Effect of dynamin-related protein 1 in rats with myocardial ischemia/reperfusion injury / 中华危重病急救医学

Objective To investigate the protective effect of dynamin-related protein 1 (Drp1) in rats with myocardial ischemia/reperfusion injury (IRI).Methods Twenty-four healthy male Wistar rats were randomly divided into three groups (n = 8 each): sham group, IRI model group, and Drp1 inhibitor group. The left anterior descending branch of coronary artery was ligated to produce myocardial ischemia for 30 minutes and reperfusion injury model. Sham group was received only threading without ligation. The Drp1 inhibitor group was injected with 1.2 mg/kg mitochondrial division inhibitor 1 (mdivi-1) at 15 minutes before operation. At 3 hours after reperfusion, hemodynamics, serum myocardial enzymes, mitochondrial membrane potential (MMP), hydrogen peroxide (H2O2), reactive oxygen species (ROS) and ATP production were measured in rats. The myocardial tissues were harvested for the determination of the area at risk (AAR) and the infarct area (AI), and the ratio of AI/AAR was calculated. The expression of Drp1 and cytochrome C (Cyt C) was determined by Western Blot.Results Compared with the sham group, the left ventricular end diastolic pressure (LVEDP), cardiac troponin I (cTnI), MB isoenzyme of creatine kinase (CK-MB), lactate dehydrogenase (LDH), AI/AAR, H2O2, ROS, protein expression of Drp1 and Cyt C were significantly increased, left ventricular end systolic pressure (LVESP), ejection fraction (EF), fractional shortening (FS), MMP, ATP generation, expression of mitochondrial Cyt C were significantly decreased in IRI model group. Compared with IRI model group, LVEDP was significantly decreased in Drp1 inhibitor group [mmHg (1 mmHg = 0.133 kPa): 8.83±1.20 vs. 16.48±1.80], LVESP, EF, FS were significantly increased [LVESP (mmHg): 116.80±9.78 vs. 87.80±8.82, EF: 0.78±0.11 vs. 0.58±0.07, FS: (48.6±4.1)％ vs. (32.4±3.2)％];myocardial enzymes, H2O2 and ROS were significantly decreased in Drp1 inhibitor group [cTnI (ng/L): 31.9±8.8 vs. 49.2±13.7, CK-MB (U/L): 4.83±1.30 vs. 7.48±2.20, LDH (U/L): 1327.80±280.20 vs. 1858.80±324.80, H2O2:6.40±1.40 vs. 8.90±1.50, ROS: 41916.3±6295.3 vs. 65182.6±3777.8], AI/AAR was significantly decreased (0.38±0.01 vs. 0.62±0.01), MMP and ATP were significantly increased [MMP: 0.78±0.13 vs. 0.38±0.07, ATP (μmol/g): 150.8±12.3 vs. 103.7±8.4], the expression of Drp1 was significantly decreased (0.50±0.02 vs. 0.79±0.05), expression of mitochondria Cyt C was significantly increased (0.64±0.04 vs. 0.21±0.01), and expression of cytoplasmic Cyt C was significantly decreased (0.48±0.03 vs. 0.78±0.04), and the differences were statistically significant (allP <0.05).Conclusions Mitochondrial fission was excessively high during IRI, and its function was significantly decreased. Drp1 inhibitor could inhibit the division of mitochondria, and improve its function and cardiac function.

Effect of dynamin-related protein 1 in rats with myocardial ischemia/reperfusion injury / 中华危重病急救医学

