Generation Of Proinflammatory Mediators Research Articles

PQQ ameliorates D-galactose induced cognitive impairments by reducing glutamate neurotoxicity via the GSK-3\u03b2/Akt signaling pathway in mouse

Oxidative stress is known to be associated with various age-related diseases. D-galactose (D-gal) has been considered a senescent model which induces oxidative stress response resulting in memory dysfunction. Pyrroloquinoline quinone (PQQ) is a redox cofactor which is found in various foods. In our previous study, we found that PQQ may be converted into a derivative by binding with amino acid, which is beneficial to several pathological processes. In this study, we found a beneficial glutamate mixture which may diminish neurotoxicity by oxidative stress in D-gal induced mouse. Our results showed that PQQ may influence the generation of proinflammatory mediators, including cytokines and prostaglandins during aging process. D-gal-induced mouse showed increased MDA and ROS levels, and decreased T-AOC activities in the hippocampus, these changes were reversed by PQQ supplementation. Furthermore, PQQ statistically enhanced Superoxide Dismutase SOD2 mRNA expression. PQQ could ameliorate the memory deficits and neurotoxicity induced by D-gal via binding with excess glutamate, which provide a link between glutamate-mediated neurotoxicity, inflammation and oxidative stress. In addition, PQQ reduced the up-regulated expression of p-Akt by D-gal and maintained the activity of GSK-3β, resulting in a down-regulation of p-Tau level in hippocampus. PQQ modulated memory ability partly via Akt/GSK-3β pathway.

Josamycin suppresses Prevotella intermedia lipopolysaccharide-induced production of nitric oxide and interleukin-1β in murine macrophages.

Josamycin has immunomodulatory properties independent of its antibacterial actions. This study was designed to explore the influences and associated mechanisms of josamycin upon the generation of proinflammatory mediators in murine macrophages activated with lipopolysaccharide (LPS) from Prevotella intermedia, a pathogenic bacterium associated with periodontal disease. LPS was purified by employing phenol-water extraction protocol. Culture supernatants were analyzed for nitric oxide (NO) and interleukin (IL)-1β. Real-time PCR and immunoblotting were conducted to quantify mRNA and protein expression, respectively. The expression levels of IL-1β were analyzed by confocal laser scanning microscopy. NF-κB-dependent SEAP levels were estimated by reporter assay. Josamycin significantly attenuated NO production elicited by P. intermedia LPS as well as induction of iNOS protein and mRNA in RAW264.7 cells. While the release of IL-1β from cells stimulated by LPS was suppressed in the presence of josamycin, josamycin failed to reduce the degree of IL-1β mRNA expression. Josamycin did not reduce the stability of IL-1β mRNA induced by P. intermedia LPS, at the same time josamycin also failed to suppress the LPS-induced intracellular IL-1β expression. Josamycin augmented HO-1 induction in cells exposed to P. intermedia LPS, and SnPP, an inhibitor of HO-1 activity, reversed the suppressive impact of josamycin upon NO generation induced by LPS. Josamycin diminished NF-κB transcriptional activity induced by P. intermedia LPS. Further, josamycin enhanced SOCS1 mRNA level in cells activated with LPS. Josamycin suppressed P. intermedia LPS-induced generation of NO and IL-1β in RAW264.7 macrophages via the inhibition of NF-κB activation as well as HO-1 and SOCS1 induction. Josamycin may have benefits as a host modulatory agent in treating periodontal disease.

Duhuo Jisheng Decoction inhibits SDF-1-induced inflammation and matrix degradation in human degenerative nucleus pulposus cells in vitro through the CXCR4/NF-κB pathway.

