Reactive oxygen species activate the group IV muscle afferents in resting and exercising muscle in rats

We tested the hypothesis that the reactive oxygen species (ROS) produced at rest and mostly during muscle contraction may stimulate the group IV muscle afferents. In rats, afferent activity was recorded in the peroneal nerve innervating the tibialis anterior muscle. Group IV afferents were identified from measurements of their conduction velocity and response to lactic acid. Comparing the group IV response to an intramuscular injection of buffered isotonic NaCl solution, we searched for the effects of a ROS donor (H₂O₂) or a ROS inhibitor (superoxide dismutase, SOD) on the baseline afferent activity in resting muscles. We also explored the consequences of a pre-treatment with SOD on the afferent nerve response to H₂O₂ injection or electrical muscle stimulation (MS). In other animals, we measured the changes in intramuscular level of a marker of oxidative stress (isoprostanes) after each test agent. H₂O₂ injection markedly activated all recorded group IV afferents. SOD injection lowered the baseline activity of 50 out of 70 afferent units, suppressed the afferent response to H₂O₂ injection, and delayed and reduced the MS-induced activation of all recorded units. Intramuscular isoprostanes level significantly increased after H₂O₂ injection or MS, the oxidative stress being absent in muscles pre-treated with SOD. We concluded that ROS influence both the spontaneous and contraction-induced activities of the group IV muscle afferents and are a potent stimulus of muscle metaboreceptors.     

Suppression of Heme Oxygenase-1 by Prostaglandin E2-Protein Kinase A-A-Kinase Anchoring Protein Signaling Is Central for Augmented Cyclooxygenase-2 Expression in Lipopolysaccharide-Stimulated RAW 264.7 Macrophages

Purpose

Prostaglandin (PG) E₂ is an immunomodulatory lipid mediator generated mainly via the cyclooxygenase-2 (COX-2) pathway from arachidonic acid at sites of infection and inflammation. A positive feedback loop of PGE₂ on COX-2 expression is critical for homeostasis during toll-like receptor (TLR)-mediated inflammatory processes. The mechanism of PGE₂-regulated COX-2 expression remains poorly understood. The low-molecular-weight stress protein heme oxygenase-1 (HO-1) contributes to the anti-inflammatory, anti-oxidant and anti-apoptotic response against environmental stress.

Methods

We explored the involvement of HO-1 on PGE₂ regulation of LPS-induced COX-2 expression in RAW 264.7 macrophages.

Results

LPS-induced COX-2 expression in RAW 264.7 macrophages was enhanced by exogenous PGE₂ or cyclic AMP (cAMP) analogue and was suppressed by a COX inhibitor (indomethacin), a protein kinase A (PKA) inhibitor (KT5720), and A kinase anchoring protein (AKAP) disruptors (Ht31 and RIAD). This result suggests that the stimulatory effects of endogenous and exogenous PGE₂ on COX-2 expression are mediated by a cAMP-PKA-AKAP-dependent pathway. The induction of HO-1 was observed in LPS-stimulated RAW 264.7 macrophages. This induction was suppressed by exogenous PGE₂ and enhanced by blockage of the endogenous PGE₂ effect by the PKA inhibitor or AKAP disruptors. In addition, HO-1 induction by the HO activator copper protoporphyrin suppressed LPS-induced COX-2 expression, which was restored by the addition of exogenous PGE₂. The induction of HO-1 inhibited LPS-induced NF-κB p-65 nuclear expression and translocation.

Conclusions

AKAP plays an important role in PGE₂ regulation of COX-2 expression, and the suppression of HO-1 by PGE₂-cAMP-PKA-AKAP signaling helps potentiate the LPS-induced COX-2 expression through a positive feedback loop in RAW 264.7 macrophages.     

Anti-inflammatory effect of quinoline alkaloid skimmianine isolated from Ruta graveolens L.

Objective

The present study evaluates the anti-inflammatory effect of the quinoline alkaloid skimmianine (SKM), isolated from Ruta graveolens L., against carrageenan-induced acute inflammation.

