NF-κB Nuclear Translocation Research Articles

Abstract 14554: Novel Upstream Targets for Atrial Fibrillation

Introduction: Atrial fibrillation (AF) represents one of the most common arrhythmias seen clinically and is associated with a significant increase in morbidity and mortality, yet, current treatment paradigms have proven largely inadequate. One of the main contributors to the pathophysiology of AF is inflammation. Hence, reduction of inflammation associated atrial remodeling represents a novel therapeutic strategy for the treatment of AF. One of the most biologically important groups of oxylipins (oxygenated lipids) is the eicosanoids that are potent modulators of immune responses and are derived from arachidonic acid, linoleic acid among others, which are metabolized through three pathways including the cytochrome P450 (CYP) pathway. The CYP products are major anti-inflammatory metabolites with several cardioprotective effects. Hypothesis: We hypothesize that lipid metabolites may represent a new therapeutic target for AF Methods: In the cross-sectional study, human atrial appendage specimens and blood from informed and consented patients were obtained in accordance with the approved UC Davis IRB protocol. Metabolomic profiling for over 100 metabolites was assessed from the plasma of patients using LC-MS/MS. We used Functional Connectome, an extension of the singular value decomposition for matrices, to determine how the metabolites were functionally related to each other. The underlying mechanisms of AF were determined using an integrated approach with molecular biology, flow cytometry, and electrophysiology. Results: Metabolomic profiling shows a significant difference in several metabolites from the arachidonic and linoleic acid pathways in the cohort with AF compared to patients in NSR. There was a significant increase in oxidative stress, endoplasmic reticulum stress, inflammatory cytokines, nuclear translocation of NF-κB, and the activation of MAPK and TGF-b pathways from patients with AF compared to NSR cohort. Conclusions: The study provides important insights into the functional relatedness of the metabolites from the CYP pathway in patients with AF and reveals the prospect of arachidonic and linoleic acid metabolites being novel therapeutic targets in the treatment of AF.

New Mechanistic Insight into the Protective Effects of Ganoderma lucidum Polysaccharides Against Palmitic Acid-Induced Cell Damage in Porcine Intestinal Epithelial Cell Line IPEC-J2

Ganoderma lucidum ( G. lucidum) is one of the well-known mushrooms in China, which has G. lucidum polysaccharides (GLP) that have been widely studied for various biological activities, such as antioxidant, antitumor, antiinflammatory, antiviral, antidiabetes, and immunomodulatory activities. A signal transducer and activator of transcription (STAT) signaling pathway is related to cell proliferation and apoptosis. The relationship between STAT and intestinal protection of GLP is still unknown. We studied the inhibitors AG490 in the STAT pathway and its downstream molecules to analyze the unique effects in the protection of GLP against palmitic acid (PA)-induced porcine intestinal epithelial cells (IPEC-J2) injury. Compared to PA treatment, GLP + PA obviously decreased Ca2+ concentration, H2O2 production, NF-E2-related factor 2 (Nrf2) nuclear translocation, STAT1 and STAT2 protein levels, and increased nuclear factor kappa-B (NF-κB) nuclear translocation and p-STAT3/STAT3 ratio in IPEC-J2 cells. After inhibition of STAT3 signaling, p-STAT3/STAT3 ratio, NF-κB nuclear translocation obviously decreased and Nrf2 nuclear translocation significantly increased in the GLP + PA group. The protection of GLP on proliferation and apoptosis of PA-induced IPEC-J2 cells was suppressed by inhibiting STAT3. The STAT3 pathway regulated the enterocyte-protective effects of GLP by modulating the nuclear translocation of Nrf2 and NF-κB. We provide new insights into the mechanism of STAT signaling for the protection of GLP on PA-induced intestinal epithelial cell injury.

6'-Sialylactose abolished lipopolysaccharide-induced inflammation and hyper-permeability in endothelial cells.

