Lipid Peroxidation, Reduced Glutathione, and Glutathione Peroxidase Levels in Intervertebral Discs of Patients with Lumbar Degenerative Disc Disease.

BACKGROUND Either a reduction in antioxidant levels or an accumulation of reactive oxygen species can heighten susceptibility to oxidative damage in disc cells. To date, no research has investigated the levels of lipid peroxidation products (thiobarbituric acid reactive substances [TBARs]), reduced glutathione (GSH), and glutathione peroxidase (GPx) in excised human lumbar disc tissues affected by degenerative disease. Therefore, this study aimed to evaluate lipid peroxidation products in excised disc tissues from patients with degenerative disc disease. MATERIAL AND METHODS Forty-two patients were enrolled. Patients were divided into lumbar disc degeneration (LDD) and nonlumbar disc degeneration (nonLDD) groups according to Pfirrmann classification. Intervertebral discs were obtained from all patients during the operation and were homogenized for analysis. TBARs levels were measured using fluorometry. GSH levels and GPx activity were quantified spectrophotometrically using a kinetic method. RESULTS TBARs levels in excised discs from LDD patients (5.18±4.14) were significantly higher than those from nonLDD patients (2.56±1.23, P=0.008). The levels of TBARs tended to increase with the severity of degeneration according to the Pfirrmann classification. However, these 2 groups showed no significant differences in reduced glutathione levels or glutathione peroxidase activity (P>0.05). Patients with LDD exhibited a worse health-related quality of life, reflected in lower utility and EQ-VAS scores and higher Oswestry disability index scores. CONCLUSIONS There was a notable increase in lipid peroxidation products in the excised intervertebral discs of patients with LDD. This finding suggests that oxidative stress may contribute to the development of disc degeneration.

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.

Antiplatelet activity of deferiprone through cyclooxygenase-1 inhibition.

Thalassemia patients are susceptible to both iron overload and thromboembolism. Deferiprone is an iron chelator that shows an antiplatelet activity and thus may alleviate platelet hyperactivation in thalassemia. Therefore, this study aimed to characterize the inhibitory effects and mechanisms of deferiprone on normal human platelets. The results illustrated that deferiprone inhibited platelet aggregation at the iron chelating concentrations (0.08-0.25 mmol/l). Deferiprone inhibited human platelet aggregation stimulated by arachidonic acid and ADP more potently than epinephrine and collagen, with the IC50 of 0.24 mmol/l and 0.25 mmol/l vs. 3.36 mmol/l and 3.73 mmol/l, respectively. Interestingly, deferiprone significantly inhibited COX-1 activity, with the IC50 of 0.33 mmol/l, and slightly increased cAMP level at the high concentration of 4 mmol/l. Moreover, the results from molecular docking showed that deferiprone interacted closely with key residues in the peroxidase active site of COX-1. These results suggested that deferiprone possessed antiplatelet activity mainly through the inhibition of COX-1 activity.

Microparticles from splenectomized ß-thalassemia/HbE patients play roles on procoagulant activities with thrombotic potential.

Thromboembolic events including cerebral thrombosis, deep vein thrombosis, and pulmonary embolism are major complications in ß-thalassemia. Damaged red blood cells and chronic platelet activation in splenectomized ß-thalassemia/HbE patients were associated with increased microparticles (MPs) releases into blood circulation. MPs are small membrane vesicles, which play important roles on coagulation. However, the role of MP in thalassemia is poorly understood. In this study, the effects of splenectomized-MPs on platelet activation and aggregation were investigated. The results showed that isolated MPs from fresh platelet-free plasma of patients and normal subjects directly induce platelet activation, platelet aggregation, and platelet-neutrophil aggregation in a dose-dependent manner. Interestingly, MPs obtained from splenectomized patients are more efficient in induction of platelet activation (P-selectin+) when compared to MPs from normal subjects (P < 0.05), tenfold lower than pathophysiological level, at 1:0.1 platelet MP ratio. Co-incubation of splenectomized-MPs with either normal-, non-splenectomized- or splenectomized-platelets at 1:10 platelet MP ratio increased platelet activation up to 5.1 ± 2.2, 5.6 ± 3.7, and 9.5 ± 3.0%, respectively, when normalized with individual baseline. These findings suggest that splenectomized patients were proned to be activated by MPs, and splenectomized-MPs could play an important role on chronic platelet activation and aggregation, leading to thrombus formation in ß-thalassemia/HbE 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.

