Effect of Carbon Monoxide-Releasing Molecules II-liberated CO on Suppressing Inflammatory Response in Sepsis by Interfering with Nuclear Factor Kappa B Activation

Sepsis continues to be a challenge in clinic. The rates of mortality in sepsis patients remain high. The present study aimed to investigate the effects and the underlying mechanisms of carbon monoxide-releasing molecules II (CORM-2)-liberated CO on suppressing inflammatory response in sepsis. It was shown that treatment of septic mice with CORM-2 attenuated PMN accumulation, downregulated cytokines production, inhibited expressions of iNOS and NF-κB activity in the lung and liver. In parallel, CORM-2 prevented activation of NF-κB in LPS-stimulated HUVEC. This was accompanied by a decrease in ROS and NO production, expression of ICAM-1 and subsequent PMN adhesion to HUVEC. These findings demonstrated that CORM-released CO attenuates inflammatory responses by interfering with NF-κB activation and therefore decreasing the expression of ICAM-1 and NO production, attenuating the oxidative stress and inflammation in sepsis.     

Hydrogen sulfide attenuated tumor necrosis factor-α-induced inflammatory signaling and dysfunction in vascular endothelial cells

Background

Hydrogen sulfide (H₂S), the third physiologically relevant gaseous molecule, is recognized increasingly as an anti-inflammatory mediator in various inflammatory conditions. Herein, we explored the effects and mechanisms of sodium hydrosulfide (NaHS, a H₂S donor) on tumor necrosis factor (TNF)-α-induced human umbilical vein endothelial cells (HUVEC) dysfunction.

Methodology and Principal Findings

Application of NaHS concentration-dependently suppressed TNF-α-induced mRNA and proteins expressions of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), mRNA expression of P-selectin and E-selectin as well as U937 monocytes adhesion to HUVEC. Western blot analysis revealed that the expression of the cytoprotective enzyme, heme oxygenase-1 (HO-1), was induced and coincident with the anti-inflammatory action of NaHS. Furthermore, TNF-α-induced NF-κB activation assessed by IκBα degradation and p65 phosphorylation and nuclear translocation and ROS production were diminished in cells subjected to treatment with NaHS.

Significance

H₂S can exert an anti-inflammatory effect in endothelial cells through a mechanism that involves the up-regulation of HO-1.     

Anesthetic propofol reduces endotoxic inflammation by inhibiting reactive oxygen species-regulated Akt/IKKβ/NF-κB signaling

Background

Anesthetic propofol has immunomodulatory effects, particularly in the area of anti-inflammation. Bacterial endotoxin lipopolysaccharide (LPS) induces inflammation through toll-like receptor (TLR) 4 signaling. We investigated the molecular actions of propofol against LPS/TLR4-induced inflammatory activation in murine RAW264.7 macrophages.

Methodology/Principal Findings

Non-cytotoxic levels of propofol reduced LPS-induced inducible nitric oxide synthase (iNOS) and NO as determined by western blotting and the Griess reaction, respectively. Propofol also reduced the production of tumor necrosis factor-α (TNF-α), interleukin (IL)-6, and IL-10 as detected by enzyme-linked immunosorbent assays. Western blot analysis showed propofol inhibited LPS-induced activation and phosphorylation of IKKβ (Ser180) and nuclear factor (NF)-κB (Ser536); the subsequent nuclear translocation of NF-κB p65 was also reduced. Additionally, propofol inhibited LPS-induced Akt activation and phosphorylation (Ser473) partly by reducing reactive oxygen species (ROS) generation; inter-regulation that ROS regulated Akt followed by NF-κB activation was found to be crucial for LPS-induced inflammatory responses in macrophages. An in vivo study using C57BL/6 mice also demonstrated the anti-inflammatory properties against LPS in peritoneal macrophages.

Conclusions/Significance

These results suggest that propofol reduces LPS-induced inflammatory responses in macrophages by inhibiting the interconnected ROS/Akt/IKKβ/NF-κB signaling pathways.     

