Biomechanical strain regulates TNFR2 but not TNFR1 in TMJ cells

We sought to examine whether cyclic tensile strain (CTS) regulates the gene expression of tumor necrosis factor (TNF)-alpha, its receptors TNFR1 and TNFR2, and inducible nitric oxide synthase (iNOS) under inflammatory conditions, and whether these effects of CTS are sustained. Rat temporomandibular joint disc cells (TDC) were exposed to CTS in the presence or absence of interleukin (IL)-1beta for 4 and 24h. Cells were also stimulated with IL-1beta for 24h while being subjected to CTS only for the initial 1, 2, 4, 8, and 12h or the entire 24h incubation time. Furthermore, cells were incubated with IL-1beta for 24, 36, or 48 h while being exposed to CTS only for the initial 8h. Gene expression of TNF-alpha, its receptors, and iNOS was analyzed by RT-PCR, whereas protein synthesis was determined by ELISA for TNF-alpha, immunofluorescence for TNFRs, and Griess reaction for nitric oxide. CTS inhibited the IL-1beta-stimulated synthesis of TNF-alpha, TNFR2, and iNOS. TNFR1 was constitutively expressed but not regulated by IL-1beta or CTS. Application of CTS for only 1 or 2h during a 24h incubation with IL-1beta was sufficient to inhibit IL-1beta-induced upregulation of TNF-alpha, TNFR2, and iNOS. However, for maximal inhibition of these genes a longer exposure of CTS was required. These findings are the first to show that biomechanical signals regulate the expression of TNFR2 but not TNFR1 under inflammatory conditions. Furthermore, the antiinflammatory effects of biomechanical signals on TDC are maintained for prolonged periods of time but are transient.     

Dynamic compression counteracts IL-1beta induced iNOS and COX-2 activity by human chondrocytes cultured in agarose constructs

*NO and PGE2 are inflammatory mediators derived from the inducible iNOS and COX enzymes and are potentially important pharmacological targets in OA. Both mechanical loading and IL-1beta will influence the release of *NO and PGE2. Accordingly, the current study examines the effect of dynamic compression on *NO and PGE2 release by human chondrocytes cultured in agarose constructs in the presence and absence of selective iNOS and COX-2 inhibitors. The current data demonstrate that IL-1beta induced nitrite and PGE2 release and inhibited [3H]-thymidine and 35SO4 incorporation. Inhibitor experiments indicate that 1400W and NS-398 either partially reversed or abolished IL-1beta induced nitrite and PGE2 release. IL-1beta induced inhibition of cell proliferation and proteoglycan synthesis was partially reversed with 1400W but was not influenced by NS-398. For the dynamic loading experiments, 1400W and NS-398 either reduced or abolished the compression-induced inhibition of *NO and PGE2 release in the presence of IL-1beta. The IL-1beta induced inhibition of cell proliferation was not influenced by 1400W or NS-398 whereas strain-induced stimulation of proteoglycan synthesis in the presence of IL-1beta was enhanced by 1400W. The data obtained using human chondrocytes demonstrate that IL-1beta induced *NO and PGE2 release via an iNOS-driven-COX-2 inter-dependent pathway. This response could be reversed by dynamic compression. These data indicate interactions exist between the NOS and COX pathways, a finding which will provide new insights in the development of pharmacological or biophysical treatments for cartilage disorders such as OA.     

