Anna Gorąca

Lipoic acid – biological activity and therapeutic potential

α-Lipoic acid (LA; 5-(1,2-dithiolan-3-yl)pentanoic acid) was originally isolated from bovine liver by Reed et al. in 1951. LA was once considered a vitamin. Subsequently, it was found that LA is not a vitamin and is synthesized by plants and animals. LA is covalently bound to the ε-amino group of lysine residues and functions as a cofactor for mitochondrial enzymes by catalyzing the oxidative decarboxylation of pyruvate, α-ketoglutarate and branched-chain α-keto acids. LA and its reduced form - dihydrolipoic acid (DHLA), meet all the criteria for an ideal antioxidant because they can easily quench radicals, can chelate metals, have an amphiphlic character and they do not exhibit any serious side effects. They interact with other antioxidants and can regenerate them. For this reason, LA is called an antioxidant of antioxidants. LA has an influence on the second messenger nuclear factor κB (NF-κB) and attenuates the release of free radicals and cytotoxic cytokines. The therapeutic action of LA is based on its antioxidant properties. Current studies support its use in the ancillary treatment of many diseases, such as diabetes, cardiovascular, neurodegenerative, autoimmune diseases, cancer and AIDS. This review was undertaken to gather the most recent information regarding the therapeutic properties of LA and its possible utility in disease treatment.

The NADPH oxidase family and its inhibitors.

The classical nicotinamide adenine dinucleotide phosphate (NADPH) oxidase was originally detected in neutrophils as a multicomponent enzyme that catalyzes the generation of superoxide from oxygen and the reduced form of NADPH. This enzyme is composed of two membrane-bound subunits (p22phox and gp91phox), three cytosolic subunits (p67phox, p47phox, and p40phox) and a small G-protein Rac (Rac1 and Rac2). Recently, it has been demonstrated that there are several isoforms of nonphagocytic NADPH oxidase. Endothelial cells, vascular smooth muscle cells or adventitial fibroblasts possess multiple isoforms of this enzyme. The new homologs, along with gp91phox are now designated the Nox family of NADPH oxidases and are key sources of reactive oxygen species in the vasculature. Reactive oxygen species play a significant role in regulating endothelial function and vascular tone. However, besides the participation in the processes of physiological cell, these enzymes can also be the perpetrator of oxidative stress that causes endothelial dysfunction. This review summarizes the current state of knowledge of the structure and functions of NADPH oxidase and NADPH oxidase inhibitors in the treatment of disorders with endothelial damage.

Effects of lipoic acid on spleen oxidative stress after LPS administration

BACKGROUNDnBacterial endotoxin (lipopolysaccharide - LPS) is a strong modulator of the immune system that plays a crucial role in the pathogenesis of endotoxic shock. The aim of the study was to investigate the effects of lipoic acid (LA) on oxidative stress markers in spleen homogenates obtained from LPS-induced endotoxic shock rats.nnnMETHODSnThe animals were treated with saline or lipoic acid (LA) (60 or 100 mg/kg b.w. iv) 30 min before or 30 min after LPS administration (30 mg/kg b.w. iv). Five hours after LPS, LA or saline administration, the animals were euthanized and their spleens were isolated for measurements.nnnRESULTSnThe LPS-treated animals developed oxidative stress, indicated by a significant increase in thiobarbituric acid-reactive substances (TBARS) and hydrogen peroxide (H2O2) concentrations (p<0.001) as well as an insignificant decrease in the level of sulfhydryl groups (-SH groups) and the glutathione redox ratio [reduced glutathione (GSH) to oxidized glutathione (GSSG) ratio] (p<0.02) as compared with control group. Treatment with LA (60 or 100 mg/kg) before or after LPS administration resulted in an increase in -SH group content (p<0.01) and a decrease in TBARS and H2O2 concentration in the spleen as compared with LPS group (p<0.001). LA (60 or 100 mg/kg) before LPS administration decreased the level of GSSG (p<0.05) and increased the level of GSH in spleen homogenates (p<0.05), resulting in an increase in the GSH/GSSG ratio compared with the LPS group (p<0.01). It also improved the LPS-induced increase in the spleen weight (SW) to body weight (BW) ratio (p<0.001 vs. control).nnnCONCLUSIONnThe present results have shown that LA is endowed with antioxidant properties that are protective in the spleen against the deleterious actions of Gram-negative bacterial endotoxin.

