Iron chelation therapy with deferiprone improves oxidative status and red blood cell quality and reduces redox-active iron in ß-thalassemia/hemoglobin E patients.

The oxidative status of twenty-three ß-thalassemia/hemoglobin E patients was evaluated after administration of 75 mg/kg deferiprone (GPO-L-ONE®) divided into 3 doses daily for 12 months. Serum ferritin was significantly decreased; the median value at the initial and final assessments was 2842 and 1719 ng/mL, respectively. Progressive improvement with significant changes in antioxidant enzyme activity, including plasma paraoxonase (PON) and platelet-activating factor acetylhydrolase (PAF-AH), and in antioxidant enzymes in red blood cells (glutathione peroxidase (GPx), catalase and superoxide dismutase (SOD)) were observed at 3-6 months of treatment. The levels of total GSH in red blood cells were significantly increased at the end of the study. Improved red blood cell membrane integrity was also demonstrated using the EPR spin labeling technique. Membrane fluidity at the surface and hydrophobic regions of the red blood cell membrane was significantly changed after 12 months of treatment. In addition, a significant increase in hemoglobin content was observed (6.6 ± 0.7 and 7.5 ± 1.3 g/dL at the initial assessment and at 6 months, respectively). Correlations were observed between hemoglobin content, membrane fluidity and antioxidant enzymes in red blood cells. The antioxidant activity of deferiprone may partly be explained by progressive reduction of redox active iron that catalyzes free radical reactions, as demonstrated by the EPR spin trapping technique. In conclusion, iron chelation therapy with deferiprone notably improved the oxidative status in thalassemia, consequently reducing the risk of oxidative-related complications. Furthermore, the improvement in red blood cell quality may improve the anemia situation in patients.

Inhibitory Effects of Erythrosine/Curcumin Derivatives/Nano-Titanium Dioxide-Mediated Photodynamic Therapy on Candida albicans.

This study focuses on the role of photosensitizers in photodynamic therapy. The photosensitizers were prepared in combinations of 110/220 µM erythrosine and/or 10/20 µM demethoxy/bisdemethoxy curcumin with/without 10% (w/w) nano-titanium dioxide. Irradiation was performed with a dental blue light in the 395-480 nm wavelength range, with a power density of 3200 mW/cm2 and yield of 72 J/cm2. The production of ROS and hydroxyl radical was investigated using an electron paramagnetic resonance spectrometer for each individual photosensitizer or in photosensitizer combinations. Subsequently, a PrestoBlue® toxicity test of the gingival fibroblast cells was performed at 6 and 24 h on the eight highest ROS-generating photosensitizers containing curcumin derivatives and erythrosine 220 µM. Finally, the antifungal ability of 22 test photosensitizers, Candida albicans (ATCC 10231), were cultured in biofilm form at 37 °C for 48 h, then the colonies were counted in colony-forming units (CFU/mL) via the drop plate technique, and then the log reduction was calculated. The results showed that at 48 h the test photosensitizers could simultaneously produce both ROS types. All test photosensitizers demonstrated no toxicity on the fibroblast cells. In total, 18 test photosensitizers were able to inhibit Candida albicans similarly to nystatin. Conclusively, 20 µM bisdemethoxy curcumin + 220 µM erythrosine + 10% (w/w) nano-titanium dioxide exerted the highest inhibitory effect on Candida albicans.

Anti-inflammatory effect of photodynamic therapy using guaiazulene and red lasers on peripheral blood mononuclear cells.

