Effects of hydroxyurea on cytotoxicity, inflammation and oxidative stress markers in neutrophils of patients with sickle cell anemia: dose-effect relationship

ABSTRACT Introduction: Although the efficacy of hydroxyurea (HU) in inhibiting erythrocyte sickling has been well demonstrated, the action of this drug on human neutrophils and the mechanism by which it improves the manifestations of the disease have not been studied thoroughly. We aimed to investigate the cell viability, along with inflammatory and oxidative markers in the neutrophils of sickle cell anemia (SCA) patients and the effects of HU therapy on these cells, by evaluating the dose-responsiveness. Methods: In the present study, 101 patients (45 men and 56 women, aged 18-69 years) with SCA were divided into groups according to the use or not of HU: the SS group (without HU treatment, n = 47) and the SSHU group (under HU treatment, n = 54). The SSHU group was further stratified into subgroups according to the daily dose of the drug that patients already used: SSHU - 0.5 g (n = 19); SSHU - 1 g (n = 26) and SSHU - 1.5-2 g (n = 9). A control group (AA) comprised 50 healthy individuals. Neutrophils isolated from whole blood were analyzed using Trypan Blue, monoiodotyrosine (MTT) and lactate dehydrogenase (LDH) toxicity assays. Myeloperoxidase (MPO), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) activities and concentrations of interleukin 10 (IL-10), tumor necrosis factor alpha (TNF-α) and malonaldehyde (MDA) were also measured. Results: Neutrophils from SCA patients showed membrane fragility and a significant decrease in cell viability when analyzed by Trypan Blue (p < 0.05), MTT (p < 0.001) and LDH (p = 0.011), compared to the AA group. Levels of inflammatory (MPO, TNF-α, and IL-10) and oxidative markers (SOD, GSH-Px, and MDA) were also altered (p < 0.05) in these cells, showing a significant difference in the SSHU-1g and SSHU - 1.5-2 g groups, compared to the SS group. Treatment with HU reverted the levels of all markers to concentrations similar to those in healthy individuals in a positive dose-effect relationship. Conclusion: The HU did not generate a cytotoxic effect on neutrophils in SCA patients, but it modulated their oxidative and inflammatory mechanisms, promoting cytoprotection with a positive dose-effect.

Effects of hydroxyurea on cytotoxicity, inflammation and oxidative stress markers in neutrophils of patients with sickle cell anemia: dose-effect relationship.

INTRODUCTION: Although the efficacy of hydroxyurea (HU) in inhibiting erythrocyte sickling has been well demonstrated, the action of this drug on human neutrophils and the mechanism by which it improves the manifestations of the disease have not been studied thoroughly. We aimed to investigate the cell viability, along with inflammatory and oxidative markers in the neutrophils of sickle cell anemia (SCA) patients and the effects of HU therapy on these cells, by evaluating the dose-responsiveness. METHODS: In the present study, 101 patients (45 men and 56 women, aged 18-69 years) with SCA were divided into groups according to the use or not of HU: the SS group (without HU treatment, n = 47) and the SSHU group (under HU treatment, n = 54). The SSHU group was further stratified into subgroups according to the daily dose of the drug that patients already used: SSHU - 0.5 g (n = 19); SSHU - 1 g (n = 26) and SSHU - 1.5-2 g (n = 9). A control group (AA) comprised 50 healthy individuals. Neutrophils isolated from whole blood were analyzed using Trypan Blue, monoiodotyrosine (MTT) and lactate dehydrogenase (LDH) toxicity assays. Myeloperoxidase (MPO), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) activities and concentrations of interleukin 10 (IL-10), tumor necrosis factor alpha (TNF-α) and malonaldehyde (MDA) were also measured. RESULTS: Neutrophils from SCA patients showed membrane fragility and a significant decrease in cell viability when analyzed by Trypan Blue (p < 0.05), MTT (p < 0.001) and LDH (p = 0.011), compared to the AA group. Levels of inflammatory (MPO, TNF-α, and IL-10) and oxidative markers (SOD, GSH-Px, and MDA) were also altered (p < 0.05) in these cells, showing a significant difference in the SSHU-1g and SSHU - 1.5-2 g groups, compared to the SS group. Treatment with HU reverted the levels of all markers to concentrations similar to those in healthy individuals in a positive dose-effect relationship. CONCLUSION: The HU did not generate a cytotoxic effect on neutrophils in SCA patients, but it modulated their oxidative and inflammatory mechanisms, promoting cytoprotection with a positive dose-effect.

