Nuclear Factor kappa-B Kinase Subunit Research Articles

Salvimulticanol from Salvia multicaulis suppresses LPS-induced inflammation in RAW264.7 macrophages: in vitro and in silico studies.

Sustained inflammatory responses can badly affect several vital organs and lead to chronic inflammation-related disorders, such as atherosclerosis, pneumonia, rheumatoid arthritis, obesity, diabetes, Alzheimer's disease, and cancers. Salvia multicaulis is one of the widely distributed plants that contains several biologically active phytochemicals and diterpenoids with anti-inflammatory effects. Therefore, finding alternative and safer natural plant-extracted compounds with good curative anti-inflammatory efficiencies is an urgent need for the clinical treatment of inflammation-related diseases. In the current study, S. multicaulis Vahl was used to extract and isolate two compounds identified as salvimulticanol and candesalvone B methyl ester to examine their effects against inflammation in murine macrophage RAW264.7 cells that were induced by lipopolysaccharide (LPS). Accordingly, after culturing RAW264.7 cells and induction of inflammation by LPS (100ng/ml), cells were exposed to different concentrations (9, 18, 37.5, 75, and 150µM) of each compound. Then, Griess assay for detection of nitric oxide (NO) levels and western blotting for the determination of inducible nitric oxide synthase (iNOS) expression were performed. Molecular docking and molecular dynamics (MD) simulation studies were employed to investigate the anti-inflammatory mechanism. Our obtained results validated that the level of NO was significantly decreased in the macrophage cell suspensions as a response to salvimulticanol treatment in a dose-dependent manner (IC50: 25.1 ± 1.2µM) as compared to the methyl ester of candesalvone B which exerted a weaker inhibition (IC50: 69.2 ± 3.0µM). This decline in NO percentage was comparable with a down-regulation of iNOS expression by western blotting. Salvimulticanol strongly interacted with both the Toll-like receptor 4 (TLR4)/myeloid differentiation factor 2 (MD-2) complex and the inhibitor of nuclear factor kappa-B (NF-κB) kinase subunit beta (IKKβ) to disrupt their inflammatory activation due to the significant hydrogen bonds and effective interactions with amino acid residues present in the target proteins' active sites. S.multicaulis is a rich natural source of the aromatic abietane diterpenoid, salvimulticanol, which exerted a strong anti-inflammatory effect through targeting iNOS and diminishing NO production in LPS-induced RAW264.7 cells in a mechanism that is dependent on the inhibition of TLR4-MD-2 and IKKβ as activators of the classical NF-κB-mediated inflammatory pathway.

Dissecting Combinational Mechanisms of Herbal Formula from a Transcriptome-based Multi-scale Network Pharmacology Model

Abstract Objective: Illumination of the integrative effects of herbs in a formula is a bottleneck that limits the development of traditional Chinese medicine (TCM). In the present study, we developed a transcriptome-based multi-scale network pharmacology model to explore the combined effects of different herbs. Materials and Methods: First, we curated gene signatures at different biological scales, from the molecular to higher tissue levels, including tissues, cells, pathological processes, biological processes, pathways, and targets. Second, using the Xiexin Tang (XXT) formula as an example, we collected transcriptomic data in response to the treatment of XXT or its three compositive herbs on Michigan cancer foundation7 cells. Third, we linked each herbal drug to different biological scales by calculating the correlation scores between herb-induced gene expression profiles and gene signatures. Finally, the combined mechanisms of the three constituent herbs in XXT were deciphered by comparing their multi-scale effects with those of the formula. Results: The results showed that although XXT or single herbs regulated a large number of signatures on each biological scale, the phenotypic effects of these herbal drugs are concentrated onto the “Blood” tissue, types of hemocytes, and hemorrhagic injury-related pathological processes. At the molecular level, these herbs consistently regulate processes such as the cell cycle and blood coagulation-related pathways, as well as protein targets related to the immunoinflammatory response and blood coagulation, such as proteinase-activated receptor 2, integrin beta-3, inhibitor of nuclear factor kappa-B kinase subunit beta, and coagulation factor XII. The analysis of the combinational modes demonstrated that different herbs can cooperate by acting on the same objects and/or regulating different objects in related functions, and cooperative behaviors change at different biological scales. Conclusions: Our model can dissect the combined effects of herbal formulae from a multi-scale perspective and should be beneficial for the development and exploitation of TCM.

N6-isopentenyladenosine inhibits aerobic glycolysis in glioblastoma cells by targeting PKM2 expression and activity.

