adenovirus. MMP1 and MMP13 expression was measured by immunohistochemistry. The mRNA and protein expression were performed by qRT-PCR and western blotting, respectively. RESULTS: A20 was down-regulated in human OA cartilage samples, and negatively correlated with the expressions of MMP1, MMP13 and IL-1ß. The IL-1ß-induced chondrocyte manifested decreased A20 with increased NF-κB p65 activity. A20 overexpression suppressed the NF-κB p65 activity in IL-1ß-induced chondrocyte. Furthermore, PDTC decreased IL-1ß-induced chondrocyte apoptosis with the upregulated COL1A1, COL2A1, COL10A1 and ACAN, as well as the down-regulated MMP1, MMP13, COX2, iNOS, IL-6, TNF-α, NO and PGE2, which was reversed by A20 siRNA. In vivo, OA mice gained higher OARSI score and Mankin's score, exhibited up-regulations of MMP1 and MMP13, and decreased NF-κB p65 activity, which was improved after injection of A20 adenovirus. CONCLUSION: A20 was reduced in OA cartilage samples, and its overexpression, by suppressing the activity of NF-κB p65, could improve IL-1ß-induced chondrocyte degradation and apoptosis in vitro, as well as mitigate the inflammation in OA mice.

Nuclear factor kappa B inhibitor suppresses experimental autoimmune neuritis in mice via declining macrophages polarization to M1 type.

Guillain-Barré syndrome (GBS) is an acute inflammatory and immune-mediated demyelinating disease of the peripheral nervous system (PNS). Macrophages play a central role in its animal model, experimental autoimmune neuritis (EAN), which has been well accepted. Additionally, nuclear factor (NF)-κB inhibitors have been used to treat cancers and have shown beneficial effects. Here, we investigated the therapeutic effect of M2 macrophage and the NF-κB pathway's correlation with macrophage activation in EAN in C57BL/6 mice. We demonstrate that M2 macrophage transfusion could alleviate the clinical symptoms of EAN by reducing the proportion of M1 macrophage in the peak period, inhibiting the phosphorylation of NF-κB p65. The NF-κB inhibitor (BAY-11-7082) could alleviate the clinical symptoms of EAN and shorten the duration of symptoms by reducing the proportion of M1 macrophages and the expression of proinflammatory cytokines. Consequently, BAY-11-7082 exhibits strong potential as a therapeutic strategy for ameliorating EAN by influencing the balance of M1/M2 macrophages and inflammatory cytokines.

MiR-520b inhibits endothelial activation by targeting NF-κB p65-VCAM1 axis.

MiR-520b belongs to the miR-373/520 family, is expressed only in human and nonhuman primates. Previous reports indicated that the expression of miR-520b was repressed in human atherosclerotic plaque tissue compared with healthy vessels. However, the role of miR-520b in coronary artery disease still remains to be uncovered. In this study, we demonstrated that endothelial cells (ECs) in human atherosclerotic plaques expressed miR-520b and aimed to elucidate the impact of miR-520b on EC activation and inflammatory response. To determine the potential targets of miR-520b, we performed RNA-seq analysis by transfecting miR-520b mimics in ECs. The quantitative real-time PCR (qPCR) validation suggested that miR-520b over-expression reduced pro-inflammatory gene expression (e.g. ICAM1, VCAM1, SELE) while the inhibition of miR-520b induced their expression. By combining bioinformatics prediction and functional assays, we identified that RELA (Nuclear Factor-κB (NF-κB) Transcription Factor P65) was a direct target of miR-520b. Moreover, miR-520b mimics attenuated monocyte adhesion and monocyte trans-endothelial migration (the initial steps of atherosclerotic formation) in response to lipopolysaccharides (LPS) stimulation. Re-expression of a non-miR-targetable version of p65 could rescue the reduced monocyte cell attachment, suggesting that this process is NF-κB p65 dependent. MiR-520b reduced the abundance of NF-κB p65 in cytoplasmic fractions without corresponding increase in nuclear fractions, indicating that this regulation is independent of p65 translocation process. MiR-520b mimics attenuated the activity of VCAM-1 promoter, whereas miR-520b inhibitor activated its activity. However, miR-520b inhibitor had no effect on promoter activity containing the mutated NF-κB p65 binding sites, strongly demonstrating that the impact of miR-520b on VCAM1 gene is mediated by NF-κB p65. Thus, we concluded that miR-520b suppressed EC inflammation and the cross-talk between monocytes and ECs by down-regulating NF-κB p65-ICAM1/VCAM1 axis and might serve as a potential therapeutic target for EC dysfunction and atherosclerosis.

