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RESUMEN Introducción: Resultados de nuestro laboratorio sugieren que la disfunción mitocondrial en el corazón precede a la falla miocárdica asociada a la hiperglucemia sostenida. Objetivo: Estudiar los eventos tempranos que ocurren en las mitocondrias de corazón en un modelo de diabetes mellitus tipo 1. Materiales y métodos: Ratas Wistar macho fueron inyectadas con estreptozotocina (STZ; 60 mg/kg, ip) y sacrificadas 10 o 14 días posinyección. Se obtuvo la fracción mitocondrial de corazón. Resultados: El consumo de O2 en estado 3 en presencia de malato-glutamato (21%) o succinato (16%) y las actividades de los complejos I-III (27%), II-III (24%) y IV (22%) fueron menores en los animales diabéticos a los 14 días posinyección. Cuando los animales se sacrificaron al día 10, solo el consumo de O2 en estado 3 en presencia de sustratos del complejo I (23%) y su control respiratorio (30%) fueron menores en las ratas inyectadas con STZ, de acuerdo con una reducción en la actividad del complejo I-III (17%). Estos cambios se acompañaron de un aumento en las velocidades de producción de H2O2 (117%), NO (30%) y ONOO- (∼225%), en la expresión de mtNOS (29%) y en la [O2 -]ss (∼150%) y [NO]ss (∼30%), junto con una disminución de la actividad de la Mn-SOD (15%) y la [GSSG+GSH]mitocondrial (28%), sin cambios en la expresión de PGC-1α. Conclusión: La disfunción del complejo I y el aumento en la generación de H2O2, NO y ONOO- pueden considerarse señales subcelulares prodrómicas del deterioro de la función mitocondrial que precede a la disfunción cardíaca en la diabetes.
ABSTRACT Background: Previous results from our laboratory suggest that heart mitochondrial dysfunction precedes myocardial failure associated with sustained hyperglycemia. Purpose: The aim of this study was to analyze the early events that take place in heart mitochondria in a type 1 diabetes mellitus (DM) model. Methods: Male Wistar rats were injected with streptozotocin (STZ; 60 mg/kg, ip.) to induce DM. They were euthanized 10 or 14 days later and the heart mitochondrial fraction was obtained. Results: State 3 O2 consumption in the presence of malate-glutamate (21%) or succinate (16%), and complex I-III (27%), II-III (24%) and IV (22%) activities were lower in diabetic animals 14 days after STZ injection. When animals were euthanized at day 10, only state 3 O2 consumption sustained by complex I substrates (23%) and its corresponding respiratory control (30%) were lower in rats injected with STZ, in agreement with reduced complex I-III activity (17%). These changes were accompanied by increased H2O2 (117%), NO (30%) and ONOO- (~225%) production rates, mtNOS expression (29%) and O2 - (~150%) and NO (~30%) steady-state concentrations, together with a decrease in Mn-SOD activity (15%) and mitochondrial [GSSG+GSH] (28%), without changes in PGC-1α expression. Conclusion: Complex I dysfunction and increased H2O2, NO and ONOO- production rates can be considered subcellular prodromal signals of the mitochondrial damage that precedes myocardial dysfunction in diabetes.
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Objective To investigate the mechanism of uptake and retention of a novel PET myocardial perfusion imaging agent 18F-MyoZone in cardiomyocytes. Methods 1) Mechanism of inhibition of mitochondrial respiratory chain enzyme activity: With mitochondrial respiratory chain enzyme complex I (MC-I) activity assay kit, a sequence of 19F-MyoZone solution (start at 15 μmol/L, 3 times dilution, 12 points) was interacted with MC-I to detect the half inhibition rate (IC50) of 19F-MyoZone inhibiting mitochondrial respiratory chain activity. 2) Autoradiography experiment of 18F-MyoZone combining with MC-I: Myocardial tissue sections of neonatal rat were hatched with normal saline and 4 known MC-I inhibitors [rotenone (4 μmol/L), 19F-Flurpiridaz (4 μmol/L), 19F-MyoZone (4 μmol/L) and pyridaben (4 μmol/L)], and then 18F-MyoZone was added to autoradiography for detecting whether 18F-MyoZone can specifically bind the cardiomyocytes MC-I; and then the myocardial tissue sections of rat were hatched for 30 min with different concentrations of rotenone or 19F-MyoZone solution (0, 20 μmol/L, 2 μmol/L and 200 nmol/L, 20 nmol/L), then the 18F-MyoZone was added and hatched for 30 min again, developing for 10 min with phosphor storage screen after cleaning the slice, and analyzing and calculating the inhibition rate of each inhibitor concentration. 3) Experiment of rotenone inhibitting the uptake of 18F-MyoZone by cardiomyocytes: Neonate rats' primary cardiomyocytes were cultured for 15 min with different concentrations of 19F-MyoZone or equivalent rotenone (start at 10 μmol/L, 3 times dilution, 12 points), then 50 μl of 18F-MyoZone (about 17 kBq) was added and culturing for 30 min. The lysate was then collected, the radioactivity was counted and the IC50 of rotenone was calculated. 