Nitric Oxide Production In J774A Research Articles

A novel mechanism of Korean Red Ginseng-mediated anti-inflammatory action via targeting caspase-11 non-canonical inflammasome in macrophages

BackgroundKorean Red Ginseng (KRG) was reported to play an anti-inflammatory role, however, previous studies largely focused on the effects of KRG on priming step, the inflammation-preparing step, and the anti-inflammatory effect of KRG on triggering, the inflammation-activating step has been poorly understood. This study demonstrated anti-inflammatory role of KRG in caspase-11 non-canonical inflammasome activation in macrophages during triggering of inflammatory responses. MethodsCaspase-11 non-canonical inflammasome-activated J774A.1 macrophages were established by priming with Pam3CSK4 and triggering with lipopolysaccharide (LPS). Cell viability and pyroptosis were examined by MTT and lactate dehydrogenase (LDH) assays. Nitric oxide (NO)-inhibitory effect of KRG was assessed using a NO production assay. Expression and proteolytic cleavage of proteins were examined by Western blotting analysis. In vivo anti-inflammatory action of KRG was evaluated with the LPS-injected sepsis model in mice. ResultsKRG reduced LPS-stimulated NO production in J774A.1 cells and suppressed pyroptosis and IL-1β secretion in caspase-11 non-canonical inflammasome-activated J774A.1 cells. Mechanistic studies demonstrated that KRG suppressed the direct interaction between LPS and caspase-11 and inhibited proteolytic processing of both caspase-11 and gasdermin D in caspase-11 non-canonical inflammasome-activated J774A.1 cells. Furthermore, KRG significantly ameliorated LPS-mediated lethal septic shock in mice. ConclusionThe results demonstrate a novel mechanism of KRG-mediated anti-inflammatory action that operates through targeting the caspase-11 non-canonical inflammasome at triggering step of macrophage-mediated inflammatory response.

Preparation of Amino Alcohols Condensed with Carbohydrates: Evaluation of Cytotoxicity and Inhibitory Effect on NO Production

This work reports the preparation of several amino alcohols condensed with d-arabinose, d-glucose, and d-galactose derivatives. These compounds were evaluated in vitro for their cytotoxicity and ability to decrease nitric oxide production in J774A.1 cells. Arabinofuranoside derivatives 5a, 5b and 5c showed a significant inhibition of nitric oxide production (>80% at 5 μg/mL), while the galactopyranoside derivative 8d showed a notable nitric oxide inhibitory activity (126% at 0.5 μg/mL).

Discoidin domain receptor 1 mediates collagen‐induced inflammatory activation of microglia in culture

Discoidin domain receptor 1 (DDR1) is a nonintegrin collagen receptor tyrosine kinase with an extracellular domain homologous to discoidin 1 of a soil-living amoeba Dictyostelium discoideum. We have previously demonstrated that DDR1 mediates collagen-induced nitric oxide production in J774A.1 murine macrophages. Because collagen is one of the main components of extracellular matrix in the central nervous system, we hypothesized that collagen also induces inflammatory activation of brain microglia, and DDR1 may mediate collagen-induced microglial activation. Using BV-2 mouse microglial cells and mouse primary microglial cultures, we have demonstrated that (1) collagen induces inflammatory activation of microglia as evidenced by production of nitric oxide, expression of inducible nitric oxide synthase, COX-2, CD40, and matrix metalloproteinase-9; (2) DDR1 is expressed in microglia and is phosphorylated by collagen treatment; and (3) collagen-induced microglial activation is abrogated by DDR1 blockade but not by integrin neutralization. We have further shown that p38 MAPK, c-Jun N-terminal kinase, and nuclear factor-kappa B are involved in the collagen-DDR1-induced microglial activation. Our results suggest that collagen can induce inflammatory activation of brain microglia and that DDR1 mediates this effect of collagen in an integrin-independent manner.

Discoidin domain receptor 1 mediates collagen-induced nitric oxide production in J774A.1 murine macrophages

Nitric oxide (NO) is an important regulator of immune responses. Effects of cytokines, such as tumor necrosis factor (TNF)-α or IFN-γ, and bacterial products, such as lipopolysaccharide, on macrophage NO production have been well documented; however, the role of the extracellular matrix proteins, including collagen, in this process remains unclear. We previously reported that discoidin domain receptor 1 (DDR1), a nonintegrin collagen receptor, was expressed in human macrophages, and its activation facilitated their differentiation as well as cytokine/chemokine production. Here, we examined the role for DDR1 in collagen-induced NO production using the murine macrophage cell line J774 cells that endogenously express DDR1. Activation of J774 cells with collagen induced the expression of inducible NO synthase (iNOS) and NO production. Inhibition of DDR1, but not β1-integrins, abolished collagen-induced iNOS and NO production. Activation of J774 cells with collagen-activated nuclear factor-κB, p38 mitogen-activated protein kinase (MAPK), and c-jun N-terminal kinase (JNK) and a pharmacological inhibitor of each signaling molecule significantly reduced collagen-induced NO production. Thus, we have demonstrated, for the first time, that the interaction of DDR1 with collagen induces iNOS expression and subsequent NO synthesis in J774 cells through activation of NF-κB, p38 MAPK, and JNK and suggest that intervention of DDR1 signaling in macrophages may be useful in controlling inflammatory diseases in which NO plays a critical role.

