Inhibition of COX-2 expression by endocannabinoid 2-arachidonoylglycerol is mediated via PPAR-γ

BACKGROUND AND PURPOSE

Endocannabinoids have both anti-inflammatory and neuroprotective properties against harmful stimuli. We previously demonstrated that the endocannabinoid 2-arachidonoylglycerol (2-AG) protects hippocampal neurons by limiting the inflammatory response via a CB₁ receptor-dependent MAPK/NF-κB signalling pathway. The purpose of the present study was to determine whether PPARγ, an important nuclear receptor, mediates 2-AG-induced inhibition of NF-κB phosphorylation and COX-2 expression, and COX-2-enhanced miniature spontaneous excitatory postsynaptic currents (mEPSCs).

EXPERIMENTAL APPROACH

By using a whole-cell patch clamp electrophysiological recording technique and immunoblot analysis, we determined mEPSCs, expression of COX-2 and PPARγ, and phosphorylation of NF-kB in mouse hippocampal neurons in culture.

KEY RESULTS

Exogenous and endogenous 2-AG-produced suppressions of NF-κB-p65 phosphorylation, COX-2 expression and excitatory synaptic transmission in response to pro-inflammatory interleukin-1β (IL-1β) and LPS were inhibited by GW9662, a selective PPARγ antagonist, in hippocampal neurons in culture. PPARγ agonists 15-deoxy-Δ^12,14-prostaglandin J₂ (15d-PGJ₂) and rosiglitazone mimicked the effects of 2-AG on NF-κB-p65 phosphorylation, COX-2 expression and mEPSCs, and these effects were eliminated by antagonism of PPARγ. Moreover, exogenous application of 2-AG or elevation of endogenous 2-AG by inhibiting its hydrolysis with URB602 or JZL184, selective inhibitors of monoacylglycerol lipase (MAGL), prevented the IL-1β- and LPS-induced reduction of PPARγ expression. The 2-AG restoration of the reduced PPARγ expression was blocked or attenuated by pharmacological or genetic inhibition of the CB₁ receptor.

CONCLUSIONS AND IMPLICATIONS

Our results suggest that CB₁ receptor-dependent PPARγ expression is an important and novel signalling pathway in endocannabinoid 2-AG-produced resolution of neuroinflammation in response to pro-inflammatory insults.

LINKED ARTICLES

This article is part of a themed issue on Cannabinoids in Biology and Medicine. To view the other articles in this issue visit http://dx.doi.org/10.1111/bph.2011.163.issue-7     

Hepatoprotective effect of withanolide-rich fraction in acetaminophen-intoxicated rat: decisive role of TNF-α, IL-1β, COX-II and iNOS

Context: Overdose of acetaminophen (APAP) is common in humans and is often associated with hepatic damage. Withania somnifera (L.) Dunal (Solanaceae) shows multiple pharmacological activities including antioxidant and anti-inflammatory potential.

Objective: To evaluate the possible mechanism of hepatoprotective activity of withanolide-rich fraction (WRF) isolated from a methanolic extract of Withania somnifera roots.

Materials and methods: Hepatotoxicity was induced by oral administration of APAP (750?mg/kg, p.o.) for 14 d. The control group received the vehicle. APAP-treated animals were given either silymarin (25?mg/kg) or graded doses of WRF (50, 100 and 200mg/kg) 2?h prior to APAP administration. Animals were killed on 15th day and blood and liver tissue samples were collected for the further analysis.

Results: In WRF-treated group, there was significant and dose-dependent (p?<?0.01 and p?<?0.001) decrease in serum bilirubin, ALP, AST and ALT levels with significant and dose-dependent (p?<?0.01 and p?<?0.001) increase in hepatic SOD, GSH and total antioxidant capacity. The level of MDA and NO decreased significantly (p?<?0.01) by WRF treatment. Up-regulated mRNA expression of TNF-α, IL-1β, COX-II and iNOS was significantly down-regulated (p?<?0.001) by WRF. Histological alternations induced by APAP in liver were restored to near normality by WRF pretreatment.

