NF-κB Nuclear Translocation Research Articles

Nuanxinkang protects against ischemia/reperfusion-induced heart failure through regulating IKKβ/IκBα/NF-κB-mediated macrophage polarization

BackgroundHeart failure (HF) is a leading cause of death worldwide. Nuanxinkang (NXK) is an effective Chinese herbal formula used in treating HF, but its underlying potential mechanisms have not been fully elucidated. PurposeTo explore the protective activities of NXK in ischemia/reperfusion (IR)-induced HF through modulating the ratio of proinflammatory (M1) and anti-inflammatory (M2) macrophage populations and leading to the alleviation of inflammation. Materials and methodsIn vivo, mice were subjected to myocardial IR to generate HF mouse models. Mice in the NXK group were treated with NXK for 28 days. Cardiac function was detected by echocardiography. Major lesions on mouse hearts were determined by hematoxylin–eosin (HE) staining, Masson staining, and TUNEL staining. Inflammatory cytokines were determined by enzyme-linked immunosorbent assay (ELISA) and qPCR examination. Flow cytometric analyses and qPCR examination were utilized for monitoring the temporal dynamics of macrophage infiltration following IR. In vitro, two polarized models were established by stimulating RAW264.7 cells with 200 ng/ml lipopolysaccharide (LPS) or 20 ng/ml interleukin-4 (IL-4). The RAW264.7 cells with nuclear factor-κB (NF-κB) overexpression was generated by transient transfection of NF-κB plasmids, and NXK intervention was conducted on this cell model to further clarify the involvement of NF-κB signaling in the NXK-mediated HF process. ResultsIn the present study, NXK was found to significantly contribute the cardiac function and ameliorate cardiac fibrosis and apoptosis after myocardial IR injury in vivo, which may be partially due to a decrease in inflammation. We therefore hypothesized that NXK reduced inflammatory damage by modulating subtypes of macrophages. And the results demonstrated that the percentage of proinflammatory macrophages infiltrated in the post-IR period was reduced with NXK treatment, and thereby blunting the wave of proinflammatory response and shifting the peak of the anti-inflammatory macrophage-mediated wound healing process towards an earlier time point. The further investigation showed that macrophage polarization was mediated by NXK through inhibiting the phosphorylation and the nuclear translocation of NF-κB. Besides, the phosphorylated IKKβ and IκBα, upstream mediators of the NF-κB pathway, also decreased by NXK. Moreover, the overexpression of NF-κB partially reversed the NXK-induced favorable activities; and successfully compensated the suppressive effect on inflammation and the phosphorylation of NF-κB. ConclusionIn conclude, our results demonstrated that NXK induced the cardioprotective effects against IR injury through a regulatory axis of IKKβ/IκBα/NF-κB-mediated macrophage polarization. The information gained from this study provide a possible natural strategy for anti-inflammatory treatment of HF.

Recombinant Prolidase Activates EGFR-Dependent Cell Growth in an Experimental Model of Inflammation in HaCaT Keratinocytes. Implication for Wound Healing.

This study was conducted to investigate the proliferative capacity of recombinant human prolidase (rhPEPD) in a human model of inflammation induced by IL-1β in HaCaT keratinocytes. In this report, we provide evidence that IL-1β stimulates keratinocyte proliferation, and rhPEPD significantly augmented this process through activation of epidermal growth factor receptor (EGFR) and downstream signaling proteins as phosphorylated Akt, ERK1/2, and STAT3, which are implicated in keratinocyte migration, proliferation, and epithelialization during the wound healing process. Inhibition of PEPD-dependent EGFR signaling by gefitinib supported the finding. Moreover, during activation of EGFR in the presence of IL-1β the epithelial-to-mesenchymal transition (EMT) occurred via downregulation of E-cadherin and upregulation of N-cadherin. The phenomenon was accompanied by an increase in the activity of matrix metalloproteinase-9 (MMP-9), suggesting extracellular matrix (ECM) remodeling during the inflammatory process. MMP-9 activation may result from nuclear translocation of NF-κB through IKK-mediated IκBα degradation. Interestingly, some mutated variants of PEPD (rhPEPD-G448R, rhPEPD-231delY, and rhPEPD-E412K) evoked the ability to induce EGFR-dependent HaCaT cell proliferation. To the best of our knowledge, this is the first report on the cross-talk between PEPD and IL-1β in the process of keratinocyte proliferation. The data suggest that both enzymatically active and inactive rhPEPD may activate EGFR-dependent cell growth in an experimental model of inflammation in HaCaT keratinocytes and the knowledge may be useful for further approaches for therapy of wound healing disorders.