Methods

SKM at a dose of 5.0 mg/kg body weight was found to be the minimal concentration for maximal edema inhibition. Carrageenan suspension was administered into the sub-plantar tissue of the right hind paw 1 h after SKM and diclofenac (20 mg/kg) administration (i.p.). Paw edema was determined 3 h after carrageenan administration. The rats were then killed and mRNA expressions of TNF-α and IL-6, levels of PGE₂ and TBARS, activities of COX-2, 5-LOX, SOD, catalase, glutathione peroxidase (GPx) and myeloperoxidase (MPO) and the level of nitrite were measured.

Results

SKM treatment resulted in a decrease in the mRNA levels of TNF-α and IL-6, which are upstream events of the inflammatory cascade. The levels of PGE₂ and NO and the activities of COX-2 and 5-LOX were also significantly reduced after SKM treatment. Neutrophil infiltration, lipid peroxidation and associated oxidative stress in the paw tissue were reduced following SKM treatment.

Conclusion

These results support the anti-inflammatory properties of skimmianine and its multi-targeted mechanism of action, suggesting its potential therapeutic efficacy in various inflammatory diseases.     

The effect of BisGMA on cyclooxygenase-2 expression,PGE2 production and cytotoxicity via reactive oxygen species- and MEK/ERK-dependent and -independent pathways

After operative restoration, some monomers released from dentin bonding agents or composite resin may induce tissue inflammation and affect the vitality of dental pulp. Whether BisGMA, a major monomer of composite resin, may induce prostaglandin release and cytotoxicity to pulp cells and their mechanisms awaits investigation. We found that BisGMA induced cytotoxicity to human dental pulp cells at concentrations higher than 0.075 mm as analyzed by 3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. BisGMA (0.1 mm) also stimulated ERK phosphorylation, PGE₂ production, COX-2 mRNA and protein expression as well as ROS production (as indicated by an increase in cellular DCF fluorescence) in dental pulp cells. Catalase (500 and 1000 U/ml) and U0126 (10 and 20 μm, a MEK inhibitor) effectively prevented the BisGMA-induced ERK activation, PGE₂ production and COX-2 expression. Moreover, catalase can protect the pulp cells from BisGMA cytotoxicity, whereas aspirin and U0126 lacked of this protective activity. These results suggest that BisGMA released from composite resin may potentially affect the vitality of dental pulp and induce pulpal inflammation via stimulation of ROS production, MEK/ERK1/2 activation and subsequent COX-2 gene expression and PGE₂ production. Cytotoxicity of BisGMA to dental pulp cells is related to ROS production, but not directly mediated by MEK activation and PGE₂ production.     

The role of PGE2-associated inflammatory responses in gastric cancer development

Accumulating evidence indicates that inflammation plays a critical role in cancer development. Cyclooxygenase-2 (COX-2) is a rate-limiting enzyme for prostanoid biosynthesis, including prostaglandin E₂ (PGE₂), and plays a key role in both inflammation and cancer. It has been demonstrated that inhibition of COX-2 and PGE₂ receptor signaling results in the suppression of tumor development in a variety of animal models. However, the molecular mechanisms underlying COX-2/PGE₂-associated inflammation in carcinogenesis have not yet been fully elucidated. In order to study the role of PGE₂-associated inflammatory responses in tumorigenesis, it is important to use in vivo mouse models that recapitulate human cancer development from molecular mechanisms with construction of tumor microenvironment. We have developed a gastritis model (K19-C2mE mice) in which an inflammatory microenvironment is constructed in the stomach via induction of the COX-2/PGE₂ pathway. We also developed a gastric cancer mouse model (Gan mice) in which the mice develop inflammation-associated gastric tumors via activation of both the COX-2/PGE₂ pathway and Wnt signaling. Expression analyses using these in vivo models have revealed novel mechanisms of the inflammatory responses underlying gastric cancer development. PGE₂-associated inflammatory responses activate epidermal growth factor receptor (EGFR) signaling through the induction of EGFR ligands and ADAMs that release EGFR ligands from the cell membrane. In Gan mice, a combination treatment with EGFR and COX-2 inhibitors significantly suppresses gastric tumorigenesis. Moreover, PGE₂-associated inflammation downregulates tumor suppressor microRNA, miR-7, in gastric cancer cells, which suppresses epithelial differentiation. These results indicate that PGE₂-associated inflammatory responses promote in vivo gastric tumorigenesis via several different molecular mechanisms.     

