OBJECTIVETo evaluate the effects of aminoguanidine on methylglyoxal-mediated oxygen-glucose deprivation (OGD) injury in the cultured human brain microvascular endothelial cells (HBMEC).

METHODSCultured HBMEC cells were pretreated with methylglyoxal before oxygen-glucose deprivation injury. Cell vitality was determined by MTT method, cell mortality was assessed by LDH release method, cell apoptosis was examined by Annexin V/PI formation method, and the advanced glycation end products (AGEs) were detected by Western-blot.

RESULTSMethylglyoxal induced HBMEC injury in a dose-dependent manner. At 2 mmol/L of methylglyoxal, the cell viability was 56.1% when methylglyoxal-pretreated cells exposed to oxygen-glucose deprivation, the cell inhibition rate was 90.0%. Aminoguanidine (1 mmol/L) inhibited methylglyoxal and OGD induced LDH release and Annexin V/PI formation. Furthermore, aminoguanidine (1 mmol/L) also decreased advanced glycation end products (AGEs) formation induced by methylglyoxal and oxygen-glucose deprivation.

CONCLUSIONAminoguanidine protected methylglyoxal mediated-oxygen-glucose deprivation injury in the cultured HBMEC, which may be associated with anti-glycation activity.

Apoptosis ; drug effects ; Cell Hypoxia ; drug effects ; Cell Survival ; drug effects ; Cells, Cultured ; Drug Antagonism ; Endothelial Cells ; drug effects ; metabolism ; pathology ; Endothelium, Vascular ; cytology ; Glycation End Products, Advanced ; metabolism ; Guanidines ; pharmacology ; Humans ; Pyruvaldehyde ; pharmacology

This study was purposed to explore the changes of possible angiogenetic factors other than VEGF after inhibition of NHE1 and their related mechanisms. The K562 cells were treated by NHE1 specific inhibitor cariporide, the angiogenesis factors after inhibition of NHE1 were screened by using protein chip, the IL-8 expression level after cariporide treatment was detected by real-time quantitative PCR; the K562 cells with stable interference of NHE1 were constructed, the IL-8 expression level after interference of NHE1 was detected by real-time quantitative PCR; the p38 phosphorylation level in K562 cells treated with cariporide was detected by Western blot. After treatment of K562 cells with p38 inhibitor SB203580, the IL-8 expression level was decreased by real-time quantitative PCR. The results of protein chip showed that IL-8 expression decreased after cariporide treatment. Real-time quantitative PCR confirmed this inhibitory effect. The p38 phosphorylation level increased after cariporide treatment. The down-regulation of IL-8 expression induced by cariporide treatment was partially restored after K562 cells were treated with p38 inhibitor SB203580. It is concluded that the inhibition of NHE1 can inhibit IL-8 expression through up-regulation of p38 phosphorylation.
Cation Transport Proteins ; antagonists & inhibitors ; Down-Regulation ; Guanidines ; pharmacology ; Humans ; Imidazoles ; pharmacology ; Interleukin-8 ; metabolism ; K562 Cells ; Phosphorylation ; drug effects ; Pyridines ; pharmacology ; Sodium-Hydrogen Exchanger 1 ; Sodium-Hydrogen Exchangers ; antagonists & inhibitors ; Sulfones ; pharmacology ; p38 Mitogen-Activated Protein Kinases ; metabolism

OBJECTIVETo explore the effects of intrathecal injection of neuronal nitric oxide synthase (nNOS) inhibitors 7-Nitroindazole (7-Ni) and inducible nitric oxide synthase(iNOS) inhibitors aminoguanidine (AG) on the behavioral changes of morphine-induced dependent and withdrawal rats; the expression of Fos, nNOS and iNOS in spinal cord.

METHODSTo set up morphine dependence model, rats were subcutaneously injected with morphine (twice a day, for 5 d). The dose of morphine was 10 mg/kg in the first day and was increased by 10 mg/ kg every day. On day 6, 4 h after the injection of morphine (50 mg/kg), morphine withdrawal syndrome was precipitated by an injection of naloxone (4 mg/kg ip). 7-Ni, an nNOS inhibitor or iNOS inhibitors AG were intrathecally injected 30 min before the administration of naloxone respectively. The scores of morphine withdrawal symptom and morphine withdrawal-induced allodynia were observed. One hour after naloxone-precipitated withdrawal, Fos protein expression was assessed by immunohistochemical analysis and Western blot was used to detect the expression of nNOS and iNOS in the rat spinal cord.

