Aminoguanidine Treatment Research Articles

PRODUCTION OF CROSS-REACTIVE AUTOANTIBODY BINDING TO BOVINE SERUM ALBUMIN IN THE D-GALACTOSE-INDUCED AGING MOUSE MODEL

The D-galactose (D-gal)-induced animal model, generated by repeated subcutaneous D-gal injections over approximately 6 weeks, has been frequently used for diabetes and aging research. However, little research has investigated the direct correlation between D-gal and autoantibody formation despite several reports on diabetes-and aging-related autoantibodies. The purpose of this study was to determine whether repetitive injection of D-gal can induce autoantibody production in mice. First, we used Bovine Serum Albumin (BSA) and Advanced Glycation End products (AGE)-BSA as the test antigens. The immunoreactivity of serum samples from mice treated with D-gal for 6 weeks was evaluated using Enzyme-Linked Immunosorbent Assay (ELISA). We found that serum samples of D-gal-treated mice had significantly high antibody titers against both BSA and AGE-BSA. Furthermore, the result showed that aminoguanidine treatment, an AGE inhibitor tended to decrease this immunoreactivity. The results of competitive inhibition ELISA using BSA and AGE-BSA as the competitors suggested that the serum samples from D-gal-treated mice contained antibodies not only against BSA but also specific to AGE-BSA. To assess whether the immunoreactivity against BSA is comparable to that against Mouse Serum Albumin (MSA), we examined the reactivity of D-gal-induced antibodies against MSA. Unexpectedly, D-gal-induced antibodies did not react with MSA. This suggests that the production of antibodies by D-gal is in response to an unknown antigen(s), aside from MSA, in mice and that this unknown antigen(s) may share similar sequences or three-dimensional structures with BSA.

Parasitological, histopathological, and immunohistochemical assessment of nitric oxide synthase inhibitor: aminoguanidine versus albendazole in the treatment of experimental murine toxocariasis

Objectives The study was intended to evaluate the effect of aminoguanidine compared with that of albendazole on mice infected with eggs of Toxocara canis by parasitological, histopathological, and immunohistochemical studies. Background T. canis is a widely distributed parasite. The nematode can cause toxocariasis in man. The infection takes place after swallowing fully embryonated eggs with larvae inside. Materials and methods The study was conducted on 117 albino mice classified into four groups: GI (the control group with subgroups Ia, Ib, and Ic); GII (infected with T. canis eggs); GIII (infected with T. canis eggs and treated with albendazole); and GIV (infected with T. canis eggs and treated with aminoguanidine). Sera from different groups of mice were collected for nitrite assay. The lung and brain tissues were taken for larval recovery and histopathological and immunohistochemical [inducible nitric oxide synthase (iNOS)] studies. Results The mean larval count decreased significantly from 7th to 45th day post infection (d.p.i), with albendazole and aminoguanidine, which was more effective in decreasing larval count. Aminoguanidine treatment caused early improvement in histopathological lesions initiating from the second d.p.i.; however, albendazole improvement was observed on the seventh d.p.i. The grade of iNOs expression in GII and GIII was high as compared with that in GIV. There was significantly positive correlation between serum nitrite and the grade of iNOS expression, especially on early d.p.i only in groups II and III. Conclusion Both albendazole and aminoguanidine treatment for T. canis-infected mice caused decrease in larval count and improvement in histopathological lesions. Aminoguanidine was more effective with early response and decreased tissue damage.

Cardioprotective effect of aminoguanidine in combination with steroid therapy after blunt chest trauma.

