Preventive effect of Ligularia fischeri on inhibition of nitric oxide in lipopolysaccharide-stimulated RAW 264. 7 macrophages depending on cooking method

BACKGROUND: Ligularia fischeri (common name Gomchwi) is known for its pharmaceutical properties and used in the treatment of jaundice, scarlet-fever, rheumatoidal arthritis, and hepatic diseases; however, little is known about its anti-inflammatory effect. In this study the influence of blanching and pan-frying on the anti-inflammatory activity of Ligularia fischeri (LF) was evaluated. RESULTS: Fresh LF and cooked LF showed no significant effect on the viability of macrophages after 24 h incubation. Fresh LF was found to be the most potent inhibitor of nitric oxide (NO) production at 100 µg/ml, while pan-fried LF showed little inhibitory effect on lipoloysaccharide (LPS) stimulated murine machrophage RAW264.7 cells. In contrast with its effect on NO production, pan-fried LF showed significant attenuation of the expression of inducible nitiric oxide synthase (iNOS) compared with fresh LF. In the cooking method of LF, PGE2 production was not affected in the LPS-induced RAW 264.7 cells. In LPS-induced RAW 264.7 cells, pretreatment by fresh and cooked LF increased COX2 mRNA expression. The 3-O-caffeoylquinic acid content of blanching and pan-frying LF increased by 4.92 and 9.7 fold with blanching and pan-frying respectively in comparison with uncooked LF. CONCLUSIONS: Regardless of the cooking method, Ligularia fischeri exhibited potent inhibition of NO production through expression of iNOS in LPS-induced RAW264.7 cells.

Noncrystalline uric acid inhibits proteoglycan and glycosaminoglycan synthesis in distal tubular epithelial cells (MDCK)

Hyperuricemia is associated with renal stones, not only consisting of uric acid (UrAc) but also of calcium oxalate (CaOx). Glycosaminoglycans (GAGs) are well-known inhibitors of growth and aggregation of CaOx crystals. We analyzed the effect of noncrystalline UrAc on GAG synthesis in tubular distal cells. MDCK (Madin-Darby canine kidney) cells were exposed to noncrystalline UrAc (80 µg/mL) for 24 h. GAGs were labeled metabolically and characterized by agarose gel electrophoresis. The expression of proteoglycans and cyclooxygenase 2 (COX-2) was assessed by real-time PCR. Necrosis, apoptosis and prostaglandin E2 (PGE2) were determined by acridine orange, HOESCHT 33346, and ELISA, respectively. CaOx crystal endocytosis was evaluated by flow cytometry. Noncrystalline UrAc significantly decreased the synthesis and secretion of heparan sulfate into the culture medium (UrAc: 2127 ± 377; control: 4447 ± 730 cpm) and decreased the expression of perlecan core protein (UrAc: 0.61 ± 0.13; control: 1.07 ± 0.16 arbitrary units), but not versican. Noncrystalline UrAc did not induce necrosis or apoptosis, but significantly increased COX-2 and PGE2 production. The effects of noncrystalline UrAc on GAG synthesis could not be attributed to inflammatory actions because lipopolysaccharide, as the positive control, did not have the same effect. CaOx was significantly endocytosed by MDCK cells, but this endocytosis was inhibited by exposure to noncrystalline UrAc (control: 674.6 ± 4.6, CaOx: 724.2 ± 4.2, and UrAc + CaOx: 688.6 ± 5.4 geometric mean), perhaps allowing interaction with CaOx crystals. Our results indicate that UrAc decreases GAG synthesis in MDCK cells and this effect could be related to the formation of UrAc and CaOx stones.

Ethyl acetate fraction from Cudrania tricuspidata inhibts IL-1β-induced rheumatoid synovial fibroblast proliferation and MMPs, COX-2 and PGE2 production

Objectives: The objective of this study is to determine the effects of Ethyl acetate fraction from Cudrania tricuspidata (EACT) on the interleukin-1b (IL-1b)-induced proliferation of rheumatoid synovial fbroblasts (RASFs) and production of matrix metalloproteinases (MMPs), cyclooxygenase (COX) and prostaglandin E2 (PGE2) by RASFs. Materials and Methods: The proliferation of RASFs was evaluated with CCK-8 reagent in the presence of IL-1b with/without EACT. The expression of MMPs, TIMP-1, COXs, PGE2 and intracellular MAPK signalings, including p-ERK, p-p38, p-JNK and NF-kB were examined by immunoblotting or semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) and ELISA in conditions as described above. Results: EACT inhibits IL-1β-induced proliferation of RASFs and MMP-1, 3, COX-2 mRNA and protein expression, PGE2 production induced with IL-1b. EACT also inhibits the phosphorylation of ERK-1/2, p38, JNK and activation of NF-kB by IL-1b. Conclusions: These results suggest that EACT might be involved in synovial fbroblast proliferation and MMPs, COX-2, and PGE2 production, which are involved in joint destruction in rheumatoid arthritis (RA), indicating that this might be a new therapeutic modality for management of rheumatoid arthritis.

