Aminoguanidine Research Articles

Effect of aminoguanidine on cell apoptosis induced by acute myocardial ischemia in rats

Objective To evaluate the effect of aminoguanidine on cell apoptosis induced by acute myocardial ischemia in rats.Methods Thirty adult male Sprague-Dawley rats,weighing 250-290 g,were randomly divided into 3 groups (n =10 each):sham operation group (group S),myocardial ischemia group (group Ⅰ),and aminoguanidine group (group AG).The model of acute myocardial ischemia was established by ligating the left anterior descending branch of the coronary artery of rats anesthetized with chloral hydrate.Aminoguanidine 100 mg/kg was intraperitoneally administrated at 6 h of ischemia in AG group,while the equal volume of normal saline was given instead of aminoguanidine in group Ⅰ.The chest was opened at 3 h after aminoguanidine administration and hearts were quickly removed for detection of apoptosis in cardiomyocytes (by TUNEL) and expression of Bcl-2 and Bax in cardiomyocytes (by immuno-histochemistry),and for microscopic examination with light microscope.Apoptotic rate was calculated.Results Compared with group S,the apoptotic rate was significantly increased,the expression of Bax was up-regulated,and the expression of Bcl-2 was downregulated and the ratio of Bcl-2/Bax was decreased in group Ⅰ.Compared with group Ⅰ,the apoptotic rate was significantly decreased,the expression of Bax was down-regulated,and the expression of Bcl-2 was up-regulated,and the ratio of Bcl-2/Bax was increased in group AG.The pathological changes of myocardial cells were significantly attenuated in AG group as compared with group Ⅰ.Conclusion Amionguanidine can inhibit apoptosis in cardiomyocytes and is helpful in mitigating injury induced by acute myocardial ischemia in rats. Key words: Guanidines; Myocardical ischemia ; Apopotosis

Hypoxia/ischemia a key player in early post stroke seizures: Modulation by opioidergic and nitrergic systems

Stroke is a leading cause of death, disability, and socioeconomic loss worldwide. All attempts at pharmacological reduction of the complications of stroke (e.g. post-stroke seizure, and brain׳s vulnerability to hypoxic/ischemic injury) have failed. Endogenous opioids and nitric oxide (NO) overproduction has been documented in brain hypoxia/ischemia (H/I), which can exert pro-convulsive effects. In this study, we aimed to examine the possible involvement of opioidergic and nitrergic pathways in the pathogenesis of post-stroke seizure. H/I was induced by right common carotid ligation and sham-operated mice served as controls. We demonstrated that right common carotid ligation decreases the threshold for clonic seizures induced by pentylenetetrazole (PTZ), a GABA antagonist. Furthermore, pro-convulsive effect of H/I following right common carotid ligation was blocked by naltrexone (NTX) (3mg/kg), NG-Nitro-l-arginine methyl ester (l-NAME) (10mg/kg), and aminoguanidine (AG) (100mg/kg) administration (P<0.001). Interestingly, co-administration of non-effective doses of NTX and l-NAME (1 and 0.5mg/kg, respectively) reverses epileptogenesis of H/I (P<0.001). In the same way, co-administration of non-effective doses of NTX and AG (1 and 5mg/kg, respectively), reverses epileptogenesis of H/I (P<0.001). Indeed, the histological studies performed on mice exposed to H/I confirmed our previous data. These findings suggest hyper-susceptibility to PTZ induced seizure following H/I is mediated by interaction of opioidergic, and iNOS/NO pathways. Therefore, our results identify new pharmacological targets and provide the rationale for a novel strategy to promote recovery after stroke and possibly other brain injuries.

Methylglyoxal increases dopamine level and leads to oxidative stress in SH-SY5Y cells.