Cardiac contusion is an important cause of mortality after blunt chest trauma (BCT). The aim of this study was to investigate the therapeutic efficacy of the usage of aminoguanidine (AG), in myocardial damage occurring after BCT, alone and in combination with methylprednisolone (MP). Thirty-five female Wistar albino rats were randomly assigned to five groups (n = 7) including: sham controls (S); only cardiac contusion (CONT); cardiac contusion treated with methylprednisolone (CONT+MP); cardiac contusion treated with aminoguanidine (CONT+AG); and cardiac contusion treated with methylprednisolone and aminoguanidine (CONT+MP+AG). Seven days following the treatments, heart and serum specimens were evaluated histopathologically, immunohistochemically, and biochemically in all groups. Serum AOPP and Tn-I levels increased significantly after cardiac contusions. Haemorrhage, tissue degeneration, and necrosis development was evident following contusions. Increased iNOS expression in myocardial tissue was significantly decreased in the CONT+AG+MP group compared to CONT+AG and CONT+MP groups (p = 0.001 and p = 0.011, respectively). The combined treatment of AG and MP increased Bcl-2 expression significantly after contusions compared to the other treatment groups. Combined usage of AG, a selective iNOS inhibitor, with MP, in cardiac contusions, showed a more powerful cardioprotective effect by increasing Bcl-2 expression and reducing iNOS expression (Tab. 3, Fig. 4, Ref. 33).

Effects of aminoguanidine, a potent nitric oxide synthase inhibitor, on myocardial and organ structure in a rat model of hemorrhagic shock.

Background:Nitric oxide (NO) has been shown to increase following hemorrhagic shock (HS). Peroxynitrite is produced by the reaction of NO with reactive oxygen species, leads to nitrosative stress mediated organ injury. We examined the protective effects of a potent inhibitor of NO synthase, aminoguanidine (AG), on myocardial and multiple organ structure in a rat model of HS.Materials and Methods:Male Sprague Dawley rats (300-350 g) were assigned to 3 experimental groups (n = 6 per group): (1) Normotensive rats (N), (2) HS rats and (3) HS rats treated with AG (HS-AG). Rats were hemorrhaged over 60 min to reach a mean arterial blood pressure of 40 mmHg. Rats were treated with 1 ml of 60 mg/kg AG intra-arterially after 60 min HS. Resuscitation was performed in vivo by the reinfusion of the shed blood for 30 min to restore normo-tension. Biopsy samples were taken for light and electron microscopy.Results:Histological examination of hemorrhagic shocked untreated rats revealed structural damage. Less histological damage was observed in multiple organs in AG-treated rats. AG-treatment decreased the number of inflammatory cells and mitochondrial swollen in myocardial cells.Conclusion:AG treatment reduced microscopic damage and injury in multiple organs in a HS model in rats.

Extract of Cassiae semen attenuates diabetic nephropathy via inhibition of advanced glycation end products accumulation in streptozotocin-induced diabetic rats

Chronic hyperglycemia leads to the formation of advanced glycation end products (AGEs), which accelerates the development of diabetic complications. Previous studies have shown that extract of Cassiae semen (CS), the seed of Cassia tora, has inhibitory activity on AGEs formation in vitro and reduces transforming growth factor-beta1 (TGF-β1) and extracellular matrix protein expression via inhibition of AGEs-mediated signaling in glomerular mesangial cells. In this study, to examine the preventive effects of CS extract on the development of diabetic nephropathy in vivo, streptozotocin (STZ)-injected diabetic rats were orally administered CS extract (200mg/kg body weight/day) for 12 weeks. Serum glucose, triglycerides, and total cholesterol in diabetic rats were significantly higher compared to control rats. CS or aminoguanidine (AG) treatment significantly reduced these factors. Proteinuria and creatinine clearance were also significantly decreased in the CS-treated group compared with the untreated diabetic group. The CS-treated group had significantly inhibited COX-2 mRNA and protein, which mediates the symptoms of inflammation in the renal cortex of diabetic rats. Furthermore, histopathological studies of kidney tissue showed that in diabetic rats, AGEs, the receptor for AGEs, TGF-β1, and collagen IV were suppressed by CS treatment. Our data suggest that oral treatment of CS can inhibit the development of diabetic nephropathy via inhibition of AGEs accumulation in STZ-induced diabetic rats.

Aminoguanidine changes hippocampal expression of apoptosis-related genes, improves passive avoidance learning and memory in streptozotocin-induced diabetic rats.