Reactive oxygen species and inflammatory mediators enhance muscle spindles mechanosensitivity in rats

We tested the hypothesis that reactive oxygen species (ROS) and inflammatory mediators affect transduction properties of muscle spindles. In rats, muscle spindles response to high-frequency vibration (HFV) was recorded before and after (1) injection of hydrogen peroxide (H2O2) in control rats and animals pre-treated with diclofenac (anti-inflammatory substance), (2) injection of bradykinin and (3) fatigue induced by muscle stimulation (MS) in control rats and rats receiving diclofenac, superoxide dismutase (SOD) or H2O2. Muscular oxidative stress and inflammation induced by H2O2 or MS were assessed by measurements of isoprostanes and IL-6 levels. In control rats, H2O2, bradykinin and MS significantly enhanced the HFV response. Pre-treatment with SOD abolished the post-MS-enhanced HFV response whereas diclofenac lowered the peak HFV response to MS and H2O2. H2O2 injection and MS elicited significant and similar increases in isoprostanes and IL-6. We report a direct modulation of muscle spindles mechanosensitivity by ROS and inflammatory mediators.     

Involvement of TLR3-Dependent PGES Expression in Immunosuppression by Human Bone Marrow Mesenchymal Stem Cells

Human mesenchymal stem cells (MSCs) are known for their prostaglandin E₂ (PGE₂)-mediated immunosuppressive function but the precise molecular mechanisms underlying PGE₂ biosynthesis during inflammation have not been completely elucidated. In this study, we have investigated the involvement of PGE₂ pathway members in PGE₂ production by bone marrow (BM)-MSCs in response to inflammatory stimuli, and their role in immunosuppression mediated by BM-MSCs. We found that IFN-γ and TNF-α increased cyclooxygenase (COX)-2 expression but not that of prostaglandin E synthase (PGES), or PGE₂ production. On the other hand, the toll like receptor 3 (TLR3) stimulant poly(I:C) increased expression of both COX-2 and PGES, resulting in a significant increase in PGE₂ levels. This effect was reversed upon COX-2 inhibition with indomethacin or PGES downregulation by siRNA. Reduced PGE₂ levels decreased MSC’s capacity to inhibit hPBMC proliferation. In addition, administration of MSCs with inhibited PGES expression into mice with graft-versus-host disease (GVHD) did not reduce mortality. In summary, the present study reveals that upregulation of PGES via TLR3 is critical for BM-MSCs-mediated immunosuppression by PGE₂ secretion via the COX-2/PGE₂ pathway. These results provide a basis for understanding the molecular mechanisms underlying the PGE₂-mediated immunosuppressive properties of MSCs.     

Effects of age and glutathione levels on oxidative stress in rats after chronic exposure to stretch-shortening contractions

We investigated effects of age and glutathione synthesis inhibition on the oxidative stress status of tibialis anterior muscles from young and old Fisher 344 × Brown Norway male rats after chronic administration of stretch-shortening contractions. Oral supplementation of l-buthionine-(S,R)-sulfoximine (BSO) inhibited glutathione synthesis. Dorsiflexor muscles in the hindlimb were exposed to 80 maximal stretch-shortening contractions (SSCs) three times per week for 4.5 weeks. We measured malondialdehyde, hydrogen peroxide (H₂O₂), and free isoprostanes to determine oxidative stress. Glutathione peroxidase activity was measured as an indicator of H₂O₂ scavenging. Glutathione measurements confirmed the effectiveness of BSO treatment. In young rats, the SSC exposure protocol prevented oxidative stress and enhanced H₂O₂ scavenging. In old rats, malondialdehyde was increased in the exposed muscle and a BSO-induced increase in H₂O₂ was not alleviated with SSC exposure as seen in young rats. In addition, glutathione peroxidase activity and total glutathione were increased in old rats relative to their young counterparts. All comparisons were significant at the 0.05 level. Overall, BSO administration was effective in decreasing total glutathione levels and increasing H₂O₂ levels in old and young rats exposed to SSCs. In addition, effects of chronic exposure to high-force resistive loading SSCs in active muscle from old animals are: (1) antioxidant capacity is enhanced similar to what is seen with endurance training and (2) oxidative stress is increased, probably as a consequence of the enhanced vulnerability due to aging.     