Role of endothelin-1 receptor blockers on hemodynamic parameters and oxidative stress

Endothelin (ET) was first isolated and described by Yanagisawa et al. and has since been described as one of the most potent known vasoconstrictor compounds. ET-1 mediates its effects via two types of receptors, ETA and ETB, which are expressed in the vascular smooth muscle cells, endothelial cells, intestines and brain. Secretion of ET-1 results in long-lasting vasoconstriction, increased blood pressure and, in turn, overproduction of free radicals. As dysregulation of the endothelin system is an important factor in the pathogenesis of several diseases including atherosclerosis, hypertension and endotoxic shock, the ETA and ETB receptors are attractive therapeutic targets for treatment of these disorders. The biosynthesis and release of ET-1 are regulated at the transcriptional level. Studies have shown that p38MAP kinase, nuclear factor kappaB (NF-kappaB), PKC/ERK and JNK/c-Jun all take part in the ROS-activated production of ET-1. Furthermore, administration of ET(A) significantly reduces the generation of free radicals. However, treatment with ETB receptor blockers does not elicit the same effect. Therefore, the effects of endothelin receptor blockers on blood pressure and the generation of free radicals remain debatable. This review summarizes recent investigations into the role of endothelin receptor blockers with respect to the modulation of hemodynamic parameters and the generation of free radicals.

Influence of specific endothelin-1 receptor blockers on hemodynamic parameters and antioxidant status of plasma in LPS-induced endotoxemia.

BACKGROUNDnThe potent vasoconstrictor endothelin-1 has been implicated in the pathogenesis of plasma oxidative stress seen in sepsis. The selective endothelin receptor blockers BQ123 and BQ788 were used to investigate the importance of selective endothelin receptor blockage in modulating oxidative stress during endotoxemia.nnnMETHODSnThe study was performed on male Wistar rats (n = 6 per group) divided into groups: (1) saline, (2) lipopolysaccharide (LPS) (15 mg/kg)-saline, (3) BQ123 (0.5 mg/kg)-LPS, (4) BQ123 (1 mg/kg)-LPS, (5) BQ788 (3 mg/kg)-LPS. The endothelin receptor type A(ETA-R) or type B (ETB-R) antagonist was injected intravenously 30 min before LPS administration. Blood pressure was monitored and blood was taken before, 90 min and 300 min after saline or LPS administration.nnnRESULTSnInjection of LPS alone resulted in a decrease in mean arterial pressure (MAP) (p < 0.05), a decrease in ferric reducing ability of plasma (FRAP) value (p < 0.01) and a marked increase in plasma tumor necrosis factor α (TNF-α) and thiobarbituric acid reactive substances (TBARS) (p < 0.001, p < 0.001, respectively). Administration of BQ123 before LPS administration deteriorated MAP in a dose dependent way. Moreover, BQ123 (1 mg/kg) decreased plasma level of TBARS and TNF-α (p < 0.01 and p < 0.05, respectively) and increased FRAP value (p < 0.001). On the contrary, BQ788 prevented LPS-induced decrease in MAP(p < 0.001) and led to a significant reduction in plasma TBARS concentration (p < 0.01).nnnCONCLUSIONSnOur study showed that blockage of ETB-R during endotoxemia improved blood hemodynamics and decreased plasma lipid peroxidation. Blockage of ETA-R improved plasma antioxidant status and decreased lipid peroxidation and TNF-α production, but it deteriorated hemodynamic conditions.

The influence of ETA and ETB receptor blockers on LPS-induced oxidative stress and NF-κB signaling pathway in heart.