More and more studies have suggested that methylglyoxal (MGO) induced by type-2 diabetes is related to Parkinson's disease (PD). However, little is known about the molecular mechanism. In this study, we explored the MGO toxicity in neuroblastoma SH-SY5Y cells. Neurotoxicity of MGO was measured by mitochondrial membrane potential, malondialdehyde, and methylthiazoletetrazolium assays. The levels of dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC), and 1-methyl-4-phenyl-1,2,3,4-tetrahydroisoquinoline (salsolinol) were detected by liquid chromatography-mass spectrometry/mass spectrometry. The expressions of tyrosine hydroxylase (TH) and dopamine transporter (DAT) were detected by reverse transcriptase polymerase chain reaction and western blot analysis. The results showed that MGO induced an increase in TH and DAT expressions in SH-SY5Y neuroblastoma cells, while the levels of dopamine, DOPAC, and endogenous neurotoxin salsolinol also increased. Aminoguanidine (AG) is an inhibitor of MGO. It was found that AG could decrease the reactive oxygen species (ROS) level induced by MGO, but could not inhibit an increase of TH, DAT and dopamine. The increase of dopamine, DOPAC and salsolinol levels could lead to high ROS and mitochondrial damage. This study suggests that ROS caused by dopamine could contribute to the damage of dopaminergic neurons when MGO is increased during the course of diabetes.

Extracts and compounds with anti-diabetic complications and anti-cancer activity from Castanea mollissina Blume (Chinese chestnut).

BackgroundCastanea mollissima Blume (Chinese chestnut), as a food product is known for its various nutrients and functional values to the human health. The present study was carried out to analyze the anti-diabetic complications and anti-cancer activities of the bioactive compounds present in C. mollissima.MethodsThe kernels (CK), shells (CS) and involucres (CI) parts of C. Blume were extracted with 90% alcohol. The water suspension of these dried alcohol extracts were extracted using EtOAc and n-BuOH successively. The n-BuOH fraction of CI (CI-B) was isolated by silica gel column, Sephadex LH 20 column and preparative HPLC. The isolated compounds were identified by 1H-NMR, 13C-NMR, HMBC, HMQC and ESI-Q-TOF MS, All the fractions and compounds isolated were evaluated on human recombinant aldose reductase (HR-AR) assay, advanced glycation end products (AGEs) formation assay and human COLO 320 DM colon cancer cells inhibitory assay.ResultsCI-B was found to show a significant inhibitory effect in above biological screenings. Six flavonoids and three polyphenolic acids were obtained from CI-B. They were identified as kaempferol (1), kaempferol-3-O-[6''-O-(E)-p-coumaroyl]-β-D-glucopyranoside (2), kaempferol-3-O-[6''-O-(E)-p-coumaroyl]-β-D-galactopyranoside (3), kaempferol-3-O-[2''-O-(E)-p-coumaroyl]-β-D-glucopyranoside (4), kaempferol-3-O-[2", 6"-di-O-(E)-p-coumaroyl]-β-D-glucopyranoside (5) and kaempferol-3-O-[2", 6"-di-O-(E)-p-coumaroyl]-β-D-galactopyranoside (6), casuariin (7), casuarinin (8) and castalagin (9). Compounds 2–9 were found to show higher activity than quercetin (positive control) in the AR assay. Compounds 3–6, 8, and 9 showed stronger inhibitory effects than amino guanidine (positive control) on AGEs production. Compounds 4–6, 7, and 8 showed much higher cytotoxic activity than 5-fluorouracil (positive control) against the human COLO 320 DM colon cancer cells.ConclusionsOur results suggest that flavonoids and polyphenolic acids possesses anti-diabetes complications and anti-cancer properties, and they were presumed to be the bioactive components of Castanea mollissima Blume.Electronic supplementary materialThe online version of this article (doi:10.1186/1472-6882-14-422) contains supplementary material, which is available to authorized users.

