Patrice Thérond

Piceatannol is more effective than resveratrol in restoring endothelial cell dimethylarginine dimethylaminohydrolase expression and activity after high-glucose oxidative stress

Abstract Glucose-induced oxidative stress is involved in endothelial dysfunction. Dimethylarginine dimethylaminohydrolase (DDAH) and arginase are regulators of the endothelial NO synthase (eNOS). This study aimed to compare the effect of two polyphenolic antioxidants, resveratrol and piceatannol, on DDAH and arginase pathways in bovine aortic endothelial cells under 25 mM glucose for 24 h. DDAH activity and expression were decreased in these cells as compared to control cells, whereas arginase activity was unchanged. DDAH inhibition led to intracellular accumulation of asymmetric dimethylarginine (ADMA), a natural inhibitor of eNOS. Under these conditions, cell pre-treatment with resveratrol (0.1–10 μM) restored basal DDAH activity and ADMA level with a dose-dependent effect. Piceatannol acted as resveratrol on DDAH pathway but at 10-fold lower concentrations. Resveratrol and piceatannol restored DDAH activity even in the presence of splitomicin, a specific inhibitor of Sirtuin 1. These results suggest potential therapeutic intervention targeting resveratrol or piceatannol administration to improve endothelial dysfunction.

Neuroinflammatory and oxidative stress phenomena in MPS IIIA mouse model: the positive effect of long-term aspirin treatment.

Sanfilippo disease (MPS IIIA) is an autosomal recessive lysosomal storage disorder resulting from sulfamidase deficiency, which is characterized by severe neurological impairment. Various tissues of MPS IIIA mice accumulate undegraded glycosaminoglycans and mimic the human neurodegenerative disorder, and are an excellent tool to both delineate disease pathogenesis and test potential therapies. The relationship between abnormal glycosaminoglycan storage and neurodysfunction remains ill defined. Pathways such as inflammation or oxidative stress have been highlighted in many neurodegenerative disorders, including lysosomal storage diseases, as major components of the neuropathology. By using quantitative polymerase chain reaction, we have compared the expression of selected genes in normal and MPS IIIA mouse cerebral tissues, focusing on inflammation, apoptosis and oxidative stress-related genes. We have identified several genes strongly over-expressed in the central nervous system of a MPS IIIA mouse, reflecting a neurological deterioration state. We have used these genes as markers to follow-up a long-term aspirin treatment. Aspirin treatment led to the normalization of inflammation- and oxidative stress-related mRNA levels in treated MPS IIIA mouse brains. A biochemical correction of an oxidative stress phenomenon both in the brain and peripheral organs of treated MPS IIIA mice was also obtained. These results suggest that anti-inflammatory intervention may be of potential benefit in MPS IIIA disease.

Resveratrol self-emulsifying system increases the uptake by endothelial cells and improves protection against oxidative stress-mediated death

The aim of the present study was to develop and characterize a resveratrol self-emulsifying drug delivery system (Res-SEDDS), and to compare the uptake of resveratrol by bovine aortic endothelial cells (BAECs), and the protection of these cells against hydrogen peroxide-mediated cell death, versus a control resveratrol ethanolic solution. Three Res-SEDDSs were prepared and evaluated. The in vitro self-emulsification properties of these formulations, the droplet size and the zeta potential of the nanoemulsions formed on adding them to water under mild agitation conditions were studied, together with their toxicity on BAECs. An optimal atoxic formulation (20% Miglyol® 812, 70% Montanox® 80, 10% ethanol 96% v/v) was selected and further studied. Pre-incubation of BAECs for 180 min with 25 μM resveratrol in the nanoemulsion obtained from the selected SEDDS significantly increased the membrane and intracellular concentrations of resveratrol (for example, 0.076±0.015 vs. ethanolic solution 0.041±0.016 nmol/mg of protein after 60 min incubation, p<0.05). Resveratrol intracellular localization was confirmed by fluorescence confocal microscopy. Resveratrol nanoemulsion significantly improved the endothelial cell protection from H2O2-induced injury (750, 1000 and 1500 μM H2O2) in comparison with incubation with the control resveratrol ethanolic solution (for example, 55±6% vs. 38±5% viability after 1500 μM H2O2 challenge, p<0.05). In conclusion, formulation of resveratrol as a SEDDS significantly improved its cellular uptake and potentiated its antioxidant properties on BAECs.

