DJ-1 mediates resveratrol to attenuate myocardial ischemia/reperfusion injury in rats by protecting activity of mitochondrial complex I / 中国病理生理杂志

AIM:From the perspective of regulating mitochondrial complex I activity by DJ-1 protein,this study aims to explore the mechanism of DJ-1-mediated resveratrol(RES)preconditioning in protecting against oxidative stress injury induced by myocardial ischemia-reperfusion(I/R)in rats.METHODS:After intramyocardial injection of lentivirus carrying DJ-1 shRNA(sh-DJ-1)or negative control(NC)shRNA,the myocardial I/R model was constructed by ligating the left anterior descending branch of the rat coronary artery.Sprague-Dawley(SD)rats were randomly divided in-to 6 groups:sham group,I/R group,RES+I/R group,NC+RES+I/R group,sh-DJ-1+RES+I/R group,and IACS-010759(mitochondrial complex I inhibitor)+RES+I/R group,with 10 rats in each group.The rats in RES treatment groups were given RES(20 mg/kg)via gavage for 7 d prior to the myocardial I/R modeling,once daily.Moreover,the rats in sham and I/R groups received an equivalent volume of normal saline via gavage.Myocardial infarction area and cardiac function were assessed by TTC staining and echocardiography,respectively.The MitoSOX fluorescent probe was used to detect levels of mitochondrial reactive oxygen species(ROS)in the myocardium.The levels of malondialdehyde(MDA),superoxide dis-mutase(SOD)and lactate dehydrogenase(LDH)in the serum were detected using kits.Western blot and co-immunopre-cipitation assays were used to observe the interaction between DJ-1 and the two subunits,ND-1 and NDUFA4,of the mito-chondrial complex I.RESULTS:Compared with I/R group,RES pretreatment significantly reduced the myocardial in-farction area,mitochondrial ROS levels,serum LDH activity,and serum MDA content(P＜0.01).It also elevated left ventricular ejection fraction,left ventricular fractional shortening and serum SOD activity(P＜0.01).Pretreatment with RES increased the expression and mitochondrial translocation of DJ-1(P＜0.01),promoted the interaction between DJ-1 and ND-1/NDUFA4,which in turn protected the activity of mitochondrial complex I(P＜0.01).However,when the ex-pression of DJ-1 was suppressed,the protective effects of RES against myocardial I/R injury were significantly inhibited compared with RES+I/R group(P＜0.05 or P＜0.01).CONCLUSION:Pretreatment with RES increases the expression and mitochondrial translocation of DJ-1,and facilitates the interaction of DJ-1 with ND1 and NDUFA4 subunits of mito-chondrial complex I,thus preserving the activity of mitochondrial complex I and attenuating myocardial I/R-induced oxida-tive stress damage.

Anti-aging effects of chlorpropamide depend on mitochondrial complex-II and the production of mitochondrial reactive oxygen species

Sulfonylureas are widely used oral anti-diabetic drugs. However, its long-term usage effects on patients' lifespan remain controversial, with no reports of influence on animal longevity. Hence, the anti-aging effects of chlorpropamide along with glimepiride, glibenclamide, and tolbutamide were studied with special emphasis on the interaction of chlorpropamide with mitochondrial ATP-sensitive K+ (mitoK-ATP) channels and mitochondrial complex II. Chlorpropamide delayed aging in Caenorhabditis elegans, human lung fibroblast MRC-5 cells and reduced doxorubicin-induced senescence in both MRC-5 cells and mice. In addition, the mitochondrial membrane potential and ATP levels were significantly increased in chlorpropamide-treated worms, which is consistent with the function of its reported targets, mitoK-ATP channels. Increased levels of mitochondrial reactive oxygen species (mtROS) were observed in chlorpropamide-treated worms. Moreover, the lifespan extension by chlorpropamide required complex II and increased mtROS levels, indicating that chlorpropamide acts on complex II directly or indirectly via mitoK-ATP to increase the production of mtROS as a pro-longevity signal. This study provides mechanistic insight into the anti-aging effects of sulfonylureas in C. elegans.