5-O-Demethylnobiletin, a polymethoxylated flavonoid, from Citrus depressa Hayata peel prevents protein glycation

Advanced glycation end products (AGEs) have been implicated in many age-related chronic diseases and the search for AGEs has intensified. In the present study, eight Okinawan edible or medicinal plants were screened for the anti-glycation activity. A known polymethoxylated flavonoid, 5-O-demethyl nobiletin (DN), was isolated from the chloroform fraction of Citrus depressa Hayata (Rutaceae) and was identified for the first time as an AGEs inhibitor. DN prevented protein glycation significantly better than aminoguanidine (AG) with the respective IC50 values of 64.2 and 484.3 µM (p = 0.01). Further studies revealed that DN prevented AGEs formation predominantly by inhibiting fructosamine adduct formation while only partly by preventing dicarbonyl generation. Together, these results suggest that further exploration on the identification of AGEs inhibitors from Okinawan plants and in-depth investigations on the AGEs inhibitions by polymethoxylated flavonoids are required.

Effect of aminoguanidine on sciatic functional index, oxidative stress, and rate of apoptosis in an experimental rat model of ischemia–reperfusion injury

This study was conducted to investigate the potential protective effects of aminoguanidine (AG) on sciatic functional index (SFI), oxidative stress status, and apoptosis index using a rat model of experimental sciatic nerve ischemia-reperfusion injury (I/R). Treatment groups received 150 mg AG/kg body mass, 24 h after the induction of ischemia. After reperfusion for 2, 4, 7, 14, and 28 days, we evaluated measured SFI, plasma antioxidant enzymes, total antioxidant capacity (TAC), malondialdehyde (MDA), and index of apoptosis. SFI was significantly improved on the 7th and 14th day of reperfusion in the AG-treated groups. AG treatment resulted in the significant reduction of MDA levels on the 7th and 14th day of reperfusion. TAC was only increased after 7 days of reperfusion compared with the untreated group. SOD activity was decreased in both the untreated and AG-treated groups by comparison with the control, but did not show a significant change. GPx activity decreased only after 7 days of reperfusion. The maximal rate of apoptosis occurred on the 7th day of reperfusion. Treatment with AG significantly reduced this enhancement. AG exhibits positive effects against sciatic nerve I/R injury, possibly in part because of the protective effects of AG against apoptosis and I/R-induced oxidative stress.

Structure, Physicochemical Properties, and Density Functional Theory Calculation of High-Energy-Density Materials Constructed with Intermolecular Interaction: Nitro Group Charge Determines Sensitivity

Four nitro-containing energetic compounds, cocrystal of AT·DNBA (1), salt of MA·DNSA (2), salt of AG·DNBA·H2O (3), and salt of DAT·DNSA H2O (4), are synthesized and structurally characterized based on supramolecular interactions (AT, 4-amino-1,2,4-triazole; DNBA, 3,5-dinitrobenzoic acid; MA, melamine; DNSA, 3,5-dinitrosalicylic acid; AG, amino guanidine; DAT, 3,5-diamino-1,2,4-triazole). The physicochemical properties of the compounds are theoretically and experimentally investigated in detail. The optimized structures, molecular total energies, frontier orbit energies, and charge densities of 1–4 are calculated by theoretical methods. The experimental results indicate that all compounds exhibit good thermostability and low sensitivity. It is worth noting that the values of impact sensitivity are measured to be 30, >40, 38, and >40 J for 1, 2, 3, and 4, respectively, which correspond well to the order of nitro group charge (QNitro) calculated by density functional theory. The detonation performances of 1–4 are discussed; in particular, 1 and 2 exhibit heats of detonation (2.191 kcal g–1 for 1 and 2.214 kcal g–1 for 2) superior to those of classical nitro-rich compounds. In addition, the nonisothermal thermokinetic parameters are obtained by Kissinger and Ozawa methods, and the standard molar enthalpies of formation are calculated from the determination of constant volume combustion energies.