Objective To investigate the protective effect of dynamin-related protein 1 (Drp1) in rats with myocardial ischemia/reperfusion injury (IRI).Methods Twenty-four healthy male Wistar rats were randomly divided into three groups (n = 8 each): sham group, IRI model group, and Drp1 inhibitor group. The left anterior descending branch of coronary artery was ligated to produce myocardial ischemia for 30 minutes and reperfusion injury model. Sham group was received only threading without ligation. The Drp1 inhibitor group was injected with 1.2 mg/kg mitochondrial division inhibitor 1 (mdivi-1) at 15 minutes before operation. At 3 hours after reperfusion, hemodynamics, serum myocardial enzymes, mitochondrial membrane potential (MMP), hydrogen peroxide (H2O2), reactive oxygen species (ROS) and ATP production were measured in rats. The myocardial tissues were harvested for the determination of the area at risk (AAR) and the infarct area (AI), and the ratio of AI/AAR was calculated. The expression of Drp1 and cytochrome C (Cyt C) was determined by Western Blot.Results Compared with the sham group, the left ventricular end diastolic pressure (LVEDP), cardiac troponin I (cTnI), MB isoenzyme of creatine kinase (CK-MB), lactate dehydrogenase (LDH), AI/AAR, H2O2, ROS, protein expression of Drp1 and Cyt C were significantly increased, left ventricular end systolic pressure (LVESP), ejection fraction (EF), fractional shortening (FS), MMP, ATP generation, expression of mitochondrial Cyt C were significantly decreased in IRI model group. Compared with IRI model group, LVEDP was significantly decreased in Drp1 inhibitor group [mmHg (1 mmHg = 0.133 kPa): 8.83±1.20 vs. 16.48±1.80], LVESP, EF, FS were significantly increased [LVESP (mmHg): 116.80±9.78 vs. 87.80±8.82, EF: 0.78±0.11 vs. 0.58±0.07, FS: (48.6±4.1)％ vs. (32.4±3.2)％];myocardial enzymes, H2O2 and ROS were significantly decreased in Drp1 inhibitor group [cTnI (ng/L): 31.9±8.8 vs. 49.2±13.7, CK-MB (U/L): 4.83±1.30 vs. 7.48±2.20, LDH (U/L): 1327.80±280.20 vs. 1858.80±324.80, H2O2:6.40±1.40 vs. 8.90±1.50, ROS: 41916.3±6295.3 vs. 65182.6±3777.8], AI/AAR was significantly decreased (0.38±0.01 vs. 0.62±0.01), MMP and ATP were significantly increased [MMP: 0.78±0.13 vs. 0.38±0.07, ATP (μmol/g): 150.8±12.3 vs. 103.7±8.4], the expression of Drp1 was significantly decreased (0.50±0.02 vs. 0.79±0.05), expression of mitochondria Cyt C was significantly increased (0.64±0.04 vs. 0.21±0.01), and expression of cytoplasmic Cyt C was significantly decreased (0.48±0.03 vs. 0.78±0.04), and the differences were statistically significant (allP <0.05).Conclusions Mitochondrial fission was excessively high during IRI, and its function was significantly decreased. Drp1 inhibitor could inhibit the division of mitochondria, and improve its function and cardiac function.

Mechanism of ethyl gallate induced the apoptosis of Lovo cells through mitochodrial pathway / 中国药学杂志

OBJECTIVE: To study the effect of ethyl gallate in inducing apoptosis of human colon carcinoma Lovo cells. METHODS: Cultured human colon carcinoma Lovo cells in vitro were used as the research object. Morphological changes of apoptotic cells were observed by inverted microscope.AnnexinV-FITC/PI assay and DNA Ladder assay were performed to study the apoptotic effects. Immunocytochemistry was used to detect the expression of cleaved caspase-9 and cleaved caspase-3 proteins. RESULTS: Ethyl gallate had growth inhibition on colon cancer Lovo cells,the density was decrease: the apoptotic rates were increased with dose increase: Agarose gel electrophoresis showed evident DNA fragmentation: the expression of cleaved caspase-9 and cleaved caspase-3 proteins was increased in treated groups. CONCLUSION: Ethyl gallate could induce the apoptosis in Lovo cells through up-regulating the expression of cleaved caspase-9 and cleaved caspase-3 protein,and then activated the mitochondrial pathway.

Effects of chronic fluorosis on 4.8 kb mitochondrial DNA in liver, kidney and brain of rats / 中国地方病学杂志