Gastrointestinal effects of standardized Brazilian phytomedicine (Arthur de Carvalho Drops®) containing Matricaria recutita, Gentiana lutea and Foeniculum vulgare.

Arthur de Carvalho Drops® (ACD) is a traditional Brazilian herbal medicine used to treat functional gastrointestinal disorders (FGIDs). ACD is a formulation of herbal extracts from Matricaria recutita (chamomile), Foeniculum vulgare (fennel) and Gentiana lutea L. (gentian). Considering the popular use for FGIDs, the aim of this work was to investigate the ACD effect on gastric and intestinal parameters with emphasis in a mechanistic approach using isolated duodenal preparations of rodents. Analytical method was developed and validated for quantify three actives principles/markers (Apigenin-7-glucoside, gentiopicroside and anethole) in ACD. The treatment with ACD significantly reduced the emetogenic stimuli induced by cisplatin in rats, showed a laxative effect, reduced the bethanechol-enhanced gastrointestinal transit and completely reversed the contraction induced by carbachol in rat duodenum. However, ACD did not alter the secretory gastric volume or total gastric acidity. The ACD affect the contractions of duodenal smooth muscle mediated by Ca2+ channels and it is also able to inhibit the contractile response mediated by the release from its intracellular store. Furthermore, the relaxant effects of ACD appear independent of the nitric oxide pathway in rat duodenum. These results suggest that ACD could be beneficial for the treatment of disorders of the gastrointestinal tract.

Alendronate-induced gastric damage in normoglycemic and hyperglycemic rats is reversed by metformin.

Alendronate is a bisphosphonate widely used for the treatment of osteoporosis; however, one of its main adverse reactions is gastric ulcer. Metformin is an oral antihyperglycemic agent that has several beneficial effects, including healing, gastroprotective and anti-tumoral action. This study aimed to evaluate the gastroprotective activity of metformin in alendronate-induced gastric damage in normoglycemic and hyperglycemic rats. The treatment with 100 mg/kg of metformin showed a significant gastroprotective effect in damage induced by alendronate (50 mg/kg) in macroscopic analysis and the analysis of light microscopy and atomic force microscopy. The results suggested metformin decreased the inflammatory response by reducing the expression of proinflammatory cytokines (TNF-α, IL-1ß and IL-6), myeloperoxidase activity, and malondialdehyde levels. Also, the results suggested that metformin induces the maintenance of basal levels of collagen and increase the production of mucus. Interestingly, with the presence of the AMPK inhibitor (Compound C), metformin presented impairment of its gastroprotective action. The gastroprotective effect of metformin might be related to the activation of the AMPK pathway. These findings revealed that metformin has a gastroprotective action and may be considered a therapeutic potential for the prevention and treatment of gastric lesions induced by alendronate.

Pharmacognostical Analysis and Protective Effect of Standardized Extract and Rizonic Acid from Erythrina velutina against 6-Hydroxydopamine-Induced Neurotoxicity in SH-SY5Y Cells.

