P65 Subunit Research Articles

TNF/iNOS/NO pathway mediates host susceptibility to endothelial-dependent circulatory failure and death induced by betacoronavirus infection

Poor disease outcomes and lethality are directly related to endothelial dysfunction in betacoronavirus infections. Here, we investigated the mechanisms underlying the vascular dysfunction caused by the betacoronaviruses MHV-3 and SARS-CoV-2. Wild-type C57BL/6 (WT) and knockout mice for inducible nitric oxide synthase (iNOS-/-) or TNF receptor 1 (TNFR1-/-) were infected with MHV-3, and K18-hACE2 transgenic mice expressing human ACE2 were infected with SARS-CoV-2. Isometric tension was used to evaluate vascular function. Protein expression was determined by immunofluorescence. Tail-cuff plethysmography and Doppler were used to assess blood pressure and flow, respectively. Nitric oxide (NO) was quantified with the DAF probe. ELISA was used to assess cytokine production. Survival curves were estimated using Kaplan-Meier. MHV-3 infection reduced aortic and vena cava contractility, arterial blood pressure, and blood flow, resulting in death. Resistance mesenteric arteries showed increased contractility. The contractility of the aorta was normalized by removing the endothelium, inhibiting iNOS, genetically deleting iNOS, or scavenging NO. In the aorta, iNOS and phospho-NF-kB p65 subunit expression was enhanced, along with basal NO production. TNF production was increased in plasma and vascular tissue. Genetic deletion of TNFR1 prevented vascular changes triggered by MHV-3, and death. Basal NO production and iNOS expression were also increased by SARS-CoV-2. In conclusion, betacoronavirus induces an endothelium-dependent decrease in contractility in macro-arteries and veins, leading to circulatory failure and death via TNF/iNOS/NO. These data highlight the key role of the vascular endothelium and TNF in the pathogenesis and lethality of coronaviruses.

Biomarkers Found in the Tumor Interstitial Fluid may Help Explain the Differential Behavior Among Keratinocyte Carcinomas

Basal cell carcinomas (BCCs) and cutaneous squamous cell carcinomas (SCCs) are the most frequent types of cancer, and both originate from the keratinocyte transformation, giving rise to the group of tumors called keratinocyte carcinomas (KCs). The invasive behavior is different in each group of KC and may be influenced by their tumor microenvironment. The principal aim of the study is to characterize the protein profile of the tumor interstitial fluid (TIF) of KC to evaluate changes in the microenvironment that could be associated with their different invasive and metastatic capabilities. We obtained TIF from 27 skin biopsies and conducted a label-free quantitative proteomic analysis comparing seven BCCs, 16 SCCs, and four normal skins. A total of 2945 proteins were identified, 511 of them quantified in more than half of the samples of each tumoral type. The proteomic analysis revealed differentially expressed TIF proteins that could explain the different metastatic behavior in both KCs. In detail, the SCC samples disclosed an enrichment of proteins related to cytoskeleton, such as Stratafin and Ladinin-1. Previous studies found their upregulation positively correlated with tumor progression. Furthermore, the TIF of SCC samples was enriched with the cytokines S100A8/S100A9. These cytokines influence the metastatic output in other tumors through the activation of NF-kB signaling. According to this, we observed a significant increase in nuclear NF-kB subunit p65 in SCCs but not in BCCs. In addition, the TIF of both tumors was enriched with proteins involved in the immune response, highlighting the relevance of this process in the composition of the tumor environment. Thus, the comparison of the TIF composition of both KCs provides the discovery of a new set of differential biomarkers. Among them, secreted cytokines such as S100A9 may help explain the higher aggressiveness of SCCs, while Cornulin is a specific biomarker for BCCs. Finally, the proteomic landscape of TIF provides key information on tumor growth and metastasis, which can contribute to the identification of clinically applicable biomarkers that may be used in the diagnosis of KC, as well as therapeutic targets.

Circ_0060967 contributes to colorectal cancer progression by sponging miR-1184 to up-regulate SRC proto-oncogene

Background and study aimsCircular RNAs (circRNAs) are closely associated with cancer pathogenesis. The purpose of our current study was to explore the role and mechanism of circ_0060967 in colorectal cancer (CRC) development. Patients and methodsHuman CRC specimens and paired healthy tissues were used to examine variable expression. The expression of circ_0060967 and microRNA (miR)-1184 was examined by quantitative reverse transcription-PCR. The protein levels of proliferating cell nuclear antigen, BCL2-associated X, apoptosis regulator (Bax), proto-oncogene nonreceptor tyrosine kinase Src (SRC), nuclear factor-κB inhibitor alpha (IκBα), phosphorylated-IκBα (p-IκBα), RELA proto-oncogene, nuclear factor-κB subunit (p65), and phosphorylated-p65 (p-p65) were determined by western blot. Proliferation and motility of HCT-116 and SW480 CRC cells were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and transwell assays, respectively. Dual-luciferase reporter assay and RNA immunoprecipitation assay were used to determine the binding relation between miR-1184 and circ_0060967 or SRC. Animal studies were used to detect the role of circ_0060967 in CRC cell tumorigenicity. ResultsCirc_0060967 abundance was enhanced in human CRC tissue samples versus paired normal colorectal tissues and in HCT-116 and SW480 CRC cells versus normal HCO cells. Decreased expression of circ_0060967 could suppress cell growth, motility, and invasiveness of CRC cells in vitro and tumor growth in vivo. Circ_0060967 sponged miR-1184, and miR-1184 targeted SRC. Furthermore, we also found circ_0060967 affected cell growth by modulating miR-1184/SRC axis in CRC. ConclusionThis study demonstrates a novel circ_0060967/miR-1184/SRC regulatory cascade in affecting CRC cell malignant behaviors, which can have a broad effect on the field of molecularly targeted therapeutics.