Role of Zn 2+ in oxidative stress caused by endotoxin challenge

The role of Zn 2+ in oxidative stress during endotoxemia was investigated. In rats fed a Zn 2+-deficient diet (Zn 2+ concentration of less than 1.5 mg/kg) for 8 weeks, the Zn 2+ level in the serum was about 62% lower than that in rats fed a Zn 2+-adequate diet (Zn 2+ concentration, 50 mg/kg). The Zn 2+ level in serum 18 h after administration of endotoxin (6 mg/kg, i.p.) to Zn 2+-deficient diet rats was markedly lower than that of the endotoxin/Zn 2+-adequate diet group. Lipid peroxide formation in the liver of Zn 2+-deficient diet rats was markedly increased 18 h after endotoxin injection compared with that in the endotoxin/Zn 2+-adequate diet group. Metallothionein in the liver of endotoxin/Zn 2+-adequate diet rats was increased more than 17-fold by endotoxin administration, while a markedly lower level of metallothionein was observed in the endotoxin/Zn 2+-deficient diet group. On the other hand, treatment with ZnSO 4 (100 μM) significantly increased endotoxin (1 μg/ml)-induced tumor necrosis factor-α (TNF-α) production in J774A.1 cells. Our results clearly demonstrated that treatment with ZnSO 4 significantly inhibited the endotoxin-induced increase in intracellular Ca 2+ level in J774A.1 cells. However, a cell membrane-permeable Zn 2+ chelator, N, N, N′, N′-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN, 1 μM), did not affect the endotoxin-induced TNF-α production or Ca 2+ level in J774A.1 cells. In addition, we investigated whether Zn 2+ can suppress nitric oxide (NO) generation and cytotoxicity in endotoxin-treated cells. Treatment with ZnSO 4 (50 μM) significantly inhibited endotoxin-induced NO production in J774A.1 cells, but did not affect endotoxin-induced cytotoxicity. These findings suggest that zinc may play an important role, at least in part, in the oxidative stress during endotoxemia.

Roles of selenium in endotoxin-induced lipid peroxidation in the rats liver and in nitric oxide production in J774A.1 cells

We examined the role of selenium (Se) in the mechanism of oxidative stress caused by endotoxin by feeding rats deficient a diet in this element. In rats fed the Se-deficient diet (concentration of Se, less than 0.027 μg g −1) for 10 weeks, Se level and glutathione peroxidase (GSH-Px) activity in the liver were about 47 and 43% lower, respectively, than those in rats fed a Se-adequate diet (Se, 0.2 μg g −1). Rat fed the Se-deficient diet and given endotoxin (6 mg kg −1, i.p.) showed a mortality rates of about 43% at 18 h. Nevertheless, no lethality was observed with endotoxin (4 mg kg −1, i.p.) challenge. Levels of serum lactate dehydrogenase and acid phosphatase leakage were significantly higher in Se-deficient rats than those in Se-adequate diet 18 h after endotoxin (4 mg kg −1, i.p.) challenge. Superoxide anion generation and lipid peroxide formation in the liver of Se-deficient rat were markedly increased 18 h after endotoxin (4 mg kg −1, i.p.) injection compared with those in the endotoxin/Se-adequate diet group, whereas non-protein sulfhydryl level in the liver after administration of endotoxin to Se-deficient rats was lower than that in Se-adequate rats treated with endotoxin. We investigated whether Se can suppress nitric oxide (NO) generation and cytotoxicity in endotoxin-treated J774A.1 cells. Treatment with Se (10 −6 M) markedly inhibited endotoxin (0.1 μg ml −1)-induced NO production in J774A.1 cells. Se induced an increased activity of GSH-Px in cells after 24 h of incubation, suggesting that the preventive effect of Se on NO production in endotoxemia is due to the induction of Se–GSH-Px activity. However, Se did not affect endotoxin-induced cytotoxicity in J774A.1 cells. These findings suggested that the oxidative stress caused by endotoxin may be due, at least in part, to changes in Se regulation during endotoxemia.

Arginase modulates nitric oxide production in activated macrophages.

In macrophages and many other cell types, L-arginine is used as a substrate by both nitric oxide synthase (NOS) and arginase to produce nitric oxide (NO) and urea, respectively. Because the availability of L-arginine is a major determinant for NO synthesis in the activated macrophage, we hypothesized that NO production may be reduced by arginase via depleting the common substrate in this cell type. To test this hypothesis, we investigated the effect of an arginase inhibitor, L-norvaline, on NO production in J774A.1 mouse macrophages activated by lipopolysaccharide (LPS, 1.0 microgram/ml) for 22 h. In the absence of LPS, macrophages produced a low level of NO. In contrast, NO production from these cells was significantly increased in the presence of LPS. Increasing extracellular levels of L-arginine (0.01-0.8 mM) produced a concomitant increase in NO production of activated macrophages. L-Norvaline (10 mM), which specifically inhibits arginase activity (i.e., reducing urea production by 50%) without altering NOS activity, enhanced NO production (by 55%) from activated macrophages. The enhancement of NO production by L-norvaline was inversely related to the extracellular level of L-arginine. A more pronounced increase in NO production was observed at the lower level of extracellular L-arginine, i.e., a 55 vs. 28% increase for 0.05 and 0.1 mM extracellular L-arginine, respectively. When the L-arginine concentration exceeded 0.5 mM, the L-norvaline effect was abolished. These results indicate that arginase can compete with NOS for their common substrate and thus inhibit NO production. This regulatory mechanism may be particularly important when the extracellular supply of L-arginine is limited.