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Background Apocynin, a main component extracted from the root of Picrorhiza kurroa Royle, was a well-knownNADPH oxidase inhibitor and reported to have effect on lung injury, liver injury, diabetes and asthma. AN-1, anitrone derivative of apocynin, was found to exhibit significant effect on treatment of acute lung injury.Aim In order to carry out further preclinical study, it is important to reveal in vivo disposition of AN-1. A simple andrapid high-performance liquid chromatography (HPLC) method was developed to disclose the tissue distributionbehavior of AN-1 in Sprague-Dawley (SD) rats.Methods A HPLC method was developed and validated to measure the concentration of AN-1 in tissue sampleswith carbamazepine as internal standard (IS). The mobile phase consisted of water and methanol (47:53, v/v), theflow rate was 1 mL/min, and an ultraviolet (UV) detector was used at wavelength of 279 nm. The tissue distributionstudy of AN-1 was performed in Sprague-Dawley (SD) rats after a single intravenous dose of 40 mg/kg.Results The developed HPLC-UV method was of good specificity, precision (< 4%), accuracy (90-97%) and recovery(88-104%) for analysis of AN-1 in tissue samples of rats. The linear range was established over a concentrationrange 0.2-50 µg/mL (r2 > 0.998) in tissues including heart, liver, spleen, lung, kidney and brain. After administration,AN-1 was rapidly distributed in tissues and reached peak concentration with time, which showed a high distribution
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Background Apocynin (4-hydroxy-3-methoxyacetophenone), an important active ingredient contained in root ofPicrorhiza kurroa Royle, has been widely investigated as an antioxidative and anti-inflammatory agent in kinds ofdisease models, and exerted certain efficacy deserving further research. However, little was known about thedisposal process of apocynin in vivo, which is important information required in drug research and development.Aim In this work, a tissue distribution study of apocynin was performed in Sprague-Dawley (SD) male rats to helpfurther understanding well the disposition of apocynin in vivo.Methods A simple HPLC-UV method was developed for measurement of apocynin concentration in rat tissues. Amixture of water-methanol (47:53, v/v) was used as solvent system, the flow rate was 1 mL/min, and the detectedwavelength was 279 nm. The method was validated and applied to the tissue distribution study of a single bolusintravenous administration of apocynin in SD male rats.Results The developed HPLC-UV method showed good specificity, precision, accuracy and extraction recovery. Thegood linearity was achieved within 0.8-32 μg/mL in tissues including heart, liver, spleen, lung, kidney and brain. Thelower limit of quantification (LLOQ) was 0.8 μg/mL. The method was well used in the study of tissue distribution ofapocynin. The results demonstrated that apocynin was distributed fast into the tested tissues and reached peakconcentration at 5 min after injection. Apocynin was mainly distributed in liver, kidney and lung within 15 minutesafter administration, and eliminated from the tissues with no sample be detected more than 2 h after dose
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O osteossarcoma (OS) é o tumor maligno primário mais comum do tecido ósseo, caracterizado pela formação de osteócitos anormais. Apesar do avanço nas terapias convencionais (quimioterapia e retirada do tumor), essas não conseguem eliminar totalmente as células tumorais e impedir a progressão da doença. Recentemente, agentes derivados de fontes naturais ganharam considerável atenção por causa de sua segurança, eficácia e disponibilidade imediata. Nesse sentido, a apocinina, inibidor do complexo NADPH-oxidase, vem sendo estudada como agente antitumoral em alguns tipos de câncer como: pâncreas, próstata, pulmão e mama. Apocinina é um pró-fármaco e sua ação parece estar relacionada à sua conversão produzindo a diapocinina, a qual se mostrou mais efetiva do que a apocinina. Portanto, o objetivo desse estudo é avaliar, in vitro, o potencial antitumoral da apocinina e diapocinina em células de osteossarcoma humano. Para isso, foram utilizados osteoblastos humanos normais (HOb) e osteossarcoma humano imortalizadas (SaOS-2) tratados ou não com apocinina e diapocinina em diversas concentrações. Foram realizados os ensaios de viabilidade celular, alterações morfológicas, apoptose celular, produção de espécies reativas de oxigênio (EROs), formação de colônias, migração, invasão e expressão do fator indutor de hipóxia-1alfa (HIF-1). Também foram conduzidos ensaios para verificar a atividade de metaloproteinase de matriz (MMP) 2 e 9. Os resultados em SaOS-2 mostraram que o tratamento com apocinina nas concentrações de 1,5 e 3 mM; e diapocinina nas concentrações de 0,75 e 1,5 mM reduziram a viabilidade; aumentaram o número de células em apoptose e diminuíram a produção de EROs; sem causar danos às células HOb. Além disso, essas mesmas concentrações inibiram a migração e invasão celular; diminuíram a expressão de HIF-1; e reduziram a atividade de MMP-2 em SaOS-2. Considerando os resultados obtidos, concluímos que a apocinina e diapocinina podem atuar como possíveis moduladores de células tumorais, sendo que a diapocinina mostrou ser mais efetiva nos parâmetros testados.(AU)
Osteosarcoma (OS) is the most common primary malignant tumor of bone tissue, characterized by the formation of abnormal osteocytes. Despite advances in conventional therapies (chemotherapy and surgery) they cannot completely eliminate tumor cells and prevent the progression of the disease. Recently, agents derived from natural sources have achieved considerable attention because of their safety, efficacy and immediate availability of therapies. In this way, apocynin, an inhibitor of the NADPH-oxidase complex, has been studied as an antitumor agent in some types of cancer, such as pancreas, prostate, lung and breast. Apocynin is a prodrug and its action indicate to be related to its conversion to diapocynin, which has been shown to be more efficient than apocynin itself. Thus, the aim of this study is to evaluate, in vitro, the antitumor potential of apocynin and diapocynin in human osteosarcoma cells. For this, normal human osteoblasts (HOb) and immortalized human osteosarcoma cells (SaOS-2) were treated or no-treated with apocynin and diapocynin in various concentrations. Cell viability assay, morphological alterations, cellular apoptosis, reactive oxygen species (ROS) production, colony formation, migration, invasion and expression of hypoxia-inducible factor-1 alpha (HIF-1) were performed. We also performed assays to verify the activity of matrix metalloproteinase (MMP) 2 and 9. The results in SaOS-2 showed that treatment with apocynin at concentrations of 1,5 e 3 mM; and diapocynin at concentrations of 0,75 e 1,5 mM reduced cell viability; increased the number of cells in apoptosis and decreased the production of ROS; without damaging HOb cells. Moreover, these same concentrations inhibited cell migration and invasion; decreased HIF-1 expression; and reduced MMP 2 activity in SaOS-2. Considering the results, we suggest that apocynin and diapocynin may act as possible modulators of tumor cells, and diapocynin has been shown to be more effective.(AU)
Subject(s)
Humans , Acetophenones/pharmacology , Antineoplastic Agents/pharmacology , Biphenyl Compounds/pharmacology , Osteosarcoma/drug therapy , Apoptosis/drug effects , Cell Movement/drug effects , Cell Survival/drug effects , Matrix Metalloproteinase 2/drug effects , Matrix Metalloproteinase 9/drug effects , Osteoblasts/drug effects , Reactive Oxygen Species/analysis , Reproducibility of Results , Tumor Cells, CulturedABSTRACT
In this study, we developed and validated a fast, specific, sensitive, precise and stability-indicating high performance liquid chromatography (HPLC) method to determine the drug apocynin in bovine serum albumin (BSA) nanoparticles. Chromatographic analyses were performed on an RP C18 column and using a photodiode array detector at a wavelength of 276 nm. Mobile phase consisted of a mixture of acetonitrile and 1%acetic acid (60:40, v/v), and it was eluted isocratically at a flow rate of 0.8 mL/min. The retention time of apocynin chromatographic peak was 1.65 min. The method was linear, precise, accurate and specific in the range of 5–100μg/mL. The intra-and inter-day precisions presented relative standard deviation (RSD) values lower than 2%. The method was robust regarding changes in mobile phase proportion, but not for flow rate. Limits of detection and quantitation were 78 ng/mL and 238 ng/mL, respectively. Apocynin was exposed to acid and alkali hydrolysis, oxidation and visible light. The drug suffered mild degradation under acid and oxidation conditions and great degradation under alkali conditions. Light exposure did not degrade the drug. The method was successfully applied to determine the encapsulation efficiency of apocynin in BSA nanoparticles.