Abstract 1482: Critical role of OGT-mediated novel NF-κB o-glcNAcylation in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) has one of the highest mortalities of all malignancies, with a mere 5-year survival of ~10%. However, the current first-line treatments have poor patient outcomes, highlighting the urgent need for innovative therapeutics. The nuclear factor κB (NF-κB) is a crucial transcription factor frequently activated constitutively in PDAC. It mediates the transcription of oncogenic and inflammatory genes that facilitate multiple PDAC phenotypes. Thus, a better understanding of the mechanistic underpinnings of NF-κB activation holds substantial promise in PDAC diagnosis and new therapeutics. The purpose of this study is to identify novel regulation of NF-κB, with the aim of providing new diagnostic and therapeutic strategies for PDAC. Here, we report protein O-GlcNAc transferase (OGT) - mediated NF-κB activation through novel serine O-GlcNAcylation of its p65 subunit. We show that overexpression of serine-to-alanine (S-A) mutant at the O-GlcNAcylation site impaired NF-κB nuclear translocation and transcriptional activity in PDAC cells. Moreover, these S-A p65 mutants downregulate NF-κB-target genes important to major cancer hallmarks and inhibit cellular proliferation, migration, and anchorage-independent growth of PDAC cells compared to WT-p65. Interestingly, this modification happens downstream of the inhibitor of NF-κB (IκBα) degradation. Collectively, we have identified OGT-mediated serine O-GlcNAcylation of NF-κB and determined its mechanistic and cellular function in driving PDAC phenotypes. Thus, our study holds great significance as it uncovers a brand-new strategy for PDAC diagnosis and effective therapeutics development. Citation Format: Aishat Motolani, Matthew Martin, Benlian Wang, Guanglong Jiang, Yunlong Liu, Tao Lu. Critical role of OGT-mediated novel NF-κB o-glcNAcylation in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1482.

5593300 INVESTIGATIONS OF KIDNEY DYSFUNCTION-RELATED GENE VARIANTS IN SICKLE CELL DISEASE PATIENTS IN CAMEROON (SUB-SAHARAN AFRICA)

Background: Renal dysfunctions are associated with increased morbidity and mortality in sickle cell disease (SCD). Early detection and subsequent management of SCD patients at risk for renal failure and dysfunctions are essential, however, predictors that can identify patients at risk of developing renal dysfunction are not fully understood. Aims: the aims of the study were: to employ a multi-factorial approach to investigate genetic factors and also to explore the contribution of socio-demographic, anthropometric, clinical and haematological variables associated to renal abnormalities in SCD patients in Cameroon. Methods: In this study, we have investigated the association of 31 known kidney dysfunctions-related variants detected in African Americans from multi-ethnic genome wide studies (GWAS) meta-analysis to kidney-dysfunctions in a group of 413 Cameroonian patients with SCD. Systems level bioinformatics analyses were performed, employing protein-protein interaction networks to further interrogate the putative associations. Results: Up to 61% of these patients had micro-albuminuria, 2.4% proteinuria, 71% glomerular hyperfiltration, and 5.9% had renal failure. Six variants are significantly associated with the two quantifiable phenotypes of kidney dysfunction (eGFR and crude-albuminuria): A1CF-rs10994860 (P = 0.02020), SYPL2-rs12136063 (P = 0.04208), and APOL1 (G1)-rs73885319 (P = 0.04610) are associated with eGFR; and WNT7A-rs6795744 (P = 0.03730), TMEM60-rs6465825 (P = 0.02340), and APOL1 (G2)-rs71785313 (P = 0.03803) observed to be protective against micro-albuminuria. We identified a protein-protein interaction sub-network containing three of these gene variants: APOL1, SYPL2, and WNT7A, connected to the nuclear factor NF-kappa-B p105 subunit (NFKB1), revealed to be essential and might indirectly influence extreme phenotypes. Interestingly, clinical variables, including body mass index (BMI), systolic blood pressure, vaso-occlusive crisis (VOC), and haemoglobin (Hb), explain better the kidney phenotypic variations in this SCD population. Conclusion: This study highlights a strong contribution of haematological indices (Hb level), anthropometric variables (BMI, blood pressure), and clinical events (i.e., vasoocclusive crisis) to kidney dysfunctions in SCD, rather than known genetic factors. Only 6/31 characterised gene-variants are associated with kidney dysfunction phenotypes in SCD samples from Cameroon. The data reveal and emphasise the urgent need to extend GWAS studies in populations of African ancestries living in Africa, and particularly for kidney dysfunctions in SCD. Keywords: sickle cell disease, kidney dysfunctions, gene variants, cameroon, Africa

New natural pro-inflammatory cytokines (TNF-α, IL-6 and IL-1β) and iNOS inhibitors identified from Penicillium polonicum through in vitro and in vivo studies.

