Acetylbritannilatone suppresses NO and PGE2 synthesis in RAW 264.7 macrophages through the inhibition of iNOS and COX-2 gene expression

In order to elucidate the mechanism of anti-inflammatory effect of 1-o-acetylbritannilatone (ABL) isolated from Inula Britannica-F, we investigated ABL for its ability to inhibit the inflammatory factor production in RAW 264.7 macrophages. The studies showed that ABL not only inhibited LPS/IFN-gamma-mediated nitric oxide (NO) production and inducible nitric synthase (iNOS) expression, but also decreased LPS/IFN-gamma-induced prostaglandin E2 (PGE2) production and cyclo-oxygenase-2 (COX-2) expression in a concentration-dependent manner. EMSA demonstrated that ABL inhibited effectively the association of NF-kappaB, which is necessary for the expression of iNOS and COX-2, with its binding motif in the promoter of target genes. These data suggest that ABL suppress NO and PGE2 synthesis in RAW 264.7 macrophages through the inhibition of iNOS and COX-2 gene expression, respectively. The anti-inflammatory effect of ABL involves blocking the binding of NF-kappaB to the promoter in the target genes and inhibiting the expression of iNOS and COX-2.     

Systemic poly(ADP-ribose) polymerase-1 activation,chronic inflammation,and oxidative stress in COPD patients

Oxidative stress and systemic inflammation in chronic obstructive pulmonary disease (COPD) strongly suggest a role for the nuclear enzyme poly(ADP-ribose) polymerase-1 (PARP-1, E.C.2.4.2.30) in the disease pathophysiology. PARP-1 is highly activated by reactive oxygen species-induced DNA strand breaks, upon which it forms extensive poly(ADP-ribose) (PAR) polymers from its substrate NAD(+). We hypothesized that in COPD, chronic inflammation and oxidative stress would lead to systemic PARP-1 activation and to a reduced NAD(+) status. In a patient-control study, systemic PARP-1 activation was assessed by immunofluorescent detection of PAR polymers in peripheral blood lymphocytes. The percentage of PAR polymer-positive lymphocytes appeared to be higher in COPD patients (27 +/- 3%) than in healthy age-matched controls (17 +/- 2%, p <.05). Trolox equivalent antioxidant capacity (TEAC) of deproteinized plasma (p <.001), plasma uric acid (p <.05), as well as blood NAD(+) (p <.01) of stable COPD patients were significantly reduced when compared to controls. In addition, levels of proinflammatory cytokines IL-6, IL-8, and sICAM-1 were increased (p <.005) in COPD patients. In this study, evidence was found for the presence of systemic inflammation, chronic oxidative stress, and systemic PARP-1 activation in stable COPD patients. These data support a contribution of oxidative stress-induced PARP-1 activation to the pathophysiology of COPD.     

Corn silk induced cyclooxygenase-2 in murine macrophages

Stimulation of murine macrophages with corn silk induced cyclooxygenase (COX)-2 with secretion of PGE2. Expression of COX-2 was inhibited by pyrolidine dithiocarbamate (PDTC), and increased DNA binding by nuclear factor kappa B (NF-kappaB), indicating that COX-2 induction proceeds also via the NF-kappaB signaling pathway. A specific inhibitor of COX-2 decreased the expression level of inducible nitric oxide synthase (iNOS) stimulated by corn silk. PGE2 elevated the expression level of iNOS, probably via EP2 and EP4 receptors on the surface of the macrophages.     

Astaxanthin inhibits nitric oxide production and inflammatory gene expression by suppressing I(kappa)B kinase-dependent NF-kappaB activation