4) Outflow experiment of 18F-MyoZone from cardiomyocytes: Cultured neonate rats' primary cardiomyocytes were interacted with 500 μl of 18F-MyoZone (about 37 kBq) for 30 min, then the cell supernatant and lysate were separated to do photon counts at the time points of 0, 10, 20, 30, 60, 90, 120 and 150 min. The ratio of cardiomyocyte outflow rate was then calculated. Results Enzyme activity studies showed that 19F-MyoZone may effectively inhibit the activity of MC-I(IC50=229.9 nmol/L) in a dose dependent manner. MyoZone could specifically bind to MC-I with binding sites in accordance with the inhibitors rotenone, pyridaben and 19F-Flurpiridaz. Experiment of rotenone inhibitting the uptake of 18F-MyoZone by cardiomyocytes showed that 18F-MyoZone could be absorbed by rat's cardiomyocytes, and with the increase of rotenone concentration, the radio uptake of cardiomyocytes decreased gradually with inhibitor IC50 as 7 nmol/L. Outflow experiment showed that rat's cardiomyocytes could uptake 18F-MyoZone and stably detain over time. The outflow rate increased gradually within 0-30 min, and then maintained constantly from 60 min to 150 min. The amount of retention was about 20% of the entire uptake. Conclusions 18F-MyoZone may specifically bind MC-I and detain for a long time in rat's cardiomyocytes. 18F-MyoZone is a valuable myocardial perfusion imaging agent with great research value.
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AIM:To investigate the integrative treatment of both coenzyme Q 10 ( CoQ10 ) and minocycline in the rats intranigrally intoxicated with 1-methyl-4-phenylpyridinium ( MPP+) .METHODS:The rat model of Parkinson disease ( PD) was established by intranigral microinjection of MPP +.The degree of microglial activation was measured by immuno-fluorescent density of OX-42 ( a microglia marker ) in the substantia nigra ( SN) .The number of viable dopaminergic neurons was determined by counting the tyrosine hydroxylase ( TH) positive neurons in the SN .The behavioral performances were re-vealed with the number of apomorphine-induced rotations , score of forelimb akinesia and vibrissae-elicited forelimb placing a-symmetry.RESULTS:Pretreatment with CoQ10 or intracerebroventricular (icv) posttreatment with minocycline alone pro-vided partial attenuation against MPP +-induced locomotor defects .Integrative therapy provided enhanced beneficial effects , and resulted in a significant attenuation of locomotor disability than any single therapy (all P<0.01).The results of immu-nohistological analysis showed that the TH positive neurons were maximally protected by integrative therapy compared with minocycline group and CoQ 10 group (P<0.01) .CONCLUSION:The integrative therapy of CoQ 10 combined with minocy-cline may offer additional therapeutic benefit to MPP +-induced hemiparkinson rat model .Such neuroprotective strategy of tar-geting different aspect of the neurodegenerative phenotypes may highlight a new therapeutic strategy for future management of PD.
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Objective: To investigate the activity of mono-tetrahydrofuran (THF) annonaceous acetogenins (ACGs) against mitochondrial complex I of rats. Methods: The inhibitory activity of mono-THF ACGs with six different chemical structures against mitochondrial complex I of rats was investigated to clarify the carbon number and substituted hydroxyl number between THF ring and lactone ring as well as the effect of the core configuration in THF ring on mitochondrial complex I of rats. Results: The results show that mono-THF ACGs can inhibit the mitochondrial complex I of rats. With analysis of the results from the structure-activity relationship between antitumoral activity and their chemical structure of mono-THF ACGs, the less the carbon number between the two rings is, the better their inhibitory activities are; The number of substituted hydroxyl groups is not the decisive factor for influencing its activity in mono-THF ACGs. Conclusion: The inhibitory activity of compound's configurations with th/t/er is better than that of the compound's configurations with th/t/th in mono-THF ACGs.
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Parkinsons disease(PD) is characterized by a selective lo ss of dopaminergic neurons in the substantia nigra pars compacta. The etiology of PD is still not full y understood. Molecular pathways such as oxidative stress, mitochondrial dysfunc tion and impairment in the ubiquitinproteasomal system, are involved in the proc ess of the dopaminergic neuronal cell death and the progress of PD. The explorat ion and elucidation of these molecular pathways will provide new potential targe ts for the drug therapy of PD.