Essential role of ERK activation in neurite outgrowth induced by α-lipoic acid

Background

Neurite outgrowth is an important aspect of neuronal plasticity and regeneration after neuronal injury. Alpha-lipoic acid (LA) has been used as a therapeutic approach for a variety of neural disorders. We recently reported that LA prevents local anesthetics-induced neurite loss. In this study, we hypothesized that LA administration promotes neurite outgrowth.

Methods

To test our hypothesis, we treated mouse neuroblastoma N2a cells and primary neurons with LA. Neurite outgrowth was evaluated by examination of morphological changes and by immunocytochemistry for β-tubulin-3. ROS production was examined, as were the phosphorylation levels of ERK and Akt. In separate experiments, we determined the effects of the inhibition of ERK or PI3K/Akt as well as ROS production on LA-induced neurite outgrowth.

Results

LA promoted significantly neurite outgrowth in a time- and concentration-dependent manner. LA stimulation significantly increased the phosphorylation levels of both Akt and ERK and transiently induced ROS production. PI3K/Akt inhibition did not affect LA-induced neurite outgrowth. However, the inhibition of ERK activation completely abolished LA-induced neurite outgrowth. Importantly, the prevention of ROS production by antioxidants attenuated LA-stimulated ERK activation and completely abolished LA-promoted neurite outgrowth.

Conclusion

Our data suggest that LA stimulates neurite outgrowth through the activation of ERK signaling, an effect mediated through a ROS-dependent mechanism.     

Beneficial effect of dl-α-lipoic acid on cyclosporine A induced hyperlipidemic nephropathy in rats

Cyclosporine A (CsA)-induced dyslipidemia is one of the most important risk factors for morbidity and mortality after solid organ transplantation. Reducing this side effect of CsA by dietary agents may be safe, cost-effective, and attractive to both patients and health professionals. Hence the present study was designed to evaluate the role of dl-α-Lipoic acid (LA) in deteriorating the lipid abnormalities induced by CsA in rat kidney. Male albino Wistar rats were divided into four groups. CsA administered at a dose of 25 mg/kg body weight, orally for 21 days showed abnormal changes in the levels of lipoprotein fractions (LDL, HDL and VLDL) and lipid profile in both plasma and renal tissue. Significant alterations were also observed in the activities of lipid metabolizing enzymes. Co-treatment with LA (20 mg/kg body weight, oral gavage, for 21 days) reverted the levels of lipid profile (P < 0.001, P < 0.01) and lipoprotein fractions (P < 0.001, P < 0.01) to near control. The activities of lipid metabolizing enzymes also showed considerable restoration on LA supplementation. The outcome of this study provides evidence that LA (a natural metabolic antioxidant) treatment acts as a potent antilipemic agent against CsA-induced lipid abnormalities.     

Chaperone-aided expression of LipA and LplA followed by the increase in α-lipoic acid production

α-Lipoic acid (LA), a naturally occurring cofactor reported to be present in a diverse group of microorganisms, plants, and animal tissues, has been widely and successfully used as a therapy for a variety of diseases, including diabetes and heart disease. However, to date, recombinant DNA technology has not been applied for higher LA production due mainly to difficulties in the functional expression of key enzymes involved in LA production. Here, we report a study for higher LA production with the aid of chaperone plasmids, DnaKJE and trigger factor (Tf). The lipA and lplA genes encoding lipoate synthase and lipoate protein ligase in Pseudomonas fluorescens, respectively, were cloned and transformed into Escherichia coli K12. When they were overexpressed in E. coli, both LipA and LplA were expressed as inclusion bodies leading to no increase in LA production. However, when chaperone plasmids DnaKJE and Tf were coexpressed with lipA and lplA, the resulting recombinant E. coli strains showed higher LA production than the wild-type E. coli by 32–111%, respectively.     

