Design, Synthesis, and Evaluation of Baicalein–Thiazole–Piperazine Hybrid As Inhibitor of NF-κB Activation for Hepatobiliary Inflammation Treatment

Serum proteins play an important role in LPS-induced cell activation. The LPS binding protein (LBP) enhances cellular responses to LPS, whereas the polymorphonuclear leukocyte product bactericidal/permeability-increasing protein (BPI) inhibits LPS-induced cell activation. In this study the influences of LBP and BPI, two proteins with opposite effects, but with considerable sequence homology, on LPS-induced mononuclear phagocytic cell cytokine release was studied. LBP was shown to enhance LPS-induced TNF-alpha, IL-6, and IL-8 release by mononuclear phagocytic cells, whereas BPI inhibited the release of these cytokines. Furthermore, the effects of LBP and BPI on LPS-induced cytokine release by mononuclear phagocytic cells were shown to be counteractive. BPI interfered with the enhancing effect of LBP on the LPS-induced cytokine release. At high LBP to BPI ratios, BPI could no longer inhibit LBP-induced enhancement. In accordance, increasing concentrations of BPI abrogated the LBP effect. Next, it was shown that LBP and BPI compete for binding to LPS by using an assay system that detects binding of free BPI to an anti-BPI mAb. LPS prevented binding of BPI to anti-BPI mAb, whereas preincubation of LPS with LBP prevented the LPS-induced inhibition. Also, it was observed that both BPI and LBP inhibited LPS activity in the chromogenic LAL assay. We conclude from this study that LBP and BPI have counteractive effects on LPS-induced mononuclear phagocytic cell cytokine release by competing for binding to LPS.

Bacterial Lipopolysaccharide Increases Interleukin-6 and Prostaglandin Release in Rat Cortical Type I Astrocytes by Different Mechanisms: Role of Anti-inflammatory Agents

Cytotoxic and inflammatory effects of contact lens solutions on human corneal epithelial cells in vitro.

Bronchial epithelial cell lines and primary nasal epithelial cells from cystic fibrosis respond differently to cigarette smoke exposure

BackgroundEndotoxemia is well known to be associated with an excessive host response to bacteria or microbial compounds, resulting in systemic inflammation and organ injury. The aim of the present study...

ObjectivesOccurring of acute coronary syndromes (ACSs) displayed circadian rhythms as well as the levels of catecholamines and pro-inflammatory cytokines in vivo. Such circadian rhythms were proved to exist also in...

BackgroundExperimental studies suggest that systemic inflammation contributes to the pathophysiology of delirium. The aim of our study was to determine blood-derived inflammatory signatures of post-stroke delirium.MethodsWe included 144 ischemic stroke patients. We assessed delirium on a daily basis during the first 7 days of hospitalization. Venous blood was collected at day 3 after the onset of stroke and stimulated ex vivo with lipopolysaccharide (LPS). We measured LPS-induced cytokine concentration (TNFα, IP-10, IL-1β, IL-6, IL-8, IL-10, and IL-12p70) as well as plasma levels of IL-6 and TNFα.ResultsDelirium was diagnosed in 21.5% of patients. After correction for monocyte count, patients with delirium had reduced LPS-induced TNFα, IP-10, IL-1β, IL-6, and IL-12 release. The plasma IL-6 level was higher in delirious patients compared to patients without delirium. After adjusting for stroke severity and infections, higher ex vivo TNFα (OR 0.29, 95%CI 0.11–0.72, P = 0.01), IP-10 (OR 0.25, 95%CI 0.08–0.73, P = 0.01), IL-1β (OR 0.42, 95%CI 0.20–0.89, P = 0.02), and IL-12 (OR 0.07, 95%CI 0.01–0.70, P = 0.02) release was associated with the reduced risk of delirium. In multivariate analysis, the higher plasma IL-6 was associated with the increased risk of delirium (OR 1.61, 95%CI 1.00–2.58, P = 0.04).ConclusionsReduced ex vivo release of pro-inflammatory cytokines after LPS stimulation and the elevated plasma IL-6 are signatures of post-stroke delirium.

