Aminoguanidine Research Articles

Comparative Evaluation of Aminoguanidine, Semicarbazide and Thiosemicarbazide Treatment for Methylglyoxal-Induced Neurological Toxicity in Experimental Models

Background: Advanced glycation end products (AGEs) are complex compounds that play a critical role in neurological disorders, including the pathogenesis of Alzheimer's disease. Methylglyoxal (MG) is recognized as the primary precursor of AGEs. Methylglyoxal is produced endogenously and also introduced through dietary exposures. Objectives: This study aimed to investigate and compare the effects of aminoguanidine (AG), semicarbazide (SC), and thiosemicarbazide (TSC) on MG-induced neurological toxicity in rats. Methods: Male Wistar rats were exposed orally to MG, MG + AG, MG + SC, and MG + TSC for 70 days. Neurobehavioral, biochemical, and histopathological changes were evaluated. Results: The findings indicated that oral administration of MG for 70 days resulted in memory impairment and increased anxiety in neurobehavioral tests. Additionally, MG elevated protein carbonylation in brain tissues. Semicarbazide was found to prevent MG-induced memory problems, while both SC and AG reduced carbonyl content in brain tissues. Advanced glycation and TSC were effective in alleviating anxiety induced by MG exposure. Histopathological analysis revealed that MG caused cell damage and neuronal necrosis in the hippocampus, particularly in the cornu ammonis 1 (CA1) and dentate gyrus (DG) regions. However, AG, SC, and TSC improved neuronal survival in these areas. Conclusions: The data suggest that SC, AG, and TSC may offer neuroprotective effects against MG-induced neurobehavioral toxicity. Further studies are required to explore the mechanisms of action of these compounds.

Crosstalk of methylglyoxal and calcium signaling in maize (Zea mays L.) thermotolerance through methylglyoxal-scavenging system

Methylglyoxal (MG) and calcium ion (Ca2+) can increase multiple-stress tolerance including plant thermotolerance. However, whether crosstalk of MG and Ca2+ exists in the formation of maize thermotolerance and underlying mechanism still remain elusive. In this paper, maize seedlings were irrigated with MG and calcium chloride alone or in combination, and then exposed to heat stress (HS). The results manifested that, compared with the survival percentage (SP, 45.3%) of the control seedlings, the SP of MG and Ca2+ alone or in combination was increased to 72.4%, 74.2%, and 83.4% under HS conditions, indicating that Ca2+ and MG alone or in combination could upraise seedling thermotolerance. Also, the MG-upraised SP was separately weakened to 42.2%, 40.3%, 52.1%, and 39.4% by Ca2+ chelator (ethylene glycol tetraacetic acid, EGTA), plasma membrane Ca2+ channel blocker (lanthanum chloride, LaCl3), intracellular Ca2+ channel blocker (neomycin, NEC), and calmodulin (CaM) antagonist (trifluoperazine, TFP). However, significant effect of MG scavengers N-acetylcysteine (NAC) and aminoguanidine (AG) on Ca2+-induced thermotolerance was not observed. Similarly, an endogenous Ca2+ level in seedlings was increased by exogenous MG under non-HS and HS conditions, while exogenous Ca2+ had no significant effect on endogenous MG. These data implied that Ca2+ signaling, at least partly, mediated MG-upraised thermotolerance in maize seedlings. Moreover, the activity and gene expression of glyoxalase system (glyoxalase I, glyoxalase II, and glyoxalase III) and non-glyoxalase system (MG reductase, aldehyde reductase, aldo-keto reductase, and lactate dehydrogenase) were up-regulated to a certain extent by Ca2+ and MG alone in seedlings under non-HS and HS conditions. The up-regulated MG-scavenging system by MG was enhanced by Ca2+, while impaired by EGTA, LaCl3, NEC, or TFP. These data suggest that the crosstalk of MG and Ca2+ signaling in maize thermotolerance through MG-scavenging system. These findings provided a theoretical basis for breeding climate-resilient maize crop and developing smart agriculture.