NF­κB is negatively associated with Nurr1 to reduce the inflammatory response in Parkinson's disease.

Parkinson's disease (PD) is one of the most disabling diseases of the central nervous system, seriously affecting health and quality of life for the elderly. The present study aimed to explore the effects of nuclear receptor subfamily 4 group A member 2 (Nurr1) and nuclear factor­κB (NF­κB) on the progression of Parkinson's disease (PD). Pheochromocytoma (PC12) cells were pretreated with the NF­κB inhibitor quinazoline (QNZ) or transfected with small interfering (si)RNA­NF­κB, followed by the addition of lipopolysaccharide (LPS). After culturing for 24 h, Cell Counting Kit­8 (CCK­8) was utilized to measure cell viability. Next, the expression levels of interleukin (IL)­1ß, IL­6 and tumor necrosis factor (TNF)­α were determined using the relevant Enzyme­linked immunosorbent assay kits. Expression levels of p65, tyrosine hydroxylase (TH), α­Synuclein (A­SYN) and Nurr1 were examined by immunofluorescence and western blotting. CCK­8 results showed that the cell viability was significantly reduced in the LPS group than in the control group (P<0.05), whereas QNZ and si­NF­κB demonstrated significantly enhanced viability induced by LPS (P<0.05). After LPS induction, the levels of IL­1ß, IL­6 and TNF­α were significantly elevated when compared with those in the control group (P<0.05), whereas QNZ and NF­κB interference partially restored their levels. Additionally, after LPS induction, the expression of p65 and A­SYN was higher, while the expression of TH and Nurr1 was lower. However, QNZ and NF­κB treatment significantly reversed the expression levels induced by LPS (P<0.05). Finally, it was observed that NF­κB may be negatively associated with Nurr1. In conclusion, inhibition of NF­κB may reduce the production of inflammatory factors by upregulating Nurr1 and TH and downregulating A­SYN, thus relieving the inflammatory response in PD.

The Inflammatory Profile of CTEPH-Derived Endothelial Cells Is a Possible Driver of Disease Progression.

Chronic thromboembolic pulmonary hypertension (CTEPH) is a form of pulmonary hypertension characterized by the presence of fibrotic intraluminal thrombi and causing obliteration of the pulmonary arteries. Although both endothelial cell (EC) dysfunction and inflammation are linked to CTEPH pathogenesis, regulation of the basal inflammatory response of ECs in CTEPH is not fully understood. Therefore, in the present study, we investigated the role of the nuclear factor (NF)-κB pro-inflammatory signaling pathway in ECs in CTEPH under basal conditions. Basal mRNA levels of interleukin (IL)-8, IL-1ß, monocyte chemoattractant protein-1 (MCP-1), C-C motif chemokine ligand 5 (CCL5), and vascular cell adhesion molecule-1 (VCAM-1) were upregulated in CTEPH-ECs compared to the control cells. To assess the involvement of NF-κB signaling in basal inflammatory activation, CTEPH-ECs were incubated with the NF-κB inhibitor Bay 11-7085. The increase in pro-inflammatory cytokines was abolished when cells were incubated with the NF-κB inhibitor. To determine if NF-κB was indeed activated, we stained pulmonary endarterectomy (PEA) specimens from CTEPH patients and ECs isolated from PEA specimens for phospho-NF-κB-P65 and found that especially the vessels within the thrombus and CTEPH-ECs are positive for phospho-NF-κB-P65. In summary, we show that CTEPH-ECs have a pro-inflammatory status under basal conditions, and blocking NF-κB signaling reduces the production of inflammatory factors in CTEPH-ECs. Therefore, our results show that the increased basal pro-inflammatory status of CTEPH-ECs is, at least partially, regulated through activation of NF-κB signaling and potentially contributes to the pathophysiology and progression of CTEPH.

