Naoxintong restores ischemia injury and inhibits thrombosis via COX2-VEGF/ NFκB signaling.

ETHNOPHARMACOLOGICAL RELEVANCE: Naoxintong (NXT) is a traditional Chinese medicine preparation that is often used in combination with aspirin in the treatment of cardiovascular diseases (CVD). One of the main symptoms of CVD is hypoxic-ischemia (HI). The purpose of this study is to find out the molecular nodes targeted by NXT and its related molecular pathways in vascular repair. MATERIALS AND METHODS: First, human vein umbilical endothelial cells (EA.hy926) were utilized to set up the Oxygen-Glucose Deprivation-Reoxygenation (OGD/R) model and treated with NXT. Cell proliferation, damage and apoptosis were detected by MTT, LDH, and flow cytometry assays. Second, transcriptional responses of OGD/R cells to NXT treatment were investigated. qRT-PCR, western blotting and inhibitor assays were performed. Third, the anti-thrombotic effect of NXT was evaluated by the zebrafish thrombosis model. Morphological observation, histological staining and qRT-PCR assays were implemented on zebrafish model to further observe in vivo the therapeutic effects of NXT on ischemia and thrombosis. RESULTS: In OGD/R EA.hy926 cells, NXT treatment could reduce ischemic vascular injury, increase cell viability and decrease the proportion of apoptosis. Through RNA-seq analysis, 183 differentially expressed genes (DEGs) were screened with 110 up-regulated genes and 73 down-regulated genes between OGD/R and OGD/R + NXT treated EA.hy926 cells. VEGF and NFκB pathways were enriched. Among these genes, COX2 was identified as one of important targets via which NXT could restore vascular injury. COX2 inhibitor (NS-398), and aspirin, a drug that prevents the development of CVD by targeting COX2, exhibited similar effects to NXT in the treatment of OGD/R EA.hy926 cells. In zebrafish thrombosis model, NXT could attenuate tail venous thrombus and recover the quantity of heart red blood cells. Furthermore, NXT could prevent the formulation of thrombosis and eliminate inflammation in zebrafish by COX2-VEGF/NFκB signaling. CONCLUSION: Our studies implicated that NXT could restore HI injury and inhibit thrombosis through COX2-VEGF/NFκB signaling, which is consistent with the molecular target of aspirin. This finding might explain the principle of NXT combined with aspirin in the treatment of cardiovascular diseases.

Doxorubicin Intracellular Release Via External UV Irradiation of Dextran-g-poly(o-nitrobenzyl acrylate) Photosensitive Nanoparticles.

In the present study, innovative doxorubicin-loaded nanoparticles (NPs) made of a photosensitive poly(o-nitrobenzyl acrylate) (PNBA) hydrophobic matrix and an hydrophilic dextran (Dex) shell were first formulated by the emulsion-solvent evaporation process. Doxorubicin (DOX), a very well-known anticancer drug, was herein chosen as the model. DOX-loaded NPs were successfully produced by covering the hydrophobic PNBA core with Dex chains either physically adsorbed or covalently linked by changing process parameters as the presence of a catalyst (CuBr or CuSO4/ascorbic acid). It was then proved that the neutralization of DOX optimized drug loading. DOX loading and release were independent of the coverage mechanism if the catalyst used to covalently link the shell to the core was correctly chosen. Second, the kinetics of DOX release were investigated by simple diffusion or light irradiation of the NPs. Experiments showed that less than 20% of DOX was released by simple diffusion after 48 h in PBS or DMEM media when 45% of DOX released after only 30 s of light irradiation of the NPs. Finally, the impact of the phototriggered DOX release on cell viability was investigated on various cell lines [Caco-2, HepG2, HCT-116, and HT-29 cells as well as murine macrophages (RAW 264.7)]. Cellular mortality was evaluated to be dependent on the cell lines tested. Our approach provided an improved DOX release toward the human liver cancer cell line, and a high internalization of the PNBA-based NPs into HepG2 cells was observed using fluorescence microscopy.

Bioenergetics impairment of Trypanosoma cruzi by the antihypertensive manidipine: A drug repurposing strategy.