Inhibitory Effects of Aqueous Ethanol Extracts of Poplar-Type Propolis on Advanced Glycation End Products and Protein Oxidation

(1) Background: The non-enzymatic glycation of proteins is a significant contributor to the formation of advanced glycation end products (AGEs) and intermediates that are responsible for diabetic complications. It is imperative to explore effective inhibitors to prevent protein glycation. (2) Methods: This study aimed to investigate the inhibitory potential of various aqueous ethanol extracts of poplar-type propolis on AGEs and oxidative modifications in bovine serum albumin (BSA)-glucose and BSA-methylglyoxal models. (3) Results: The results revealed that these propolis extracts exhibited significant effectiveness in inhibiting the formation of total AGEs, pentosidine, and Nε-carboxymethyllysine (CML). Furthermore, the investigation discovered that these propolis extracts can effectively inhibit oxidative modification, based on measuring the levels of carbonyl and thiol groups and analyzing tryptophan fluorescence quenching. Notably, 75% ethanol extracts of propolis (EEP) exhibited the highest inhibitory activity, surpassing the chemical inhibitor aminoguanidine (AG). (4) Conclusions: The remarkable anti-glycation potency of aqueous ethanol extracts of poplar-type propolis can be attributed to their elevated contents of phenolic compounds, especially abundant flavonoids, which inhibit the formation of AGEs by scavenging free radicals, decreasing the levels of reactive oxygen species (ROS), and capturing reactive carbonyl species (RCS) in the protein glycation process. Our results indicate that poplar-type propolis may be a potential AGE inhibitor and could be used to develop functional foods and nutraceuticals to prevent diabetic complications.

Inhibition and Mechanism of Protein Nonenzymatic Glycation by Lactobacillus fermentum.

Lactobacillus fermentum (L. fermentum) was first evaluated as a potential advanced glycation end-product (AGE) formation inhibitor by establishing a bovine serum albumin (BSA) + glucose (glu) glycation model in the present study. The results showed that the highest inhibition rates of pentosidine and total fluorescent AGEs by L. fermentum were approximately 51.67% and 77.22%, respectively, which were higher than that of aminoguanidine (AG). Mechanistic analysis showed that L. fermentum could capture methylglyoxal and glyoxal, inhibit carbonyl and sulfhydryl oxidation, reduce the binding of glucose and amino groups, increase total phenolic content and antioxidant activity, and release intracellular substances to scavenge free radicals; these abilities were the basis of the antiglycation mechanism of L. fermentum. In addition, L. fermentum significantly prevented conformational changes in proteins during glycation, reduced protein cross-linking by 35.67%, and protected the intrinsic fluorophore. Therefore, the inhibition of L. fermentum on glycation mainly occurs through antioxidation, the capture of dicarbonyl compounds, and the protection of the BSA structure. These findings collectively suggest that Lactobacillus is an inhibitor of protein glycation and AGE formation and has the potential for nutraceutical applications.

Attenuation of albumin glycation and oxidative stress by minerals and vitamins: An in vitro perspective of dual-purpose therapy.

Nonenzymatic glycation of proteins is accelerated in the context of elevated blood sugar levels in diabetes. Vitamin and mineral deficiencies are strongly linked to the onset and progression of diabetes. The antiglycation ability of various water- and fat-soluble vitamins, along with trace minerals like molybdenum (Mo), manganese (Mn), magnesium (Mg), chromium, etc., have been screened using Bovine Serum Albumin (BSA) as in vitro model. BSA was incubated with methylglyoxal (MGO) at 37°C for 48h, along with minerals and vitamins separately, along with controls and aminoguanidine (AG) as a standard to compare the efficacy of the minerals and vitamins. Further, their effects on renal cells' (HEK-293) antioxidant potential were examined. Antiglycation potential is measured by monitoring protein glycation markers, structural and functional modifications. Some minerals, Mo, Mn, and Mg, demonstrated comparable inhibition of protein-bound carbonyl content and ß-amyloid aggregation at maximal physiological concentrations. Mo and Mg protected the thiol group and free amino acids and preserved the antioxidant potential. Vitamin E, D, B1 and B3 revealed significant glycation inhibition and improved antioxidant potential in HEK-293 cells as assessed by estimating lipid peroxidation, SOD and glyoxalase activity. These results emphasize the glycation inhibitory potential of vitamins and minerals, indicating the use of these micronutrients in the prospect of the therapeutic outlook for diabetes management.

Inhibitory Potential of Carnosine and Aminoguanidine Towards Glycation and Fibrillation of Albumin: In-vitro and Simulation Studies.