Lower back pain (LBP) is the most common disease in orthopedic clinics world-wide. A classic Fangji of traditional Chinese medicine, Duhuo Jisheng Decoction (DHJSD), has been proven clinically effective for LBP but its therapeutic mechanisms remain unclear. We hypothesized that DHJSD might relieve LBP through inhibiting the exaggerated proinflammatory cytokines and extracellular matrix (ECM) degradation. Thus, we studied the effects of DHJSD on stromal cell-derived factor-1 (SDF-1)-induced inflammation and ECM degradation in human nucleus pulposus cells (hNPCs). The primary hNPCs were isolated from either degenerated human intervertebral disc (HID) of LBP patients or normal HID of lumbar vertebral fracture patients, and cultured in vitro. The cells were treated with SDF-1 (10 ng/mL) and subsequently with different concentrations (100-500 μg/mL) of DHJSD for 24 h, respectively. We found that application of DHJSD significantly antagonized the SDF-1-induced production of proinflammatory cytokines and reduction of aggrecan and type II collagen in the hNPCs. DHJSD also markedly reduced the SDF-1-induced increase of CXCR4 and p-p65 and inhibited the nuclear translocation of p65 in the hNPCs. DHJSD, CXCR4-siRNA, and NF-κB inhibitor (BAY11-7082) caused the same inhibition of exaggerated proinflammatory cytokines in the SDF-1-treated hNPCs. These results provided compelling evidence that DHJSD may inhibit the generation of proinflammatory mediators and ECM degradation of HID through an orchestrated targeting at multiple molecules in the SDF-1/CXCR4/NF-κB pathway, thus offered novel mechanistic insights into the clinical effectiveness of DHJSD on LBP.

In-vitro antioxidative, antiinflammatory properties of Aurea helianthus leaf extract a Korean traditional medicinal plant.

The leaf of Aurea helianthus (A. helianthus Jinhuakui) is popularly used in China traditional medicine, however, scientific evidence on its antioxidant properties rarely studied. In this study, biological activities of A. helianthus leave’s 80% ethanol extract (AHL) were investigated. The measured total polyphenol and flavonoid content of AHL was 184.24 ± 5.01 mg GAE/g and 102.53 ± 0.98 mg NAR/g. AHL showed the highest α, α-diphenyl-β-picrylhydrazyl (DPPH) and 2,2′-azino-bis-3-ethylbenzo-thiazoline-6-sulfonic acid (ABTS) radical scavenging activities of 98.30 ± 0.18% at 1000 µg/mL. DPPH and ABTS radical scavenging activities significantly increased in a AHL concentration-dependent manner. AHL treatment significantly suppressed the generation of pro-inflammatory mediators, including nitric oxide (NO), in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. AHL demonstrated strong anti-inflammatory activity that reduced NO production in LPS-stimulated RAW 264.7 cells. To test the potential protective effect of AHL, the antioxidant capacity, on the cell growth, viability of a human hepatoma cell (HepG2) and Raw 264.7 cell were investigated. AHL also enhanced cytotoxicity on the proliferation of HepG2 cells and was capable of inhibiting 56% against LPS at 400 µg/mL. The results of this study the potential of AHL as an excellent antioxidant substance for inhibiting inflammatory mediators. Therefore, AHL may be used as a therapeutic approach to various inflammatory diseases.

Effect of nitric oxide-releasing derivative of indomethacin on Prevotella intermedia lipopolysaccharide-induced production of proinflammatory mediators in murine macrophages

The purpose of this study was to investigate the influences of NCX 2121, a nitric oxide (NO)-releasing derivative of indomethacin, upon the generation of proinflammatory mediators using murine macrophages activated by lipopolysaccharide (LPS) isolated from Prevotella intermedia, which is one of the pathogens implicated in periodontal diseases. Inducible NO synthase (iNOS)-derived NO, IL-1β and IL-6 as well as their relevant mRNA were significantly attenuated by NCX 2121 in RAW264.7 cells activated by P. intermedia LPS. NCX 2121 was much more effective than the parental compound indomethacin in reducing these proinflammatory mediators. NCX 2121 triggered induction of heme oxygenase-1 (HO-1) in cells exposed to P. intermedia LPS, and its inhibitory influence upon P. intermedia LPS-elicited NO generation was notably blocked by SnPP treatment. NCX 2121 attenuated NF-κB-dependent SEAP release induced by P. intermedia LPS. NCX 2121 did not display inhibitory action towards IκB-α degradation triggered by LPS. Instead, it significantly diminished nuclear translocation as well as DNA-binding action of NF-κB p50 subunit elicited by P. intermedia LPS. Further, NCX 2121 significantly up-regulated SOCS1 mRNA expression in cells challenged with P. intermedia LPS. In summary, NCX 2121 down-regulates P. intermedia LPS-elicited generation of NO, IL-1β and IL-6 in murine macrophages in a mechanism that involves anti-inflammatory HO-1 induction as well as decrement of NF-κB activation, which may be associated with SOCS1 expression. NCX 2121 may have potential benefits as a host immunomodulatory agent for the therapy of periodontal disease.