Disruption of the endothelial barrier function and reduction in cell migration leads to endothelial dysfunction. One of the most abundant human milk oligosaccharides, 6'-sialylactose (6'-SL), is reported to exert various biological functions related to inflammatory responses. In this study, we evaluated the effects of 6'-SL on lipopolysaccharide (LPS)-induced inflammation caused by endothelial barrier damage. Our results showed that LPS at 500ng/mL strongly not only abolished cell migration but also hyperactivated MAPK and NF-κB pathways. 6'-SL suppressed LPS-induced endothelial inflammation via ERK1/2, p38, and JNK MAPK pathways. 6'-SL supported endothelial junctions by upregulating PECAM-1 expression and mRNA levels of tight junctions, such as ZO-1 and occludin, which were downregulated by LPS stimulation. It significantly inhibited the nuclear translocation of NF-κB, along with the downregulation of inflammatory cytokines, including TNF-α, IL-1β, MCP-1, VCAM-1, and ICAM-1. Furthermore, 6'-SL abolished NF-κB-mediated STAT3 in controlling endothelial migration and hyperpermeability via downregulating STAT3 activation and nuclear translocation. Finally, LPS induced over-expression of VCAM-1 and ZO-1 disassembly in both atheroprone and atheroprotective areas of mouse aorta, which were reversed by 6'-SL treatment. Altogether, our findings suggest that 6'-SL is a potent therapeutic agent for modulating inflammatory responses and endothelial hyperpermeability.

The involvement of peroxisome proliferator-activated receptor gamma (PPARγ) in anti-inflammatory activity of N-stearoylethanolamine

BackgroundN-stearoylethanolamine (NSE) is a bioactive lipid amine with a wide range of biological activities. Anti-inflammatory properties of NSE were previously confirmed on multiple animal models. However, the molecular mechanisms of anti-inflammatory action of NSE remain unclear. In the current study, we examined the involvement of nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ) in the NF-kB –dependent pathway of anti-inflammatory action of NSE using different methodological approaches. MethodsMolecular modeling calculated the possibility of NSE binding PPAR. Ex vivo experiment, using selective agonist of PPARα/γ - LY-171883 and antagonist of PPARγ - GW9662, examined the role of PPARα/γ in the NSE’s effect on nuclear NF-kB translocation in LPS-activated rat peritoneal macrophages. Finally, the NSE’s action on mRNA level of PPARγ-dependent genes was studied in the liver of insulin-resistant rats. ResultsThe results of molecular docking showed that NSE could bind to PPARγ and compete for the binding site with antagonist GW9662 and agonist LY-171883. These data was supported by in vitro study where pre-treatment with NSE prevented further LPS-induced NF-kB translocation into the nuclei of rat peritoneal macrophages. NSE treatment before GW9662 and LPS addition normalized the level of NF-kB translocation and IL-1β content. This finding confirmed a competitive binding of NSE with GW9662 for the ligand-binding domain of PPARγ. Additional in vivo study showed that NSE administration changed the mRNA expression of several PPARγ target genes, including SLC27A1 encoding fatty acid transport protein-1 and IL1RN - interleukin-1 receptor antagonist in insulin resistant rats. ConclusionNSE suppressed nuclear translocation of NF-κB in LPS-stimulated peritoneal macrophages via PPARγ and changed hepatic mRNA expression of PPARγ target genes (SLC27A1, IL1RN) in insulin resistant rats.

Santolina pinnata Viv. Exerts Promising Antitumor Activity against Breast Cancer Cells and Anti-Inflammatory Effects in LPS-Stimulated RAW 264.7 Cells.

