Histone deacetylase inhibitors valproic acid and sodium butyrate enhance prostaglandins release in lipopolysaccharide-activated primary microglia.

Modifications of histone deacetylases (HDACs) may be involved in microglia-driven neuroinflammatory responses. Recent studies suggest that several inflammatory molecules can regulate the extent of neurodegeneration and regeneration in the central nervous system (CNS). In the present study, we investigated the effects of HDAC inhibitors (HDACi) valproic acid (VPA) and sodium butyrate (NaBut) on the release of prostaglandins (PGs) in lipopolysaccharide (LPS)-activated microglia. We found that VPA and NaBut significantly enhanced LPS-induced release of PGE2, PGD2 and 8-iso-PGF2α. In addition, both compounds increased cyclooxygenase-2 and microsomal prostaglandin E synthase immunoreactivity and gene expression in LPS-stimulated microglia. Interestingly, treatment of activated microglia with HDACi also enhanced the gene expression and the release of different pro-inflammatory cytokines. Microglia activation with LPS leads to IκB-α degradation, as well as p38, ERK1/2 and JNK MAPKs phosphorylation and thus activation, which is not affected by treatment with VPA and NaBut. Furthermore, VPA and NaBut treatment induced histone acetylation at H3-K18 in microglia. We suggest that VPA and NaBut-driven increase in PGs release in LPS-activated microglia might be regulated at the transcriptional level and involves histone hyperacetylation. Our data demonstrate that VPA and NaBut are able to modulate microglia responses to inflammatory insults and thus possibly can regulate the CNS degenerative and regenerative processes.

Micro-array profiling exhibits remarkable intra-individual stability of human platelet micro-RNA.

Platelets play an important role in haemostasis and thrombus formation. Latest research identified platelets harbouring so called microRNAs (miRNA). MiRNAs are short single-stranded RNAs modulating gene expression by targeting mRNAs. Limited data exist on inter-individual variability of platelet miRNA profile while no data are available on intra-individual variability. We assessed platelet miRNA profile in five volunteers at five time points over a time course of 10 days; 24 hours prior to the last blood sampling, subjects took 500 mg acetylsalicylic acid (ASA). Platelet miRNA was isolated from leucocyte-depleted platelet-rich plasma, and miRNA array-analysis was performed. Temporal patterns and ASA effect were explored by a linear mixed effects model for each miRNA. For the 20 most abundantly expressed platelet miRNAs, target gene search was performed and an annotation network was created. MiRNA expression profiling of 1,281 human miRNAs revealed relevant expression of 221 miRNAs consistently expressed in all samples at all time points. Correlation of platelet miRNA ranks was highly significant to other studies. Global distribution of miRNA expression was relatively similar in all subjects. No miRNA exhibited a significant effect of time at level 0.05. After 24 hours, no significant effect of ASA was found. Concerning functional implications of the 20 most abundantly expressed miRNAs, we found six functional themes. In conclusion, platelet miRNA profile is remarkably stable over the time period studied. Single-point analysis of platelet miRNA profile is reasonable when inter-individual differences are studied. The functional annotation network points toward extra-platelet effects of platelet miRNAs.

Assessing medicinal plants from South-Eastern Spain for potential anti-inflammatory effects targeting nuclear factor-Kappa B and other pro-inflammatory mediators.

AIM OF THE STUDY: Identification of plants with anti-inflammatory activity can be successfully based on information gained through knowledge on their traditional use. This is particularly true for biodiversity-rich regions of the world such as the Mediterranean. While such approaches are often single target based, here we used a multitarget, cell-based approach focusing on the pro-inflammatory signaling cascade and especially the NF-kappaB (NF-kappaB) pathway. MATERIALS AND METHODS: The plants from South-Eastern Spain were chosen on the basis that they were recorded as having a traditional use against an indication related to inflammation. The primary target was the transcription factor NF-kappaB (using a luciferase-based assay in HeLa cells). In addition extracts were tested in vitro for effects on cytokines (IL-6, IL-8, TNF-alpha) or PGE(2) in monocytes and for potential cytotoxic/pro-apoptotic action as well as for their influence on the cell cycle. RESULTS: Overall, 64 medicinal plant drugs from 61 species were assessed as potential inhibitors of inflammatory mediators to levels of 100-10 microg/ml. Three plants showed the highest level of activity (50 microg/ml) in inhibiting the activation of NF-kappaB in 5.1 cells: Helichrysum stoechas (Asteraceae), Dorycnium pentaphyllum (Fabaceae, s.l.) and Phlomis almeriensis (Lamiaceae). In the tests against the cytokines it was particularly striking to find that a number of species, Bupleurum fruticosum, Chamaespartium tridentatum, Genista ramosissima, Helichrysum stoechas, Mercurialis tomentosa, Ononis ramosissima, Peganum harmala, Picnomon acarna, Retama sphaerocarpa and Santolina viscosa showed extracts that were active at inhibiting TNF-alpha (10 microg/ml). CONCLUSIONS: Overall, this project has identified a series of species with an activity profile which merits further phytochemical-pharmacological investigation.