Molecular Mechanism of Cinnamomum cassia against Gastric Damage and Identification of Active Compounds.

Cinnamomum cassia is a natural product found in plants that has been used as a folk remedy for inflammation. In this study, we investigated the mechanism underlying the anti-inflammatory and antioxidant properties of C. cassia extract (ECC) in lipopolysaccharide (LPS)-induced murine RAW 264.7 cells, in comparison with 4-hydroxycinnamaldehyde, a C. cassia extract component. ECC and 4-hydroxycinnamaldehyde inhibited the production of nitrite oxide in a dose-dependent manner and did not show any change in cellular toxicity when treated with the same dose as that used in the nitrite assay. Moreover, they attenuated ROS accumulation after lipopolysaccharide (LPS) stimulation. ECC and 4-hydroxycinnamaldehyde decreased the mRNA and protein expression levels of inflammatory mediators (iNOS and COX-2) and cytokines such as TNF and IL-6. We also found that ECC and 4-hydroxycinnamaldehyde mitigated the phosphorylation of ERK, JNK, and transcription factors, such as NF-κB and STAT3, suppressing NF-κB nuclear translocation in LPS-activated macrophages. In addition, administration of ECC in a Sprague Dawley rat model of acute gastric injury caused by indomethacin significantly increased the gastric mucus volume. Analysis of serum and tissue levels of inflammatory mediators revealed a significant decrease in serum PGE2 and myeloperoxidase levels and a reduction in gastric iNOS, COX-2, and p65 protein levels. Collectively, these results suggest that ECC has antioxidant and anti-inflammatory effects and is a potential candidate for curing gastritis.

Phenethyl Isothiocyanate Suppresses the Proinflammatory Cytokines in Human Glioblastoma Cells through the PI3K/Akt/NF-κB Signaling Pathway In Vitro.

Phenethyl isothiocyanate (PEITC), extracted from cruciferous vegetables, showed anticancer activity in many human cancer cells. Our previous studies disclosed the anticancer activity of PEITC in human glioblastoma multiforme (GBM) 8401 cells, including suppressing the cell proliferation, inducing apoptotic cell death, and suppressing cell migration and invasion. Furthermore, PEITC also inhibited the growth of xenograft tumors of human glioblastoma cells. We are the first to investigate PEITC effects on the receptor tyrosine kinase (RTK) signaling pathway and the effects of proinflammatory cytokines on glioblastoma. The cell viability was analyzed by flow cytometric assay. The protein levels and mRNA expressions of cytokines, including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6), were determined by enzyme-linked immunosorbent assay (ELISA) reader and real-time polymerase chain reaction (PCR) analysis, respectively. Furthermore, nuclear factor-kappa B- (NF-κB-) associated proteins were evaluated by western blotting. NF-κB expression and nuclear translocation were confirmed by confocal laser microscopy. NF-κB binding to the DNA was examined by electrophoretic mobility shift assay (EMSA). Our results indicated that PEITC decreased the cell viability and inhibited the protein levels and expressions of IL-1β, IL-6, and TNF-α genes at the transcriptional level in GBM 8401 cells. PEITC inhibited the binding of NF-κB on promoter site of DNA in GBM 8401 cells. PEITC also altered the protein expressions of protein kinase B (Akt), extracellular signal-regulated kinase (ERK), and NF-κB signaling pathways. The inflammatory responses in human glioblastoma cells may be suppressed by PEITC through the phosphoinositide 3-kinase (PI3K)/Akt/NF-κB signaling pathway. Thus, PEITC may have the potential to be an anti-inflammatory agent for human glioblastoma in the future.

Anti-Inflammatory Effects of Ginsenoside Rb3 in LPS-Induced Macrophages Through Direct Inhibition of TLR4 Signaling Pathway.