Background/Aims: Lipopolysaccharide (LPS) plays a critical role in excessive inflammatory cytokine production during sepsis. Previously, artesunate (AS) was reported to protect septic mice by reducing LPS-induced pro-inflammatory cytokine release. In the present study, the possible mechanism of the anti-inflammatory effect of AS was further investigated. Methods: An enzyme-linked immunosorbent assay was used to detect TNF-α and IL-6 release from macrophages. Specific small interfering RNAs (siRNAs) were used to knockdown the mRNA expression of target genes. Transmission electron microscopy and laser confocal microscopy were used to observe changes in autophagy. Western blotting was performed to detect the protein levels of tumor necrosis factor receptor-associated factor6 (TRAF6), Beclin1, phosphatidylinositol 3-kinase class III (PI3KC3), autophagy-related protein 5 (ATG5), and sequestosome 1. Immunoprecipitation (IP) and fluorescent co-localization were used to detect the interactions between TRAF6–Beclin1 and Beclin1–PI3KC3, and the ubiquitination of Beclin1. Results: AS inhibited TNF-α and IL-6 release from RAW264.7 cells, mouse bone marrow-derived monocytes (BMDMs) and peritoneal macrophages (PMs) induced by LPS. However, the inhibition by AS of LPS-induced cytokine release decreased when autophagy was inhibited using 3-MA, bafilomycin A1, or a siRNA targeting the Atg5 gene. Notably, AS showed an inhibition of LPS-induced autophagic activation not degradation. Whereas, these effects of AS were lost in macrophages lacking TLR4 and decreased in macrophages with down-regulated TRAF6, indicating that AS inhibited LPS-induced cytokine release and autophagic activation via TLR4-TRAF6 signaling. Western blotting results showed AS could reduce the levels of TRAF6, Beclin1, and PI3KC3. Importantly, the IP results showed AS only inhibited K63-linked ubiquitylation not total ubiquitylation of Beclin1 by acting on TRAF6. This interrupted the TRAF6–Beclin1 interaction and subsequent the formation of Beclin1– PI3KC3 core complex of the PI3K-III complex. Conclusion: AS inhibited LPS-induced cytokine release from macrophages by inhibiting autophagic activation. This effect was tightly related to blockade of the TRAF6-Beclin1-PI3KC3 pathway via decreasing K63-linked ubiquitination of Beclin1 and then interrupting the formation of Beclin1-PI3KC3 core complex of the PI3K-III complex. Our findings reveal the mechanism of AS’s anti-inflammatory effect and is significant for future targeted investigations of sepsis treatment.

TGFβ signaling through Smad3 plays a multifunctional role within the liver, promoting myofibroblast production of collagen and inducing hepatocellular apoptosis. The purpose of the current study was to evaluate the importance of Smad3 signaling during chronic cholestasis. Wild type (wt) or Smad3−/− mice were subjected to ligation of the common bile duct (BDL) or sham surgery and allowed to recover. Twenty one days following BDL, wt mice presented with significant increases in serum transaminases and hepatocellular apoptosis as assessed by terminal UTP nick end labeling (TUNEL) as well as significant myofibroblast activation and hepatic collagen deposition. Smad3−/− mice subjected to the same duration of cholestasis present with significant reductions in hepatocellular injury as assessed by serum aspartate aminotransferase levels and reduced hepatocellular apoptosis, findings which correlated with reduced collagen deposition but similar stellate cell activation. Together, these data demonstrate the importance of Smad3 signaling not only in the production of collagen by intrahepatic myofibroblasts but also in the induction of hepatocellular apoptosis following chronic cholestasis. Interruption of TGFβ signaling may therefore represent an important therapeutic target for the treatment of both hepatocellular injury and tissue fibrosis. This work was supported by NIH grant AA0014243.

