Anna Rosa Ciampa

Nitric oxide mediates anti‐inflammatory action of extracorporeal shock waves

Here, we show that extracorporeal shock waves (ESW), at a low energy density value, quickly increase neuronal nitric oxide synthase (nNOS) activity and basal nitric oxide (NO) production in the rat glioma cell line C6. In addition, the treatment of C6 cells with ESW reverts the decrease of nNOS activity and NO production induced by a mixture of lipopolysaccharides (LPS), interferon‐γ (IFN‐γ) plus tumour necrosis factor‐α (TNF‐α). Finally, ESW treatment efficiently downregulates NF‐κB activation and NF‐κB‐dependent gene expression, including inducible NOS and TNF‐α. The present report suggests a possible molecular mechanism of the anti‐inflammatory action of ESW treatment.

STAT1 as a new molecular target of anti-inflammatory treatment.

Cyclooxygenase (COX) is widely considered as the molecular target of non-steroid anti-inflammatory drugs (NSAIDs). However, due to the harmful side effect frequently observed following chronic use, the development of new anti-inflammatory agents is the matter of many studies. Signal transducers and activators of transcription (STAT) are a family of nuclear proteins mediating the action of a number of cytokines. Among them, STAT1 plays a critical role in the signal transduction pathway of interferon-gamma (IFN-gamma) and growth hormone. STAT1 cascade is one major signalling pathway converting the IFN-gamma signal into gene expression, such as inducible nitric oxide synthase (iNOS), COX, vascular cell adhesion molecules (VCAM) and intercellular cell adhesion molecules (ICAM), critically involved in different pathologies correlated to the inflammatory process. This review focuses the attention on an alternative approach to the development of novel drugs based on inhibition of STAT1 pathway. In this context, a growing interest has been focused on natural compounds. We have recently reported a several data indicating that green tea extract (GTE), St. Johns Wort extract and epigallocatechin-3-gallate (EGCG) exhibit a specific and strong anti-STAT1 activity which is independent of their acclaimed strong anti-oxidant activity. More recently, GTE has been shown to protect heart damage from ischaemia/reperfusion in rats, suggesting that the protective effect of green tea might be correlated to its anti-STAT1 activity. The present review is aimed at providing data that STAT1 may potentially be claimed as a new molecular target of an anti-inflammatory treatment and that among natural compounds there are a number of anti-STAT1 substances.

Targeting STAT1 by myricetin and delphinidin provides efficient protection of the heart from ischemia/reperfusion-induced injury.

Flavonoids exhibit a variety of beneficial effects in cardiovascular diseases. Although their therapeutic properties have been attributed mainly to their antioxidant action, they have additional protective mechanisms such as inhibition of signal transducer and activator of transcription 1 (STAT1) activation. Here, we have investigated the cardioprotective mechanisms of strong antioxidant flavonoids such as quercetin, myricetin and delphinidin. Although all of them protect the heart from ischemia/reperfusion‐injury, myricetin and delphinidin exert a more pronounced protective action than quercetin by their capacity to inhibit STAT1 activation. Biochemical and computer modeling analysis indicated the direct interaction between STAT1 and flavonoids with anti‐STAT1 activity.

Protective effect of Arbutus unedo aqueous extract in carrageenan-induced lung inflammation in mice.

In the present study, we show that an aqueous extract of Arbutus unedo L. (AuE), a Mediterranean endemic plant widely employed as an astringent, diuretic and urinary anti-septic, in vitro down-regulates the expression of some inflammatory genes, such as those encoding inducible nitric oxide synthase (iNOS) and intracellular adhesion molecule-(ICAM)-1, exerting a inhibitory action on both IFN-gamma-elicited STAT1 activation and IL-6-elicited STAT3 activation. To evaluate further the effect of AuE in animal models of acute inflammation, we examined whether AuE administration attenuates inflammatory response of murine induced by intrapleural injection of carrageenan. For this purpose we studied: (1) STAT1/3 activation, (2) TNF-alpha, IL-1beta and IL-6 production in pleural exudate, (3) lung iNOS, COX-2 and ICAM-1 expression, (4) neutrophil infiltration, (5) the nitration of cellular proteins by peroxynitrite, (6) lipid peroxidation, (7) prostaglandin E2 and nitrite/nitrate levels and (8) lung injury. We show that AuE strongly down-regulates STAT3 activation induced in the lung by carrageenan with concomitant attenuation of all parameters examined associated with inflammation, suggesting that STAT3 should be a new molecular target for anti-inflammatory treatment. This study demonstrates that acute lung inflammation is significantly attenuated by the treatment with AuE.