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Objective To observe the variation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and microglia in the comorbidity of neuropathic pain and depression and discuss the related mechanism.Methods The spared nerve injury model was used.Forty-four male adult Sprague Dawley rats were randomly divided into the following four groups (n=11 each): sham+vehicle group (group SV), sham+APO group (group SA), SNI+vehicle group (group SNV), SNI+APO group (group SNA).In groups SA and SNA, rats were intraperitoneally injected with apocynin (APO) 15 mg/kg 24 hours and 1 hour before SNI and continued once daily until the 14th day.The rats in the other two groups received the equal volume of vehicle.The mechanical withdrawal threshold (MWT) was tested 1 day before SNI and 7 days and 14 days after SNI, and the open field test, the forced swimming test and the sucrose preference test were performed 14 days after SNI.The prefrontal cortex were collected 2 hour after the behavior tests.The expression of gp91phox was detected by Western blot and the expression of Iba1 and gp91phox were detected by double-immunofluorescance staining.Results The reduced MWT, the increased immobility time, the decreased sucrose consumption and the increased content of gp91phox were observed in group SNV compared with groups SV, SA and SNA (P<0.05).The expression of Iba1 and gp91phox were increased in group SNV.The total travel distance in the open field test and the total liquid consumption in the sucrose preference test had no significant difference among the four groups.Conclusion Neuropathic pain may induce depressive behaviors and activate NADPH oxidase in the prefrontal cortex.Moreover, the inhibition of NADPH oxidase by APO can alleviate neuropathic pain and depression, which is potentially related to the activation of microglia.
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BACKGROUND: The dysfunction of the proteasome system has been implicated in neuronal degeneration. Apocynin, a specific inhibitor for nicotinamide adenine dinucleotide phosphate oxidase, has anti-inflammatory and anti-oxidant effects. However, the effect of apocynin on the neuronal cell death induced by proteasome inhibition has not been studied. METHODS: Using differentiated PC12 cells, in the respect of cell death process the suppressive effect of apocynin on the proteasome inhibition-mediated apoptosis was examined. RESULTS: The proteasome inhibitors MG132 and MG115 induced a decrease in Bid and Bcl-2 protein levels, an increase in Bax and p53 levels, mitochondrial depolarization, efflux of cytochrome c into cytosol and increase in caspases (-8, -9 and -3) activities. Treatment with apocynin attenuated the proteasome inhibitor-induced changes in the apoptosis-related protein levels, formation of reactive oxygen species, glutathione (GSH) depletion and cell death. CONCLUSIONS: Apocynin may attenuate the proteasome inhibitor-mediated apoptosis in differentiated PC12 cells by inhibiting the activation of the mitochondria-mediated pathway and the caspase-8- and Bid-dependent pathways. The preventive effect of apocynin appears to be attributed to inhibition of the production of reactive oxygen species and the depletion of cellular GSH contents.