Cancer is one of the largest causes of mortality in the world, and due to its incidence, the discovery of novel anticancer drugs is of great importance. Many successful anticancer drugs used in clinical practices are derived from natural products. The genus Santolina is a group of species distributed in the Mediterranean area and used in traditional medicine for their biological properties. The aim of this work was to investigate, for the first time, the multi-target biological potential of Italian Santolina pinnata in relation to their chemical profile, by which an interesting natural source of valuable phytochemicals endowed with anticancer and anti-inflammatory features could be assessed. n-Hexane (EHSP) and methanol (EMSP) extracts were investigated by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) and ultra-high-performance liquid chromatography (UHPLC), respectively. Anti-proliferative activity was analyzed on MCF-7 and MDA-MB-231 breast cancer cells, as well as on non-tumorigenic MCF-10A cells, by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Apoptotic death was assessed by comet assay. Cell motility and invasive features were examined in highly invasive MDA-MB-231 by wound-healing scratches, while, in both breast cancer cell lines, by gel-zymography experiments. The anti-inflammatory potential was analyzed by nitric oxide (NO) production and the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) staining experiments in bacterial lipopolysaccharides (LPS) which stimulated RAW 264.7 cells. EHSP and EMSP extracts exhibited anticancer activity against breast cancer cells, promoting apoptotic death, as well as decreasing cell migration and invasive behaviours. The highest activity (IC50 of 15.91 μg/mL) was detected against MDA-MB-231 cells, a highly invasive breast cancer cell line. Both extracts were also able to promote anti-inflammatory effects (IC50 values ranging from 27.5 to 61.14 μg/mL), as well as to reduce NO levels by inducing inhibitory effects on NF-κB nuclear translocation in LPS-stimulated RAW 264.7 cells. The different biological behaviours found between the extracts could be related to their different chemical compositions. Herein, the multi-target biological potential of S. pinnata in inducing antitumor and anti-inflammatory effects was comprehensively demonstrated. These findings will provide important stepping-stones for further investigations and may lead to the development of highly effective S. pinnata extract-based treatments for breast cancer and inflammatory processes.

Memantine nitrate MN-08 suppresses NLRP3 inflammasome activation to protect against sepsis-induced acute lung injury in mice

Sepsis is a life-threatening organ dysfunction with devastating consequences, prominent among which is lung damage. Memantine, an N-methyl-D-aspartic acid receptor (NMDAR) antagonist, is able to alleviate acute lung injury (ALI). Nitric oxide (NO) suppresses NLRP3 inflammasome activation against lipopolysaccharide (LPS)-induced septic shock. MN-08, a novel nitrate derivative of memantine, possesses both the ability to antagonize NMDAR and release NO. In the present study, we aimed to investigate the protective effects of MN-08 against LPS-induced systemic inflammation and septic lung injury in mice, and to explore the underlying mechanisms of MN-08 in LPS-induced mice and THP-1 macrophages. MN-08 significantly increased the survival rate of septic mice, alleviated LPS-induced sepsis in mice via improving systemic inflammatory response syndrome and immune dysfunction, and attenuated pulmonary injury and inflammatory infiltration. More importantly, the therapeutic benefit of MN-08 for sepsis was greater than that of memantine and dexamethasone. Mechanistically, MN-08 exerted anti-inflammatory activity through inhibiting nuclear translocation of NF-κB, activation of the MAPK signaling pathway and the signaling transduction of STAT3/NF-κB. In addition, MN-08 suppressed NLRP3 inflammasome activation. Taken together, our studies demonstrate that MN-08 may be a promising therapeutic agent for sepsis-induced acute lung injury.

Ginsenoside F2 enhances glucose metabolism by modulating insulin signal transduction in human hepatocarcinoma cells

BackgroundGinsenoside F2 (GF2), a minor component of Panax ginseng, has been reported to possess a wide variety of pharmacological activities. However, its effects on glucose metabolism have not yet been reported. Here, we investigated the underlying signaling pathways involved in its effects on hepatic glucose. MethodsHepG2 cells were used to establish insulin-resistant (IR) model and treated with GF2. Cell viability and glucose uptake-related genes were also examined by real-time PCR and immunoblots. ResultsCell viability assays showed that GF2 up to 50 μM did not affect normal and IR-HepG2 cell viability. GF2 reduced oxidative stress by inhibiting phosphorylation of the mitogen-activated protein kinases (MAPK) signaling components such as c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase 1/2 (ERK1/2), and p38 MAPK, and reducing the nuclear translocation of NF-κB. Furthermore, GF2 activated PI3K/AKT signaling, upregulated the levels of glucose transporter 2 (GLUT-2) and GLUT-4 in IR-HepG2 cells, and promoted glucose absorption. At the same time, GF2 reduced phosphoenolpyruvate carboxykinase and glucose-6-phosphatase expression as well as inhibiting gluconeogenesis. ConclusionOverall, GF2 improved glucose metabolism disorders by reducing cellular oxidative stress in IR-HepG2 cells via MAPK signaling, participating in the PI3K/AKT/GSK-3β signaling pathway, promoting glycogen synthesis, and inhibiting gluconeogenesis.