Dynamic compression inhibits the synthesis of nitric oxide and PGE(2) by IL-1beta-stimulated chondrocytes cultured in agarose constructs

Both mechanical loading and interleukin-1beta (IL-1beta) are known to regulate metabolic processes in articular cartilage through pathways mediated by nitric oxide ((*)NO) and PGE(2). This study uses a well-characterized model system involving isolated chondrocytes cultured in agarose constructs to test the hypothesis that dynamic compression alters the synthesis of (*)NO and PGE(2) by IL-1beta-stimulated articular chondrocytes. The data presented demonstrate for the first time that dynamic compression counteracts the effects of IL-1beta on articular chondrocytes by suppressing both (*)NO and PGE(2) synthesis. Inhibitor experiments indicated that the dynamic compression-induced inhibition of PGE(2) synthesis and stimulation of proteoglycan synthesis were (*)NO mediated, while compression-induced stimulation of cell proliferation was (*)NO independent. The inhibition of (*)NO and PGE(2) by dynamic compression is a finding of major significance that could contribute to the development of novel strategies for the treatment of cartilage-degenerative disorders.     

Effect of cyclosporin-A on the blood--retinal barrier permeability in streptozotocin-induced diabetes

BACKGROUND: Our previous results showed that in retinas from streptozotocin (STZ)-induced diabetic rats there is an increased level of interleukin-1beta (IL-1beta). This cytokine may be involved in the expression of the inducible isoform of the nitric oxide synthase (iNOS), with consequent synthesis of large amounts of NO and blood-retinal barrier (BRB) breakdown. AIMS: The aim of this work was to examine whether the administration of cyclosporin-A (Cs-A) to STZ-induced diabetic rats inhibits the synthesis of IL-1beta and the expression of the inducible proteins, iNOS and cyclo-oxygenase-2 (COX-2) in retinal cells, and whether the activity of these proteins contribute to BRB breakdown. METHODS: The level of IL-1beta was evaluated by ELISA and the NO production by L-[3H]-citrulline formation. Expression of iNOS and COX-2 proteins was determined by two methods, western blot and immunohistochemistry. The permeability of the BRB was assessed by quantification of the vitreous protein. RESULTS AND DISCUSSION: Our results indicated that the levels of IL-1beta and NO in retinas from Cs-A-treated diabetic rats are significantly reduced, as compared to that in non-treated diabetic rats. The treatment of diabetic rats with Cs-A also significantly inhibited the expression of the inducible proteins, iNOS and COX-2. The evaluation of the vitreous protein content revealed that Cs-A also reduces the BRB permeability. Taken together, these results suggest that the increased production of the inflammatory mediators, IL-1beta and NO, in diabetes may affect the BRB permeability and therefore contribute to the development of diabetic retinopathy.     

Green tea polyphenol epigallocatechin-3-gallate inhibits the IL-1 beta-induced activity and expression of cyclooxygenase-2 and nitric oxide synthase-2 in human chondrocytes

We have previously shown that green tea polyphenols inhibit the onset and severity of collagen II-induced arthritis in mice. In the present study, we report the pharmacological effects of green tea polyphenol epigallocatechin-3-gallate (EGCG), on interleukin-1 beta (IL-1 beta)-induced expression and activity of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) in human chondrocytes derived from osteoarthritis (OA) cartilage. Stimulation of human chondrocytes with IL-1 beta (5 ng/ml) for 24 h resulted in significantly enhanced production of nitric oxide (NO) and prostaglandin E(2) (PGE(2)) when compared to untreated controls (p <.001). Pretreament of human chondrocytes with EGCG showed a dose-dependent inhibition in the production of NO and PGE(2) by 48% and 24%, respectively, and correlated with the inhibition of iNOS and COX-2 activities (p <.005). In addition, IL-1 beta-induced expression of iNOS and COX-2 was also markedly inhibited in human chondrocytes pretreated with EGCG (p <.001). Parallel to these findings, EGCG also inhibited the IL-1 beta-induced LDH release in chondrocytes cultures. Overall, the study suggests that EGCG affords protection against IL-1 beta-induced production of catabolic mediators NO and PGE(2) in human chondrocytes by regulating the expression and catalytic activity of their respective enzymes. Furthermore, our results also indicate that ECGC may be of potential therapeutic value for inhibiting cartilage resorption in arthritic joints.     