Considering the lack of effective and safe therapy for the treatment of Chagas disease, the antihypertensive drug manidipine (MDP) was in vitro evaluated against Trypanosoma cruzi. The bioenergetics of trypomastigotes was studied in the presence of the drug using fluorimetric and luminescent assays. Manidipine showed a potent antiparasitic activity, with IC50 values of 0.1 µM (intracellular amastigotes) and 3 µM (trypomastigotes), resulting in a promising selectivity index against the amastigotes (>1459). Using fluorimetric analysis, the drug showed depolarisation of the electric potential of the plasma membrane with no alteration of the permeability. A decrease in ATP levels suggested a bioenergetic alteration of the mitochondria, which was confirmed by the depolarisation of the mitochondrial membrane potential and a slight increase of the ROS levels. This is the first study to show the promising in vitro effectiveness of the antihypertensive MDP against T. cruzi, which may represent a candidate for future investigations in animal models.

A synthetic Nitraria alkaloid, isonitramine protects pancreatic ß-cell and attenuates postprandial hyperglycemia.

OBJECTIVE: The extracts of Nitraria genus are composed of Nitraria alkaloids and have been used traditionally as a hypoglycemic medicine. However, the efficacy and precise mechanism of Nitraria alkaloids remain largely unknown. METHODS: Previously, we reported the total synthesis of (+)-isonitramine, one of Nitraria alkaloids. In this study, we investigated the anti-diabetic potential of isonitramine in diabetes mellitus and its underlying molecular mechanism in carbohydrate catabolism in vitro and in vivo. RESULTS: Isonitramine exerted significant inhibitory effect on α-glucosidases but not α-amylase in vitro. In zebrafish, isonitramine alleviated the streptozotocin (STZ)-induced postprandial hyperglycemia and protected the pancreatic damages against alloxan-induced oxidative stress in vivo. Also, isonitramine induced insulin without any toxicities and downregulated phosphoenolpyruvate carboxykinase (PEPCK), which catalyzes the first committed step in gluconeogenesis. CONCLUSION: Taken together, isonitramine inhibited α-glucosidase activity and PEPCK expression, while increased insulin expression, resulting in attenuating the postprandial hyperglycemia. Also, isonitramine protected the pancreas from ROS-mediated toxicities. Therefore, isonitramine may be a new drug candidate for the treatment of diabetes mellitus.

Treatment with high dose of atorvastatin reduces vascular injury in diabetic rats.

BACKGROUND: Previous reports showed conflicting results regarding the treatment effects of statin on Diabetes mellitus (DM). We investigated how treatment with high dose of atorvastatin affects the impaired vascular function in diabetic rats. METHODS: Atorvastatin (80mg/kg/day, oral gavage, 4 weeks) or its vehicle was administered to male control or streptozotocin (STZ)-induced diabetic rats. Aortic segments were used to investigate the vascular reactivity, protein expression of cyclooxygenase-2 (COX-2) and nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase) 1 (NOX1) and superoxide anions levels. RESULTS: Atorvastatin treatment did not affect glycemia levels. In diabetic rats, the vascular reactivity to phenylephrine increased compared with controls and the atorvastatin treatment reduced this response. Removal of the endothelium increased the response to phenylephrine in control rats, but not in the diabetic group. Atorvastatin increased the endothelial modulation in diabetic rats. L-NAME (100µM) increased the reactivity in all groups, but this effect was greater in atorvastatin-treated diabetic rats. Indomethacin (10µM) and NS398 (1µM) decreased the contractile response in diabetic rats and atorvastatin reversed these effects, without changing COX-2 expression. Apocynin (30µM) decreased the phenylephrine response in diabetic rats, which also showed increased NOX1 and superoxide anions; these effects were prevented by atorvastatin treatment. CONCLUSIONS: The results suggest that treatment with high dose of atorvastatin, independent of glycemia, improves endothelial function in aortas from diabetic rats by reducing the constrictor prostanoids derived from COX-2 and by reducing the oxidative stress by NADPH oxidase, as well as a possible increasing of nitric oxide participation.