RESULTSIntrathecal administration of nNOS inhibitor 7-Ni and iNOS inhibitors AG decreased the scores of morphine withdrawal, attenuated morphine withdrawal-induced allodynia and also inhibited the increase of Fos protein expression in the spinal cord of morphine withdrawal rats. nNOS and iNOS positive neurons in dorsal horn in nNOS group and iNOS group were significantly lower than that in withdrawal group. Compared with withdrawal group, level of nNOS and iNOS protein in spinal cord in nNOS group and iNOS group were significantly lower.

CONCLUSIONIt is suggested that nNOS and iNOS in the spinal cord may contribute to naloxone-precipitated withdrawal in rats and may play different roles in the above-mentioned effect.

Animals ; Guanidines ; pharmacology ; Indazoles ; pharmacology ; Male ; Morphine Dependence ; metabolism ; Naloxone ; pharmacology ; Nitric Oxide Synthase Type I ; antagonists & inhibitors ; metabolism ; Nitric Oxide Synthase Type II ; antagonists & inhibitors ; metabolism ; Rats ; Rats, Sprague-Dawley ; Spinal Cord ; drug effects ; metabolism ; Substance Withdrawal Syndrome ; metabolism

OBJECTIVETo investigate the effects of aminoguanidine cream on the proliferation of keratinocytes (KC), content of advanced glycosylation end products (AGE) and oxidative stress in skin tissue of rats with diabetes.

METHODSStearic acid, liquid paraffin, vaseline, lanolin, isopropyl myristate fat, glycerol, 50 g/L alcohol paraben, aminoguanidine hydrochloride etc. were mixed in certain proportion to make aminoguanidine cream, and cream without aminoguanidine was used as matrix. The dorsal skin of normal rats were harvested and treated by aminoguanidine cream with dose of 5, 10 g/L, or 5 g/L together with 10 g/L azone. The transdermal effect was respectively measured at post treatment hour 2, 4, 7, 10, 12, 24. Thirty SD rats were divided into normal control (NC, n = 6), diabetes (D, n = 8), aminoguanidine cream-interfered (AI, n = 8), matrix cream-interfered groups (MI, n = 8) according to the random number table. Diabetes was reproduced by intraperitoneal injection of STZ (65 mg/kg) in rats of D, AI, and MI groups, and rats in NC group were injected with 0.05 mmol/L citrate buffer as control. One week later, dorsal skin of rats in AI and MI groups were respectively treated with 10 g/L aminoguanidine cream and matrix cream by external use for 4 weeks. AGE content was determined with fluorescence detection from skin collagen extract. KC cell cycle was detected by flow cytometry. Skin tissue specimens were obtained for determination of levels of superoxide dismutase (SOD), malondialdehyde (MDA), myeloperoxidase (MPO), and total antioxidant capacity. Data were processed with t test.

RESULTSTransdermal effect of aminoguanidine cream with dose of 10 g/L was better than that with 5 g/L or 5 g/L + 10 g/L azone cream. One rat was not induced successfully in MI group. Four weeks after model reproduction, 4 rats died in D group and 1 rat died in AI group. The AGE content in D group was obviously higher than that in NC group [(36.8 +/- 2.6), (24.6 +/- 2.7) U per milligram hydroxyproline, respectively, t = 7.2, P < 0.01], and that in AI group [(28.6 +/- 3.7) U per milligram hydroxyproline] was also lower as compared with that in D group (t = -3.9, P < 0.05). There was no significant difference in AGE content between MI [(32.2 +/- 5.2) U per milligram hydroxyproline] and D groups (t = 1.6, P > 0.05). The percentage of KC in S phase was obviously lower in D group than in NC group [(5.3 +/- 0.6)%, (7.6 +/- 0.9)%, respectively, t = 4.50, P < 0.01], while that in MI group [(9.2 +/- 1.5)%] was higher as compared with that in D group ( t = 4.90, P < 0.01). It was more higher in AI group than in D group on KC percentage in S and G2/M phase (with t value respectively 6.80, 3.17, P values all below 0.01). The oxidative stress indexes of skin tissue in D group were all higher than those in NC group, in which levels of MPO and SOD showed statistical difference (with t value respectively 4.4, 3.7, P values all below 0.05). The oxidative stress indexes were all lower in AI group than in D group, especially in SOD level (t = -1.4, P < 0.05). Levels of MAD, MPO in MI group were significantly lower than those in D group (with t value respectively 2.6, 2.9, P values all below 0.05).