INTRODUCTION: Photodynamic therapy improves oral mucositis treatment. The reactive oxygen species (ROS) generated from this reaction could contribute to an anti-inflammatory effect by suppressing inflammatory cells. OBJECTIVE: To evaluate the anti-inflammatory effect of photodynamic therapy using guaiazulene and a red laser in peripheral blood mononuclear cells (PBMCs). METHODS: Guaiazulene solutions (1, 2, 5, 25, 35, and 100 µM in 99.8 % methanol) were irradiated with red laser light (625 nm, 146.2 mW/cm2) in continuous mode at 0, 4, and 8 J/cm2 in black 96-well plates. ROS were measured using spin trapping technique with electron spin resonance (ESR) spectroscopy and fluorescence. The two highest concentrations were tested using cell viability (PrestoBlue®) and anti-inflammation (RANTES and PGE2 ELISA) assay kits. Kruskal-Wallis and Dunn Bonferroni tests were used for statistical analyses with significant differences at p-value < 0.05. RESULTS: Guaiazulene solutions between 2 and 5 µM exposed to red laser light at 4-8 J/cm2 generated significantly more singlet oxygen compared to the no guaiazulene group (p < 0.01) and reduced RANTES and PGE2 levels in TNF-α-inflamed peripheral blood mononuclear cells without affecting cell viability. CONCLUSION: Photodynamic activation of guaiazulene generated singlet oxygen and suppressed inflammatory markers in PBMCs.

MRI imaging and histopathological study of brain iron overload of ß-thalassemic mice.

Brain iron overload is chronic and slow progressing and plays an important role in the pathogenesis of neurodegenerative disorders. Magnetic resonance imaging (MRI) is a useful noninvasive tool for determining liver iron content, but it has not been proven to be adequate for evaluating brain iron overload. We evaluated the usefulness of MRI-derived parameters to determine brain iron concentration in ß-thalassemic mice and the effects of the membrane permeable iron chelator, deferiprone. Sixteen ß-thalassemic mice underwent 1.5T MRI of the brain that included a multiecho T2*-weighted sequence. Brain T2* values ranged from 28 to 31ms for thalassemic mice. For the iron overloaded thalassemic mice, brain T2* values decreased, ranging from 8 to 12ms, which correlated with the iron overload status of the animals. In addition, brain T2* values increased in the group with the treatment of deferiprone, ranging from 18 to 24ms. Our results may be useful to understand brain pathology in iron overload. Moreover, data could lead to an earlier diagnosis, assist in following disease progression, and demonstrate the benefits of iron chelation therapy.

Photodynamic therapy of Curcuma longa extract stimulated with blue light against Aggregatibacter actinomycetemcomitans.

BACKGROUND AND OBJECTIVE: Curcumin, one of an established curcuminoid substances extracted from Curcuma longa, has been used as a photosensitizer (PS) in photodynamic therapy (PDT). Curcuminoid substances has been reported to have benefits in treating dental chronic infection and inflammation diseases, such as chronic periodontitis. The purpose of this study was to find the optimum concentration of Curcuma longa (CL) extract, containing all curcuminoid substances, and the power density of blue light (BL) in photodynamic therapy against periodontally pathogenic bacteria, A. actinomycetemcomitans. METHODS: Antibacterial activity of various concentrations of CL extract against A. actinomycetemcomitans was determined. Exponentially growing bacteria were combined with 2-fold dilution of CL extract solution ranging from 25 to 0.098 µg/ml. Co-culture bacteria treated with 0.12% chlorhexidine (CHX) served as the positive control. The effect of photostimulation with light emitting diode (LED) 420-480 nm at 16.8 J/cm2 for 1 min on the selected concentration of CL extract was examined. Bacteria viability was determined by plate counting technique. In addition, production of free radicals was tested by electron spin resonance spectroscope (ESR) with 5,5-dimethyl-1-pyrroline N-oxide (DMPO). RESULTS: The antibacterial activity of CL extract was dose dependent. Without BL, 25 µg/ml CL extract showed 6.03 ±â€¯0.39 log10A. actinomycetemcomitans. Interestingly, the combination of BL and 0.78 µg/ml CL extract solution showed complete absence of A. actinomycetemcomitans. Peak signal intensity of hydroxyl radical production was also detected with the combination of BL and CL. CONCLUSIONS: CL extract not only had antimicrobial activity but also could be used as an effective PS when stimulated with BL in PDT. The optimal antibacterial effect of CL extract with BL was equal to the standard oral disinfectant, 0.12% CHX.

Iron distribution and histopathological study of the effects of deferoxamine and deferiprone in the kidneys of iron overloaded ß-thalassemic mice.