Carnosine is beta-alanyl histidine, a dipeptide, endogenously produced in our body by the carnosine synthase enzyme. It is an antioxidant, thus protecting from the deleterious effect of advanced glycation end products (AGEs). Similarly, aminoguanidine (AG) also prevents AGEs formation by scavenging free radicals such as reactive oxygen species (ROS)/reactive carbonyl species (RCS). This study used experimental and computational techniques to perform a comparative analysis of carnosine and AG and their inhibiting properties against glycated human serum albumin (HSA). Fructose-mediated glycation of albumin produced fluorescent structures, such as pentosidine and malondialdehyde. These AGEs were significantly reduced by carnosine and AG. At 20mM, carnosine and AG quenches pentosidine fluorescence by 66% and 83%, respectively. A similar inhibitory effect was observed for malondialdehyde. Protein hydrophobicity and tryptophan fluorescence were restored in the presence of carnosine and AG. Aminoguanidine decreased fibrillation in HSA, while carnosine did not significantly affect aggregation/fibrillation. In addition, molecular docking study observed binding scores of -5.90kcal/mol and -2.59kcal/mol by HSA-aminoguanidine and HSA-carnosine complex, respectively. Aminoguanidine forms one conventional hydrogen bond with ARG A:10 and a salt bridge with ASP A:13, ASP A:259, ASP A:255, and ASP A:256 from the amine group. Similarly, carnosine forms only hydrogen bonds with GLU A:501 and GLN A:508 from the amine and hydroxy group. The root mean square deviation (RMSD) calculated from simulation studies was 1nm upto 70ns for the HSA-aminoguanidine complex and the spectrum of HSA-carnosine was significantly deviated and not stabilized. The superior inhibitory activity of aminoguanidine could be due to additional salt bridge bonding with albumin. Conclusively, aminoguanidine can be the better treatment choice for diabetes-associated neurological diseases.

Novel mechanism of the COVID-19 associated coagulopathy (CAC) and vascular thromboembolism

Previous studies from our laboratory revealed that SARS-CoV-2 spike protein (SP) administration to a genetically engineered model expressing the human angiotensin-converting enzyme 2; ACE2 receptor (i.e., hACE2 humanized mouse) mimicked the coronavirus disease-19 (COVID-19) pathology. In humans the cause of high morbidity, and mortality is due to ‘cytokine-storm’ led thromboembolism; however, the exact mechanisms of COVID-19 associated coagulopathy (CAC) have yet to be discovered. Current knowledge suggests that CAC is distinct from the standard coagulopathy, in that the intrinsic and extrinsic thrombin-dependent coagulation factors, and the pathway(s) that are common to coagulopathy, are not recruited by SARS-CoV-2. Findings from patients revealed that there is little change in their partial thromboplastin, or the prothrombin time coupled with a significant decline in platelets. Further, there appears to be an endothelial dysfunction during COVID-19 suggesting an interaction of the endothelia with immune cells including neutrophils. There are also reports that inflammatory NGAL is elevated during COVID-19. Furthermore, the levels of NPT are also increased indicating an increase in inflammatory M1 macrophage iNOS which sequesters BH4; an essential enzyme co-factor that acts as a potent antioxidant thus causing damage to endothelia. SARS-CoV-2 entry into the host cells is facilitated by a co-operative action between TMPRSS2 and the main ACE2 receptor. Interestingly, after infection ADAMTS13; a von Willebrand factor; VWF cleaving enzyme is found to be decreased. Based on these facts, we hypothesize that vascular thromboembolism is associated with serine and metalloproteinase, and in that context, we opine that inhibition of iNOS might help mitigate COVID-19 harmful effects. To test this hypothesis, we administered SP to the hACE2 mice that were subsequently treated with amino guanidine (AG; a potent inhibitor of glycoxidation, lipoxidation and oxidative vicious cycles). Our results revealed increase in TMPRSS2, and NGAL by SP but treatment with AG mitigated their levels. Similarly, levels of MMP-2, and -9 were increased; however, AG treatment normalized these levels. Our findings suggest that occurrence of CAC is influenced by TMPRSS2, ADAMTS13, NGAL and MMP- 2, and -9 factors, and an intervention with iNOS blocker helped mitigate the CAC condition in experimental settings.

Crosstalk between Aldosterone and Glycation through Rac-1 Induces Diabetic Nephropathy.