CONCLUSIONSAminoguanidine cream can promote KC proliferation and appropriately reduce oxidative stress through inhibiting AGE formation to a certain extent in skin tissue of rats with diabetes. Signal use of matrix cream can also reduce oxidative stress in skin tissue of rats with diabetes.

Administration, Cutaneous ; Animals ; Cell Proliferation ; Diabetes Mellitus, Experimental ; metabolism ; pathology ; Glycation End Products, Advanced ; metabolism ; Guanidines ; administration & dosage ; pharmacology ; Keratinocytes ; drug effects ; Male ; Ointments ; administration & dosage ; pharmacology ; Oxidative Stress ; drug effects ; Rats ; Rats, Sprague-Dawley ; Skin ; drug effects ; metabolism ; pathology

This experimental study was designed to explore the possible mechanisms of Cariporide, a kind of Na+/H+ exchanger inhibitor, for protecting the lung from warm ischemia/reperfusion injury (WI/RI) of isolated rat lung model. Thirty isolated rat lungs were established on the Langendorff apparatus and randomly divided to three groups (n = 10, each): control group (C group), ischemia/reperfusion group (IR group) and Cariporide group (CP group). Mean pulmonary artery pressure (MPAP) and peak airway pressure (pAwP) were monitored continuously. At the end of reperfusion, right bronchoalveolar lavage was performed, bronchoalveolar lavage fluid (BALF) recovery rate (BALFRR) was recorded, and protein content in BALF was measured. Lung water content (LWC), malondialdehyde (MDA) and superoxide dismutase (SOD)of left lung tissue were measured; histomorphology evaluation was performed under light microscope and transmission electron microscope. In comparison with the data from IR group, BALF protein concentration, LWC, MDA content and MPAP content of reperfusion were significantly decreased, but SOD activity was increased in CP group. Histomorphologic feature also showed that pathological change significantly reduced in CP group. In this rat WI/RI model, the mechanism by which the selective Na+/H+ exchanger inhibitor (Cariporide) attenuates lipid peroxidation induced by WI/IR may be: preventing Ca2+ overload via inhibiting the transport of Na+/H2 exchanger-1 (NHE1) in the context of the coupled exchanger, thereby reducing the activation of xanthine oxidase pathway and oxygen free radical liberation which is dependent on certain intracellular Ca2+ concentration, and lastly promoting the endogenous antioxidative mechanism.
Animals ; Antioxidants ; pharmacology ; Guanidines ; pharmacology ; In Vitro Techniques ; Ischemic Preconditioning ; Lung ; blood supply ; Male ; Rats ; Rats, Sprague-Dawley ; Reperfusion Injury ; etiology ; prevention & control ; Sodium-Hydrogen Exchangers ; antagonists & inhibitors ; Sulfones ; pharmacology ; Superoxide Dismutase ; metabolism ; Warm Ischemia

It has been demonstrated that inhibitors of advanced glycation end products (AGE), such as aminoguanidine, can suppress peritoneal AGE in rats on peritoneal dialysis (PD). However, it is unknown whether late administration of a putative crosslink breaker, alagebrium, could reverse peritoneal AGE. We therefore compared alagebrium with aminoguanidine in their ability to reverse peritoneal AGE in rats on PD. Male Sprague-Dawley rats were randomly divided into 3 groups: group I dialyzed with 4.25% glucose solution for all exchanges; group II dialyzed with 4.25% glucose solution containing aminoguanidine, and group III dialyzed with 4.25% glucose solution containing alagebrium for last 8 weeks of 12-week dialysis period. Dialysis exchanges were performed 2 times a day for 12 weeks. Immunohistochemistry was performed using a monoclonal anti-AGE antibody. One-hour PET was performed for comparison of transport characteristics. The immunolabelling of AGE in peritoneal membrane was markedly decreased in the alagebrium group. Consistent with this, the alagebrium group exhibited significantly higher D/Do glucose and lower D/P urea, suggesting low peritoneal membrane transport. But there were no significant differences between the control and the aminoguanidine group. These results suggest that the alagebrium may be the optimal therapeutic approach, compared with treatment with inhibitors of AGE formation, in rats on PD.
Animals ; Biological Transport ; Body Weight ; Cell Membrane/metabolism ; Glycosylation End Products, Advanced/*metabolism ; Guanidines/metabolism ; Immunohistochemistry/methods ; Male ; Peritoneal Dialysis/*methods ; Peritoneum/metabolism/*pathology ; *Permeability ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo investigate the impact of intracellular acidification (IA) on drug resistance of leukemia cells with high P-glycoprotein (P-gp) expression, and to provide a new method for the reversing of multidrug resistance (MDR).