Overexpression of pro-inflammatory cytokines and iNOS have been found to be concomitant with several chronic inflammatory diseases and hence targeting their inhibition would be a useful therapy for inflammation. In view of this, study on discovery of natural pro-inflammatory cytokines inhibitory lead molecules from Penicillium polonicum, an endophytic fungus isolated from the fresh fruits of Piper nigrum was performed. When the culture broth extract of P. polonicum (EEPP) was subjected to LPS-induced cytokines expression (ELISA in RAW 264.7 cells), it exhibited inhibition of TNF-α, IL-6 and IL-1β and this encouraged us to do chemical investigation on EEPP to explore the bioactive components. Four compounds isolated and characterised as 3,5-di-tert-butyl-4-hydroxy-phenyl propionic acid (1), 2,4-di-tert-butyl phenol (2), indole 3-carboxylic acid (3) and tyrosol (4) were tested for their effect on the production of TNF-α, IL-1β and IL-6 in RAW 264.7 cells (ELISA). All the compounds exhibited a highly significant (P<0.0001) inhibition effect, particularly against IL-1β (IC50: 4-0.91μM, 1-2.81μM, 3-4.38μM, and 2-5.54μM). Tyrosol (4) was most active with IC50 values of 0.91, 2.67 and 4.60μM against IL-1β, IL-6 and TNF-α, respectively. On observing the potential activity of the compounds, two compositions C1 and C2 were prepared by mixing equimolar concentrations of compounds 1, 2, 3 & 4 (C1) and compounds 1, 2, 3, 4 & piperine (C2) in equal ratio. A synergistic effect was observed with C1 exhibiting potential suppression of IL-6 secretion (IC50 1.91μM) and C2 against IL-1β (IC50 5.98μM). Also, the individual compounds and C1 were effective in controlling iNOS expressions in RAW 264.7 cells (RTPCR). Further, the in vivo performance of the compounds and compositions were studied under two in vivo inflammatory models (LPS-induced endotoxaemia and carrageenan-induced paw oedema). Compounds 1, 2, 3, 4, C1 and C2 at 50mg/kg oral dose showed a significant control over the LPS-stimulated TNF-α, IL-1β and IL-6 levels in plasma. C1, C2 and 1 exhibited>50% pan-cytokine inhibition effect. Under the carrageenan-induced anti-inflammatory model, a significant reduction in the paw oedema measured in terms of the difference in the paw thickness was observed. Further, attenuation of pro-inflammatory cytokines levels following ELISA and RT-PCR experiments in the paw tissue homogenate was in agreement with paw thickness results. All compounds and C1 decreased the iNOS gene expression levels, and also the MPO activity and NO production in the paw tissue homogenate with tyrosol (4) as the most active molecule. Further, the mechanism of action was explored by testing the effect of the compounds on the expression of inflammatory markers using western blot analysis (in vitro). They were found to regulate the expression of pro-form and matured-form of IL-1β by inhibiting NFκB. Also, the compounds reduced the translocation of the NF-κB subunit p65 to the nucleus. Thus, compounds 3,5-di-tert-butyl-4-hydroxy-phenyl propionic acid (1), 2,4-di-tert-butyl phenol (2), indole 3-carboxylic acid (3) and tyrosol (4) are reported as new natural multiple pro-inflammatory cytokines inhibitory leads. The interesting results of C1 might lay a footing for the development of a new anti-inflammatory composition.

NFKB2 haploinsufficiency identified via screening for IFN-α2 autoantibodies in children and adolescents hospitalized with SARS-CoV-2–related complications

Autoantibodies against type I IFNs occur in approximately 10% of adults with life-threatening coronavirus disease 2019 (COVID-19). The frequency of anti-IFN autoantibodies in children with severe sequelae of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is unknown. We quantified anti-type I IFN autoantibodies in a multicenter cohort of children with severe COVID-19, multisystem inflammatory syndrome in children (MIS-C), and mild SARS-CoV-2 infections. Circulating anti-IFN-α2 antibodies were measured by a radioligand binding assay. Whole-exome sequencing, RNA sequencing, and functional studies of peripheral blood mononuclear cells were used to study any patients with levels of anti-IFN-α2 autoantibodies exceeding the assay's positive control. Among 168 patients with severe COVID-19, 199 with MIS-C, and 45 with mild SARS-CoV-2 infections, only 1 had high levels of anti-IFN-α2 antibodies. Anti-IFN-α2 autoantibodies were not detected in patients treated with intravenous immunoglobulin before sample collection. Whole-exome sequencing identified a missense variant in the ankyrin domain of NFKB2, encoding the p100 subunit of nuclear factor kappa-light-chain enhancer of activated B cells, aka NF-κB, essential for noncanonical NF-κB signaling. The patient's peripheral blood mononuclear cells exhibited impaired cleavage of p100 characteristic of NFKB2 haploinsufficiency, an inborn error of immunity with a high prevalence of autoimmunity. High levels of anti-IFN-α2 autoantibodies in children and adolescents with MIS-C, severe COVID-19, and mild SARS-CoV-2 infections are rare but can occur in patients with inborn errors of immunity.