Subject(s)
Animals , Antioxidants , Apoptosis , Caspases , Cell Death , Cytochromes c , Cytosol , Glutathione , NADP , Neurons , Oxidoreductases , PC12 Cells , Proteasome Endopeptidase Complex , Proteasome Inhibitors , Reactive Oxygen SpeciesABSTRACT
Objective To investigate the optimal dose of apocynin to protect severe acute pancreatitis (SAP) and SAP caused intestinal injury in rats. Methods A total of 53 SPF male Wistar rats were randomly allocated into five groups:sham operation group (SO group, n=10), SAP group (n=12), low-dose apocynin group (25 mg/kg,n=11), medium-dose apocynin group (50 mg/kg, n=10) and high-dose apocynin group (100 mg/kg,n=10). SAP model was prepared by retrograde infusing 5%sodium taurocholate (1 mL/kg) into biliopancreatic duct of rat. At thirty minutes before modeling, apocynin was injected into rat to intervention. The survival condition was recorded at 12 h after modeling, and blood samples were obtained for detecting serum amylase (AMY), alanine aminotransferase (ALT) and creatinine (Cr). Pancreatic and ileal tissue samples were obtained for HE staining and pathological examination. Results Two rats died in SAP group and one died in low-dose apocynin group. The quantity of ascites, the levels of AMY, ALT, Cr and pancreatic and intestinal pathologic scores were significantly increased in SAP group than those in SO group (P<0.05). Except the levels of Cr and intestinal pathologic score, there was no significant difference between low-dose apocynin group and SAP group. The quantity of ascites ascites, levels of AMY, Cr and pancreatic and intestinal pathologic scores were significantly lower in medium-dose and high-dose apocynin groups than those in SAP group (P<0.05). The levels of ALT and Cr were significantly higher in high-dose apocynin group than those of medium-dose apocynin group (P<0.05). Conclusion Apocynin improves SAP symptoms and reduces SAP caused intestinal injury in rats, which may be related to the inhibition of NOX activity, and 50 mg/kg of apocynin is the optimal dose.
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Aim To determine the protective effect of NADPH oxidase against focal cerebral ischemia/reper-fusion ( I/R) injury in rats. Method A thread occlu-sion method was used to make a middle cerebral artery occlusion ( MCAO ) model. Apocynin ( 2. 5 mg · kg-1 ) was injected by tail vein 15 min before ischemi-a. Then, the neurological behavior, cerebral infarction volume, pathological morphological changes and the expression of Cx36, PKC, Bax, Bcl-2 of rats were de-tected after ischemia for 2 h, followed by reperfusion for 24 h. Results Compared with cerebral I/R group, administration of apocynin significantly reduced the neurological behavior scores and the cerebral in-farction volume percentage, alleviated the pathological morphological damage, increased the protein expres-sion of Cx36 and PKC, and reduced the Bax/Bcl-2 ra-tio of rats with focal cerebral I/R injury. Compared with apocynin group , apocynin combined with PKC inhibitor significantly reduced above protective effects. Conclusion Inhibition of NADPH oxidase could alle-viate cerebral I/R injury, increase the levels of Cx36, PKC proteins and reduce the Bax/Bcl-2 ratio.
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Objective To clarify whether the NADPH oxidases (NOXs) family contributed to the reactive oxygen species (ROS) production and subsequent interstitial fibrosis in unilateral ureter obstruction (UUO) rats.Methods Male Wistar rats were randomly divided into sham operation group (n =8),sham operation + apocynin treatment group (n =8),UUO operation group (n =8) and UUO operation+apocynin treatment group (n =8).Either vehicle or apocynin (100 mg/kg per day) were given by gavage for 7 days after surgery.Rats were sacrificed at 7th day.ELISA was used to detect the activity of superoxide dismutase (SOD) and catalase (CAT),and the level of 8-iso-prostaglandin F2alpha (8-isoPGF2α) in renal tissue.Western blotting was used to detect the protein expressions of NADPH oxidase subunit NOX2 and NOX4,α-smooth muscle actin(α-SMA),collagen Ⅰ (COL-Ⅰ) and the level of ERK1/ 2 phosphorylation (p-ERK1/2).Results UUO rats with vehicle displayed increased oxidative stress,as measured by renal tissue 8-iso-PGF2α,accompanied with increased renal expression of NADPH oxidases (NOX2,1.5-fold and NOX4,1.7-fold,respectively),compared with sham-operated rats (P ＜0.05).Furthermore,vehicle treated UUO rats showed increased renal COL-Ⅰ and α-SMA levels,compared with sham-operated rats (P ＜ 0.05).ERK1/2 was also activated as detected by p-ERK1/2 expression in UUO rats with vehicle (P ＜ 0.05).Apocynin treatment significantly decreased renal tissue 8-iso-PGF2α level and expressions of NOX2 (-28.7％) and NOX4 (-31.0％) in UUO rats,respectively,compared with vehicle treated rats (P ＜ 0.05).And significant decrease of COL-Ⅰ (-26.4％) and α-SMA expression (-80.0％) were also observed (P ＜ 0.05).The activation of ERK1/2 in UUO rats was greatly inhibited by apocynin treatment (P ＜ 0.01).Despite the pronounced dysregulation of pro-oxidative NOXs family,no compensatory increase of antioxidative enzyme activities occurred.Conclusion The NOXs family contributes largely to the production of ROS and subsequent interstitial fibrosis after ureter ligation,and inhibition of the NOXs family may be a choice for preventing interstitial fibrosis.