The variations during the circadian cycle of liver CD1d-unrestricted NK1.1+TCRγ/δ+ cells lead to successful ageing. Role of metallothionein/IL-6/gp130/PARP-1 interplay in very old mice

Abstract NKT cells derive from the thymus and home to the liver. Liver NKT cells can be divided in two groups: ‘classical’ and ‘non-classical’. The first is CD1d-restricted, the second is CD1d-unrestriced. NKT cells (classical and non-classical) co-express T-cell receptor (TCR) and NK-cell marker (NK1.1), display cytotoxicity and produce IFN-γ under IL-12 stimulation affecting, thereby, Th1 response and innate immunity. NK1.1 + TCRα/β + cells belong to both groups. NK1.1 + TCRγ/δ + cells belong to the second group. Anyway, both NKT cell subtypes, via IFN-γ production, protect against viruses and bacteria from early in life. Immune variations as well as zinc rhythmicity during the circadian cycle confer the immune plasticity, which is essential for successful ageing. Liver NK1.1 + TCRγ/δ + cells, rather than TCRα/β + , from young and very old mice display ‘in vitro’ (under IL-12 stimulation) nocturnal peaks in cytotoxicity and IFN-γ production. The acrophase of liver NK1.1 + TCRγ/δ + cells is present in young and very old mice, not in old. The interplay among zinc-bound metallothionein (MT)/IL-6/gp130/poly(ADP-ribose) polymerase-1 (PARP-1) may be involved in conferring plasticity to liver NK1.1 + TCRγ/δ + cells. IL-6, via sub-unit receptor gp130, induces MTmRNA. At night, gene expressions of MT, IL-6, gp130 are lower in very old mice than old and young MT-I transgenic mice (MT-I ∗ ). In very old mice, this phenomenon allows limited sequester of intracellular zinc from MT leading to good free zinc ion bioavailability for immune efficiency and zinc-dependent PARP-1 activity. Indeed (1) in vitro, high IL-6 provokes strong accumulation of MT, impaired cytotoxicity and low zinc ion bioavailability in liver NK1.1 + TCRγ/δ + cells exclusively from old and MT-I ∗ mice. (2) The ratio total/endogen PARP-1 activity is higher in very old than in old and MT-I ∗ mice, suggesting a higher capacity of PARP-1 in base excision DNA-repair in very old age thanks to low zinc-bound MT. Cytotoxicity and IFN-γ production from liver NK1.1 + TCRγ/δ + cells are thus preserved leading to successful ageing. In conclusion, MT/IL-6/gp130/PARP-1 interplay may confer plasticity to liver CD1d-unrestricted NK1.1 + TCRγ/δ + cells, where MT, IL-6, gp130 are the main upstream protagonists, and PARP-1 is the main downstream protagonist in immunosenescence.

Regulation of serine racemase activity by d-serine and nitric oxide in human glioblastoma cells

In the human glioblastoma cell line U87, the activity of serine racemase (SR), catalyzing the isomerisation of serine, was inversely regulated by D-serine and nitric oxide (NO), a neuromodulator and a neurotransmitter, respectively. SR activity was dose-dependently enhanced up to five times in cells treated with 10 mM D-serine, whereas it was inhibited by NO. Furthermore, D-serine was found to induce the denitrosylation of SR purified from mouse brain. These results suggest that serine racemase activity in astrocyte is regulated inversely by d-serine and NO. SR should be inhibited through nitrosylation by NO and activated through denitrosylation elicited by D-serine.