Background: Advanced glycation end products (AGEs) interaction with its receptor (RAGE) and aldosterone (Aldo) through the mineralocorticoid receptor (MR) activates Rac-1 and NF-κB independently in diabetic nephropathy (DN). However, the crosstalk of Aldo with AGEs-RAGE is still unresolved. Our study examined the impact of the AGEs-Aldo complex on renal cells and its effect on the RAGE-MR interaction. Methods and results: Glycation of human serum albumin (HSA) (40 mg/mL) with methylglyoxal (10 mM) in the presence of Aldo (100 nM) and aminoguanidine (AG) (100 nM) was performed. Glycation markers such as fructosamine and carbonyl groups and fluorescence of AGEs, pentosidine, and tryptophan followed by protein modification were measured. Renal (HEK-293T) cells were treated with the glycated HSA-Aldo (200 μg/mL) along with FPS-ZM1 and spironolactone antagonists for RAGE and Aldo, respectively, for 24 h. Glycation markers and esRAGE levels were measured. Protein and mRNA levels of RAGE, MR, Rac-1, and NF-κB were estimated. Glycation markers were enhanced with Aldo when albumin was only 14-16% glycated. AGEs-Aldo complex upregulated RAGE, MR, Rac-1 and NF-κB expressions. However, FPS-ZM1 action might have activated the RAGE-independent pathway, further elevating MR, Rac-1, and NF-κB levels. Conclusion: Our study concluded that the presence of Aldo has a significant impact on glycation. In the presence of AGEs-Aldo, RAGE-MR crosstalk exerts inflammatory responses through Rac-1 in DN. Insights into this molecular interplay are crucial for developing novel therapeutic strategies to alleviate DN in the future.

Antiglycation, antifibrillation and antioxidative effects of para coumaric acid and vitamin D; an in-vitro and in-silico comparative-cum-synergistic approach

Diabetes Mellitus is a metabolic disorder that results in impaired utilization of carbohydrates, lipids, and proteins. Severe hyperglycemia is its principal clinical symptom. Human serum albumin (HSA) is used as a model protein since it is viewed as a sign of glycaemic management because it is more likely to get glycated in diabetic people than other proteins. Para-coumaric acid (pCA), a phenolic acid, and Vitamin D (vit-D) are used as protective agents. In the present work, we deduce a synergistic-cum-comparative effect of pCA and vit-D, expecting some improvement in the efficacy of pCA when combined with vit-D. Methods employed are DPPH radical scavenging activity, In-vitro glycation of HSA, UV–vis spectroscopy, fluorescence analysis, and circular dichroism measurement. After treatment, increasein the absorbance and fluorescence intensity were reduced along with normalization of CD value. . The glycation-mediated fibrillation assessed by Congo-Red and Thioflavin T (ThT) were found to be diminishedwhen HSA was treated with equimolar concentration of p-CA and vit-D- treatment. Fructosamine adduct formation and lysine modificationwas also decreased, while inhibition to hemolysis and lipid peroxidation was found to increase upon treatment. The reactive oxygen species generation detection was also performed in lymphocytes treated with various protein samples. Docking results further confirmed theblocking some glycation-prone amino acids by both compounds. The study shows that the combination in the ratio of 1:1 has provided higher overall protection comparable to aminoguanidine (AG), the molecule which is utilized as a positive control.

Anti-skin glycation and collagen level stimulation of brown seaweed extracts and their compositional characteristics

This study aimed to investigate anti-skin aging potential of the extracts from three Southern Australian brown seaweeds: Ecklonia radiata (ER), Cystophora moniliformis (CM), and Cystophora siliquosa (CS). These extracts and two positive controls (Aminoguanidine (AG), a drug candidate for anti-glycation, and phloroglucinol (PG)) were tested for their inhibition of glycation reaction using in vitro non-cellular bovine serum albumin (BSA)-glucose and methylglyoxal (MGO) models. The safe doses were also established by toxicity test with human skin fibroblast cell by MTT assay. Among three extracts studied, ER showed the highest toxic, but far less than both positive controls. The CM and CS extracts at 500 μg/mL showed inhibition of glycation activity, approximately 65–90 % compared to AG (15–40 %) and PG (15–20 %) at 20 μg/mL. In addition, the stimulation of collagen and elastin levels using an in vitro cell-based assay was evaluated. Relative to the control (water without samples), the CM and CS extracts at concentration greater than or equal to 250 μg/mL significantly increased the collagen levels of human skin fibroblast cells up to ∼16.5 folds, while there was no significant increase in elastin levels by most seaweed extracts. Furthermore, compositions of all extracts were characterized. Apart from high polysaccharide contents, phlorotannin which accounts for 17–23 % of CM and CS might be responsible for their anti-skin aging benefits. These findings demonstrate that the brown seaweed extracts CM and CS have high potential to be used as skin anti-aging supplements with anti-glycation and collagen stimulation benefits, worthy of further in vivo studies.

The Effect of Treatment With Aminoguanidine, an Advanced Glycation End Product Inhibitor, on Streptozotocin-Induced Diabetic Rats and Its Effects on Physiological and Renal Functions.