Cognitive dysfunction occurs in patients with diabetes mellitus. The objective of this study was to examine whether bilateral intrahippocampal CA1 (intra-CA1) injection of aminoguanidine (AG) can either affect the Bcl-2 family gene expression or reduce the diabetic imposing abnormalities of passive avoidance learning (PAL) and memory. Rats were divided into five groups: control (C), control treated with normal saline (CS), control treated with AG (S-AG), diabetics (D), and diabetics treated with AG (D-AG). Diabetes mellitus was induced by a single intraperitoneal injection of streptozotocin (STZ) (50 mg/kg). AG (30 μg/rat) or vehicle was administered intra-CA1 bilaterally at the onset of hyperglycemia. PAL was assessed 7 weeks later. Animals were killed, and hippocampus was dissected following the behavioral test. The expressions of Bax, Bcl-2, and Bcl-xl mRNAs were measured using semiquantitative RT-PCR technique. The result of passive avoidance task showed that AG significantly improved the cognitive performance in diabetic rats. Moreover, AG treatment decreased the levels of Bcl-2 and Bcl-xL expressions in diabetic group. The ratio of Bax/Bcl-2 and Bax/Bcl-xL decreased significantly in AG-treated diabetic animals. In conclusion, initial treatment with AG by intra-CA1 micro-injection improves the impaired passive avoidance task in STZ-induced diabetic rats which may be related to the decreased Bax/Bcl-2 and Bax/Bcl-xL ratios.

Sequence analysis of diamine oxidase gene from fava bean and its expression related to γ‐aminobutyric acid accumulation in seeds germinating under hypoxia‐NaCl stress

γ-Aminobutyric acid (GABA) is synthesized via the polyamine degradation pathway in plants, with diamine oxidase (DAO) being the key enzyme. In this study the cDNA of DAO in fava bean was cloned and its expression in seeds germinating under hypoxia-NaCl stress was investigated. Fava bean DAO cDNA is 2199 bp long and contains 2025 bp of open reading frame that encodes 675 amino acid peptides with a calculated molecular weight of 76.31 kDa and a pI of 5.41. Hypoxia and hypoxia-NaCl stress enhanced DAO activity and resulted in GABA accumulation in germinating fava bean. However, DAO gene expression was down-regulated under hypoxia compared with non-stress condition, while its expression in the cotyledon and shoot was up-regulated under hypoxia-NaCl. In addition, DAO expression could be promoted to enhance GABA accumulation after increasing the stress intensity using NaCl. DAO gene expression was significantly inhibited by aminoguanidine treatment under hypoxia but increased under hypoxia-NaCl. Under hypoxia, GABA accumulation due to NaCl was mainly concentrated in the cotyledon. The GABA content increase under hypoxia did not result from DAO gene expression, but DAO existing in seeds was activated under hypoxia. DAO gene expression was up-regulated to enhance GABA accumulation after increasing the stress intensity.

Nitro-oxidative stress in experimental periodontitis

In chronic inflammations nitro-oxidative stress may induce secondary pathological processes. Therefore, in the present study we aimed to evaluate nitro-oxidative stress in animals with chronic experimental periodontitis. A rat model of ligature-induced experimental periodontitis was used. The study groups (n = 12) were: group I - negative control of healthy rats; group II – periodontitis (PER); group III - periodontitis + aminoguanidine (AG) (50 mg/kg/d i.p.); group IV – periodontitis + NG-nitro-L-arginine methyl ester (NAME) (5 mg/kg/d i.p.); group V – periodontitis + L-arginine (ARG) (100 mg/kg/d i.p.); group VI – periodontitis + Trolox (20 mg/kg/d i.p). Periodontitis was induced in 2 weeks and the treatments were administrated for one week. Blood samples were harvested at completion the study. Nitro-oxidative stress was assessed by total oxidative status (TOS), total antioxidative capacity (TAC), oxidative stress index (OSI) and total serum nitrates and nitrites (NOx). Systemic NOx, TOS and OSI increased significantly. AG, NAME and Trolox treatments reduced NOx, TOS and OSI. TAR was very low at PER animals, and increased significantly after AG, NAME and Trolox. ARG increased NOx, TOS and OSI, but did not influence TAR. In conclusion in rat experimental periodontitis systemic nitro-oxidative stress was important, and iNOS was the main enzyme involved in NO synthesis. NO synthesis was not involved in the antioxidant activity.

Effects of aminoguanidine on retinal apoptosis in mice with oxygen-induced retinopathy.