Conclusion: WRF may exert its hepatoprotective action by alleviating inflammatory and oxido-nitrosative stress via inhibition of TNF-α, IL-1β, COX-II and iNOS.     

Anticonvulsive activity of (1S)-(−)-verbenone involving RNA expression of BDNF,COX-2, and c-fos

Naunyn-Schmiedeberg's Archives of Pharmacology - (1S)-(−)-verbenone (VRB) is a monoterpene present in the essential oils of many plants which has shown therapeutic effect; however, its...     

Modulation of motor activity by α1 and α2 stimulation in mice

Summary The influence of two -adrenoceptor agonists, clonidine and B-HT 920, on motor activity was tested in mice. Both, clonidine and B-HT 920 (2-amino-6-allyl-5,6,7,8-tetrahydro-4H-thiazolo-[4,5-d]-azepine) in the dose range 30–300 g/kg s.c. equieffectively inhibited exploratory activity. On the other hand only clonidine, which stimulates ₂- and ₂-adrenoceptors increased locomotor activity in mice treated with reserpine (5 mg/kg) and apomorphine (3 mg/kg) in the doses of 0.3 and 1 mg/kg i.p. The highly selective ₂-agonist B-HT 920 was ineffective under these conditions up to 30 mg/kg i.p. It is concluded, that in mice sedative -adrenoceptors are of the ₂- and excitatory of the ₁-type.     

α-Adrenoceptor activity of arylalkylimidazoles is improved byα-methylation and impaired byα-hydroxylation

Summary To investigate the effects of hydroxyl and methyl substitution of the alkyl bridge bond on the-adrenoceptor activity of arylalkylimidazole derivatives, the cardiovascular effects of the molecules were studied in anaesthetized and pithed rats. The compounds studied were 4(5)-substituted imidazole derivatives with a methano, ethano or etheno bridge between the imidazole and the 2-, 2,3- or 2,6-methyl substituted phenyl rings. The hypotensive and bradycardic activities of the molecules in the anaesthetized rat were always reduced by-hydroxylation and usually augmented by-methylation of the bridge between the imidazole and phenyl rings. Hydroxylation was associated with a consistent, marked decrease in vasopressor and sympatho-inhibitory activity in the cardiovascular system of the pithed rat, but a methyl moiety as a bulky substituent in the-position of the alkyl bridge did not decrease but even caused an increase in-adrenoceptor activity in this test system. The detrimental effect of-hydroxylation of the compounds at ₁- and ₂-adrenoceptors supports the notion that the interaction of the imidazoles at-adrenoceptor is different from that of the classical, noradrenaline-like phenethylamines. The results also suggest that the alkyl bridge between the phenyl and imidazole rings of the imidazoles may contribute directly to the binding process.     

Stereoselective inhibition of monoamine oxidase and semicarbazide-sensitive amine oxidase by 4-dimethylamino-2,α-dimethylphenethylamine (FLA 336)

Summary The in vitro inhibition by amiflamine [FLA 336(+)] and related compounds of the activity of rat monoamine oxidase (MAO)-A and-B, rat semicarbazide sensitive amine oxidase (SSAO) and human platelet poor plasma benzylamine oxidase was studied. Amiflamine was an MAO-A selective inhibitor, but also inhibits SSAO with both a reversible (competitive, K _i=200 mol/l) and a small time-dependent component which was irreversible in nature. The optical isomer FLA 336(–) was ten times less potent towards MAO-A. However, this compound was much more potent an inhibitor of SSAO (competitive, K _i=4.6 mol/l). The amiflamine metabolites FLA 788(+) and FLA 668(+) inhibited SSAO, but only at concentrations considerably higher than required for MAO-A inhibition. Ex vivo experiments indicated that there was no significant irreversible inhibition of rat heart and lung SSAO after both single and repeated administration of amiflamine at doses up to 20 times higher than required for inhibition of MAO-A within central serotoninergic neurones.     