Escin inhibits angiogenesis by suppressing interleukin­8 and vascular endothelial growth factor production by blocking nuclear factor­κB activation in pancreatic cancer cell lines.

Pancreatic cancer (PaCa) is one of the most aggressive types of cancer. Thus, the development of new and more effective therapies is urgently required. Escin, a pentacyclic triterpenoid from the horse chestnut, has been reported to exhibit antitumor potential by reducing cell proliferation and blocking the nuclear factor­κB (NF­κB) signaling pathway in several types of cancer. Our previous study reported that NF­κB enhanced the secretion of interleukin (IL)­8 and vascular endothelial growth factor (VEGF), thereby inducing angiogenesis in PaCa cell lines. In the present study, it was examined whether escin inhibited angiogenesis by blocking NF­κB activation in PaCa. It was initially confirmed that escin, at concentrations >10 µM, significantly inhibited the proliferation of several PaCa cell lines. Next, using immunocytochemical staining, it was found that escin inhibited the nuclear translocation of NF­κB. Furthermore, ELISA confirmed that NF­κB activity in the escin­treated PaCa cells was significantly inhibited and reverse transcription­quantitative PCR showed that the mRNA expression levels of tumor necrosis factor­α­induced IL­8 and VEGF were significantly suppressed following escin treatment in the PaCa cell lines. ELISA also showed that escin decreased the secretion of IL­8 and VEGF from the PaCa cells. Furthermore, tube formation in immortalized human endothelial cells was inhibited following incubation with the supernatants from escin­treated PaCa cells. These results indicated that escin inhibited angiogenesis by reducing the secretion of IL­8 and VEGF by blocking NF­κB activity in PaCa. In conclusion, escin could be used as a novel molecular therapy for PaCa.

Involvement of TLR2/4­MyD88­NF­κB signaling pathway in the pathogenesis of intracranial aneurysm.

Toll­like receptor (TLR) 2/4 serves an important regulatory role in nerve tissue injury. However, the downstream and potential mechanisms remain to be elucidated. The present study was designed to investigate the roles of the TLR2/4­major myeloid differentiation response gene 88 (MyD88)­NF­κB signaling pathway in the development of intracranial aneurysm. The expression of TLR2, TLR4 and MyD88 in the blood of normal controls and patients with intracranial aneurysm were detected by quantitative PCR and ELISA. Human brain vascular smooth muscle cells were treated by Angiotensin II (Ang II) to evaluate the involvement of TLR2/4­MyD88­NF­κB signaling pathway in the process. The in vitro experiment was divided into four groups: The control group, an Ang â…¡ group, an Ang â…¡ + small interfering (si)RNA control group and an Ang â…¡ + TLR2­group. Cell viability, migration, apoptosis and expression of TLR2, TLR4, MyD88, NF­κB and phosphorylated (p­)p65 expression were detected. The results demonstrated that the expression of TLR2, TLR4, MyD88 and NF­κB at mRNA and protein levels in patients with intracranial aneurysm was significantly higher compared with corresponding protein in normal controls (P<0.05). In vitro experiments demonstrated that Ang â…¡ treatment increased the cell proliferation and migration rate but reduced the apoptotic rate compared with the control (P<0.05). The expression of TLR2, TLR4, MyD88, NF­κB and p­p65 was significantly increased in the Ang II group (vs. control; P<0.05). By contrast, TLR2­short interfering RNA reduced the cell proliferation and migration rate, and reduced the expression of TLR2, TLR4, MyD88, NF­κB and p­p65 (vs. Ang â…¡ + short interfering RNA control; P<0.05). In conclusion, the data of the present study indicated that the TLR2/4­MyD88­NF­κB signaling pathway is involved in the pathogenesis of intracranial aneurysm.

PDTC ameliorates neuropathic pain by inhibiting microglial activation via blockage of the TNFα-CX3CR1 pathway.