A simple method to detect plant based inhibitors of glycation induced protein cross-linking

Background: Glycation induced cross-linking of proteins are associated with chronic diabetic complications. Inhibition of protein glycation is one of the therapeutic approaches to prevent the progression of diabetic complications. Objective: Objective of this study was to establish a simple method to identify medicinal plants which can inhibit glycation induced protein cross-linking. Methods: Lysozyme was incubated at 37°C up to 4 weeks with different concentrations of glucose, fructose and ribose in sodium phosphate buffer (pH 7.4). Appropriate controls and blanks were carried out. Aminoguanidine (AG) was used as the standard inhibitor. Water extracts of Bryophyllum pinnatum leaves, Coriandrum sativum seed and Murraya koenigi leaves were used as potential inhibitors. Aliquots were removed from the incubation mixtures at intervals and analyzed for the presence of AGE induced protein cross-links, using SDS-PAGE. Appearance and the intensity of high molecular weight products were compared. Results: Extent of cross-linking was dependent on the sugar concentration. Cross-linking was slowest in the presence of glucose and fastest in the presence of ribose. AG inhibited glycation induced protein cross-linking in the presence of all three sugars. B. pinnatum leaves, C. sativum seeds and M. koenigi leaves inhibited protein cross-linking in the presence of sugar. This inhibition was greater than that of AG. Conclusions: We have established a simple SDS-PAGE method to identify medicinal plants which inhibit glycation induced protein cross-linking. We also demonstrated the effectiveness of B. pinnatum leaves, C. sativum seed and M. koenigi leaves in inhibiting glycation induced protein cross-linking in vitro. DOI: http://dx.doi.org/10.3126/ajms.v6i1.10181 Asian Journal of Medical Sciences Vol.6(1) 2015 28-33

Glycoxidation of biological macromolecules: a critical approach to halt the menace of glycation.

Glycation is the result of covalent bonding of a free amino group of biological macromolecules with a reducing sugar, which results in the formation of a Schiff base that undergoes rearrangement, dehydration and cyclization to form a more stable Amadori product. The final products of nonenzymatic glycation of biomacromolecules like DNA, proteins and lipids are known as advanced glycation end products (AGEs). AGEs may be generated rapidly or over long times stimulated by distinct triggering mechanisms, thereby accounting for their roles in multiple settings and disease states. Both Schiff base and Amadori glycation products generate free radicals resulting in decline of antioxidant defense mechanisms and can damage cellular organelles and enzymes. This critical review primarily focuses on the mechanistic insight of glycation and the most probable route for the formation of glycation products and their therapeutic interventions. Furthermore, the prevention of glycation reaction using therapeutic drugs such as metformin, pyridoxamine and aminoguanidine (AG) are discussed with special emphasis on the novel concept of the bioconjugation of these drugs like, AG with gold nanoparticles (GNPs). At or above 10 mM concentration, AG is found to be toxic and therefore has serious health concerns, and the study warrants doing this novel bioconjugation of AG with GNPs. This approach might increase the efficacy of the AG at a reduced concentration with low or no toxicity. Using the concept of synthesis of GNPs with abovementioned drugs, it is assumed that toxicity of various drugs which are used at high doses can be minimized more effectively.

Anti-Apoptotic Effects of Aminoguanidine Against Liver Damage on Experimental Diabetes in Rats

Objective: This study was designed to investigate the antiapoptotic and improving effects of aminoguanidine on the histological alterations in liver in streptozotocin (STZ)-induced diabetic rat model. Material and Methods: 32 male Sprague Dawley rats were divided into the following 4 groups: Control, Aminoguanidine (AMG), Streptozotocin (STZ), Streptozotocin + Aminoguanidine (STZ+AMG). The animals in group STZ and STZ+AMG were made diabetic by intraperitoneal injection of streptozotocin 45 mg/kg. Histochemical and immunohistochemical staining methods were applied to sections obtained from paraffin blocks and preparations were examined by using Leica DFC-280 light microscope. Results: The sections taken from the control and AMG groups were normal in histological appearance. However, multifocal nodules containing inflammatory cells were observed in the diabetic group. Moreover necrotic hepatocytes and hemorrhagic areas were observed among intact hepatocyte of lobul in the STZ group. Glycogen loss was observed in the hepatocytes localized at the periphery of lobule. In addition, the numbers of cells with positive staining by caspase-3 were significantly increased. On the other hand, it was seen that, in the groups administered with STZ, histological injury in the liver was attenuated by the administration of AMG. Moreover, it was found that the number of caspase-3 positive cells was significantly decreased in the STZ+AMG group. Conclusion: We concluded that chronic aminoguanidine administration reduced liver injury in STZ- induced diabetic rats. Thus, we suggest that aminoguanidine may be used to prevent the development of diabetic liver damage. Key Words: Aminoguanidine; Diabetes mellitus; Streptozotocin; Rat.