Agomelatine, a MT1/MT2 melatonergic receptor agonist with serotonin 5-HT2C receptor antagonistic properties, suppresses Prevotella intermedia lipopolysaccharide-induced production of proinflammatory mediators in murine macrophages

ObjectiveThis study was performed in an attempt to examine the influence of agomelatine in mitigating the generation of proinflammatory mediators in RAW264.7 murine macrophages exposed to lipopolysaccharide (LPS) obtained from Prevotella intermedia, a gram-negative anaerobic bacterium that is related with various types of periodontal diseases, and the molecular mechanisms behind its effects. DesignLPS from P. intermedia strain ATCC 25611 was prepared employing the conventional phenol-water procedure. Conditioned culture media were analyzed for the levels of nitric oxide (NO), interleukin-1β (IL-1β) and IL-6. Real-time PCR analysis was carried out to determine the mRNA levels of inducible NO synthase (iNOS), IL-1β, IL-6 and SOCS1. Protein expression levels were evaluated by immunoblot test. NF-κB-dependent SEAP reporter assay was performed using a reporter cell line. DNA-binding activities of NF-κB subunits were analyzed utilizing the ELISA-based kits. ResultsAgomelatine was found to down-regulate significantly the generation of iNOS-derived NO, IL-1β and IL-6 as well as the expression of their mRNAs in cells activated with P. intermedia LPS. Agomelatine decreased NF-κB-dependent SEAP release caused by P. intermedia LPS. Agomelatine did not inhibit NF-κB transcription induced by LPS at the level of IκB-α degradation. Instead, LPS-induced nuclear translocation and DNA binding of NF-κB p50 subunit was blocked by agomelatine. P. intermedia LPS-elicited activation of STAT1 and STAT3 was reduced notably by co-treatment with agomelatine. Agomelatine showed a tendency to enhance mRNA level of SOCS1 in LPS-activated cells as well. ConclusionsAgomelatine merits further evaluation to reveal its usefulness on the host modulation of periodontal disease.

Possible involvement of iNOS and TNF-α in nutritional intervention against nicotine-induced pancreatic islet cell damage.

Nicotine is the more abundant and most significant components of cigarette smoke. Epidemiological evidence strongly suggests an association between cigarette smoking and pancreatic injury. Although effects of smoking on endocrine pancreas are still controversial Here, we examined the impact and underlying mechanisms of action of folic acid and vitamin B12 on nicotine induced damage in pancreatic islets of rats. Male Wistar rats were treated with nicotine (3mg/kg body weight/day, intraperitonealy) with or without folic acid (36μg/kg body weight/day, orally) and vitamin B12 (0.63μg/kg body weight/day, orally) for 21days. Supplementation with folic acid and vitamin B12 suppressed the nicotine induced changes in HbA1c, insulin, TNF-α, IL-6, generation of reactive oxygen species, and attenuated the changes in markers of oxidative stress. Moreover, folic acid and vitamin B12 also counteracted the increased expression of protein and mRNA contents of TNF-α and iNOS produced by nicotine. Further, folic acid and vitamin B12 in combination limits the nicotine induced changes in cell cycle and excessive apoptosis of the pancreatic β-cells and also successfully blunted the nicotine induced alteration in loss of mitochondrial membrane potential. In conclusion, data demonstrate that folic acid and vitamin B12 may be possible nutritional intervention against cellular oxidative stress, which is a critical step in nicotine-mediated islet injury, and improves islet cell functional status by scavenging free radicals and by inhibiting the generation of pro-inflammatory mediators.