Previous studies have suggested that pyrrolidine dithiocarbamate (PDTC), a nuclear factor κB (NF-κB) inhibitor, play a role in deterring nerve injury-induced neuropathic pain (NP) The activation of NF-κB pathway may contribute to spinal microglial activation, CX3CR1 and tumor necrosis factor-alpha (TNF-a) up-regulation. The aim of this study was to clarify whether PDTC could inhibit the development of neuropathic pain via decreasing TNF-a-induced CX3CR1 up-regulation. Sprague-Dawley rats were randomly divided into sham group and NP group. Rats in each group were treated with intrathecal infusion of PDTC (100 or 1000 pmol/d) or saline. The sciatic nerve chronic constriction injury (CCI) model was used to induce NP in rats. Mechanical stimuli and radiant heat were used to evaluate mechanical allodynia and thermal hyperalgesia. Spinal microglial marker OX42 and TNF-a were detected by immunohistochemistry. In vitro BV-2 microglia activation was induced by TNF-a incubation, and the levels of CX3CR1 were assessed by Western blot and reverse transcription-polymerase chain reaction. Pain behavior and immunohistochemistry results showed that intrathecal infusion of PDTC at 100 or 1000 pmol/d prevented the development of mechanical and thermal hyperalgesia, spinal microglial activation and TNF-a expression induced by sciatic nerve CCI in rats. In vitro experiment results showed that PDTC inhibited the TNF-a-induced CX3CR1 up-regulation in BV-2 microglial cells. In conclusion, intrathecal infusion of PDTC could attenuate the pain-related behaviors induced by sciatic nerve CCI through suppressing the spinal microglia activation and TNF-a up-regulation in rats. The NF-κB activation might be responsible for TNF-a-induced CX3CR1 up-regulation in microglia.

Glaucocalyxin A induces cell cycle arrest and apoptosis via inhibiting NF-κB/p65 signaling pathway in melanoma cells.

AIMS: Melanoma is a malignant tumor of the skin with a high metastasis rate and poor prognosis. Glaucocalyxin A (GLA), isolated from Rabdosia japonica, is a diterpenoid compound with anticancer properties. Here, we investigated the anticancer properties and explored the mechanisms underlying GLA activity in melanoma cells in vitro and in vivo. MAIN METHODS: Cell Counting Kit-8 and colony formation assays were used to assess the effects of GLA on cell proliferation. Flow cytometry was used to evaluate the cell cycle, apoptosis, mitochondrial membrane potential (MMP), and reactive oxygen species (ROS), and western blot analysis and immunofluorescence staining were used to examine protein expression. Immunohistochemical analysis was performed to examine animal tissues and tumors in mice. KEY FINDINGS: GLA could effectively inhibit cell proliferation and induce cell apoptosis. GLA induced an overproduction of cellular ROS, decreased MMP, and upregulated the Bax/Bcl-2 ratio, which is an indicator of apoptosis. Phosphorylation of nuclear factor κB (NF-κB)/p65 and NF-κB/p65 nuclear expression decreased after GLA treatment in vitro and in vivo, suggesting that the anticancer effects of GLA are mediated through the NF-κB/p65 pathway. Moreover, we observed that GLA was effective in inhibiting tumor growth without obvious toxicity to major organs in mice. SIGNIFICANCE: This is the first study to show that GLA inhibits cell proliferation, arrests the cell cycle in the G2/M phase, and induces mitochondrial apoptosis via the NF-κB/p65 pathway in melanoma cells. Overall, our results demonstrate that GLA may be a potential anticancer agent for the treatment of melanoma.

Promoted inhibition of TLR4/miR-155/ NFkB p65 signaling by cannabinoid receptor 2 agonist (AM1241), aborts inflammation and progress of hepatic fibrosis induced by thioacetamide.