Antiapoptotic effect of aminoguanidine on doxorubicin-induced apoptosis

Doxorubicin (DOX) is a broad-spectrum anthracycline that has cardiotoxicity as a major side effect. Reactive oxygen species (ROS) and reactive nitrogen species generations have been proposed to be an important mechanism of DOX-induced cardiotoxicity and cardiomyocyte apoptosis, which may be mediated by p53 protein. Aminoguanidine (AG) is an effective antioxidant due to its free radical scavenger activity. A549 lung cell line was incubated with various concentrations of AG (100-1,000 μM) wit/without 0.25 μM DOX for 24 h. The expression of p53 and its transcriptional target p21 were analyzed by Western blot. Apoptosis was analyzed with Annexin V assay. JC1 and H2AX immunofluorescence were used to assess mitochondrial and nuclear DNA damage, respectively. This study demonstrated that AG has a dose-dependent antiapoptotic effect on DOX-induced apoptosis. Thus, these data further identify AG as a potential chemopreventive agent to reduce ROS and nitric oxide synthase damage generated by DOX.

Anti-Aging Effect of Adipose-Derived Stem Cells in a Mouse Model of Skin Aging Induced by D-Galactose

IntroductionGlycation products accumulate during aging of slowly renewing tissue, including skin, and are suggested as an important mechanism underlying the skin aging process. Adipose-derived cells are widely used in the clinic to treat ischemic diseases and enhance wound healing. Interestingly, adipose-derived stem cells (ASCs) are also effective in anti-aging therapy, although the mechanism underlying their effects remains unknown. The purpose of the present study was to examine the anti-aging effect of ASCs in a D-galactose-induced aging animal model and to clarify the underlying mechanism.Materials and MethodsSix-week-old nude mice were subcutaneously injected with D-gal daily for 8 weeks. Two weeks after completion of treatment, mice were randomized to receive subcutaneous injections of 106 green fluorescent protein (GFP)-expressing ASCs, aminoguanidine (AG) or phosphate-buffered saline (PBS). Control mice received no treatment. We examined tissue histology and determined the activity of senescence-associated molecular markers such as superoxide dismutase (SOD) and malondialdehyde (MDA).ResultsTransplanted ASCs were detectable for 14 days and their GFP signal disappeared at day 28 after injection. ASCs inhibited advanced glycation end product (AGE) levels in our animal model as well as increased the SOD level and decreased the MDA level, all of which act to reverse the aging phenotype in a similar way to AG, an inhibitor of AGE formation. Furthermore, ASCs released angiogenic factors in vivo such as vascular endothelial growth factor, suggesting a skin trophic effect.ConclusionsThese results demonstrate that ASCs may contribute to the regeneration of skin during aging. In addition, the data shows that ASCs provide a functional benefit by glycation suppression, antioxidation, and trophic effects in a mouse model of aging.

Aminoguanidine Inhibits the AGE–RAGE Axis to Modulate the Induction of Periodontitis but Has Limited Effects on the Progression and Recovery of Experimental Periodontitis: A Preliminary Study