Effects of diphenyl diselenide on oxidative stress induced by sepsis in rats

Sepsis is a potentially deadly complication that can be caused by different factors. Actually, it is known that oxidative stress is involved in the pathogenesis of sepsis. Thus, the aim of this study was to evaluate the effect of diphenyl diselenide (PhSe)₂, an emergent compound, on oxidative stress parameters induced by sepsis in rats. Animals were pre-injected with (PhSe)₂ or vehicle. Twenty-four hours later, sepsis was induced by cecal ligation puncture (CLP). After 12 h, liver was taken for thiobarbituric acid reactive species (TBARS) measurement, δ-aminolevunic acid dehydratase (δ-ALA-D), Cu/Zn superoxide dismutase (Cu/Zn SOD) and catalase (CAT) activities assay. The sepsis increased TBARS, inhibited δ-ALA-D, activated Cu/Zn SOD and had a tendency to decrease CAT activity. However, (PhSe)₂ prevented the TBARS formation, but did not prevent the inhibition of δ-ALA-D activity in the animals with damage. Thus, this study showed that (PhSe)₂ partially prevents the oxidative stress induced by sepsis, indicating the potential of this compound as a treatment for this pathology. Nevertheless, more tests should be performed to confirm the hypothesis suggested here.     

Separate and combined effects of heat stress and exercise on circulatory markers of oxidative stress in euhydrated humans

Combined heat stress, dehydration, and exercise is associated with enhanced oxidative stress in humans, but the separate and combined effects of heat stress and exercise on circulatory markers of oxidative stress without the influence of dehydration remain uncertain. The purpose of this study was to determine the effects of whole body heat stress alone and in combination with exercise on blood markers of oxidative stress in euhydrated humans. Eight males wore a water-perfused suit at rest and during 6 min of one-legged knee extensor exercise under control and heat stress conditions while maintaining euhydration. Following the control trial and a 15 min resting period, hot water was perfused through the suit in order to increase core, skin, and mean body temperatures by ~1, ~6, and ~2°C, respectively. Blood samples were taken to measure reduced glutathione (GSH), oxidized glutathione (GSSG), superoxide dismutase (SOD) and plasma isoprostanes. Heat stress alone did not alter GSH, SOD activity, or plasma isoprostanes, but increased GSSG leading to a reduction in the GSH/GSSG ratio. No changes in these variables were observed with exercise alone. Conversely, combined heat stress and exercise increased both GSH and GSSG, decreased SOD activity, but did not alter GSH/GSSG ratio or isoprostanes. In conclusion, these findings suggest that heat stress, independently of dehydration, induces non-radical oxidative stress at rest but not during moderate exercise because an increase in antioxidant defense compensates the heat stress-induced non-radical oxidative stress.     

Renal and vascular oxidative stress and salt‐sensitivity of arterial pressure

Oxidative stress occurs in a tissue or in the whole body when the total oxidant production exceeds the antioxidant capacity. Recent studies in human essential hypertension indicate that free radical production is increased and antioxidant levels are decreased, and more than one‐half of these hypertensives have a salt‐sensitive type of hypertension with progressive renal damage. Increased oxidative stress may also play a critical role in animal models of salt‐sensitive hypertension. The stroke‐prone spontaneously hypertensive rats (SHRSP) exhibits salt‐sensitivity, vascular release of superoxide is increased, and total plasma antioxidant capacity is decreased. The superoxide release in the SHRSP rats inactivates nitric oxide, and superoxide dismutase (SOD) administration returns the bioactive nitric oxide levels to normal. The deoxycorticosterone acetate (DOCA)‐salt hypertensive rat is salt‐sensitive, aortic superoxide production is increased, and renal inflammation is significant. Treatment of the DOCA‐salt rats with apocynin, an NADPH oxidase inhibitor, decreased aortic superoxide production and decreased arterial pressure. The Dahl salt‐sensitive (S) rat has increased mesenteric microvascular and renal superoxide production and increased plasma levels of H₂O₂. The renal protein expression of SOD is decreased in the kidney of Dahl S rats, and long‐term administration of Tempol, a superoxide mimetic, significantly decreased arterial pressure and renal damage. In conclusion, both human hypertension and experimental models of salt‐sensitive hypertension have increased superoxide release, decreased antioxidant capacity and elevated renal damage.     