Glioblastoma (GBM) is a primary tumor in the central nervous system with poor prognosis. It exhibits elevated glucose uptake and lactate production. This metabolic state of aerobic glycolysis is known as the Warburg effect. N6-isopentenyladenosine (iPA), a natural cytokine modified with an isopentenyl moiety derived from the mevalonate pathway, has well-established anti-tumor activity. It inhibits cell proliferation in glioma cells, inducing cell death by apoptosis and/or necroptosis. In the present study, we found that iPA inhibits aerobic glycolysis in unmodified U87MG cells and in the same cell line engineered to over-express wild-type epidermal growth factor receptor (EGFR) or EGFR variant III (vIII), as well as in a primary GBM4 patient-derived cell line. The detection of glycolysis showed that iPA treatment suppressed ATP and lactate production. We also evaluated the response of iPA treatment in normal human astrocyte primary cells, healthy counterpart cells of the brain. Aerobic glycolysis in treated normal human astrocyte cells did not show significant changes compared to GBM cells. To determine the mechanism of iPA action on aerobic glycolysis, we investigated the expression of certain enzymes involved in this metabolic pathway. We observed that iPA reduced the expression of pyruvate kinase M2 (PKM2), which plays a key role in the regulation of aerobic glycolysis, promoting tumor cell proliferation. The reduction of PKM2 expression is a result of the inhibition of the inhibitor of nuclear factor kappa-B kinase subunit, beta/nuclear factor-kappa B pathway upon iPA treatment. In conclusion, these experimental results show that iPA may inhibit aerobic glycolysis of GBM in stabilized cell lines and primary GBM cells by targeting the expression and activity of PKM2.

IKBKE promotes the ZEB2-mediated EMT process by phosphorylating HMGA1a in glioblastoma

IKBKE (Inhibitor of Nuclear Factor Kappa-B Kinase Subunit Epsilon) is an important oncogenic protein in a variety of tumors, which can promote tumor growth, proliferation, invasion and drug resistance, and plays a critical regulatory role in the occurrence and progression of malignant tumors. HMGA1a (High Mobility Group AT-hook 1a) functions as a cofactor for proper transcriptional regulation and is highly expressed in multiple types of tumors. ZEB2 (Zinc finger E-box Binding homeobox 2) exerts active functions in epithelial mesenchymal transformation (EMT). In our current study, we confirmed that IKBKE can increase the proliferation, invasion and migration of glioblastoma cells. We then found that IKBKE can phosphorylate HMGA1a at Ser 36 and/or Ser 44 sites and inhibit the degradation process of HMGA1a, and regulate the nuclear translocation of HMGA1a. Crucially, we observed that HMGA1a can regulate ZEB2 gene expression by interacting with ZEB2 promoter region. Hence, HMGA1a was found to promote the ZEB2-related metastasis. Consequently, we demonstrated that IKBKE can exert its oncogenic functions via the IKBKE/HMGA1a/ZEB2 signalling axis, and IKBKE may be a prominent biomarker for the treatment of glioblastoma in the future.

Lactate-lactylation-HSPA6 axis promotes PRRSV replication by impairing IFN-β production.

Lactate, traditionally considered a metabolic by-product, has recently been identified as a substrate for the induction of lactylation, a newly identified epigenetic modification that plays an important role in the regulation of host gene expression. Our previous study showed that lactate levels were significantly elevated in cells infected with the porcine reproductive and respiratory syndrome virus (PRRSV), an Arterivirus that has devastated the swine industry worldwide for over 30 years. However, the role of elevated lactate in PRRSV infections remains unknown. In this study, we found that lactate was required for optimal PRRSV proliferation, and PRRSV infection increased cellular lactylation in a dose-dependent manner. Using the Cleavage Under Targets and Tagmentation (CUT&Tag) combined with RNA sequencing (RNA-seq) to screen the downstream genes regulated by lactylation in PRRSV-infected cells, we found that PRRSV-induced lactylation activated the expression of heat shock 70 kDa protein 6 (HSPA6). Follow-up experiments showed that HSPA6 is important for PRRSV proliferation by negatively modulating interferon (IFN)-β induction. Mechanistically, HSPA6 impeded the interaction between TNF-receptor-associated factor 3 (TRAF3) and inhibitor of nuclear factor kappa-B kinase subunit epsilon (IKKε), thereby hindering the production of IFN-β. Taken together, these results indicate that the activated lactate-lactylation-HSPA6 axis promotes viral growth by impairing IFN-β induction, providing new therapeutic targets for the prevention and control of PRRSV infection. The results presented here also link lactylation to the virus life cycle, improving our understanding of epigenetic regulation in viral infection.IMPORTANCEAs a newly identified epigenetic modification, lactate-induced lactylation has received attentions because it plays important roles in gene expression and contributes to tumorigenesis and the innate immune response. Previous studies showed that many viruses upregulate cellular lactate levels; however, whether virus-elevated lactate induces lactylation and the subsequent biological significance of the modification to viral infection have not been reported. In this study, we demonstrated that porcine reproductive and respiratory syndrome virus (PRRSV) infection induced cellular lactylation, which, in turn, upregulated the expression of HSPA6, an IFN-negative regulator. We also dissected the mechanism by which HSPA6 negatively regulates IFN-β production. To our knowledge, this is the first report to study virus-induced lactylation and establish the relationship between lactylation and virus infection.