Both p38alpha(MAPK) and JNK/SAPK pathways are important for induction of nitric-oxide synthase by interleukin-1beta in rat glomerular mesangial cells

Interleukin 1beta (IL-1beta) induces expression of the inducible nitric-oxide synthase (iNOS) with concomitant release of nitric oxide (NO) from glomerular mesangial cells. These events are preceded by activation of the c-Jun NH(2)-terminal kinase/stress-activated protein kinase (JNK/SAPK) and p38(MAPK). Our current study demonstrates that overexpression of the dominant negative form of JNK1 or p54 SAPKbeta/JNK2 significantly reduces the iNOS protein expression and NO production induced by IL-1beta. Similarly, overexpression of the kinase-dead mutant form of p38alpha(MAPK) also inhibits IL-1beta-induced iNOS expression and NO production. In previous studies we demonstrated that IL-1beta can activate MKK4/SEK1, MKK3, and MKK6 in renal mesangial cells; therefore, we examined the role of these MAPK kinases in the modulation of iNOS induced by IL-1beta. Overexpression of the dominant negative form of MKK4/SEK1 decreases IL-1beta-induced iNOS expression and NO production with inhibition of both SAPK/JNK and p38(MAPK) phosphorylation. Overexpression of the kinase-dead mutant form of MKK3 or MKK6 demonstrated that either of these two mutant kinase inhibited IL-1beta-induced p38(MAPK) (but not JNK/SAPK) phosphorylation and iNOS expression. Interestingly overexpression of wild type MKK3/6 was associated with phosphorylation of p38(MAPK); however, in the absence of IL-1beta, iNOS expression was not enhanced. This study suggests that the activation of both SAPK/JNK and p38alpha(MAPK) signaling cascades are necessary for the IL-1beta-induced expression of iNOS and production of NO in renal mesangial cells.     

Endogenous nitric oxide proves not to be involved in the inhibition by IL-1beta of TGF-alpha-stimulated proliferation of RGM1 cells.

Combined treatment of isolated rat gastric mucosal (RGM1) cells with interleukin (IL)-1beta and transforming growth factor (TGF)-alpha resulted in expression of inducible nitric oxide synthase (iNOS) mRNA and iNOS protein 24 hr after the treatment. Constitutive NOS (nNOS) protein was not proved in the cells and not activated by IL-1beta+TGFalpha. Although IL-1beta and TGF-alpha alone exerted little or no effect on NO2 production, their combination gradually increased NO2- production from 12 to 24 hr following treatment. NO2- production stimulated by IL-1beta + TGFalpha was significantly reduced by N(G)-nitro-L-arginine methyl ester (L-NAME) or aminoguanidine, yet not by D-NAME. S-nitroso-N-acetyl-D,L-penicillamine and sodium nitropruside significantly inhibited both spontaneous and TGF-alpha stimulated DNA synthesis. Nonetheless, L-NAME did not affect the inhibition by IL-1beta of TGF-alpha-stimulated proliferation of RGM1 cells, eliminating the possibility of involvement of NO in the underlying mechanisms.     

Juxtacrine effects of IL-1 alpha precursor promote iNOS expression in vascular smooth muscle cells