Effects of coffees before and after special treatment procedure on cell membrane potentials in stomach cells.

Coffee, one of the most excessively used beverages worldwide, commences the risk of gastroesophageal reflux (GER), which may lead to gastric ulcers and increase the risk of gastric cancer. Many attempts have been made by the coffee industry to diminish the irritating effect on mucosa by means of altering the extraction methods concerning gerbic acids and the roasting processes. This paper describes the effect of differently produced coffees involving two brands of Darboven and two brands of other coffee roasters. The aim of this study was to prove the results of gastric potential measurements we found in literature by using human AGS gastric epithelial cells (human adenocarcinoma). All four coffee extracts tested differentially affected the membrane resting potential of AGS cells. Coffees no. 1 and no. 2 depolarized the cells, presumably by increasing the cation entry into the cytosol. In marked contrast, coffee no. 4 hyperpolarizes the cells, possibly by H(+) extrusion and/or Cl(-) influx, suggesting that this coffee might increase acidity in the stomach, which might negatively affect the stomach, especially in people with gastroesophageal reflux symptoms. Overall, our data suggest that different roasting methods of coffees affect the membrane potentials of AGS stomach cells, resulting in increased influx of H+ possibly resulting in decreased stomach acidity and thus reducing GER. These results are in good accordance with clinical pharmacological results from potential difference measurements in healthy volunteers we found in the literature.

[5-HT3 receptor antagonist als analgetics in rheumatic diseases]. / 5-HT3-Rezeptor-Antagonisten als Analgetika bei rheumatischen Erkrankungen.

Various rheumatic diseases like fibromyalgia, systemic inflammatory rheumatic disorders and localized diseases, such as arthritides and activated arthroses, tendinopathies and periarthropathies, as well as trigger points can be improved considerably by treatment with the 5-HT3 receptor antagonist tropisetron. Particularly in the latter group of diseases, local injections have done surprisingly rapid analgesic action. This effect matches that of local anesthetics, but lasts considerably longer and is comparable to local injections of local anesthetics combined with corticosteroids. The action of the 5-HT3 receptor antagonists can be attributed to an antinociceptive effect that occurs at the same time as an antiphlogistic and probably also an immunosuppressive effect. Whereas an inhibited release of substance P from the nociceptors, and possibly some other neurokins as well, seems to be the most likely explanation for the antinociceptive action, the antiphlogistic effect is primarily due to an inhibited formation of various different phlogistic substances; in some conditions, like systemic inflammatory rheumatic diseases, for example, the 5-HT3 receptor antagonists may exert an immunosuppressive effect in addition to this.

Petasites hybridus extracts in vitro inhibit COX-2 and PGE2 release by direct interaction with the enzyme and by preventing p42/44 MAP kinase activation in rat primary microglial cells.

Rhizomes of butterbur, Petasites hybridus L. (Asteraceae), have been used since ancient times for the treatment of inflammatory diseases. In the present study, the effects of lipophilic extracts from rhizomes of Petasites hybridus on the formation and release of prostaglandin E2 were investigated. The extracts had different contents of petasin and isopetasin: A: 2.1 % and 0.4 %, B: 0.2 % and 0.1 %, C: 12.1 % and 6.1 % and D: 21.9 % and 9.4 %, respectively. Direct inhibition of cyclooxygenase (COX) -1 and -2 isoenzymes and inhibition of the expression of COX-2 and p42/44 MAP kinase in rat primary microglial cells were tested. All extracts were found to be only weak direct inhibitors of COX-1 (IC50> 400 microg/mL). However, most extracts revealed a strong inhibitory activity against the inducible isoform COX-2 ( A: IC50=30.4 microg/mL; B: IC50=60.6 microg/mL; C: IC50=22.6 microg/mL; D: IC50=20.0 microg/mL). This activity was not correlated to the content of petasin and isopetasin. Pure petasin and isopetasin neither inhibited COX-1 nor COX-2 (IC50 > 400 microM for both compounds and enzymes). Petasites extracts dose-dependently inhibited LPS-induced and thus COX-2-mediated PGE2 release in primary rat microglial cells (A: IC50= 2.4 microg/mL; C: IC50=5.8 microg/mL and D: IC50=4.6 microg/mL). Also this effect was independent from the petasin and isopetasin content. COX-2 synthesis in microglia was totally blocked with 5 microg/mL of C whereas COX-1 synthesis was not influenced. C and D did not affect the LPS-induced activation of p38 MAPK and IkappaBalpha, but they prevented the LPS-induced activation of p42/44 MAPK. Therefore, these Petasites hybridus extracts can be regarded as natural selective inhibitors of COX-2 and its expression, an effect which is independent from the petasin content.