Astaxanthin, a carotenoid without vitamin A activity, has shown anti-oxidant and anti-inflammatory activities; however, its molecular action and mechanism have not been elucidated. We examined in vitro and in vivo regulatory function of astaxanthin on production of nitric oxide (NO) and prostaglandin E2 (PGE2) as well as expression of inducible NO synthase (iNOS), cyclooxygenase-2, tumor necrosis factor-alpha (TNF-alpha), and interleukin-1beta (IL-1beta). Astaxanthin inhibited the expression or formation production of these proinflammatory mediators and cytokines in both lipopolysaccharide (LPS)-stimulated RAW264.7 cells and primary macrophages. Astaxanthin also suppressed the serum levels of NO, PGE2, TNF-alpha, and IL-1beta in LPS-administrated mice, and inhibited NF-kappaB activation as well as iNOS promoter activity in RAW264.7 cells stimulated with LPS. This compound directly inhibited the intracellular accumulation of reactive oxygen species in LPS-stimulated RAW264.7 cells as well as H2O2-induced NF-kappaB activation and iNOS expression. Moreover, astaxanthin blocked nuclear translocation of NF-kappaB p65 subunit and I(kappa)B(alpha) degradation, which correlated with its inhibitory effect on I(kappa)B kinase (IKK) activity. These results suggest that astaxanthin, probably due to its antioxidant activity, inhibits the production of inflammatory mediators by blocking NF-kappaB activation and as a consequent suppression of IKK activity and I(kappa)B-alpha degradation.     

Lipoteichoic acid-induced nitric oxide synthase expression in RAW 264.7 macrophages is mediated by cyclooxygenase-2, prostaglandin E2, protein kinase A, p38 MAPK, and nuclear factor-kappaB pathways

We recently reported that lipoteichoic acid (LTA), a cell wall component of the gram-positive bacterium Staphylococcus aureus, stimulated inducible nitric oxide synthase (iNOS) expression, nitric oxide (NO) release, and cyclooxygenase-2 (COX-2) expression in RAW 264.7 macrophages. This study was carried out to further investigate the roles of COX-2 and prostaglandin E2 (PGE2) in LTA-induced iNOS expression and NO release in RAW 264.7 macrophages. Treatment of RAW 264.7 macrophages with LTA caused a time-dependent increase in PGE2 release. LTA-induced iNOS expression and NO release were inhibited by a non-selective COX inhibitor (indomethacin), a selective COX-2 inhibitor (NS-398), an adenylyl cyclase (AC) inhibitor (dideoxyadenosine, DDA), and a protein kinase A (PKA) inhibitor (KT-5720). Furthermore, both PGE2 and the direct PKA activator, dibutyryl-cAMP, also induced iNOS expression in a concentration-dependent manner. Stimulation of RAW 264.7 macrophages with LTA, PGE2, and dibutyryl-cAMP all caused p38 MAPK activation in a time-dependent manner. LTA-mediated p38 MAPK activation was inhibited by indomethacin, NS-398, and SB 203580, but not by PD 98059. The PGE2-mediated p38 MAPK activation was inhibited by DDA, KT-5720, and SB 203580, but not by PD 98059. LTA caused time-dependent activation of the nuclear factor-kappaB (NF-kappaB)-specific DNA-protein complex formation. The LTA-induced increase in kappaB-luciferase activity was inhibited by indomethacin, NS-398, KT-5720, and a dominant negative mutant of p38 alphaMAPK (p38 alphaMAPK DN). These results suggest that LTA-induced iNOS expression and NO release involve COX-2-generated PGE2 production, and AC, PKA, p38 MAPK, and NF-kappaB activation in RAW 264.7 macrophages.     

Cross-regulation of iNOS and COX-2 by its products in murine macrophages under stress conditions.

Exposure of macrophages to heat shock induces rapid synthesis of heat shock proteins (HSPs) which are important for cell homeostasis. Prostaglandins (PGs) and nitric oxide (NO) are important cell regulatory molecules. We have therefore investigated the interactions between these molecules in the LPS-induced expression of iNOS and COX-2 and in the mitochondrial activity of macrophages. Cultures of the murine macrophage cell line, J774, were exposed to heat shock (43 degrees C, 30 min) and stimulated with LPS (1 microg/ml), concomitantly or after 8h of cell recovery. NO production was measured by Griess reaction; PGE(2) by ELISA; HSP70, iNOS and COX-2 by immunobloting; mitochondrial activity by MTT assay. Heat shock induced HSP70, but not iNOS or COX-2 whereas LPS induced iNOS and COX-2 but not HSP70. When heat shock and LPS were given concomitantly, iNOS but not COX-2 expression was reduced. When a period of 8h was given between heat shock and LPS stimulation, iNOS, COX-2, PGE(2) and NO levels were significantly increased. Under these conditions, the expression of COX-2 was reduced by L-NAME (NO-synthesis inhibitor) and of iNOS by nimesulide (PGs-synthesis inhibitor). Such cross-regulation was not observed in cells at 37 degrees C. These treatments significantly reduced MTT levels in cells at 37 degrees C but not in cells submitted to heat shock. These results suggest that HSPs and cross-regulation of iNOS and COX-2 by their products might be of relevance in the control of cell homeostasis during stress conditions.     