Suppressors of cytokine signaling 3 is essential for FcγR-mediated inflammatory response via enhancing CCAAT/enhancer-binding protein δ transcriptional activity in macrophages

Compelling evidence indicates that suppressor of cytokine signaling 3 (SOCS3) plays a pivotal regulatory role in inflammation. However, the function of SOCS3 in inflammatory responses mediated by Fcγ receptor (FcγR) remains largely unknown. In the current study, we found that SOCS3 expression was greatly enhanced in peritoneal macrophages treated with IgG immune complex (IgG IC). By over-expressing SOCS3 in macrophages, we observed that SOCS3 promoted IgG immune complex-induced production of inflammatory mediators, including IL-6, TNF-α, MIP-2, and MIP-1α. In contrast, SOCS3-defective peritoneal macrophages generated less inflammatory cytokines and chemokines when compared with their wild type counterparts during IgG IC-induced inflammatory responses. We further demonstrated that CCAAT/enhancer-binding protein (C/EBP) δ transcription factor was the major downstream target of SOCS3 in macrophages. These data suggested that SOCS3 was an inflammatory enhancer in IgG IC-treated macrophages by increasing C/EBPδ activity. To elucidate the role for myeloid-derived SOCS3 in IgG IC-induced inflammation in vivo, LysM-cre SOCS3fl/fl mice lacking SOCS3 in macrophages and neutrophils were generated. We found that SOCS3 deficiency greatly alleviated IgG IC-induced generation of pro-inflammatory mediators in lungs, consistent with the in vitro data. Our current findings may provide a new theoretical basis for designing drugs for treatment of IgG IC-associated diseases.

PPAR-α activation reduced LPS-induced inflammation in alveolar epithelial cells

ABSTRACTPurpose of the study: Acute respiratory distress syndrome (ARDS) represents a major cause of mortality in intensive care patients. Activation of peroxisome proliferator-activated receptor-α (PPAR-α) by fibrates, such as WY-14643 (WY), has been described to beneficially influence inflammation and experimental lung injury. The impact of PPAR-α activation on alveolar epithelial cells (AEC) has not been studied yet. Materials and Methods: To investigate the effect of PPAR-α activator WY in wild-type (WT) and in PPAR-α knockout (PPAR-α−/−) animals, mice were treated in different regimes: mice received chow enriched with or without WY for 14 days prior AEC isolation (in-vivo treatment). Furthermore, isolated AEC from both groups were subsequently cultured with or without WY (in-vitro treatment). AEC were stimulated with lipopolysaccharide (LPS). Cell culture supernatant and cell lysate were used for analysis of pro-inflammatory mediators. Results: AEC challenged with LPS showed a significantly increased generation of pro-inflammatory mediators. After in-vivo WY-exposure, AEC displayed significantly reduced concentration of TNF-α, MIP-2, and TxB2 after LPS stimulation. This beneficial effect was abrogated in PPAR-α−/− animals. Interestingly, sole in-vitro application of WY-14643 failed to reduce levels of pro-inflammatory mediators whereas we found an additive effect of a combined in-vivo and in-vitro PPAR-α activation. PGE2 concentration remained high after LPS challenge and was unaffected by WY treatment. Conclusion: PPAR-α activation by in-vivo exposure to fibrates reduced the inflammatory response in isolated AEC. These findings may facilitate further studies investigating the translation of pharmacological PPAR-α activation into clinical therapy of ARDS.

Artesunate Attenuates Lipopolysaccharide-Stimulated Proinflammatory Responses by Suppressing TLR4, MyD88 Expression, and NF-κB Activation in Microglial Cells.