Hypericin-an inhibitor of proteasome function.

Hypericin is the presumed active moiety within Saint John's wort. Extracts of Saint John's wort are widely used as an effective treatment for depression. Available as "over-the-counter" drugs, they are frequently part of the self-medication of patients undergoing radiation therapy for malignant diseases. In addition to antidepressive properties, hypericin has been shown to be able to induce apoptosis and radiosensitize tumor cells, and to have antiinflammatory and phototoxic skin effects. However, the underlying mechanisms are not clear. In this study, we investigated possible inhibitory effects of hypericin on proteasome function and related pathways. Extracts from U373 human glioma cells were incubated with different concentrations of hypericin. Three proteasome activities were monitored using a fluorogenic peptide assay. Activity of the transcription factor NF-kappaB and protein levels of p65, p50, IkappaBalpha and caspase-3 were investigated by EMSA and Western blotting, respectively. Hypericin caused a dose-dependent and photoactivation-independent inhibition of proteasome function. Hypericin treatment (6.25-50 microM) inhibited NF-kappaB, caused accumulation of phosphorylated IkappaBalpha, decreased p50 protein levels and induced cleavage of p65 protein in U373 cells. These effects were observed in MCF-7 cells only at higher concentrations of hypericin (12.5-50 microM). Additionally, inhibition of NF-kappaB activity in U373 cells by hypericin was prevented by caspase inhibition. Although hypericin clearly inhibits proteasome function, its effect NF-kappaB DNA-binding activity was not exclusively proteasome-dependent. The underlying mechanism might also involve caspase activation, a consequence of proteasome inhibition.

Expression of 5-HT3A receptors in cells of the immune system.

There is evidence from both human and animal research that 5-hydroxytryptamine3 (5-HT3) receptor antagonists, particularly tropisetron, exert analgesic and antiinflammatory effects. However, the underlying mechanisms of these effects including the expression of 5-HT3 receptors in cells of the immune system have not yet been investigated in detail. Therefore, we investigated the expression of the 5-HT3A receptor in primary human monocytes, chondrocytes, T-cells, dendritic cells, and synovial tissue. We found that 5-HT3A receptors are expressed in monocytes, chondrocytes, T-cells, and synovial tissue but not in dendritic cells. Our data show that 5-HT3A receptors are widely expressed in cells of the immune system and that they might play an important role in inflammatory events and in the observed antiphlogistic effects of 5-HT3 receptor antagonists.

Antiinflammatory effects of 5-HT3 receptor antagonists in lipopolysaccharide-stimulated primary human monocytes.

There is evidence from both human and animal research that 5-hydroxytryptamine (5-HT)3 receptor antagonists, particularly tropisetron, exert analgesic and antiinflammatory effects. However, the underlying mechanisms of these effects have not yet been investigated in detail. Therefore, the antiinflammatory effects of tropisetron and ondansetron were investigated in human monocytes. In human monocytes, both lipopolysaccharide (LPS)-stimulated tumour necrosis factor (TNF)-alpha and interleukin (IL)-1beta secretion were dose-dependently inhibited by tropisetron starting at a concentration of 5 microg/mL and reaching maximal levels at 25 microg/mL (IC50: 32 microg/mL and 12 microg/mL, respectively). LPS-induced IL-6 and PGE2 release was only slightly inhibited at high doses, whereas LPS-induced release of IL-8 and matrix metalloprotease (MMP)-9 was not affected. In conclusion, our data show that the binding of tropisetron to 5-HT3 receptors results in antiinflammatory effects through inhibition of TNF-alpha/IL-1beta, which might explain the antiphlogistic effects of 5-HT3 antagonists.

Influence of tropisetron on the serum substance P levels in fibromyalgia patients.