After injury to the blood vessel wall, vascular smooth muscle cells (SMC) synthesize interleukin (IL)-1 and inducible nitric oxide (NO) synthase (iNOS). The present study tested whether endogenous production of IL-1 alpha stimulates iNOS expression in vascular SMC, and assessed whether IL-1 alpha exerts autocrine effects on the cells producing IL-1 alpha or juxtacrine effects on cells that contact the IL-1 alpha producing cells. Rat aortic SMC were transiently transfected with expression plasmids encoding either IL-1 alpha precursor, which localizes to the plasma membrane, or mature IL-1 alpha, which remains cytosolic. iNOS mRNA levels, determined by RT-PCR, and production of nitrite, a stable oxidation product of NO, were markedly elevated in SMC overexpressing IL-1 alpha precursor, and modestly elevated in SMC overexpressing mature IL-1 alpha, relative to SMC transfected with vector alone. Exposure to exogenous IL-1 beta or TNF-alpha further stimulated iNOS gene expression in SMC producing IL-1 alpha; low levels of IL-1 beta (20 pg/ml) were effective in SMC transfected with IL-1 alpha precursor plasmid, whereas SMC transfected with mature IL-1 alpha plasmid or vector alone required higher concentrations of IL-1 beta (200 and 2,000 pg/ml, respectively). The increases in iNOS mRNA levels and NO production in SMC overexpressing IL-1 alpha precursor were prevented by exogenous IL-1 receptor antagonist, suggesting that these effects were mediated by the type I IL-1 receptor. Immunostaining studies indicated that IL-1 alpha precursor stimulates iNOS gene expression via cell-cell contact. Expression of iNOS was enhanced in cells that were in contact with a cell overexpressing IL-1 alpha precursor (identified by coexpression of green fluorescent protein), and in cells that were overexpressing IL-1 alpha themselves, but only when the cell contacted another cell. Together these results indicate that IL-1 alpha precursor acts by cell-cell contact as an autocrine and juxtacrine enhancer of iNOS gene expression, inducing moderate iNOS expression on its own, and markedly augmenting the responsiveness of rat aortic SMC to exogenous cytokines.     

Role of mitogen-activated protein kinases and tyrosine kinases on IL-1-Induced NF-kappaB activation and iNOS expression in bovine articular chondrocytes.

Nitric oxide (NO), produced by the inducible isoform of the NO synthase (iNOS), plays an important role in the pathophysiology of arthritic diseases. This work aimed at elucidating the role of the mitogen-activated protein kinases (MAPK), p38MAPK and p42/44MAPK, and of protein tyrosine kinases (PTK) on interleukin-1beta (IL-1)-induced iNOS expression in bovine articular chondrocytes. The specific inhibitor of the p38MAPK, SB 203580, effectively inhibited IL-1-induced iNOS mRNA and protein synthesis, as well as NO production, while the specific inhibitor of the p42/44MAPK, PD 98059, had no effect. These responses to IL-1 were also inhibited by treatment of the cells with the tyrosine kinase inhibitors, genistein and tyrphostin B42, which also prevented IL-1-induced NF-kappaB activation. The p38MAPK inhibitor, SB 203580, had no effect on IL-1-induced NF-kappaB activation. Finally, the p42/44MAPK inhibitor, PD 98059, prevented IL-1-induced AP-1 activation in a concentration that did not inhibit iNOS expression. In conclusion, this study shows that (1) PTK are part of the signaling pathway that leads to IL-1-induced NF-kappaB activation and iNOS expression; (2) the p38MAPK cascade is required for IL-1-induced iNOS expression; (3) the p42/44MAPK and AP-1 are not involved in IL-1-induced iNOS expression; and (4) NF-kappaB and the p38MAPK lie on two distinct pathways that seem to be independently required for IL-1-induced iNOS expression. Hence, inhibition of any of these two signaling cascades is sufficient to prevent iNOS expression and the subsequent production of NO in articular chondrocytes.     

Development of selective tolerance to interleukin-1beta by human chondrocytes in vitro