To explore the protective effects of aminoguanidine (AG) on retinal apoptosis in mice with oxygen-induced retinopathy (OIR). A total of 80 C57BL/6J mice, aged 7 days, were randomly divided into four groups: normal, high oxygen, high oxygen saline and high oxygen treated with AG. In the normal group, mice were housed in normoxic conditions from postnatal day P7 to P17. Mice in the other 3 groups were placed under hyperoxic conditions (75±2%O2) in an oxygen-regulated chamber for 5 days and subsequently placed in normoxic conditions for 5 days. Mice in the AG group were treated once daily, from P12 to P17, with AG hemisulfate (100mg/kg body weight, intraperitoneally) dissolved in physiological saline. An equivalent amount of 0.9% physiological saline was administered, as above, to mice in the high oxygen saline group. Ten mice were randomly selected from each group on P14 and on P17, euthanized and the retinas examined. Apoptotic cells in the retina were detected using the terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) method. The expression of nitric oxide synthase (iNOS) in the retina was detected by immunohistochemistry and changes in rod cells were observed using electron microscopy. TUNEL-positive cells and iNOS immunoreactive neurons were present in the inner nuclear and ganglion cell retinal layers of mice in the high oxygen group. The number of TUNEL-positive cells was significantly greater in the high oxygen group compared with the normal group (t=-20.81, P 14d <0.05; t=-15.05, P 17d <0.05). However, the number of TUNEL-positive cells in the AG treatment group was significantly lower (t=-13.21, P 14d<0.05; t=-6.61, P 17d <0.05) compared with the high oxygen group. The expression of iNOS was significantly higher in the high oxygen group compared with the normal group (t=-21.95, P 14d<0.05; t=-17.30, P 17d<0.05). However, the expression of iNOS in the AG treatment group was significantly lower (t=-12.17, P 14d<0.05; t=-10.30, P 17d<0.05) compared with the high oxygen group. The outer segments of the rods were disorganized and short in the high oxygen group. Rod morphology appeared to be slightly improved in the AG group. AG may protect retinal neurons in OIR by inhibiting apoptosis. The mechanism may be related to iNOS.

Aging-Dependent Reduction in Glyoxalase 1 Delays Wound Healing

Methylglyoxal (MG), the major dicarbonyl substrate of the enzyme glyoxalase 1 (GLO1), is a reactive metabolite formed via glycolytic flux. Decreased GLO1 activity in situ has been shown to result in an accumulation of MG and increased formation of advanced glycation endproducts, both of which can accumulate during physiological aging and at an accelerated rate in diabetes and other chronic degenerative diseases. To determine the physiological consequences which result from elevated MG levels and the role of MG and GLO1 in aging, wound healing in young (≤12 weeks) and old (≥52 weeks) wild-type mice was studied. Old mice were found to have a significantly slower rate of wound healing compared to young mice (74.9 ± 2.2 vs. 55.4 ± 1.5% wound closure at day 6; 26% decrease; p < 0.0001). This was associated with decreases in GLO1 transcription, expression and activity. The importance of GLO1 was confirmed in mice by inhibition of GLO1. Direct application of MG to the wounds of young mice, decreased wound healing by 24% compared to untreated mice, whereas application of BSA modified minimally by MG had no effect. Treatment of either young or old mice with aminoguanidine, a scavenger of free MG, significantly increased wound closure by 16% (66.8 ± 1.6 vs. 77.2 ± 3.1%; p < 0.05) and 64% (40.4 ± 7.9 vs. 66.4 ± 5.2%; p < 0.05), respectively, by day 6. As a result of the aminoguanidine treatment, the overall rate of wound healing in the old mice was restored to the level observed in the young mice. These findings were confirmed in vitro, as MG reduced migration and proliferation of fibroblasts derived from young and old, wild-type mice. The data demonstrate that the balance between MG and age-dependent GLO1 downregulation contributes to delayed wound healing in old mice.