Microglia are considered as a major target in the prevention of neuroinflammation by modulating the production of pro-inflammatory mediators. Artesunate, a water-soluble artemisinin derivative, exerts an anti-inflammatory effect. In the present study, we showed artesunate dose-dependently suppressed the lipopolysaccharide (LPS)-induced production of nitric oxide (NO), inducible nitric oxide synthase (iNOS), and interleukin-1beta (IL-1β) in BV2 microglial cells. In addition, artesunate inhibited LPS-induced expression of Toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), and activation of nuclear factor kappa B (NF-κB) by blockade of inhibitor of NF-κB (IκB) degradation. This data indicate that artesunate attenuates the generation of proinflammatory mediators on LPS-stimulated BV-2 microglial cells. And this effect may be associated with the suppression of TLR4/MyD88/NF-κB signaling pathways. Therefore, artesunate may be a potential anti-neuroinflammatory agent.

Absence of Annexin A1 impairs host adaptive immunity against Mycobacterium tuberculosis in vivo

The role of Annexin A1 (ANXA1) in counter-regulating the activities of innate immune cells, such as the migration of neutrophils and monocytes, and the generation of pro-inflammatory mediators in various models of inflammatory and autoimmune diseases is well documented. However, while ANXA1 has been proposed as an important mediator of the adaptive immune response, its involvement in this respect has been less studied. Furthermore, while there have been numerous studies on the role of ANXA1 in inflammatory diseases, less has been reported on its influence in immunity against infection. A recent study reported a link between ANXA1 and tuberculosis, and proposed a model in which Mycobacterium tuberculosis exerts its virulence by manipulating the ANXA1-mediated host apoptotic response. This has prompted us to further investigate the role of ANXA1 in the pathogenesis of tuberculosis in vivo. Here, we show that ANXA1−/− mice are more susceptible to M. tuberculosis infection, as evidenced by a transient increase in the pulmonary bacterial burden, and exacerbated and disorganized granulomatous inflammation. These pathological manifestations correlated with an impaired ability of ANXA1−/− dendritic cells to activate naïve T cells, thereby supporting a role for ANXA1 in shaping the adaptive immunity against M. tuberculosis.

Enriched endogenous omega-3 fatty acids in mice protect against global ischemia injury

Transient global cerebral ischemia, one of the consequences of cardiac arrest and cardiovascular surgery, usually leads to delayed death of hippocampal cornu Ammonis1 (CA1) neurons and cognitive deficits. Currently, there are no effective preventions or treatments for this condition. Omega-3 (ω-3) PUFAs have been shown to have therapeutic potential in a variety of neurological disorders. Here, we report that the transgenic mice that express the fat-1 gene encoding for ω-3 fatty acid desaturase, which leads to an increase in endogenous ω-3 PUFAs and a concomitant decrease in ω-6 PUFAs, were protected from global cerebral ischemia injury. The results of the study show that the hippocampal CA1 neuronal loss and cognitive deficits induced by global ischemia insult were significantly less severe in fat-1 mice than in WT mice controls. The protection against global cerebral ischemia injury was closely correlated with increased production of resolvin D1, suppressed nuclear factor-kappa B activation, and reduced generation of pro-inflammatory mediators in the hippocampus of fat-1 mice compared with WT mice controls. Our study demonstrates that fat-1 mice with high endogenous ω-3 PUFAs exhibit protective effects on hippocampal CA1 neurons and cognitive functions in a global ischemia injury model.