Diabetes is a multifactorial syndrome that affects the functioning of the renin-angiotensin system (RAS). The role of advanced glycation end products (AGEs) in diabetes is well known. In the present study, we hypothesized that the prevention of AGE accumulation or abrogation of AGE synthesis using an AGE inhibitor, aminoguanidine (AG), in streptozotocin (STZ)-induced diabetic animal modelswould affect the progression of diabetes and its related complications. We determined the effects of aminoguanidine (AG), an AGE inhibitor, in STZ-induced diabetic rats by determining various indices of RAS and renal functions. Additionally, we also investigated the effect of the drug, AG, on various hemodynamic and physiological functions in the body of the animals. Male Sprague Dawley rats weighing 200-250 g were assigned to four groups (n = 4-6): Vehicle, Vehicle+AG, STZ-induced, and STZ-induced+AG rats. Type 1 diabetes was induced by a single intraperitoneal (IP) injection of streptozotocin (55 mg/kg) dissolved in sodium citrate buffer. The control groups (Vehicle) were injected with buffer. The blood glucose levels were measured after 48 hours, and animals with blood glucose levels > 300 mg/dL were included in the study. Blood glucose levels in the vehicle rats were also determined to ensure non-diabetic conditions. After confirmation, AG was administrated at a dose of 1 g/L in drinking water for two weeks. Urine was collected to measure the glomerular filtration rate (GFR), and the immune reactivity for AT1 and AT2 proteins was analyzed by immunoblotting. Data were expressed as mean ± standard error of the mean(SEM), and a p-value < 0.05 was considered statistically significant. Diabetic rats had a significant drop in body weight, accompanied by increased food and water consumption. The diabetic rats exhibited significantly increased urine flow and GFR. These phenotypes were significantly or considerately reversed by AG treatment in the STZ+AG-treated diabetic rats. Aminoguanidineprevented the increase in blood sugar levels compared to STZ-induced diabetic rats alone (295.9 ± 50.69 versus 462.3 ± 18.6 mg/dL (p < 0.05)). However, it did not affect the glomerular filtration rate (GFR) and glomerular damage, as assessed by the renal histopathological studies. The STZ-induced diabetic rats had an increased sodium excretion (3.24 ± 0.40 mmol) and significantly increased expression of the AT2 receptor and that of the AT1 receptor, which was slightly reversed by the treatment with AG. Treatment with AG decreased sodium excretion (2.12 ± 0.63, as compared to the diabetic rats). These rats also had modestly decreased expression of the AT2 receptor (0.99 ± 0.07 versus 1.12 ± 0.08, as compared to the STZ-induced diabetic rats), while theAT1 receptors showed a slight increase in the STZ+AG-treated rats compared to the STZ-induced diabetic rats (1.1 ± 0.19 versus 1.08 ± 0.12). This study highlights the action of the drugAG in not exacerbating any damage in diabetic rats. Employing AG as a pharmacological intervention to prevent an increase in blood sugar adds a new dimension to controlling increased blood sugar and preventing diabetic complications. The employability and pharmacological intervention of the drug AG, in diabetes, therefore, need a renewed and further investigation.

Licofelone Attenuates Acetic Acid-Induced Colitis in Rats Through Suppression of the Inflammatory Mediators.

Licofelone is a dual Cyclooxygenase 1,2 (COX1,2)/5-lipoxygenase) 5-LOX (inhibitor with analgesic and anti-inflammatory effects with possible functions on inflammatory bowel disease (IBD), which is a chronic recurrent condition with no particular treatment. This study evaluated the anti-inflammatory effects of licofelone on acetic acid-induced colitis in rats. Ten groups of male Wistar rats (n = 6) were used. Sham, control group, licofelone at doses of 2.5, 5, and 10 mg/kg, L-NG-nitro arginine methyl ester (L-NAME) (10 mg/kg, i.p.), aminoguanidine (AG) (100 mg/kg, i.p.), 30 min before using licofelone (10 mg/kg). Also, three groups received L-NAME, aminoguanidine, or dexamethasone. Macroscopic, microscopic, and biochemical analysis of myeloperoxidase (MPO), and nuclear factor-kappa B (NF-κB), tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β), superoxide dismutase (SOD), reactive oxygen species (ROS), and Toll-like receptor 4 (TLR-4) were assessed in colon tissue. Licofelone at a dose of 10 mg/kg attenuated colitis, increased SOD activity, and significantly reduced colonic levels of the abovementioned inflammatory factors. In addition, licofelone improved macroscopic and microscopic symptoms in the acetic acid-induced colitis model. Moreover, the concurrent use of nitric oxide synthase (NOS) inhibitors with 10 mg/kg of licofelone reversed the observed positive effects, demonstrating the function of nitric oxide in IBD pathogenesis and the probable mechanism for licofelone in the healing process of induced colitis. A reduced level of inflammatory factors confirmed the anti-inflammatory activity of licofelone as a dual COX1,2/5-LOX inhibitor. Furthermore, outcomes revealed the protective role of licofelone in treating experimental colitis. The findings are suggestive of the potential use of licofelone in IBD.