BACKGROUND: Erythrina velutina is a tree common in the northeast of Brazil extensively used by traditional medicine for the treatment of central nervous system disorders. OBJECTIVE: To develop a standardized ethanol extract of E. velutina (EEEV) and to investigate the neuroprotective potential of the extract and rizonic acid (RA) from E. velutina on neuronal cells. MATERIALS AND METHODS: The plant drug of E. velutina previously characterized was used for the production of EEEV. Three methods were evaluated in order to obtain an extract with higher content of phenols. The neuroprotective effect of standardized EEEV (HPLC-PDA) and RA was investigated on SH-SY5Y cell exposure to the neurotoxin 6-hydroxydopamine (6-OHDA). RESULTS: The powder of the plant drug was classified as moderately coarse and several quality control parameters were determined. EEEV produced by percolation gave the highest phenol content when related to others extractive methods, and its HPLC-PDA analysis allowed to identify four flavonoids and RA, some reported for the first time for the species. EEEV and RA reduced significantly the neurotoxicity induced by 6-OHDA in SH-SY5Y cells determined by the MTT assay and the nitrite concentration. EEEV also showed a free radical scavenging activity. CONCLUSION: This is the first pharmacological study about E. velutina which used a controlled standardized extract since the preparation of the herbal drug. This extract and RA, acting as an antioxidant, presents a neuroprotective effect suggesting that they have potential for future development as a therapeutic agent in neurodegenerative disease as Parkinson. SUMMARY: The powder of Erythrina velutina was classified as moderately coarse and several quality-control parameters were determined.Ethanolic extract from E. velutina (EEEV) produced by percolation gave the highest phenol content when related to others extractive methods and its HPLC-PDA analysis of EEEV allowed to identify four flavonoids and rizonic acid (RA), some reported for the first time for the species.The EEEV and RA reduced significantly the neurotoxicity induced by 6-OHDA in SH-SY5Y cells determined by the MTT assay and the nitrite concentration.The EEEV also showed a free radical scavenging activity. Abbreviations used: ±: More or less, %: Percentage, °C: Degree Celsius, <: Less than, µg: Microgram, µL: Microliter, µM: Micromol, [1D] MNR: One-dimensional nuclear magnetic resonance spectroscopy, [2D] MNR:Two-dimensional nuclear magnetic resonance spectroscopy, 6-OHDA: [6-] Hydroxydopamine. Abs: Absorbance, CFU: Colony forming units, CH2Cl2: Dichloromethane, CHCl3: Chloroform cmCentimeter, DMEM/F12: Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-12. DMSO: Dimethyl sulfoxide, DPPH: 1,1-Diphenyl-2-picrylhydrazyl, EAG: Gallic acid equivalents, EEEV: Ethanolic extract of Erythrina velutina, EtOAc: Ethyl acetate, g: Gram, h: Hour, H2O: Water, HPLC: High-performance liquid chromatography, H REIMS: Hydrogen rapid evaporative ionization mass spectrometry, Kg: Kilogram M: Molar, m: Metro, MeOH: Methanol, mg: Milligram, min: Minute, mL: Milliliter, mm: Millimeter, MTT: Bromide 3 [4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazolium, N: Normal, NBT: Nitroblue tetrazolium, nm: Nanometer, PDA: Photodiode array detector, TPC: Total polyphenol content, RA: Rizonic acid, RP: Reverse phase, SOD: Superoxide dismutase, v/v: Volume per volume, Vs: Versus W: Watts.

Pentoxifylline Neuroprotective Effects Are Possibly Related to Its Anti-Inflammatory and TNF-Alpha Inhibitory Properties, in the 6-OHDA Model of Parkinson's Disease.

Pentoxifylline (PTX) is a phosphodiesterase inhibitor with anti-TNF-alpha activity, associated with its anti-inflammatory action. Considering Parkinson's disease (PD) as a neuroinflammatory disorder, the objectives were to evaluate PTX neuroprotective properties, in a model of PD. Male Wistar rats, divided into sham-operated (SO), untreated 6-OHDA, and 6-OHDA treated with PTX (10, 25, and 50 mg/kg) groups, received a unilateral 6-OHDA injection, except the SO group administered with saline. Treatments started 24 h after surgery and continued for 15 days when the animals were submitted to apomorphine-induced rotations, open field, and forced swimming tests. At the next day, they were euthanized and their striata processed for neurochemical (DA and DOPAC determinations), histological, and immunohistochemical (Fluoro-Jade, TH, DAT, OX-42, TNF-alpha, COX-2, and iNOS) studies. PTX reversed the behavioral changes observed in the untreated 6-OHDA animals. Furthermore, PTX partially reversed the decrease in DA contents and improved neuronal viability. In addition, decreases in immunostaining for TH and dopamine transporter (DAT) were reversed. The untreated 6-OHDA group showed intense OX-42, TNF-alpha, COX-2, and iNOS immunoreactivities, which were attenuated by PTX. In conclusion, we demonstrated a neuroprotective effect of PTX, possibly related to its anti-inflammatory and antioxidant actions, indicating its potential as an adjunct treatment for PD.