Nesfatin-1 alleviates gastric damage via direct antioxidant mechanisms

BackgroundIndomethacin is a nonsteroidal anti-inflammatory drug, which is known to produce serious side effects, causing ulcerative lesions. Nesfatin-1, a newly identified anorexigenic peptide, was recently shown to have neuroprotective effects. The aim of the study was to investigate the anti-inflammatory effects of nesfatin-1 on indomethacin-induced gastric ulcer. Materials and methodsAfter a 24-h starvation period, ulcer was induced in Sprague–Dawley rats by subcutaneous administration of indomethacin (25 mg/kg), whereas control group received vehicle. Fifteen minutes after ulcer induction, rats were treated with either saline or nesfatin-1 (0.1, 0.3, or 1 μg/kg, intraperitoneally). At the fourth hour, all rats were decapitated and their trunk blood was collected for tumor necrosis factor (TNF)-α and interleukin (IL)-6 measurements. Stomach samples were examined microscopically and analyzed for myeloperoxidase (MPO) activity, malondialdehyde (MDA), glutathione (GSH), luminol-, and lucigenin-enhanced chemiluminescence (CL) levels. ResultsUlcer induction increased serum TNF-α; and IL-6 levels, gastric CL and MDA levels and MPO activity but decreased gastric GSH content (P < 0.05–0.001). On the other hand, 0.1 μg/kg dose of nesfatin-1 reduced microscopic and macroscopic damage scores, decreased MPO activity and MDA levels, CL and IL-6 levels, whereas gastric GSH was replenished (P < 0.01). However, indomethacin-induced increase in TNF-α level was abolished at only 1 μg/kg dose of nesfatin-1 (P < 0.01). ConclusionsNesfatin-1 alleviated indomethacin-induced gastric injury, suggesting that the anti-inflammatory and gastroprotective effects of nesfatin-1 on oxidative gastric damage could be implemented by supporting the balance in oxidant and antioxidant systems while inhibiting the generation of pro-inflammatory mediators.

Antagonism by Bioactive Polyphenols Against Inflammation: A Systematic View

Through pattern recognition receptors, infections and tissue injuries drive innate immune cells to trigger inflammation with elevated cytokines, chemokines, growth factors, and other mediators. Inflammation resolves upon removal of pathogenic signals and the presence of pro-resolving conditions including combating adaptive immunity. Failure of resolution progresses into chronic inflammation, manifesting as detrimental disease development known as inflammatory diseases including cardiovascular diseases, diabetes, obesity, cancers, etc. Inflammation typically involves activations of many intracellular signaling pathways such as PI3K/AkT/mTORC1, PI3K/AkT/IKK(JNK), Ras/Raf/MEK/ERK, JAK/STAT, etc.; these pathways could in turn mediate the upregulations of proinflammatory transcription factors (e.g., NFκB, activator protein 1 (AP-1), HIF, signal transducer and activator of transcription (STAT), etc.). Furthermore, the resulting FOXO inactivation ensures inflammatory proceeding. This review provides a systematic view that polyphenols target multiple inflammatory components and reinforce anti-inflammatory mechanisms by antioxidant potentials, AMPK activation, PI3K/AkT inhibition, IKK/JNK inhibition, mTORC1 inhibition, JAK/STAT inhibition, TLR suppression, and ACE inhibition. As a result, polyphenols readily lead to NFκB, AP-1, HIF, and STAT inactivations with reduced proinflammatory mediator generation. In conclusion, polyphenols sustain resolution of inflammation and antagonize against proinflammation, which is readily consistent with diverse anti-inflammatory actions. The promoted, restored, and maintained tissue homeostasis beyond its anti-inflammatory effects also extends to diverse health benefits for disease preventions and interventions.

Cloning and expression analysis of a novel high-mobility group box 2 homologue from Lampetra japonica

High-mobility group box 2 (HMGB2) is a nonhistone architectural protein that plays important roles in many biological processes. In this study, we cloned a homologue of the HMGB2 from the lymphocyte-like cells of Lampetra japonica (L. japonica). Sequence analysis reveals that L. japonica HMGB2 contains two highly conserved motifs and shares more than 70 % identity with the homologues from other vertebrate species. Subsequently, Lj-HMGB2 was subcloned into the pET-28a(+) and pIRES2 AcGFP1-Nuc vector and expressed in Rosetta blue (DE3) and Hela cell lines, respectively. The recombinant L. japonica HMGB2 (rLj-HMGB2) with apparent molecular mass of 22 kDa was further purified by His-Bind affinity chromatography. Real-time quantitative PCR indicates that the expression level of Lj-HMGB2 was particularly up-regulated in intestines after challenged with lipopolysaccharide, while up-regulated in lymphocyte-like cells and heart after challenged with concanavalin A in vivo. In addition, rLj-HMGB2 could induce the generation of proinflammatory mediators in the activated human acute monocytic leukemia cell line (THP1), which suggested that Lj-HMGB2 may participate in the immune response of the lampreys.