The aim of this study was to assess whether an endothelin-A receptor (ETA-R) blocker, BQ123, or an endothelin-B (ETB-R) receptor blocker, BQ788, influences nuclear factor kappa beta (NF-κB) pathway, free radical generation, tumor necrosis factor-alpha (TNF-α) concentration, and glutathione redox system in hearts obtained from lipopolysaccharide (LPS)-induced endotoxic rats. The study was performed on rats divided into groups: 1) saline, 2) saline + LPS (15 mg/kg), 3) BQ123 (1 mg/kg b.w.) + LPS, 4) BQ123 (0.5 mg/kg b.w.) + LPS, 5) BQ788 (3 mg/kg b.w.) + LPS. The ETA-R and ETB-R antagonists were injected i.v. 30 min before LPS administration. In rats, BQ123 caused a significant decrease in TBARS (p < 0.05) but not in H2O2 concentration. It also decreased tissue protein level and improved tissue redox status (p < 0.01). Only a dose of 1 mg/kg decreased TNF-α concentration (p < 0.05). BQ788 lowered TBARS, H2O2 and protein concentration (p < 0.05; p < 0.02; p < 0.001, respectively), however, it did not affect TNF-α concentration. Neither ETA-R nor ETB-R blockers influenced LPS-induced increase in p65 subunit level and activation of NF-κB pathway. Our results demonstrated that ETA-R blockage is more effective in inhibiting free radical generation and improving heart antioxidant properties than ETB-R blockage under oxidative stress. NF-κB pathway is not incorporated in ETA-R and ETB-R influence on ROS production.

ET-1 mediates the release of reactive oxygen species and TNF-α in lung tissue by protein kinase C α and β1

BACKGROUNDnThe aim of this study was to determine the involvement of protein kinase C (PKC) in the ET-1 induced generation of reactive oxygen species and TNF-α in rat lungs.nnnMETHODSnExperiments were performed on 6 groups of rats: Group I: saline-treated control; Group II: saline followed by endothelin-1 (ET-1) (3μg/kg); Group III: saline followed by selective PKC αβ1 inhibitor (Gö6976) (2μg/kg); Group IV: Gö6976 (2μg/kg) administered 30min before ET-1 (3μg/kg); Group V: saline followed by the PKC activator phorbol 12-myristate 13-acetate (PMA) (50μg/kg); Group VI: Gö6976 (2μg/kg) administered 30min before PMA (50μg/kg). After 5h, the animals were euthanized and their lungs were isolated for measurements.nnnRESULTSnET-1 resulted in increase in thiobarbituric acid reactive substances (TBARS) and hydrogen peroxide (H2O2) levels and lung edema, as well as a decrease in GSH/GSSG ratio compared to the controls. The level of TNF-α also was elevated in the presence of ET-1. Administration of Gö6976 30min before ET-1 injection significantly decreased lung edema, as well as the concentrations of TBARS, H2O2 and TNF-α, but increased the GSH/GSSG redox ratio compared to ET-1. Conversely, PMA elevated lung edema and TBARS, H2O2 and TNF-α concentrations, but decreased the GSH/GSSG redox ratio compared to the control group. Treatment with Gö6976 significantly ameliorated the PMA-induced oxidative stress parameters, decreased tissue TNF-α level, and lung edema.nnnCONCLUSIONnEndothelin-1 induces ROS generation, increases TNF-α level and lung edema via activation of PKC αβ1.

Influence of NADPH oxidase inhibition on oxidative stress parameters in rat hearts