Valproic Acid Neuroprotection in the 6-OHDA Model of Parkinson's Disease Is Possibly Related to Its Anti-Inflammatory and HDAC Inhibitory Properties.

Parkinson's disease is a neurodegenerative disorder where the main hallmark is the dopaminergic neuronal loss. Besides motor symptoms, PD also causes cognitive decline. Although current therapies focus on the restoration of dopamine levels in the striatum, prevention or disease-modifying therapies are urgently needed. Valproic acid (VA) is a wide spectrum antiepileptic drug, exerting many biochemical and physiological effects. It has been shown to inhibit histone deacetylase which seems to be associated with the drug neuroprotective action. The objectives were to study the neuroprotective properties of VA in a model of Parkinson's disease, consisting in the unilateral striatal injection of the neurotoxin 6-OHDA. For that, male Wistar rats (250 g) were divided into the groups: sham-operated (SO), untreated 6-OHDA-lesioned, and 6-OHDA-lesioned treated with VA (25 or 50 mg/kg). Oral treatments started 24 h after the stereotaxic surgery and continued daily for 2 weeks, when the animals were subjected to behavioral evaluations (apomorphine-induced rotations and open-field tests). Then, they were sacrificed and had their mesencephalon, striatum, and hippocampus dissected for neurochemical (DA and DOPAC determinations), histological (Fluoro-Jade staining), and immunohistochemistry evaluations (TH, OX-42, GFAP, TNF-alpha, and HDAC). The results showed that VA partly reversed behavioral and neurochemical alterations observed in the untreated 6-OHDA-lesioned rats. Besides, VA also decreased neuron degeneration in the striatum and reversed the TH depletion observed in the mesencephalon of the untreated 6-OHDA groups. This neurotoxin increased the OX-42 and GFAP immunoreactivities in the mesencephalon, indicating increased microglia and astrocyte reactivities, respectively, which were reversed by VA. In addition, the immunostainings for TNF-alpha and HDAC demonstrated in the untreated 6-OHDA-lesioned rats were also decreased after VA treatments. These results were observed not only in the CA1 and CA3 subfields of the hippocampus, but also in the temporal cortex. In conclusion, we showed that VA partly reversed the behavioral, neurochemical, histological, and immunohistochemical alterations observed in the untreated 6-OHDA-lesioned animals. These effects are probably related to the drug anti-inflammatory activity and strongly suggest that VA is a potential candidate to be included in translational studies for the treatment of neurodegenerative diseases as PD.

Neuroprotective Properties of the Standardized Extract from Camellia sinensis (Green Tea) and Its Main Bioactive Components, Epicatechin and Epigallocatechin Gallate, in the 6-OHDA Model of Parkinson's Disease.

Camellia sinensis (green tea) is largely consumed, mainly in Asia. It possesses several biological effects such as antioxidant and anti-inflammatory properties. The objectives were to investigate the neuroprotective actions of the standardized extract (CS), epicatechin (EC) and epigallocatechin gallate (EGCG), on a model of Parkinson's disease. Male Wistar rats were divided into SO (sham-operated controls), untreated 6-OHDA-lesioned and 6-OHDA-lesioned treated for 2 weeks with CS (25, 50, or 100 mg/kg), EC (10 mg/kg), or EGCG (10 mg/kg) groups. One hour after the last administration, animals were submitted to behavioral tests and euthanized and their striata and hippocampi were dissected for neurochemical (DA, DOPAC, and HVA) and antioxidant activity determinations, as well as immunohistochemistry evaluations (TH, COX-2, and iNOS). The results showed that CS and catechins reverted behavioral changes, indicating neuroprotection manifested as decreased rotational behavior, increased locomotor activity, antidepressive effects, and improvement of cognitive dysfunction, as compared to the untreated 6-OHDA-lesioned group. Besides, CS, EP, and EGCG reversed the striatal oxidative stress and immunohistochemistry alterations. These results show that the neuroprotective effects of CS and its catechins are probably and in great part due to its powerful antioxidant and anti-inflammatory properties, pointing out their potential for the prevention and treatment of PD.