Histone methylation is an important epigenetic mechanism that plays an essential role in regulating gene expression in mammalian cells. To understand its influence on inflammation, methylation of H3K4, H3K9, H3K36, H3K79, and H4K20, the most common histones methylated in the inflammatory response was analyzed in murine RAW264.7 cells and bone marrow-derived macrophages (BMDMs) upon lipopolysaccharide (LPS) stimulation. LPS stimulation resulted in enhanced methylation at H3K4 and H3K9 in both RAW264.7 and BMDMs. To further confirm whether LPS-stimulated H3K4me2 and H3K9me2 were responsible for subsequent proinflammatory cytokine expression, the recruitment of H3K4me2 and H3K9me2 at the promoters of interleukin (IL)-6 and tumor necrosis factor-α (TNF-α) was assessed. H3K4me2, but not H3K9me2, was enriched at the promoters of both IL-6 and TNF-α. Furthermore, LPS-stimulated gene expression and release of IL-6 and TNF-α were markedly suppressed in macrophages by MTA, a specific inhibitor of H3K4 methylation. These results demonstrate that histone methylation, in particular H3K4me2, plays a critical role in the regulation of LPS-induced expression and release of IL-6 and TNF-α.

Sepsis is associated with myocardial injury and early mortality. The innate immune receptor Toll-like receptor 4 (TLR4) can recognize pathogen-associated-molecular-patterns (PAMPs) and damage-associated molecular patterns (DAMPs); the latter are released during tissue injury. We hypothesized that TLR4 inhibition reduces proinflammatory signaling and cytokine release in: (1) LPS or Escherichia coli-treated isolated mouse heart; (2) LPS-treated mouse primary adult cardiomyocytes; and (3) the isolated heart during ischemia-reperfusion. Isolated C57BL/6N male mouse hearts were perfused for 120 min, with either LPS, E. coli, with and without CLI-095 (TLR4 inhibitor). Primary adult mouse cardiomyocytes were treated with LPS or LPS + CLI-095. Isolated hearts, exposed to 35 min of global ischemia, were treated with either vehicle or CLI-095 during reperfusion. Infarct size was quantified by triphenyltetrazolium staining. Cytokine expression was analyzed with ELISA, western blot analysis, and qPCR. In isolated hearts, E. coli increased the expression of proinflammatory cytokines (IL-6 and CXCL2), which was not attenuated with TLR4 inhibition. TLR4 inhibition reduced expression (p = 0.004) and release of IL-6 (p < 0.0001) in LPS-exposed isolated hearts. LPS activated the nuclear-factor κ-light-chain-enhancer of activated B cells signaling pathway (NF-κB) in primary adult cardiomyocytes. Moreover, TLR4 inhibition reduced LPS-induced mRNA expression and release of IL-6 in primary adult cardiomyocytes. Isolated hearts treated with CLI-095 during reperfusion after ischemia (induced DAMPs release) showed reduced infarct size (39 ± 17% to 26 ± 8%, p = 0.034) and decreased IL-6 release (p = 0.006). Inhibition of TLR4 reduced proinflammatory signaling and cytokine release in LPS-treated and ischemia-reperfused isolated mouse hearts and in primary adult murine cardiomyocytes.

Adenoid hypertrophy (AH) is a common pediatric disease caused by inflammatory stimulation. The pro-inflammatory cytokine IL-32 has been reported to promote airway inflammation and also be involved in the pyroptosis pathway. However, whether IL-32 can contribute to AH by mediating pyroptosis remains to be elucidated. The present study aimed to investigate the role of IL-32 in AH and determine the potential underlying mechanisms. Adenoid tissues were collected from healthy children and children with AH, and the expression of IL-32, NACHT LRR and PYD domains-containing protein 3 (NLRP3) and IL-1β in normal and hypertrophic tissues were measured. Human nasal epithelial cells (HNEpCs) were exposed to a series of IL-32 concentrations. HNEpCs with or without IL-32 silencing were stimulated with lipopolysaccharide (LPS), and cell proliferation, cell apoptosis, gasdermin D (GSDMD) activation, production of inflammatory cytokines and the expression levels of proteins related to the potential mechanisms were evaluated by Cell Counting Kit-8, flow cytometry, immunofluorescence staining, ELISA and western blot assays, respectively. The results showed that IL-32, NLRP3 and IL-1β exhibited higher expression in adenoid tissues with AH compared with normal tissues. In HNEpC cells, treatment with IL-32 (2 and 10 ng/ml) promoted cell proliferation, while 50 ng/ml IL-32 inhibited cell proliferation at 12, 24 and 48 h post-treatment. IL-32 (2, 10 and 50 ng/ml) also resulted in differing degrees of apoptosis, GSDMD activation, release of IL-1β, IL-6 and TNF-α, and increased protein expression levels of NLRP3, cleaved-caspase-1, activated GSDMD, nucleotide-binding oligomerization domain-containing protein (NOD) 1/2 and Toll-like receptor (TLR)4 in a concentration-dependent manner. In addition, compared with the LPS group, IL-32 knockdown significantly inhibited LPS-induced enhancement of cell proliferation, cell apoptosis, GSDMD activation and production of inflammatory cytokines, and reversed the increased protein expression of NLRP3, cleaved-caspase-1, activated GSDMD, NOD1/2 and TLR4. In conclusion, IL-32 may play a role in the progression of AH via promoting inflammation, and the potential mechanism may involve the activation of NLRP3-mediated pyroptosis.