Temporal expression of cyclooxygenase-2 in peritoneal macrophages of rats and effects of panax notoginseng saponins

Objective:  To explore the temporal expression pattern of cyclooxygenase (COX)-2 and effects of panax notogensing saponins (PNS) in peritoneal macrophages of rats. Materials and methods:  Phagocytosis function of peritoneal macrophages was measured by chicken red blood cell phagocytosis assay in vitro. Expression of COX-2 mRNA and protein, PGE₂ and PGD₂ production were determined with real-time PCR, Western blotting and radioimmunoassay, respectively. Results:  Phagocytosis function of macrophages increased significantly after stimulation and reached peak during 2–3h. Expression of COX-2 mRNA and its protein increased markedly after stimulation and reached the first peak at 2 h and 3h, respectively; and then decreased to reach a minimum at 24h. The second peak appeared at 36h. PNS (50, 100, 200 mg/kg) increased the phagocytosis function obviously at 2h, decreased the expression level of COX-2 and PGE₂ production at 2 h and elevated COX-2 expression and PGD₂ production at 36 h, respectively. Conclusion:  COX-2 expression in peritoneal macrophages has a double-hump feature after stimulation. PNS enhanced phagocytosis, inhibiting COX-2 expression at an early stage and elevating it at a later stage. Received 29 February 2008; returned for revision 27 March 2008; received from final revision 14 May 2008; accepted by G. Wallace 9 June 2008 The first two authors contributed equally to this work.     

The Role of Inflammation and COX-Derived Prostanoids in the Effects of Bradykinin on Isolated Rat Aorta and Urinary Bladder

Bradykinin, a vasoactive peptide, increases during inflammation and induces the formation of prostaglandins through specific receptor activation. Two types of receptors mediate the biological effects of bradykinin, B₁ and B₂ receptors. Although B₂ receptors are present in most tissues, B₁ receptors are expressed after inflammatory stimuli or tissue injury. Bradykinin has a high affinity for B₂ and a low affinity for B₁ receptors, whereas the opposite occurs for des-Arg⁹-bradykinin. Recently, it has been reported that nonsteroidal anti-inflammatory drugs have different inhibitory activities on cyclooxygenase isozymes, COX-1, COX-2, and COX-3. In the present study, we have investigated the contributions of different COX isozyme inhibitions and inflammation on bradykinin-induced effects of isolated rat aorta and urinary bladder smooth muscle contractions. Male Sprague–Dawley rats weighing 200–250 g were used in the study. The vasodilatory responses to bradykinin (1 nM–1 μM) were studied on isolated rat aorta rings contracted with norepinephrine (0.1 μM) following incubation with dipyrone (100, 700, and 2,000 μM). The relaxant responses of dipyrone (100, 700, and 2,000 μM) were also compared on the isolated rat urinary bladder contracted with bradykinin (n = 8). A bacterial lipopolysaccharide was used for the induction of inflammation (n = 8). The levels of PGE₂, PGF_1α, TXB₂, nitric oxide synthase (NOS), IL-10, and TNF-α were all determined in both the plasma and the perfusate of the aorta preparations (n = 5). The vasodilatory activities of bradykinin and des-Arg9-bradykinin were significantly increased upon the inhibition of COX-3 (dipyrone at 100 μM). These effects disappeared in the inflamed group. PGE₂, PGF1α, and TXB₂ were significantly high, but NOS activity was low in the aorta perfusate after the inhibition of COX-3. Dipyrone showed the relaxant activity of the urinary bladder contracted with bradykinin. The vasodilatory activity of des-Arg⁹-bradykinin was in the inflamed group but not in the non-inflamed group. Bradykinin did not contract urinary bladder in inflamed group. The results suggest that COX-induced products may play an important role in the bradykinin-induced rat aortic smooth muscle relaxations.     

Activation of PPAR-γ by Carbon Monoxide from CORM-2 Leads to the Inhibition of iNOS but not COX-2 Expression in LPS-Stimulated Macrophages

The effect of CO on the expression of iNOS and COX-2 was investigated by using a CO-releasing molecule (CORM)-2 in LPS-activated RAW 264.7 cells in vitro. Interestingly, CORM-2 significantly inhibited iNOS (NO) but not COX-2 (PGE₂) expression. PPAR-γ activators such as troglitazone, GW1929, and 15-deoxy-Δ12, 14- prostaglandin J₂ showed preferential inhibitory effect on iNOS over COX-2 expression in LPS-activated macrophages. The same effect was shown in lung tissues (iNOS, COX-2) and serum (NO, PGE₂) when administered of CORM-2 in LPS-induced septic mice, indicating that CO derived from CORM-2 differentially regulates iNOS and COX-2 through PPAR-γ activation under inflammation state.     