Panax ginseng has therapeutic effects on various inflammation-related diseases. Ginsenoside Rb3 (GRb3), a natural compound with anti-inflammatory and immunomodulatory properties, is one of the main active panaxadiol extracted from Panax ginseng. We explored whether GRb3 inhibited LPS-mediated inflammation through TLR4/NF-κB/MAPK signaling in macrophages. GRb3 attenuated NO and PGE2 production by attenuating iNOS and COX2 expression. GRb3 also suppressed pro-inflammatory cytokines levels, including IL-1β, IL-6, and TNF-α. Moreover, GRb3 administration significantly suppressed NF-κB (p65) nuclear translocation and the phosphorylation levels of p65, IκBα, JNK, p38, and ERK dose-dependently. Molecular docking demonstrated that GRb3 could dock onto the hydrophobic binding site of TLR4/MD2 complex, with a binding energy of −8.79 kcal/mol. Molecular dynamics (MD) displayed stable TLR4-MD2-GRb3 modeling. GRb3 dose-dependently inhibited LPS binding to cell membranes and blocked TLR4 expression. Surface plasmon resonance imaging (SPRi) revealed that GRb3 had an excellent binding affinity to TLR4/MD2 complex. Notably, resatorvid (TAK242), a selective TLR4 inhibitor, did not increase the repressive influence of GRb3 in RAW264.7 macrophages. Moreover, TLR4 overexpression partially reversed the repressive roles of GRb3 on the NF-κB/MAPK pathway and inflammatory mediators. Collectively, our study strongly indicated that GRb3 attenuated LPS-mediated inflammation through direct inhibition of TLR4 signaling. A novel insight into the underlying mechanism of anti-inflammatory effects of GRb3 in macrophages was confirmed.

Dihydrohomoplantagin and Homoplantaginin, Major Flavonoid Glycosides from Salvia plebeia R. Br. Inhibit oxLDL-Induced Endothelial Cell Injury and Restrict Atherosclerosis via Activating Nrf2 Anti-Oxidation Signal Pathway.

Oxidized low-density lipoprotein (oxLDL)-induced endothelium injury promotes the development of atherosclerosis. It has been reported that homoplantaginin, a flavonoid glycoside from the traditional Chinese medicine Salvia plebeia R. Br., protected vascular endothelial cells by inhibiting inflammation. However, it is undetermined whether homoplantaginin affects atherosclerosis. In this study, we evaluated the effect of homoplantaginin and its derivative dihydrohomoplantagin on oxLDL-induced endothelial cell injury and atherosclerosis in apoE-/- mice. Our results showedthat both dihydrohomoplantagin and homoplantaginin inhibited apoptosis and the increased level of ICAM-1 and VCAM-1 in oxLDL-stimulated HUVECs and the plaque endothelium of apoE-/- mice. Additionally, both of them restricted atherosclerosis development of apoE-/- mice. Mechanistic studies showed that oxLDL-induced the increase in ROS production, phosphorylation of ERK and nuclear translocation of NF-κB in HUVECs was significantly inhibited by the compounds. Meanwhile, these two compounds promoted Nrf2 nuclear translocation and increased the anti-oxidation downstream HO-1 protein level in HUVECs and plaque endothelium. Notably, knockdown of Nrf2 by siRNA abolished the cell protective effects of compounds and antagonized the inhibition effects of them on ROS production and NF-κB activation in oxLDL-stimulated HUVECs. Collectively, dihydrohomoplantagin and homoplantaginin protected VECs by activating Nrf2 and thus inhibited atherosclerosis in apoE-/- mice.

Ebosin Attenuates the Inflammatory Responses Induced by TNF-α through Inhibiting NF-κB and MAPK Pathways in Rat Fibroblast-Like Synoviocytes.