Mutant p53 Drives the Development of Myelodysplastic Syndromes Via Dysregulating Pre-mRNA Splicing in Hematopoietic Stem and Progenitor Cells

Despite the clinical importance of TP53 mutations, to date there have been no studies on the functional impact of TP53 mutations on the initiation and progression of MDS. We found that heterozygous p53 mutant mice ( p53 R248W/+) show extended survival and died within 15-20 months after birth. p53 mutant mice had hypercellular bone marrow and displayed cytopenia, thrombocytopenia, and anemia. Approximately 60% of p53 R248W/+ mice developed MDS based upon pathological analysis of bone marrow (BM) and peripheral blood smears, while the remaining p53 R248W/+ mice developed lymphoma and sarcoma. We further showed that mutant p53-driven MDS are transplantable. Thus, we have generated a mouse model of MDS that has some features of human MDS with TP53 mutations. To understand how mutant p53 drives MDS development, we performed RNA-seq studies to compare gene expression in hematopoietic stem and progenitor cells (HSPCs). GSEA analysis revealed that spliceosome genes were significantly downregulated in middle-aged p53 R248W/+ HSPCs compared to age-matched p53 +/+ HSPCs. We confirmed that the expression of splicing factors, such as Prpf3 and Prpf4, was significantly downregulated in middle-aged p53 R248W/+ HSPCs. We conducted alternative splicing analysis utilizing MISO (Mixture of Isoforms) algorithm to identify differences in exon inclusion ratio between WT and mutant HSPCs. We observed differential splicing of all classes of alternative splicing events, including Cassette Exons (CE), Competing 5′ and 3′ splice sites (A5' or A3'SS), Mutually Exclusive Exons (MXE), and Retained Introns (RI) in p53 R248W/+ cells compared to that of the WT HSPCs. Notably, we found that murine p53 mutant HSPCs exhibited aberrant splicing of key regulators of NFκBactivation, such as USP15. USP15 overexpression promotes NFκB expression through inhibiting its ubiquitination, whereas NFκB promotes USP15 expression. p53 mutant HSPCs tend to skip exon 7 and express high levels of short isoform of Usp15. The short isoform of Usp15, but not the long isoform of Usp15, activates NFκB in HSPCs, manifested by increased phosphorylation of p65. To determine the impact of mutant p53 on pre-mRNA splicing in human HSPCs, we ectopically expressed GFP or p53 R248W in human cord blood CD34 + cells and performed RNA-seq in transduced cells (GFP +). Human HSPCs expressing mutant p53 displayed alterations in pre-mRNA splicing compared to HSPCs expressing GFP. We found that human HSPCs expressing mutant p53 display aberrant splicing in key regulator of NFκB such as IKBKE (Inhibitor of nuclear factor kappa-B kinase subunit epsilon). Human HSPCs expressing mutant p53 tend to include exon 7 and express high levels of long isoform of IKBKE. The long isoform of IKBKE, but not the short isoform of IKBKE, activates NFκB in human HSPCs. Many proinflammatory cytokine genes are NFκB targets and we observed increased levels of pro-inflammatory cytokines, including IL-1β and IL-6, in the BM of middle-aged p53 mutant mice. Notably, we observed increased levels of IL-1β and IL-6 in the BM of MDS or AML patients with TP53 mutations. TP53 mutations co-occur with splicing factor mutations in MDS. To determine whether TP53 and SRSF2 mutations cooperate in pathogenesis of MDS, we have generated p53 R248W/+SRSF2 P95H/+ mice. We found that the survival of recipient mice repopulated with p53 R248W/+SRSF2 P95H/+ BM cells was significantly decreased compared to recipients repopulated with WT, p53 R248W/+, or SRSF2 P95H/+ BM cells. All recipient mice repopulated with p53 R248W/+SRSF2 P95H/+ BM cells died within 6 months and developed MDS or leukemia. Thus, we demonstrate that TP53 and SRSF2 mutations cooperate in MDS or leukemia development. In summary, we discovered that mutant p53 dysregulates pre-mRNA splicing in key regulators of inflammatory response during aging, thereby generating a chronic inflammatory microenvironment to drive MDS pathogenesis.

Dihydrokaempferol attenuates LPS-induced inflammation and apoptosis in WI-38 cells.

Globally, pneumonia has been associated as a primary cause of mortality in children aged less than 5 years. Dihydrokaempferol (DHK) has been proposed for being correlated with the process of various diseases. Nevertheless, whether DHK has a role in the progression of infantile pneumonia remains unclear. This study aimed at exploring whether DHK was involved in the progression of infantile pneumonia. Human fibroblast cells WI-38 were treated with lipopolysaccharide (LPS). The viability of WI-38 cells was measured via Cell counting kit-8. Reverse transcription-quantitative polymerase chain reaction was used to evaluate the levels of interleukin (IL)-1β, IL-6, and tumor necrosis factor-α (TNF-α). Western blot analysis revealed the protein levels of IL-1β, IL-6, TNF-α, Bax, and cleaved-caspase 3. Flow cytometry was applied for exploring the apoptosis of WI-38 cells. The concentrations of IL-1β, IL-6, and TNF-α were assessed via enzyme-linked-immunosorbent serologic assay. DHK modulated the viability of WI-38 cells in infantile pneumonia. Furthermore, we identified that DHK treatment inversely changed LPS induction-mediated elevation on the levels of inflammation biomarkers. Besides, DHK counteracted LPS-induced production of reactive oxygen species (ROS) in WI-38 cells. DHK also decreased LPS-induced elevation of WI-38 cells apoptosis and mediated the levels of apoptosis-associated indexes. Moreover, modulating sirtuin-1 (SIRT1) protein level was lowered by the induction of LPS, and was reversed by DHK treatment. In addition, DHK counteracted LPS induction-mediated elevation of p-p65 and phosphorylated inhibitor of nuclear factor kappa-B kinase subunit alpha (p-IκBα) protein levels. DHK alleviated LPS-induced WI-38 cells inflammation injury in infantile pneumonia through SIRT1/NF-κB pathway. The results shed light on the implications of DHK on the prevention and treatment of infantile pneumonia.