Isovitexin Exerts Anti-Inflammatory and Anti-Oxidant Activities on Lipopolysaccharide-Induced Acute Lung Injury by Inhibiting MAPK and NF-κB and Activating HO-1/Nrf2 Pathways

Oxidative damage and inflammation are closely associated with the pathogenesis of acute lung injury (ALI). Thus, we explored the protective effect of isovitexin (IV), a glycosylflavonoid, in the context of ALI. To accomplish this, we created in vitro and in vivo models by respectively exposing macrophages to lipopolysaccharide (LPS) and using LPS to induce ALI in mice. In vitro, our results showed that IV treatment reduced LPS-induced pro-inflammatory cytokine secretion, iNOS and COX-2 expression and decreased the generation of ROS. Consistent findings were obtained in vivo. Additionally, IV inhibited H₂O₂-induced cytotoxicity and apoptosis. However, these effects were partially reversed following the use of an HO-1 inhibitor in vitro. Further studies revealed that IV significantly inhibited MAPK phosphorylation, reduced NF-κB nuclear translocation, and upregulated nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase 1 (HO-1) expression in RAW 264.7 cells. In vivo, pretreatment with IV attenuated histopathological changes, infiltration of polymorphonuclear granulocytes and endothelial activation, decreased the expression of ICAM-1 and VCAM-1, reduced the levels of MPO and MDA, and increased the content of GSH and SOD in ALI. Furthermore, IV treatment effectively increased Nrf2 and HO-1 expression in lung tissues. Therefore, IV may offer a protective role against LPS-induced ALI by inhibiting MAPK and NF-κB and activating HO-1/Nrf2 pathways.     

The complex effects of the slow‐releasing hydrogen sulfide donor GYY4137 in a model of acute joint inflammation and in human cartilage cells

The role of hydrogen sulfide (H₂S) in inflammation remains unclear with both pro- and anti-inflammatory actions of this gas described. We have now assessed the effect of GYY4137 (a slow-releasing H₂S donor) on lipopolysaccharide (LPS)-evoked release of inflammatory mediators from human synoviocytes (HFLS) and articular chondrocytes (HAC) in vitro. We have also examined the effect of GYY4137 in a complete Freund''s adjuvant (CFA) model of acute joint inflammation in the mouse. GYY4137 (0.1–0.5 mM) decreased LPS-induced production of nitrite (NO₂⁻), PGE₂, TNF-α and IL-6 from HFLS and HAC, reduced the levels and catalytic activity of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) and reduced LPS-induced NF-κB activation in vitro. Using recombinant human enzymes, GYY4137 inhibited the activity of COX-2, iNOS and TNF-α converting enzyme (TACE). In the CFA-treated mouse, GYY4137 (50 mg/kg, i.p.) injected 1 hr prior to CFA increased knee joint swelling while an anti-inflammatory effect, as demonstrated by reduced synovial fluid myeloperoxidase (MPO) and N-acetyl-β-D-glucosaminidase (NAG) activity and decreased TNF-α, IL-1β, IL-6 and IL-8 concentration, was apparent when GYY4137 was injected 6 hrs after CFA. GYY4137 was also anti-inflammatory when given 18 hrs after CFA. Thus, although GYY4137 consistently reduced the generation of pro-inflammatory mediators from human joint cells in vitro, its effect on acute joint inflammation in vivo depended on the timing of administration.     

alpha-lipoic acid inhibits endotoxin-stimulated expression of iNOS and nitric oxide independent of the heat shock response in RAW 264.7 cells

The heat shock response protects against sepsis-induced mortality, organ injury, cardiovascular dysfunction, and apoptosis. Several inducers of the heat shock response, such as hyperthermia, sodium arsenite, and pyrollidine dithiocarbonate, inhibit NF-κB activation and nitric oxide formation. The antioxidant lipoic acid (LA) has recently been found to inhibit NF-κB activation and nitric oxide formation. We therefore tested the hypothesis that LA induces a heat shock response. To test this hypothesis, we determined whether exposure to LA affects expression of both heat shock protein 70 (HSP-70) and nuclear heat shock factor-1 (HSF-1) in lipopolysaccharide (LPS) stimulated macrophages. LA and hyperthermia attenuated LPS-induced increases in nuclear NF-κB, iNOS protein, and media nitrite concentrations. LPS and hyperthermia increased HSP-70 concentrations 8-fold and 20-fold, respectively. No effect of LA treatment alone on HSP-70 protein expression was detected. Likewise, no effect of LA on HSF-1 protein expression was detected. These data suggest that LA inhibits LPS-induced activation of iNOS in macrophages independent of the heat shock response.     