Suppression of inducible nitric oxide synthase and cyclooxygenase-2 by cell-permeable superoxide dismutase in lipopolysaccharide-stimulated BV-2 microglial cells

Oxidative stress plays a pivotal role in uncontrolled neuro-inflammation leading to many neurological diseases including Alzheimer’s. One of the major antioxidant enzymes known to prevent deleterious effects due to oxidative stress is Cu,Zn-superoxide dismutase (SOD). In this study, we examined the regulatory function of SOD on the LPS-induced signaling pathways leading to NF-kappaB activation, expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), in BV-2 cells using cell-permeable SOD. Treatment of BV-2 cells with cell-permeable SOD led to a decrease in LPS-induced reactive oxygen species (ROS) generation and significantly inhibited protein and mRNA levels of iNOS and COX-2 upregulated by LPS. Production of NO and PGE2 in LPS stimulated BV-2 cells was significantly abrogated by pretreatment with a cell-permeable SOD fusion protein. Furthermore, cell-permeable SOD inhibited LPS-induced NF-kappaB DNA-binding activity and activation of MAP kinases including ERK, JNK, and p38 in BV-2 cells. These data indicate that SOD has a regulatory function for LPS-induced NF-kappaB activation leading to expression of iNOS and COX-2 in BV-2 cells and suggest that cell-permeable SOD is a feasible therapeutic agent for regulation of ROS-related neurological diseases.     

Poly(ADP-ribose) polymerase 1 (PARP-1) binds to 8-oxoguanine-DNA glycosylase (OGG1)

Human 8-oxoguanine-DNA glycosylase (OGG1) plays a major role in the base excision repair pathway by removing 8-oxoguanine base lesions generated by reactive oxygen species. Here we report a novel interaction between OGG1 and Poly(ADP-ribose) polymerase 1 (PARP-1), a DNA-damage sensor protein involved in DNA repair and many other cellular processes. We found that OGG1 binds directly to PARP-1 through the N-terminal region of OGG1, and this interaction is enhanced by oxidative stress. Furthermore, OGG1 binds to PARP-1 through its BRCA1 C-terminal (BRCT) domain. OGG1 stimulated the poly(ADP-ribosyl)ation activity of PARP-1, whereas decreased poly(ADP-ribose) levels were observed in OGG1(-/-) cells compared with wild-type cells in response to DNA damage. Importantly, activated PARP-1 inhibits OGG1. Although the OGG1 polymorphic variant proteins R229Q and S326C bind to PARP-1, these proteins were defective in activating PARP-1. Furthermore, OGG1(-/-) cells were more sensitive to PARP inhibitors alone or in combination with a DNA-damaging agent. These findings indicate that OGG1 binding to PARP-1 plays a functional role in the repair of oxidative DNA damage.     

Ca2+/calmodulin-dependent protein phosphatase calcineurin mediates the expression of iNOS through IKK and NF-kappaB activity in LPS-stimulated mouse peritoneal macrophages and RAW 264.7 cells

Ca(2+) and Ca(2+)/calmodulin-dependent protein phosphatase calcineurin (CN) have been known to play crucial roles in immune response and inflammation. Using mouse peritoneal macrophages and RAW 264.7 macrophage cells, we demonstrated that LPS mobilized intracellular free Ca(2+) and induced CN phosphatase activity. iNOS expression and NO secretion in response to LPS were suppressed by Ca(2+) antagonists (TMB-8, BAPTA/AM, and nifedipine) and CN inhibitor (cyclosporin A). Transient expression of constitutively active CN in mouse peritoneal macrophages and RAW 264.7 macrophages strongly activated NF-kappaB, a key mediator of iNOS expression. We also found that CN mediates NF-kappaB activation via IkappaB-alpha hyperphosphorylation and degradation. Overexpression of dominant negative mutant of IKKalpha and -beta demonstrates that only IKKbeta is the target for CN. These results indicate that CN is required for full iNOS expression and the effective activation of NF-kappaB in RAW 264.7 and peritoneal macrophages.