METHODSReal-time PCR was used to determine the expression level of mdr1 gene, and the leukemia cells with high P-gp expression were selected. The specific inhibitor of Na+/H+ exchanger 1 and the "high K+" buffer were used to acidify the cells, and the confocal laser microscopy was used to determine the intracellular pH (pHi) and effect of IA on the accumulation of doxorubicin. The MTT method was used to determine the effect of IA on the cell viability. The flow cytometry was used to detect the effect of IA on the P-gp function, and Western blotting was used to determine the effect of IA on the expression of P-gp.

RESULTSThe pHi was decreased to 7.0, and compared with that of control the mdr1 mRNA expression was decreased to (53.2+/-11.0)% after 1 h, and to (16.6+/-7.0)% after 3 h treatment. The P-gp expression was decreased to (56.0+/-9.0)% of the control after 3 h treatment. The accumulation of Rh123 was 71.03+/-0.47 at pHi 7.0, which was increased obviously as compared to the control group 20.07+/-0.39. The increased accumulation of doxorubicin was also observed by confocal laser microscopy.

CONCLUSIONThe expression and function of P-gp on the patients cells are inhibited by IA.

ATP Binding Cassette Transporter, Sub-Family B ; ATP-Binding Cassette, Sub-Family B, Member 1 ; genetics ; metabolism ; Cell Survival ; drug effects ; Doxorubicin ; pharmacokinetics ; Drug Resistance, Neoplasm ; drug effects ; Guanidines ; pharmacology ; Humans ; Hydrogen-Ion Concentration ; drug effects ; Leukemia ; drug therapy ; metabolism ; RNA, Messenger ; genetics ; Sulfones ; pharmacology ; Tumor Cells, Cultured

Connective tissue growth factor (CTGF) is a known profibrotic cytokine. The purpose of the present study was to explore the effects of inhalation of aminoguanidine (AG) on the up-regulation of CTGF in fibrotic lungs of rats. Sprague-Dawley rats received single intratracheal instillation of bleomycin (BLM) or instillation of the same volume of normal saline (NS) as control. From day 1 to day 30 after intratracheal BLM instillation, the rats inhaled AG (2, 10 or 50 mmol/L, 5 min each time) twice a day or inhaled the same volume of NS as vehicle control. The change of nitric oxide (NO) content in lungs was evaluated by nitrite/nitrate (NO₂(-)/NO₃(-)) content in out-flowing pulmonary plasma (OPP). The degree of fibrosis in lung was evaluated by the content of hydroxyproline (chloramine T method) and area of collagen (Masson stain) in lung. The CTGF expression in lung was detected by Western blot and RT-PCR. The contents of NO₂(-)/NO₃(-) were increased in OPP of rats on day 14 after the instillation of BLM, compared with those in the rats with instillation of NS [(156+/-21) mumol/L vs (51+/-15) mumol/L, P<0.01]. The content of hydroxyproline, the area of collagen, and the levels of CTGF protein and mRNA were increased in lungs of rats on day 30 after intratracheal instillation of BLM, compared with those in the rats with instillation of NS [hydroxyproline, (51+/-10) mg/g lung vs (20+/-5) mg/g lung; area of collagen, (38.7+/-8.8)% vs (5.7+/-1.5)%; CTGF protein, (1+/-0.25) vs (0.3+/-0.1); CTGF mRNA, (0.8+/-0.2) vs (0.15+/-0.03), P<0.01]. The above-mentioned indices were ameliorated by the inhalation of AG (10 or 50 mmol/L) (NO₂(-)/NO₃(-) content, P<0.01; other indices, P<0.05). It is therefore concluded that the inhalation of AG prevented the up-regulation of CTGF in fibrotic lungs of rats suffering from BLM instillation, which might be one of the mechanisms of the anti-fibrosis of AG in lungs.
Administration, Inhalation ; Animals ; Bleomycin ; adverse effects ; Connective Tissue Growth Factor ; metabolism ; Guanidines ; pharmacology ; Lung ; metabolism ; pathology ; Pulmonary Fibrosis ; drug therapy ; metabolism ; RNA, Messenger ; Rats ; Rats, Sprague-Dawley ; Up-Regulation