Mutation of the RelA(p65) Thr505 phosphosite disrupts the DNA replication stress response leading to CHK1 inhibitor resistance.

Mutation of the RelA(p65) Thr505 phosphosite disrupts the DNA replication stress response leading to CHK1 inhibitor resistance.

Pyrrolidine Dithiocarbamate-loaded Electrospun Membranes for Peritendinous Anti-adhesion through Inhibition of the Nuclear Factor-κB Pathway

Peritendinous adhesion is a major cause of limb dysfunction and disability in clinical practice. Numerous studies suggest that activation of nuclear factor-κB (NF-κB) pathway in macrophages could be the pivotal figure in excessive collagen synthesis and thus peritendinous adhesion formation. In this study, we assumed this pathological process could be suppressed by inhibiting NF-κB phosphorylation and nuclear translocation using pyrrolidine dithiocarbamate (PDTC), a specific NF-κB inhibitor with the ability to penetrate cell membranes, in macrophages. Then, we conducted electrospinning process to incorporate PDTC into poly(L-lactic) acid (PLA) electrospinning membranes, that is, the PDTC-PLA membranes. Further, with integral film quality and stable drug release property, the PDTC-PLA membranes were subsequently analyzed in the capability and mechanism of preventing adhesion formation both in vitro and in vivo. Our results showed inhibition of macrophage proliferation as well as NF-κB pathway activation from in vitro assays and outstanding promotion in inhibiting NF-κB p65 phosphorylation and reducing adhesion formation from in vivo assays of PDTC-PLA compared to PLA membranes. In conclusion, our findings suggested that PDTC-PLA as an alternative therapeutic approach alleviated inflammation and peritendinous adhesion formation through NF-κB signaling pathway. Statement of significancePyrrolidine dithiocarbamate (PDTC) can be blended into poly-L-lactic acid (PLA) fibrous membranes by electrospinning process. This incorporation of PDTC into PLA is an effective way to inhibit proinflammatory activation of macrophages and to achieve advanced anti-adhesion outcome after tendon repair.

Bupivacaine inhibits the TLR4- and TLR2-Myd88/NF-κB pathways in human leukocytes.

Local anesthetics have anti-inflammatory effects. Because most previous experiments were performed with supra-therapeutic concentrations, we measured the effects of clinically relevant concentrations of bupivacaine on the Toll like receptor 4 (TLR4)- and TLR2-myeloid differentiation primary response 88 (MyD88)-nuclear factor kappa-light-chain-enhancer of activated B cell (NF-κB) pathways. We measured tumor necrosis factor alpha (TNF-α) and prostaglandin E2 (PGE2) release, p38 mitogen-activated protein kinase (MAP-kinase) phosphorylation and translocation of NF-κB in human peripheral blood mononuclear cells (hPBMCs) and human monocytes challenged with lipopolysaccharide (LPS) or tripalmitoylated lipopeptide Pam3CysSerLys4 (Pam3CSK4) in the presence or absence of bupivacaine. Similarly, we measured the effect of bupivacaine on HEK293 cells expressing the hTLR4 and the hTLR2 genes and challenged with LPS or Pam3CSK4. Finally, molecular docking simulations of R(+)- and S(-)-bupivacaine binding to the TLR4-myeloid differentiation protein 2 (MD-2) complex and to the TLR2/TLR1 heterodimer were performed. In PBMCs, bupivacaine from 0.1 to 100 μM inhibited LPS-induced TNF-α and PGE2 secretion, phosphorylation of p38 and nuclear translocation of NF-κB in monocytes. Bupivacaine similarly inhibited the effects of Pam3CSK4 on TNF-α secretion. Bupivacaine inhibited the effect of LPS on HEK293 cells expressing the human TLR4 receptor and the effect of Pam3CSK4 on HEK293 cells expressing the human TLR2 receptor. Molecular docking showed that bupivacaine binds to the MD-2 co-receptor of TLR4 and to the TLR2 receptor. Contrary to numerous experiments performed with supratherapeutic doses, our results were obtained with concentrations of bupivacaine as low as 0.1μM. We conclude that bupivacaine modulates the inflammatory reactions such as those observed after surgery or trauma, at least partly by inhibiting the TLR4- and TLR2-NF-κB pathways.