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.

산양삼(Korean Panax ginseng) 추출물의 항산화 효과 및 LPS로 염증이 활성화된 RAW 264.7 대식세포에서의 염증매개물질 억제효과

Biological activities of Korean Panax ginseng 55% ethanol extract (KPGE) were investigated. The measured total polyphenol content of KPGE was 357.45 mg/100 g. KPGE showed the highest α,α-diphenyl-β-picrylhydrazyl (DPPH) and 2,2-azino-bis-3-ethylbenzo-thiazoline-6-sulfonic acid (ABTS) radical scavenging activities of 80% and 86% at 1,000 μg/mL, respectively. DPPH and ABTS radical scavenging activities significantly increased in a KPGE concentration-dependent manner. SOD-like activity of KPGE (1, 10, and 100 μg/mL) increased from 22% up to 33% at KPGE concentrations of 500 and 1,000 μg/mL. KPGE treatment significantly suppressed the generation of pro-inflammatory mediators, including nitric oxide (NO), prostaglandin E2 (PGE2), and cytokines (tumor necrosis factor-alpha: TNF-α, interleukin-6: IL-6, interleukin-1β: IL-1β), in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. KPGE demonstrated strong anti-inflammatory activity that reduced NO and PGE 2 production in LPS-stimulated RAW 264.7 cells. Even low concentrations of KPGE (1 and 10 μg/mL) reduced PGE 2 and NO production in RAW 264.7 macrophages without LPS-stimulation, respectively. At concentrations of 100, 500, and 1,000 μg/mL, TNF-α, IL-1β and IL-6 production were significantly suppressed. The results of our study suggest the potential of Korean Panax ginseng as an excellent antioxidant substance for inhibiting inflammatory mediators. Therefore, Korean Panax ginseng (KPGE) may be used as a therapeutic approach to various inflammatory diseases.

Caffeine suppresses lipopolysaccharide-stimulated BV2 microglial cells by suppressing Akt-mediated NF-κB activation and ERK phosphorylation

Since the anti-inflammatory effect of caffeine is unclear in microglial cells, we performed whether caffeine attenuates the expression of pro-inflammatory mediators in lipopolysaccharide (LPS)-stimulated BV2 microglial cells. Caffeine substantially suppressed the LPS-induced pro-inflammatory mediators nitric oxide (NO), prostaglandin E2 (PGE2) and tumor necrosis factor-α (TNF-α) in BV2 microglial cells. These effects resulted from the inhibition of their regulatory genes inducible NO synthase (iNOS), cycloxygenase-2 (COX-2) and TNF-α. In addition, caffeine significantly decreased LPS-induced DNA-binding activity of nuclear factor-κB (NF-κB) by suppressing the nuclear translocation of p50 and p65 subunits. A specific NF-κB inhibitor, pyrrolidine dithiocarbamate (PDTC), attenuated the LPS-induced expression of iNOS, COX-2 and TNF-α genes. In addition, we elucidated that inhibition of Akt phosphorylation plays a crucial role in caffeine-mediated NF-κB regulation in LPS-stimulated BV2 microglial cells. Caffeine also attenuated the LPS-induced phosphorylation of extracellular signal-regulated kinase (ERK) and a specific inhibitor of ERK, PD98059, subsequently downregulated the expression of the pro-inflammatory genes iNOS, COX-2 and TNF-α. Taken together, our data indicate that caffeine suppresses the generation of pro-inflammatory mediators, such as NO, PGE2 and TNF-α as well as their regulatory genes in LPS-stimulated BV2 microglial cells by inhibiting Akt-dependent NF-κB activation and the ERK signaling pathway.