Effect of fed dietary yeast (Rhodotorula paludigena CM33) on shrimp growth, gene expression, intestinal microbial, disease resistance, and meat composition of Litopenaeus vannamei

Yeast is a health-promoting and bio-therapeutic probiotic that is commonly used in aquaculture. Rhodotorula paludigena CM33 can accumulate amounts of intracellular carotenoids and lipid, which are regarded as nutritionally beneficial compounds in various aspects. The aim of this study was to evaluate the impact of different levels of R. paludigena CM33 (RD) incorporated in a dietary composition at 0% (control), 1% (1% RD), 2% (2% RD), and 5% (5% RD) on the growth of shrimp (Litopenaeus vannamei), their immune-related gene expression, intestinal health, resistance to Vibrio parahaemolyticus (VPAHPND) infection, and meat composition. The results showed significant improvements in the specific growth rate, weight gain, and survival of shrimp fed with 1% RD, 2% RD, and 5% RD, which were higher than the control group after 4 weeks of administration. The administration of 5% RD group resulted in a decrease in cumulative mortality upon VPAHPND challenge when compared to the control group. Furthermore, the expression levels of immune-responsive genes, including proPO system (prophenoloxidase-2: PO2), antioxidant enzyme (superoxide dismutase: SOD, glutathione peroxidase: GPX, and catalase: CAT), JAK/STAT pathway (signal transducer and activator of transcription: STAT, gamma interferon inducible lysosomal thiol reductase: GILT), IMD pathway (inhibitor of nuclear factor kappa-B kinase subunit beta and epsilon: IKKb and IKKe), and Toll pathway (Lysozyme) genes, were up-regulated in the 5% RD group. In the context of microbiota, microbiome analysis revealed that the main phyla in shrimp intestines were Proteobacteria, Firmicutes, Bacteroidota, Campilobacterota, Actinobacteriota, and Verrucomicrobiota. At the genus level, Vibrio was found to be reduced in the 5% RD group, whereas the abundance of potentially beneficial bacteria Bifidobacterium was increased. The 5% RD group showed a significant increase in the levels of crude protein and crude lipid, both of which are essential nutritious components. Our results show the capability of R. paludigena CM33 as a probiotic supplement in shrimp feed in improving growth, antimicrobial responses against VPAHPND, and meat quality by increasing protein and lipid content in shrimp.

A dahlia flower extract has antidiabetic properties by improving insulin function in the brain

Abstract Butein, a rare chalcone found in the toxic plant Toxicodendron vernicifluum, has been shown to regulate glucose homeostasis via inhibition of the nuclear factor kappa-B kinase subunit beta (IKKβ)/nuclear factor kappa B (NF-κB) pathway in the brain. Here, we investigated whether the nonpoisonous plant Dahlia pinnata could be a source of butein as a potential treatment for type 2 diabetes (T2D). In mice fed a high-fat diet (HFD) to induce glucose intolerance, an oral D. pinnata petal extract improved glucose tolerance at doses of 3.3 mg/kg body weight and 10 mg/kg body weight. Surprisingly, this effect was not mediated by butein alone but by butein combined with the closely related flavonoids, sulfuretin and/or isoliquiritigenin. Mechanistically, the extract improved systemic insulin tolerance. Inhibition of phosphatidylinositol 3-kinase to block insulin signaling in the brain abrogated the glucoregulatory effect of the orally administered extract. The extract reinstated central insulin signaling and normalized astrogliosis in the hypothalamus of HFD-fed mice. Using NF-κB reporter zebrafish to determine IKKβ/NF-κB activity, a potent anti-inflammatory action of the extract was found. A randomized controlled crossover clinical trial on participants with prediabetes or T2D confirmed the safety and efficacy of the extract in humans. In conclusion, we identified an extract from the flower petals of D. pinnata as a novel treatment option for T2D, potentially targeting the central regulation of glucose homeostasis as a root cause of the disease.

Intermittent food restriction upregulates critical hypothalamic genes involved in energy regulation imbalance

The effect of intermittent food restriction (IFR) on the Central Nervous System is unclear, especially when alternated with an obesity-inducing diet (DIO). This study aimed to evaluate key genes involved in energy-regulation imbalance in the hypothalamus after IFR and DIO alternation. Therefore, 45-d-old female Wistar rats were divided into 4 groups: standard control (ST-C), fed with an ad libitum standard diet; DIO control (DIO-C), fed with a DIO in the first and last 15 d of the intervention and a standard diet between the 16th and 45th day; standard restricted (ST-R), fed with a standard diet in the first and last 15 d of the intervention followed by IFR at 50% of the ST-C diet between the 16th and 45th day; and DIO restricted (DIO-R), fed with a DIO in the first and last 15 d of the intervention and subjected to IFR under the same conditions as the ST-R group. At 105 d of age, animals were euthanized, and the hypothalamus was removed for quantitative polymerase chain reaction analysis. The ST-R and DIO-R groups showed higher inhibitor of nuclear factor kappa-B kinase subunit beta (P < 0.001; P = 0.029) and nuclear factor kappa B (P < 0.001; P = 0.029) gene expression when compared with the ST-C group. The same held true for the JNK (P = 0.001; P = 0.003) and PPARα genes (both P < 0.001). Instead, the DIO-R group exhibited higher CCL5 gene expression than the ST-C (P = 0.001) and DIO-C (P < 0.001) groups, whereas all groups had higher SOCS3 gene expression than did the ST-C group. These data together suggest that IFR, whether combined with DIO or not, alters the expression of critical genes involved in energy regulation imbalance in the hypothalamus, which warrants caution and more research, because long-term usage might be hazardous.