Renal glomerular and tubular dysfunctions have been reported with high prevalence in ß-thalassemia. Iron toxicity is implicated in the kidney damage, which may be reversed by iron chelation therapy. To mimic heavy iron overload and evaluate the efficacy of iron chelators in the patients, iron dextran (180mg iron/mouse) was intraperitoneally (i.p.) injected in heterozygous ß-globin knockout mice ((mußth-3/+), BKO) and wild type mice (C57BL/6J, WT) over a period of 2 weeks, followed by daily i.p. injection of deferoxamine (DFO) or deferiprone (L1) for 1 week. In BKO mice, iron preferentially accumulated in the proximal tubule with a grading score of 0-1 and increased to grade 3 after iron loading. In contrast, iron mainly deposited in the glomerulus and interstitial space in iron overloaded WT mice. Increased levels of kidney lipid peroxidation, glomerular and medullar damage and fibrosis in iron overloaded mice were reversed by treatment with iron chelators. L1 showed higher efficacy than DFO in reduction of glomerular iron, which was supported by a significantly decreased the amount of glomerular damage. Notably, DFO and L1 demonstrated a distinct pattern of iron distribution in the proximal tubule of BKO mice. In conclusion, chelation therapy has beneficial effects in iron-overloaded kidneys. However, the defect of kidney iron metabolism in thalassemia may be a determining factor of the treatment outcome in individual patients.

Morus alba L. Stem Extract Attenuates Pain and Articular Cartilage Damage in the Anterior Cruciate Ligament Transection-Induced Rat Model of Osteoarthritis.

AIM: This study was designed to investigate the anti-nociceptive effect of Morus alba stem extract as well as its cartilage protective effect in the anterior cruciate ligament transection (ACLT)-induced rat model of osteoarthritis (OA). METHODS: The anti-nociceptive effect of this plant extract was determined by measuring hind limb weight bearing, while the severity of cartilage damage to the knee joints was evaluated using the modified Mankin grading system. RESULTS: Oral administration of M. alba stem extract (56 and 560 mg/kg) significantly attenuated joint pain as indicated by a significant (p < 0.05) increase in the values of percent weight borne on the operated hind limb for the OA-induced groups that received M. alba stem extract at 56 and 560 mg/kg when compared to those of the vehicle-treated OA-induced group. In addition, a significant improvement in the Mankin score was also observed in rats treated with 560 mg/kg M. alba stem extract, which was in agreement with its pain-relieving effect. CONCLUSION: The results showed that M. alba stem extract exhibited an anti-nociceptive effect as well as cartilage protection in the ACLT-induced rat model of OA, supporting its potential use as a therapeutic treatment for OA.

Effects of Iron Chelators on Pulmonary Iron Overload and Oxidative Stress in ß-Thalassemic Mice.

AIM: To evaluate the effect of iron chelators on iron-related pulmonary pathology and oxidative stress in an animal model of ß-thalassemia. METHODS: Pulmonary iron overload was induced in heterozygous ß-globin knockout mice (mußth-3/+, BKO). Over a period of 2 weeks, 180 mg of iron/mouse was loaded by intraperitoneal injection of iron dextran, and subsequently treated daily via intraperitoneal with either deferoxamine (DF) or deferiprone (L1) at an equimolar concentration of iron binding (0.2 and 0.6 µmol/g body weight, respectively) for 7 days. RESULTS: Iron loading resulted in iron deposition in peribronchial regions, septa and also in alveolar macrophages with a grading score of 3. This iron burden resulted in lung epithelial injuries, fibrosis and corresponded with increased lipid peroxidation and decreased tissue catalase activity. Treatment with DF or L1 resulted in a reduction of iron-laden alveolar macrophages and decreased oxidative stress and tissue damage, showing the iron mobilizing ability of both compounds. CONCLUSION: Iron chelation therapy, with DF and L1, may protect against pulmonary damage by sequestering catalytic iron and improving oxidative status. It may be beneficial in the prevention of pulmonary complications in thalassemia.

Iron distribution and histopathological characterization of the liver and heart of ß-thalassemic mice with parenteral iron overload: Effects of deferoxamine and deferiprone.