TNF-α Impairs Pericyte-Mediated Cerebral Microcirculation via the NF-κB/iNOS Axis after Experimental Traumatic Brain Injury.

Secondary structural and functional abnormalities of the neurovascular unit are important pathological mechanisms following traumatic brain injury (TBI). The neurovascular unit maintains blood-brain barrier and vascular integrity through interactions among glial cells, pericytes and endothelial cells. Trauma-induced neuroinflammation and oxidative stress may act as initiating factors for pathological damage after TBI, which in turn impairs cerebral microcirculatory function. Studies have shown that the tumor necrosis factor α (TNF-α)/nuclear factor-κB (NF-κB) pathway regulates inflammation and oxidative damage, but its role in pericyte-mediated cerebral microcirculation are currently unknown. Herein, we assessed TNF-α/NF-κB signaling and inducible nitric oxide synthase (iNOS), and the effects of the TNF-α inhibitor infliximab after TBI. Whether pericyte damage is dependent on the TNF-α/NF-κB/iNOS axis was also evaluated to explore the mechanisms underlying disturbances in the microcirculation after TBI. Microglia are activated after TBI to promote inflammatory factors and free radical release, and upregulate NF-κB and iNOS expression. After lipopolysaccharide treatment, the activity of TNF-α/NF-κB/iNOS in BV2 cells was also upregulated. Inhibition of TNF-α using infliximab reduced NF-κB phosphorylation and nuclear translocation and downregulated iNOS expression, which attenuated the inflammation and oxidative damage. Meanwhile, inhibition of TNF-α reversed pericyte marker loss, and improved pericyte function and microcirculation perfusion after TBI. In conclusion, our study suggests that microglia released TNF-α after TBI, which promoted neuroinflammation and oxidative stress by activating downstream NF-κB/iNOS signals, and this led to pericyte-mediated disturbance of the cerebral microcirculation.

Tristaenone A: A New Anti-Inflammatory Compound Isolated from the Australian Indigenous Plant Tristaniopsis laurina.

Inspired by ethnopharmacological knowledge, we conducted a bioassay-guided fractionation of the leaves of Tristaniopsis laurina which led to the discovery of a new anti-inflammatory compound, tristaenone A (1). The structure was elucidated by detailed spectroscopic data analysis, and the absolute configuration was established using X-ray crystallography analysis. Tristaenone A (1) suppressed LPS and IFN-γ-induced NO, TNF-α and IL-6 production in RAW 264.7 cells with IC50 values of 37.58 ± 2.45 μM, 80.6 ± 5.82 μM and 125.65 ± 0.34 μM, respectively. It also inhibited NF-κB nuclear translocation by 52.93 ± 14.14% at a concentration of 31.85 μM.

Carvacrol Ameliorates DSS-Induced Intestinal Inflammation in Rats by Suppressing the TLR4/NF-κB Pathway

The present study aimed to explore the therapeutic effects of carvacrol on the ulcerative colitis (UC) inflammation model induced by DSS (dextran sulfate sodium) in rats. Forty rats were randomly divided into the blank, model, low-, middle-, and high-dose carvacrol groups. The rats drank 4.5% DSS for 5 consecutive days to induce the UC model, except for the blank group. Different concentrations of either carvacrol or purified water were supplied by intragastric administration for 7 consecutive days to assess the protective effect of carvacrol on inflammation in UC rats involving the TLR4/NF-κB signaling pathway. In vivo administration of carvacrol was found to ameliorate the symptoms of colitis in rats. Carvacrol significantly improved the pathological symptoms of colitis in the model group and reduced the DAI and pathological scores of rats. At the same time, carvacrol can reduce the expression of IL-6 and TNF-α induced by DSS, inhibit the activation of TLR4, hinder the nuclear translocation of NF-κB (P65), reduce the cleavage of the apoptosis-related protein cleaved-caspase 3, and protect colon cell apoptosis. The results of in vitro experiments showed that carvacrol has a significant inhibitory effect on cell apoptosis caused by the activation of TLR4 induced by LPS and pcDNA3.1tlr4. Research results revealed that carvacrol has a certain protective effect against inflammation-induced injury in ulcerative colitis rats via inhibiting the activation of the TLR4/NF-κB signaling pathway.