Kinetic resolution of α-lipoic acid via enzymatic differentiation of a remote stereocenter

Kinetic resolution of α-lipoic acid, a case of remote stereocenter discrimination, was accomplished using lipase from Aspergillus oryzae WZ007. Performance of this lipase was investigated for enantioselective esterification of (S)-α-lipoic acid, leaving the target product (R)-α-lipoic acid in unreacted form. The effects of chain length of alcohol, type of solvent, molar ratio of alcohol:acid, and reaction temperature were studied. The optimum reaction conditions were found to be esterification with n-octanol at 50°C in heptane with an alcohol:acid molar ratio of 5:1. The conversion rate of α-lipoic acid was 75.2%, with an enantiomeric excess of 92.5% towards unreacted substrate in a reaction time of 48 h.     

Salt-induced hypertension in WKY rats: Prevention by α-lipoic acid supplementation

There is strong evidence that points to excess dietary salt as a major factor contributing to the development of hypertension. Salt sensitivity is associated with glucose intolerance and insulin resistance in both animal models and humans. In insulin resistance, impaired glucose metabolism leads to elevated endogenous aldehydes which bind to vascular calcium channels, increasing cytosolic [Ca²⁺]_i and blood pressure. In an insulin resistant animal model of hypertension, spontaneously hypertensive rats (SHRs), dietary supplementation with lipoic acid lowers tissue aldehydes and plasma insulin levels and normalizes blood pressure. The objective of this study is to examine the effects of a high salt diet on tissue aldehydes, cytosolic [Ca²⁺]_i and blood pressure in WKY rats and to investigate whether dietary supplementation with lipoic acid can prevent a salt induced increase in blood pressure. Starting at 7 weeks of age, WKY rats were divided into three groups of six animals each and treated for 10 weeks with diets as follows: WKY-normal salt (0.7% NaCl); WKY-high salt (8% NaCl); WKY-high salt + lipoic acid (8% NaCl diet + lipoic acid 500 mg/Kg feed). At completion, animals in the high salt group had elevated systolic blood pressure, platelet [Ca²⁺]_i, and tissue aldehyde conjugates compared with the normal salt group and showed smooth muscle cell hyperplasia in the small arteries and arterioles of the kidneys. Dietary -lipoic acid supplementation in high salt-treated WKY rats normalized systolic blood pressure and cytosolic [Ca²⁺]_i and aldehydes in liver and aorta. Kidney aldehydes and renal vascular changes were attenuated, but not normalized.     

Study of the therapeutic effects of Lactobacillus and α-lipoic acid against dimethylnitrosamine-induced liver fibrosis in rats

Lactobacillus (LB) and α-lipoic acid (ALA) were investigated to compare their protective effects against dimethylnitrosamine (DMN)-induced liver fibrosis in rats. Animals were either injected intraperitoneally with DMN to induce hepatic fibrosis, or were left untreated (negative control). For the DMN + LB and DMN + ALA treatment groups, at two weeks of DMN treatment LB or ALA was added to the feed and supplementation continued until the experimental endpoint at sixty days. At the study endpoint, expression of IL-1β, IL-6, IL-10, TNF-α, IFN-γ, TGF-β1, COL1-α1 genes and the concentration of glutathione and malondialdehyde were measured in liver tissues, while GOT, GPT, and ALP concentrations were measured in blood. Body weights remained higher in NC and DMN + LB groups compared to DMN and DMN + ALA groups, while activity of GOT and GPT in serum was lower in DMN + LB and DMN + ALA groups compared to the DMN group. Compared to other treatment groups, in the DMN group expression of both TGF-β1 and, COL1-α1 mRNAs and pro-inflammatory cytokines increased, while that of 1L-10 decreased. Furthermore, LB and ALA treatments increased antioxidant activity of glutathione and decreased malondialdehyde in comparison to the DMN group. Between LB and ALA treatments, glutathione concentration was higher in the DMN + LB group, while malondialdehyde was lower. Our results indicate that both LB and ALA exert hepatoprotective effects against DMN-induced liver fibrosis. Their beneficial effects may be partly associated with down-regulation of both TGF-β1 and COL1-α1 signaling, which may be accounted for reduction of increased oxidative stress and TNF-α production.     