Protective effect of post-ischemic treatment with trans-resveratrol on cytokine production and neutrophil recruitment by rat liver

Oxidative and inflammatory processes are elicited during hepatic post-ischemic reperfusion and generate liver damage. This study investigated the early anti-inflammatory effect of trans-resveratrol (T-res) and its consequences on the late self-aggravating inflammatory process in liver ischemia-reperfusion (I/R). Partial hepatic ischemia was initiated in rats for 1 h and T-res (0.02 and 0.2 mg/kg) was administered intravenously 5 min before starting reperfusion for 3 h. Plasma levels of aminotransferases and cytokines (tumour necrosis factor (TNF)-alpha, interleukin (IL)-1beta, IL-6) and hepatic neutrophil recruitment were assessed. Hepatic expression of stress protein (heat-shock protein (HSP-70), heme oxygenase-1(HO-1)) and cytokine (TNF-alpha, IL-1beta, keratinocyte chemoattractant (KC)) mRNA was investigated. I/R caused an increase in aminotransferase levels and increased polymorphonuclear cell infiltration. Post-ischemic treatment with T-res (0.02 and 0.2 mg/kg) resulted in a significant decrease in aminotransferase, IL-1beta and IL-6 plasma levels by about 40%, 60% and 40%, respectively, compared to the vehicle I/R group. Post-ischemic treatment with T-res (0.02 mg/kg) also significantly decreased hepatic neutrophil recruitment. TNF-alpha, IL-1beta, KC and HO-1 hepatic mRNA expression was reduced by T-res without any change in HSP-70 mRNA. This T-res mediated decrease in early release of cytokines and neutrophil recruitment led to a reduction in the late inflammatory process. T-resveratrol might be useful in the prevention of inflammation secondary to hepatic surgery or liver transplantation.

Peroxiredoxin 6 fails to limit phospholipid peroxidation in lung from Cftr-knockout mice subjected to oxidative challenge.

Oxidative stress plays a prominent role in the pathophysiology of cystic fibrosis (CF). Despite the presence of oxidative stress markers and a decreased antioxidant capacity in CF airway lining fluid, few studies have focused on the oxidant/antioxidant balance in CF cells. The aim of the current study was to investigate the cellular levels of reactive oxygen species (ROS), oxidative damage and enzymatic antioxidant defenses in the lung of Cftr-knockout mice in basal conditions and as a response to oxidative insult. The results show that endogenous ROS and lipid peroxidation levels are higher in Cftr −/− lung when compared to wild-type (Cftr +/+) in basal conditions, despite a strong enzymatic antioxidant response involving superoxide dismutases, glutathione peroxidases and peroxiredoxin 6 (Prdx6). The latter has the unique capacity to directly reduce membrane phospholipid hydroperoxides (PL-OOH). A dramatic increase in PL-OOH levels in Cftr −/− lung consecutive to in vivo oxidative challenge by paraquat (PQ) unmasks a susceptibility to phospholipid peroxidation. PQ strongly decreases Prdx6 expression in Cftr −/− mice compared to Cftr +/+. Similar results were obtained after P. aeruginosa LPS challenge. Two-dimensional gel analysis of Prdx6 revealed one main molecular form in basal conditions and a PQ-induced form only detected in Cftr +/+ lung. Mass spectrometry experiments suggested that, as opposed to the main basal form, the one induced by PQ is devoid of overoxidized catalytic Cys47 and could correspond to a fully active form that is not induced in Cftr −/− lung. These results highlight a constitutive redox imbalance and a vulnerability to oxidative insult in Cftr −/− lung and present Prdx6 as a key component in CF antioxidant failure. This impaired PL-OOH detoxification mechanism may enhance oxidative damage and stress-related signaling, contributing to an exaggerated inflammatory response in CF lung.

Chain-breaking activity of resveratrol and piceatannol in a linoleate micellar model

This study investigated the in vitro protective effects of three derivatives of resveratrol, i.e., piceatannol (trans-3,5,3,4-tetrahydroxystilbene), PDM2 (1,3-dichloro-5-[(1E)-2-(4-chlorophenyl)ethenyl]-benzene) and PDM11 ((E)-5-[2-(4-chlorophenyl)ethenyl]-1,3-dimethoxyphenyl-ethene), compared with resveratrol as reference compound, against oxidation of linoleate micelles (10(-2)M) initiated by radiolysis-generated hydroxyl radicals. Lipid peroxidation was monitored by conjugated dienes (differential absorbance at 234nm), and by hydroperoxides (reverse phase HPLC with chemiluminescence detection). The higher the concentration of resveratrol or piceatannol (from 10(-5)M to 10(-4)M), the stronger the antioxidant ability. Piceatannol, with the presence of an additional hydroxyl group, showed a better antioxidant effect than resveratrol for a given concentration (competition with the fatty acid to scavenge lipid peroxyl radicals LOO), whereas PDM2 and PDM11, without any hydroxyl group, did not exhibit any significant protective effect. A lower limit for the LOO rate constant has been estimated for piceatannol (>/=1.4x10(5)M(-1)s(-1)) and for resveratrol (>/=0.3x10(5)M(-1)s(-1)).