BACKGROUND: Substance P is found at an elevated level in the cerebrospinal fluid of fibromyalgia (FM) patients. Treatment with tropisetron leads in a subgroup of FM patients to pain reduction. The question arises of whether the substance P level in the serum can be changed by tropisetron treatment. METHOD: Twenty patients with FM diagnosed according to the ACR criteria were treated for 5 days with a 5 mg tropisetron intravenous (i.v.) bolus injection daily. Before the first injection, 3 h later, and before and 3 h after the last injection, the serum levels of substance P were determined. The determination of this substance was carried out by means of an immunoassay from Assay Design Biotrend, Cologne. To evaluate the success of the tropisetron treatment, patients made a global assessment as 'clearly better', 'better', 'unchanged', or 'poor'. Patients who answered 'clearly better' and 'better' were regarded as responders. RESULTS: Of the 20 patients, ten reported a good or very good influence on their pain (responders). In these responders, the means of the serum substance P levels were elevated in comparison with the non-responders, though the difference was not significant. In responders, the 5-HT3 receptor antagonist tropisetron produced a significant decrease in the serum substance P levels, while this did not occur in the non-responders. CONCLUSION: It is possible that the responders to tropisetron represent a subgroup of FM patients for whom substance P and 5-HT3 receptors play key roles in the development of the pain symptoms.

Serum levels of substance P and response to antidepressant pharmacotherapy.

Substance P (SP) is possibly involved in the etiopathology of affective disorders. Here we investigated the relationship of SP serum levels and response to antidepressant drug therapy. SP serum levels were determined before and during a 9-week drug trial in 40 depressed patients treated with paroxetine in combination with either lamotrigine (n = 20) or placebo (n = 20). Responders (n = 18) and non-responders (n = 22) significantly differed in SP serum levels: responders started with higher SP levels that decreased during drug therapy, whereas non-responders had lower SP levels that increased at the beginning. There were no differences between patients with adjunct lamotrigine or placebo. These preliminary data indicate that SP serum levels might be related to response to antidepressant drug therapy. Further studies have to substantiate this finding.

Phenylpropanoid NF-kappaB inhibitors from Bupleurum fruticosum.

Two phenylpropanoids from Bupleurum fruticosum (Apiaceae/Umbelliferae) were shown to inhibit the transcriptional activity induced by PMA or TNFalpha of an NF-kappaB-controlled reporter gene. Western blot experiments indicated that the phenylpropanoids did not prevent IkappaBalpha degradation, suggesting that their molecular target is at a post-IKB degradation level. Both compounds prevented cytokine (IL-1, IL-6, TNF, IL-8) release and prostaglandin E2 synthesis.

Microglial expression of prostaglandin EP3 receptor in excitotoxic lesions in the rat striatum.

Prostaglandins (PG) are produced by the enzymatic activity of cyclooxygenase (COX). PGs and COX have been implicated in the pathophysiology of excitotoxicity and neurodegeneration in the central nervous system (CNS). The PGE2 receptor EP3 is the most abundantly expressed PGE2 receptor subtype in the brain. So far, in the innate rat brain EP3 receptors have been found exclusively in neurons. The aim of this study was to investigate whether EP3 expression in the brain changes under neurodegenerative circumstances such as an acute excitotoxic lesion. Intrastriatal injection of quinolinic acid (QUIN) resulted in a loss of EP3-positive striatal neurons, while simultaneously small glial-shaped EP3-positive cells appeared. Five days after lesioning, 63% of the glial-shaped EP3-positive cells could be identified as ED-1 expressing microglial cells. This percentage increased to 82% after 10 days, suggesting that most of the EP3-positive ED-1-negative cells on day 5 may be microglia which did not yet express ED-1. ED-1-positive microglia also expressed COX-1. These experiments show for the first time that activated microglial cells in excitotoxic lesions express in vivo the PGE2 receptor EP3 and the PGE2 synthesizing enzyme COX-1. Activation of EP3 receptor downregulates cAMP formation and may counteract the upregulation of cAMP formation via EP2 receptors, which has been linked to the anti-inflammatory effects of PGs. This change in EP3-receptor expression in microglia might participate in acute or chronic microglial activation in a variety of brain diseases such as ischemia or Alzheimer's disease (AD). Investigation of the expression of different PGE2 receptor subtypes might promote a better understanding of the pathophysiology of these diseases as well as leading to a modulation of microglial activation by a more specific interference with selective EP receptors than can be achieved by inhibiting global PG synthesis by selective or non-selective COX inhibitors.

Effects of an ethanolic salix extract on the release of selected inflammatory mediators in vitro.