Gamma-aminobutyric acid (GABA) suppresses hemocyte phagocytosis by binding to GABA receptors and modulating corresponding downstream pathways in blood clam, Tegillarca granosa

Although accumulating data demonstrated that gamma-aminobutyric acid (GABA), an inhibitory neurotransmitter, plays an important regulatory role in immunity of vertebrates, its immunomodulatory function and mechanisms of action remain poorly understood in invertebrates such as bivalve mollusks. In this study, the effect of GABA on phagocytic activity of hemocytes was evaluated in a commercial bivalve species, Tegillarca granosa. Furthermore, the potential regulatory mechanism underpinning was investigated by assessing potential downstream targets. Data obtained demonstrated that in vitro GABA incubation significantly constrained the phagocytic activity of hemocytes. In addition, the GABA-induced suppression of phagocytosis was markedly relieved by blocking of GABAA and GABAB receptors using corresponding antagonists. Hemocytes incubated with lipopolysaccharides (LPS) and GABA had significant higher K+-Cl- cotransporter 2 (KCC2) content compared to the control. In addition, GABA treatment led to an elevation in intracellular Cl−, which was shown to be leveled down to normal by blocking the ionotropic GABAA receptor. Treatment with GABAA receptor antagonist also rescued the suppression of GABAA receptor-associated protein (GABARAP), KCC, TNF receptor associated factor 6 (TRAF6), inhibitor of nuclear factor kappa-B kinase subunit alpha (IKKα), and nuclear factor kappa B subunit 1 (NFκB) caused by GABA incubation. Furthermore, incubation of hemocytes with GABA resulted in a decrease in cAMP content, an increase in intracellular Ca2+, and downregulation of cAMP-dependent protein kinase (PKA), calmodulin kinase II (CAMK2), calmodulin (CaM), calcineurin (CaN), TRAF6, IKKα, and NFκB. All these above-mentioned changes were found to be evidently relieved by blocking the metabotropic G-protein-coupled GABAB receptor. Our results suggest GABA may play an inhibitory role on phagocytosis through binding to both GABAA and GABAB receptors, and subsequently regulating corresponding downstream pathways in bivalve invertebrates.

Effects of tumor necrosis factor-α on glucose uptake in human granulosa cells under high androgen conditions.

Hyperandrogenism is a key pathological characteristic of polycystic ovary syndrome (PCOS). Tumor necrosis factor α (TNF-α) is both an adipokine and a chronic inflammatory factor, which has been proven to be involved in the pathologic process of PCOS. This study aimed to determine how TNF-α affects glucose uptake in human granulosa cells in the presence of high testosterone concentration. KGN cell line was treated with testosterone and TNF-α alone or co-culture combination for 24 hr, or starved for 24 hr. Quantitative real-time polymerase chain reaction (qPCR) and western blot were performed to measure glucose transporter type 4 (GLUT4) message RNA (mRNA) and protein expression in treated KGN cells. Glucose uptake and GLUT4 expression were detected by immunofluorescence (IF). Furthermore, western blot was performed to measure the contents in the nuclear factor kappa-B (NF-κB) pathway. Meantime, upon addition of TNF-α receptor II (TNFRII) inhibitor or Inhibitor of nuclear factor kappa-B kinase subunit beta (IKKβ) antagonist to block the TNFRII-IKKβ-NF-κB signaling pathway, the glucose uptake in KGN cells and GLUT4 translocation to cytomembrane were detected by IF, and related proteins in TNFRII-IKKβ-NF-κB were detected by western blot. The glucose uptake in Testosterone + TNF-α group was lowered significantly, and Total GLUT4 mRNA and proteins were reduced significantly. GLUT4 translocation to cytomembrane was tarnished visibly; concurrently, the phosphorylated proteins in the TNFRII-IKKβ-NF-κB signaling pathway were enhanced significantly. Furthermore, upon addition of TNFRII inhibitor or IKKβ inhibitor to block the TNFRII-IKKβ-NF-κB signaling pathway, the glucose uptake of treated granulosa cells was improved. TNFRII and IKKβ antagonists may improve glucose uptake in granulosa cells induced by TNF-α by blocking the TNFRII-IKKβ-NF-κB signaling pathway under high androgen conditions.

Antioxidant and Anti-Inflammatory Actions of Polyphenols from Red and White Grape Pomace in Ischemic Heart Diseases.