NF-KB P65-subunit activity and T-helpers 1 / T-helpers 2 ratio in pregnant women with placental disorders and premature labor

Objectives: to study the levels of the total, phosphorylated p65-subunit of the nuclear factor NF-kB, activity of p65 and the relation with the level and ratio of T-helpers type I and II in pregnant women with placental dysfunction and different clinical types of the course of preterm labor (with preterm premature rupture of membranes (pPROM) and without it).Materials and methods. The case-control study included 60 pregnant women: 40 women with placental disorders and spontaneous premature labor in the period of 24–34 weeks (group I – 20 women with premature labor and timely discharge of amniotic fluid, group II – 20 women with pPROM) and 20 women of the control group (CG) with normal timely delivery in the head position of a fetus without complications.The value of the total NF-kB p65 subunit and its phosphorylated fraction was determined in all women using ELISA in placenta lysates. On this basis the p65 subunit activity was calculated; number of T-helper I (Th1) and T-helper II (Th2) was determined using flow cytometry in a whole blood sample, with afterward calculation of the Th1/Th2 ratio.Results. Elevated levels of total p65 and its phosphorylated fraction were found in women with placental dysfunction (p &lt; 0.01 in groups I and II compared with CG), as well as the activity p65 (p &lt; 0.01 in group I, p = 0.04 in group II compared with CG). The difference in the Th1 value and the Th1/Th2 ratio was significantly higher in both groups (p &lt; 0.01 in group I, p = 0.03 in group II for Th1; p &lt; 0.01 in both groups for Th1/Th2), the number of Th2 differed significantly only in group I (p &lt; 0.01 compared with CG). A strong positive correlation between p65 activity and Th1/Th2 was also established (r = 0.8).Conclusions. Obtained data indicates the increased NF-kB p65-subunit activity in women with placental disorders and spontaneous premature labor without pPROM, which is impact on the increase of the Th1/Th2 ratio due to the Th1 increase. This mechanism might be considered to be the leading cause of the premature birth in this group of pregnant women. However, for women with the preterm labor activity with pPROM, the difference with GC has a lower level of significance, which may indicate the existence of another leading mechanism for the initiation of premature labor in this group.

Innate Immune Zonation in the Liver: NF-κB (p50) Activation and C-Reactive Protein Expression in Response to Endotoxemia Are Zone Specific.

Hepatic innate immune function plays an important role in the pathogenesis of many diseases. Importantly, a growing body of literature has firmly established the spatial heterogeneity of hepatocyte metabolic function; however, whether innate immune function is zonated remains unknown. To test this question, we exposed adult C57BL/6 mice to endotoxemia, and hepatic tissue was assessed for the acute phase response (APR). The zone-specific APR was evaluated in periportal and pericentral/centrilobular hepatocytes isolated using digitonin perfusion and on hepatic tissue using RNAscope and immunohistochemistry. Western blot, EMSA, chromatin immunoprecipitation, and immunohistochemistry were used to determine the role of the transcription factor NF-κB in mediating hepatic C-reactive protein (CRP) expression. Finally, the ability of mice lacking the NF-κB subunit p50 (p50-/-) to raise a hepatic APR was evaluated. We found that endotoxemia induces a hepatocyte transcriptional APR in both male and female mice, with Crp, Apcs, Fga, Hp, and Lbp expression being enriched in pericentral/centrilobular hepatocytes. Focusing our work on CRP expression, we determined that NF-κB transcription factor subunit p50 binds to consensus sequence elements present in the murine CRP promoter. Furthermore, pericentral/centrilobular hepatocyte p50 nuclear translocation is temporally associated with zone-specific APR during endotoxemia. Lastly, the APR and CRP expression is blunted in endotoxemic p50-/- mice. These results demonstrate that the murine hepatocyte innate immune response to endotoxemia includes zone-specific activation of transcription factors and target gene expression. These results support further study of zone-specific hepatocyte innate immunity and its role in the development of various disease states.

Aspirin prevents estrogen deficiency-induced bone loss by inhibiting osteoclastogenesis and promoting osteogenesis

BackgroundAspirin is a commonly used antipyretic, analgesic, and anti-inflammatory drug. Numerous researches have demonstrated that aspirin exerts multiple biological effects on bone metabolism. However, its spatiotemporal roles remain controversial according to the specific therapeutic doses used for different clinical conditions, and the detailed mechanisms have not been fully elucidated. Hence, in the present study, we aimed to identify the dual effects of different aspirin dosages on osteoclastic activity and osteoblastic bone formation in vitro and in vivo.MethodsThe effects of varying doses of aspirin on osteoclast and osteoblast differentiation were evaluated in vitro. The underlying molecular mechanisms were detected using quantitative real-time polymerase chain reaction, western blotting, and immunofluorescence techniques. An ovariectomized rat osteoporosis model was used to assess the bone-protective effects of aspirin in vivo.ResultsAspirin dose-dependently suppressed RANKL-induced osteoclasts differentiation and bone resorption in vitro and reduced the expression of osteoclastic marker genes, including TRAP, cathepsin K, and CTR. Further molecular analysis revealed that aspirin impaired the RANKL-induced NF-κB and MAPK signaling pathways and prevented the nuclear translocation of the NF-κB p65 subunit. Low-dose aspirin promoted osteogenic differentiation, whereas these effects were attenuated when high-dose aspirin was administered. Both low and high doses of aspirin prevented bone loss in an ovariectomized rat osteoporosis model in vivo.ConclusionAspirin inhibits RANKL-induced osteoclastogenesis and promotes osteogenesis in a dual regulatory manner, thus preventing bone loss in vivo. These data indicate that aspirin has potential applications in the prevention and treatment of osteopenia.