Remedial effect of DL-α-lipoic acid against adriamycin induced testicular lipid peroxidation

Adriamycin (ADR), a cytotoxic antineoplastic drug is used in the treatment of various solid tumors. However, its efficacy continues to be challenged by significant toxicities including testicular toxicity. In the present study, the effect of lipoic acid, a "universal antioxidant" was investigated on ADR induced peroxidative damages in rat testis. Adult male albino rats of Wistar strain were administered ADR (1 mg/kg body weight, i.v.) once a week for 10 weeks. ADR injected rats showed a significant decline in the activities of enzymic antioxidants (catalase, superoxide dismutase, glutathione peroxidase, glutathione reductase and glutathione-S-transferase) and non-enzymic antioxidants (reduced glutathione, Vitamin A, Vitamin C and Vitamin E) with high malondialdehyde levels. The extent of testicular toxicity was evident from the decreased activities of testicular marker enzymes (sorbitol dehydrogenase and glucose-6-phosphate dehydrogenase). Treatment with lipoic acid (35 mg/kg body weight, i.p.) one day prior to ADR administration, maintained near normal activities of the enzymes and significantly reduced lipid peroxidation, thereby proving it to be an effective cytoprotectant.     

Insight into the role of DL-α-lipoic acid against cyclophosphamide induced alterations in calcium sensitivity of cardiac myofilaments

Cyclophosphamide (CP), a potent antitumor drug, is known to cause severe cardiotoxicity. The present study is aimed at evaluating the role of DL-α-Lipoic acid (LA) on the calcium responsiveness of cardiac myofilaments isolated from CP treated rats. Adult male Wistar rats were divided into four treatment groups. Two groups received single intraperitoneal injection of CP (200 mg/kg b.wt.) to induce cardiotoxicity, one of these groups received LA treatment (25 mg/kg b.wt. for 10 days). A vehicle treated control group and a LA drug control were also included. Cardiotoxicity was evident from increased levels of cardiac Troponin I in serum of CP treated rats. The pCa-actomyosin ATPase relationship of myofilaments demonstrated a rightward shift indicating diminished responsiveness in CP treated rats. The hill coefficient was reduced and the myofibrillar myosin Ca²⁺-ATPase and K⁺-(EDTA) activities were also significantly (P < 0.05) reduced. Ultrastuctural observations were also in agreement with the above abnormal changes, wherein loss of myofilaments occurred. LA effectively normalized these abnormalities and restored the cardiac function in CP administered rats.     

The effects of α-lipoic acid on immature rats with traumatic brain injury

Abstract

Traumatic brain injury (TBI) is a leading cause of morbidity and mortality during childhood. TBI enhances formation of reactive oxygen species that cause neuron damage and apoptosis. α-Lipoic acid (LA) is a free radical scavenger and biological antioxidant. We investigated the effects of LA treatment on the parietal and prefrontal cortex, and on the hippocampal regions of the brain in 7-day-old rat pups that had been subjected to contusion injury. Forty-two male rats were divided randomly into a control group, a TBI group and a TBI + LA treated group. LA was administered 30 min after TBI through an intragastric tube once daily for 2 days. Forty-eight hours after TBI, the animals were sacrificed and tissues were examined for apoptosis and density of neurons. Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) and active caspase-3 immunostaining were used to detect apoptosis. Glutathione peroxidase (GPx), superoxide dismutase (SOD) activity and malondialdehyde (MDA) levels also were measured. Histological evaluation showed that LA treatment significantly reduced TBI-induced neuronal death in the hippocampus, prefrontal and parietal cortex; TUNEL- and caspase-3-positive cells also were decreased in the same regions. In addition, LA administration increased GPx and SOD activity in the prefrontal cortex. It appears that LA may be beneficial for TBI in rats.     