Grape pomace (GP) represents a very reliable source of polyphenols because it could be found globally as a remnant of the wine industry. During the winemaking process, two types of GP are generated: red GP and white GP, according to the produced wine, red or white. Grape pomace represents a viable source of polyphenols, mainly flavanols, procyanidins anthocyanins, and resveratrol which possess antioxidant and anti-inflammatory activities. Multiple differences were observed between red and white GP in terms of their antioxidant and anti-inflammatory activity in both in vitro and in vivo studies. Although most studies are focused on the antioxidant and anti-inflammatory effect of red grape pomace, there are still many variables that need to be taken into consideration, as well as extensive study of the white GP. It was observed that in both in vitro and in vivo studies, the GP polyphenols have a direct antioxidant activity by acting as a free radical scavenger or donating a hydrogen atom. It also possesses an indirect antioxidant and anti-inflammatory activity by reducing mitochondrial reactive oxygen species (ROS) generation, malondialdehyde (MDA), tumor necrosis factor-alpha (TNF-α), interleukin-1-beta (IL-1β), interleukin-6 (IL-6), nuclear factor kappa-light-chain-enhancer of activated B cells (NF- κβ), and inhibitor of nuclear factor kappa-B kinase subunit beta (Iκκβ) levels or nitrate oxide-4 (NOX4) expression and by increasing the levels of antioxidants enzymes like superoxide dismutase (SOD), catalase (CAT) glutathione reductase (GRx) and glutathione peroxidase(GPx). Besides these activities, many beneficial effects in ischemic heart diseases were also observed, such as the maintenance of the ventricular function as close as possible to normal, and the prevention of infarcted area extension. In this context, this review intends to present the actual knowledge of grape pomace’s potential antioxidant and anti-inflammatory activity in ischemic heart disease, knowledge gathered from existing in vitro and in vivo studies focused on this.

HM-Chromanone, a Major Homoisoflavonoid in Portulaca oleracea L., Improves Palmitate-Induced Insulin Resistance by Regulating Phosphorylation of IRS-1 Residues in L6 Skeletal Muscle Cells

This study investigated the effect of (E)-5-hydroxy-7-methoxy-3-(2-hydroxybenzyl)-4-chromanone (HM-chromanone) on palmitate-induced insulin resistance and elucidated the underlying mechanism in L6 skeletal muscle cells. Glucose uptake was markedly decreased due to palmitate-induced insulin resistance in these cells; however, 10, 25, and 50 µM HM-chromanone remarkably improved glucose uptake in a concentration-dependent manner. HM-chromanone treatment downregulated protein tyrosine phosphatase 1B (PTP1B) and phosphorylation of c-Jun N-terminal kinase (JNK) and inhibitor of nuclear factor kappa-B kinase subunit beta (IKKβ), which increased because of palmitate mediating the insulin-resistance status in cells. HM-chromanone promoted insulin receptor substrate-1 (IRS-1) tyrosine phosphorylation and suppressed palmitate-induced phosphorylation of IRS-1 serine. This activated phosphoinositide 3-kinase (PI3K) and stimulated protein kinase B (AKT) phosphorylation. Phosphorylated AKT promoted the translocation of Glucose transporter type 4 to the plasma membrane and significantly enhanced glucose uptake into muscle cells. Additionally, HM-chromanone increased glycogen synthesis through phosphorylating glycogen synthase kinase 3 alpha/beta (GSK3 α/β) via AKT. Consequently, HM-chromanone may improve insulin resistance by downregulating the phosphorylation of IRS-1 serine through inhibition of negative regulators of insulin signaling and inflammation-activated protein kinases in L6 skeletal muscle cells.

Abstract 1570: Macrophage mediated cell-kill evasion by human prostate cancer cells

Abstract Macrophages can adopt a cytotoxic M1 phenotype which can eliminate cancer cells. However, despite the presence of macrophages in the prostate, prostate cancer (PCa) is a prevalent cancer in men. This suggests that PCa cells can evade M1 macrophage mediated cell-kill. We have shown that human THP-1 derived M1-like macrophages readily kill human prostate cancer LNCap cells, while in testosterone proficient conditions (Fraction Growth over Mono-culture (FGM): 0.1). However, in testosterone deprived conditions LNCap cells survive (FGM: 1.1). In addition, when exposed to M1-like macrophage conditioned medium, LNCap cells are killed in testosterone proficient conditions and not in depleted conditions (FGM: 0.2 and 0.7, respectively). This suggests that M1 macrophage mediated PCa cell-kill depends on Androgen Receptor (AR) signaling and the release of soluble factors by macrophages. This is supported by cell-kill of castration-resistant LNCap-abl cells with an AR amplification (FGM: 0.1) in testosterone deprived conditions and no cell-kill in both testosterone deprived and proficient conditions of castration resistant PC3 cells without AR expression when co-cultured with M1-like macrophages (FGM: 0.7 and 0.7. respectively). To dissect the mechanism of M1 macrophage mediated cell-kill evasion, a genome wide CRISPR knock-out screen was performed in LNCap cells in co-culture with M1-like macrophages. LNCap cells were transfected with the Brunello library and cultured in testosterone proficient conditions. After 72 hours of co-culture, cells were harvested and DNA was isolated and submitted for sequencing. After quality check analysis, five gene knock-outs with a significant (log2FcMedian &amp;gt;1.5) survival advantage were identified, AR, PRKCD (Protein Kinase C Delta), IKBKA (Inhibitor of nuclear factor kappa-B kinase subunit α:), IKBKB (Inhibitor of nuclear factor kappa-B kinase subunit β) and IKBKG (Inhibitor of nuclear factor kappa-B kinase subunit γ). The latter three genes are components of the IkB kinase (IKK) complex, which is essential for activation of members of the nuclear factor-kB (NFkB) family of transcription factors. The hits as identified in the CRISPR knock-out screen, were validated in single guide knock-out LNCap cells in co-culture with M1-like macrophages. Additionally, we have shown that AR signaling enhances PRKCD expression, both at the RNA and protein level. Moreover, expression of PRKCD is significantly lower in prostatectomy specimen of 56 patients treated for 3 months with the AR signalling inhibitor enzalutamide prior to surgery, than untreated matched controls. Furthermore, LNCap cells lacking PRKCD, IKBKB and IKBKG were significantly less affected in their growth by reactive oxygen species (H2O2) than non-targeting LNCap cells. Ongoing studies are aimed to dissect how these critical factors are interconnected in LNCap cells, M1 macrophage mediated cell-kill evasion as a result. Citation Format: Anniek Zaalberg, Emma Minnee, Wilbert Zwart, Andries M. Bergman. Macrophage mediated cell-kill evasion by human prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1570.