Madecassoside suppresses proliferation and invasiveness of HGF-induced human hepatocellular carcinoma cells via PKC-cMET-ERK1/2-COX-2-PGE2 pathway.

Recent studies showed that Madecassoside (MAD), a pentacyclic triterpene isolated from Centella asitica (L.), was used as a therapeutic agent in wound healing and also as an anti-inflammatory, anti-oxidative activities and anti-aging agent. However, its role in cancer has not been elucidated. In our present study, hepatocyte growth factor (HGF) induced the phosphorylation of its corresponding receptor cMET, increased expression of cyclo-oxygenase-2 (COX-2) and prostaglandin E2 (PGE2) in human hepatocellular carcinoma (HCC) cells lines (HepG2 and SMMC-77), and this effect was inhibited by MAD in a dose-dependent manner. In addition, MAD exhibited significant anti-proliferative and anti-invasive effect in HGF-induced HepG2 and SMMC-77 cells. Moreover, MAD inhibited the phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1/2) and the protein kinase C (PKC) activity in HGF-induced HepG2 and SMMC-77 cells. This conclusion was consistent with the effect of selective COX-2 inhibitor (NS-398) and knockdown of COX-2 by siRNA on attenuating the proliferation and invasiveness potential, and over-expression of COX-2 on abolishing the effects of MAD on proliferation and invasiveness potential, and was also in parallel with the effect of PKC inhibitor (Bisindolylmaleimide) on inhibiting PKC activity, MEK/ERK1/2 inhibitor (PD98059) inhibited MEK/ERK1/2 pathways in HGF-induced HepG2 and SMMC-77 cells. Collectively, MAD could inhibit the HGF-activated proliferation and invasiveness of HCC cells via regulating the activation of cMET-PKC-ERK1/2-COX-2-PGE2 cascade, which indicated that MAD might help control HGF-linked HCC.

Endothelial Dysfunction in Rheumatoid Arthritis: Mechanistic Insights and Correlation with Circulating Markers of Systemic Inflammation.

OBJECTIVES: To determine mechanisms involved in endothelial dysfunction (ED) during the course of arthritis and to investigate the link between cytokines, chemokines and osteoprotegerin. APPROACH AND RESULTS: Experiments were conducted on aortic rings at day 4 (preclinical), day 11 (onset of disease), day 33 (acute disease) and day 90 (chronic disease) after adjuvant-induced arthritis (AIA) in Lewis rats. At day 4, the unique vascular abnormality was a reduced norepinephrine-induced constriction. At day 11, endothelial function assessed by the relaxation to acetylcholine was normal despite increased cyclo-oxygenase-2 activity (COX-2) and overproduction of superoxide anions that was compensated by increased nitric oxide synthase (NOS) activity. At day 33, ED apparition coincides with the normalization of NOS activity. At day 90, ED was only observed in rats with a persisting imbalance between endothelial NOS and COX-2 pathways and higher plasma levels of IL-1ß and TNFα. Plasma levels of IL-1ß, TNFα and MIP-1α negatively correlated with Ach-induced relaxation throughout the course of AIA. CONCLUSIONS: Our data identified increased endothelial NOS activity as an important compensatory response that opposes the ED in the early arthritis. Thereafter, a cross-talk between endothelial COX-2/NOS pathways appears as an important element for the occurrence of ED. Our results encourage determining the clinical value of IL-1ß, TNFα and MIP-1α as biomarkers of ED in RA.

Significance of αThr-349 in the catalytic sites of Escherichia coli ATP synthase.