Objective To investigate the deletion pattern of 4.8 kb mitochondrial DNA(mito-DNA) in liver,kidney,and brain of rats with chronic fluorosis and to explore the significance of mitochondria in the pathogenesis of chronic fluorosis.Methods Sixty SD rats were randomly divided into 3 groups according to body mass (20 in each group):control,low-fluoride and high-fluoride groups,and they were fed with different concentrations of fluoride in drinking water (0,10,50 mg/L,respectively) for 6 months.Mito-DNA in liver,kidney and brain was detected by real-time PCR.Results The amounts of 4.8 kb mito-DNA in liver(2.1 × 10-3,1.6 × 10-3),kidney (1.7 × 10-3,1.4 × 10-4) and brain cortex (1.5 × 10-5,1.3 × 10-5) in low-and high-fluoride groups were significantly reduced,as compared with that of control group (2.9 × 10-3,2.0 × 10-3,1.1 × 10-4,all P ＜ 0.05).The amount of 4.8 kb mito-DNA in kidney in high-fluoride group was lower than that in low-fluoride group (P ＜ 0.05).Conclusions Excessive fluoride intake can result in missing of 4.8 kb mito-DNA in liver,kidney and brain cortex.The abnormal of mito-DNA might be related to the dysfunction of mitochondrial respiratory chain.

Changed transcription level of mitochondrial fission and fusion gene loci in cortical neurons of rats with chronic fluorosis / 中国地方病学杂志

ObjectiveTo investigate the transcriptional changes of nitochondria fission and fusion gene loci and reactive oxygen species (ROS) level in cortical neurons of rats with chronic fluorosis,and to reveal their roles in mitochondria damage due to chronic fluorosis.MethodsSD rats were fed with different doses of fluoride through drinking water[＜ 0.5(control),10,50 mg/L,respectively] for 3 and 6 months.The level of ROS and mRNA contents of mitochondria fission gene loci Drp1/Fis1 and fusion gene locus Mfn1 in the cortical neurons of rat brains were detected with ROS fluorescent probe and real-time PCR,respectively.ResultsAs compared with control group [10.43 ± 5.98,(3.4 ± 0.6) × 103,(8.8 ± 1.4) × 10,(1.2 ± 0.2) × 102] at the experiment period of 3 months,the level of ROS and mRNA contents of mitochondria fusion gene locus Mfn1 and fission gene loci Drp1/Fis1 in the cortical neurons were obviously increased in the rats fed with 50 mg/L fluoride through drinking water[25.48 ± 6.09,(1.0 ± 0.2) × 1011,(3.0 ± 1.6) × 103,(8.9 ± 3.6) × 102,all P ＜ 0.05],whereas no significant changes were found in the rats fed with 10 mg/L fluoride[11.67 ± 3.49,(3.1 ± 0.3) × 104,(6.7 ± 2.7) × 10,(5.0 ± 0.9) × 10,all P ＞0.05].Furthermore,at 6 months of the experiment the increases in ROS level(63.02 ± 8.15,65.60 ± 7.40) and mRNA contents of mitochondria fission gene loci Drp1/Fis1 [(2.0 ± 0.8) × 106,(4.0 ± 0.6) × 105,(3.8 ± 1.3) × 103,(1.3 ± 0.2) × 103] and the decrease in mitochondrial fusion gene locus Mfn1[(3.0 ± 0.4) × 106、(4.0 ± 0.9) × 104]were observed in the cortical neurons of the rats fed with 10 mg/L and 50 mg/L fluoride as compared with the control group[25.26 ± 6.41,(3.0 ± 0.8) × 109,(5.1 ± 0.8) × 103,(2.8 ± 0.7) × 102,all P ＜ 0.05].Conclusions Excessive intake of fluorine leads to elevated ROS levels,and decreased transcription of mitochondrial fusion gene loci Mfn1,which is positively correlated with the time and dose-exposed to fluoride.The changes of mitochondrial fission and fusion gene loci in the cortical neurons may be related to high level of oxidative stress induced by chronic fluorosis.

Clinical Spectrum and Prognostic Factors of Acute Necrotizing Encephalopathy in Children

This study was conducted to investigate the etiology, the clinical characteristics and prognosis of acute necrotizing encephalopathy (ANE) in Korean children. Six children (1 yr to 7 yr) patients with ANE were enrolled. They were diagnosed by clinical and radiological characteristics and their clinical data were retrospectively analyzed. In a search of clinically plausible causes, brain MRI in all patients, mitochondrial DNA studies for mitochondrial encephalomyopathy, lactic acidosis, and strokelike episodes (MELAS) and myoclonus epilepsy and ragged red fibers (MERRF) in four patients, and genomic typing on HLA DRB/HLA DQB genes in three patients were performed. All had precedent illnesses and the main initial symptoms included mental change (83%), seizures (50%), and focal deficits (50%). MRI revealed increased T2 signal density in the bilateral thalami and/or the brainstem in all patients. Mitochodrial DNA studies for MELAS and MERRF were negative in those children and HLA-DRB1*1401, HLA-DRB3*0202, and HLA-DQB1*0502 seemed to be significant. A high dose steroid was given to all patients, which seemed to be partly effective except for 2 patients. In conclusion, ANE is relatively rare, but can result in serious neurological complication in children. Early detection and appropriate treatment may lead to a better neurological outcome.