A cypovirus encoded microRNA negatively regulates the NF-κB pathway to enhance viral multiplication in Silkworm, Bombyx mori

MicroRNAs (miRNAs) are small non-coding RNAs that function as novel gene expression regulators at the post-transcriptional level. Not with standing that the biogenesis and function of miRNAs are well-understood in eukaryotes, little is known about RNA virus-encoded miRNAs. Bombyx mori cypovirus (BmCPV) is a double-stranded RNA virus with a segmented genome that causes cytoplasmic polyhedrosis disease in silkworm larvae. To date, the interaction between BmCPV and silkworm remains largely unclear. 22 candidate BmCPV-encoded miRNAs were identified in this study through small RNA sequencing, stem-loop RT-PCR and qRT-PCR. Then, generation and function analyses were conducted on one of the candidate miRNAs, BmCPV-miR-1, in the BmN cells and the silkworm larvae by RNA interference, quantitative PCR, dual-luciferase assay. Our results revealed that BmCPV-miR-1 was encoded by BmCPV genome RNA rather than the degraded fragments of the viral genome. Its generation depended on Dicer-1 and might also be correlated with Dicer-2, Argonaute-1 and Argonaute-2. Moreover, BmCPV-miR-1 could suppress the expression of the target gene, B. mori inhibitor of nuclear factor kappa-B kinase subunit beta (BmIKKβ), via binding to the target mRNA 3′-untranslated region, which fine-tuned the host NF-κB signaling pathway and consequently enhanced viral replication. Our results provide new evidence supporting the hypothesis that RNA viruses could generate miRNAs to modulate antiviral host defense.

IKKβ mediates homeostatic function in inflammation via competitively phosphorylating AMPK and IκBα

Inhibitor of nuclear factor kappa-B kinase subunit beta (IKKβ) is one of important kinases in inflammation to phosphorylate inhibitor of nuclear factor kappa-B (IκBα) and then activate nuclear factor kappa-B (NF-κB). Inhibition of IKKβ has been a therapeutic strategy for inflammatory and autoimmune diseases. Here we report that IKKβ is constitutively activated in healthy donors and healthy IkkβC46A (cysteine 46 mutated to alanine) knock-in mice although they possess intensive IKKβ–IκBα–NF-κB signaling activation. These indicate that IKKβ activation probably plays homeostatic role instead of causing inflammation. Compared to IkkβWT littermates, lipopolysaccharides (LPS) could induce high mortality rate in IkkβC46A mice which is correlated to breaking the homeostasis by intensively activating p-IκBα–NF-κB signaling and inhibiting phosphorylation of 5ʹ adenosine monophosphate-activated protein kinase (p-AMPK) expression. We then demonstrated that IKKβ kinase domain (KD) phosphorylates AMPKα1 via interacting with residues Thr183, Ser184, and Thr388, while IKKβ helix–loop–helix motifs is essential to phosphorylate IκBα according to the previous reports. Kinase assay further demonstrated that IKKβ simultaneously catalyzes phosphorylation of AMPK and IκBα to mediate homeostasis. Accordingly, activation of AMPK rather than inhibition of IKKβ could substantially rescue LPS-induced mortality in IkkβC46A mice by rebuilding the homeostasis. We conclude that IKKβ activates AMPK to restrict inflammation and IKKβ mediates homeostatic function in inflammation via competitively phosphorylating AMPK and IκBα.