Carbon Monoxide (CO) Released from Tricarbonyldichlororuthenium (II) Dimer (CORM-2) in Gastroprotection against Experimental Ethanol-Induced Gastric Damage

The physiological gaseous molecule, carbon monoxide (CO) becomes a subject of extensive investigation due to its vasoactive activity throughout the body but its role in gastroprotection has been little investigated. We determined the mechanism of CO released from its donor tricarbonyldichlororuthenium (II) dimer (CORM-2) in protection of gastric mucosa against 75% ethanol-induced injury. Rats were pretreated with CORM-2 30 min prior to 75% ethanol with or without 1) non-selective (indomethacin) or selective cyclooxygenase (COX)-1 (SC-560) and COX-2 (celecoxib) inhibitors, 2) nitric oxide (NO) synthase inhibitor L-NNA, 3) ODQ, a soluble guanylyl cyclase (sGC) inhibitor, hemin, a heme oxygenase (HO)-1 inductor or zinc protoporphyrin IX (ZnPPIX), an inhibitor of HO-1 activity. The CO content in gastric mucosa and carboxyhemoglobin (COHb) level in blood was analyzed by gas chromatography. The gastric mucosal mRNA expression for HO-1, COX-1, COX-2, iNOS, IL-4, IL-1β was analyzed by real-time PCR while HO-1, HO-2 and Nrf2 protein expression was determined by Western Blot. Pretreatment with CORM-2 (0.5–10 mg/kg) dose-dependently attenuated ethanol-induced lesions and raised gastric blood flow (GBF) but large dose of 100 mg/kg was ineffective. CORM-2 (5 mg/kg and 50 mg/kg i.g.) significantly increased gastric mucosal CO content and whole blood COHb level. CORM-2-induced protection was reversed by indomethacin, SC-560 and significantly attenuated by celecoxib, ODQ and L-NNA. Hemin significantly reduced ethanol damage and raised GBF while ZnPPIX which exacerbated ethanol-induced injury inhibited CORM-2- and hemin-induced gastroprotection and the accompanying rise in GBF. CORM-2 significantly increased gastric mucosal HO-1 mRNA expression and decreased mRNA expression for iNOS, IL-1β, COX-1 and COX-2 but failed to affect HO-1 and Nrf2 protein expression decreased by ethanol. We conclude that CORM-2 released CO exerts gastroprotection against ethanol-induced gastric lesions involving an increase in gastric microcirculation mediated by sGC/cGMP, prostaglandins derived from COX-1, NO-NOS system and its anti-inflammatory properties.     

API2-MALT1 oncoprotein promotes lymphomagenesis via unique program of substrate ubiquitination and proteolysis

Lymphoma of mucosa-associated lymphoid tissue(MALT lymphoma) is the most common extranodal B cell tumor and accounts for 8% of non-Hodgkin's lymphomas. Gastric MALT lymphoma is the best-studied example and is a prototypical neoplasm that occurs in the setting of chronic inflammation brought on by persistent infection or autoimmune disease. Cytogenetic abnormalities are commonly acquired during the course of disease and the most common is chromosomal translocation t(11;18)(q21;q21), which creates the API2-MALT1 fusion oncoprotein. t(11;18)-positive lymphomas can be clinically aggressive and have a higher rate of dissemination than t(11;18)-negative tumors. Many cancers, including MALT lymphomas, characteristically exhibit deregulated over-activation of cellular survival pathways, such as the nuclear factor-κB(NF-κB) pathway. Molecular characterization of API2-MALT1 has revealed it to be a potent activator of NF-κB, which is required for API2-MALT1-induced cellular transformation, however the mechanisms by which API2-MALT1 exerts these effects are only recently becoming apparent. The API2 moiety of the fusion binds tumor necrosis factor(TNF) receptor associated factor(TRAF) 2 and receptor interacting protein 1(RIP1), two proteins essential for TNF receptor induced NF-κB activation. By effectively mimicking ligand-bound TNF receptor, API2-MALT1 promotes TRAF2-dependent ubiquitination of RIP1, which then acts as a scaffold for nucleating and activating the canonical NF-κB machinery. Activation occurs, in part, through MALT1 moiety-dependent recruitment of TRAF6, which can directly modify NF-κB essential modulator, the principal downstream regulator of NF-κB. While theintrinsic MALT1 protease catalytic activity is dispensable for this canonical NF-κB signaling, it is critical for noncanonical NF-κB activation. In this regard, API2-MALT1 recognizes NF-κB inducing kinase(NIK), the essential upstream regulator of non-canonical NF-κB, and cleaves it to generate a stable, constitutively active fragment. Thus, API2-MALT1 harnesses multiple unique pathways to achieve deregulated NF-κB activation. Emerging data from our group and others have also detailed additional gain-of-function activities of API2-MALT1 that extend beyond NF-κB activation. Specifically, API2-MALT1 recruits and subverts multiple other signaling factors, including LIM domain and actin-binding protein 1(LIMA1) and Smac/DIABLO. Like NIK, LIMA1 represents a unique substrate for API2-MALT1 protease activity, but unlike NIK, its cleavage sets in motion a major NF-κB-independent pathway for promoting oncogenesis. In this review, we highlight the most recent results characterizing these unique and diverse gain-of-function activities of API2-MALT1 and how they contribute to lymphomagenesis.     