Electron Paramagnetic Resonance Study of the Free Radical Scavenging Capacity of Curcumin and Its Demethoxy and Hydrogenated Derivatives.

The quantitative free radical scavenging capacity of curcumin and its demethoxy derivatives (demethoxycurcumin (Dmc) and bisdemethoxycurcumin (Bdmc)) and hydrogenated derivatives (tetrahydrocurcumin (THC), hexahydrocurcumin (HHC) and octahydrocurcumin (OHC)) towards 1,1-diphenyl-2-picryl hydrazyl (DPPH), nitric oxide radical (NO), hydroxyl radical (HO(·)) and superoxide anion radical (O2(·)) were investigated by electron paramagnetic resonance (EPR) spectroscopy. One mole of the hydrogenated derivatives scavenged about 4 mol of the DPPH radical, while curcumin and Dmc scavenged about 3 mol of the DPPH radical. Curcumin and THC showed moderate scavenging activity towards NO, yielding 200 mmol of NO scavenged per 1 mol of the scavenger. In contrast, curcumin and its derivatives showed very low scavenging activity towards HO(·) and O2(·), yielding approximately only 3-12 mmol scavenged per 1 mol of the tested compounds. Our results suggest that curcumin and its derivatives principally act as chain breaking antioxidants rather than as direct free radical scavengers. Furthermore, we showed that the ortho-methoxyphenolic group and the heptadione linkage of these molecules greatly contributed to their DPPH and NO scavenging activity.

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.

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.

Flavonoid Metabolites in Serum and Urine after the Ingestion of Selected Tropical Fruits.

The serum concentration and urinary excretion of flavonoids after the ingestion of guava, pineapple, and pomelo were determined using liquid chromatography-mass spectroscopy (LC-MS/MS). Each group of healthy volunteers was given 200 g of fresh fruit after overnight fasting and a 24-h flavonoid-free diet. The results demonstrate that only the glucuronic-conjugated metabolites of luteolin, quercetin, kaempferol, and myricetin were detected after fruit ingestion. The metabolites were first detected after 2 h, with the time to maximum concentration (Tmax) at 6 h. The most abundant metabolites for guava, pineapple, and pomelo were the glucuronide metabolites of quercetin (AUC0-8 5.4 ± 1.3 µg·h/mL), kaempferol (AUC0-8 9.9 ± 2.3 µg·h/mL), and luteolin (AUC0-8 6.4 ± 1.1 µg·h/mL), respectively. The flavonoids found in the 24-h urinary excretions were glucuronic- and mainly sulfate-conjugated metabolites. Quercetin metabolites were the most abundant after guava and pineapple ingestion, accounting for 900 and 700 µg, respectively. Luteolin metabolites were the most abundant after pomelo ingestion, accounting for 450 µg. The serum and urinary metabolite profiles suggested that guava and pineapple are good sources of quercetin, pineapple is a good source of kaempferol, and pomelo is a good source of luteolin. The study of flavonoid profiles may provide information for the selection of fruits as functional foods for their health benefits to help with various health conditions.

Functional Roles of Furin in Cardio-Cerebrovascular Diseases.