The liver and heart are the major target organs for iron accumulation and iron toxicity in ß-thalassemia. To mimic the phenomenon of heavy iron overload resulting from repeated blood transfusions, a total of 180 mg of iron dextran was intraperitoneally injected into C57BL/6J mice (WT) and heterozygous ß-globin knockout mice ((mu)ß(th-3/+), BKO). The effects of deferiprone and deferoxamine in this model were investigated. The iron was distributed homogenously throughout the 4 liver lobes (left, caudate, right and median) and was present in hepatocytes, Kupffer cells and the sinusoidal space. Iron accumulation in phagocytic macrophages, recruitment of hepatic lymphocytes and nucleus membrane degeneration were observed as a result of iron overload in the WT and BKO mice. However, the expansion of hepatic extramedullary hematopoiesis was observed only in the BKO mice with iron overload. In the heart, the iron accumulated in the cardiac interstitium and myocytes, and moderate hypertrophy of the myocardial fibers and cardiac myocyte degeneration were observed. Although the total liver iron was not significantly altered by iron chelation therapy, image analysis demonstrated a difference in the efficacies of two iron chelators. The major site of chelation was the extracellular compartment, but treatment with deferiprone also resulted in intracellular iron chelation. Interestingly, iron chelators reversed the pathological changes resulting from iron overload in WT and BKO mice despite being used for only a short treatment period. We suggest that some of these effects may be secondary to the anti-inflammatory activity of the chelators.

Curcumin I mediates neuroprotective effect through attenuation of quinoprotein formation, p-p38 MAPK expression, and caspase-3 activation in 6-hydroxydopamine treated SH-SY5Y cells.

6-Hydroxydopamine (6-OHDA) selectively enters dopaminergic neurons and undergoes auto-oxidation resulting in the generation of reactive oxygen species and dopamine quinones, subsequently leading to apoptosis. This mechanism mimics the pathogenesis of Parkinson's disease and has been used to induce experimental Parkinsonism in both in vitro and in vivo systems. In this study, we investigated the effects of curcumin I (diferuloylmethane) purified from Curcuma longa on quinoprotein production, phosphorylation of p38 MAPK (p-p38), and caspase-3 activation in 6-OHDA-treated SH-SY5Y dopaminergic cells. Pretreatment of SH-SY5Y with curcumin I at concentrations of 1, 5, 10, and 20 µM, significantly decreased the formation of quinoprotein and reduced the levels of p-p38 and cleaved caspase-3 in a dose-dependent manner. Moreover, the levels of the dopaminergic neuron marker, phospho-tyrosine hydroxylase (p-TH), were also dose-dependently increased upon treatment with curcumin I. Our results clearly demonstrated that curcumin I protects neurons against oxidative damage, as shown by attenuation of p-p38 expression, caspase-3-activation, and toxic quinoprotein formation, together with the restoration of p-TH levels. This study provides evidence for the therapeutic potential of curcumin I in the chemoprevention of oxidative stress-related neurodegeneration.

Comparison of pharmacokinetics and urinary iron excretion of two single doses of deferiprone in ß-thalassemia/hemoglobin E patients.

Dose-related pharmacokinetics and urinary iron excretion (UIE) of an orally active iron chelator, deferiprone (L1), was investigated in 12 severe ß-thalassemia/hemoglobin E patients. The patients received two single doses of 25 and 50 mg/kg with a 2-week washout period. Deferiprone was rapidly absorbed and reached maximum concentration (C(max)) within 1 h after administration. Pharmacokinetic parameters including C(max) and area under concentration time curve from time zero to infinity (AUC(0-∞)) as well as urinary excretion of non-conjugated and glucuronide-conjugated deferiprone (L1 and L1-G) increased proportionally with the dose of deferiprone. A constant ratio of AUC(0-∞) of L1-G to L1 and a percentage of urinary excretion of L1-G indicated that increasing the dosage does not influence deferiprone biotransformation. Longer terminal elimination half-lifeand higher volume of distribution of L1 were observed with the high dose and correlated with deferiprone-chelated iron in serum. Unexpectedly, UIE did not show a linear relationship with the increased dose of deferiprone. The correlation between UIE and creatinine clearance suggested the possibility of L1-iron complex redistribution in patients with renal impairment treated with high-dose deferiprone.