OBJECTIVETo study the relation between the nitric-oxide synthase (NOS) expression and nitric oxide (NO) content in the skeletal muscles and the injury condition of soft tissue in the 3rd lumbar vertebrae syndrome model rats, and to observe the effect of acupotomology therapy.

METHODSOne hundred and twenty-eight adult SD rats were allocated to 4 groups randomly: normal group, model group, aminoguanidin group and acupotomology treatment group, 32 rats in each group. NOS expression, NO content and injury of the soft tissue in the 3rd lumbar vertebra were observed on the 1st, 3rd, 7th and 14th day after the acupotomology treatment and aminoguanidine intervention.

RESULTS1) Inducible NOS (iNos) activity and NO content in model group was significantly higher (F = 522.860, P < 0.01), in acupotomology group and aminoguanidine group was significantly lower than the model group (FiNOS = 28.894, P < 0.01), and iNOS activity and NO content in all groups was in competence with the condition of soft tissue injuries. 2) Endothelium NOS (eNOS) expression raised in model group and acupotomology group, and achieve peak on the 7th day. There was significant difference between the eNOS expression in acupotomology group and the model group (FeNOS = 3.454, P < 0.05). 3) The expression of neuron NOS (nNOS) in the model group, aminoguanidine group and acupotomology group had no significant (FnNOS = 0.962, P > 0.05).

CONCLUSIONAcupotomology treatment can restrain the development of high content NO, release the inflammatory reaction and injury condition, improve microcirculation, prevent the development of scar tissue of the injured soft tissue, and has significant recovering effectiveness in the soft tissue injured model rats.

Animals ; Disease Models, Animal ; Gene Expression Regulation, Enzymologic ; Guanidines ; therapeutic use ; Lumbar Vertebrae ; drug effects ; metabolism ; pathology ; surgery ; Male ; Muscle, Skeletal ; drug effects ; metabolism ; pathology ; Nitric Oxide ; metabolism ; Nitric Oxide Synthase ; metabolism ; Rats ; Rats, Sprague-Dawley ; Syndrome ; Time Factors

OBJECTIVETo investigate the effect of a selective inducible nitric oxide synthase(iNOS) inhibitor, aminoguanidine (AG), on the proliferation and apoptosis of human colorectal cancer (CRC) Lovo cell line, and explore its possible mechanism.

METHODSMTT assay was used to detect the inhibition of Lovo cell growth by aminoguanidine. Apoptosis and cell cycle of Lovo cells were examined by flow cytometry (FCM). Morphologic change of Lovo cell treated by AG was observed with AO/EB staining.

RESULTSThere were significant differences in 0.5 mmol/L and 1.0 mmol/L AG groups as compared to the control group (P<0.05). The absorbance (A) values of Lovo cells in each time point were significantly different (P<0.05). Growth of Lovo cells was inhibited by aminoguanidine in a dose- and time-dependent manner. FCM analysis showed that the cell ratio of G(0)/G(1) phase increased with the increasing of the concentration of aminoguanidine, but the cell ratio of S-and G(2)/M phase decreased correspondingly (P<0.05). S phase fraction and proliferation index (PI) decreased remarkably, and the apoptotic rate of Lovo cells increased. After AG treatment, AO/EB staining revealed some apoptotic morphological features such as cell shrinkage, nuclear condensation, DNA fragmentation, and formation of apoptosis bodies.

CONCLUSIONSAminoguanidine inhibits the proliferation and facilitates the apoptosis of human CRC Lovo cells. One of the mechanisms may be explained as blocking the progress of cell cycle of CRC Lovo cells by aminoguanidine.

Apoptosis ; drug effects ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Colorectal Neoplasms ; metabolism ; Drug Resistance, Neoplasm ; drug effects ; Enzyme Inhibitors ; pharmacology ; Guanidines ; pharmacology ; Humans ; Nitric Oxide Synthase Type II ; antagonists & inhibitors