ERK-1/-2 and p38 Kinase Oppositely Regulate 15-deoxy-delta(12,14)-prostaglandinJ2-Induced PPAR-gamma Activation That Mediates Dedifferentiation But Not Cyclooxygenase-2 Expression in Articular Chondrocytes

Peroxisome proliferator-activated receptor gamma (PPAR-gamma) is a ligand-activated transcription factor and plays an important role in growth, differentiation, and inflammation in different tissues. In this study, we investigated the effects of 15d-PGJ2, a high-affinity ligand of PPAR-gamma, on dedifferentiation and on inflammatory responses such as COX-2 expression and PGE2 production in rabbit articular chondrocytes with a focus on ERK-1/-2, p38 kinase, and PPAR-gamma activation. We report here that 15d-PGJ2 induced dedifferentiation and/or COX-2 expression and subsequent PGE2 production. 15d-PGJ2 treatment stimulated activation of ERK-1/-2, p38 kinase, and PPAR-gamma. Inhibition of ERK-1/-2 with PD98059 recovered 15d-PGJ2-induced dedifferentiation and enhanced PPAR-gamma activation, whereas inhibition of p38 kinase with SB203580 potentiated dedifferentiation and partially blocked PPAR-gamma activation. Inhibition of ERK-1/-2 and p38 kinase abolished 15d-PGJ2-induced COX-2 expression and subsequent PGE2 production. Our findings collectively suggest that ERK-1/-2 and p38 kinase oppositely regulate 15d-PGJ2-induced dedifferentiation through a PPAR-gamma-dependent mechanism, whereas COX-2 expression and PGE2 production is regulated by ERK-1/-2 through a PPAR-gamma-independent mechanism but not p38 kinase in articular chondrocytes. Additionally, these data suggest that targeted modulation of the PPAR-gamma and mitogen-activated protein kinase pathway may offer a novel approach for therapeutic inhibition of joint tissue degradation.

Interaction between NO and oxytocin: influence on LHRH release

Nitric oxide synthase (NOS)-containing neurons have been localized in various parts of the CNS. These neurons occur in the hypothalamus, mostly in the paraventricular and supraoptic nuclei and their axons project to the neural lobe of the pituitary gland. We have found that nitric oxide (NO) controls luteinizing hormone-releasing hormone (LHRH) release from the hypothalamus acting as a signal transducer in norepinephrine (NE)-induced LHRH release. LHRH not only releases LH from the pituitary but also induces sexual behavior.On the other hand, it is known that oxytocin also stimulates mating behavior and there is some evidence that oxytocin can increase NE release. Therefore, it occurred to us that oxytocin may also stimulate LHRH releave via NE and NO. To test this hypothesis, we incubated medial basal hypothalamic (MBH) explants from adult male rats in vitro. Following a preincubation period of 30 min, MBH fragments were incubated in Krebs-Ringer bicarbonate buffer in the presence of various concentrations of oxytocin. Oxytocin relesed LHRH at concentrations ranging from 0.1 nM to 1muM with a maximal stimulatory effect (P<0.001) at 0.1 muM, but with no stimulatory effect at 10 muM. That these effects were mediated by NO was shown by the fact that incubation of the tissues with NG-monomethyl-L-arginine (NMMA), a competitive inhibitor of NOS, blocked the stimulatory effects. Furthermore, the release of LHRH by oxytocin was also blocked by prazocin, an alpha1-adrenergic receptor antagonist, indicating that NE mediated this effect. Oxytocin at the same concentrations also increased the activity of NOS (P<0.01) as measured by the conversion of [14C]arginine to citrulline, which is produced in equimolar amounts with NO by the action of NOS. The release of LHRH induced by oxytocin was also accompanied by a significant (P<0.02) increase in the release of prostaglandin E2 (PGE2), a mediator of LHRH release that is released by NO. On the other hand, incubation of neural lobes with vaious concentrations of sodium nitroprusside (NP) (300 or 600 muM), a releaser of NO, revealed that NO acts to suppres (P<0.01) the release of oxytoxin. Therefore, our results indicate that oxytocin releases LHRH by stimulating NOS via NE, resulting in an increased release of NO, which increases PGE2 release that in turn induces LHRH release. Furthermore, the released NO can act back on oxytocinergic terminals to suppress the release of oxytocin in an ultrashort-loop negative feedback.