Differential effects of selective and non-selective nitric oxide synthase inhibitors on the blood perfusion of ischemia-reperfused myocardium in dogs

BackgroundNitric oxide (NO) is protective for the cardiovascular system, and excessive NO exerts negative effects on the circulatory system. This study aimed to compare the effects of selective or non-selective NO synthase (NOS) inhibitors on blood flow perfusion of ischemia-reperfused myocardium.Materials/MethodsMale mongrel dogs were randomly assigned to 4 groups: only ischemia-reperfusion (control), ischemia-reperfusion plus Nω-nitro-L-arginine methyl ester (NAME) treatment, ischemia-reperfusion plus aminoguanidine (AMD) treatment, and sham operation group. Myocardial contrast echocardiography (MCE) was performed. Blood samples were taken for measurement of NO. Background-subtracted peak videointensity (PVI) and PVI ratio in myocardium were measured.ResultsIn the NAME-treated group, the PVI at 5 min reperfusion did not significantly differ from pre-LAD-occlusion, but declined to and retained at a level obviously lower than the pre-LAD-occlusion. In the AMD-treated group, the PVI at 5 min reperfusion was significantly higher than at pre-LAD-occlusion, and then restored to and remained at the pre-LAD-occlusion level. The changes of PVI ratios in the 3 groups were similar to PVI values. In the AMD-treated group, the curve width increased in the early reperfusion, but returned to the pre-LAD-occlusion level at 90 min reperfusion. The plasma NO concentration in the NAME-treated group greatly decreased and remained low during the whole period of reperfusion. In the AMD-treated group, there were only slight increases in NO concentrations during reperfusion.ConclusionsNAME totally inhibited NO production and attenuated myocardial blood flow perfusion. Aminoguanidine significantly relieved the increase in NO production and alleviated the congestion of reperfused myocardium. Selective inhibitors of iNOS might be useful in the management of certain diseases associated with ischemia-reperfusion.

Original article Suppression of the lipid peroxidation process in the CNS reduces neurological expression of experimentally induced autoimmune encephalomyelitis

Here we report the influence of malondialdehyde (MDA) as a measure of the lipid peroxidation process (LP), on multiple sclerosis (MS) pathogenesis and its neurological signs, during the treatment with aminoguanidine (AG) - a selective inducible nitric oxide synthase inhibitor and N-Acetyl cysteine (NAC) - an oxidative scavenger, in the experimental autoimmune encephalomyelitis (EAE), an animal model for studying MS. Encephalomyelitis induction by the subcutaneous injection of myelin basic protein of bovine type, dissolved in phosphate buffered saline (PBS) emulsified in equal volume of the complete Freund's adjuvant (CFA), was described in detail in our earlier published papers. Each of animals was randomly assigned to seven groups - control (PBS), EAE, CFA, EAE + AG, AG, EAE + NAC and NAC group. In each animal, the development of neurological signs of EAE was scored, these results were published earlier. MDA was evaluated in the central nervous system (CNS) structure - cerebellums and spinal cords. The obtained results show that the AG and NAC treatment significantly reduces the MDA level in both examined tissues (p < 0.05) ameliorating at the same time EAE clinical signs (p < 0.05). Taking together our present and earlier findings we conclude that LP may provoke and promote MS, while blocking of this process results in amelioration of the clinical onset and disease activity. These results may be useful as a new insight into mechanisms and potential targets for therapeutic strategies in MS.

Beneficial effects of aminoguanidine against streptozotocin-induced pathological changes in diabetic mice kidney

To investigate whether aminoguanidine ameliorates streptozotocin-induced renal oxidative stress and inflammation in diabetic mice. Diabetic nephropathy is characterized by structural and functional changes in both glomerular, tubular and finally impairment of renal function. Streptozotocin [100mg/kg body weight dissolved in freshly prepared citrate buffer and administered through tail vein injection] was used to induce diabetes in male Swiss albino male mice and received standard diet throughout study period [8weeks]. Diabetic mice group received aminoguanidine [100mg/kg/day]. We studied streptozotocin-induced renal changes by noticing body weight, kidney weight and their ratio, histological and electron microscopic studies. Antioxidant activities like glutathione, superoxide dismutase and catalase were analyzed. RT-PCR gene expression studies were used to analyze the inhibitory effect of aminoguanidine against diabetes-induced IL-1β, IL-6 and TNF-α. STZ-diabetic mice showed significantly increased oxidative stress indices like melonaldehyde and nitric oxide levels, reduced endogenous antioxidant enzyme activities, whereas aminoguanidine treatment normalizes these pathological changes and may play a crucial role in oxidative stress-related changes during STZ-induced diabetic mice. Aminoguanidine treatment restored diabetes-induced structural and biochemical alterations. RT-PCR gene expression studies suggests that aminoguanidine protect STZ-induced renal pathological changes probably by attenuating diabetes-induced IL-1β, IL-6 and TNF-α gene expression.