Guanidine-modified nanoparticles as robust BTZ delivery carriers and activators of immune responses

Dendritic cells (DCs), the primary antigen-presenting cells in the immune system, play a critical role in regulating tumor immune responses. However, the tumor immunosuppressive microenvironment severely impedes the process of antigen-presenting and DC maturation, thereby limiting the efficacy of cancer immunotherapy. In this work, a pH-responsive polymer nanocarrier (PAG) modified with aminoguanidine (AG) was constructed for the efficient delivery of bortezomib (BTZ) through bidentate hydrogen bonds and electrostatic adsorption formed between guanidine groups of PAG and boronic acid groups of BTZ. The obtained PAG/BTZ nanoparticles exhibited pH-responsive release of BTZ and AG in the acidic tumor microenvironment. On the one hand, BTZ induced potent immune activation by eliciting immunogenic cell death (ICD) and releasing damage-associated molecular patterns. On the other hand, the cationic AG significantly promoted antigen uptake by DCs and activated DC maturation. As a result, PAG/BTZ significantly stimulated tumoral infiltration of cytotoxic T lymphocytes (CTLs) and triggered robust antitumor immune responses. Thus, it showed potent antitumor efficacy when synergizing with an immune checkpoint-blocking antibody.

Synthesis of Fluorinated Hydrazinylthiazole Derivatives: A Virtual and Experimental Approach to Diabetes Management.

A novel series of fluorophenyl-based thiazoles was synthesized following the Hanztsch method. All of the compounds were initially verified with physical parameters (color, melting point, retardation factor (R f)), which were further confirmed by several spectroscopic methods, including ultraviolet-visible (UV-visible), Fourier-transform infrared (FTIR), 1H, 13C, 19F NMR, and high-resolution mass spectrometry (HRMS). The binding interactions of all compounds were studied using a molecular docking simulation approach. Furthermore, each compound was evaluated for its alpha(α)-amylase, antiglycation, and antioxidant potentials. The biocompatibility of all compounds was checked with an in vitro hemolytic assay. All synthesized scaffolds were found biocompatible with minimal lysis of human erythrocytes as compared to the standard Triton X-100. Among the tested compounds, the analogue 3h (IC50 = 5.14 ± 0.03 μM) was found to be a highly potent candidate against α-amylase as compared to the standard (acarbose, IC50 = 5.55 ± 0.06 μM). The compounds 3d, 3f, 3i, and 3k exhibited excellent antiglycation inhibition potential with their IC50 values far less than the standard amino guanidine (IC50 = 0.403 ± 0.001 mg/mL). The antidiabetic potential was further supported by docking studies. Docking studies revealed that all synthesized compounds exhibited various interactions along enzyme active sites (pi-pi, H-bonding, van der Waals) with varied binding energies.

Nitric oxide and salt resistance in Dahl rats: no role of inducible NO synthase.

Inducible NO synthase (NOS II) was proposed to play an important role in salt resistance of Dahl salt-resistant (SR/Jr) rats. Its chronic inhibition by specific inhibitors was accompanied by blood pressure (BP) elevation in animals subjected to high salt intake. The aim of our study was to evaluate 1) whether such inhibitors affect BP and/or its particular components (sympathetic tone and NO-dependent vasodilation) only under the conditions of high salt intake, and 2) whether similar BP effects are elicited after systemic or intracerebroventricular (icv) application of these inhibitors. Wistar rats fed Altromin diet (0.45 % NaCl) and SR/Jr rats fed either a low-salt (LS, 0.3 % NaCl) or a high-salt (HS, 4 % NaCl) diet were studied. Aminoguanidine (AMG) and 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (AMT) were used as NOS II inhibitors. BP and its responses to acute blockade of renin-angiotensin system (captopril), sympathetic nervous system (pentolinium) and NO synthase (L-NAME) were measured in conscious cannulated rats. There were no significant changes of BP or its components in either Wistar rats or SR/Jr rats subjected to chronic inhibition of NOS II by peroral aminoguanidine administration (50 mg/kg/day for 4 weeks). This was true for SR/Jr rats fed either LS or HS diets. Furthermore, we have studied BP effects of chronic icv administration of both NOS II inhibitors in SR/Jr rats fed HS diet, but we failed to find any BP changes elicited by such treatment. In conclusion, inducible NO synthase does not participate in the resistance of SR/Jr rats to hypertensive effects of excess salt intake.