Salix extracts are in current use for the treatment of pain and inflammation. In order to obtain an insight into the mechanism(s) of action of the ethanolic Salix extract 1520L--which is essentially similar to an extract for which clinical studies have demonstrated analgesic effectiveness--its effects were evaluated in an established in vitro assay test system using primary human monocytes. The IC50-values obtained for the inhibition of lipopolysaccharide (LPS)-induced release of prostaglandin E2 (PGE2) reflecting cyclooxygenase (COX)-2-mediated PGE2 release were 47 microg/ml and 0.6 microg/ml, for the Salix extract 1520L and rofecoxib-like research compound L745337, respectively. There was no effect on COX-1 and COX-2 activity. The Salix extract inhibited the LPS-induced release of tumor necrosis factor-alpha, interleukin-1beta and interleukin-6 with IC50-values of 180.0, 33.0 and 86.0 microg/ml, respectively. Both, salicin and salicylate, had no effect in any of the parameters. Our results indicate that Salix extract 1520L inhibits COX-2-mediated PGE2 release through compounds other than salicin or salicylate. Our data further suggest that the proprietary Salix extract is a weak inhibitor of proinflammatory cytokines.

Pathways of inflammatory activation in Alzheimer's disease: potential targets for disease modifying drugs.

In the human brain several cell types are capable of initiating and amplifying a brain specific inflammatory response involving the synthesis of cytokines, prostaglandins and oxygen free radicals. In Alzheimer's disease (AD), signs of an inflammatory activation of microglia and astroglia are present inside and outside amyloid deposits. Cell culture and animal models suggest an interactive relationship between inflammatory activation, reduced neuronal functioning and deposition of amyloid. The activation of inflammation-associated enzymes such as p38 mitogen-activated protein kinase (p38 MAPK) and cycloxygenase-2 (COX-2) is not restricted to glial cells but also found in neurons and may contribute to intraneuronal damage. Epidemiological studies have shown a reduced risk of AD among users of anti-inflammatory drugs. Therefore, anti-inflammatory drugs have become the focus of several new treatment strategies. Small clinical trials with non-steroidal anti-inflammatory drugs (NSAIDs) such as indomethacin and diclofenac showed a trend for a disease modifying effect, while clinical trials with steroids did not show a beneficial effect. NSAIDs may not only act on COX-2 but also inhibit COX-1 activity or activate peroxisome proliferator-activated receptor gamma (PPAR gamma). Among promising new strategies to reduce the inflammatory activation in the CNS interfering with intracellular pro-inflammatory pathways has been shown to be effective in various cell culture and animal models. Inhibitors of p38MAPK and PPAR gamma agonists may be suitable agents to suppress inflammatory activation in AD.

Mechanisms of prostaglandin E2-induced interleukin-6 release in astrocytes: possible involvement of EP4-like receptors, p38 mitogen-activated protein kinase and protein kinase C.

The expression of cyclooxygenase-2 (COX-2) and the synthesis of prostaglandin E2 (PGE2) as well as of cytokines such as interleukin-6 (IL-6) have all been suggested to propagate neuropathology in different brain disorders such as HIV-dementia, prion diseases, stroke and Alzheimer's disease. In this report, we show that PGE2-stimulated IL-6 release in U373 MG human astroglioma cells and primary rat astrocytes. PGE2-induced intracellular cAMP formation was mediated via prostaglandin E receptor 2 (EP2), but inhibition of cAMP formation and protein kinase A or blockade of EP1/EP2 receptors did not affect PGE2-induced IL-6 synthesis. This indicates that the cAMP pathway is not part of PGE2-induced signal transduction cascade leading to IL-6 release. The EP3/EP1-receptor agonist sulprostone failed to induce IL-6 release, suggesting an involvement of EP4-like receptors. PGE2-activated p38 mitogen-activated kinase (p38 MAPK) and protein kinase C (PKC). PGE2-induced IL-6 synthesis was inhibited by specific inhibitors of p38 MAPK (SB202190) and PKC (GF203190X). Although, up to now, EP receptors have only rarely been linked to p38 MAPK or PKC activation, these results suggest that PGE2 induces IL-6 via an EP4-like receptor by the activation of PKC and p38 MAPK via an EP4-like receptor independently of cAMP.

Inhibition of substance P-induced cytokine synthesis by St. John's wort extracts.

We tested the hypothesis that extracts from St. John's wort interfere with protein synthesis induced by substance P (SP), a neuropeptide which has been implicated in the etiopathology of depression and anxiety. Using human astrocytoma cells, which express functional neurokinin (NK)-1-receptors, we investigated whether extracts from St. John's wort are able to inhibit SP-induced synthesis of the cytokine interleukin-6 (IL-6). We found a potent and dose-dependent inhibition of SP-induced IL-6 synthesis by various extracts from St. John's wort. These results do not only give further evidence of the anti-inflammatory effects of St. John's wort, but also lend support to the hypothesis that the antidepressant effect of St. John's wort is, at least in part, a result of its inhibitory effects on SP-induced protein synthesis.