Tumor necrosis factor-α (TNF-α) lies at the apex of signal transduction cascades that results in induced destruction of joints in rheumatoid arthritis. It is therefore of great medicinal interest to modulate the cellular responses to TNF-α. Ebosin, a novel exopolysaccharide derived from Streptomyces sp, has been demonstrated to have remarkable therapeutic actions on collagen-induced arthritis in rats, while it also suppressed the production of IL-1β, TNF-α, and IL-6 at both mRNA and protein levels in cultured fibroblast-like synoviocytes. In order to further understand the potential mechanisms involved in the anti-inflammatory effects of ebosin at molecular level, we investigated the impact of it on the activation of MAPK and NF-κB pathways following TNF-α induced in fibroblast-like synoviocytes (FLS). The results showed that the phosphorylation levels of TNF-α-induced p38, JNK1, JNK2, IKKα, IKKβ, and IκB, as well as NF-κB nuclear translocation, were reduced significantly in FLS cells in response to ebosin. Furthermore, we proved that ebosin decreased the level of NF-κB in the nucleus and blocked the DNA-binding ability of NF-κB using electrophoresis mobility gel shift assay. Besides, low levels of matrix metalloproteinases (MMP-1 and MMP-3) and chemokines (interleukin-8 and RANTES) were found in TNF-α-stimulated fibroblast-like synoviocytes treated with ebosin. These results indicate that ebosin can suppress a range of activities in both MAPK and NF-κB pathways induced by TNF-α in rat fibroblast-like synoviocytes, which provides a rationale for examining the use of ebosin as a potential therapeutic candidate for rheumatic arthritis.

Formononetin attenuates Aβ25-35-induced adhesion molecules in HBMECs via Nrf2 activation

Brain vascular inflammation plays a crucial role in the pathogenesis of Alzheimer’s disease (AD). As a central pathogenic factor in AD, the extracellular buildup of amyloid-β (Aβ) induces brain microvascular endothelial cells activation, impairs endothelial structure and function. Formononetin (FMN) has been reported to protect against Alzheimer’s disease (AD) and attenuates vascular inflammation in atherosclerosis. However, its involvement in regulating vascular inflammation of AD has not been investigated. In the study, we found that FMN significantly attenuates Aβ25–35-induced expression of adhesion molecules, including intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in the human brain microvascular endothelial cells (HBMECs), suggesting that FMN inhibits Aβ25–35-induced brain endothelial cells inflammatory response. Moreover, we observed that FMN attenuates Aβ25–35-induced translocation of NFκB (p65) into the nucleus of HBMECs, and found that FMN treatment induces Nrf2 expression and attenuates Nrf2-Keap1 association in a dose-dependent manner in HBMECs. Furthermore, we demonstrated that Nrf2 silencing significantly attenuates FMN-reduced NFκB (p65) activation and nuclear translocation. Lastly, our results showed that FMN treatment attenuates Aβ25–35-induced adhesion of THP-1 cell to endothelial cell monolayer. Collectively, these findings suggest that FMN attenuates Aβ25–35-induced activation in human brain microvascular endothelial cells, which at least in part was mediated through Nrf2 pathways.

Hsa_circ_0087352 promotes the inflammatory response of macrophages in abdominal aortic aneurysm by adsorbing hsa-miR-149-5p

Abdominal aortic aneurysms (AAA) is a common cardiovascular disease with the risk of rupture. Macrophage depletion can significantly limit the formation of experimental AAA. However, how macrophages in the arterial wall affect the focal distribution and progression of AAA remains unclear. Here, we aimed to evaluate whether circRNAs characterized by stable structure and high tissue specific expression can regulate the inflammatory response of macrophages in AAA. First, we applied bioinformatics to analyze circRNA expression profile in human AAA specimens, and screened out hsa_circ_0087352, which is up-regulated in human AAA specimens and related to inflammatory response of THP-1 macrophages induced by LPS. Besides, hsa_circ_0087352 is stably expressed in THP-1 and mainly distributed in the nucleus. Then, we constructed ceRNA network of circRNA-miRNA-mRNA (IL-6/CCL2/NF-κB) in AAA and found that hsa_circ_0087352 promotes IL-6 transcription and the secretion of inflammatory cytokines by sponging endogenous hsa-miR-149-5p in macrophages. Dual luciferase reporter gene and RNA pull-down suggested hsa_circ_0087352 directly binds to hsa-miR-149-5p. Fluorescence in situ hybridization assay showed the localization of hsa_circ_0087352 and hsa-miR-149-5p in the nucleus of macrophages. Further, western blot demonstrated hsa_circ_0087352 expands the signal transduction of ERK/NF-κB pathway, then IκB phosphorylation promotes NF-κB p65 phosphorylation and nuclear translocation. In addition, hsa_circ_0087352 overexpression in macrophages induces human vascular smooth muscle cells (VSMC) apoptosis in macrophage-VSMC coculture system via the release of proapoptotic cytokines, such as IL-6, TNF-α and IL-1β. Overall, this study provides experimental evidence that hsa_circ_0087352 can be used as a new biomarker and therapeutic target for abdominal aortic aneurysm.

