OBJECTIVES: Myocardial ischemia reperfusion injury (IRI) occurs occasionally in the process of ischemic heart disease. Sevoflurane preconditioning has an effect on attenuating IRI. Preserving the structural and functional integrity of mitochondria is the key to reduce myocardial IRI. Silent information regulator 3 (SIRT3), a class of nicotinamide adenine dinucleotide (NAD⁺) dependent deacetylases, is an important signal-regulating molecule in mitochondria. This study aims to explore the role of mitochondrial NAD⁺-SIRT3 pathway in attenuating myocardial IRI in rats by sevoflurane preconditioning. METHODS: A total of 60 male Sprague Dawley (SD) rats were randomly divided into 5 groups (n=12): A sham group (Sham group), an ischemia reperfusion group (IR group), a sevoflurane preconditioning group (Sev group, inhaled 2.5% sevoflurane for 30 min), a sevoflurane preconditioning+SIRT3 inhibitor 3-TYP group (Sev+3-TYP group, inhaled 2.5% sevoflurane for 30 min and received 5 mg/kg 3-TYP), and a 3-TYP group (5 mg/kg 3-TYP). Except for the Sham group, the IR model in the other 4 groups was established by ligating the left anterior descending coronary artery. The size of myocardial infarction was determined by double staining. Serum cardiac troponin I (cTnI) level was measured. The contents of NAD⁺ and ATP, the activities of mitochondrial complexes I, II, and IV, the content of MDA, the activity of SOD, and the changes of mitochondrial permeability were measured. The protein expression levels of SIRT3, SOD2, catalase (CAT), and voltage dependent anion channel 1 (VDAC1) were detected by Western blotting. The ultrastructure of myocardium was observed under transmission electron microscope. MAP and HR were recorded immediately before ischemia (T0), 30 min after ischemia (T1), 30 min after reperfusion (T2), 60 min after reperfusion (T3), and 120 min after reperfusion (T4). RESULTS: After ischemia reperfusion, the content of NAD⁺ in cardiac tissues and the expression level of SIRT3 protein were decreased (both P<0.01), and an obvious myocardial injury occurred, including the increase of myocardial infarction size and serum cTnI level (both P<0.01). Correspondingly, the mitochondria also showed obvious damage on energy metabolism, antioxidant function, and structural integrity, which was manifested as: the activities of mitochondrial complexes I, II, and IV, ATP content, protein expression levels of SOD2 and CAT were decreased, while MDA content, VDAC1 protein expression level and mitochondrial permeability were increased (all P<0.01). Compared with the IR group, the content of NAD⁺ in cardiac tissues and the expression level of SIRT3 protein were increased in the Sev group (both P<0.01); the size of myocardial infarction and the level of serum cTnI were decreased in the Sev group (both P<0.01); the activities of mitochondrial complexes I, II, and IV, ATP content, protein expression levels of SOD2 and CAT were increased, while MDA content, VDAC1 protein expression level, and mitochondrial permeability were decreased in the Sev group (all P<0.01). Compared with the Sev group, the content of NAD⁺ in cardiac tissues and the expression level of SIRT3 protein were decreased in the Sev+3-TYP group (both P<0.01); the size of myocardial infarction and the level of serum cTnI were increased in the Sev+3-TYP group (both P<0.01); the activities of mitochondrial complexes I, II, and IV, ATP content, protein expression levels of SOD2 and CAT were decreased, while MDA content, VDAC1 protein expression level, and mitochondrial permeability were increased in the Sev+3-TYP group (all P<0.01). CONCLUSIONS Sevoflurane preconditioning attenuates myocardial IRI through activating the mitochondrial NAD⁺-SIRT3 pathway to preserve the mitochondrial function.
Adenosine Triphosphate/metabolism* ; Animals ; Male ; Mitochondria/metabolism* ; Myocardial Infarction/metabolism* ; Myocardial Reperfusion Injury/metabolism* ; NAD/metabolism* ; Rats ; Rats, Sprague-Dawley ; Sevoflurane/metabolism* ; Sirtuin 3/metabolism* ; Voltage-Dependent Anion Channel 1/metabolism*