Influence of process conditions on the physicochemical characteristics of cumaru (Amburana cearensis) powder produced by spray drying

The aim of the work was to study the spray-drying of ethanolic extract from Amburana cearensis (Allemão) A.C. Sm., Fabaceae, in order to obtain powders with better pharmacological and technological properties for herbal medicine. A 2³ fractional factorial statistical design was used to find adequate spray-drying operating conditions (inlet air temperature; feed flow rate and air flow rate) to produce A. cearensis powder with adequate concentration of active principles (amburoside and coumarin), low moisture content and high process yield. The HPLC analyses showed that the spray-drying powder of A. cearensis production did not cause alterations in the chromatographic profile when related to the fluid extract. The most significant factor that affected the amburoside concentration was air flow rate, while the concentration of coumarin, a thermolabile molecule, was influenced mainly by inlet air temperature. The moisture content of the spray-drying powder of A. cearensis varied from 3.72 to 5.85% (w/w), while the maximal process yield was 41.1% (w/w). The present study demonstrates for the first time the best operating conditions to produce A. cearensis extract powder that were adequate when related to the coumarin and amburoside concentrations and moisture content. However, additional studies are still needed to improve mainly it technological characteristics.

Piperine decreases pilocarpine-induced convulsions by GABAergic mechanisms.

Piperine, an alkaloid present in the Piper genus, was shown to have an anticonvulsant activity, evaluated by the pilocarpine-induced model, in mice. Pilocarpine (350mg/kg, i.p.) was administered 30min after piperine (2.5, 5, 10 and 20mg/kg, i.p.) which significantly increased latencies to 1st convulsion and to death, and percentage of survivals. These parameters were also increased in the pilocarpine groups pretreated with atropine plus piperine (10 and 2.5mg/kg, respectively), as related to the pilocarpine group. However, they were not altered in the pilocarpine groups pretreated with memantine (a NMDA-type glutamate receptors blocker, 2mg/kg, p.o.) or nimodipine (a calcium channel blocker, 10mg/kg, p.o.), both associated with piperine (1 or 2.5mg/kg), as compared to the piperine plus pilocarpine group. Moreover, the pilocarpine group pretreated with diazepam (which binds to the GABAA receptor, 0.2 and 0.5mg/kg, i.p.) plus piperine (1 and 2.5mg/kg) significantly increased latency to the 1st convulsion, as related to the pilocarpine group, suggesting that the GABAergic system is involved with the piperine action. Furthermore, the piperine effect was blocked by flumazenil (2mg/kg, i.p.), a benzodiazepine antagonist. Untreated P350 animals showed decreased striatal DA and increased DOPAC and HVA levels that were not affected in the piperine plus pilocarpine groups. Piperine increased striatal levels of GABA, glycine and taurine, and reversed pilocarpine-induced increases in nitrite contents in sera and brain. Hippocampi from the untreated pilocarpine group showed an increased number of TNF-α immunostained cells in all areas, as opposed to the pilocarpine group pretreated with piperine. Taken together, piperine anticonvulsant effects are the result of its anti-inflammatory and antioxidant actions, as well as TNF-α reduction. In addition, piperine effects on inhibitory amino acids and on the GABAergic system may certainly contribute to the drug anticonvulsant activity.