Complejo I, H2O2 y NO mitocondriales como señales prodrómicas de la disfunción cardíaca en diabetes tipo 1 / Mitochondrial Complex I, H2O2 and NO as Prodromal Signals of Cardiac Dysfunction in Type 1 Diabetes

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.

Mitochondrial complex Ⅰ deficiency due to NDUFAF5 gene mutations: report of 2 cases and literature review / 中华实用儿科临床杂志

Objective:To analyze the clinical features and genotypes of mitochondrial complex Ⅰ deficiency due to NDUFAF5 gene mutations.Methods:Clinical data of 2 cases with mitochondrial complex Ⅰ deficiency due to NDUFAF5 gene mutations admitted in the Department of Pediatrics, Peking University First Hospital from February 2015 to July 2018 were retrospectively reviewed and followed up.Reported cases of mitochondrial complex Ⅰ deficiency due to NDUFAF5 gene mutations were searched in online databases, including the PubMed, Wanfang, Chinese Journal Full-Text Database and VIP database from January 1975 to February 2020 with " NDUFAF5" as the key word.Through literature review, clinical features and genotypes of mitochondrial complex Ⅰ deficiency due to NDUFAF5 gene mutations were summarized.Results:Case 1 showed mentor and mental regression after infection at the age of 1 year and 4 months.The condition of case 1 remained stable at the age of 5 year and 6 months at the last follow-up.Brain magnetic resonance imaging (MRI) showed multiple lesions in the white matter of the frontal and parieto-occipital lobes, basal ganglia, thalamus, cerebellum, brain stem and corpus callosum.Case 2 showed rapidly bilateral visual impairment at the age of 7 years and 4 months.The patient′s vision moderately recovered at the age of 8 years and 8 months.Brain MRI showed midbrain, periaqueductal gray, medulla oblongata and putamen lesions.Spinal MRI showed continuous lesions in the cervical cord 1-4.Genetic test showed NDUFAF5 gene c. 764C>T (p.Ala255Val) and c. 508C>T (p.Arg170Trp), homozygous c. 836T>G (p.Met279Arg) mutations in case 1 and case 2 respectively.Through online searching, 6 reports involving 14 cases were retrieved.The most common clinical phenotype was Leigh syndrome.Two cases had disease onset during the neonatal period, and their disease progressed rapidly and died within 1 year old.Eleven cases had onset during the infantile period, and 72.7% (8/11 cases) of them had a normal development.The common initial symptoms were mental or motor regression, feeding difficulty and dystonia.Seventy-two point seven percent (8/11 cases) had acute/subacute onset after infection, showing paroxysmal deterioration, and died in infancy or childhood.One patient developed dystonia in childhood and visual impairment in adulthood.Conclusions:The onset age ranged from neonatal period to childhood in patients with NDUFAF5 gene mutations, and their clinical phenotypes vary a lot.The main clinical phenotype is Leigh syndrome.Disease onset during the infantile period is frequent, and mostly presents paroxysmal deterioration after infection, while disease onset in childhood is rare.

Mechanism of uptake and retention of 18F-MyoZone in cardiomyocytes: A novel PET myocardial perfusion imaging agent / 解放军医学杂志

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.

A familial case of mitochondrial leukoencephalopathy related to NADH dehydrogenase (ubiquinone)flavoprotein 2 gene mutation / 中华实用儿科临床杂志