Supramolecular structure features and immunomodulatory effects of exopolysaccharide from Paecilomyces cicadae TJJ1213 in RAW264.7 cells through NF-κB/MAPK signaling pathways

This study investigated the supramolecular structure features and immunomodulatory effects of two exopolysaccharide fractions (EPS1 and EPS2) from Paecilomyces cicada TJJ1213 in vitro. AFM images revealed that EPS1 and EPS2 displayed different morphological features at different concentrations. Congo red and XRD assay further proved that EPS1 and EPS2 mainly exhibited amorphous structure with random coil conformation in solution. Furthermore, the immunomodulatory effect of EPSs was investigated on RAW264.7 cells. Results showed that EPS1 and EPS2 could enhance the phagocytic activity and induce the NO production and could also significantly up-regulate the mRNA expression of iNOS, TNF-α, IL-6, IL-1β, IFN-γ and IL-4. Western blot assay analysis demonstrated that EPSs increased protein expression of TLR4 and the nuclear translocation of NF-κB p50/p65. Additionally, the phosphorylation levels of MAPKs proteins (p38, ERK and JNK) were also remarkably increased. Thus, EPSs could active TLR4-NF-κB/MAPKs signaling pathways to exert the immunomodulatory effect on macrophages.

Dehydrocostus Lactone Suppresses Dextran Sulfate Sodium-Induced Colitis by Targeting the IKKα/β-NF-κB and Keap1-Nrf2 Signalling Pathways.

Dehydrocostus lactone (DCL) is a major sesquiterpene lactone isolated from Aucklandia lappa Decne, a traditional Chinese herbal medicine that used to treat gastrointestinal diseases. This study aimed to examine the therapeutic effects of DCL on dextran sulfate sodium (DSS)-induced colitis with a focus on identifying the molecular mechanisms involved in DCL-mediated anti-inflammatory activity in macrophages. First, oral administration of DCL (5–15 mg/kg) not only ameliorated symptoms of colitis and colonic barrier injury, but also inhibited the expression of proinflammatory cytokines and myeloperoxidase in colon tissues in DSS-challenged mice. Furthermore, DCL also exhibited significant anti-inflammatory activity in LPS/IFNγ-stimulated RAW264.7 macrophages. Importantly, DCL significantly suppressed the phosphorylation and degradation of IκBα and subsequent NF-κB nuclear translocation, and enhanced the nuclear accumulation of Nrf2 in LPS/IFNγ-treated RAW264.7 cells. Mechanistically, DCL could directly interact with IKKα/β and Keap1, thereby leading to the inhibition of NF-κB signalling and the activation of Nrf2 pathway. Furthermore, DCL-mediated actions were abolished by dithiothreitol, suggesting a thiol-mediated covalent linkage between DCL and IKKα/β or Keap1. These findings demonstrated that DCL ameliorates colitis by targeting NF-κB and Nrf2 signalling, suggesting that DCL may be a promising candidate in the clinical treatment of colitis.

Cyclin D1 mediated by the nuclear translocation of nuclear factor kappa B exerts an oncogenic role in lung cancer

ABSTRACT The relevance of cyclin D1 (CCND1) has been implicated in lung cancer progression. Nevertheless, the mechanism by which CCND1 supports lung cancer development is yet to be expounded. Here, we established that CCND1 is overexpressed in clinical lung cancer specimens and various lung cancer cells. Importantly, CCND1 overexpression enhanced lung cancer cell proliferation, invasion and migration, and arrested the cell cycle at the S phase. In vivo, overexpression of CCND1 promoted lung cancer growth and metastasis. The nuclear translocation of nuclear factor kappa B (NF-κB) promoted p65 protein expression and CCND1 transcription. Meanwhile, PI3K/AKT pathway activity was significantly reduced when NF-κB nuclear translocation was decreased. PI3K/AKT pathway activity was significantly elevated upon CCND1 overexpression. Inhibition of PI3K/AKT pathway activity or suppression of NF-κB translocation in cells with high CCND1 expression was found to significantly reduce the activity of lung cancer cells in vitro and in vivo. Our data revealed that NF-κB/CCND1/PI3K/AKT axis could act as a prospective diagnostic biomarker and a therapeutic option for lung cancer.