Alpha-mangostin inhibits viral replication and suppresses nuclear factor kappa B (NF-κB)-mediated inflammation in dengue virus infection

Severe dengue virus (DENV) infection results from viral replication and dysregulated host immune response, which trigger massive cytokine production/cytokine storm. The result is severe vascular leakage, hemorrhagic diathesis, and organ dysfunction. Subsequent to previously proposing that an ideal drug for treatment of DENV infection should efficiently inhibit both virus production and cytokine storm, we discovered that α-mangostin (α-MG) from the pericarp of the mangosteen fruit could inhibit both DENV infection and cytokine/chemokine production. In this study, we investigated the molecular mechanisms underlying the antiviral and anti-inflammatory effects of α-MG. Time-of-drug-addition and time-of-drug-elimination studies suggested that α-MG inhibits the replication step of the DENV life cycle. α-MG inhibited polymerization activity of RNA-dependent RNA polymerase (RdRp) with IC50 values of 16.50 μM and significantly reduced viral RNA and protein syntheses, and virion production. Antiviral and cytokine/chemokine gene expression profiles of α-MG-treated DENV-2-infected cells were investigated by polymerase chain reaction array. α-MG suppressed the expression of 37 antiviral and cytokine/chemokine genes that relate to the NF-κB signaling pathway. Immunofluorescence and immunoblot analyses revealed that α-MG inhibits NF-κB nuclear translocation in DENV-2-infected cells in association with reduced RANTES, IP-10, TNF-α, and IL-6 production. These results suggest α-MG as a potential treatment for DENV infection.

Glucogallin Attenuates the LPS-Induced Signaling in Macrophages and Protects Mice against Sepsis.

The anti-oxidant and anti-inflammatory effect of beta-glucogallin (BGG), a plant-derived natural product, was evaluated in both in vitro and in vivo studies. For the in vitro study, the ability of BGG pre-treatment to quench LPS-induced effects compared to LPS alone in macrophages was investigated. It was found that BGG pre-treatment showed a significant decrease in ROS, NO, superoxide, and pro-inflammatory cytokines (TNF-alpha, IL-4, IL-17, IL-1β, and IL-6) and increased reduced glutathione coupled with the restoration of mitochondrial membrane potential. Gene profiling and further validation by qPCR showed that BGG pre-treatment downregulated the LPS-induced expression of c-Fos, Fas, MMP-9, iNOS, COX-2, MyD88, TRIF, TRAF6, TRAM, c-JUN, and NF-κB. We observed that BGG pre-treatment reduced nuclear translocation of LPS-activated NF-κB and thus reduced the subsequent expressions of NLRP3 and IL-1β, indicating the ability of BGG to inhibit inflammasome formation. Molecular docking studies showed that BGG could bind at the active site of TLR4. Finally, in the LPS-driven sepsis mouse model, we showed that pre-treatment with BGG sustained toxic shock, as evident from their 100% survival. Our study clearly showed the therapeutic potential of BGG in toxic shock syndrome.

Hsa-microRNA-27b-3p inhibits hepatocellular carcinoma progression by inactivating transforming growth factor-activated kinase-binding protein 3/nuclear factor kappa B signalling