Wuzi-Yanzong-Wan (WZYZW) is a classic prescription for male infertility. Our previous investigation has demonstrated that it can inhibit sperm apoptosis via affecting mitochondria, but the underlying mechanisms are unclear. The purpose of the present study was to explore the actions of WZYZW on mitochondrial permeability transition pore (mPTP) in mouse spermatocyte cell line (GC-2 cells) opened by atractyloside (ATR). At first, WZYZW-medicated serum was prepared from rats following oral administration of WZYZW for 7 days. GC-2 cells were divided into control group, model group, positive group, as well as 5%, 10%, 15% WZYZW-medicated serum group. Cyclosporine A (CsA) was used as a positive control. 50 μmol·L^-1 ATR was added after drugs incubation. Cell viability was assessed using CCK-8. Apoptosis was detected using flow cytometry and TUNEL method. The opening of mPTP and mitochondrial membrane potential (MMP) were detected by Calcein AM and JC-1 fluorescent probe respectively. The mRNA and protein levels of voltage-dependent anion channel 1 (VDAC1), cyclophilin D (CypD), adenine nucleotide translocator (ANT), cytochrome C (Cyt C), caspase 3, 9 were detected by RT-PCR (real time quantity PCR) and Western blotting respectively. The results demonstrated that mPTP of GC-2 cells was opened after 24 hours of ATR treatment, resulting in decreased MMP and increased apoptosis. Pre-protection with WZYZ-medicated serum and CsA inhibited the opening of mPTP of GC-2 cells induced by ATR associated with increased MMP and decreased apoptosis. Moreover, the results of RT-qPCR and WB suggested that WZYZW-medicated serum could significantly reduce the mRNA and protein levels of VDAC1 and CypD, Caspase-3, 9 and CytC, as well as a increased ratio of Bcl/Bax. However, ANT was not significantly affected. Therefore, these findings indicated that WZYZW inhibited mitochondrial mediated apoptosis by attenuating the opening of mPTP in GC-2 cells. WZYZW-medicated serum inhibited the expressions of VDAC1 and CypD and increased the expression of Bcl-2, which affected the opening of mPTP and exerted protective and anti-apoptotic effects on GC-2 cell induced by ATR.
1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine ; Animals ; Atractyloside/pharmacology* ; Cyclophilin D ; Male ; Matrix Metalloproteinases ; Mice ; Mitochondrial Membrane Transport Proteins/metabolism* ; Mitochondrial Permeability Transition Pore ; RNA, Messenger ; Rats

OBJECTIVE: To explore the characteristics of SLC25A13 gene variants in 16 infants with neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD). METHODS: The infants were subjected to high-throughput DNA sequencing for coding exons and flanking regions of the target genes. Suspected variants were verified by Sanger sequencing and bioinformatic analysis. RESULTS: Among the 16 NICCD cases, 15 were found to harbor pathogenic variants. Among these, IVS14-9A>G, c.1640G>A, c.762T>A, c.736delG, c.1098Tdel and c.851G>A were previously unreported. CONCLUSION Six novel SLC25A13 variants were found by high-throughput sequencing, which has enriched the spectrum of SLC25A13 gene variants and provided a basis for genetic counseling and prenatal diagnosis.
Calcium-Binding Proteins/genetics* ; Cholestasis, Intrahepatic/genetics* ; Citrullinemia/genetics* ; Humans ; Infant ; Infant, Newborn ; Mitochondrial Membrane Transport Proteins/genetics* ; Mutation ; Organic Anion Transporters/genetics* ; Protein Deficiency

OBJECTIVE: To determine the carrier rate for 21 inherited metabolic diseases among a Chinese population of childbearing age. METHODS: A total of 897 unrelated healthy individuals (including 143 couples) were recruited, and DNA was extracted from their peripheral blood samples. Whole exome sequencing (WES) was carried out to screen potential variants among 54 genes associated with 21 inherited metabolic diseases. Pathogenic and likely pathogenic variants and unreported loss-of-function variants were analyzed. RESULTS: One hundred fourty types of pathogenic/likely pathogenic variants (with an overall number of 183) and unreported loss-of-function variants were detected, which yield a frequency of 0.20 per capita. A husband and wife were both found to carry pathogenic variants of the SLC25A13 gene and have given birth to a healthy baby with the aid of preimplantation genetic diagnosis. The detected variants have involved 40 genes, with the most common ones including ATP7B, SLC25A13, PAH, CBS and MMACHC. Based on the Hardy-Weinberg equilibrium, the incidence of the 21 inherited metabolic diseases in the population was approximately 1/1100, with the five diseases with higher incidence including citrullinemia, methylmalonic acidemia, Wilson disease, glycogen storage disease, and phenylketonuria. CONCLUSION This study has preliminarily determined the carrier rate and incidence of 21 inherited metabolic diseases among a Chinese population of childbearing age, which has provided valuable information for the design of neonatal screening program for inherited metabolic diseases. Pre-conception carrier screening can provide an important measure for the prevention of transmission of Mendelian disorders in the population.
Asians/genetics* ; China ; Exome ; Female ; Humans ; Infant, Newborn ; Metabolic Diseases/genetics* ; Mitochondrial Membrane Transport Proteins/genetics* ; Oxidoreductases/genetics* ; Whole Exome Sequencing