Interleukin-1 induces release of NO and PGE(2) and production of matrix degrading enzymes in chondrocytes. In osteoarthritis (OA), IL-1 continually, or episodically, acts on chondrocytes in a paracrine and autocrine manner. Human chondrocytes in chondron pellet culture were treated chronically (up to 14 days) with IL-1beta. Chondrons from OA articular cartilage were cultured for 3 weeks before treatment with IL-1beta (0.05-10 ng/ml) for an additional 2 weeks. Spontaneous release of NO and IL-1beta declined over the pretreatment period. In response to IL-1beta (0.1 ng/ml), NO and PGE(2) release was maximal on Day 2 or 3 and then declined to near basal level by Day 14. Synthesis was recovered by addition of 1 ng/ml IL-1beta on Day 11. Expression of inducible nitric oxide synthase (iNOS), detected by immunofluorescence, was elevated on Day 2 and declined through Day 14, which coordinated with the pattern of NO release. On the other hand, IL-1beta-induced MMP-13 synthesis was elevated on Day 3, declined on Day 5, and then increased again through Day 14. IL-1beta increased glucose consumption and lactate production throughout the treatment. IL-1beta stimulated proteoglycan degradation in the early days and inhibited proteoglycan synthesis through Day 14. Chondron pellet cultures from non-OA cartilage released the same amount of NO but produced less PGE(2) and MMP-13 in response to IL-1beta than OA cultures. Like the OA, IL-1beta-induced NO and PGE(2) release decreased over time. In conclusion, with prolonged exposure to IL-1beta, human chondrocytes develop selective tolerance involving NO and PGE(2) release but not MMP-13 production, metabolic activity, or matrix metabolism.     

Interleukin-1beta up-regulates nitrite production: effects on ovarian function.

We have previously reported that Interleukin-1beta (IL-1beta) affects ovarian function in the rat, modulating prostaglandin and progesterone (P) production. As IL-1beta effects were associated to nitric oxide (NO) synthesis, in the present work we have further examined the role of ovarian NOS-system, in IL-1beta antisteroidogenic action. Mid-luteal explants from rats were incubated for 4 h in the presence of IL-1beta (1-35 ng/ml)-alone or in combination with NOS-inhibitors-and then assayed for P and nitrite production. IL-1beta treatment reduced P levels in a dose-dependent manner, returning to basal levels at 35 ng/ml. This reduction in steroid synthesis was paralleled by a dose-dependent increase in nitrite levels, reaching a maximum at 25 ng/ml but without effect at 35 ng/ml. L-Arginine (1 and 2 mM) was able to mimic IL-1beta actions and the NOS blocker L-Nitro-Arginin-Methyl Ester reverted these effects. Moreover, the selective iNOS inhibitor, 1400 W, completely abolished IL-1beta antisteroidogenic effect, therefore confirming the dependence of IL-1beta action upon iNOS activation. Finally, IL-1beta did not affect eNOS expression but up-regulated iNOS mRNA and protein levels. Our results suggest an interaction between IL-1beta and the NOS-system. Thus, we may conclude that in the rat iNOS-derived NO production, induced by IL-1beta, affects ovarian P biosynthesis and hence NO may be a major effector molecule of ovarian IL-1 system.     

Suppression of interleukin-1 beta-induced nitric oxide production in RINm5F cells by inhibition of glucose-6-phosphate dehydrogenase

In rat pancreatic islets and insulin-producing cell lines, IL-1beta induces expression of inducible nitric oxide synthase and NO production leading to impairment of glucose-stimulated insulin release and decreased cell survival. NADPH is an obligatory cosubstrate for iNOS synthesis of NO. We hypothesized that IL-1beta stimulates an increase in activity of NADPH-producing enzyme(s) prior to NO production and that this increase is necessary for NO production. Using rat insulin-secreting RINm5F cells, we found that (1) IL-1beta caused a biphasic change in the NADPH level (increased by 6 h and decreased after prolonged incubation in the presence of 2 ng/mL IL-1beta); (2) IL-1beta stimulated increased activity of glucose-6-phosphate dehydrogenase (G6PD) in a time- and dose-dependent manner, and G6PD expression was increased by about 80% after exposure to 2 ng/mL IL-1beta for 18 h: (3) IL-1beta-stimulated NO production was positively correlated with increased G6PD activity; (4) IL-1beta did not cause any significant change in enzyme activity of another NADPH-producing enzyme, malic enzyme; (5) IL-1beta-induced NO production was significantly reduced either by inhibiting G6PD activity using an inhibitor of G6PD (dehydroepiandrosterone) or by inhibiting G6PD expression using an antisense oligonucleotide to G6PD mRNA; and (6) IL-1beta stimulated a decrease in the cAMP level. 8-Bromo-cAMP caused decreased G6PD activity, and the protein kinase A inhibitor H89 led to a increase in G6PD activity in RINm5F cells. In conclusion, our data show that IL-1beta stimulated G6PD activity and expression level, providing NADPH that is required by iNOS for NO production in RINm5F cells. Also, inhibition of the cAMP-dependent PKA signal pathway is involved in an IL-1beta-stimulated increase in G6PD activity.     