THE INFLUENCE OF SPERMINE AND AMINOGUANIDINE ON THE MORPHOFUNCTIONAL CHARACTERISTICS OF HUMAN PROSTATE CANCER CELL LINE LNCaP

Summary. Aim: to investigate the inhibitory effect of spermine (Spn) and the modifying effect of aminoguanidine (AG) on the structural and functional characteristics of human prostate cancer cells of the LNCaP line, depending on the mode of their use. Object and methods: studies were conducted in vitro on human prostate cancer (PC) cells of the hormone-dependent LNCaP line. Cell survival was determined by the trypan blue exclusion. The concentration of Spn used in the study was 1.5 and 5.0 mM, and AG in combination with Spn 1.5 mM. Changes in the morphology of LNCaP cells were evaluated under microscopr on the fixed cytological specimens of cells grown on coverslips and stained with hematoxylin and eosin. Results: Spn at a concentration of 1.5 mM exhibites a pronounced inhibitory effect on the growth of LNCaP cells reducing their survival. Cells die at a Spn concentration of 5.0 mM. The presence of AG (1.5 mM) in the culture medium contributes to a significant recovery of cell survival. Conclusions: the increase in Spn concentration from 1.5 mM to 5.0 mM seems to result in apoptotic death of LNCaP cells. In sublethal concentrations, Spn reduces the synthetic and suppresses the mitotic activity of LNCaP cells, while AG shows a certain protective effect.

Anti-glycation and anti-inflammatory activities of anthocyanins from purple vegetables.

Anthocyanins may be effective bioactive constituents to reduce the potential risk of chronic diseases induced by glycation and inflammation. In the present study, the anti-glycation and anti-inflammatory activities of anthocyanins derived from purple cabbage (PCA), purple sweet potato (PSP), purple corn (PCO) and gynura bicolor (GB) were evaluated. According to the results from the bovine serum albumin (BSA)-fructose and BSA-methylglyoxal (MGO) model, the inhibition effects of anthocyanins on non-enzymatic glycosylation not only acted on the intermediate stage, but also played a certain role in the entire non-enzymatic glycosylation process, among which anthocyanins from PCA exhibited the best inhibitory effect. The anthocyanins from all four purple vegetables could trap MGO effectively (p > 0.05). The anthocyanins also presented a good inhibitory effect on amyloid beta peptide (Aβ)1-42 fibrillation, even better than that of aminoguanidine (AG), in a thermal induction assay. Furthermore, anthocyanins from PCA, PSP, PCO and GB showed significant anti-inflammatory effects, inhibiting pro-inflammatory factor (i.e., NO and TNF-α) production, among which the anthocyanins from PCA and PSP exhibited a higher inhibition effect than the others. This is probably due to the suppression of the TLR4-mediated MyD88 signaling pathway in the lipopolysaccharide (LPS)-induced BV2 cells based on the western blot analysis. Anthocyanins from purple vegetables could be used as a value-added food ingredient for the food industry. Food fortification with anthocyanins might be a promising way to protect humans against various chronic diseases caused by glycation and inflammation.

Inhibition of nitric oxide synthase or cystathionine gamma-lyase abolishes leptin-induced fever in male rats

Leptin is an adipokine that regulates energy balance and immune function. Peripheral leptin administration elicits prostaglandin E₂-dependent fever in rats. The gasotransmitters nitric oxide (NO) and hydrogen sulfide (H₂S) are also involved in lipopolysaccharide (LPS)-induced fever response. However, there is no data in the literature indicating if these gasotransmitters have a role in leptin-induced fever response. Here, we investigate the inhibition of NO and H₂S enzymes neuronal nitric oxide synthase (nNOS), inducible nitric oxide synthase (iNOS), and cystathionine γ-lyase (CSE) in leptin-induced fever response, respectively. Selective nNOS inhibitor 7-nitroindazole (7-NI), selective iNOS inhibitor aminoguanidine (AG), and CSE inhibitor dl-propargylglycine (PAG) were administered intraperitoneally (ip). Body temperature (Tb), food intake, and body mass were recorded in fasted male rats. Leptin (0.5 mg/kg ip) induced a significant increase in Tb, whereas AG (50 mg/kg ip), 7-NI (10 mg/kg ip), or PAG (50 mg/kg ip) caused no changes in Tb. AG, 7-NI, or PAG abolished leptin increase in Tb. Our results highlight the potential involvement of iNOS, nNOS, and CSE in leptin-induced febrile response without affecting anorexic response to leptin in fasted male rats 24 h after leptin injection. Interestingly, all the inhibitors alone had the same anorexic effect induced by leptin. These findings have implications for understanding the role of NO and H₂S in leptin-induced febrile response.