Histone Deacetylase Inhibitors Activate NF-��B in Human Leukemia Cells through an ATM/NEMO-related Pathway

Mechanisms underlying histone deacetylase inhibitor (HDACI)-mediated NF-κB activation were investigated in human leukemia cells. Exposure of U937 and other leukemia cells to LBH-589 induced reactive oxygen species (ROS) followed by single strand (XRCC1) and double strand (γ-H2AX) DNA breaks. Notably, LBH-589 lethality was markedly attenuated by small interfering RNA (siRNA) knockdown of the DNA damage-linked histone, H1.2. LBH-589 triggered p65/RelA activation, NF-κB-dependent induction of Mn-SOD2, and ROS elimination. Interference with LBH-589-mediated NF-κB activation (e.g. in IκBα super-repressor transfected cells) diminished HDACI-mediated Mn-SOD2 induction and increased ROS accumulation, DNA damage, and apoptosis. The Mn-SOD2 mimetic TBAP (manganese(III)-tetrakis 4-benzoic acid porphyrin) prevented HDACI-induced ROS and NF-κB activation while dramatically attenuating DNA damage and cell death. In contrast, TRAF2 siRNA knockdown, targeting receptor-mediated NF-κB activation, blocked TNFα- but not HDACI-mediated NF-κB activation and lethality. Consistent with ROS-mediated DNA damage, LBH-589 exposure activated ATM (on serine 1981) and increased its association with NEMO. Significantly, siRNA NEMO or ATM knockdown blocked HDACI-mediated NF-κB activation, resulting in diminished MnSOD2 induction and enhanced oxidative DNA damage and cell death. In accord with the recently described DNA damage/ATM/NEMO pathway, SUMOylation site mutant NEMO (K277A or K309A) cells exposed to LBH-589 displayed diminished ATM/NEMO association, NEMO and p65/RelA nuclear localization/activation, and MnSOD2 up-regulation. These events were accompanied by increased ROS production, γ-H2AX formation, and cell death. Together, these findings indicate that in human leukemia cells, HDACIs activate the cytoprotective NF-κB pathway through an ATM/NEMO/SUMOylation-dependent process involving the induction of ROS and DNA damage and suggest that blocking NF-κB activation via the atypical ATM/NEMO nuclear pathway can enhance HDACI antileukemic activity.     

Proinflammatory Cytokine and Nitric Oxide Production by Human Macrophages Stimulated with Trichomonas vaginalis

Trichomonas vaginalis commonly causes vaginitis and perhaps cervicitis in women and urethritis in men and women. Macrophages are important immune cells in response to T. vaginalis infection. In this study, we investigated whether human macrophages could be involved in inflammation induced by T. vaginalis. Human monocyte-derived macrophages (HMDM) were co-cultured with T. vaginalis. Live, opsonized-live trichomonads, and T. vaginalis lysates increased proinflammatory cytokines, such as TNF-α, IL-1β, and IL-6 by HMDM. The involvement of nuclear factor (NF)-κB signaling pathway in cytokine production induced by T. vaginalis was confirmed by phosphorylation and nuclear translocation of p65 NF-κB. In addition, stimulation with live T. vaginalis induced marked augmentation of nitric oxide (NO) production and expression of inducible NO synthase (iNOS) levels in HMDM. However, trichomonad-induced NF-κB activation and TNF-α production in macrophages were significantly inhibited by inhibition of iNOS levels with L-NMMA (NO synthase inhibitor). Moreover, pretreatment with NF-κB inhibitors (PDTC or Bay11-7082) caused human macrophages to produce less TNF-α. These results suggest that T. vaginalis stimulates human macrophages to produce proinflammatory cytokines, such as IL-1, IL-6, and TNF-α, and NO. In particular, we showed that T. vaginalis induced TNF-α production in macrophages through NO-dependent activation of NF-κB, which might be closely involved in inflammation caused by T. vaginalis.     