BackgroundMicroRNAs (miRNAs) play crucial roles in the development of hepatocellular carcinoma (HCC). Hsa-microRNA-27b-3p (hsa-miR-27b) is involved in the formation and progression of various cancers, but its role and clinical value in HCC remain unclear.MethodsThe expression of hsa-miR-27b in HCC was examined by quantitative real-time PCR (qRT-PCR) and in situ hybridization (ISH) assays of clinical samples. Cell Counting Kit-8 assays (CCK-8), 5-ethynyl-2′-deoxyuridine (EdU) incorporation assays, Transwell assays, filamentous actin (F-actin) staining and western blot analyses were used to determine the effects of hsa-miR-27b on HCC cells in vitro. Subcutaneous xenograft and lung metastatic animal experiments were conducted to verify the role of hsa-miR-27b in HCC in vivo. In silico prediction, qRT-PCR, western blot, anti-Argonaute 2 (AGO2) RNA immunoprecipitation (RIP) and dual luciferase reporter assays were applied to identify the target genes of hsa-miR-27b. To detect the impacts of hsa-miR-27b on nuclear factor kappa B (NF-кB) signalling cascades mediated by transforming growth factor-activated kinase-binding protein 3 (TAB3), we performed qRT-PCR, western blot assays, immunofluorescence staining, immunohistochemistry (IHC) and dual-luciferase reporter assays. Recombinant oncolytic adenovirus (OncoAd) overexpressing hsa-miR-27b was constructed to detect their therapeutic value in HCC.ResultsThe expression of hsa-miR-27b was lower in HCC than in adjacent non-tumourous tissues (ANTs), and the reduced expression of hsa-miR-27b was associated with worse outcomes in patients with HCC. Hsa-miR-27b significantly inhibited the proliferation, migration, invasion, subcutaneous tumour growth and lung metastasis of HCC cells. The suppression of hsa-miR-27b promoted the nuclear translocation of NF-κB by upregulating TAB3 expression. TAB3 was highly expressed in HCC compared with ANTs and was negatively correlated with the expression of hsa-miR-27b. The impaired cell proliferation, migration and invasion by hsa-miR-27b overexpression were recovered by ectopic expression of TAB3. Recombinant OncoAd with overexpression of hsa-miR-27b induced anti-tumour activity compared with that induced by negative control (NC) OncoAd in vivo and in vitro.ConclusionsBy targeting TAB3, hsa-miR-27b acted as a tumour suppressor by inactivating the NF-кB pathway in HCC in vitro and in vivo, indicating its therapeutic value against HCC.Graphical

Sonidegib Suppresses Production of Inflammatory Mediators and Cell Migration in BV2 Microglial Cells and Mice Treated with Lipopolysaccharide via JNK and NF-κB Inhibition.

Our structure-based virtual screening of the FDA-approved drug library has revealed that sonidegib, a smoothened antagonist clinically used to treat basal cell carcinoma, is a potential c-Jun N-terminal kinase 3 (JNK3) inhibitor. This study investigated the binding of sonidegib to JNK3 via 19F NMR and its inhibitory effect on JNK phosphorylation in BV2 cells. Pharmacological properties of sonidegib to exert anti-inflammatory and anti-migratory effects were also characterized. We found that sonidegib bound to the ATP binding site of JNK3 and inhibited JNK phosphorylation in BV2 cells, confirming our virtual screening results. Sonidegib also inhibited the phosphorylation of MKK4 and c-Jun, the upstream and downstream signals of JNK, respectively. It reduced the lipopolysaccharide (LPS)-induced production of pro-inflammatory factors, including interleukin-1β (IL-1β), IL-6, tumor necrosis factor-α (TNF-α), and nitric oxide (NO), and the expression of inducible NO synthase and cyclooxygenase-2. The LPS-induced cell migration was suppressed by sonidegib. Sonidegib inhibited the LPS-induced IκBα phosphorylation, thereby blocking NF-κB nuclear translocation. Consistent with these findings, orally administered sonidegib attenuated IL-6 and TNF-α levels in the brains of LPS-treated mice. Collectively, our results indicate that sonidegib suppresses inflammation and cell migration in LPS-treated BV2 cells and mice by inhibiting JNK and NF-κB signaling. Therefore, sonidegib may be implicated for drug repurposing to alleviate neuroinflammation associated with microglial activation.

A Peptide HEPFYGNEGALR from Apostichopus japonicus Alleviates Acute Alcoholic Liver Injury by Enhancing Antioxidant Response in Male C57BL/6J Mice.

Liver-related disease caused by alcohol is a frequent disorder of the hepatic tract. Heavy consumption of alcohol in a short period causes oxidative damage to the liver. Sea cucumber is abundant in nutrients and its various extracts have been studied for antioxidant properties. One peptide was isolated and identified from Apostichopus japonicus in our recent study. We investigated the benefits of the peptide in a model of acute ethanol-induced male C57BL/6J mice. Dietary intake of the peptide could attenuate hepatomegaly, hepatitis and the accumulation of lipid droplets, and increase antioxidant enzyme activities in mice with acute alcoholic liver injury. The results indicated that a 20 mg/kg peptide supplement could activate the Nrf2/HO-1 pathway and block the nuclear translocation of NF-κB to alleviate oxidative stress and inflammation. In addition, the preventive effects of peptide supplementation may be related to autophagy. This study suggests that dietary supplementation with a sea cucumber-derived peptide is one of the potential candidates to alleviate acute alcoholic liver injury.