Vanillic Acid Inhibited the Induced Glycation Using In Vitro and In Vivo Models.

Glycation is implicated in the pathophysiology of many diseases, including diabetes, cancer, neurodegenerative diseases, and aging. Several natural and synthetic compounds were investigated for their antiglycation activity. We evaluated the antiglycation effect of vanillic acid (VA) using in vitro and in vivo experimental models. In vitro, bovine serum albumin (BSA) (50 mg/ml) was incubated with glucose (50 mM) with or without VA at 1.0-100 mM for 1 week at 37°C, and then, excitation/emission fluorescence was measured at 370/440 nm to determine glycation inhibition. The cytoprotective effect of VA was evaluated using RAW 264.7 cells incubated with or without VA at 7.8-500 μM along with 100-400 μM of methylglyoxal for 48 hours, and cell viability was determined using the MTT assay. Aminoguanidine (AMG) was used as a positive control in both in vitro and cell culture experiments. In vivo, 52 streptozotocin-induced diabetic rats were randomly assigned to 4 groups and treated with 0, 1.5, 4.5, or 15 mg/kg VA for four weeks. Serum fructosamine and blood glycosylated hemoglobin (HbA1c) were then measured, and advanced glycation end-products (AGEs) were detected in the kidneys and the skin of deboned tails using an immunohistochemistry assay. VA caused a concentration-dependent effect against BSA glycation (IC50 of 45.53 mM vs. 5.09 mM for AMG). VA enhanced cell viability at all concentrations of VA and methylglyoxal. VA did not affect serum fructosamine or blood HbA1c levels, although it markedly decreased AGEs in the kidney in a dose-dependent manner and decreased AGEs in the skin of deboned tail tissues. VA had significant antiglycation activity at cellular and long-term glycation.

Abstract 13187: Inhibition of Advanced Glycation End Products Formation and Serum Protein Infiltration in Bioprosthetic Heart Valves: Investigations of Anti-Glycation Agents

Introduction: Bioprosthetic heart valves (BHV) fabricated from glutaraldehyde pretreated bovine pericardium (BP), are the most commonly used heart valve replacements. However, BHV durability is limited by structural valve degeneration (SVD) resulting from both calcification and advanced glycation end product (AGE) deposition together with serum protein infiltration. This study investigated, for the first time, the application of anti-glycation agents as a way to mitigate AGE-related structural degeneration of BHV. Hypothesis: We investigated the hypothesis that anti-AGE agents (Aminoguanidine (AG), Pyridoxamine (PYR), and N-Acetylcysteine (NAC)) could mitigate BP glycation in model studies, both in vitro and in vivo. Methods: To model AGE accumulation in BHV, we used well-established accelerated in vitro glycation models using BP incubation with or without inhibitors. Incorporation of AGE in BP was monitored using radiolabeled glycation precursors. Changes in collagen structure were examined by two photon confocal microscopy and differential scanning calorimetry. In vivo studies used a juvenile rat subdermal BP implantation model with drug administration via drinking water. Endpoints were AGE formation, collagen structure, thermal denaturation temperature, serum albumin uptake, calcification, and serum glycation marker levels. Results: In vitro studies demonstrated that each of these agents significantly inhibited AGE formation in BP. However, after 28-day rat subdermal BP implantation in vivo, only PYR demonstrated significant inhibition of both AGE and serum albumin accumulation per immunostaining. BHV calcification was not mitigated by PYR, AG or NAC. Conclusions: This study is the first to demonstrate the use of AGE inhibitors in mitigating BHV glycation. PYR demonstrated significant efficacy both in vitro and in vivo, mitigating AGE formation and albumin infiltration in BP. Thus, this study provides a rationale for investigating PYR as a possible candidate for improving the durability and biocompatibility of clinically used BHV, providing the solution for this unmet clinical need.