Normal rat hepatic stellate cells respond to endotoxin in LBP-independent manner to produce inhibitor(s) of DNA synthesis in hepatocytes

Endotoxin is implicated in the pathology of acute liver failure. The mechanisms of its actions on quiescent hepatic stellate cells (qHSCs) and their implications in hepatocyte injury are incompletely understood. We investigated effects of endotoxin (bacterial lipopolysaccharide; LPS) on qHSCs and subsequently on hepatocytes. After overnight culture following their isolation, qHSCs were incubated with or without endotoxin for 24 h. The cells and the culture supernatant were analyzed for cytokines and nitric oxide (NO) synthesis. The effects of qHSC-conditioned media on hepatocytes were then determined. LPS increased inducible NO synthase expression, stimulated NO synthesis, and inhibited DNA synthesis in qHSCs. qHSC-conditioned medium inhibited DNA synthesis in hepatocytes without affecting NO synthesis, while LPS (1-1,000 ng/ml)-conditioned qHSC medium stimulated NO synthesis and caused further inhibition of DNA synthesis and apoptosis. These effects of LPS were more pronounced when qHSCs were incubated with serum, but not with LPS-binding protein (LBP) although CD14 (a receptor for LPS-LBP complex) was found in qHSCs. LPS stimulated the synthesis of TNF-alpha, interleukin (IL)-6, and IL-1beta but not of TGF-beta in qHSCs. Individually or together, L-N(G)-monomethylarginine and antibodies to IL-1beta, IL-6, and TNF-alpha only partly reversed qHSC + LPS-conditioned medium-induced inhibition of DNA synthesis in hepatocytes. These results suggest that the effects of LPS on qHSCs are novel, occurring without the aid of LBP/CD14. They also indicate that other factors, in addition to NO, TGF-beta, TNF-alpha, IL-1beta, and IL-6 are involved in the mechanisms of the growth inhibitory effects of qHSCs on hepatocytes.     

Isoprenoid depletion by statins antagonizes cytokine-induced down-regulation of endothelial nitric oxide expression and increases NO synthase activity in human umbilical vein endothelial cells.

Endothelial dysfunction and atherosclerosis are associated with an inflammation-induced decrease in endothelial nitric oxide synthase (eNOS) expression. Based on the differences between hydrophobic and hydrophilic statins in their reduction of cardiac events, we analyzed the effects of rosuvastatin and cerivastatin on eNOS and inducible NO synthase (iNOS) expression and NOS activity in TNF-alpha-stimulated human umbilical vein endothelial cells (HUVEC). Both statins reversed down-regulation of eNOS mRNA and protein expression by inhibiting HMG-CoA reductase and isoprenoid synthesis. Cerivastatin tended to a more pronounced effect on eNOS expression compared to rosuvastatin. NOS activity - measured by conversion of [(3)H]-L-arginine to [(3)H]-L-citrulline - was enhanced under treatment with both drugs due to inhibition of HMG-CoA reductase. Statin-treatment reduced iNOS mRNA expression under normal conditions, but had no relevant effects on iNOS mRNA expression in cytokine-treated cells. Rosuvastatin and cerivastatin reverse the detrimental effects of TNF-alpha-induced down-regulation in eNOS protein expression and increase NO synthase activity by inhibiting HMG-CoA reductase and subsequent blocking of isoprenoid synthesis. These results provide evidence that statins have beneficial effects by increasing eNOS expression and activity during the atherosclerotic process.     