Purple Corn Silk Extract Attenuates UVB-Induced Inflammation in Human Keratinocyte Cells

UVB is a causative factor for severe skin damage, such as cell aging, death, and inflammation. UVB easily permeates into the epidermis layer of human skin, which is mainly composed of keratinocyte cells. In previous results, we found that purple corn silk (PCS) extract showed the potential to inhibit keratinocyte damages of UVB-treated cells. Thus, in this study, we aimed to evaluate the preventive effects of PCS extract against the inflammation of UVB-induced keratinocyte cells using the HaCaT cell line. HaCaT cells were treated with PCS extract at various concentrations for 1 h, then exposed to 25 mJ/cm2 UVB before subsequent experiments. Fragmented DNA was observed using flow cytometry. The inflammatory response was investigated through NF-κB activity by immunofluorescence staining and related protein expression by Western blotting. The results demonstrated that PCS extract decreased the sub-G1 DNA content. Interestingly, PCS extract attenuated NF-κB activity via suppressed NF-κB nuclear translocation and protein expression. Moreover, PCS extract remarkably decreased c-Jun phosphorylation and decreased proinflammatory cytokines, along with iNOS and COX-2 levels in UVB-treated cells compared to the UVB-control group. This finding exhibited that PCS extract minimized inflammation in keratinocyte cells induced by UVB radiation.

Upregulation of TRPA1 and reduction of NF-κB translocation could be part of the immunomodulatory process during primary tooth inflammation.

Transient receptor potential ankyrin 1 (TRPA1) is expressed on neurons and immune and endothelial cells, and acts as a chemosensor that is activated by bacterial components and endotoxins. This study aimed to better understand how TRPA1 responds to inflammation induced by bacterial-related stimuli during dental caries. The pulp of 40 primary teeth and 46 permanent teeth were categorized into three stages of carious progression: intact dentin, exposed dentin and exposed pulp. We measured the percentages of cells with NF-κB nuclear translocation to verify the severity of inflammation, and then assessed TRPA1 expression by immunofluorescence technique in the pulpal horn, subodontoblastic and mid-coronal regions of the dental pulp samples at various stages of caries. We found NF-κB nuclear translocation gradually reduced during the progression of caries in all three areas of the dental pulp in both primary and permanent teeth. TRPA1 was gradually upregulated in the pulp of primary teeth as caries progressed, but did not significantly vary during caries in the pulp of permanent teeth. This study of TRPA1 expression demonstrates primary and permanent teeth exhibit distinct responses when undergoing carious infection.

Oligomeric α-Synuclein induces skin degeneration in reconstructed human epidermis

Aged and photoaged skin exhibit fine wrinkles that are signs of epidermal inflammation and degeneration. It has been shown that healthy elderly skin expresses amyloidogenic proteins, including α-Synuclein, which are known to oligomerize and trigger inflammation and neurodegeneration. However, little is known about their putative role in skin physiology and sensitivity. To unravel this possible role, we investigated the impact of oligomeric α-Synuclein (Oα-Syn) in 2D and 3D keratinocyte human models. Exogenous Oα-Syn caused degeneration of reconstructed human epidermis (RHE) by diminishing proliferation and thickness of the stratum basale. Oα-Syn also increased NF-kB nuclear translocation in keratinocytes and triggered inflammation in the RHE, by increasing expression of interleukin-1β and tumor necrosis factor-alpha, and the release of tumor necrosis factor-alpha in a time-dependent manner. Dexamethasone and an IL-1β inhibitor partially diminished RHE degeneration caused by Oα-Syn. These findings suggest that Oα-Syn induces epidermal inflammation and decreases keratinocyte proliferation, and therefore might contribute to epidermal degeneration observed in human skin aging.

RIG-I Promotes Cell Viability, Colony Formation, and Glucose Metabolism and Inhibits Cell Apoptosis in Colorectal Cancer by NF-κB Signaling Pathway.