Anti-inflammatory activities of the hydroalcoholic extracts from Erythrina velutina and E. mulungu in mice

This work studied the anti-inflammatory activities of the hydroalcoholic extracts (HAEs) from Erythrina velutina Willd. (Ev) and E. mulungu Mart. ex Benth. (Em) in the carrageenan- and dextran-induced mice hind paw edema models. These medicinal plants belonging to the Fabaceae family are used in some Brazilian communities to treat pain, inflammation, insomnia and disorders of the central nervous system. In the present work, the extracts were administered orally in male mice at the doses of 200 or 400 mg/kg. In the carrageenan-induced test, only Em showed anti-inflammatory activity, decreasing the paw edema, at the doses of 200 and 400 mg/kg. No effect was observed with Ev in this model. On the other hand, in the dextran model, Ev demonstrated anti-inflammatory effect, showing decrease of the paw edema at the 1, 2, 3, 4 and 24th h. Em (200 or 400 mg/kg) presented anti-inflammatory effect at the 2, 3 and 4th h after administration of dextran, as compared to control. In conclusion, the work showed that Ev and Em present anti-edematous actions, which possibly occurs by distinct mechanisms. While Ev seems to interfere especially in inflammatory processes in which mast cells have an important role, Em exerts greater activity in the inflammatory process that depends mainly on polymorphonuclear leucocytes. However, further studies are needed to determine the exact mechanism of action of the species investigated.

Anti-inflammatory properties of doxycycline and minocycline in experimental models: an in vivo and in vitro comparative study.

AIMS AND METHODS: Minocycline (Mino) and doxycycline (Dox) are second generation tetracyclines known to present several other effects, which are independent from their antimicrobial activities. We studied in a comparative way the anti-inflammatory effects of Mino and Dox, on acute models of peripheral inflammation in rodents (formalin test and peritonitis in mice, and carrageenan-induced paw oedema in rats). Immunohistochemical assays for TNF-alpha and iNOS in rat paws of carrageenan-induced oedema were also carried out as well as in vitro assays for myeloperoxidase (MPO) and lactate dehydrogenase (LDH). Furthermore, antioxidant activities were evaluated by the DPPH assay. RESULTS: In the formalin test although Mino and Dox (1, 5, 10 and 25 mg/kg, i.p.) inhibited the first phase, they acted predominantly on the second phase of the test, where inhibition of the licking time close to 80% were observed. Mino and Dox were very efficacious in reducing the carrageenan-induced paw oedema in rats (10, 25 and 50 mg/kg, i.p.) and carrageenan-induced leucocyte migration (1 and 5 mg/kg, i.p.) to mice peritoneal cavities. Besides, they also significantly inhibited MPO and LDH releases at doses ranging from 0.001 to 1 µg/ml. Thus, in general, the anti-inflammatory activity of Dox was higher as compared to that of Mino, although the radical scavenging activity of Mino was of a magnitude 10 times higher. CONCLUSIONS: Our data indicate that anti-inflammatory and antioxidant effects, involve the inhibition of iNOS and TNF-alpha, among other properties, and these encourage clinical studies of these compounds for new therapeutic applications, especially those were inflammation plays a role.

K+(ATP) channels-independent analgesic action of Crotalus durissus cumanensis venom.

The effect was investigated of the K+ channel blocker, glibenclamide, on the ability of Crotalus durissus cumanensis venom (CDCM) to promote peripheral antinociception. This was measured by formalin-induced nociception in male Swiss mice. CDCM (200 and 300 microg/kg) produced an antinociceptive effect during phase 2 in the formalin test. The effect of CDCM (200 microg/kg) was unaffected by the ATP-sensitive K+ channel blocker glibenclamide (2 mg/kg). These results suggest that CDCM is effective against acute pain. However, the ATP-sensitive K+ channels pathway is not contributable to the antinociceptive mechanism of CDCM.

Antiproliferative effects of several compounds isolated from Amburana cearensis A. C. Smith.

Amburana cearensis a common tree found in Northeastern Brazil is widely used in folk medicine. The present work evaluated the cytotoxicity of kaempferol, isokaempferide, amburoside A and protocatechuic acid isolated from the ethanol extract of the trunk bark of A. cearensis. The compounds were tested for their cytotoxicity on the sea urchin egg development, hemolysis assay and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay using tumor cell lines. Isokaempferide and kaempferol, but not amburoside A and protocatechuic acid, inhibited the sea urchin egg development as well as tumor cell lines, but in this assay isokaempferide was more potent than kaempferol. Protocatechuic acid was the only compound able to induce hemolysis of mouse erythrocytes, suggesting that the cytotoxicity of kaempferol and isokaempeferide was not related to membrane damage.