Objective To analyze the clinical and imaging features of 2 siblings with leukoencephalopathy due to NADH dehydrogenase (ubiquinone)flavoprotein 2 (NDUFV2) gene mutation,in order to better understand and diagnose it earlier.Methods Clinical and follow-up data of the proband and his brother were collected.Clinical features including symptoms,signs and cranial magnetic resonance imaging (MRI) were analyzed,and 2 patients were followed up for a long time.Sanger sequencing,targeted next generation sequencing,and whole exome sequencing were performed to identify potential genetic variations in the 2 patients and their parents.Results (1) Clinical characteristics and follow-up:ages of onset were 4 months and 1 year respectively.Both of the patients presented rapid motor regression hyperinyotonia,positive pathological character.During the follow-up the condition became stable,motor function and cognition improved gradually after cocktail therapy.(2) Brain MRI of the 2 patients showed prominent abnormalities in deep cerebral white matter,presenting T1 hypointense,T2 and fluid attenuated inversion recovery (FLAIR) hyperintense in the periventricular area.FLAIR images revealed that the abnormal white matter was partially rarefied and cavitated.Diffusion weighted images (DWI) showed high signals along the periphery of the involved areas.The follow-up MRI showed the cavitation still existed and even expanded,and DWI showed regional linear or spotty high signals around the original lesions.(3) Novel mutations in NDUFV2 gene,c.467T>A and c.404G>C,were identified in proband and his brother.The former inherited from his father,while the latter inherited from his mother,which was the new mutation not reported in the international.Conclusions The clinical features of the brothers presented subacute leukoencephalopathy with relatively stable or improved outcome.This was distinctive from the phenotypic features reported in 12 cases with hypertrophic cardiomyopathy or Leigh syndrome.The finding expanded the phenotypic spectrum of NDUFV2 mutations.Pathogenic gene of these patients which is the basis of genetic counseling for this family was determined.

Mitochondrial Complexes I and II Are More Susceptible to Autophagy Deficiency in Mouse beta-Cells

BACKGROUND: Damaged mitochondria are removed by autophagy. Therefore, impairment of autophagy induces the accumulation of damaged mitochondria and mitochondrial dysfunction in most mammalian cells. Here, we investigated mitochondrial function and the expression of mitochondrial complexes in autophagy-related 7 (Atg7)-deficient beta-cells. METHODS: To evaluate the effect of autophagy deficiency on mitochondrial function in pancreatic beta-cells, we isolated islets from Atg7(F/F):RIP-Cre+ mice and wild-type littermates. Oxygen consumption rate and intracellular adenosine 5'-triphosphate (ATP) content were measured. The expression of mitochondrial complex genes in Atg7-deficient islets and in beta-TC6 cells transfected with siAtg7 was measured by quantitative real-time polymerase chain reaction. RESULTS: Baseline oxygen consumption rate of Atg7-deficient islets was significantly lower than that of control islets (P<0.05). Intracellular ATP content of Atg7-deficient islets during glucose stimulation was also significantly lower than that of control islets (P<0.05). By Oxygraph-2k analysis, mitochondrial respiration in Atg7-deficient islets was significantly decreased overall, although state 3 respiration and responses to antimycin A were unaffected. The mRNA levels of mitochondrial complexes I, II, III, and V in Atg7-deficient islets were significantly lower than in control islets (P<0.05). Down-regulation of Atg7 in beta-TC6 cells also reduced the expression of complexes I and II, with marginal significance (P<0.1). CONCLUSION: Impairment of autophagy in pancreatic beta-cells suppressed the expression of some mitochondrial respiratory complexes, and may contribute to mitochondrial dysfunction. Among the complexes, I and II seem to be most vulnerable to autophagy deficiency.

Integrative therapy using coenzyme Q10 and minocycline in 1-methyl-4-phenylpyridinium-induced hemiparkinson rats / 中国病理生理杂志

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.

Effect of monotetrahydrofuran annonaceous acetogenins on mitochondrial complex I of rats / 中草药

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.

Effects of repeated hypoxic exposures on glycolysis, mitochondrial oxidative phosphorylation and energy charge in mouse brain / 基础医学与临床

Objective To study the effects of repeated hypoxic exposures (HEs) on glycolysis, mitochondrial oxi-dative phosphorylation and energy charge in mouse brain. Methods Adult BALB/c mice were repeatedly exposed to hypoxia for 5 times and the standard tolerant time and body temperature were recorded. The activities of PFK, PK and mitochondrial complex Ⅰ in the brain were assayed. Phosphoadenosines and energy charge were measured. Results Repeated HEs prolonged the hypoxic tolerance and reduced the body temperature. The activities of PFK and PK experienced regular changes, with an increase in 1st and 3rd HEs and a decline to control levels in 5th HE. The complex Ⅰ activity continued to decrease during HEs. The energy charge was stable. Conclusion HEs lead to a regular change of glycolysis, a continued inhibition of mitochondrial oxidative phosphorylation, and a main-tained energy charge in the brains of mouse.

Molecular Pathway in parkinsons disease: An update on progress / 中国药理学通报

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.