BACKGROUNDnThe aim of this study was to assess whether apocynin, an nicotinamide adenine dinucleotide phosphate (NADPH) oxidase blocker, influences lipid peroxidation TBARS, hydrogen peroxide (H2O2) content, protein level, heart edema, tumor necrosis factor α (TNF-α) concentration or the glutathione redox system in heart homogenates obtained from endothelin 1 (ET-1)-induced oxidative stress rats.nnnMETHODSnExperiments were carried out on adult male Wistar-Kyoto rats. The animals were divided into 4 groups: Group I: saline-treated control; Group II: saline followed by ET-1 (3 μg/kg b.w., iv); Group III: apocynin (5 mg/kg b.w., iv) administered half an hour before saline; Group IV: apocynin (5 mg/kg b.w., iv) administered half an hour before ET-1 (3 μg/kg b.w., iv).nnnRESULTSnInjection of ET-1 alone showed a significant (p < 0.001) increase in thiobarbituric acid reactive substances (TBARS) and the hydrogen peroxide level (p < 0.01) vs. control, as well as a decrease (p < 0.001) in the GSH level. Apocynin significantly decreased TBARS (p < 0.001) and H2O2 (p < 0.05) level (vs. control) as well as improved protein level (p < 0.001) in the heart. Apocynin also prevented ET-1-induced heart edema (p < 0.05). The presence of ET-1 increased the concentration of TNF-α (p < 0.05) while apocynin decreased it (p < 0.05). Our results indicate that ET-1 may induce oxidative stress in heart tissue by reducing the GSH/GSSG ratio, stimulating lipid peroxidation and increasing TNF-α concentration. Apocynin diminished these measures of oxidative stress and TNF-α.nnnCONCLUSIONnET-1-induced formation of ROS in the heart is at least partially regulated via NADPH oxidase.

Estimate of antioxidant capacity and lipid peroxidation in plasma of healthy subjects

Serum contains various antioxidant molecules that may provide important protection against free radical attack. The aim of this work was to assess the total antioxidant capacity of plasma and a marker of lipid per oxidation [(thiobarbituric acid-reactive substances (TBARS)] in plasma of healthy smoking and non-smoking young and elderly subjects. In addition, we investigated plasma concentrations of α-tocopherol, β-carotene, and ascorbic acid. In in vitro experiments, the effects of exogenous compounds (ascorbic acid, uric acid, Trolox) on total ferric-reducing activity of plasma (FRAP) were also tested. We demonstrated that total antioxidant capacity of plasma obtained from healthy non-smoking young subjects was significantly higher than plasma antioxidant capacity of smoking elderly subjects. The concentration of TBARS in young non-smoking volunteers was lower than that in young smokers. The concentration of TBARS in elderly non-smoking volunteers was lower than in elderly smokers. Plasma concentrations of alpha-tocopherol, beta-carotene and ascorbic acid were significantly lower in elderly smoker than in elderly non-smokers of the same age. No difference in the plasma levels of alpha-tocopherol, beta-carotene and ascorbic acid were found in 22-year-old smoking and non-smoking subjects. In vitro addition of ascorbic acid, uric acid, or Trolox to plasma samples significantly increased their total antioxidant capacity. Decrease of FRAP values and increase of TBARS concentrations is a significant physiologic condition of the aging process. Supplementation of antioxidants could be useful for the enhancement of antioxidant screen in plasma.

BQ123 Stimulates Skeletal Muscle Antioxidant Defense via Nrf2 Activation in LPS-Treated Rats

Little is understood of skeletal muscle tissue in terms of oxidative stress and inflammation. Endothelin-1 is an endogenous, vasoconstrictive peptide which can induce overproduction of reactive oxygen species and proinflammatory cytokines. The aim of this study was to evaluate whether BQ123, an endothelin-A receptor antagonist, influences the level of TNF-α, IL-6, SOD-1, HO-1, Nrf2 mRNA, and NF-κB subunit RelA/p65 mRNA in the femoral muscle obtained from endotoxemic rats. Male Wistar rats were divided into 4 groups (n = 6) and received iv (1) saline (control), (2) LPS (15u2009mg/kg), (3) BQ123 (1u2009mg/kg), (4) BQ123 (1u2009mg/kg), and LPS (15u2009mg/kg, resp.) 30u2009min later. Injection of LPS led to significant increase in levels of RelA/p65 mRNA, TNF-α, and IL-6, while content of SOD-1, HO-1, and Nrf2 mRNA was unchanged. Administration of BQ123 prior to LPS challenge resulted in a significant reduction in RelA/p65 mRNA, TNF-α, and IL-6 levels, as well as markedly elevated concentrations of SOD-1, HO-1, and Nrf2 mRNA. BQ123 appears to enhance antioxidant defense and prevent production of TNF-α and IL-6 in skeletal muscle of LPS-treated rat. In conclusion, endothelin-A receptor antagonism exerts significant impact on the skeletal muscle favouring anti-inflammatory effects and protection against oxidative stress.