The endocannabinoid system plays a pivotal role, whether it is promoting or dampening hepatic fibrosis. This study investigated the role of Cannabinoid receptor 2 (CB2) activation by the synthetic analog (AM1241) on revoking the progress of liver fibrosis. Thioacetamide (TAA) was used to induce liver fibrosis in rats for three weeks followed by its concurrent administration with AM1241 at two different doses for another three weeks. Markers for liver function and oxidative stress, hepatic TNF-α, IL-1ß and IL-6, qRT-PCR expression of Toll like receptor 4 (TLR4), TGF-ß1, α-SMA and microRNA-155 (miR-155) genes, Western blot for protein levels of Vimentin and E-cadherin, immunohistochemical expression of NFκB p65 and histopathology of liver tissue were all investigated. AM1241 administration significantly maintained liver function markers and decreased; malondialdehyde, Vimentin, TLR4, TGF-ß1, α-SMA and miR-155 genes expression, NFκB p65 immune-expression and pro-inflammatory cytokines (TNF-α, IL-1ß and IL-6). Additionally, AM1241 significantly increased E-Cadherin level, GSH and SOD content. Histologically, AM1241 limited fibroplasia extension, and broke the itinerary of bridging fibrosis. In conclusion, activation of the CB2 receptors by AM1241 promoted liver regeneration and overrun the progression of liver fibrosis through; inhibition of TLR4/miR-155/NFκB p65 pathway, suppression of pro-inflammatory IL-6, IL-1ß and TNF-α, reducing TGF-ß1, α-SMA, Vimentin and up-regulating E-Cadherin.

Pristimerin Protects Against OVX-Mediated Bone Loss by Attenuating Osteoclast Formation and Activity via Inhibition of RANKL-Mediated Activation of NF-κB and ERK Signaling Pathways.

INTRODUCTION: Osteoporosis is an osteolytic bone condition characterized by decreased bone strength and increased bone fragility. It is the result of elevated formation or activity of bone-resorbing osteoclasts. Although current therapeutic agents are efficacious against osteoclast-mediated bone loss, detrimental side effects preclude the long-term use of these agents. Pristimerin (PRI) is a naturally occurring quinone-methide triterpenoid that has been revealed to exert anti-inflammatory and anti-tumor effects via regulating various signaling cascades including NF-κB and MAPK activation. METHODS: The bone marrow macrophages were used to confirm the anti-osteoclastic and anti-resorptive functions of PRI in vitro. An in vivo ovariectomy (OVX) model was applied to verify the function of PRI protecting bone loss. RESULTS: PRI abolished the early activation of NF-κB and ERK MAPK signal cascades thereby thwarting the downstream expression of c-Fos and NFATc1, which prevented the production of mature osteoclasts. In vivo, PRI protects mice against ovariectomy (OVX)-mediated bone loss by diminishing osteoclast formation and bone resorptive activity. CONCLUSION: Our study shows that PRI demonstrates therapeutic potential in the effective treatment against osteoclast-induced osteolytic diseases like osteoporosis.

Chlorogenic acid induces apoptosis, inhibits metastasis and improves antitumor immunity in breast cancer via the NF­κB signaling pathway.

Breast cancer which is the most common type of diagnosed cancer among women worldwide possesses metastatic potential, multi­drug resistance, and high mortality. The NF­κB signaling pathway has been revealed to be abnormally activated in breast cancer cells and closely associated with high metastasis and poor prognosis. In the present study, it was reported that chlorogenic acid (CGA), a potent NF­κB inhibitor derived from coffee, exerted antitumor activity in breast cancer. MTT and colony formation assays were conducted and it was revealed that CGA inhibited viability and proliferation in breast cancer cells. Additionally, CGA significantly induced apoptosis and suppressed migration and invasion in breast cancer cells. Notably, immunofluorescence analysis confirmed that CGA could efficiently suppress nuclear transcription of NF­κB p65. In addition, results of western blotting demonstrated that CGA markedly impaired the NF­κB and EMT signaling pathways. The antitumor effect of CGA was evaluated in a subcutaneous tumor mouse model of 4T1 cells, and the results revealed that CGA markedly retarded tumor growth and prolonged the survival rate of tumor­bearing mice. Notably, CGA inhibited pulmonary metastasis of 4T1 cells by enhancing the proportion of CD4+ and CD8+ T cells in spleens of mice, which indicated an improvement of antitumor immunity. In conclusion, the present present study demonstrated that CGA improved antitumor immunity, exerting antitumor and anti­metastatic effects by impairing the NF­κB/EMT signaling pathway, suggesting that CGA may serve as a potential candidate for therapy of breast cancer.