Hepatic reduction of carbamoyl-PROXYL in ferric nitrilotriacetate induced iron overloaded mice: an in vivo ESR study.

Reduction of a nitroxyl radical, carbamoyl-PROXYL in association of free radical production and hepatic glutathione (GSH) was investigated in iron overloaded mice using an in vivo L-band electron spin resonance (ESR) spectrometer. Significant increases in hepatic iron, lipid peroxidation and decrease in hepatic GSH were observed in mice intraperitoneally (i.p.) administrated with ferric nitrilotriacetate (Fe(III)-NTA, a total 45 µmol/mouse over a period of 3 weeks). Free radical production in iron overloaded mice was evidenced by significantly enhanced rate constant of ESR signal decay of carbamoyl-PROXYL, which was slightly reduced by treatment with iron chelator, deferoxamine. Moreover, the rate constant of ESR signal decay was negatively correlated with hepatic GSH level (r=-0.586, p<0.001). On the other hand, hepatic GSH-depletion (>80%) in mice through daily i.p. injection and drinking water supplementation of L-buthionine-[S,R]-sulfoximine (BSO) significantly retarded ESR signal decay, while there were no changes in serum aspartate aminotransferase and liver thiobarbituric acid-reactive substances levels. In conclusion, GSH plays two distinguish roles on ESR signal decay of carbamoyl-PROXYL, as an antioxidant and as a reducing agent, dependently on its concentration. Therefore, it should be taken into account in the interpretation of free radical production in each specific experimental setting.

Vitamin E supplement improves erythrocyte membrane fluidity of thalassemia: an ESR spin labeling study.

BACKGROUND: Beta-thalassemia/Hemoglobin E (beta-thal/Hb E) is prevalent in Thailand. The imbalance of globin chains in red blood cells is the primary cause of this anemic disease. The excess alpha-globin in beta-thal/Hb E causes typical damage(s) to membrane of erythroblasts and erythrocytes. By using three paramagnetic labeled compounds (5-, 12-, and 16-spin labeled stearic acids, SLS), the changes of the molecular motion in the lipid bilayer of thalassemic RBCs that have structural modification can be detected. OBJECTIVE: to investigate erythrocyte membrane fluidity and the effect of vitamin E treatment in beta-thalassemia/Hemoglobin E patients by using spin labeling techniques. MATERIAL AND METHOD: The erythrocyte membrane fluidity was investigated in nine splenectomized and five non-splenectomized beta-thalassemia/hemoglobin E (beta-thal/Hb E) patients using EPR spin labeling techniques. To determine the effect of vitamin E on erythrocyte membrane fluidity, only the splenectomized patients were enrolled. Patients were divided into two groups. The first group received 350 mg vitamin E daily for a period of 1 month (n = 5) and the second group received placebo for an equal period (n = 4). Three paramagnetic fatty acid, 5-, 12-, and 16-doxyl stearic acids, (5-, 12- and 16-DS) were used to label phospholipids layer near both the surface (5-DS) and the deeper hydrophobic region of membrane (12-and 16-DS). Lipid peroxidation (TBARs) was measured using a colorimetric method. Vitamin E was measured with high performance liquid chromatography (HPLC). RESULTS: Significantly higher values of erythrocyte membrane fluidity were revealed with 12-, 16-DS in splenectomized patients, as compared with non-splenectomized patients and normal subjects. In 3-thal/Hb E patients, fluidity values, both outer hyperfine splitting (2T(//)) and order parameter (S) of 12-DS showed inverse correlation with serum TBARs. There was no significant difference between the fluidity values measured with 5-DS. After vitamin E supplementation, the erythrocyte membrane fluidity was decreased in almost all patients. In contrast to the vitamin E supplementation group, increased erythrocyte membrane fluidity was demonstrated in the placebo group. Vitamin E supplementation also had effect on other clinical parameters such as increased plasma vitamin E, decreased serum TBARs and no change in hemoglobin. CONCLUSION: The present results suggested the abnormal motion of lipid in the deeper phospholipids region of membrane. In addition, vitamin E supplementation may have a role in the prevention of erythrocyte membrane damage of these patients.