Involvement of protein kinase C in the inhibition of lipopolysaccharide-induced nitric oxide production by thapsigargin in RAW 264.7 macrophages

This study explored the effects of inhibition of endoplasmic reticulum (ER) Ca²⁺-ATPase on lipopolysaccharide (LPS)-induced protein kinase C (PKC) activation, nuclear factor-κB (NF-κB) translocation, inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production in RAW 264.7 macrophages. Thapsigargin (TG) irreversibly inhibits ER Ca²⁺-ATPase and LPS-induced NO production is reduced even after washout. TG also attenuated LPS-stimulated iNOS expression by using immunoblot analysis. However, another distinct fully reversible ER Ca²⁺-ATPase inhibitor, 2,5-di-tert-butylhydroquinone (DBHQ), ionophore A23187 and ionomycin could exert a similar effect to TG in increasing intracellular calcium concentration; however, these agents could not mimic TG in reducing iNOS expression and NO production. LPS increased PKC- and -β activation, and TG pretreatment attenuated LPS-stimulated PKC activation. Not did pretreatment with DBHQ, A23187 and ionomycin reduce LPS-stimulated PKC activation. Furthermore, NF-κB-specific DNA–protein-binding activity in the nuclear extracts was enhanced by treatment with LPS, and TG pretreatment attenuated LPS-stimulated NF-κB activation. None of DBHQ, A23187 and ionomycin pretreatment reduced LPS-stimulated NF-κB activation. These data suggest that persistent inhibition of ER Ca²⁺-ATPase by TG would influence calcium release from ER Ca²⁺ pools that was stimulated by the LPS activated signal processes, and might be the main mechanism for attenuating PKC and NF-κB activation that induces iNOS expression and NO production.     

Ubiquitin-specific Peptidase 21 Inhibits Tumor Necrosis Factor ��-induced Nuclear Factor ��B Activation via Binding to and Deubiquitinating Receptor-interacting Protein 1

Ubiquitination and deubiquitination of receptor-interacting protein 1 (RIP1) play an important role in the positive and negative regulation of the tumor necrosis factor α (TNFα)-induced nuclear factor κB (NF-κB) activation. Using a combination of functional genomic and proteomic approaches, we have identified ubiquitin-specific peptidase 21 (USP21) as a deubiquitinase for RIP1. USP21 is constitutively associated with RIP1 and deubiquitinates RIP1 in vitro and in vivo. Notably, knockdown of USP21 in HeLa cells enhances TNFα-induced RIP1 ubiquitination, IκB kinase β (IKKβ), and NF-κB phosphorylation, inhibitor of NF-κB α (IκBα) phosphorylation and ubiquitination, as well as NF-κB-dependent gene expression. Therefore, our results demonstrate that USP21 plays an important role in the down-regulation of TNFα-induced NF-κB activation through deubiquitinating RIP1.     

Immunostimulating activity of maysin isolated from corn silk in murine RAW 264.7 macrophages

Corn silk (CS) has long been consumed as a traditional herb in Korea. Maysin is a major flavonoid of CS. The effects of maysin on macrophage activation were evaluated, using the murine macrophage RAW 264.7 cells. Maysin was isolated from CS by methanol extraction, and preparative C₁₈ reverse phase column chromatography. Maysin was nontoxic up to 100 μg/ml, and dose-dependently increased TNF-α secretion and iNOS production by 11.2- and 4.2-fold, respectively, compared to untreated control. The activation and subsequent nuclear translocation of NF-κB was substantially enhanced upon treatment with maysin (1-100 μg/ml). Maysin also stimulated the phosphorylation of Akt and MAPKs (ERK, JNK). These results indicated that maysin activates macrophages to secrete TNF-α and induce iNOS expression, via the activation of the Akt, NF-κB and MAPKs signaling pathways. These results suggest for the first time that maysin can be a new immunomodulator, enhancing the early innate immunity. [BMB Reports 2014; 47(7): 382-387]     

Salicortin suppresses lipopolysaccharide-stimulated inflammatory responses via blockade of NF-κB and JNK activation in RAW 264.7 macrophages

We isolated the phenolic glucoside salicortin from a Populus euramericana bark extract, and examined its ability to suppress inflammatory responses as well as the molecular mechanisms underlying these abilities, using lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Salicortin inhibited iNOS expression and the subsequent production of NO in a dose-dependent manner in the LPS-stimulated RAW 264.7 cells. Salicortin significantly suppressed LPS-induced signal cascades of NF-κB activation, such as IKK activation, IκBα phosphorylation and p65 phosphorylation in RAW 264.7 cells. In addition, salicortin inhibited the LPS-induced activation of JNK, but not ERK or p38 MAPK. Furthermore, salicortin significantly inhibited production of pro-inflammatory cytokines, such as TNF-α, IL-1β and IL-6 in the LPS-stimulated RAW 264.7 cells. These findings suggest that salicortin may show its anti-inflammatory activity by suppressing the LPS-induced expression of pro-inflammatory mediators through inhibition of NF-κB and JNK MAPK signaling cascades in macrophages. [BMB Reports 2014; 47(6): 318-323]