Radical-induced oxidation of trans-resveratrol.

trans-Resveratrol (RVT) (3,5,4-trihydroxystilbene), a polyphenolic constituent of red wine, is thought to be beneficial in reducing the incidence of cardiovascular diseases, partly via its antioxidant properties. However, the mechanism of action by which trans-resveratrol displays its antioxidant effect has not been totally unravelled. This study aimed at establishing a comprehensive scheme of the reaction mechanisms of the direct scavenging of HO(*) and O(2)(*-) radicals generated by water gamma radiolysis. Aerated aqueous solutions of trans-RVT (from 10 to 100μmolL(-1)) were irradiated with increasing radiation doses (from 25 to 400Gy) and further analyzed by UV-visible absorption spectrophotometry for detection of trans-RVT oxidation products. Separation and quantification of RVT and its four oxidation products previously identified by mass spectrometry, i.e., piceatannol (PCT), 3,5-dihydroxybenzoic acid (3,5-DHBA), 3,5-dihydroxybenzaldehyde (3,5-DHB) and para-hydroxybenzaldehyde (PHB), were performed by HPLC/UV-visible spectrophotometry. Determination of the radiolytic yields of trans-RVT consumption and oxidation product formation has allowed us to establish balance between trans-RVT disappearance and the sum of oxidation products formation. Under our conditions, O(2)(-) radicals seemed to poorly initiate oxidation of trans-RVT, whereas the latter, whatever its initial concentration, quantitatively reacted with HO() radicals, via a dismutation mechanism. Two reaction pathways involving HO()-induced trans-RVT primary radicals have been proposed to explain the formation of the oxidation end-products of trans-RVT.

Penetration of resveratrol into bovine aortic endothelial cells (BAEC): a possible passive diffusion.

Several studies have demonstrated that, in a context of oxidative stress, resveratrol, a polyphenol found in wine, could act as a protective agent on endothelial cells by various mechanisms but without showing that it could penetrate inside the cell. The aim of this study was to detect for the first time resveratrol inside bovine endothelial aortic cells and to determine which kind of transport mechanism was involved. Intracellular and membrane concentrations of resveratrol have been measured by high performance liquid chromatography after incubation of several concentrations of resveratrol with endothelial cells for 24h. Concentrations of resveratrol in the culture media have been determined by UV spectrophotometry and experiments of transport mechanisms have been performed. Our results showed that, for the concentrations tested (1, 5, 10 and 50 μM), resveratrol was detected inside the cells and suggested that it was able to penetrate into the cells through a passive diffusion mechanism.

Experimental evidence of the reciprocal oxidation of Bovine Serum Albumin and Linoleate in aqueous solution, initiated by HO* free radicals.

An investigation of radiation-induced oxidation of aqueous bovine serum albumin (BSA) in the presence of linoleate (LH) at pH 10.5 has been carried out in order to better understand the respective oxidative processes involved in both lipid and protein phases. Solutions containing BSA (15 micromol L(-1)) and linoleate (15-600 micromol L(-1)) below the critical micellar concentration (cmc=2000 micromol L(-1)), have been irradiated by gamma-rays (137Cs) at radiation doses ranging from 10 to 400 Gy (dose rate 9.5 Gy min(-1)). It can be noticed that, in the absence of BSA, the main hydroperoxides formed from HO*-induced linoleate oxidation below the cmc, do not exhibit a conjugated dienic structure. This was also verified in the presence of BSA. Selected chemical markers of oxidation have been monitored: non-conjugated dienic hydroperoxides and conjugated dienes (without hydroperoxide function) for linoleate oxidation, and carbonyl groups for BSA oxidation. We have shown that for the lowest linoleate concentration (15 micromol L(-1)) in the presence of BSA (15 micromol L(-1)), the formation of conjugated dienes was not observed, meaning that LH was not exposed to HO* radicals attack. However, non-conjugated dienic lipid hydroperoxides were simultaneously detected, indicating that LH was secondarily oxidised by BSA oxidised species. Moreover, the oxidation of linoleate was found to be enhanced by the presence of BSA. For the highest linoleate concentration (600 micromol L(-1)), the expected protection of BSA by LH was not observed, even if LH monomers were responsible for the total scavenging of HO* radicals. In this latter case, the formation of non-conjugated dienic lipid hydroperoxides was lower than expected. Those results showed that BSA was not oxidised by the direct action of HO* radicals but was undergoing a secondary oxidation by non-dienic lipid hydroperoxides and/or lipid radical intermediates, coming from the HO*-induced linoleate oxidation.