Participation of autonomic nervous system in the pathogenesis of Chagas disease

Evidences accumulated over the last decade give adequate proof for the existence of circulating antibodies in Chagas disease which binds to ß adrenergic and muscarinic cholinergic receptor of lymphocytes and myocardium. The interaction of the antibodies with lymphocytes and cardiac neurotransmitter receptors behaving as an agonist, triggers in the cells intracellular signal transductions that alter the physiological behaviour of this cells. These events converted the cells in pathologically active cells. Thus, antibodies activating ß adrenergic receptors of T helper (Th) lymphocytes increase cAMP and releases PGE2 by T suppressor/cytotoxic (Ts/c) cell, inducing in this way, immunosuppression by simultaneous inhibition of Th and stimulation of Ts/c cell function. All these antibodies actions were mimetized by parasite's membranes. On the other hand, the interaction of antibodies against heart ß adrenergic and cholinergic receptors trigger physiologic, morphologic, enzymatic and molecular alterations, that leading to cardiac damage. The analysis of the prevalence and distribution of these antibodies shows a strong association with seropositive asymptomatic patients with autonomic dysfunction in comparison with those asymptomatic without alteration of the heart autonomic disorders: pointing to that the presence of these antibodies may partially explain the cardiomyoneuropathy of Chagas disease, in which the sympathetic and parasympathetic systems are affected. The deoposit of autoantibodies on the myocardial neurotransmitter receptors, behaving like an agonist, could induced desensitization and/or down regulation of the receptors. This in turn, could led to a progressive blockade of myocardium neurotransmitter receptors, with sympathetic and parasympathetic dennervation, a phenomenon that has been described in the course of Chagas cardioneuropathy

Influence of the estrous cycle on the norepinephrine-induced contraction of rat aorta: relationship to vascular prostanoids biosynthesis

Since ovarian sex steroids (estradiol and progesterone) may affect both blood pressure and prostanoids synthesis, and because prostaglandin-E2 (PGE2) and prostacyclin (PGI2) can modulate the vascular action of pressor hormones, we investigated the vascular reactivity to norepinephrine during the estrous cycle of the rat. In addition, we determined the vascular biosynthesis of PGE2 and 6-keto-PGF1 alpha (the stable metabolite of PGI2) at different stages of the estrous cycle. Cumulative dose-response curves were obtained by a stepwise increase in the concentration of norepinephrine. The contraction of thoracic aortic rings induced by norepinephrine did not change significantly between estrus, metestrus and diestrus. However, aortic rings obtained on proestrus showed a significant reduction in the maximal contraction (Emax) induced by norepinephrine (p < 0.001). In addition, we found significant increases in vascular synthesis of PGE2 and PGI2 on proestrus (p < 0.001). These results indicate that vascular reactivity and vascular prostanoids synthesis are influenced by the hormonal changes occurring during the estrous cycle of normal female rats. It is possible that prostanoids generated locally may play an important role in the regulation of vasomotor tone in the systemic vascular bed throughout the estrous cycle

Prostaglandin biosynthesis inhibitory activity of some indian-1 acids in relation to their anti-inflammatory activity and ulcerogenic potency.

Continuation of our work towards development of some newer non-steroidal anti-inflammatory agents led us to some substituted indian-1-acids with low ulcerogenic liability. Prostaglandin biosynthesis inhibitory activity of these indian acids and their acid dissociation constants were evaluated in view of their activity profile.

The effect of immunotherapy on the in vitro productions of histamine, prostaglandin E2 and leukotriene C4 in asthmatic children.

In order to elucidate the working mechanisms of immunotherapy (IT), the in vitro productions of histamine, prostaglandin E2 (PGE2) and leukotriene C4 (LTC4) were studied in 18 newly diagnosed and 20 hyposensitized (greater than 2 yr) asthmatic children. All were sensitive to house dust and dust mites. (D. pteronyssinus). Ten age-matched normal children were included as control. Polymorphonuclear (PMNs) and mononuclear (MNCs) leukocytes were separated by density gradient centrifugation and dextran sedimentation. PMNs (2 x 10(7) cells/ml) and MNCs (2 x 10(7) cells/ml) were stimulated with mite allergen (10 micrograms/ml) and calcium ionophore A23187 (1 microgram/ml) for 15 minutes. The plasma and culture supernatant (sup) histamine levels and sup PGE2 and LTC4 were measured by RIA. The results showed; 1) When compared to new patients, the treated patients had much lower plasma and sup histamine (p less than 0.001), no matter whether PMNs and MNCs were stimulated with allergen or A23187 and the normals had the lowest histamine level among 3 groups; 2) LTC4 in A23187-stimulated sup was lower in treated patients (p less than 0.05); 3) The PGE2 in allergen-stimulated sup was markedly increased in treated patients as compared to new patients (p less than 0.01) and the PGE2 in sup of normals was also much higher than that of new patients. Thus, immunotherapy is able to reverse the abnormal secretory pattern of inflammatory mediators of allergic patients, and this change may account, partly, for its clinical effectiveness.