This paper describes the role of α-subunit VISIT-DG sequence residue αThr-349 in the catalytic sites of Escherichia coli F1Fo ATP synthase. X-ray structures show the highly conserved αThr-349 in the proximity (2.68 Å) of the conserved phosphate binding residue ßR182 in the phosphate binding subdomain. αT349A, -D, -Q, and -R mutations caused 90-100-fold losses of oxidative phosphorylation and reduced ATPase activity of F1Fo in membranes. Double mutation αT349R/ßR182A was able to partially compensate for the absence of known phosphate binding residue ßR182. Azide, fluoroaluminate, and fluoroscandium caused insignificant inhibition of αT349A, -D, and -Q mutants, slight inhibition of the αT349R mutant, partial inhibition of the αT349R/ßR182A double mutant, and complete inhibition of the wild type. Whereas NBD-Cl (7-chloro-4-nitrobenzo-2-oxa-1,3-diazole) inhibited wild-type ATPase and its αT349A, -D, -R, and -Q mutants essentially completely, ßR182A ATPase and double mutant αT349A/ßR182A were inhibited partially. Inhibition characteristics supported the conclusion that NBD-Cl reacts in ßE (empty) catalytic sites, as shown previously by X-ray structure analysis. Phosphate protected against NBD-Cl inhibition in the wild type, αT349R, and double mutant αT349R/ßR182A but not in αT349A, αT349D, or αT349Q. The results demonstrate that αThr-349 is a supplementary residue involved in phosphate binding and transition state stabilization in ATP synthase catalytic sites through its interaction with ßR182.

IL-1ß/HMGB1 complexes promote The PGE2 biosynthesis pathway in synovial fibroblasts.

PGE2 is a potent lipid mediator of pain and oedema found elevated in RA. Microsomal prostaglandin E synthase-1 (mPGES-1) is a terminal enzyme of the PGE2 pathway inducible by proinflammatory cytokines. mPGES-1 is markedly upregulated in RA synovial tissue despite antirheumatic treatments, suggesting that multiple inflammatory stimuli contribute to its induction. High-mobility group box chromosomal protein 1 (HMGB1) is known to induce inflammation both by direct interaction with TLR4 and by enhancement of other proinflammatory molecules signalling, through complex formation. The high expression of extracellular HMGB1 within the inflamed synovium, implies its pro-arthritogenic role in RA. We aimed to investigate the effects of IL-1ß/HMGB1 complexes on mPGES-1 and other enzymes of the PGE2 pathway in synovial fibroblasts (SFs) from patients with arthritis. Furthermore, we studied the effect of COX-2 inhibition and IL-1RI antagonism on prostanoid and cytokine production by SFs. Stimulation of SFs with HMGB1 in complex with suboptimal amounts of IL-1ß significantly increased mPGES-1 and COX-2 expressions as well as PGE2 production, as compared to treatment with HMGB1 or IL-1ß alone. Furthermore, NS-398 reduced the production of IL-6 and IL-8, thus indicating that IL-1ß/HMGB1 complexes modulate cytokine production in part through prostanoid synthesis. Treatment with IL-1RA completely abolished the induced PGE2 and cytokine production, suggesting an effect mediated through IL-1RI. IL-1ß/HMGB1 complexes promote the induction of mPGES-1, COX-2 and PGE2 in SF. The amplification of the PGE2 biosynthesis pathway by HMGB1 might constitute an important pathogenic mechanism perpetuating inflammatory and destructive activities in rheumatoid arthritis.

Cyclooxygenase-2-related signaling in the hypothalamus plays differential roles in response to various acute stresses.

We previously suggested that cyclooxygenase (COX)-2 plays a role as a common mediator of stresses in the brain. In the present study, we evaluated the possible involvement of COX-2-related signaling in the activation of the hypothalamic-pituitary-adrenal (HPA) axis under three different stress conditions, namely infectious (lipopolysaccharide, LPS), hypoglycemic (2-deoxy-d-glucose, 2DG) and restraint (1h) stresses in rats. Both an unselective COX inhibitor (indomethacin) and a selective COX-2 inhibitor (NS-398) significantly attenuated the increase of serum corticosterone levels after LPS and restraint stresses, but not after 2DG injection. COX-2 and microsomal prostaglandin E synthase (mPGES)-1 mRNA levels in the hypothalamus were significantly increased after LPS injection in intact rats. In adrenalectomized (ADX) rats, the expression of both genes was significantly increased after 2DG and restraint stresses, which was blocked by treatment with corticosterone. Interleukin-1ß (IL-1ß) mRNA levels in the hypothalamus in intact rats were increased only by LPS injection, though those in ADX rats were increased by all three stress stimuli. These results suggest that the relationship between COX-2-related signaling and activation of the HPA axis is stress-specific, and that COX-2-related signaling preferably mediates infectious and restraint stresses. Furthermore, the expression of COX-2 and mPGES-1 mRNA under the infectious stress condition was not negatively regulated by endogenous glucocorticoids, likely due to an increase in IL-1ß levels.