Production of an invertebrate lysozyme of Scylla paramamosain in E.coli and evaluation of its antibacterial, antioxidant and anti-inflammatory effects.

Lysozymes, which are secreted in many organisms, including invertebrates, mammals, plants, bacteria and fungus, exhibit antimicrobial, antiviral, antioxidant, and anti-inflammatory activities. Splys-i is an invertebrate-type (i-type) lysozyme isolated from Scylla paramamosain in 2017 and is involved in immune defense against bacteria. However, the antibacterial, antioxidant, and anti-inflammatory activities of Splys-i remain to be elucidated. In the current study, the expression parameters (including IPTG concentration, induction temperature, and induction duration) of Splys-i in Escherichia coli were optimized to achieve high-level yield through shake-flask cultivation with approximately 120 mg of Splys-i obtained from 1 L of LB medium. The purified Splys-i displayed low cytotoxicity to RAW264.7 macrophage cells and low hemolytic activity against erythrocytes of mouse, rat, and rabbit, respectively, and exhibited potent antibacterial activity against both Gram-positive and -negative bacteria with minimum concentrations ranging from 15 to 90 µg/mL. The antibacterial property of Splys-i was also unaffected when treated with various temperature, pHs, and salinity, respectively, and Splys-i showed resistance to proteinase digestion. Radical-scavenging rate assay (including ABTS+, DPPH, hydroyl free radical, and superoxide anion) indicated that Splys-i was an efficient antioxidant. Splys-i also exerted anti-inflammatory effect through the inhibition of IκBα and NF-κB(P65) phosphorylation, thereby reducing the secretion of pro-inflammatory cytokines. All these results suggested that Splys-i can be prepared from E. coli with potent biological property.

Attenuation of canonical NF-κB signaling maintains function and stability of human Treg.

Nuclear factor 'κ-light-chain-enhancer' of activated B cells (NF-κB) signaling is a signaling pathway used by most immune cells to promote immunostimulatory functions. Recent studies have indicated that regulatory T cells (Treg) differentially integrate TCR-derived signals, thereby maintaining their suppressive features. However, the role of NF-κB signaling in the activation of human peripheral blood (PB) Treg has not been fully elucidated so far. We show that the activity of the master transcription factor forkhead box protein 3 (FOXP3) attenuates p65 phosphorylation and nuclear translocation of the NF-κB proteins p50, p65, and c-Rel following activation in human Treg. Using pharmacological and genetic inhibition of canonical NF-κB signaling in FOXP3-transgenic T cells and PB Treg from healthy donors as well as Treg from a patient with a primary NFKB1 haploinsufficiency, we validate that Treg activation and suppressive capacity is independent of NF-κB signaling. Additionally, repression of residual NF-κB signaling in Treg further enhances interleukin-10 (IL-10) production. Blockade of NF-κB signaling can be exploited for the generation of in vitro induced Treg (iTreg) with enhanced suppressive capacity and functional stability. In this respect, dual blockade of mammalian target of rapamycin (mTOR) and NF-κB signaling was accompanied by enhanced expression of the transcription factors FOXP1 and FOXP3 and demethylation of the Treg-specific demethylated region compared to iTreg generated under mTOR blockade alone. Thus, we provide first insights into the role of NF-κB signaling in human Treg. These findings could lead to strategies for the selective manipulation of Treg and the generation of improved iTreg for cellular therapy.

Clinacanthus nutans Mitigates Neuronal Death and Reduces Ischemic Brain Injury: Role of NF-κB-driven IL-1ß Transcription.