Ethanol Extract of Rosa laevigata Michx. Fruit Inhibits Inflammatory Responses through NF-κB/MAPK Signaling Pathways via AMPK Activation in RAW 264.7 Macrophages.

The fruit of Rosa laevigata Michx. (FR), a traditional Chinese herb utilized for the treatment of a variety diseases, has notably diverse pharmacological activities including hepatoprotective, anti-oxidant, and anti-inflammatory effects. Despite ongoing research on illustrating the underlying anti-inflammatory mechanism of FR, the principal mechanism remained inadequately understood. In this study, we investigated in depth the molecular mechanism of the anti-inflammatory actions of the ethanol extract of FR (EFR) and its potential targets using lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages in vitro. We showed that EFR effectively ameliorated the overproduction of inflammatory mediators and cytokines, as well as the expression of related genes. It was further demonstrated that LPS-induced activation of nuclear factor kappa B (NF-κB) and mitogen-activated protein kinases (MAPKs) were significantly inhibited by pretreatment with EFR, accompanied by a concomitant decrease in the nuclear translocation of the p65 subunit of NF-κB and activator protein 1 (AP-1). In addition, EFR pretreatment potently prevented LPS-induced decreased phosphorylation of adenosine monophosphate-activated protein kinase (AMPK). Our data also revealed that the activation of AMPK and subsequent inhibition of the mammalian target of the rapamycin (mTOR) signaling pathway was probably responsible for the inhibitory effect of EFR on LPS-induced inflammatory responses, evidenced by reverse changes observed under the condition of AMPK inactivation following co-treatment with the AMPK-specific inhibitor Compound C. Finally, the main components with an anti-inflammatory effect in EFR were identified as madecassic acid, ellagic acid, quinic acid, and procyanidin C1 by LC-MS and testified based on the inhibition of NO production and inflammatory mediator expression. Taken together, our results indicated that EFR was able to ameliorate inflammatory responses via the suppression of MAPKs/NF-κB signaling pathways following AMPK activation, suggesting the therapeutic potential of EFR for inflammatory diseases.

Brain vitamin D3-auto/paracrine system in relation to structural, neurophysiological, and behavioral disturbances associated with glucocorticoid-induced neurotoxicity.

Vitamin D3 (VD3) is a potent para/autocrine regulator and neurosteroid that can strongly influence nerve cell function and counteract the negative effects of glucocorticoid (GC) therapy. The aim of the study was to reveal the relationship between VD3 status and behavioral, structural-functional and molecular changes associated with GC-induced neurotoxicity. Female Wistar rats received synthetic GC prednisolone (5 mg/kg b.w.) with or without VD3 (1000 IU/kg b.w.) for 30 days. Behavioral, histological, physiological, biochemical, molecular biological (RT-PCR, Western blotting) methods, and ELISA were used. There was no difference in open field test (OFT), while forced swim test (FST) showed an increase in immobility time and a decrease in active behavior in prednisolone-treated rats, indicative of depressive changes. GC increased the perikaryon area, enlarged the size of the nuclei, and caused a slight reduction of cell density in CA1-CA3 hippocampal sections. We established a GC-induced decrease in the long-term potentiation (LTP) in CA1-CA3 hippocampal synapses, the amplitude of high K+-stimulated exocytosis, and the rate of Ca2+-dependent fusion of synaptic vesicles with synaptic plasma membranes. These changes were accompanied by an increase in nitration and poly(ADP)-ribosylation of cerebral proteins, suggesting the development of oxidative-nitrosative stress. Prednisolone upregulated the expression and phosphorylation of NF-κB p65 subunit at Ser311, whereas downregulating IκB. GC loading depleted the circulating pool of 25OHD3 in serum and CSF, elevated VDR mRNA and protein levels but had an inhibitory effect on CYP24A1 and VDBP expression. Vitamin D3 supplementation had an antidepressant-like effect, decreasing the immobility time and stimulating active behavior. VD3 caused a decrease in the size of the perikaryon and nucleus in CA1 hippocampal area. We found a recovery in depolarization-induced fusion of synaptic vesicles and long-term synaptic plasticity after VD3 treatment. VD3 diminished the intensity of oxidative-nitrosative stress, and suppressed the NF-κB activation. Its ameliorative effect on GC-induced neuroanatomical and behavioral abnormalities was accompanied by the 25OHD3 repletion and partial restoration of the VD3-auto/paracrine system. GC-induced neurotoxicity and behavioral disturbances are associated with increased oxidative-nitrosative stress and impairments of VD3 metabolism. Thus, VD3 can be effective in preventing structural and functional abnormalities in the brain and behavior changes caused by long-term GC administration.

Epstein–Barr virus envelope glycoprotein 110 inhibits NF-κB activation by interacting with NF-κB subunit p65

Epstein-Barr virus (EBV) is a member of the lymphotropic virus family and is highly correlated with some human malignant tumors. It has been reported that envelope glycoprotein 110 (gp110) plays an essential role in viral fusion, DNA replication, and nucleocapsid assembly of EBV. However, it has not been established whether gp110 is involved in regulating the host's innate immunity. In this study, we found that gp110 inhibits tumor necrosis factor α-mediated NF- κB promoter activity and the downstream production of NF- κB-regulated cytokines under physiological conditions. Using dual-luciferase reporter assays, we showed that gp110 might impede the NF-κB promoter activation downstream of NF-κB transactivational subunit p65. Subsequently, we used coimmunoprecipitation assays to demonstrate that gp110 interacts with p65 during EBV lytic infection, and that the C-terminal cytoplasmic region of gp110 is the key interaction domain with p65. Furthermore, we determined that gp110 can bind to the N-terminal Rel homologous and C-terminal domains of p65. Alternatively, gp110 might not disturb the association of p65 with nontransactivational subunit p50, but we showed it restrains activational phosphorylation (at Ser536) and nuclear translocation of p65, which we also found to be executed by the C-terminal cytoplasmic region of gp110. Altogether, these data suggest that the surface protein gp110 may be a vital component for EBV to antagonize the host's innate immune response, which is also helpful for revealing the infectivity and pathogenesis of EBV.