Amelioration of lipid abnormalities by α-lipoic acid through antioxidative and anti-inflammatory effects

Recent data have revealed that oxidative products and inflammatory mediators are increased in the insulin‐resistant states of obesity and type 2 diabetes mellitus (T2DM). Obese patients with impaired glucose tolerance (IGT) are at high risk for developing T2DM and have high incidence of dyslipidemia. α‐Lipoic acid (ALA) is a potent antioxidant with insulin sensitizing activity. However, it is not clear whether ALA is effective on lipid parameters in humans. This study has investigated 22 obese subjects with IGT (obese‐IGT), 13 of whom underwent 2‐week ALA treatment, 600 mg intravenously once daily. Before and after the treatment, euglycemic‐hyperinsulinemic clamps were used to measure insulin sensitivity. Meanwhile, plasma lipids, oxidative products, and chronic inflammatory markers were measured. After treatment of ALA in obese‐IGT patients, insulin sensitivity was improved, insulin sensitivity index (ISI) impressively enhanced by 41%. Plasma levels of free fatty acids (FFAs), triglyceride (TG), total cholesterol (T‐Chol), low density lipoprotein‐cholesterol (LDL‐Chol), small dense LDL‐Chol (sd‐LDL), oxidized LDL‐Chol (ox‐LDL‐Chol), very low density lipoprotein‐cholesterol (VLDL‐Chol) were all significantly decreased (P < 0.01). At the same time, both plasma oxidative products (malondialdehyde (MDA), 8‐iso‐prostaglandin) and inflammatory markers (tumor necrosis factor‐α (TNF‐α), interleukin‐6 (IL‐6)) were remarkably decreased (P < 0.01), while adiponectin was increased (P < 0.01). There are significant negative correlations between ISI and plasma FFAs, sd‐LDL‐Chol, ox‐LDL‐Chol, MDA, 8‐iso‐prostaglandin, TNF‐α, and IL‐6, and positive correlations with HDL‐Chol and adiponectin in obese‐IGT patients. The results indicate that short‐term treatment with ALA can improve insulin sensitivity and plasma lipid profile possibly through amelioration of oxidative stress and chronic inflammatory reaction in obese patients with IGT.     

Effects of α-lipoic acid supplementation on maternal diabetes-induced growth retardation and congenital anomalies in rat foetuses

The mechanism of diabetic embryopathy is not known. Excessive reactive oxygen species (ROS) produced in diabetes may be causally related to foetal anomalies. The objective of this study was to determine whether supplementation with the antioxidant lipoic acid (LA) could prevent maternal diabetes-related foetal malformations and intrauterine growth retardation (IUGR) in rats. Pregnant rats were non-treated (Group I) or made diabetic on gestation day (GD) 2 by injecting streptozotocin (Group II). Group III was injected with 20 mg kg^–1 of LA daily starting on GD 6 and continued through GD 19. Group IV was administered only Tris buffer on the corresponding days. Group V was a set of STZ-treated animals, which were supplemented with a daily dose of 20 mg kg^–1 of LA from GD 6 through GD 19. All fetuses were collected on GD 20. Lipoic acid did not affect the blood sugar levels of diabetic animals significantly but improved their body weight gain and reduced food and water consumption. Diabetic group had a high incidence of embryonic resorption, IUGR, craniofacial malformations, supernumerary ribs and skeletal hypoplasia. Lipoic acid significantly reduced these abnormalities. These data support the hypothesis that ROS are causally related to fetal maldevelopment and IUGR associated with maternal diabetes in the rat. They also highlight the possible role of antioxidants in the normal processes of embryo survival, growth and development. (Mol Cell Biochem 261: 123–135, 2004)     

The aqueous solubility and thermal stability of α-lipoic acid are enhanced by cyclodextrin

We investigated the effects of various cyclodextrins (CDs) on the aqueous solubility and thermal stability of α-lipoic acid, a compound with low water solubility. α-CD, β-CD, and γ-CD had little effect on the aqueous solubility of α-lipoic acid. In contrast, 6-O-α-maltosyl-CDs increased it in a concentration-dependent manner, 6-O-α-maltosyl-β-CD enhancing solubility the most. The thermal stability of α-lipoic acid in the solid state was improved by the addition of G2-β-CD(?), a commercial product of 6-O-α-maltosyl-β-CD. The thermal stability of α-lipoic acid was also improved by the addition of β-CD. Analysis by differential scanning calorimetry showed that G2-β-CD(?), a mixture of maltosyl-β-CDs, and β-CD efficiently formed complexes with α-lipoic acid. These results suggest that the sizes and shapes of these β-CD compounds are compatible with complexation with α-lipoic acid. Moreover, both the formation of an aqueous complex with G2-β-CD(?) and an insoluble complex with β-CD increased the thermal stability of α-lipoic acid.     