In vivo inhibition of inducible nitric oxide synthase by aminoguanidine influences free radicals production and macrophage activity in Trichinella spiralis infected low responders (C57BL/6) and high responders (BALB/c) mice

Abstract The influence of aminoguanidine (AG) — inhibitor of inducible nitric oxide synthase (iNOS), on macrophage activity and free radicals level was examined during Trichinella spiralis infection in two strains of mice: C57BL/6 and BALB/c. AG was administered either between 1–5 days post infection (dpi) for intestinal phase examinations or between 16–29 dpi for muscle phase examinations. The number of peritoneal macrophages and level of nitric oxide NO) and hydrogen peroxide (H2O2) in biological fluids were determined in both strains after infection or infection together with AG treatment as well as in control uninfected mice. The performed studies have proved, that free radicals play role in host immune response during intestinal and muscle phase of T.spiralis infection in mice. Inflammatory response in peritoneal cavity was delayed during infection in low responders C57BL/6 mice in comparison with high responders BALB/c mice. C57BL/6 mice are Th-1 like strain and react stronger to AG in contrary to BALB/c being Th-2 like strain. It was manifested as changes and fluctuations of free radicals levels and in the number of peritoneal macrophages after AG treatment in C57BL/6 mice. A weak or no reaction on AG injection in BALB/c mice is responsible for more stable and more effective defense response of the host to T. spiralis infection.

The effect of aminoguanidine on the kidney of diabetic albino Balb/c mice

The aim of this study is to find out how activated inducible nitric oxide synthase (iNOS) and nitric oxide (NO) affect kidney tissue in streptozotocin (STZ)-induced diabetic mice and whether its influence can be prevented by aminoguanidine (AG), a specific iNOS inhibitor. Twenty-four male mice were divided into four study groups (n=6) receiving a daily dose of 100 mg.kg-1 AG for 90 days (Group AG), a single dose of 150 mg.kg-1 STZ (Group STZ), a single dose of 150 mg.kg-1 STZ followed by daily administration of 100 mg.kg-1 AG for 90 days (Group STZ-AG), and intraperitoneally injections of saline only (Group Control) for 90 days. Dispersion of NADPH-diaphorase (NADPH-d) was stronger in the kidney sections of STZ-treated animals compared with the controls. STZ treatment caused disruption of continuity of the brush borders in proximal tubules, glomerular endothelial damage, and considerable renin granules in the juxtaglomerular cells. AG administration following STZ treatment partly prevented histological and cytological changes in kidney cortex, and renin dispersion was similar to that in control animals. We found that increased inducible nitric oxide (iNO) caused kidney tissue degeneration that could be prevented to some extent by AG treatment. There is a possible relationship between increased iNOS and dispersion of renin granules in juxtaglomerular cells in diabetes.

Polycystic ovary syndrome (PCOS)-like phenotypes in the d-galactose-induced aging mouse model

The d-galactose (d-gal)-induced animal model, which is established by consecutive subcutaneous d-gal injections for approximately 6weeks, has been frequently used for aging research. This animal model has been shown to accelerate aging of the brain, kidneys, liver, and blood cells. However, aging of the female reproductive organs in this animal model has not been reported. The aim of this study was to investigate changes in the ovary in the d-gal-induced aging mouse model. First, we evaluated anti-Müllerian hormone (AMH) as a marker of ovarian aging in blood plasma. We speculated there would be lower AMH levels in d-gal-treated mice because ovarian aging would be induced by d-gal, as reported for other tissues. However, the results showed that AMH levels in d-gal-treated mice were approximately four-fold higher than control mice. Abnormally high AMH levels are detected in ovarian cancer and polycystic ovary syndrome (PCOS) patients. Therefore, we examined PCOS-related markers in this mouse model. Total testosterone levels were high and abnormal estrous cycles were induced in d-gal-treated mice. These changes, including AMH levels, in d-gal-treated mice were inhibited by aminoguanidine treatment, an advanced glycation end product reducer. In addition, ovarian cysts were observed in some d-gal-treated mice. These results indicate that with respect to female reproduction, d-gal-treated mice are suitable for PCOS studies, rather than aging studies.