Serum amyloid A and interleukin -1β facilitate LDL transcytosis across endothelial cells and atherosclerosis via NF-κB/caveolin-1/cavin-1 pathway

Background and aimsInflammatory molecules play important roles in atherosclerosis. We aimed to illustrate the roles of serum amyloid A (SAA), and interleukin (IL)-1β in low density lipoproteins (LDL) transcytosis and atherosclerosis. MethodsEffects of SAA and IL-1β on transcytosis of LDL were measured by an in vitro LDL transcytosis model. NF-κB/caveolin-1/cavin-1 pathway activation was investigated by Western blots and ELISA. Effects of SAA and IL-1β on the retention of LDL in aorta of C57BL/6J mice were detected by IVIS spectrum. Effects of SAA and IL-1β on atherosclerosis in Apoe−/− mice were examined by Oil Red O staining. ResultsSAA and IL-1β stimulated LDL transcytosis across endothelial cells (ECs), which was accompanied by an increase in LDL uptake by ECs. SAA and IL-1β enhanced the activity of nuclear factor (NF)-κB, consequently facilitating an up-regulation of proteins involved in caveolae formation, including caveolin-1 and cavin-1, along with an assembly of NLRP3 inflammasome. Furthermore, SAA- and IL-1β-induced effects were blocked by NF-κB subunit p65 siRNA. Meanwhile, SAA- and IL-1β-induced LDL transcytosis were effectively blocked by caveolin-1 siRNA or cavin-1 siRNA. Interestingly, SAA and IL-1β facilitated LDL entering into the aorta of C57BL/6J mice. In Apoe−/− mice, SAA and IL-1β increased the areas of lipid-rich atherosclerotic lesions in the both ascending and root of aorta. Furthermore, a significant increase in the NLRP3 inflammasome, accompanied by accumulation of cavin-1 and caveolin-1, was observed in the aortic endothelium of Apoe−/− mice. ConclusionsSAA and IL-1β accelerated LDL transcytosis via the NF-κB/caveolin-1/cavin-1 axis.

The impact of SGLT2 inhibitors on αKlotho in renal MDCK and HK-2 cells.

αKlotho is a transmembrane protein predominantly expressed in the kidney serving as a co-receptor for phosphate homeostasis-regulating hormone FGF23 and has an extracellular domain that can be cleaved off and is a hormone. αKlotho deficiency results in accelerated aging and early onset of aging-associated diseases while its overexpression strongly expands the lifespan of mice. Moreover, αKlotho exerts health-beneficial anti-inflammatory, anti-neoplastic, anti-fibrotic, and anti-oxidant effects. Higher αKlotho levels are associated with better outcomes in renal and cardiovascular diseases. SGLT2 inhibitors are novel drugs in the treatment of diabetes by inhibiting renal glucose transport and have additional nephro- and cardioprotective effects. We explored whether SGLT2 inhibitors affect αKlotho gene expression and protein secretion. Experiments were performed in renal MDCK and HK-2 cells, and αKlotho transcripts were determined by qRT-PCR and Klotho protein by ELISA. SGLT2 inhibitors canagliflozin, sotagliflozin, and dapagliflozin enhanced whereas empagliflozin reduced αKlotho gene expression in MDCK cells. By the same token, canagliflozin, sotagliflozin, dapagliflozin, but not empagliflozin down-regulated p65 subunit of pro-inflammatory NFκB. In HK-2 cells, all SGLT2 inhibitors reduced αKlotho transcripts. Canagliflozin and sotagliflozin, however, increased Klotho protein concentration in the cell culture supernatant, an effect paralleled by up-regulation of ADAM17. Taken together, our investigations demonstrate complex effects of different SGLT2 inhibitors on αKlotho gene expression and protein secretion in renal MDCK and HK-2 cells.

MicroRNA-181a contributes to gastric hypersensitivity in rats with diabetes by regulating TLR4 expression.