Dexmedetomidine post-conditioning protects blood-brain barrier integrity by modulating microglia/macrophage polarization via inhibiting NF-κB signaling pathway in intracerebral hemorrhage.

Intracerebral hemorrhage (ICH) is one of the most devastating forms of stroke. Dexmedetomidine (DEX) has shown certain neuroprotective roles in ICH. Nevertheless, the details concerning the underlying molecular mechanism of DEX’s protective effects still need further elucidation. Herein, a model of ICH was established. The rats were randomly divided into the sham group, the ICH group, and the ICH + DEX group. Neurological outcomes, neuronal injury, and apoptosis were evaluated. Brain water content, Evans blue extravasation, and the expression of tight junction-associated proteins were also detected to assess the blood-brain barrier (BBB) integrity. Subsequently, the microglia/macrophage polarization state and inflammatory cytokine levels were observed. To further explore the underlying mechanism, NF-κB signaling pathway-associated proteins were detected. The results showed that DEX exerted neuroprotective effects against ICH-induced neurological deficits. DEX significantly increased the numbers of the surviving neurons and ameliorated neuronal cell loss and apoptosis in ICH. The rats that received the DEX displayed a lower level of brain water content and EB extravasation, moreover, ZO-1, occludin, and claudin-5 were markedly increased by DEX. Additionally, DEX facilitated M2 microglia/macrophage polarization, the M1-associated markers were reduced by DEX, while the M2-associated identification significantly increased. We found that DEX dramatically diminished pro-inflammatory cytokines expression, simultaneously promoting anti-inflammatory cytokines expression. DEX inhibited nuclear translocation of NF-κB in ICH rats. Our data suggest that DEX post-conditioning protects BBB integrity by modulating microglia/macrophage polarization via inhibiting the NF-κB signaling pathway in ICH.

Salidroside Regulates Mitochondrial Homeostasis After Polarization of RAW264.7 Macrophages.

Salidroside has anti-inflammatory and antiatherosclerotic effects, and mitochondrial homeostasis imbalance is closely related to cardiovascular disease. The aim of this study was to investigate the effect of salidroside on mitochondrial homeostasis after macrophage polarization and elucidate its possible mechanism against atherosclerosis. RAW264.7 cells were stimulated with 1 μg·mL -1 Lipopolysaccharide and 50 ng·mL -1 IFN-γ establish M1 polarization and were also pretreated with 400 μM salidroside. The relative expression of proinflammatory genes was detected by RT-PCR whereas that of mitochondrial homeostasis-related proteins and nuclear factor kappa-B (NF-κB) was detected by WB. Levels of intracellular reactive oxygen species (ROS), mitochondrial membrane potential, and mass were measured by chemifluorescence whereas that of NF-κB nuclear translocation was detected by immunofluorescence. Compared with the Mφ group, the M1 group demonstrated increased mRNA expression of interleukin-1β , inductible nitric oxide synthase (iNOS), and tumor necrosis factor-α ; increased protein expression of iNOS, NOD-like receptor protein 3, putative kinase 1 , and NF-κB p65 but decreased protein expression of MFN2, Tom20, and PGC-1α; decreased mitochondrial membrane potential and mass; and increased ROS levels and NF-κB p65 nuclear translocation. Salidroside intervention decreased mRNA expression of interleukin-1β and tumor necrosis factor-α compared with the M1 group but did not affect that of iNOS. Furthermore, salidroside intervention prevented the changes in protein expression, mitochondrial membrane potential and mass, ROS levels, and NF-κB p65 nuclear translocation observed in the M1 group. In summary, salidroside ultimately inhibits M1 macrophage polarization and maintains mitochondrial homeostasis after macrophage polarization by increasing mitochondrial membrane potential, decreasing ROS levels, inhibiting NF-κB activation, and in turn regulating the expression of proinflammatory factors and mitochondrial homeostasis-associated proteins.