Influence of aminoguanidine on retina morphology and expression of nitric oxide and inducible nitric oxide synthase after retina ischemia-reperfusion injury

Background Many eye diseases such as central retinal artery occlusion,glaucoma and ischemic optic neuropathy,etc.lead to retinal ischemia-reperfusion injury (RIRI) and furthmore visual functional damage.It is neeessary to study the treatment of RIRI.Objective This study was to observe and discuss the influence of aminoguanidine on the retina morphological changes and its mechanism after RIRI.Methods Eighty clean healthy male Japanese white rabbits were randomly divided into normal injury group,RIRI group and aminoguanidine (AG)treated group.The model of RIRI was established by infusing saline solution into the anterior chamber to elevate intraocular pressure (IOP) in both RIRI group and AG group.AG was intraperitoneally injected in the models of the AG group,and normal saline solution was used at the same method in tbe normal group and the RIRI group.The fundus photography and fundus fluorescein angiography(FFA) were pertormed on the rabbits at the moment of retina ischemia and 6,24 and 72 hours after reperfusion.The parts of rabbits were sacrificed 1,6,24 and 72 hours after reperfusion,followed by the enucleation of the eyeballs.Retinal section was prepared for TUNEL examination to evaluate the apoptosis of retinal cells.Nitric oxide (NO) concentration in retina was detected with nitrate reductase,and the activity of inducible nitric oxide synthase (iNOS) was measured by colorimetric detection.The use of the animals followed the Regulations for the Administration of Affairs Concerning Experimental Animals by State Science and Technology Commission.Results The fundus photography and FFA showed that the retinal edema was more mild,and the percentage of vascular occlusion was lower in the AG treatment group than that in RIRI group and the amount and area of fluorescein leakage were also smaller than the treatment group.The numbers of TUNEL positive cells in the AG treatment group were less than those in the RIRI model group at 1,6,24 and 72 hours after experiment (F分组 =2762.37,P =0.00 ; F时间 =894.24,P =0.00).Numbers of TUNEL positive cells between adjacent time points were significantly different in both RIRI model group and AG treatment group (RIRI group:q =24.475,36.591,-20.37,P＜0.05;AG group:q =20.94,16.79,-6.92,P＜0.05),with the peak value at 24 hours after experiment.NO contents were significantly higher in the RIRI model group compared to AG group at various time points(q =3.84,4.01,8.91,3.75,P＜0.05),and those between adjacent time points showed significant differences (RIRI group:q=4.77,13.403,-10.29,P＜0.05;AG group:q=4.55,9.05,-5.08,P＜0.05).iNOS activity was significantly elevated in the RIRI model group compared with AG group(q=-3.74,-4.94,-6.53,-3.98,P＜0.05),and obvious differences also were seen between the adjacent time points in both two groups(RIRI group:q =8.43,6.71,-6.39,P＜0.05 ;AG group:q =4.16,5.08,-3.93,P＜0.05).Conclusions Aminoguanidine can protect the retinal function and morpbology from oxidative stress damage after RIRI by reducing the NO level and inhibiting the iNOS activity in retina. Key words: Retina ischemia-reperfusion injury; Aminoguanidine; Apoptosis; Nitric oxide; Inducible nitric oxide synthase

Aminoguanidine prevents testicular damage-induced-2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in male rats

In this study, it was aimed to determinate protective effects of aminoguanidine (AG) against reproductive toxicity caused by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), an environmental contaminant. Thirty-two rats were equally divided into four groups; the first group was kept as control and given corn oil as carrier. In second and third groups, TCDD and AG were orally administered at the dose of 2μgkg(-1) per week and 100mgkg(-1) per day for 45days, respectively. In fourth group, TCDD and AG were given together at the same doses. Although TCDD significantly increased the formation of TBARS, it caused a significant decline in the levels of GSH, CAT, GPx and SOD in rats. On the other hand, AG, given together TCDD, reversed TCDD effects on TBARS SOD, GSH, GPx and CAT. In addition, sperm characteristics negatively affected and histopathological deformation occurred with TCDD exposure. However, AG treatment partly prevented these toxic effects of TCDD on spermatological parameters and histopathological changes. In conclusion, TCDD exposure induces testicular damage (oxidative stress, histopathological damage and sperm parameters), and AG treatment reversed TCDD-induced testicular damage in rats. Thus, AG may be useful for the prevention and treatment of TCDD-induced male infertility problems.