Cytotoxic aggregates of α-lactalbumin induced by unsaturated fatty acid induce apoptosis in tumor cells

The effects of three fatty acids on cytotoxic aggregate formation of Ca²⁺-depleted bovine α-lactalbumin (apo-BLA) have been studied by UV absorbance spectroscopy and transmission electron microscopy. The experimental results demonstrate that two unsaturated fatty acids, oleic acid and linoleic acid, and one saturated fatty acid, stearic acid, induce the intermediate of apo-BLA at pH 4.0-4.5 to form amorphous aggregates in time- and concentration-dependent manners. These aggregates are dissolved under physiological conditions at 37 °C and further characterized by fluorescence spectroscopy, circular dichroism and time-of-flight mass spectrometry. Our data here indicate that the structural characteristics of these aggregates are similar to those of HAMLET/BAMLET (human/bovine α-lactalbumin made lethal to tumor cells), a complex of the partially unfolded α-lactalbumin with oleic acid. Cell viability experiments indicate the aggregates of apo-BLA induced by oleic acid and linoleic acid show significant dose-dependent cytotoxicity to human lung tumor cells of A549 but those induced by stearic acid have no toxicity to tumor cells. Furthermore, the cytotoxic aggregates of apo-BLA induced by both unsaturated fatty acids induce apoptosis of human lung cancer cell line A549, suggesting that such cytotoxic aggregates of apo-BLA could be potential antitumor drugs. The present study provides insight into the mechanism of fatty acid-dependent oligomerization and cytotoxicity of α-lactalbumin, and will be helpful in the understanding of the molecular mechanism of HAMLET/BAMLET formation.     

Bioorganic synthesis, characterization and antioxidant activity of esters of natural phenolics and α-lipoic acid

Chemo-enzymatic synthesis of six esters of natural phenolics and α-lipoic acid was carried to produce novel compounds with potential bioactivity. The synthetic route was mild, simple, and efficient with satisfactory yields. The synthesized compounds were screened for antioxidant activities. The prepared derivatives exhibited very good antioxidant activities as determined by DPPH radical scavenging assay and inhibition of lipid oxidation in fish oil emulsion system. Among the prepared derivatives, three compounds exhibited radical scavenging activity similar to the reference antioxidants, BHT and alpha-tocopherol in the DPPH radical scavenging assay, where as in fish oil emulsion system, two derivatives showed activity, which was similar to the reference antioxidants.     

Role of α-glucosidase in fetal lung maturation

The role of lysosomal enzyme acid α-glucosidase in fetal lung development was investigated with the aid of a specific inhibitor, the pseudosaccharide acarbose. The drug was added to a Waymouth culture medium of fetal rat lung expiants cultivated for 48 h from gestational stage 19.5 days, an in vitro system previously shown to allow morphological and biochemical maturation of alveolar epithelium. Glycogenolysis was reduced by 40% as compared with tissue cultivated on control medium, which means that α-glucosidase could account for as much as 40% of fetal lung glycogenolysis, the remaining 60% being presumably achieved by cytosolic phosphorylase and by a microsomal neutral α-glucosidase. By the same time, the increase of phospholipids of surfactant fraction extracted from cultivated expiants was partially inhibited: total and saturated phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol and phosphatidylinositol were about 30–40% lower than in lungs cultivated on control medium. It should be emphasized that DNA concentration and increases in non-surfactant phospholipids were unchanged by the drug. α-Glucosidase activity was evidenced in the lysosomal fraction, in the microsomal fraction and, although in lower amounts, in the surfactant fraction extracted from term fetal lung. The results suggest that lysosomal α-glucosidase plays a major role in lung maturation and could facilitate glycogenolysis for the specific use of glycogen stores in providing substrates for surfactant phospholipid biosynthesis.     