BackgroundCompelling evidence has implicated neuroinflammation in the pathogenesis of a number of neurodegenerative conditions. Chronic activation of both astrocytes and microglia leads to excessive secretion of proinflammatory molecules such as TNFα, IL-6 and IL-1β with potentially deleterious consequences for neuronal viability. Many signaling pathways involving the mitogen-activated protein kinases (MAPKs), nuclear factor κB (NFκB) complex and the Janus kinases (JAKs)/signal transducers and activators of transcription (STAT)-1 have been implicated in the secretion of proinflammatory cytokines from glia. We sought to identify signaling kinases responsible for cytokine production and to delineate the complex interactions which govern time-related responses to lipopolysaccharide (LPS).MethodsWe examined the time-related changes in certain signaling events and the release of proinflammatory cytokines from LPS-stimulated co-cultures of astrocytes and microglia isolated from neonatal rats.ResultsTNFα was detected in the supernatant approximately 1 to 2 hours after LPS treatment while IL-1β and IL-6 were detected after 2 to 3 and 4 to 6 hours, respectively. Interestingly, activation of NFκB signaling preceded release of all cytokines while phosphorylation of STAT1 was evident only after 2 hours, indicating that activation of JAK/STAT may be important in the up-regulation of IL-6 production. Additionally, incubation of glia with TNFα induced both phosphorylation of JAK2 and STAT1 and the interaction of JAK2 with the TNFα receptor (TNFR1). Co-treatment of glia with LPS and recombinant IL-6 protein attenuated the LPS-induced release of both TNFα and IL-1β while potentiating the effect of LPS on suppressor of cytokine signaling (SOCS)3 expression and IL-10 release.ConclusionsThese data indicate that TNFα may regulate IL-6 production through activation of JAK/STAT signaling and that the subsequent production of IL-6 may impact on the release of TNFα, IL-1β and IL-10.

We had demonstrated that the intragastric administration of distilled water increased male rat jejunal lymph flow and interleukin-22 levels in the lymph, resulting in the promotion of innate immunity. However, there is no information concerning whether the water intake regulates mucosal immunity in the jejunum of male rats. Based on the evidence, we investigated the effects of distilled water intake on gut immunity in the rat jejunum. Water intake significantly stimulated the release of IL-1β and IL-6 in mesenteric lymph. However, the concentration of IL-10 was not changed by water intake. Pretreatment with clodronate significantly decreased the lymph flow at 120-180 min after the intake. Clodronate significantly reduced water intake-mediated release of IL-1β and decreased, but not significantly, the release of IL-6. MyD88 inhibitor significantly decreased water intake-mediated release of IL-1β and IL-6. However, clodronate and MyD88 inhibitor did not significantly change IL-10 in the lymph. The expression levels of the macrophage markers CD68, F4/80, and CD169 in the jejunal villi significantly decreased due to pretreatment with clodronate. These findings suggest that water intake acts as a trigger for starting mucosal immunity in the jejunum via macrophage and MyD88-mediated activation of the release of IL-1β and IL-6.

Bradykinin- and lipopolysaccharide-induced bradykinin B2 receptor expression, interleukin 8 release and “nitrosative stress” in bronchial epithelial cells BEAS-2B: Role for neutrophils