Background Retinoic acid-inducible gene-I (RIG-I) has crucial effects on various cancers, while RIG-I's detailed roles and mechanism in colorectal cancer (CRC) are uncovered. Methods qRT-PCR was used to detect the expression of RIG-I in CRC, adjacent nontumor specimens, and five cell lines. CCK-8, colony formation, and flow cytometry assays were conducted to study CRC cell viabilities. Extracellular acidification rates, lactate analysis, and ATP analysis were conducted to study the cell viabilities and glucose metabolism of CRC cells. Western blot is used to determine the proteins of NF-κBp65 in the nucleus and cytoplasm. Results This study revealed the upregulation of RIG-I in CRC tissues and cells and that high RIG-I expression was correlated with poor prognosis of CRC patients. In addition, silencing RIG-I inhibited cell viability as well as colony formation and promoted cell apoptosis in CRC cells, while RIG-I knockdown suppressed transplanted tumor growth and facilitated apoptosis in nude mice. Moreover, silencing RIG-I inhibited glucose metabolism by decreasing extracellular acidification rate, lactate production, adenosine triphosphate, and content of hypoxia-inducible factor 1α and pyruvate kinase isoform. 2.2-Deoxy-d-glucose, a glycolysis inhibitor, reduced the growth of CRC cells and promoted apoptosis in vitro and in vivo. In addition, RIG-I knockdown decreased NF-κB nuclear translocation. Besides, inhibiting NF-κB effectively eliminated RIG-I overexpression roles in cell viability and glucose metabolism in CRC cells. Conclusion In summary, this study revealed that RIG-I mediated CRC cell proliferation, apoptosis, and glucose metabolism at least partly by NF-κB signaling pathway.

Khasianine ameliorates psoriasis-like skin inflammation and represses TNF-α/NF-κB axis mediated transactivation of IL-17A and IL-33 in keratinocytes

Ethno-pharmacological relevanceKhasianine is recently identified as a bioactive compound from Solanum nigrum L. (SNL) which is a traditional Chinese herb (named LongKui in China) and has been clinically applied for treating psoriasis in China but with limited knowledge about the active ingredients. Aim of the studyThis study tried to explore the bioactivity of Khasianine and showed that Khasianine possessed highly anti-inflammatory bioactivity which rapidly alleviated psoriasis-like mice skin inflammation. Materials and methodsImiquimod induced psoriasis-like mouse model, and human keratinocytes were employed in this study. In vivo, immunohistochemistry and immunofluorescence were performed to evaluate the pathological improvement in psoriatic lesions after Khasianine treatment. In vitro, tumor necrosis factor α (TNF-α) treated HaCaT cells with or without Khasianine, were used to analyze the expression and cellular location of NF-κB p65, the expression of IL-17A and IL-33, and the binding intensity of NF-κB p65 on the promoter of IL-17A and IL-33 to understand the molecular mechanism of Khasianine mediated anti-inflammatory effect. ResultsKhasianine reduced infiltration of CD4+ T helper cells (Th cells) and macrophages in mice psoriatic lesions. Immunohistochemistry analysis revealed that Khasianine reduced TNF-α levels in lesions and suppressed NF-κB p65 activation as well as expression of IL-17A and IL-33 in mice epidermal keratinocytes. Further studies in human keratinocytes demonstrated that Khasianine inhibited TNF-α-induced transcriptional activation (transactivation) of NF-κB p65 such as evicting NF-κB p65 binding from the promoter regions of IL-17A and IL-33 and preventing NF-κB nuclear translocation. ConclusionsOur results suggested that Khasianine is a potent anti-inflammatory compound with the bioactivity of NF-κB inhibition and is a promising candidate for psoriasis topical therapy.

Novel IL1RAP mutation associated with schizophrenia interferes with neuronal growth and related NF-κB signal pathways

Schizophrenia is a complex, severe psychiatric disorder with a high heritability that affects approximately 1% of the world’s population. Numerous schizophrenia-related risk genes have been reported in large-scale studies, but the role of most genetic abnormalities in the pathogenesis of the disease is still obscure. In this study, using whole-exome sequencing, we identified a novel nonsense mutation c.1324C > T in the Interleukin 1 receptor accessory protein (IL1RAP) gene in four affected individuals with schizophrenia of a Chinese family. IL1RAP was found involved in initiating the immune responses and regulating synaptic formation. Considering that schizophrenia has been hypothesized to be neurodevelopment disorder for decades, we further explored the influence of altered expression of IL1RAP gene on neuronal growth, and assessed whether this mutation affects the function of IL1RAP protein in IL-1 signaling pathway. We used lentivirus-mediated shRNA to knockdown the IL1RAP gene expression, which suppressed the axon and dendrites growth of cultured mouse cortical neurons. These defects can be recovered by human IL1RAP wild type construct, but not the R442* mutant construct. Furthermore, this mutant even inhibited neuronal growth and IL-1β-induced JNK phosphorylation when overexpressed in cortical neurons. Although overexpression of this mutant in HePG2 cells did not change IL1RAP protein expression, it partially prohibited the IL-1β-induced nuclear translocation of transcript factor NF-κB, indicating that IL1RAP c.1324C > T is a loss-of-function mutation. Our findings show that IL1RAP plays an important role in early stages of neurodevelopment, and the mutation c.1324C > T may contribute to the pathogenesis of schizophrenia.