Elimination of cells with abnormal nuclei in human epidermoid carcinoma cell line A431 by α-lipoic acid

Skin is usually exposed to adverse environmental conditions that may cause pathological cell proliferation and malignant transformation. Antioxidants are able to affect these processes and eliminate transformed cells. The purpose of this work was to investigate the effect of α-lipoic acid (ALA) on human epidermoid carcinoma cell line A431. It was found that 100, 200, 300, 500 μM ALA added for 24, 48, 72 h inhibited cell proliferation and stimulated apoptosis. Most dying cells have abnormal nuclei (micronuclei, giant nuclei, nuclei with buds). Electron microscopy showed that cells with normal nuclear phenotypes after treatment with 200 μM ALA for 48 h had ultrastructural organizations typical for control cells. Thus, α-ALA not only triggers the apoptosis of carcinoma cells, but it may also activate the mechanism for eliminating cells with abnormal numbers of chromosomes.     

Quantum-chemical investigation of the structure and the antioxidant properties of α-lipoic acid and its metabolites

Quantum-chemical computations were used to investigate the structure-antioxidant parameter relationships of α-lipoic acid and its natural metabolites bisnorlipoic acid and tetranorlipoic acid in their oxidized and reduced forms. The enantiomers of lipoic and dihydrolipoic acid were optimized using the B3LYP/6-311+G(3df,2p), B3LYP/aug-cc-pVDZ and MP2(full)/6-31+G(d,p) levels of theory as isolated molecules and in the presence of water. The geometries of the metabolites and the values of their antioxidant parameters (proton affinity, bond dissociation enthalpy, adiabatic ionization potential, spin density, and the highest occupied molecular orbital energy) were calculated at the B3LYP/6-311+G(3df,2p) level of theory. The results obtained reveal similarities between these structures: a pentatomic, nonaromatic ring is present in the oxidized forms, while an unbranched aliphatic chain (as found in saturated fatty acids) is present in both the oxidized and the reduced forms. Analysis of the spin density and the highest occupied molecular orbital energy revealed that the SH groups exhibited the greatest electron-donating activities. The values obtained for the proton affinity, bond dissociation enthalpy and adiabatic ionization potential indicate that the preferred antioxidant mechanisms for α-lipoic acid and its metabolites are sequential proton loss electron transfer in polar media and hydrogen atom transfer in vacuum.     

Effect of ethanol on the response of the rat urinary bladder to in vitro ischemia: Protective effect of α-lipoic acid

Purpose: Ethanol exposure has been used to demonstrate the increase of oxidative stress to a variety of tissues. We studied the effect of ethanol on the response of isolated strips of rat bladder to In vitro hypoxia in the absence of glucose (In vitro ischemia). Secondly, we determined if -lipoic acid (LA) could alter the response to ethanol + In vitro ischemia.Methods: Sixty-four rats were used for the these experiments. Each rat was anesthetized and its urinary bladder excised. The bladder body was cut into two longitudinal strips and each strip mounted in individual baths filled with oxygenated Tyrodes solution containing glucose at 37 C. Ethanol (0.3%, 1%, or 3%) was placed in the first six baths (two strips at each concentration). The last two baths did not receive ethanol. Each strip was incubated for 1 h and then stimulated with field stimulation at 2, 8, and 32 Hz. Each strip was stimulated with 10 M carbachol, washed three times with fresh oxygenated buffer and ethanol re-added to their respective baths. Each strip was then stimulated with 120 mM KCl and washed three times as before. Strips were then subjected to 1 h In vitro ischemia (incubation in the absence of glucose with Tyrodes equilibrated with nitrogen instead of oxygen). During the ischemic period, each strip was stimulated for 5 s every 10 min by 32 Hz FS to simulate hyperreflexia. At the end of the hour, the tissues were incubated for an additional hour in the presence of oxygen + glucose and subjected to a second series of stimulations as before. At all times, ethanol was maintained in baths 1–6. In set 2, 1% ethanol was added to the first six baths. LA was added to every other bath, and the experiments performed as mentioned earlier.Results: (a) Ethanol at 0.3% or 1% had no effect on the contractile responses prior to exposure to In vitro ischemia; 3% was inhibitory. (b) In vitro ischemia mediated a significant decrease in the contractile responses to all forms of stimulation except for carbachol. (c) Ethanol mediated a dose-response enhancement of the contractile dysfunctions caused by In vitro ischemia. (d) LA completely reversed the effects of ethanol on contractile responses following In vitro ischemia except for carbachol.Conclusions: The results demonstrate that direct exposure to ethanol significantly enhanced contractile dysfunctions mediated by In vitro ischemia followed by re-oxygenation and that the presence of LA significantly inhibits this effect of ethanol. (Mol Cell Biochem 271: 133–138, 2005)This material is based upon work supported in part by the Office of Research and Development, Department of Veterans Affairs, and NIH RO-1-DK067114     