Neuronal overexpression of cyclooxygenase-2 does not alter the neuroinflammatory response during brain innate immune activation

Neuroinflammation is a critical component in the progression of several neurological and neurodegenerative diseases and cyclooxygenases (COX)-1 and -2 are key regulators of innate immune responses. We recently demonstrated that COX-1 deletion attenuates, whereas COX-2 deletion enhances, the neuroinflammatory response, blood–brain barrier permeability and leukocyte recruitment during lipopolysaccharide (LPS)-induced innate immune activation. Here, we used transgenic mice, which overexpressed human COX-2 via neuron-specific Thy-1 promoter (TgCOX-2), causing elevated prostaglandins (PGs) levels. We tested whether neuronal COX-2 overexpression affects the glial response to a single intracerebroventricular injection of LPS, which produces a robust neuroinflammatory reaction. Relative to non-transgenic controls (NTg), 7 month-old TgCOX-2 did not show any basal neuroinflammation, as assessed by gene expression of markers of inflammation and oxidative stress, neuronal damage, as assessed by Fluoro-JadeB staining, or systemic inflammation, as assessed by plasma levels of IL-1β and corticosterone. Twenty-four hours after LPS injection, all mice showed increased microglial activation, as indicated by Iba1 immunostaining, neuronal damage, mRNA expression of cytokines (TNF-α, IL-6), reactive oxygen expressing enzymes (iNOS and NADPH oxidase subunits), endogenous COX-2, cPLA₂ and mPGES-1, and hippocampal and cortical IL-1β levels. However, the increases were similar in TgCOX-2 and NTg. In NTg, LPS increased brain PGE₂ to the levels observed in TgCOX-2. These results suggest that PGs derived from neuronal COX-2 do not play a role in the neuroinflammatory response to acute activation of brain innate immunity. This is likely due to the direct effect of LPS on glial rather than neuronal cells.     

Positive feedback effect of PGE2 on cyclooxygenase-2 expression is mediated by inhibition of Akt phosphorylation in human follicular dendritic cell-like cells

Prostaglandins (PGs) are bioactive lipid mediators generated from the phospholipids of cell membrane in response to various inflammatory signals. To understand the potential role of PGs in PG production itself during immune inflammatory responses, we examined the effect of PGE₂, PGF_2α, and beraprost on COX-2 expression using follicular dendritic cell (FDC)-like HK cells isolated from human tonsils. Those three PGs specifically augmented COX-2 protein expression in a dose-dependent manner after 4 or 8 h of treatment. The enhancing effect was also reflected in the actual production of PGs and the viable cell recovery of germinal center B-cells. To investigate the underlying molecular mechanism, we examined the impact of PI3K inhibitors on PG-induced COX-2 expression. Interestingly, COX-2 induction by PGE₂ and beraprost, but not PGF_2α, was enhanced by wortmannin and LY294002. In line with this result, Akt phosphorylation was inhibited by PGE₂ and beraprost but not by PGF_2α. The distinct effect of PGE₂ and beraprost from PGF_2α was reproduced in Akt-knockdowned HK cells. Our current findings imply that PGE₂ and PGI₂ stimulate COX-2 expression in FDC by inhibiting Akt phosphorylation. Additional studies are warranted to determine the potential role of Akt as a therapeutic target in patients with inflammatory disorders.     