PHLDA1 Suppresses TLR4-Triggered Proinflammatory Cytokine Production by Interaction With Tollip.

Pleckstrin homology-like domain, family A, member 1 (PHLDA1) has been reported to be expressed in many mammalian tissues and cells. However, the functions and exact mechanisms of PHLDA1 remain unclear. In this study, we found that PHLDA1 expression was significantly altered in macrophages after exposure to lipopolysaccharide (LPS) in vitro, suggesting that PHLDA1 may be involved in the regulation of TLR4 signaling pathway activated by LPS. PHLDA1 attenuated the production of LPS-stimulated proinflammatory cytokines (TNF-α, IL-6, and IL-1β). Further research showed that the phosphorylation levels of some important signal molecules in TLR4/MyD88-mediated MAPK and NF-κB signaling pathways were reduced by PHLDA1, which in turn impaired the transcription factors NF-κB and AP1 nuclear translocation and their responsive element activities. Furthermore, we found that PHLDA1 repressed LPS-induced proinflammatory cytokine production via binding to Tollip which restrained TLR4 signaling pathway. A mouse model of endotoxemia was established to confirm the above similar results. In brief, our findings demonstrate that PHLDA1 is a negative regulator of LPS-induced proinflammatory cytokine production by Tollip, suggesting that PHLDA1 plays an anti-inflammatory role through inhibiting the TLR4/MyD88 signaling pathway with the help of Tollip. PHLDA1 may be a novel therapeutic target in treating endotoxemia.

Natural bear bile powder suppresses neuroinflammation in lipopolysaccharide-treated mice via regulating TGR5/AKT/NF-κB signaling pathway

Ethnopharmacological relevanceAccording to the Tang Dynasty classics Dietetic Material Medica and the Ming Dynasty classics Compendium of Materia Medica records, bear bile powder (BBP) has been used to treat a variety of diseases, such as febrile seizures, the pathogenesis of which is associated to neuroinflammation. However, the mechanism of BBP on alleviating neuroinflammation remains unclear. Aims of the studyMicroglia can be activated by peripheral lipopolysaccharide (LPS) and play an important role in the pathogenesis of neuroinflammation. The purpose of this study is to investigate the effects and mechanism of BBP in inhibiting LPS-induced microglia inflammation in vitro and in vivo. Materials and methodsThe anti-microglia inflammatory effects and mechanism of BBP were assessed in LPS-treated BV2 microglial cells and in LPS-treated mice. The mRNA expression levels of the inflammatory factor and the protein expressions of cyclooxygenase-2 (COX2), inducible nitric oxide synthase (iNOS), takeda G-protein coupled receptor 5 (TGR5), nuclear factor-κB (NF-κB), inhibitor of NF-κB (IκBɑ), protein kinase B (AKT) in BV2 cells, mouse hippocampus and cortex were detected. The NF-κB transcription activity and NF-κB nuclear translocation were observed. ResultsOur findings showed that BBP reduces branched process retraction and NO in LPS-treated BV2 cells, inhibits the protein expression of ionized calcium binding adaptor molecule 1 in the hippocampus of LPS-treated mice. Moreover, we observed that BBP decreases tumor necrosis factor α, interleukin (IL)-6 and IL-1β mRNA levels, deceases iNOS and COX-2 protein levels, increases TGR5 protein levels, suppresses the phosphorylation of AKT, NF-κB and IκBɑ protein in microglia both in vitro and in vivo. Further, we found that triamterene, the inhibitor of TGR5, abolishes the effects of BBP in LPS- treated BV2 cells. ConclusionBBP inhibits LPS-induced microglia activation, and the mechanism of its action is partly through TGR5/AKT/NF-κB signaling pathway.