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Background: NADPH oxidase is a source of reactive oxygen species that may contribute to insulin resistance (IR). Aim: To assess the effect of a single oral dose of vanillin (a putative inhibitor of the enzyme) on IR in humans. Material and Methods: Using a crossover, random, double-blind design, eight lean and 10 obese males ingested 600 mg of vanillin or placebo followed by the ingestion of 75g of glucose. Serum/plasma glucose, free-fatty acids, insulin, glutathione, C reactive protein concentrations and red blood cell glutathione concentration were determined. Insulin resistance was estimated by the Matsuda index. Results: Under fasting conditions, obese individuals had higher glucose and insulin and lower red blood cell glutathione levels than their lean counterparts (p < 0.01). Serum free-fatty acids, total and oxidized plasma glutathione concentrations were similar in both groups. After glucose ingestion, obese individuals had a lower red blood cell total glutathione concentration and increased plasma oxidized glutathione concentration than their lean counterparts (p < 0.05). In addition, obese participants had a higher level of IR (p < 0.001) and impaired serum free-fatty acid suppression (p < 0.001) than their lean counterparts. Ingestion of vanillin did not modify any of these variables when compared with placebo in obese individuals. In lean volunteers a reduction in Matsuda index was detected when vanillin was administered, compared to placebo (4.3 +/- 0.6 and 3.6 +/- 0.6 respectively; p < 0.05). Conclusions: IR was ameliorated after vanillin ingestion among lean but not obese participants.
Subject(s)
Humans , Male , Adult , Antioxidants/administration & dosage , Benzaldehydes/administration & dosage , Obesity , Insulin Resistance/physiology , Acetophenones , Fatty Acids, Nonesterified/analysis , Benzaldehydes/adverse effects , Double-Blind Method , Blood Glucose , Glutathione/analysis , Inflammation , NADPH Oxidases , Oxidative Stress , C-Reactive Protein/analysisABSTRACT
BACKGROUND: Based on the assertion that apocynin diminishes acute lung injury (ALI) by inhibition of NADPH oxidase, the effect of apocynin was tested in interleukin-1alpha (IL-1)-induced ALI in rats. METHODS: IL-1 was insufflated into the trachea of Sprague-Dawley rats to induce ALI, and apocynin (8 mg/kg) was given intravenously for inhibition of NADPH oxidase. In addition, we determined whether apocynin inhibited generation of superoxide anions from isolated human neutrophils. Five hours after IL-1 instillation, lung injury parameters, expression of cytosolic phospholipase A2 (cPLA2) by cells from bronchoalveolar lavage (BAL), an index of oxidative stress in lung tissues (gamma-glutamyltranspeptidase, activity), and ultrastructure of alveolar type II (AT II) cells were evaluated. RESULTS: Apocynin decreased the generation of free radicals from phorbol myristate (PMA)-activated neutrophils in vitro, but did not ameliorate ALI. IL-1 induced enhancement of the expression of cPLA2 on neutrophils was not altered by apocynin. CONCLUSION: Apocynin induced suppression of the generation of superoxide anions from neutrophils by inhibition of NADPH oxidase does not attenuate IL-1-induced ALI in rats.
