Lamberto Re

Role of protein synthesis in the protection conferred by ozone-oxidative-preconditioning in hepatic ischaemia/reperfusion

The liver is damaged by sustained ischaemia during liver transplantation, and the reperfusion after ischaemia results in further functional impairment. Ozone oxidative preconditioning (OzoneOP) protected the liver against ischaemia/reperfusion (I/R) injury through different mechanisms. The aim of this study was to investigate the influence of the inhibition of protein synthesis on the protective actions conferred by OzoneOP in hepatic I/R. Rats were treated with cycloheximide (CHX) in order to promote protein synthesis inhibition after OzoneOP treatment. Plasma transaminases, malondialdehyde and 4‐hydroxyalkenals and morphological characteristics were measured as an index of hepatocellular damage; Cu/Zn‐superoxide dismutase (SOD), Mn‐SOD, catalase, total hydroperoxides and glutathione levels as markers of endogenous antioxidant system. OzoneOP increased Mn‐SOD isoform and ameliorated mitochondrial damage. CHX abrogated the protection conferred by OzonoOP and decreased Mn‐SOD activity. Cellular redox balance disappeared when CHX was introduced. Protein synthesis is involved in the protective mechanisms mediated by OzoneOP. Ozone treatment preserved mitochondrial functions and cellular redox balance.

Ozone oxidative preconditioning is mediated by A1 adenosine receptors in a rat model of liver ischemia/ reperfusion.

The liver is damaged by sustained ischemia in liver transplantation, and the reperfusion after ischemia results in further functional impairment. Ozone oxidative preconditioning (OzoneOP) protected the liver against ischemia/reperfusion (I/R) injury. The aim of this study was to investigate the role of A1 adenosine receptor on the protective actions conferred by OzoneOP in hepatic I/R. By using a specific agonist and antagonist of the A1 subtype receptor (2‐chloro N6 cyclopentyladenosine, CCPA and 8‐cyclopentyl‐1,3‐dipropylxanthine, DPCPX respectively), we studied the role of A1 receptor in the protective effects of OzoneOP on the liver damage, nitiric oxide (NO) generation, adenosine deaminase activity and preservation of the cellular redox balance. Immunohistochemical analysis of nuclear factor‐kappa B (NF‐κB), tumor necrosis factor alpha (TNF‐α) and heat shock protein‐70 (HSP‐70) was performed. OzoneOP prevented and/or ameliorated ischemic damage. CCPA showed a similar effect to OzoneOP + I/R group. A1AR antagonist DPCPX blocked the protective effect of OzoneOP. OzoneOP largely reduced the intensity of the p65 expression, diminished TNF‐α production, and promoted a reduction in HSP‐70 immunoreactivity. In summary, OzoneOP exerted protective effects against liver I/R injury through activation of A1 adenosine receptors (A1AR). Adenosine and .NO produced by OzoneOP may play a role in the pathways of cellular signalling which promote preservation of the cellular redox balance, mitochondrial function, glutathione pools as well as the regulation of NF‐κB and HSP‐70.

Effects of ozone therapy on haemostatic and oxidative stress index in coronary artery disease

Coronary artery disease (CAD) is the most common cause of sudden death, and death of people over 20 years of age. Because ozone therapy can activate the antioxidant system and improve blood circulation and oxygen delivery to tissue, the aim of this study was to investigate the therapeutic efficacy of ozone in patients with CAD, treated with antithrombotic therapy, Aspirin and policosanol. A randomized controlled clinical trial was performed with 53 patients divided into two groups: one (n=27) treated with antithrombotic therapy and other (n=26) treated with antithrombotic therapy plus rectal insufflation of O(3). A parallel group (n=50) age and gender matched was used as reference for the experimental variables. The efficacy of the treatments was evaluated by comparing hemostatic indexes and biochemical markers of oxidative stress in both groups after 20 day of treatment. Ozone treatment significantly (P<0.001) improved prothrombin time when compared to the antithrombotic therapy only group, without modifying bleeding time. Combination antithrombotic therapy+O(3) improved the antioxidant status of patients reducing biomarkers of protein and lipid oxidation, enhancing total antioxidant status and modulating the level of superoxide dismutase and catalase with a 57% and 32% reduction in superoxide dismutase and catalase activities respectively, moving the redox environment to a status of low production of O(2)(•-) with an increase in H(2)O(2) detoxification. No side effects were observed. These results show that medical ozone treatment could be a complementary therapy in the treatment of CAD and its complications.

Is ozone pre-conditioning effect linked to Nrf2/EpRE activation pathway in vivo? A preliminary result

The present preliminary study has been focused on verifying whether ozone preconditioning may be linked to Nrf2/EpRE (nuclear factor erythroid 2/electrophile-responsive element) activation pathway in vivo. Healthy volunteers received a total of three Major Auto-Hemotherapy (MAH) treatments, with treatments administered every second day. The amount of blood used for each subject was standardized to the value obtained multiplying the subject׳s body weight by 1.3 in order to ensure the same ozone concentrations for each subject. A parallel group (n=50) age and gender matched was used as reference for the experimental variables related to the oxidative stress parameters. Levels of Nrf2 and oxidative stress index were measured throughout the study. Levels of Nrf2 (P<0.01) in peripheral blood mononuclear cells (PBMC) were found to increase immediately after ozone/oxygen exposure (35µg/ml, prior to reinfusion). This effect was still detected (P<0.05) in total circulating PBMC when measured 30min following reinfusion. After a series of 3 MAH, Nrf2 returned back to the basal level. At the end of the experiment the activities of superoxide dismutase and catalase were increased (P<0.05). These data demonstrate for the first time in vivo the activation of the Nrf2 pathway by a low dose of ozone and the promotion of the feedback mechanism that induces the synthesis of proteins which collectively favors cell survival.