Advanced glycation end products (AGE) have been found in inflamed gingival tissue and have been shown to interfere with the integrity of extracellular matrix and cell-matrix interactions. This study aims to investigate the modulatory effect of aminoguanidine (AG), an AGE inhibitor, in various stages of experimental periodontitis. Thirty-six Sprague-Dawley rats were used. AG or normal saline (NS) was systemically administered in the induction, progression, and recovery phases of ligature-induced periodontitis. Dynamic changes of the periodontium were evaluated by microcomputed tomography, histology, and immunohistochemistry of the receptor for AGE (RAGE). Molecular mechanisms were evaluated by myeloperoxidase activity, gene expression of RAGE, and markers associated with tissue repair and homeostasis, including vascular endothelial growth factor (VEGF), type I collagen, fibronectin, and periostin. AG appeared to inhibit the degradation of the collagen matrix in the induction phase but promoted collagen reorganization in the progression and recovery phases of experimental periodontitis. In the induction sites, periodontal bone loss was significantly reduced (P <0.05), with significantly reduced RAGE (P <0.05) and significantly elevated fibronectin and periostin levels (P <0.01). No significant alterations in the levels of myeloperoxidase, VEGF, and collagen were noted. In the progression and recovery sites, similar trends were observed, with insignificant differences relative to NS-treated animals. AG reduced periodontal bone loss during the induction of experimental periodontitis, and the effects appeared to be insignificant in the progression and recovery phases. This modulation was related to the inhibition of the AGE-RAGE axis to resume cell-matrix interactions and maintain tissue integrity.

CYP2E1-mediated oxidative stress regulates HO-1 and GST expression in maneb- and paraquat-treated rat polymorphonuclear leukocytes.

Cytochrome P4502E1 (CYP2E1), glutathione-S-transferase A4-4 (GSTA4-4), and inducible nitric oxide synthase (iNOS) are implicated in maneb- and paraquat-induced toxicity leading to various pathological conditions. The study aimed to investigate the role of CYP2E1 in maneb- and paraquat-induced oxidative stress in rat polymorphonuclear leukocytes (PMNs) and its crosstalk with iNOS-mediated nitrosative stress and GSTA4-4-linked protective effect, if any and their consequent links with the nuclear factor erythoid 2-related factor 2 (Nrf2) activation and heme oxygenase-1 (HO-1) expression. Rats were treated with/without maneb and/or paraquat for 1, 2, and 3 weeks along with vehicle controls. Subsets of rats were also treated with diallyl sulfide (DAS) or aminoguanidine (AG) along with the respective controls. Maneb and paraquat augmented the reactive oxygen species (ROS), lipid peroxidation (LPO) and 4-hydroxy nonenal (4-HNE) contents, and superoxide dismutase (SOD) activity in the PMNs. However, maneb and paraquat attenuated the reduced glutathione (GSH) level and the expression/activity of total GST and GST-pi. Maneb and paraquat increased the expression/activity of CYP2E1, GSTA4-4, iNOS, Nrf2 and HO-1, and nitrite content. CYP2E1 inhibitor, DAS noticeably alleviated maneb- and paraquat-induced ROS, LPO, 4-HNE, SOD, Nrf2 and HO-1, GST, GSH, and GST-pi while iNOS, nitrite content and GSTA4-4 levels were unchanged. Conversely, AG, an iNOS inhibitor, attenuated maneb- and paraquat-directed changes in nitrite, LPO, iNOS but it did not alter ROS, GSH, SOD, GST, GST-pi, Nrf2, HO-1, CYP2E1, and GSTA4-4. The results demonstrate that CYP2E1 induces iNOS-independent free radical generation and subsequently modulates the Nrf2-dependent HO-1 and 4-HNE-mediated GST expression in maneb- and paraquat-treated PMNs.

The detrimental effects of acute hyperglycemia on myocardial glucose uptake

AimsAlthough acute hyperglycemic (AHG) episodes are linked to lower glucose uptake, underlying mechanisms remain unclear. We hypothesized that AHG triggers reactive oxygen species (ROS) production and increases non-oxidative glucose pathway (NOGP) activation, i.e. stimulation of advanced glycation end products (AGE), polyol pathway (PP), hexosamine biosynthetic pathway (HBP), PKC; thereby decreasing cardiac glucose uptake. Main methodsH9c2 cardiomyoblasts were exposed to 25mM glucose for 24h vs. 5.5mM controls±modulating agents during the last hour of glucose exposure: a) antioxidant #1 for mitochondrial ROS (250μM 4-OHCA), b) antioxidant #2 for NADPH oxidase-generated ROS (100μM DPI), c) NOGP inhibitors — 100μM aminoguanidine (AGE), 5μM chelerythrine (PKC); 40μM DON (HBP); and 10μM zopolrestat (PP). ROS levels (mitochondrial, intracellular) and glucose uptake were evaluated by flow cytometry. Key findingsAHG elevated ROS, activated NOGPs and blunted glucose uptake. Transketolase activity (pentose phosphate pathway [PPP] marker) did not change. Respective 4-OHCA and DPI treatment blunted ROS production, diminished NOGP activation and normalized glucose uptake. NOGP inhibitory studies identified PKCβII as a key downstream player in lowering insulin-mediated glucose uptake. When we employed an agent (benfotiamine) known to shunt flux away from NOGPs (into PPP), it decreased ROS generation and NOGP activation, and restored glucose uptake under AHG conditions. SignificanceThis study demonstrates that AHG elicits maladaptive events that function in tandem to reduce glucose uptake, and that antioxidant treatment and/or attenuation of NOGP activation (PKC, polyol pathway) may limit the onset of insulin resistance.