Cigarette-smoke-induced priming of neutrophils from smokers and non-smokers for increased oxidative burst response is mediated by TNF-α

In vitro treatment of human peripheral blood neutrophils from smokers and non-smokers with an aqueous cigarette smoke (CS) extract resulted in a concentration-dependent increase in surface expression of CD11b and CD66b and a corresponding decrease of CD62L, together with a concentration-dependent release of MMP-8, MMP-9, and lactoferrin, indicating considerable activation and degranulation. However, the burst response to N-formyl-methionyl-leucyl-phenylalanine (fMLP) was unchanged in CS-stimulated neutrophils from both smokers and non-smokers. When supernatants from CS-treated monocytic MonoMac-6 (MM6) cells were used for activation of neutrophils, concentration-dependent changes in surface marker expression, granule protein release, and the oxidative burst response to fMLP were observed, again with no major differences between smokers and non-smokers. CS-treated MM6 cells released significant amounts of IL-8 and TNF-α into the culture supernatant. However, antibody blocking experiments showed that only TNF-α mediated the increased burst response in neutrophils. These data show that, in the presence of secondary cells, CS is able to prime neutrophils for an increased burst response to fMLP which is mediated by TNF-α, released from the secondary cells in response to CS. Following stimulation with priming agents, peripheral blood neutrophils from healthy smokers show an equal burst response compared to those from non-smokers.     

Evidence for thromboxane receptor mediated contraction of guinea-pig and human airways in vitro by prostaglandin (PG) D2, 9α,11β-PGF2 and PGF2α

Summary The rank orders of potency of prostaglandin D₂, prostaglandin F₂, 9,11-prostaglandin F₂ and the stable thromboxane A₂ mimetics U-46619 and ONO-11113 were determined in guinea-pig trachea and human bronchus in vitro. In both tissues the thromboxane mimetics were markedly more potent than the other prostanoids with EC₅₀ values in the nanomolar range. The prostanoid antagonists BW-245C, EP-092 and GR-32191 attenuated the contractile responses to all of the prostanoid agonists and TXA₂ mimetics tested in guinea-pig tracheal spirals, although agonist selectivity was seen. Contractile responses to methacholine in the guinea-pig trachea were unaffected by any of the antagonists employed. BW-245C antagonised the effects of all prostanoid agonists tested in human bronchial spirals, the pA₂ values obtained were similar to those seen in the guinea-pig trachea when U-46619 and 9,11-PGF₂ were employed as the agonists. However, significant differences were found between the two tissues when PGD₂ and PGF₂. were tested against BW245C. EP-092 produced pA₂ values against prostanoid agonists in the human bronchus similar to those seen in the guinea-pig trachea, as did GR-32191. It is concluded that whilst the contractile responses of guinea-pig and human airways smooth muscle to prostaglandin D₂, and the other prostanoids are mediated predominantly via thromboxane (TP) receptors, it can be inferred that other receptor populations may contribute to the contractile response. The presence of these minor subpopulations may account for the agonist selectivity seen both within and between tissues from different species. Send offprint requests to: R. L. Featherstone at the above address     

α2-Adrenoceptor-mediated inhibition of platelet adenylate cyclase activity in heroin addicts in abstinence

The effects of corticotropin-releasing factor (CRF) and previously published effect of stress on the locomotor activity of mice in different regions of an open field were compared. Intracerebroventricular (ICV) administration of 0.2 g CRF, like stress, significantly increased center region activity; this effect was reversed by the benzodiazepine diazepam (DZP) at a dose of DZP having no significant effect alone. A dose of DZP that antagonized CRF-potentiated center region activity did not block amphetamine-stimulated center area activity. These results suggest that CRF may normally be responsible for many behavioral changes during conditions of stress.     