Aim: The aim of this study is to investigate the mechanism and interaction of microRNA-181a (miR-181a), toll-like receptor 4 (TLR4) and nuclear factor-kappa B (NF-κB) in gastric hypersensitivity in diabetic rats. Methods: Diabetes was induced by a single intraperitoneal injection of streptozotocin (STZ; 65mg/kg) in female SD rats. Gastric balloon distension technique was used to measure diabetic gastric hypersensitivity. Gastric-specific (T7-T10) dorsal root ganglion (DRG) neurons were acutely dissociated to measure excitability with patch-clamp techniques. Western blotting was employed to measure the expressions of TLR4, TRAF6 and NF-κB subunit p65 in T7-T10 DRGs. The expressions of microRNAs in T7-T10 DRGs were measured with quantitative real-time PCR and fluorescence in situ hybridization. Dual-luciferase reporter gene assay was used to detect the targeting regulation of microRNAs on TLR4. Results: (1) Diabetic rats were more sensitive to graded gastric balloon distention at 2 and 4weeks. (2) The expression of TLR4 was significantly up-regulated in T7-T10 DRGs of diabetic rats. Intrathecal injection of CLI-095 (TLR4-selective inhibitor) attenuated diabetic gastric hypersensitivity, and markedly reversed the hyper-excitability of gastric-specific DRG neurons. (3) The expressions of miR-181a and miR-7a were significantly decreased in diabetic rats. MiR-181a could directly regulate the expression of TLR4, while miR-7a couldn't. (4) Intrathecal injection of miR-181a agomir down-regulated the expression of TLR4, reduced the hyper-excitability of gastric-specific neurons, and alleviated gastric hypersensitivity. (5) p65 and TLR4 were co-expressed in Dil-labeled DRG neurons. (6) Inhibition of p65 attenuated diabetic gastric hypersensitivity and hyper-excitability of gastric-specific DRG neurons. (7) The expression of TRAF6 was significantly up-regulated in diabetic rats. CLI-095 treatment also reduced the expression of TRAF6 and p65. Conclusion: The reduction of microRNA-181a in T7-T10 DRGs might up-regulate TLR4 expression. TLR4 activated NF-κB through MyD88-dependent signaling pathway, increased excitability of gastric-specific DRG neurons, and contributed to diabetic gastric hypersensitivity.

DNA methylation regulates Sirtuin 1 expression in osteoarthritic chondrocytes

PurposeSirtuin 1 (SIRT1) comprises a major anti-aging longevity factor with multiple protective effects on chondrocyte homeostasis. Previous studies have reported that downregulation of SIRT1 is linked to osteoarthritis (OA) progression. In this study, we aimed to investigate the role of DNA methylation on SIRT1 expression regulation and deacetylase activity in human OA chondrocytes. Materials and methodsMethylation status of SIRT1 promoter was analyzed in normal and OA chondrocytes using bisulfite sequencing analysis. CCAAT/enhancer binding protein alpha (C/EBPα) binding to SIRT1 promoter was assessed by chromatin immunoprecipitation (ChIP) assay. Subsequently, C/EBPα′s interaction with SIRT1 promoter and SIRT1 expression levels were evaluated after treatment of OA chondrocytes with 5-Aza-2′-Deoxycytidine (5-AzadC). Acetylation and nuclear levels of nuclear factor kappa-B p65 subunit (NF-κΒp65) and expression levels of selected OA-related inflammatory mediators, interleukin 1β (IL-1β) and IL-6 and catabolic genes (metalloproteinase-1 (MMP-1) and MMP-9) were evaluated in 5-AzadC-treated OA chondrocytes with or without subsequent transfection with siRNA against SIRT1. ResultsHypermethylation of specific CpG dinucleotides on SIRT1 promoter was associated with downregulation of SIRT1 expression in OA chondrocytes. Moreover, we found decreased binding affinity of C/EBPα on the hypermethylated SIRT1 promoter. 5-AzadC treatment restored C/EBPα′s transcriptional activity inducing SIRT1 upregulation in OA chondrocytes. Deacetylation of NF-κΒp65 in 5-AzadC-treated OA chondrocytes was prevented by siSIRT1 transfection. Similarly, 5-AzadC-treated OA chondrocytes exhibited decreased expression of IL-1β, IL-6, MMP-1 and MMP-9 which was reversed following 5-AzadC/siSIRT1 treatment. ConclusionsOur results suggest the impact of DNA methylation on SIRT1 suppression in OA chondrocytes contributing to OA pathogenesis.

Role of p65 NF-κB, caspase-3 activities and VEGF gene polymorphisms on the development of preterm labor in women with placental dysfunction

The objective: to study the activity of the p65 subunit of the nuclear factor – NF-κB and effector caspase-3, as well as the presence of a single nucleotide polymorphism of the VEGF gene (936C/T) in pregnant women with placental disorders and various clinical types of the course of premature labor (with premature rupture of the membranes and without it).Materials and methods. At the clinical base of the Department of Obstetrics and Gynecology No. 1 of O. O. Bogomolets National Medical University, which is located in the non-commercial enterprise “Perinatal Center of Kyiv”, during 2019–2022, a case-control study was conducted with the involvement of 90 pregnant women: 60 women of the main group with placental disorders and the development of spontaneous premature labor in the period of 24–34 weeks of gestation (I group – 30 women with premature labor and premature rupture of membranes, II group – 30 women with premature rupture of the membranes) and 30 women of control group (CG) with term normal delivery and the physiological course of the pregnancy.In pregnant women, the number of total and activated fractions of the p65 subunit of nuclear factor NF-κB and caspase-3 was determined by enzyme-linked immunosorbent assay (ELISA) in placenta lysates with further calculation of their activity based on these data, as well as the presence of a single nucleotide polymorphism of the VEGF gene (936C/T) using the polymerase chain reaction.Results. In women with placental disorders and preterm birth a significantly higher values of fraction content and activity of p65 NF-κB and caspase-3 were found, with some peculiarities within the groups compared to the control group. Women of the I group were characterized by a higher activity of p65-subunit of nuclear factor (I group — 61.6 % with 95 % CI 59.7–64.2; II group — 33.8 pg/ml with 95 % CI 31.2-35.2; CG — 27.3 pg/ml, 95 % CI 26.4–28.6; p&lt;0.05). Pregnant women of the II group had higher values of caspase-3 activity (II group — 59.2 % with 95 % CI 57.4–59.8, I group — 39.5 % with 95 % CI 38.5–40.5, CG — 31.2 %, 95 % CI 30.4–31.9; p&lt;0.01). It was established that the presence of the T allele at position 936 of VEGF gene polymorphism is a risk factor for the development of placental disorders with the development of premature labor, the rate of this allele in the main group was 11.7 % versus 1.7 % – in the control cohort (p&lt;0.05).Conclusions. Pregnant women with placental disorders and the development of premature birth are characterized by an increased activity of the p65 subunit of nuclear factor κB and caspase-3 compared to the control group: in women without premature rupture of the fetal membranes, a significant 2.2-fold increase in p65 NF-κB activity was found, and caspase-3 activity – by 1.3 times; in the group of pregnant women with preterm premature rupture of membranes the level of caspase-3 activity exceeded the control group by 1.9 times, and the level of p65-subunit of nuclear factor activity — by 1.2 times (p&lt;0.01 for all groups).936(C/T) single nucleotide polymorphism of the vascular endothelial growth factor gene was determined significantly more often in women with placental disorders and preterm birth (11.7 % in the study group versus 1.7 % in CG, p&lt;0.05), the T-allele on the VEGF gene carriers may be associated with the development of these pregnancy complications. However, a study on a larger sample of women is needed to obtain definitive results.