Cholinergic effects of cimetidine and ranitidine

Cimetidine and ranitidine were submitted to eight in vitro assays: their intrinsic activity was evaluated on guinea-pig auricles and ileum, acetylcholine synergism on guinea-pig tracheal muscle, frog rectus abdominis, guinea-pig vas and rat phrenic nerve diaphragm, and antagonism on guinea-pig auricles and rat jejunum estimated at a range of concentrations. Ranitidine alone opposed 1-hyoscyamine antagonism in guinea-pig ileum when acetylcholine was the agonist, but not when the agonist was bethanechol.

Sodium-activated potassium current in mouse diaphragm

The mouse diaphragm muscle fiber was studied using the loose patch clamp technique. The voltage gated sodium currents were evoked by step pulses from a holding potential of about −70 mV. Following the activation of the sodium current, a very large and fast outward current was evoked. The sensitivity of this current to 4‐aminopyridine and tetraethylammonium indicates the potassium ion as the possible carrier for the channel. Furthermore, the sensitivity to tetrodotoxin and extracellular sodium demonstrated the sodium dependence of this current.

Effects of Hypericum extract on the acetylcholine release: a loose patch clamp approach

The St. Johns Wort (Hypericum perforatum) extract (Hp) represents one of the most useful natural therapeutic agents in the treatment of moderate and mild depression. The antidepressant effects of Hp are different, by a molecular mechanism point of view, when compared to those of other antidepressant drugs and, we think, a further pharmacological characterization is needed. It is suggested that the neurochemical effects of Hp could be bind either to its activity on the uptake of some mediators in the central nervous system or to the inhibition of some enzymatic activity at the receptor level. The present study carried out with the loose patch clamp (LPC) in the mouse neuromuscular junction, indicates a potentiation of the acetylcholine (ACh) action at the mouse neuromuscular junction. The spontaneous release of ACh was unaffected by Hp indicating that neither presynaptic nor postsynaptic function are modified by Hp. Indeed, both the frequency and the amplitude of the miniature end-plate currents (mepcs) were unmodified by Hp. Furthermore, the mepcs decay time (tau), i.e. the apparent cholinergic channel life time, was significantly increased after Hp treatment. The other parameter affected was the amplitude of the evoked end-plate currents (epcs) which was constantly and in a dose dependent manner increased by Hp. These findings suggest a possible action of Hp on the acetylcholinesterase (AChE) in terms of a reduction of the degradation rate of ACh.

Computerized estimation of spontaneous and evoked acetylcholine release at the neuromuscular junction

A new and automated on-line analysis of the parameters related to the neuromuscular activity is shown. The procedure used to obtain in real time the statistical evaluation of the recorded electrical signals is presented and discussed. The hardware is composed of a PDP-11/73 microcomputer equipped with an analog input/output board. The software is written principally in FORTRAN 77 and also uses MACRO/ASSEMBLY language.

Ozone oxidative preconditioning prevents atherosclerosis development in New Zealand White rabbits.

Abstract: Atherosclerosis is a major cause of death in the Western World. It is known that Lipofundin 20% induces atherosclerotic lesions, whereas ozone at low doses has been satisfactorily used in the prevention of oxidative stress–associated pathologies, such as coronary artery diseases. The aim of the present work was to evaluate the effects of ozone therapy on Lipofundin-induced atherosclerotic lesions in New Zealand White rabbits. Ozone (1 mg), mixed with oxygen as passive carrier, was administered by rectal insufflation during 15 sessions in 5 weeks. Then, the animals were intravenously treated with 2 mL/kg of Lipofundin, daily during 8 days. Animals were euthanized and eosin and hematoxylin staining was used for aortic histopathological analysis. The biomarkers of oxidative stress and lipid profile in serum were determined by spectrophotometric techniques. The results demonstrated that ozone induced inhibitory effects on aortic lesions formation. On the other hand, a reduction of biomolecular damage and an increase of antioxidant systems were observed at the end of the experiment. The serum lipids profiles were not modified after only 1 cycle of ozone treatment. Our results reinforced the hypotheses that antioxidant effects induced by ozone in the context of atherosclerosis demonstrate the antiatherogenic properties of the gas in the experimental conditions of this study.

The usefulness, in pharmacological classification, of complementary pattern-recognition techniques and structure modelling as afforded by the iterative collation of multiple-trial data in data banks☆

In this emerging information age no significant limits can be envisaged to the immense resources of knowledge that pharmacological sciences can draw upon by systematically applying multivariate pattern-recognition techniques to those data banks which can be organized internationally with better standardization of descriptors, i.e. by parametrizing observations and evaluating monitoring in experimental, biological assays, clinical trials and postmarketing surveillance. Even conventionally or habitually adopted references and communalities such as traditional drug profiles and receptor models may be iteratively re-checked and suitably adapted so as to take account of more adequate, up-to-date analytical techniques, fresh biological ideas and new advances in terms of physiological refinements. An attempt may also be made to modify the chemicophysical relationships and patterns currently traced on the basis of what are held to be quantitative structure-activity oversimplifications. The present paper focuses upon specifying a number of standardization criteria in conventional assays and upon submitting multiple biological features to monitoring; in addition, it gives some picture of the new trends the approach can offer and draws attention to the more relevant, innovative literature references.