Subject(s)
Animals , Humans , Rats , Acetophenones , Acute Lung Injury , Bronchoalveolar Lavage , Cytosol , Free Radicals , Interleukin-1 , Interleukin-1alpha , Lung , Lung Injury , Myristic Acid , NADPH Oxidases , Neutrophils , Oxidative Stress , Phorbols , Phospholipases A2 , Rats, Sprague-Dawley , Superoxides , TracheaABSTRACT
Objective To investigate the protective effect of apocynin against renal oxidative injury in a rat model of hyperoxaluria. Methods Animal model of hyperoxaluria was established by administration of 0.8% ethylene glycol (EG) to adult male Sprague-Dawley rats in administration were performed in the rats. Markers of oxidative stress(OS) state, urinary H2O2 and 8-(so-prostaglandin IP), and renal injury were assessed at the end of the study. Expression and localization of NADPH oxidase subunits (p47phox, gp91phox, Nox-1) in kidneys were examined by immunohistochemistry, real-time PCR and Western blot, respectively. Results p47phox expressed widely in kidneys of model rats, including renal cortex, inner medulla and outer medulla. Compared with the control, OS and renal injury occurred in rats receiving EG, in accordance with the up-regulated expression of NADPH oxidase subunits in kidneys. Treatment with apocynin significantly reduced the excretion of urinary H2O2 and 8-IP, improved the creatinine clearance and the kidney/body weight, with the down-reguLated expression of NADPH oxidase subunits (except gp91phox mRNA) in kidneys, but the levels of OS markers in apocynin-treated rats were still higher than thoset of normal controls. Conclusions The increased expression of NADPH oxidase subunits is suggested to be partially accounted for the development of renal OS in this rat model of hyperoxaluria. Apocynin treatment is effective for renal protection in this model.
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AIM: To investigate the roles of angiotensin Ⅱ and NADPH oxidase in the development of renal oxidative stress (OS) in a rat model of hyperoxaluria. METHODS:Animal model of hyperoxaluria was established in a-dult male Sprague - Dawley rats by administration of 0.8% ethylene glycol (EC) in drinking water for 4 weeks. Simultaneous treatment with apocynin (0.2g·kg~(-1)·d~(-1))or losartan (30 mg·kg~(-1)·d~(-1) ) by intragastric administration were performed in rats, respectively. At the end of the study, markers for the state of oxidative stress (OS) , urinary 8 - IP and the enzymatic activity of superoxide dismutase ( SOD) in kidney homogenates were assessed. The concentration of angiotensin H in kidney homogenates was determined using radioimmunoassay method. Expression of NADPH oxidase subunit p47phox in kidney was localized and evaluated by immunohistochemistry and real time - PCR, respectively. RESULTS: p47phox expressed widely in the kidneys of this rat model, including renal cortex, inner medulla and outer medulla. Compared with the control, OS developed significantly in rats received EG, with increased expression of p47phox mRNA in kidneys. Renal angiotensin Ⅱ also increased significantly. Treatment with apocynin or losartan significantly reduced the excretion of urinary 8 - IP, restored the SOD activity, with decrease in the expression of p47phox mRNA in kidney, but the levels of those OS markers in apocynin or losartan treated rats were still higher than those in normal controls. CONCLUSION: Results suggest that renal Ang Ⅱ and its stimulation of NADPH oxidase may partially account for the development of OS in kidney in this rat model of hyperoxaluria.
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Objective To investigate the influence of NO mixture(L-arginine,apocynin and sodium nitroferricyanide) on wound healing of diabetes mellitus(DM) mice.Methods Fifty adult male Kunming mice were induced by streptozotocin to establish type I DM model.They were then randomly divided into control group,L-arginine group(150 g/L),apocynin group(1?10-4 mol/L),sodium nitroferricyanide group(0.1 mmol/L) and NO mixture group(above components were mixed with equal ratio).Full-thickness skin wound was made and injected with corresponding drugs of 0.15 ml once every 2 days.The wounds were digitally photographed to calculate the percentage of wound closure using computer image analysis software in 1,3,5,7 and 10 d post-injury.The density of fibroblasts,the content of collagenous fibers and the neovascularization in the wound samples were measured with the aid of HE staining.Results From the third day post-injury,the wound healing rate of NO mixture group was significantly higher than that of all the other groups(P