Involvement of hypothalamic cyclooxygenase-2, interleukin-1ß and melanocortin in the development of docetaxel-induced anorexia in rats.

Docetaxel, a taxane derivative, is frequently used for the treatment of advanced breast cancer, non-small cell lung cancer, and metastatic prostate cancer. Clinical reports demonstrated that docetaxel-based chemotherapy often induces anorexia, but the etiology is not completely understood. To elucidate possible mechanisms, we investigated the involvement of central interleukin (IL)-1ß, cyclooxygenase (COX)-2, and pro-opiomelanocortin (POMC) in the development of docetaxel-induced anorexia in rats. Rats received docetaxel (10mg/kg, i.p.) with or without pretreatment with selective COX-2 inhibitors, NS-398 (10 and 30 mg/kg, i.g.) or celecoxib (10 and 30 mg/kg, i.g.), and a non-selective COX inhibitor, indomethacin (10mg/kg, i.g.), then food intake was monitored for 24h after administration. We also examined expression of IL-1ß, COX-2, and POMC mRNA in hypothalamus of docetaxel-treated rats and the effect of a COX-2 inhibitor on docetaxel-induced POMC mRNA expression. Food consumption in rats was significantly decreased 24h after administration of docetaxel and anorexia was partially reversed by all COX inhibitors. Administration of docetaxel increased IL-1ß, COX-2, and POMC mRNA expression in the hypothalamus of rats. The time required to increase these gene expressions was comparable to the latency period of docetaxel-induced anorexia in rats. In addition, pretreatment with COX-2 inhibitors suppressed docetaxel-induced expression of POMC mRNA. These results suggest that IL-1ß and COX-2 mRNA expression and subsequent activation of POMC in the hypothalamus may contribute to the development of docetaxel-induced anorexia in rats.

Hyperbaric oxygen preconditioning protects cortical neurons against oxygen-glucose deprivation injury: role of peroxisome proliferator-activated receptor-gamma.

Ischemic stroke is one of the leading causes of mortality and disability worldwide. Our previous studies have shown that hyperbaric oxygen (HBO) preconditioning can afford significant neuroprotection against cerebral ischemia-reperfusion (I/R) injury in rats. However, it is still unknown whether HBO preconditioning can directly protect primary cultured cortical neurons against oxygen-glucose deprivation (OGD). Peroxisome proliferator-activated receptor-gamma (PPAR γ) plays a central role in the regulation of apoptosis, oxidative stress and inflammation as well as affords significant neuroprotection against cerebral I/R injury. 15-deoxy-∆(12,14)-prostaglandin J(2) (15d-PGJ(2)) is an endogenous ligand with a high affinity for PPAR γ. Recently, some studies demonstrate that activation of PPAR γ mediates lipopolysaccharide and anesthetic preconditioning. In the present study, we firstly found that OGD exposure caused the significant damage of cultured cortical neurons evaluated by cell viability, lactate dehydrogenase (LDH) release and caspase-3 activity, which were significantly ameliorated by HBO preconditioning. Furthermore, HBO preconditioning significantly increased the levels of PPAR γ mRNA and protein, PPAR γ DNA binding activity, 15d-PGJ(2) and antioxidant enzymatic activities in primary cultured cortical neurons with OGD exposure. Moreover, PPAR γ antagonist GW9662 dose-dependently abolished the protection of HBO preconditioning in OGD-exposed neurons. GW9662 blocked the increase of PPAR γ DNA binding activity and antioxidant enzymatic activities, but did not influence the 15d-PGJ(2) level in OGD-exposed neurons with HBO preconditioning. However, the cyclooxygenase (COX)-2 inhibitor NS-398 blocked the production of 15d-PGJ(2) in OGD-exposed neurons with HBO preconditioning. In addition, 15d-PGJ(2) preconditioning could also protect cultured neurons against OGD injury. These results demonstrate that HBO preconditioning has directly beneficial effects on ODG-exposed cortical neurons by the activation of PPAR γ subsequent to the production of 15d-PGJ(2), which in turn increases the downstream antioxidant enzymatic activities.