PGE2 production is suppressed by chemically-synthesized Δ7-eicosatrienoic acid in macrophages through the competitive inhibition of COX-2

Δ7-Eicosatrienoic acid (Δ7-ETrA; Δ7,11,14–20:3), an elongation metabolite of pinolenic acid (PNA; Δ5,9,12–18:3), is a rare polyunsaturated fatty acid (PUFA) originally from pine seeds. Incorporation of PNA and Δ7-ETrA into murine macrophages inhibited lipopolysaccharide (LPS)-stimulated prostaglandin E₂ (PGE₂) production. Due to the lack of availability of the naturally-occurring fatty acid, we synthesized Δ7-ETrA and demonstrated it was capable of suppressing PGE₂ production. Using laboratory synthetic techniques involving 2-carbon elongation and argentated column chromatography, Δ7-ETrA was synthesized and isolated. Its identity and purity (>98%) were confirmed by gas chromatography (GC)/GC–mass spectroscopy. Incubation of murine RAW264.7 cells or rat primary peritoneal macrophages with Δ7-ETrA reduced PGE₂ production by up to 84%, but slightly down-regulated type-2 cyclooxygenase (COX-2) expression. Δ7-ETrA blocked nuclear factor-kappa B (NF-κB) translocation into nucleus and inactivated mitogen-activated protein kinases (MAPK), however, these results might not directly account for its inhibitory effect. Furthermore, PGE₂ production reduced by Δ7-ETrA was highly correlated with the extent of Δ7-ETrA incorporation into cellular phospholipids and appeared to be the result of competition between this unusual fatty acid and arachidonic acid (AA) for COX-2. In conclusion, Δ7-ETrA incorporation suppresses PGE₂ production by macrophages through competition between Δ7-ETrA and AA for COX-2.     

Anti-inflammatory activity of bark of Dioscorea batatas DECNE through the inhibition of iNOS and COX-2 expressions in RAW264.7 cells via NF-κB and ERK1/2 inactivation

We identified a bioactive herbal medicine with anti-inflammatory activity from an ethanol extract derived from the bark of Dioscorea batatas DECNE (BDB) in RAW264.7 cells. We examined the effects of BDB on nitric oxide (NO) and prostaglandin E₂ (PGE₂) production in LPS-induced RAW264.7 cells. BDB consistently inhibited both NO and PGE₂ production in a dose-dependent manner, with an IC₅₀ of 87–71 μg/ml, respectively. The reduction of NO and PGE₂ production were accompanied by a reduction in iNOS and COX-2 protein expression, as evaluated by Western blotting. To evaluate the action mode of BDB and its ability to inhibit iNOS and COX-2 protein expression, we assessed the effects of BDB on nuclear factor-κB (NF-κB) DNA-binding activity, NF-κB-dependent reporter gene activity, inhibitory factor-κB (IκB) phosphorylation and degradation, and p65 nuclear translocation. BDB suppressed DNA-binding activity and reporter gene activity as well as translocation of the NF-κB p65 subunit. BDB also down-regulated IκB kinase (IKK), thus inhibiting LPS-induced both phosphorylation and the degradation of IκBα. In addition, BDB also inhibited the LPS-induced activation of ERK1/2.     

Induction of Fas receptor and Fas ligand by nodularin is mediated by NF-κB in HepG2 cells

Nodularin is a natural toxin with multiple features, including inhibitor of protein phosphatases 1 and 2A as well as tumor initiator and promoter. One unique feature of nodularin is that this chemical is a hepatotoxin. It can accumulate into the liver after contact and lead to severe damage to hepatocyte, such as apoptosis. Fas receptor (Fas) and Fas ligand (FasL) system is a critical signaling network triggering apoptosis. In current study, we investigated whether nodularin can induce Fas and FasL expression in HepG2 cell, a well used in vitro model for the study of human hepatocytes. Our data showed nodularin induced Fas and FasL expression, at both mRNA and protein level, in a time- and dose-dependent manner. We also found nodularin induced apoptosis at the concentration and incubation time that Fas and FasL were significantly induced. Neutralizing antibody to FasL reduced nodularin-induced apoptosis. Further studies demonstrated that nodularin promoted nuclear translocation and activation of p65 subunit of NF-κB. By applying siRNA targeting p65, which knocked down p65 in HepG2 cells, we successfully impaired the activation of NF-κB by nodularin. In these p65 knockdown cells, we observed that Fas and FasL expression and apoptosis induced by nodularin were significantly reduced. These findings suggest the induction of Fas and FasL expression and thus cell apoptosis in HepG2 cells by nodularin is mediated through NF-κB pathway.