P12 The complex effects of a slow-releasing hydrogen sulifde donor (GYY4137) in a model of inflammatory arthritis and in primary human cartilage cells

Synthesis of H 2 S is markedly increased in knee joint synovial fluid (SF) joints from patients with inflammatory joint disease (IJD) compared to paired plasma and SF from age-matched disease controls. SF levels also negatively correlated with disease activity, SF total white cell and neutrophil count suggesting a possible anti-inflammatory role for induced H 2 S synthesis in the joint. These observations also highlight the pharmacological potential for H 2 S donor molecules in (IJD). We have assessed the effect of GYY4137 (a slow-releasing H 2 S donor) on lipopolysaccharide (LPS)-evoked release of inflammatory mediators from human synoviocytes (HFLS) and articular chondrocytes (HAC) in vitro . We also examined the effects of GYY4137 on human recombinant iNOS, COX-1, COX-2 and tumour necrosis factor- α converting enzyme (TACE). We further examined the effect of GYY4137 in a complete Freund’s adjuvant (CFA) model of arthritis in the mouse. GYY4137 (0.1–0.5 mM) decreased LPS-induced production of nitrite ( NO 2 - ) , PGE 2 , TNF- α and IL-6 from HFLS and HAC, reduced the expression and catalytic activity of iNOS, COX-2, and reduced LPS-induced NF-kB activation in vitro . Using recombinant human enzymes, GYY4137 inhibited the activity of COX-1, COX-2, iNOS and TACE. In the CFA treated mouse, adjuvant induced a two fold increase in synovial H 2 S concentrations. GYY4137 (50 mg/kg, i.p.) injected 1 h prior to CFA increased knee joint swelling whilst an anti-inflammatory effect, as evidenced by reduced synovial fluid myeloperoxidase (MPO) and N -acetyl- β - d -glucosaminidase (NAG) activity and decreased TNF- α , IL-1 β , IL-6 and IL-8 concentration, was apparent when GYY4137 was injected 6 h after CFA. GYY4137 was also anti-inflammatory when given 18 h after CFA. The effects of H 2 S on the inflammatory process are highly complex. Not only is the anti-inflammatory effect determined by the donor used (’fast’ vs ’slow’ release donors) but also determined in vivo by the timing of the donor administration. Thus, whilst GYY4137 consistently reduced the generation of pro-inflammatory mediators from human joint cells in vitro , its effect on arthritis in vivo depended on the timing of administration.

Microglial LOX-1 reacts with extracellular HSP60 to bridge neuroinflammation and neurotoxicity

Chronic neurodegeneration is in part caused by a vicious cycle of persistent microglial activation and progressive neuronal cell loss. However, the driving force behind this cycle remains poorly understood. In this study, we used medium conditioned by necrotic differentiated-PC12 cells to confirm that damaged neurons can release soluble injury signals, including heat shock protein 60 (HSP60), to efficiently promote the neurotoxic cycle involving microglia. Since lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) has previously been identified as a novel receptor for HSP60, we hypothesize that LOX-1 through binding to extracellular HSP60 promotes microglia-mediated neuroinflammation. In this study, we observed that LOX-1 expression is induced upon toxic microglial activation, and discovered that LOX-1 is necessary in microglia for sensing soluble neuronal injury signal(s) in the conditioned medium to induce generation of pro-inflammatory mediators (IL-1β, TNF-α, NO and ROS) that promote neurotoxicity. Employing a unique eukaryotic HSP60-overexpression method, we further demonstrated that extracellular HSP60 acts on microglial LOX-1 to boost the production of pro-inflammatory factors (IL-1β, NO and ROS) in microglia and to propagate neuronal damage. These results indicate that LOX-1 is essential in microglia for promoting an inflammatory response in the presence of soluble neuronal-injury signals such as extracellular HSP60, thereby linking neuroinflammation and neurotoxicity.