Aminoguanidine normalizes ET-1-induced aortic contraction in type 2 diabetic Otsuka Long–Evans Tokushima Fatty (OLETF) rats by suppressing Jab1-mediated increase in ETA-receptor expression

Circulating levels of endothelin (ET)-1 are increased in the diabetic state, as is endogenous ETA-receptor-mediated vasoconstriction. However, the responsible mechanisms remain unknown. We hypothesized that ET-1-induced vasoconstriction is augmented in type 2 diabetes with hyperglycemia through an increment in advanced glycation end-products (AGEs). So, we investigated whether treatment with aminoguanidine (AG), an inhibitor of AGEs, would normalize the ET-1-induced contraction induced by ET-1 in strips of thoracic aortas isolated from OLETF rats at the chronic stage of diabetes. In such aortas (vs. those from age-matched genetic control LETO rats): (1) the ET-1-induced contraction was enhanced, (2) the levels of HIF1α/ECE1/plasma ET-1 and plasma CML-AGEs were increased, (3) the ET-1-stimulated ERK phosphorylation mediated by ETA-R was increased, (4) the expression level of Jab1-modified ETA-R protein was reduced, and (5) the expression level of O-GlcNAcylated ETA-R protein was increased. Aortas isolated from such OLETF rats that had been treated with AG (50mg/kg/day for 10 weeks) exhibited reduced ET-1-induced contraction, suppressed ET-1-stimulated ERK phosphorylation accompanied by down-regulation of ETA-R, and increased modification of ETA-R by Jab1. Such AG-treated rats exhibited normalized plasma ET-1 and CML-AGE levels, and their aortas exhibited decreased HIF1α/ECE1 expression. However, such AG treatment did not alter the elevated levels of plasma glucose or insulin, or systolic blood pressure seen in OLETF rats. These data from the OLETF model suggest that within the timescale studied here, AG normalizes ET-1-induced aortic contraction by suppressing ETA-R/ERK activities and/or by normalizing the imbalance between Jab1 and O-GlcNAc in type 2 diabetes.

INOS‐Derived Nitric Oxide Stimulates Osteoclast Activity and Alveolar Bone Loss in Ligature‐Induced Periodontitis in Rats

Inflammatory stimuli activate inducible nitric oxide synthase (iNOS) in a variety of cell types, including osteoclasts (OC) and osteoblasts, resulting in sustained NO production. In this study, we evaluate the alveolar bone loss in rats with periodontitis under long-term iNOS inhibition, and the differentiation and activity of OC from iNOS-knockout (KO) mice in vitro. Oral aminoguanidine (an iNOS inhibitor) or water treatment was started 2 weeks before induction of periodontitis. Rats were sacrificed 3, 7, or 14 days after ligature placement, and alveolar bone loss was evaluated. In vitro OC culture experiments were also performed to study the differentiation of freshly isolated bone marrow cells from both iNOS KO and wild-type C57BL/6 mice. OC were counted 6 days later after tartrate-resistant acid phosphatase staining (a marker of osteoclast identity), and bone resorption activity was assessed by counting the number of resorption pits on dentin disks. Rats with ligature showed progressive and significant alveolar bone loss compared to sham animals, and aminoguanidine treatment significantly inhibited ligature-induced bone loss at 7 and 14 days after the induction. In comparison to bone marrow cells from wild-type mice, cells from iNOS KO mice showed decreased OC growth and the resulting OC covered a smaller culture dish area and generated fewer resorption pit counts. Our results demonstrate that iNOS inhibition prevents alveolar bone loss in a rat model of ligature-induced periodontitis, thus confirming that iNOS-derived NO plays a crucial role in the pathogenesis of periodontitis, probably by stimulating OC differentiation and activity.