Furin plays a major role in post-translational modification of several biomolecules, including endogenous hormones, growth factors, and cytokines. Recent reports have demonstrated the association of furin and cardio-cerebrovascular diseases (CVDs) in humans. This review describes the possible pathogenic contribution of furin and its substrates in CVDs. Early-stage hypertension and diabetes mellitus show a negative correlation with furin. A reduction in furin might promote hypertension by decreasing maturation of B-type natriuretic peptide (BNP) or by decreasing shedding of membrane (pro)renin receptor (PRR), which facilitates activation of the renin-angiotensin-aldosterone system (RAAS). In diabetes, furin downregulation potentially leads to insulin resistance by reducing maturation of the insulin receptor. In contrast, the progression of other CVDs is associated with an increase in furin, including dyslipidemia, atherosclerosis, ischemic stroke, myocardial infarction (MI), and heart failure. Upregulation of furin might promote maturation of membrane type 1-matrix metalloproteinase (MT1-MMP), which cleaves low-density lipoprotein receptor (LDLR), contributing to dyslipidemia. In atherosclerosis, elevated levels of furin possibly enhance maturation of several substrates related to inflammation, cell proliferation, and extracellular matrix (ECM) deposition and degradation. Neuronal cell death following ischemic stroke has also been shown to involve furin substrates (e.g., MT1-MMP, hepcidin, and hemojuvelin). Moreover, furin and its substrates, including tumor necrosis factor-α (TNF-α), endothelin-1 (ET-1), and transforming growth factor-ß1 (TGF-ß1), are capable of mediating inflammation, hypertrophy, and fibrosis in MI and heart failure. Taken together, this evidence provides functional significance of furin in CVDs and might suggest a potential novel therapeutic modality for the management of CVDs.

Naturally occurring quercetin and myricetin as potent inhibitors for human ectonucleotide pyrophosphatase/phosphodiesterase 1.

Ecto-nucleotide pyrophosphatases/phosphodiesterases 1 (ENPP1) is a key enzyme in purinergic signaling pathways responsible for cell-to-cell communications and regulation of several fundamental pathophysiological processes. In this study, Kyoto Green, a rapid chemical sensor of pyrophosphate, was employed to screen for effective ENPP1 inhibitors among five representative flavonoids (quercetin, myricetin, morin, kaempferol, and quercetin-3-glucoside), five nucleosides (adenosine, guanosine, inosine, uridine, and cytidine), and five deoxynucleosides (2'- and 3'-deoxyadenosine, 2'-deoxyguanosine, 2'-deoxyinosine, and 2'-deoxyuridine). Conventional colorimetric, fluorescence, and bioluminescence assays revealed that ENPP1 was effectively inhibited by quercetin (Ki ~ 4 nM) and myricetin (Ki ~ 32 nM) when ATP was used as a substrate at pH 7.4. In silico analysis indicated that the presence of a chromone scaffold, particularly one containing a hydroxyl group at the 3' position on the B ring, may promote binding to the active site pocket of ENPP1 and enhance inhibition. This study demonstrated that the naturally derived quercetin and myricetin could effectively inhibit ENPP1 enzymatic activity and may offer health benefits in arthritis management.

Dasatinib Ointment Promotes Healing of Murine Excisional Skin Wound.