Houttuynia cordata, a novel and selective COX-2 inhibitor with anti-inflammatory activity.

ETHNOPHARMACOLOGICAL RELEVANCE: Houttuynia cordata Thunb. (Saururaceae; HC) has been long used in traditional oriental medicine for the treatment of inflammation diseases. Modern research has implicated inducible cyclooxygenase-2 (COX-2) as a key regulator of the inflammatory process. AIM OF THE STUDY: In the present study, we aimed to investigate the effect of HC on COX-2. We examined the effects of HC on lipopolysaccharide (LPS)-induced prostaglandin (PG) E(2) production, an indirect indicator of COX-2 activity, and COX-2 gene and protein expression in mouse peritoneal macrophages. MATERIALS AND METHODS: LPS-induced mouse peritoneal macrophages were employed as an in vitro model system. LPS-induced PGE(2) production was assessed by enzyme-linked immunosorbant assay and COX-2 protein expression was assessed by Western blot assay. RESULTS: The results showed that HC was able to inhibit the release of LPS-induced PGE(2) from mouse peritoneal macrophages (IC50 value: 44.8 µg/mL). Moreover, the inhibitory activity of HC essential oil elicited a dose-dependent inhibition of COX-2 enzyme activity (IC50 value: 30.9 µg/mL). HC was also found to cause reduction in LPS-induced COX-2 mRNA and protein expression, but did not affect COX-1 expression. The non-steroidal anti-inflammatory drug (NSAID) and specific COX-2 inhibitor NS398 functioned similarly in LPS-induced mouse peritoneal macrophages. CONCLUSION: Taken together, our data suggest HC mediates inhibition of COX-2 enzyme activity and can affect related gene and protein expression. HC works by a mechanism of action similar to that of NSAIDs. These results add a novel aspect to the biological profile of HC.

Nitric oxide regulates stretch-induced proliferation in C2C12 myoblasts.

Mechanical stretch of skeletal muscle activates nitric oxide (NO) production and is an important stimulator of satellite cell proliferation. Further, cyclooxygenase (COX) activity has been shown to promote satellite cell proliferation in response to stretch. Since COX-2 expression in skeletal muscle can be regulated by NO we sought to determine if NO is required for stretch-induced myoblast proliferation and whether supplemental NO can counter the effects of COX-2 and NF-kappaB inhibitors. C2C12 myoblasts were cultured for 24 h, then switched to medium containing either the NOS inhibitor, L-NAME (200 microM), the COX-2 specific inhibitor NS-398 (100 microM), the NF-kappaB inhibiting antioxidant, PDTC (5 mM), the nitric oxide donor, DETA-NONOate (10-100 microM) or no supplement (control) for 24 h. Subgroups of each treatment were exposed to 1 h of 15% cyclic stretch (1 Hz), and were then allowed to proliferate for 24 h before fixing. Proliferation was measured by BrdU incorporation during the last hour before fixing, and DAPI stain. Stretch induced a twofold increase in nuclear number compared to control, and this effect was completely inhibited by L-NAME, NS-398 or PDTC (P < 0.05). Although DETA-NONOate (10 microM) did not affect basal proliferation, the NO-donor augmented the stretch-induced increase in proliferation and rescued stretch-induced proliferation in NS-398-treated cells, but not in PDTC-treated cells. In conclusion, NO, COX-2, and NF-kappaB are necessary for stretch-induced proliferation of myoblasts. Although COX-2 and NF-kappaB are both involved in basal proliferation, NO does not affect basal growth. Thus, NO requires the synergistic effect of stretch in order to induce muscle cell proliferation.

Stimulatory effects of areca nut extracts on prostaglandin E2 production by human polymorphonuclear leukocytes.