Therapeutic potential of beta-caryophyllene against aflatoxin B1-Induced liver toxicity: biochemical and molecular insights in rats

Aflatoxin B1 (AFB1) is a mycotoxin highly toxic and carcinogenic to humans due to its potential to induce oxidative stress. The Beta-caryophyllene (BCP) have been highlighted for its broad spectrum of pharmacological effects. The present study aimed to investigate the beneficial effects of BCP against the susceptibility of hepatic and renal tissues to AFB1 toxicity, in biochemical parameters to assess organ function, tissue oxidation, and the immunocontent of oxidative and inflammatory proteins. Male Wistar rats was exposed to AFB1 (250 μg/kg, i.g.) and/or BCP (100 mg/kg, i.p.) for 14 successive days. It was found that exposure to AFB1 did not change the measured renal toxicity parameters. Also, AFB1 increased liver injury biomarkers (gamma glutamyl transferase and alkaline phosphatase) and reduced levels of non-enzymatic antioxidant defenses (ascorbic acid and non-protein thiol), however did not cause changes in the lipid peroxidation levels. Moreover, AFB1 interfered in oxidative pathway regulated by Kelch-like ECH-associated protein (Keap1)/nuclear factor (erythroid-derived 2)-like 2 (Nrf2), overacting Glutathione-S-Transferase (GST) activity. Lastly, a main effect of AFB1 on the total interleukin 1 beta (IL-1β) was observed. Remarkably, the associated treatment of AFB1 + BCP improved altered liver parameters. In addition, BCP and AFB1 + BCP groups showed an increase in the levels of inhibitor of nuclear factor kappa-B kinase subunit beta (IKKβ). Thus, these results indicated that BCP has potential protective effect against AFB1 induced hepatotoxicity.

β-Sitosterol Circumvents Obesity Induced Inflammation and Insulin Resistance by down-Regulating IKKβ/NF-κB and JNK Signaling Pathway in Adipocytes of Type 2 Diabetic Rats.

β-sitosterol (SIT), the most abundant bioactive component of vegetable oil and other plants, is a highly potent antidiabetic drug. Our previous studies show that SIT controls hyperglycemia and insulin resistance by activating insulin receptor and glucose transporter 4 (GLUT-4) in the adipocytes of obesity induced type 2 diabetic rats. The current research was undertaken to investigate if SIT could also exert its antidiabetic effects by circumventing adipocyte induced inflammation, a key driving factor for insulin resistance in obese individuals. Effective dose of SIT (20 mg/kg b.wt) was administered orally for 30 days to high fat diet and sucrose induced type-2 diabetic rats. Metformin, the conventionally used antidiabetic drug was used as a positive control. Interestingly, SIT treatment restores the elevated serum levels of proinflammatory cytokines including leptin, resistin, tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) to normalcy and increases anti-inflammatory adipocytokines including adiponectin in type 2 diabetic rats. Furthermore, SIT decreases sterol regulatory element binding protein-1c (SREBP-1c) and enhances Peroxisome Proliferator–activated receptor-γ (PPAR-γ) gene expression in adipocytes of diabetic rats. The gene and protein expression of c-Jun-N-terminal kinase-1 (JNK1), inhibitor of nuclear factor kappa-B kinase subunit beta (IKKβ) and nuclear factor kappa B (NF-κB) were also significantly attenuated in SIT treated groups. More importantly, SIT acts very effectively as metformin to circumvent inflammation and insulin resistance in diabetic rats. Our results clearly show that SIT inhibits obesity induced insulin resistance by ameliorating the inflammatory events in the adipose tissue through the downregulation of IKKβ/NF-κB and c-Jun-N-terminal kinase (JNK) signaling pathway.

NF-κB activation in retinal microglia is involved in the inflammatory and neovascularization signaling in laser-induced choroidal neovascularization in mice

PurposeTo evaluate Nuclear Factor NF-κB (NF-κB) signaling on microglia activation, migration, and angiogenesis in laser-induced choroidal neovascularization (CNV). MethodsNine-week-old C57BL/6 male mice were randomly assigned to IMD-0354 treated or untreated groups (5 mice, 10 eyes per group). CNV was induced with a 532-nm laser. Laser spots (power 250 mW, spot size 100 μm, time of exposure 50 ms) were created in each eye using a slit-lamp delivery system. Selective inhibitor of nuclear factor kappa-B kinase subunit beta (IKK2) inhibitor IMD-0354 (10 μg) was delivered subconjunctivally; vehicle-treated mice were the control. The treatment effect on CNV development was assessed at five days post-CNV induction in vivo in C57BL/6 and Cx3cr1gfp/wt mice by fluorescent angiography, fundus imaging, and ex vivo by retinal flatmounts immunostaining and Western blot analysis of RPE/Choroidal/Scleral complexes (RCSC) lysates. In vitro evaluations of IMD-0354 effects were performed in the BV-2 microglial cell line using lipopolysaccharide (LPS) stimulation. ResultsIMD-0354 caused a significant reduction in the fluorescein leakage and size of the laser spot, as well as a reduction in microglial cell migration and suppression of phospho-IκBα, Vascular endothelial growth factor (VEGF-A), and Prostaglandin-endoperoxide synthase 2 (COX-2). In vivo and ex vivo observations demonstrated reduced lesion size in mice, CD68, and Allograft inflammatory factor 1 (IBA-1) positive microglia cells migration to the laser injury site in IMD-0354 treated eyes. The data further corroborate with GFP-positive cells infiltration of the CNV site in Cx3cr1wt/gfp mice. In vitro IMD-0354 (10–25 ng/ml) treatment reduced NF-κB activation, expression of COX-2, caused decreased Actin-F presence and organization, resulting in reduced BV-2 cells migration capacity. ConclusionThe present data indicate that NF-κB activation in microglia and it's migration capacity is involved in the development of laser CNV in mice. Its suppression by NF-κB inhibition might be a promising therapeutic strategy for wet AMD.