Dasatinib, a tyrosine kinase inhibitor, has been shown to produce anti-inflammatory activity and impair vascular integrity in vivo, including during skin wound healing, potentially promoting the repair process. Given that dasatinib is a lipophilic small molecule capable of penetrating skin, topical dasatinib might provide benefits in wound healing. In the present study, we investigated the impact of dasatinib ointments in skin wound healing in mice. A full thickness excisional skin wound (4 mm diameter) was generated on the shaved dorsum of eight-week-old C57BL/6 mice. Dasatinib ointment (0.1 or 0.2% w/w) or ointment base was applied twice daily (every 12 h) for 10 days. Elizabethan collars were used to prevent animal licking. The wound size was monitored daily for 14 days. The results showed that dasatinib ointments, particularly 0.1% dasatinib, promoted a 16-23% reduction in wound size (p < 0.05) during day 2 to day 6 postinjury compared to controls. Immunohistochemistry analyses demonstrated a reduction in wound neutrophils (38% reduction, p = 0.04), macrophages (47% reduction, p = 0.005), and tumor necrosis factor-α levels (73% reduction, p < 0.01), together with an induction of vascular leakage-mediated fibrin(ogen) accumulation (2.5-fold increase, p < 0.01) in the wound during day 3 postinjury (an early phase of repair) in 0.1% dasatinib-treated mice relative to control mice. The anti-inflammatory and vascular hyperpermeability activities of dasatinib were associated with an enhanced healing process, including increased keratinocyte proliferation (1.8-fold increase in Ki67+ cells, p < 0.05) and augmented angiogenesis (1.7-fold increase in CD31+ area, p < 0.05), compared to the ointment base-treated group. Following treatment with 0.2% dasatinib ointment, minor wound bleeding and scab reformation were observed during the late phase, which contributed to delayed healing. In conclusion, our data suggest that dasatinib ointment, mainly at 0.1%, promotes the repair process by reducing inflammation and producing a local and temporal vascular leakage, leading to an increase in fibrin(ogen) deposition, re-epithelialization, and angiogenesis. Therefore, topical dasatinib might be a potential novel candidate to facilitate skin wound healing.

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.

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.

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.

Sustained AT1R stimulation induces upregulation of growth factors in human cardiac fibroblasts via Gαq/TGF-ß/ERK signaling that influences myocyte hypertrophy.

Stimulation of angiotensin II receptor (ATR) with angiotensin II (Ang II) accelerates cardiac fibroblast activation, resulting in upregulation of cytokines and growth factors. Growth factors were strongly upregulated in animal models of myocardial fibrosis and hypertrophy as well as patients with heart failure. Nevertheless, the signal transduction of ATR for upregulation of growth factors in human cardiac fibroblasts contributing to myocyte hypertrophy have not fully understood. Long-term Ang II treatment of human cardiac fibroblasts provokes the synthesis and secretion of connective tissue growth factor (CTGF), transforming growth factor beta1 (TGF-ß1), and vascular endothelial growth factor (VEGF) through the AT1R subtype. Blockade of Gαq, not Gαi or Gα12/13, protein signaling inhibited AT1R-mediated upregulation of CTGF, TGF-ß1, and VEGF. In addition, AT1R overstimulation induced upregulation of growth factors via the TGF-ß-dependent and ERK1/2-dependent pathways. Growth factors secreted from cardiac fibroblasts are necessary for the induction of hypertrophic markers, atrial natriuretic peptide (ANP) and ß-myosin heavy chain (ß-MHC), resulting in myocyte hypertrophy. Candesartan, irbesartan, and valsartan had greater effects than losartan for blockade of fibrotic and hypertrophic effects of Ang II. Our data support the concept whereby sustained AT1R stimulation contributes to the development of myocardial fibrosis and hypertrophy, and advances understanding of this complex AT1R signaling, including fibroblasts-myocytes communication during pathological conditions.

Deferiprone, an iron chelator, alleviates platelet hyperactivity in patients with ß-thalassaemia/HbE.

Background: Hyperfunctional platelets play important roles in thromboembolism in patients with ß-thalassaemia/ haemoglobin E (ß-thal/HbE). Our previous study revealed ex vivo inhibitory effects of deferiprone on normal platelets. Herein, we aimed to investigate the in vivo effects on platelets in patients with ß-thal/HbE. Methods: A prospective, self-controlled clinical study on 30 patients with ß-thal/HbE who had received therapeutic deferiprone (20.8-94.5 mg/kg/day) was conducted. The study included a 4-week washout period followed by 4 and 12 weeks of deferiprone treatment. Platelet aggregation was performed by a turbidimetric method. Levels of deferiprone and soluble platelet (sP)-selectin in serum were measured by high-performance liquid chromatography (HPLC) and enzyme-linked immunosorbent assay (ELISA) kit, respectively. Results: The washout period significantly enhanced platelet hyperactivity both in patients who had undergone splenectomy and in those who had not. At 2 hours following the administration of a single dose of deferiprone, platelet sensitivity to ADP and arachidonic acid was significantly reduced. The inhibitory effects of deferiprone were gradually increased over the period of 4 and 12 weeks. Deferiprone also depressed sP-selectin levels, but the effect was stable over longer follow-up periods. Correlation analysis demonstrated the relationship between serum levels of deferiprone, sP-selectin, and platelet activities induced by ADP and arachidonic acid. Conclusion: We first demonstrated the in vivo antiplatelet effect and benefit of short-term treatment of deferiprone in patients with ß-thal/HbE. The impact on thrombotic outcomes deserves further study.