<Emphasis Type="Italic">Toxoplasma gondii</Emphasis> induces prostaglandin E<Subscript>2</Subscript> synthesis in macrophages via signal pathways for calcium-dependent arachidonic acid production and PKC-dependent induction of cyclooxygenase-2

In this study, the intracellular signaling pathway of PGE₂ synthesis in macrophages (RAW264.7) induced by Toxoplasma gondii was investigated. The T. gondii-induced PGE₂ production in macrophages increased in a time-dependent manner, as PGE₂ induction began at 4 h, peaked at 12 h, and then plateaued at a high level. COX-2 mRNA in macrophages was detectable as early as 4 h after treatment; the maximal expression was observed at 8 h. The earliest induction of COX-2 protein occurred at 4 h and peaked at 16 h; meanwhile, COX-1 mRNA level and protein production remained unchanged throughout. Indomethacin and nimesulide inhibited tachyzoite-induced PGE₂ production and COX-2 mRNA expression in macrophages but they had no significant effect on COX-2 protein expression. EGTA, TFP and BAPTA/AM inhibited both arachidonic acid (AA) and PGE₂ production without effecting COX-2 protein expression, but verapamil inhibited neither AA nor PGE₂ production. H7 was found to inhibit PGE₂ production, and COX-2 mRNA expression and protein expression by tachyzoite or LPS stimulated macrophages in a dose-dependent manner. Our results demonstrate that T. gondii induces PGE₂ biosynthesis in RAW264.7 macrophages by regulating AA production through a calcium-dependent pathway and induction of COX-2 expression by a PKC-dependent pathway.     

阿魏酸钠对结肠炎大鼠结肠巨噬细胞功能的影响

目的:探讨阿魏酸钠在整体水平下对结肠炎大鼠结肠巨噬细胞功能的影响及其机制。方法:建立大鼠免疫性结肠炎模型。阿魏酸钠(SF)灌肠用药21d后检测结肠组织MDA、NO、PGE₂含量,SOD、IL-1、TNF-α、MPO活性及NF-κBp65表达水平。结果:阿魏酸钠(200、400、800mg/kg)灌肠用药剂量依赖性降低模型组大鼠显著升高的MDA、NO、PGE₂含量,IL-1、TNF-α、MPO活性及NF-κBp65表达水平,同时升高显著降低的SOD活性。结论:SF整体水平下减弱结肠炎大鼠结肠活化巨噬细胞的生物活性,缓解结肠炎症反应,机制可能与抑制NF-κB表达有关。     

Induction of heme oxygenase-1 attenuates lipopolysaccharide-induced cyclooxygenase-2 expression in mouse brain endothelial cells

Background  

Prostaglandin E₂ (PGE₂), an arachidonic acid metabolite converted by cyclooxygenase-2 (COX-2), plays important roles in the regulation of endothelial functions in response to bacterial infection. The enzymatic activity of COX-2 can be down-regulated by heme oxygenase-1 (HO-1) induction. However, the mechanisms underlying HO-1 modulating COX-2 protein expression are not known.     

Prostaglandin production via induction of cyclooxygenase-2 by human gingival fibroblasts stimulated with lipopolysaccharides

The purpose of the present study was to investigate the involvement of cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) in PGE₂ production by human gingival fibroblasts stimulated with lipopolysaccharides (LPS) from periodondopathogenic bacteria. LPS were isolated fromPorphyromonas gingivalis (P. gingivalis), Actinobacillus actinomycetemcomitans (A. actinomycetemcomitans) andEschericia coli (E. coli) by the phenol-water procedure. The three LPS preparations produced PGE₂ up to 48 h in a time-dependent manner in human gingival fibroblasts.P. gingivalis-LPS was the most potent stimulator of PGE₂ production and, to a lesser extent,A. actinomycetemcomitans- andE. coli-LPS. Treatment of the cells with indomethacin, a non selective COX-1/COX-2 inhibitor and NS-398, a selective COX-2 inhibitor, completely depressed PGE₂ production. Treatment of dexamethasone, known to inhibit COX-2 expression, also significantly prevented PGE₂ production. Immunohistochemical staining of COX-2 protein demonstrated that expression of COX-2 protein was increased at 24 h afterP. gingivalis-LPS stimulation, while expression of COX-1 protein was not affected byP. gingivalis-LPS. In order to investigate the regulation of PGE₂ production,P. gingivalis-LPS-stimulated cells were treated with herbimycin A and genistein, both inhibitors of tyrosine kinases. Both the inhibitors significantly inhibited PGE₂ production. Herbimycin A treatment depressed expression of COX-2 protein. These data suggest that human gingival fibroblasts stimulated with LPS from periodontopathogenic bacteria mainly produce PGE₂ not by COX-1, but by COX-2, induction of which may be regulated by tyrosine kinase and that the produced PGE₂ may be involved in the pathogenesis of periodontal diseases.