Macrolide antibiotics protect against immune complex-induced lung injury in rats: role of nitric oxide from alveolar macrophages.

Macrolide antibiotics have unique immunomodulatory actions apart from antimicrobial properties. We studied the effects of macrolides on IgG immune complex (IgG-ICx)-induced lung injury in rats in vivo and in vitro. Intrapulmonary deposition of IgG-ICx produced a time-dependent increase in the concentration of NO in exhaled air. There were corresponding increases in the number of neutrophils accumulated into alveolar spaces, and lung wet-to-dry weight ratio. All of these changes were inhibited by pretreatment with erythromycin or josamycin, but not by amoxicillin or cephaclor. Incubation of cultured pulmonary alveolar macrophages caused up-regulation of NO production and expression of inducible NO synthase mRNA, an effect that was dose dependently inhibited by erythromycin, roxithromycin, or josamycin. The macrolides also reduced IgG-ICx-induced release of IL-1beta and TNF-alpha, but did not alter the release of NO induced by exogenously added IL-1beta and TNF-alpha. These results suggest that macrolide antibiotics specifically inhibit immune complex-induced lung injury presumably by inhibiting cytokine release and the resultant down-regulation of inducible NO synthase gene expression and NO production by rat pulmonary alveolar macrophages.     

Piceatannol prevents lipopolysaccharide (LPS)-induced nitric oxide (NO) production and nuclear factor (NF)-kappaB activation by inhibiting IkappaB kinase (IKK)

The effect of piceatannol on lipopolysaccharide (LPS)-induced nitric oxide (NO) production was examined. Piceatannol significantly inhibited NO production in LPS-stimulated RAW 264.7 cells. The inhibition was due to the reduced expression of an inducible isoform of NO synthase (iNOS). The inhibitory effect of piceatannol was mediated by down-regulation of LPS-induced nuclear factor (NF)-kappaB activation, but not by its cytotoxic action. Piceatannol inhibited IkappaB kinase (IKK)-alpha and beta phosphorylation, and subsequently IkappaB-alpha phosphorylation in LPS-stimulated RAW 264.7 cells. On the other hand, piceatannol did not affect activation of mitogen-activated protein (MAP) kinases including extracellular signal regulated kinase 1/2 (Erk1/2), p38 and stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK). Piceatannol inhibited the phosphorylation of Akt and Raf-1 molecules, which regulated the activation of IKK-alpha and beta phosphorylation. The detailed mechanism of the inhibition of LPS-induced NO production by piceatannol is discussed.     

Amomum xanthoides extract prevents cytokine-induced cell death of RINm5F cells through the inhibition of nitric oxide formation

We previously showed that Amomum xanthoides extract prevented alloxan-induced diabetes through the suppression of NF-kappaB activation. In this study, the preventive effects of A. xanthoides extract on cytokine-induced beta-cell destruction were examined. Cytokines produced by immune cells infiltrating pancreatic islets are important mediators of beta-cell destruction in insulin-dependent diabetes mellitus. A. xanthoides extract completely protected interleukin-1beta (IL-1beta) and interferon-gamma (IFN-gamma)-mediated cytotoxicity in rat insulinoma cell line (RINm5F). Incubation with A. xanthoides extract resulted in a significant reduction in IL-1beta and IFN-gamma-induced nitric oxide (NO) production, a finding that correlated well with reduced levels of the inducible form of NO synthase (iNOS) mRNA and protein. The molecular mechanism by which A. xanthoides extract inhibited iNOS gene expression appeared to involve the inhibition of NF-kappaB activation. Our results revealed the possible therapeutic value of A. xanthoides extract for the prevention of diabetes mellitus progression.