BACKGROUND: Areca quid chewing increases the prevalence of periodontal diseases. Areca nut extract (ANE) inhibits the defensive functions of human polymorphonuclear leukocytes (PMNs). This in vitro study investigates the effects of ANE on the production of cyclooxygenase (COX)-2 and the inflammatory mediator prostaglandin E(2) (PGE(2)) by PMNs. METHODS: The possible effects of ANE on the production of COX-2 were examined using Western blotting analysis. The viability and production of PGE(2) of treated PMNs were determined using the propidium iodide staining method and the competition enzyme assay, respectively. The possible pathways involved were also examined using the COX-2 inhibitor (NS398), the intracellular calcium chelator 1,2-bis(2-aminophenoxy)ethane-N, N, N', N'-tetraacetic acid tetrakis (acetoxymethyl ester) (BAPTA-AM), the p38 mitogen-activated protein kinase (MAPK) inhibitor (SB203580), and the extracellular signal-regulated protein kinase (ERK) inhibitor (U0126). The effects of ANE on the viability or PGE(2) production were statistically assessed using a one-way analysis of variance and Tukey multiple-comparison intervals with alpha = 0.05. RESULTS: ANE significantly induced the production of PGE(2) in a time- and concentration-dependent manner. This induction resulted from an increased expression of COX-2. Moreover, the application of BAPTA-AM, SB203580, and U0126 statistically significantly suppressed the induction of PGE(2). CONCLUSIONS: ANE induced the production of PGE(2). The activation of the intracellular calcium concentrations, p38 MAPK, and ERK may be involved in the inducing effects of ANE on PMNs. The findings suggest that areca nut chewing may induce an inflammatory response and affect the periodontal health of consumers.

Suppression of migratory/invasive ability and induction of apoptosis in adenomyosis-derived mesenchymal stem cells by cyclooxygenase-2 inhibitors.

OBJECTIVE: To investigate if stem or progenitor cells are found in adenomyosis and to characterize the role of cyclooxygenase-2 (COX-2) in adenomyosis-derived mesenchymal stem cell (AMSC)-related pathogenesis of adenomyosis. DESIGN: Experimental clinical study. SETTING: University hospital. PATIENT(S): Ten patients with adenomyosis. INTERVENTION(S): Hysterectomy. MAIN OUTCOME MEASURE(S): The gene expression of AMSCs and endometrial mesenchymal stem cells (EMSCs) were analyzed by microarray, quantitative polymerase chain reaction and Western blot. Methylthiazol tetrazolium, proliferation, apoptosis, and migration/invasion assays of AMSCs and EMSCs were evaluated after COX-2 inhibitor treatment. RESULT(S): We isolated nine EMSCs from normal endometrium (n=10) and six AMSCs (n=10) from adenomyosis. The morphology, phenotype, and potential of multilineage differentiation between EMSCs and AMSCs were not significantly different. Using complementary DNA microarrays, the expression profiles of EMSCs are related to those of bone marrow-derived mesenchymal stem cells (BMSCs), but AMSCs are different from EMSCs and BMSCs in the gene profiles. We validated the microarray results and showed that there is increased COX-2 expression in AMSCs compared with EMSCs. Treatment with a COX-2 inhibitor suppressed migration and invasion and induced apoptotic capabilities of AMSCs, but not of EMSCs. CONCLUSION(S): Overexpression of COX-2 in AMSCs may play an important role in the pathogenesis of adenomyosis. COX-2 could be a possible target for treatment and prevention of adenomyosis.

Wogonin inhibits osteoclast formation induced by lipopolysaccharide.

To evaluate the inhibitory activity of wogonin against lipopolysaccharide (LPS)-induced bone resorption, we investigated the effect of wogonin on osteoclastogenesis induced by LPS. Wogonin inhibited LPS-induced osteoclastogenesis in co-cultures of mouse calvaria-derived osteoblasts and bone marrow-derived pre-osteoclasts. Wogonin also suppressed osteoclastogenesis in LPS-injected mouse calvaria. In osteoblasts, the upregulation of receptor activator of nuclear factor-kappaB (RANKL) expression and the downregulation of osteoprotegerin (OPG) expression by LPS were inhibited by wogonin. Wogonin and NS-398, a COX-2 inhibitor, suppressed LPS-stimulated PGE(2) production in osteoblasts. NS-398 inhibited the effect of LPS on RANKL and OPG expression in osteoblasts. These results suggest that wogonin acts as an inhibitor of LPS-induced osteoclastogenesis through downregulation of RANKL and upregulation of OPG expression via blockage of PGE(2) production. Based on these results, wogonin has potential for use as a therapeutic agent in bacteria-induced bone resorption.