Neuroinflammation has been shown to exacerbate ischemic brain injury, and is considered as a prime target for the development of stroke therapies. Clinacanthus nutans Lindau (C. nutans) is widely used in traditional medicine for treating insect bites, viral infection and cancer, due largely to its anti-oxidative and anti-inflammatory properties. Recently, we reported that an ethanol extract from the leaf of C. nutans could protect the brain against ischemia-triggered neuronal death and infarction. In order to further understand the molecular mechanism(s) for its beneficial effects, two experimental paradigms, namely, in vitro primary cortical neurons subjected to oxygen-glucose deprivation (OGD) and in vivo rat middle cerebral artery (MCA) occlusion, were used to dissect the anti-inflammatory effects of C. nutans extract. Using promoter assays, immunofluorescence staining, and loss-of-function (siRNA) approaches, we demonstrated that transient OGD led to marked induction of IL-1ß, IL-6 and TNFα, while pretreatment with C. nutans suppressed production of inflammatory cytokines in primary neurons. C. nutans inhibited IL-1ß transcription via preventing NF-κB/p65 nuclear translocation, and siRNA knockdown of either p65 or IL-1ß mitigated OGD-mediated neuronal death. Correspondingly, post-ischemic treatment of C. nutans attenuated IκBα degradation and decreased IL-1ß, IL-6 and TNFα production in the ischemic brain. Furthermore, IL-1ß siRNA post-ischemic treatment reduced cerebral infarct, thus mimicking the beneficial effects of C. nutans. In summary, our findings demonstrated the ability for C. nutans to suppress NF-κB nuclear translocation and inhibit IL-1ß transcription in ischemic models. Results further suggest the possibility for using C. nutans to prevent and treat stroke patients.

NF-κB Blockade by NEMO Binding Domain Peptide Ameliorates Inflammation and Neurobehavioral Sequelae After Cranial Radiation Therapy in Juvenile Mice.

PURPOSE: Cranial radiation therapy (CRT) is a common treatment for pediatric brain tumor patients. However, side effects include significant neurobehavioral dysfunction in survivors. This dysfunction may in part be caused by inflammation, including increased production of tumor necrosis factor alpha (TNFα) and its receptor TNFR1, which can activate the nuclear factor kappa light-chain enhancer of activated B cells (NF-κB). The TNFα blockade abrogates this inflammatory response, although it presents immunologic risks. Thus, modulation of pathway subsets may be preferable. Here, we test whether inhibition of NF-κB activation using an NF-κB essential modulator binding domain (NBD) peptide mitigates CRT-induced neuroinflammation and improves behavioral outcomes. METHODS AND MATERIALS: Male C57BL/6J 28-day old mice were randomized to saline (sham), 5 Gy whole-brain CRT, or CRT + NBD-peptide. Brain tissue was collected after 4 hours or 3 months for Western blot or immunohistochemistry. The cortex, corpus callosum (CC), and dentate gyrus were variably imaged for NF-κB-p65, IκBα, proliferation, apoptosis, necroptosis, TNFα, TNFR1, IBA-1, doublecortin, CD11c, and GFAP. Neurobehavioral changes were assessed by open field and elevated plus maze tests 3 months post-CRT. RESULTS: NF-κB expression increased in whole and nuclear fractions 4 hours after CRT and was abrogated by NBD treatment. Cell death increased and proliferation decreased after CRT, including within neuronal progenitors, with some loss mitigated by NBD. Increased levels of TNFα, IBA-1, and GFAP were found in the CC and cortex months after CRT and were limited by NBD. The anti-NF-κB peptide also improved neurobehavioral assessments, yielding improvements in anxiety and exploration. CONCLUSIONS: Results suggest a role for NF-κB modulation by NBD peptide in the reduction of neuroinflammation and mitigation of behavioral complications after pediatric radiation therapy.

Identification of a Selective RelA Inhibitor Based on DSE-FRET Screening Methods.

Nuclear factor-κB (NF-κB) is an important transcription factor involved in various biological functions, including tumorigenesis. Hence, NF-κB has attracted attention as a target factor for cancer treatment, leading to the development of several inhibitors. However, existing NF-κB inhibitors do not discriminate between its subunits, namely, RelA, RelB, cRel, p50, and p52. Conventional methods used to evaluate interactions between transcription factors and DNA, such as electrophoretic mobility shift assay and luciferase assays, are unsuitable for high-throughput screening (HTS) and cannot distinguish NF-κB subunits. We developed a HTS method named DNA strand exchange fluorescence resonance energy transfer (DSE-FRET). This assay is suitable for HTS and can discriminate a NF-κB subunit. Using DSE-FRET, we searched for RelA-specific inhibitors and verified RelA inhibition for 32,955 compounds. The compound A55 (2-(3-carbamoyl-6-hydroxy-4-methyl-2-oxopyridin-1(2H)-yl) acetic acid) selectively inhibited RelA-DNA binding. We propose that A55 is a seed compound for RelA-specific inhibition and could be used in clinical applications.