Young and aging human embryonic lung diploid cell line WI-38: A comparison of mitochondrial content, relative amount of mtDNA, and mitochondrial functions / 第二军医大学学报

Objective: To compare the mitochondrial content, the relative amount of mtDNA, and mitochondrial functions between the young and aging WI-38 cells, so as to investigate the correlation between mitochondrial and aging. Methods: Human embryonic lung diploid cell line WI-38 was cultured and its viability was assayed by MTT assay; the content of mitochondrial protein was determined using BCA-100 Protein Quantitative Analysis Kit after mitochondria were fractionated by differential centrifugation; mtDNA relative content was measured by a competitive polymerase chain reaction (PCR) method; mitochondrial membrane potential was measured by flow cytometry; and NADH oxidase activity was measured by spectrophotometry. Results: Compared with the young cells, the aging cells had a longer time to form a monolayer, an obviously decreased cell viability and mitochondrial membrane potential (by 50%), and a decreased NADH oxidase activity, with the maximal reaction speed declining from 66.73 nmol/(mg protein · min) to 36. 01 nmol/(mg protein · min). Mitochondrial content in the aging cells([0.78 ± 0.02] mg/ml) was higher than that in the young cells([0.56 ± 0.03] mg/ml). Using 18S rDNA of nuclear as an internal reference, the relative amount of mtDNA in the aging cells (1.557 ± 0.072) was found to be obviously higher than that in the young cells (1.292 ± 0.068). Conclusion: The increase of mitochondrial contents and mtDNA relative amounts in aging cells may be one of the compensatory mechanisms for decreased mitochondrial function, which may provide an evidence for studying the correlation between mitochondrial and aging.

Mitochondrial Intestinal Pseudo-Obstruction with Neurogenic Bladder Syndrome: Point Mutation at T8356C: A New Mitochondrial Disease?

We describe a unique patient with progressive external ophthalmoplegia, intestinal pseudo-obstruction, and neurogenic bladder. Genetic study in this patient shows point mutation at T8356C, the locus known as that of myoclonic epilepsy with ragged-red fibers. To the best of our knowledge, this is the first report of a mitochondrial syndrome consisting of intestinal pseudo-obstruction, neurogenic bladder, and progressive external ophthalmoplegia, point mutation at T8356C. We suggest that this could comprise a new mitochondrial disease rather than a new variant of mitochondrial neurogastrointestinal encephalomyopathy.

Mitochondrial DNA partly point mutations in praecox Parkinson's disease / 中华神经科杂志

Objective To test the association between mitochondrial DNA(mtDNA) point mutations and praecox Parkinson's disease (PPD),and to investigate the characteristics of mtDNA mutations in Chinese patients with PPD. Methods Screening mtDNA A4336C, G5460A,A10398G,A13780G point mutations in 40 patients with PPD and 48 in control group was carried out by using Polymerase Chain Reaction (PCR), dot blotting, radiant developing. And sequencing was given to the nucleotide position (np)10256～np10577mtDNA of 20 patients with PPD and 20 subjects in control group.Results Out of the 40 patients with PPD, 20 (50%)had A10398G mutation. 6 (15%) had G5460A, 5(12.5%) had A13780G, 2 (5%)had A4336C, 19 (47.5%)had C10400T mutation. Out of the 48 controls, 7(14.6%) had A10398G, 24.2% had G5460A, 1 (2.1%)had A13780G and 20 (41.7%)had C10400T, but no any A4336C mutation was found in the controls. Thus, the ratios of A10398G,G5460A,A4336C,A13780G in patients with PPD were separately higher than those in the control group. Moreover significant difference was found in A10398G point mutation (P