Smilax china L. Polyphenols Improves Insulin Resistance and Obesity in High-fat Diet-induced Mice Through IRS/AKT-AMPK and NF-κB Signaling Pathways.

Smilax china L. is an important herb used in traditional Chinese medicine. In this study, the mechanism of Smilax china L. polyphenols (SCP) on insulin resistance and anti-obesity in mice induced by a high-fat diet (HFD) was investigated. Fifty female mice were randomly divided into five groups: control, HFD and low, medium, and high doses of SCP for 70 d. SCP significantly decreased intraperitoneal adipose tissue index, body weight gain, liver lipids, and serum inflammatory factor levels. Blood glucose and insulin concentrations, as well as insulin resistance index in SCP, were significantly lower than those in HFD. In addition, SCP markedly up-regulated the gene expression of glucose transporter 4 (GLUT4), insulin receptor substrate 1 (IRS1), insulin receptor substrate 2 (IRS2), serine-threonine kinase (AKT), Acyl-CoA oxidase (ACO), and protein kinase A (PKA), and down-regulated the expression of mammalian target of rapamycin complex 1 (mTORC1), sterol-responsive element-binding protein-1c (SREBP1c), fatty acid synthase (FAS), 3-hydroxy-3-methyl glutaryl coenzyme A reductase (HMGCR), and forkhead box protein O1 (FOXO1). SCP significantly increased the protein expression of AKT, GLUT4, AMP-activated protein kinase (AMPK), phosphorylated-AMPK (p-AMPK), phosphorylated-AKT (p-AKT), and uncoupling protein 1 (UCP-1), and decreased the expression of SREBP1c, FAS, HMGCR, phosphorylation of IKBα (p-IKBα), and nuclear factor kappa B subunit p65 (P65) in the liver. Overall, SCP effectively reduced HFD-induced insulin resistance and obesity in mice, partly through NF-κB and IRS/AKT-AMPK signaling pathways to regulate inflammatory factors. Therefore, SCP may improve lifestyle diseases.

The Atypical MAP Kinase MAPK15 Is Required for Lung Adenocarcinoma Metastasis via Its Interaction with NF-κB p50 Subunit and Transcriptional Regulation of Prostaglandin E2 Receptor EP3 Subtype

Studying the relatively underexplored atypical MAP Kinase MAPK15 on cancer progression/patient outcomes and its potential transcriptional regulation of downstream genes would be highly valuable for the diagnosis, prognosis, and potential oncotherapy of malignant tumors such as lung adenocarcinoma (LUAD). Here, the expression of MAPK15 in LUAD was detected by immunohistochemistry and its correlation with clinical parameters such as lymph node metastasis and clinical stage was analyzed. The correlation between the prostaglandin E2 receptor EP3 subtype (EP3) and MAPK15 expression in LUAD tissues was examined, and the transcriptional regulation of EP3 and cell migration by MAPK15 in LUAD cell lines were studied using the luciferase reporter assay, immunoblot analysis, qRT-PCR, and transwell assay. We found that MAPK15 is highly expressed in LUAD with lymph node metastasis. In addition, EP3 is positively correlated with the expression of MAPK15 in LUAD tissues, and we confirmed that MAPK15 transcriptionally regulates the expression of EP3. Upon the knockdown of MAPK15, the expression of EP3 was down-regulated and the cell migration ability was decreased in vitro; similarly, the mesenteric metastasis ability of the MAPK15 knockdown cells was inhibited in in vivo animal experiments. Mechanistically, we demonstrate for the first time that MAPK15 interacts with NF-κB p50 and enters the nucleus, and NF-κB p50 binds to the EP3 promoter and transcriptionally regulates the expression of EP3. Taken together, we show that a novel atypical MAPK and NF-κB subunit interaction promotes LUAD cell migration through transcriptional regulation of EP3, and higher MAPK15 level is associated with lymph node metastasis in patients with LUAD.