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.

Kinetics of lipid radical formation in lipoproteins from ß-thalassemia: Implication of cholesteryl esters and α-tocopherol.

Vascular complications in ß-thalassemia are associated with oxidative modification of lipoproteins under high oxidative stress. The lipid components of lipoproteins are oxidized via lipid peroxidation and produce lipid radicals (L•) as the key initial intermediates. Modification of lipid components, therefore, might result in alterations in the rate and products of lipid peroxidation. In this study, the kinetics of L• formation during the 2,2'-Azobis(2-amidinopropane) dihydrochloride (AAPH)- and hemin-induced oxidation of low-density and high-density lipoproteins (LDL and HDL) from ß-thalassemia patients and healthy volunteers were investigated using a specific and sensitive fluorescence probe for L•. Kinetic parameters, including initial lag time, propagation rate and total L• production, were calculated by monitoring a fluorescence-active NBD-Pen-L• adduct. Oxidation of thalassemia lipoproteins exhibited a significantly shorter lag time but a slower propagation rate of L• formation when compared with healthy lipoproteins. LDL showed higher resistance to oxidation during the initiation phase but higher L• formation than HDL. Our results indicated that the levels of α-tocopherol determined the initial lag time, whereas the levels of core lipids and cholesteryl esters, especially cholesteryl linoleate (CL), determined the propagation rate and total L• production. The difference in potency of AAPH and hemin supported that hemin preferentially targeted core lipids. Moreover, analysis of 13-hydroxyoctadecadienoic acid cholesteryl ester (13-HODE-CE)/CE ratio indicated that thalassemia lipoproteins have higher susceptibility to oxidation than healthy lipoproteins. In conclusion, our findings suggested that CL and α-tocopherol were implicated in the susceptibility of lipoproteins to lipid peroxidation in physiological and pathological conditions of ß-thalassemia.

Antioxidative systems defense against oxidative stress induced by blood meal in Aedes aegypti.

The release of iron from hemoglobin via the digestion of a blood meal in female mosquitoes can potentially induce oxidative damage and even death. These mosquitoes need an effective antioxidant to prevent this. We carried out this study to determine the antioxidant activities of ferritin, glutathione peroxidase (GPx), glutathione S-transferase (GST) and catalase, and glutathione (GSH). These enzymes had their greatest activity among 4 day old virgin female mosquitoes. Using a single blood feed model, groups of female mosquitoes were tested at 4, 7 and 20 days post-emergence. They were allowed to feed on a hamster for 1 hour. The engorged mosquitoes were collected at 48 and 72 hours after their blood meal. There were no changes in GSH, GPx, GST or catalase levels, but ferritin levels increased markedly (about 2-3 fold) by 48 hours post blood-feed in all mosquito age groups. On repeated blood-feed experiments, mosquitoes aged 4 days were blood fed, once every 3 days and were collected 48 hours after their most recent blood meal. A significant decrease in GSH and GPx activity and a further increase in ferritin, were detected. Ferritin levels were 0.19+/-0.03 and 0.14+/-0.02 ng/microg protein in the repeat and single blood-feed groups, respectively. These results suggest ferritin is an inducible, sensitive defense system protecting against oxidative stress caused by iron derived from blood meals in Aedes aegypti mosquitoes.