Cyclo-oxygenase-2 mediates hyperbaric oxygen preconditioning-induced neuroprotection in the mouse model of surgical brain injury.

BACKGROUND AND PURPOSE: We investigated the role of cyclo-oxygenase-2 (COX-2) in mechanisms of hyperbaric oxygen preconditioning (HBO-PC) in the mouse model of surgical brain injury (SBI). METHODS: C57BL mice were administered 100% oxygen for 1 hour at 2.5 atmosphere absolute for 5 consecutive days and subjected to SBI. Neurological status and brain edema were evaluated at 24 hours and 72 hours after the brain insult. Fluorescent immunostaining and Western blotting were performed to study hypoxia-inducible factor-1alpha and COX-2, respectively. Two doses of COX-2 inhibitor, NS398 (3 mg/kg and 10 mg/kg) were used to verify the role of COX-2 signaling pathway in the mechanism of HBO-PC. RESULTS: HBO-PC improved neurological status and decreased brain edema at 24 hours and 72 hours after SBI. HBO-PC by itself and SBI independently increased COX-2 levels by 2-fold and 4-fold, respectively. HBO-PC, however, reduced increase in hypoxia-inducible factor-1alpha and COX-2 expression after SBI. The HBO-PC-induced improvement in neurological status and brain edema was reversed by a suboptimal dose of the COX-2 inhibitor, NS398 (10 mg/kg intraperitoneally; 1/4th of dose shown to provide neuroprotection), which itself had no effect on investigated end points. CONCLUSIONS: HBO-PC attenuates postoperative brain edema and improves neurological outcomes after SBI. The HBO-PC-induced neuroprotection is mediated through COX-2 signaling pathways.

The effect of linolenic Acid on bovine oocyte maturation and development.

Dietary polyunsaturated fatty acids can influence reproductive performance. In dairy cattle, some high-fat diets resulted in higher blastocyst rates and improved embryo quality. These effects may partly be mediated by a direct action of fatty acids on oocyte development. The present study investigated the effect of linolenic acid (ALA; 18:3 n-3) supplementation on bovine oocyte maturation and early embryo development in vitro. Treatment of cumulus-oocyte complexes (COCs) with 50 muM ALA significantly increased the percentage of oocytes at the metaphase II (MII) stage compared with untreated controls (95% +/- 2% vs. 84% +/- 2%, respectively). Higher doses of ALA were detrimental. Treatment of COCs with 50 muM ALA compared with controls also resulted in a significantly higher percentage of cleaved embryos (77% +/- 9% vs. 69% +/- 9%, respectively) and blastocyst rate (36% +/- 4% vs. 23% +/- 5%, respectively) and better-quality embryos. Furthermore, COCs treated with ALA had significant increases compared with controls in: 1) prostaglandin E(2) (PGE(2)) concentration (233% +/- 41%) in the medium, 2) intracellular cAMP at 3 h of maturation, and 3) phosphorylation of the mitogen-activated protein kinases (MAPKs) during the first 6 h of maturation. Moreover, ALA overcame the suppressive effects of the prostaglandin-endoperoxide synthase 2 inhibitor (NS-398) on oocyte maturation and partially improved the maturation rate in the presence of the MAPK kinase inhibitor (U-0126). Linolenic acid could not, however, recover maturation in the presence of both inhibitors. In conclusion, treatment of bovine COCs with ALA during oocyte maturation affects the molecular mechanisms controlling oocyte nuclear maturation, leading to an increased number of MII-stage oocytes and improved subsequent early embryo development. This effect is mediated both directly through MAPK pathway and indirectly through PGE(2) synthesis.