OBJECTIVE: To explore the clinical characteristics and genetic findings of patients with infantile intrahepatic cholestasis. METHODS: The clinical data were collected in children who were admitted to the Department of Gastroenterology in Children's Hospital, Capital Institute of Pediatrics from June 2017 to June 2019 and were suspected of inherited metabolic diseases. Next generation sequencing based on target gene panel was used for gene analysis in these children. Sanger sequencing technology was used to verify the genes of the members in this family. RESULTS: Forty patients were enrolled. Pathogenic gene variants were identified in 13 patients (32%), including CONCLUSIONS The etiology of infantile intrahepatic cholestasis is complex. Next generation sequencing is helpful in the diagnosis of infantile intrahepatic cholestasis.
Alagille Syndrome/genetics* ; Child ; Cholestasis, Intrahepatic/genetics* ; Citrullinemia ; Genetic Testing ; High-Throughput Nucleotide Sequencing ; Humans ; Mitochondrial Membrane Transport Proteins ; Mutation

OBJECTIVES: To study the physical and neuropsychological development of children with Citrin deficiency (CD). METHODS: A total of 93 children, aged 1.9-59.8 months, who were diagnosed with CD by RESULTS: For the 93 children with CD, the incidence rate of failure to thrive was 25% (23 children) and the proportion of small for gestational age was 47% (44 children). For the 100 cases of CD, the incidence rates of growth retardation, underweight, emaciation, overweight, and microcephalus were 23% (23 cases), 14% (14 cases), 4% (4 cases), 8% (8 cases), and 9% (9 cases), respectively. The incidence rate of neuropsychological developmental delay was 25% (25 cases), and the incidence rates of development delay in the five domains of adaptability, gross motor, fine motor, language, and social ability were 7% (7 cases), 15% (15 cases), 7% (7 cases), 9% (9 cases), and 7% (7 cases), respectively. CONCLUSIONS Physical and neuropsychological developmental delay can be observed in children with CD, and physical and neuropsychological development should be regularly assessed.
Child ; Citrullinemia ; Humans ; Infant ; Mitochondrial Membrane Transport Proteins ; Retrospective Studies

OBJECTIVE: To explore the clinical and genetic features of an infant with citrin deficiency (CD). METHODS: Clinical data of the patient was collected and analyzed. Genomic DNA was extracted from peripheral blood samples collected from the patient and her parents. Targeted exome sequencing was performed to explore the genetic cause, and Sanger sequencing was used to confirm the detected variants. SLC25A13 mRNA was extracted from peripheral blood lymphocytes of the infant. The effect of novel mutation of SLC25A13 was analyzed by reverse transcription-PCR, cDNA cloning and Sanger sequencing. RESULTS: The SLC25A13 genotype of the patient was determined as c.845_c.848+1delG/c.1841+3_1841+4delAA, with the latter having not been reported. The mutation has affected the splicing of the SLC25A13 mRNA, giving rise to an aberrant transcript [r.1841_1842ins1841+1_1841+67; 1841+3_c.1841+4del]. CONCLUSION A novel SLC25A13 mutation c.1841+3_1841+4delAA and the resultant abnormal splicing variant were discovered by combined DNA sequencing and cDNA cloning. The finding has enabled definite diagnosis of CD and enriched the spectrum of SLC25A13 mutations.
Base Sequence ; Citrullinemia ; Female ; Humans ; Mitochondrial Membrane Transport Proteins ; genetics ; Mutation ; Pedigree

Polyamines (putrescine, spermidine, and spermine) are essential polycations that play important roles in various physiological and pathophysiological processes in mammalian cells. The study was to investigate their role in cardioprotection against ischemia/reperfusion (I/R) injury and the underlying mechanism. Isolated hearts from male Sprague-Dawley rats were Langendorff-perfused and cardiac I/R was achieved by 30 min of global ischemia followed by 120 min of reperfusion. Different concentrations of polyamines (0.1, 1, 10, and 15 μmol/L of putrescine, spermidine, and spermine), cyclosporin A (0.2 μmol/L), or atractyloside (20 μmol/L) were given 10 min before the onset of reperfusion. The hemodynamics were monitored; the lactate dehydrogenase (LDH) levels in the coronary effluent were measured spectrophotometrically; infarct size was determined by the 2,3,5-triphenyltetrazolium chloride staining method; and mitochondrial permeability transition pore (MPTP) opening was determined spectrophotometrically by the Ca-induced swelling of isolated cardiac mitochondria. The results showed that compared to I/R alone, 0.1 and 1 μmol/L polyamines treatment improved heart function, reduced LDH release, decreased infarct size, and these effects were inhibited by atractyloside (MPTP activator). In isolated mitochondria from normal rats, 0.1 and 1 μmol/L polyamines treatment inhibited MPTP opening. However, 10 and 15 μmol/L polyamines treatment had the opposite effects, and these effects were inhibited by cyclosporin A (MPTP inhibitor). Our findings showed that polyamines may have either protective or damaging effects on hearts suffering from I/R by inhibiting or activating MPTP opening.
Animals ; Cyclosporine ; pharmacology ; Male ; Mitochondria, Heart ; physiology ; Mitochondrial Membrane Transport Proteins ; physiology ; Myocardial Reperfusion Injury ; physiopathology ; Polyamines ; metabolism ; Rats ; Rats, Sprague-Dawley