DFT study of the mechanism of the reaction of aminoguanidine with methylglyoxal

We have studied the mechanism of the reaction between aminoguanidine (AG) and methylglyoxal (MG) by carrying out Dmol3/DFT calculations, obtaining intermediates, transition-state structures, and free-energy profiles for all of the elementary steps of the reaction. Designed models included explicit water solvent, which forms hydrogen-bond networks around the reactants and intermediate molecules, facilitating intramolecular proton transfer in some steps of the reaction mechanism. The reaction take place in four steps, namely: (1) formation of a guanylhydrazone-acetylcarbinol adduct by condensation of AG and MG; (2) dehydration of the adduct; (3) formation of an 1,2,4-triazine derivative by ring closure; and (4) dehydration with the formation of 5-methyl 3-amino-1,2,4-triazine as the final product. From a microkinetic point of view, the first dehydration step was found to be the rate-determining step for the reaction, with the reaction having an apparent activation energy of 12.65 kcal mol⁻¹. Additionally, some analogous structures of intermediates and transition states for the reaction between AG and 2,3-dicarbonyl-phosphatidylethanolamine, a possible intermediate in Amadori-glycated phosphatidylethanolamine (Amadori-PE) autooxidation, were obtained to evaluate the reaction above a phosphatidylethanolamine (PE) surface. Our results are in agreement with experimental results obtaining by other authors, showing that AG is efficient at trapping dicarbonyl compounds such as methylglyoxal, and by extension these compounds joined to biomolecules such as PE in environments such as surfaces and their aqueous surroundings.

Role of Nitric Oxide Synthases in Early Blood-Brain Barrier Disruption following Transient Focal Cerebral Ischemia

The role of nitric oxide synthases (NOSs) in early blood-brain barrier (BBB) disruption was determined using a new mouse model of transient focal cerebral ischemia. Ischemia was induced by ligating the middle cerebral artery (MCA) at its M2 segment and reperfusion was induced by releasing the ligation. The diameter alteration of the MCA, arterial anastomoses and collateral arteries were imaged and measured in real time. BBB disruption was assessed by Evans Blue (EB) and sodium fluorescein (Na-F) extravasation at 3 hours of reperfusion. The reperfusion produced an extensive vasodilation and a sustained hyperemia. Although expression of NOSs was not altered at 3 hours of reperfusion, L-NAME (a non-specific NOS inhibitor) abolished reperfusion-induced vasodilation/hyperemia and significantly reduced EB and Na-F extravasation. L-NIO (an endothelial NOS (eNOS) inhibitor) significantly attenuated cerebral vasodilation but not BBB disruption, whereas L-NPA and 7-NI (neuronal NOS (nNOS) inhibitors) significantly reduced BBB disruption but not cerebral vasodilation. In contrast, aminoguanidine (AG) (an inducible NOS (iNOS) inhibitor) had less effect on either cerebral vasodilation or BBB disruption. On the other hand, papaverine (PV) not only increased the vasodilation/hyperemia but also significantly reduced BBB disruption. Combined treatment with L-NAME and PV preserved the vasodilation/hyperemia and significantly reduced BBB disruption. Our findings suggest that nNOS may play a major role in early BBB disruption following transient focal cerebral ischemia via a hyperemia-independent mechanism.