The in vitro and ex vivo antioxidant properties, hypolipidaemic and antiatherosclerotic activities of water extract of Moringa oleifera Lam. leaves.

UNLABELLED: Moringa oleifera is used in Thai traditional medicine as cardiotonic. Recent studies demonstrated its hypocholesterolaemic effect. However, to be clinically useful, more scientific data are needed. AIM OF THE STUDY: We investigated the antioxidant, hypolipidaemic and antiatherosclerotic activities of Moringa oleifera leaf extract. MATERIALS AND METHODS: Scavenging activity of the extract on 1,1-diphenyl-2-picrylhydrazyl radicals (DPPH), and the inhibitory effect on Cu(2+)-induced low-density lipoprotein (LDL) oxidation were determined in in vitro experiment. The effects of the extract on cholesterol levels, conjugated diene (CD) and thiobarbituric acid reactive substances (TBARS) and plaque formations in cholesterol-fed rabbits were investigated. RESULTS: We found that in scavenging DPPH radicals the extract and Trolox had IC(50) of 78.15+/-0.92 and 2.14+/-0.12microg/ml, respectively. The extract significantly (P<0.05) prolonged the lag-time of CD formation and inhibited TBARS formation in both in vitro and ex vivo experiments in a dose-dependent manner. In hypercholesterol-fed rabbits, at 12 weeks of treatment, it significantly (P<0.05) lowered the cholesterol levels and reduced the atherosclerotic plaque formation to about 50 and 86%, respectively. These effects were at degrees comparable to those of simvastatin. CONCLUSIONS: The results indicate that this plant possesses antioxidant, hypolipidaemic and antiatherosclerotic activities and has therapeutic potential for the prevention of cardiovascular diseases.

Hypocholesterolemic and antioxidant effects of aqueous extracts from the dried calyx of Hibiscus sabdariffa L. in hypercholesterolemic rats.

The present study was designed to investigate the hypolipidemic effects and antioxidant effects of Hibiscus sabdariffa L. (roselle) with regard to protection of LDL oxidation in vivo and ex vivo in rats made hypercholesterolemic by continuous cholesterol feeding. Administering the dried calyx extracts of roselle at doses of 500 and 1,000 mg/kg together with continuous cholesterol feeding to hypercholesterolemic rats for 6 weeks significantly decreased serum cholesterol level by 22% and 26%, respectively (p<0.001); serum triglycerides level by 33% and 28%, respectively (p<0.05); serum LDL level by 22% and 32%, respectively (p<0.05). However, serum HDL level was not affected. LDL was extracted from plasma of the hypercholesterolemic rats and the effects of the dried calyx extracts of roselle on the oxidation of LDL in vivo and ex vivo were examined. Six-week treatment with 250, 500 and 1,000 mg/kg of the extracts significantly decreased thiobarbituric acid reactive substances (TBARs) formation (p<0.05) while the formation of conjugated dienes during the oxidation of LDL induced by CuSO(4) was reduced, but not significantly different. These lines of evidence suggest that the aqueous extracts from the dried calyx of roselle possess both antioxidant effects against LDL oxidation and hypolipidemic effects in vivo. However, its mechanism(s) of action remains to be elucidated.

Antioxidant effects of aqueous extracts from dried calyx of Hibiscus sabdariffa Linn. (Roselle) in vitro using rat low-density lipoprotein (LDL).

The present study quantitatively investigated the antioxidant effects of the aqueous extracts from dried calyx of Hibiscus sabdariffa LINN. (roselle) in vitro using rat low-density lipoprotein (LDL). Formations of the conjugated dienes and thiobarbituric acid reactive substances (TBARs) were monitored as markers of the early and later stages of the oxidation of LDL, respectively. Thus, we demonstrated that the dried calyx extracts of roselle exhibits strong antioxidant activity in Cu(2+)-mediated oxidation of LDL (p<0.05) in vitro. The inhibitory effect of the extracts on LDL oxidation was dose-dependent at concentrations ranging from 0.1 to 5 mg/ml. Moreover, 5 mg/ml of roselle inhibited TBARs-formation with greater potency than 100 microM of vitamin E. In conclusion, this study provides a quantitative insight into the potent antioxidant effect of roselle in vitro.