Oxidant induced injury of erythrocyte—Role of green tea leaf and ascorbic acid

Oxidant and free radical-generating system were used to promote oxidative damage in erythrocytes. Among the oxidants used, phenylhydrazine represents one of the most investigated intracellular free radical-generating probes, which in the presence of haemoglobin autooxidises and give rise to hydroxyl radical, a marker for cellular damage. Erythrocyte, as a single cell, is a good model to be used for studying the haemolytic mechanism of anaemia. Our present investigations reveal increased lipid peroxidation of erythrocyte using phenylhydrazine as well as other oxygen-generating systems (hydrogen peroxide, iron with hydrogen peroxide). It has further been observed that not only lipid peroxidation, phenylhydrazine causes significant elevation in methemoglobin formation, catalase activity and turbidity, in the above system, which are the typical characteristics of haemolytic anaemia. However, exogenous administration of green tea leaf extract and ascorbic acid as natural antioxidants and free radical scavengers were shown to protect separately increased lipid peroxidation caused by phenylhydrazine, though the degree of protection is more in case of green tea leaf extract than ascorbic acid. Results suggest that oxidative damage in vivo due to haemolytic disease may be checked to some extent by using natural antioxidants. (Mol Cell Biochem 276: 205–210, 2005)     

Neurochemical effects of the R form of α-lipoic acid and its neuroprotective mechanism in cellular models of Parkinson’s disease

ObjectiveParkinson’s disease (PD) is a common neurodegenerative disease. This study aimed to investigate the effects of the R form of α-lipoic acid (RLA) in cellular models of PD induced by 6-hydroxydopamine (6-OHDA) or 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP).MethodsCell viability and apoptosis were detected using CCK8 and Annexin V-FITC assays, respectively. Intracellular reactive oxygen species (ROS) were detected by fluorescence staining. ELISA assays were performed to detect the levels of dopamine and α-synuclein. To evaluate the effects of RLA on mitochondrial function, cytotoxicity, ATP levels, and mitochondrial gene expression were assayed. Additionally, the expression levels of autophagy-related proteins, including Parkin, PINK1, p62, ATG12, and LC3, were analyzed by western blot, and cell autophagy was visualized by immunofluorescence.ResultsRLA increased cell viability and decreased apoptosis, intracellular ROS, and cytotoxicity, and induced cell autophagy in PD models induced by 6-OHDA and MPTP. RLA also reversed the decreased dopamine and increased α-synuclein expression induced by 6-OHDA and MPTP. The mitochondrial regulatory protein PGC-1α was significantly up-regulated by RLA. The expression levels of autophagy-related proteins, including Parkin, PINK1, p62, and ATG12, were down-regulated after RLA treatment, while LC3 expression was up-regulated.ConclusionsRLA has a protective effect against cellular damage induced by 6-OHDA and MPTP. The neuroprotective mechanism of RLA may be associated with improvement of mitochondrial function and autophagy. Therefore, RLA may serve as a promising potential adjuvant for PD treatment.