The aim of this study was to determine the effects of extremely low frequency electromagnetic field (ELF-EMF) on energy metabolism and oxidative stress in Caenorhabditis elegans (C. elegans). Worms in three adult stages (young adult stage, egg-laying stage and peak egg-laying stage) were investigated under 50 Hz, 3 mT ELF-EMF exposure. ATP levels, ATP synthase activity in vivo, reactive oxygen species (ROS) content, and changes of total antioxidant capacity (TAC) were detected, and worms' oxidative stress responses were also evaluated under ELF-EMF exposure. The results showed that ATP levels were significantly increased under this ELF-EMF exposure, and mitochondrial ATP synthase activity was upregulated simultaneously. In young adult stage, worms' ROS level was significantly elevated, together with upregulated TAC but with a decreased ROS-TAC score indicated by principal component analysis. ROS level and TAC of worms had no significant changes in egg-laying and peak egg-laying stages. Based on these results, we concluded that ELF-EMF can enhance worm energy metabolism and elicit oxidative stress, mainly manifesting as ATP and ROS level elevation together with ATP synthase upregulation and ROS-TAC score decrease in young adult C. elegans.
Adenosine Triphosphate ; metabolism ; Animals ; Caenorhabditis elegans ; radiation effects ; Electromagnetic Radiation ; Energy Metabolism ; Mitochondrial Proton-Translocating ATPases ; metabolism ; Oxidative Stress ; Reactive Oxygen Species ; analysis

OBJECTIVE: To investigate the effects of salvianolic acid A (SAA) on cardiomyocyte apoptosis and mitochondrial dysfunction in response to hypoxia/reoxygenation (H/R) injury and to determine whether the Akt signaling pathway might play a role. METHODS: An in vitro model of H/R injury was used to study outcomes on primary cultured neonatal rat cardiomyocytes. The cardiomyocytes were treated with 12.5, 25, 50 μg/mL SAA at the beginning of hypoxia and reoxygenation, respectively. Adenosine triphospate (ATP) and reactive oxygen species (ROS) levels were assayed. Cell apoptosis was evaluated by flow cytometry and the expression of cleaved-caspase 3, Bax and Bcl-2 were detected by Western blotting. The effects of SAA on mitochondrial dysfunction were examined by determining the mitochondrial membrane potential (△Ψm) and mitochondrial permeability transition pore (mPTP), followed by the phosphorylation of Akt (p-Akt) and GSK-3β (p-GSK-3β), which were measured by Western blotting. RESULTS: SAA significantly preserved ATP levels and reduced ROS production. Importantly, SAA markedly reduced the number of apoptotic cells and decreased cleaved-caspase 3 expression levels, while also reducing the ratio of Bax/Bcl-2. Furthermore, SAA prevented the loss of △Ψm and inhibited the activation of mPTP. Western blotting experiments further revealed that SAA significantly increased the expression of p-Akt and p-GSK-3β, and the increase in p-GSK-3β expression was attenuated after inhibition of the Akt signaling pathway with LY294002. CONCLUSION SAA has a protective effect on cardiomyocyte H/R injury; the underlying mechanism may be related to the preservation of mitochondrial function and the activation of the Akt/GSK-3β signaling pathway.
Adenosine Triphosphate ; analysis ; Animals ; Animals, Newborn ; Caffeic Acids ; pharmacology ; Cell Hypoxia ; Cells, Cultured ; Glycogen Synthase Kinase 3 beta ; physiology ; Lactates ; pharmacology ; Mitochondria, Heart ; drug effects ; physiology ; Mitochondrial Membrane Transport Proteins ; drug effects ; Myocytes, Cardiac ; drug effects ; Proto-Oncogene Proteins c-akt ; physiology ; Rats ; Rats, Sprague-Dawley ; Reactive Oxygen Species ; metabolism ; Signal Transduction ; physiology