Effect of atorvastatin on AGEs-induced injury of cerebral cortex via inhibiting NADPH oxidase -NF-κB pathway in ApoE-/- mice.

Advanced glycation end products (AGEs) are a group of modified proteins and/or lipids with damaging potential. AGEs-RAGE pathway plays a critical role to induce neurodegenerative encephalopathy. Statins can reduce the expression of AGEs-induced AGEs receptor (RAGE) in the aorta. It is not clear whether statins have potential benefits on AGEs-induced cognitive impairment. In this study, the effects of atorvastatin (ATV) on inflammation and oxidation stress in the cerebral cortex were investigated, and the underlying mechanisms were explored. Apolipoprotein E (ApoE)-/- male mice were divided into four groups: control, AGEs, AGEs + ALT711 (Alagebrium chloride) and AGEs + ATV. ß-amyloid (Aß) formation in the cerebral cortex was assessed through Congo red staining and the functional state of neurons was evaluated by Nissl's staining. Immunostaining was performed to assess the accumulation of AGEs in the cerebral cortex. The expressions of mRNA and protein of RAGE, Nuclear factor kappa B (NF-κB) p65 and Nicotinamide adenine dinucleotide phosphate-oxidase (NADPH oxidase) p47phox were detected by real-time polymerase chain reaction (PCR) and western blot. There were significant increases in AGEs deposit, Aß formation, and the expressions of RAGE, NF-κB p65, and NADPH oxidase p47phox, and a decrease Nissl body in AGEs group compared with control group. ALT711 group recovered above change compared with AGEs group. Atorvastatin reduced Aß formation and suppressed AGEs-induced expressions of NF-κB p65 and NADPH oxidase p47phox. Atorvastatin has little effects on AGEs deposit and RAGE expressions. Atorvastatin alleviates AGEs-induced neuronal impairment by alleviating inflammation and oxidative stress via inhibiting NADPH oxidase-NF-κB pathway.

The protective effect of piperine against isoproterenol-induced inflammation in experimental models of myocardial toxicity.

Myocardial infarction (MI) eventually exacerbates inflammatory response due to the release of inflammatory and pro-inflammatory factors. The aim of this study is to explore the protective efficacy of piperine supplementation against the inflammatory response in isoproterenol (ISO)-induced MI. Masson Trichome staining was executed to determine myocardial tissue architecture. Immunohistochemistry was performed for IL-6, TNF-α. RT-PCR studies were performed to ascertain the gene expression of IL-6, TNF-α, iNOS, eNOS, MMP-2, MMP-9, and collagen-III. Western blotting was performed to determine expression of HIF-1α, VEGF, Nrf-2, NF-ƙB, Cox-2, p-38, phospho-p38, ERK-1/2, phospho-ERK-1/2, and collagen-I. HIF-1α, VEGF, and iNOS expression were significantly upregulated with concomitant decline in eNOS expression in the heart myocardial tissue of rats received ISO alone whereas piperine pretreatment prevented these changes in ISO administered rats. Current results revealed ROS-mediated activation of MAPKs, namely, p-p38, p-ERK1/2 in the heart tissue of ISO administered group. Piperine pretreatment significantly prevented these changes in ISO treated group. NF-κB is involved in the modulation of gene expressions responsible for tissue repair. ISO-induced NF-κB-p65 expression was significantly reduced in the group pretreated with piperine and mitigated extent of myocardial inflammation. A significant increase in cardiac fibrosis upon ISO treatment was reported due to the increased hydroxyproline content, MMP-2 & 9 and upregulation of collagen-I protein compared to control group. All these cardiac hypertrophy markers were decreased in 'piperine pretreated ISO administered group' compared to group received ISO injection. Current findings concluded that piperine as a nutritional intervention could prevent inflammation of myocardium in ISO-induced MI.