Aminoguanidine treatment ameliorates inflammatory responses and memory impairment induced by amyloid-beta 25–35 injection in rats

Alzheimer disease (AD) is a neurodegenerative disorder caused by accumulation of the amyloid-beta peptide (Aβ) in neuritic plaques. Its neurotoxic mechanisms are associated with inflammatory responses and nitrosative stress generation that promote expression of inducible nitric oxide synthase (iNOS) and increased nitric oxide causing neuronal death and memory impairment. Studies suggest that treatment with anti-inflammatory and anti-oxidant agents decreases the risk of developing AD. Aminoguanidine (AG) is an iNOS inhibitor with anti-inflammatory and anti-oxidant effects. In this study, we evaluated the effects of systemic administration of AG (100mg/kg/day for 4days) on spatial memory and inflammatory responses induced by an injection of Aβ25–35 [100μM] into the temporal cortex (TCx) of rats. A significant improvement of spatial memory was evident in the Aβ25–35-treated group at day 30 post-injection subjected to AG treatment; this effect was correlated with decreases in reactive gliosis, IL-1β, TNF-α, and nitrite levels, as well as a reduction in neurodegeneration in the TCx and hippocampus (Hp). These results suggest that AG treatment inhibited glia activation and cytokine release, which may help to counteract neurodegenerative events induced by the toxicity of Aβ.

Anti-Inflammatory Effect of &lt;i&gt;Allium ursinum&lt;/i&gt;

The aim of the present study was to evaluate Allium ursinum leaves and flowers extract anti-inflammatory effect. Plant extract 1:1 (w:v) was prepared from A. ursinum leaves by a modified Squibb repercolation method. The in vivo anti-inflammatory effects were evaluated on a rat turpentine oil-induced inflammation (i.m. 6 mL/kg BW). The animals were randomly assigned to nine groups (n=8): negative control, inflammation, A. ursinum flower extract (AUF), A. ursinum leaves extract (AUL), indomethacin (INDO) (20 mg/kg BW), aminoguanidine (AG) (50 mg/kg b.w./d i.p.) as a selective NOS2 inhibitor, NG-nitro L-arginine methyl ester (NAME) (5 mg/kg b.w./d i.p.) as a nonselective NOS inhibitor, L-arginine (ARG) (100 mg/kg b.w./d i.p.), NO synthesis substrate, and Trolox (20 mg/kg b.w./d i.p) as an antioxidant. At 24h from inflammation induction total oxidative status (TOS), oxidative stress index (OSI), nitric oxide (NOx) and in vitro phagocytosis test were reduced and the total antioxidative reactivity (TAR) was increased by the testes plant extracts. AUF had a better inhibitory effect than AUL. In conclusion, we provided evidence for the hypothesis that A. ursinum leaves and flowers extract exerts anti-inflammatory activity by inhibiting the phagocytosis through the reduction of the nitro-oxidative stress.

Effects of the combination of methylprednisolone with aminoguanidine on functional recovery in rats following spinal cord injury.

Methylprednisolone (MP), a synthetic glucocorticoid, has been widely used as a standard therapeutic agent for the treatment of spinal cord injury (SCI). The combination of MP and other pharmacological agents aimed at enhancing functional recovery is desirable as the beneficial effects of MP are controversial, due to a variety of side-effects. Aminoguanidine (AG), a small water-soluble compound, is potentially useful in the treatment of acute SCI. The aim of the present study was to determine the effects of MP and AG, administered in combination, following SCI in adult rats. In rats with SCI, the combination therapy group treated with AG (75 mg/kg) and MP (0.75 mg/kg) exhibited significantly reduced levels of cytokine expression and cell apoptosis compared with those in the control group. In addition, the data demonstrated that the combination therapy significantly enhanced the recovery of limb function. These data clearly suggest that treatment with a combination of MP and AG represents a promising strategy of clinically applicable pharmacological therapy for the rapid initiation of neuroprotection following SCI.