An inflammatory response in the central nervous system mediated by activation of microglia is a key event in the early stages of the development of neurodegenerative diseases. Silymarin is a polyphenolic flavanoid derived from milk thistle that has anti-inflammatory, cytoprotective and anticarcinogenic effects. In this study, we first investigated the neuroprotective effect of silymarin against lipopolysaccharide (LPS)-induced neurotoxicity in mesencephalic mixed neuron-glia cultures. The results showed that silymarin significantly inhibited the LPS-induced activation of microglia and the production of inflammatory mediators, such as tumour necrosis factor-alpha and nitric oxide (NO), and reduced the damage to dopaminergic neurons. Therefore, the inhibitory mechanisms of silymarin on microglia activation were studied further. The production of inducible nitric oxide synthase (iNOS) was studied in LPS-stimulated BV-2 cells as a model of microglia activation. Silymarin significantly reduced the LPS-induced nitrite, iNOS mRNA and protein levels in a dose-dependent manner. Moreover, LPS could induce the activation of p38 mitogen-activated protein kinase (MAPK) and c-jun N-terminal kinase but not extracellular signal-regulated kinase. The LPS-induced production of NO was inhibited by the selective p38 MAPK inhibitor SB203580. These results indicated that the p38 MAPK signalling pathway was involved in the LPS-induced NO production. However, the activation of p38 MAPK was not inhibited by silymarin. Nevertheless, silymarin could effectively reduce LPS-induced superoxide generation and nuclear factor kappaB (NF-kappaB) activation. It suggests that the inhibitory effect of silymarin on microglia activation is mediated through the inhibition of NF-kappaB activation.

Delta(9)-Tetrahydrocannabinol (Delta(9)-THC) is the major psychoactive component of marijuana and elicits pharmacological actions via cannabinoid receptors. Anandamide (AEA) and 2-arachidonoyl-glycerol (2-AG) are endogenous ligands for cannabinoid receptors, which because of their structural similarities to arachidonic acid (AA), AEA, and 2-AG could serve as substrates for lipoxygenases and cyclooxygenases (COXs) that metabolize polyunsaturated fatty acids to potent bioactive molecules. In this study, we have compared the effects of Delta(9)-THC, AEA, 2-AG, and another cannabinoid agonist, indomethacin morpholinylamide (IMMA), on lipopolysaccharide (LPS)-induced NO, IL-6, and PGE(2) release from J774 macrophages. Delta(9)-THC, IMMA, and AEA diminish LPS-induced NO and IL-6 production in a concentration-dependent manner. 2-AG inhibits the production of IL-6 but slightly increases iNOS-dependent NO production. Delta(9)-THC and IMMA also inhibit LPS-induced PGE(2) production and COX-2 induction, while AEA and 2-AG have no effects. These discrepant results of 2-AG on iNOS and COX-2 induction might be due to its bioactive metabolites, AA and PGE(2), whose incubation cause the potentiation of both iNOS and COX-2 induction. On the contrary, the AEA metabolite, PGE(2)-ethanolamide, influences neither the LPS-induced NO nor IL-6 production. Taken together, direct cannabinoid receptor activation leads to anti-inflammatory action via inhibition of macrophage function. The endogenous cannabinoid, 2-AG, also serves as a substrate for COX-catalyzing PGE(2) production, which in turn modulates the action of CB2.

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Peroxisome proliferator-activated receptor γ (PPARγ) ligands have been reported to exert anti-inflammatory activities in macrophages by competition for transcriptional coactivators with some transcriptional factors, including NF-κB. In the present study the influence of PPARγ activators on IFN-γ-elicited macrophage stimulation and signaling cascades was investigated. The results show that IFN-γ-induced inducible NO synthase (iNOS) gene transcription, iNOS protein induction, and NO production are more sensitive to inhibition by 15-deoxy-Δ12,14-prostaglandin J2 (15dPGJ2) than by the other two PPARγ agonists, GW1929 and ciglitazone. Delayed addition of 15dPGJ2 for 2 h resulted in reduced inhibition, suggesting action by 15dPGJ2 on the upstream signaling cascades. Immunoblotting, DNA binding, and reporter gene assays consistently revealed the inhibitory ability of 15dPGJ2, but not GW1929 or ciglitazone, on IFN-γ-elicited signaling cascades, including tyrosine phosphorylation of Janus tyrosine protein kinase 2 and STAT1, DNA binding, and IFN regulatory factor-1 trans-activation of STAT1. These effects of 15dPGJ2 were not abrogated by the PPARγ antagonist, bisphenol A diglycidyl ether, indicating the PPARγ-independent actions. 15dPGJ2 also attenuated IL-6-induced tyrosine phosphorylation of STAT1 and STAT3 in Hep3B hepatoma cells. Consistent with the inhibitory effect of reactive oxygen species on STAT1 signaling, STAT1 inhibition by 15dPGJ2 was abrogated by N-acetylcysteine, glutathione, superoxide dismutase, and catalase. Furthermore, 15dPGJ2-induced inhibition of STAT1 phosphorylation and NO production still occurred in the presence of peroxovanadate, ruling out the action mechanism of 15dPGJ2 on tyrosine phosphatase. Taken together, for the first time in this study we demonstrate that 15dPGJ2 can inhibit cytokine-stimulated Janus kinase 2-STAT signaling through a PPARγ-independent, reactive oxygen species-dependent mechanism. These data provide a novel molecular mechanism of iNOS inhibition by 15dPGJ2 and confirm its physiological role in anti-inflammation.

Background and purpose: The aim of this study was to elucidate the mechanism of YC-1{3-(5 0 -hydroxy methyl-2 0 -furyl)-1-benzylindazole}-induced human renal carcinoma cells apoptosis and to evaluate the potency of YC-1 in models of tumour growth in mice. Experimental approach: YC-1-mediated apoptosis was assessed by analysis of MTT, SRB, DAPI staining and flow cytometry analysis. Knockdown of JNK protein was achieved by transient transfection using siRNA. The mechanisms of action of YC-1 on different signalling pathways involved were studied using western blot. Fas clustering was analysed by confocal microscopy and in vivo efficacy was examined in a A498 xenograft model. Key results: YC-1 displayed cytotoxicity in renal carcinoma cells at 10 À7 -10 À8 M. Increased condensation of chromatin was observed and an increase in the cell population in subG1 phase. Moreover, YC-1 triggered mitochondria-mediated and caspase-dependent pathways. YC-1 significantly induced Fas ligand expression, but did not modify either the protein levels of death receptors or ligands. In addition, Fas clustering in cells responsive to YC-1 was observed, suggesting involvement of a Fas-mediated pathway. Furthermore, YC-1 markedly induced phosphorylation of JNK and a JNK inhibitor, SP600125, and siRNA JNK1/2 significantly reversed YC-1-induced cytotoxicity and protein expression. We suggest that YC-1 induced JNK phosphorylation, the upregulation of FasL and Fas receptor clustering to promote the activation of caspases 8 and 3, resulting in apoptosis. Finally, we demonstrated the antitumour effect of YC-1 in vivo. Conclusions and implications: These data suggest that YC-1 is a good candidate for development as an anticancer drug.