Mutual regulation between serine and nitric oxide metabolism in human glioblastoma cells

D-Serine indirectly caused dose- and time-dependent inhibition of neuronal nitric oxide synthase (nNOS) without affecting endothelial nitric oxide synthase (eNOS) in human glioblastoma cell line U87. Activity of D-amino acid oxidase (DAAO), catalyzing the oxidative deamination of d-amino acid, was enhanced by NO in a dose-dependent manner. Recently, we have reported that serine racemase (SR) is inhibited by NO and activated by D-serine through nitrosylation and denitrosylation, respectively [K. Shoji, S. Mariotto, A.R. Ciampa, H. Suzuki, Regulation of serine racemase activity by D-serine and nitric oxide in human glioblastoma cells, Neurosci. Lett., in press]. Thus, the metabolism of both d-serine and NO in U87 cells is functionally correlated in a complex manner. Suppression of NO production by d-serine in U87 cells contrasts its known action in enhancing nNOS in neurons.

Epigallocatechin-3-gallate reduces allergen-induced asthma-like reaction in sensitized guinea pigs.

In this study, we have evaluated the effects of the polyphenol epigallocatechin-3-gallate (EGCG), an antioxidant molecule that also enhances constitutive nitric-oxide synthase (NOS) activity, on antigen-induced asthma-like reaction in sensitized guinea pigs. For comparison, we used epicatechin, which shares antioxidant but not NOS-modulating properties with EGCG. Ovalbumin-sensitized guinea pigs placed in a respiratory chamber were challenged with ovalbumin. EGCG (25 mg/kg b.wt.) or epicatechin (25 mg/kg b.wt.) was given i.p. 20 min before ovalbumin challenge. We analyzed latency time for the onset of respiratory abnormalities, cough severity, duration of dyspnea, lung tissue histopathology, mast cell activation (by granule release), leukocyte/eosinophilic infiltration (by major basic protein and myeloperoxidase), oxygen free radical-mediated injury (by nitrotyrosine and 8-hydroxy-2-deoxyguanosine and superoxide dismutase), NOS activity, and bronchial inflammatory response [by tumor necrosis factor-α in bronchoalveolar lavage (BAL)]. In the sensitized animals, severe respiratory abnormalities appeared soon after the antigen challenge, accompanied by bronchoconstriction, alveolar inflation, and a marked increase in the assayed parameters of inflammatory cell recruitment, free radical lung injury, and release of proinflammatory molecules in BAL fluid. This was associated with marked depression of constitutive NOS activity. Pretreatment with EGCG, but not epicatechin, significantly reduced all the above parameters and sustained endothelial-type NOS activity. These findings provide evidence that EGCG, probably by modulating NOS activity, can counteract allergic asthma-like reaction in sensitized guinea pigs and suggest its possible future use for the treatment of asthma.

Aqueous extract of Arbutus unedo inhibits STAT1 activation in human breast cancer cell line MDA-MB-231 and human fibroblasts through SHP2 activation.

Arbutus unedo L. has been for a long time employed in traditional and popular medicine as an astringent, diuretic, urinary anti-septic, and more recently, in the therapy of hypertension and diabetes. Signal transducer and activator of transcription 1 (STAT1) is a fascinating and complex protein with multiple yet contrasting transcriptional functions. Although activation of this nuclear factor is finely regulated in order to control the entire inflammatory process, its hyper-activation or time-spatially erroneous activation may lead to exacerbation of inflammation. The modulation of this nuclear factor, therefore, has recently been considered as a new strategy in the treatment of inflammatory diseases. In this study, we present data showing that the aqueous extract of Arbutus unedos leaves exerts inhibitory action on interferon-gamma (IFN-gamma) elicited activation of STAT1, both in human breast cancer cell line MDA-MB-231 and in human fibroblasts. This down-regulation of STAT1 is shown to result from a reduced tyrosine phosphorylation of STAT1 protein. Evidence is also presented indicating that the inhibitory effect of this extract may be mediated through enhancement of tyrosine phosphorylation of SHP2 tyrosine phosphatase. The modulation of this nuclear factor turns out into the regulation of the expression of a number of genes involved in the inflammatory response such as inducible nitric oxide synthase (iNOS) and intercellular adhesion molecule-1 (ICAM-1). Taken together, our results suggest that the employment of the Arbutus unedo aqueous extract is promising, at least, as an auxiliary anti-inflammatory treatment of diseases in which STAT1 plays a critical role.