Objective To explore the effects of drugs on functions of mitochondria in retinal nerve cells, and to lay a foundation of the investigation of drug protection for retinal nerve cells. Methods Cultivation of the retinal nerve cells of 8 eyes of neonatal calves was performed. The changes of fluorescent density of the mitochondria of cultured cells labeled by dye rhodamine 123 (Rh123) before and after the activation of the medicines, including ferulic acid (FA), arginine, glycine,taurine, vitamine E and brain derived neurotrophic factor( BDNF) respectively, were detected by laser-scanning confocal microscopy. Results FA with the concentration of 500 μg/ml led the diphasic variation of the fluorescent intensity of mitochondria. After scanning for 60.772 seconds when treated with FA firstly, the fluorescent intensity decreased rapidly (from 45.425±4.153 to 22.135±5.293); while after 112.774 seconds when treated secondly, the in tensity increased obviously (from 19.655±4.383 to 28.247±4.764), and after 168.773 seconds when treated thirdly the intensity still increased. After scanning for 56.457 seconds when treated with vitamin E (12.5 mg/ml), the fluorescent in tensity increased obviously (from 88.255±5.039 to 111.273±4.529), which suggested that vitamin E with the concentration of 12.5 mg/ml strengthen the fluorescent intensity. After scanning for 58.147 and 134.148 seconds when treated with BDNF(50 ng/ml) respectively, the fluorescent intensity increased obviously (from 69.115±5.038 to 77.225±5.131) which suggested that BDNF with the concent ration of 50 ng/ml led the increase of the fluorescent intensity. Glycine (2.5 mg/ml) and arginine(30 mg/ml) didn’t affect the fluorescent intensity of mitochondria, and taurine (6.25 mg/ml) caused the appreciable decrease of the fluorescent intensity . Conclusion FA, BDNF and vitamin E may promote the metabolism of retinal nerve cells via the path of mitochondria, while amino acids may adjust the activation of retinal nerve cells through other ways. (Chin J Ocul Fundus Dis，2004，20：229-232)
Objective\ To study the mechanism of myocardial protection of exogenous creatine phosphate (CP) against ischemia reperfused injury in modified isolated perfused working rat heart model.\ Methods\ Seventy two rats were divided into five groups.The rat hearts of five groups undergone Langendorff perfused were arrested and made totally ischemic for 40 minutes at 37℃ and reperfused for 20 minutes. St.Thomas cardioplegic solution wasn’t used in group A;It was used immediately after ischemia in group B and grou...
Objective To investigate the role of mitochondrial adenosine triphosphatesensitive potassium channel(mitoKATP) in immature myocardial ischemic preconditioning, and to provide evidence for immature myocardial protection. Methods Langendorff isolated heart infused model was used in the experiment. Twentyfour rabbits (aged from 14 to 21 days) were randomly divided into 4 groups：ischemiareperfusion group（I/R group）, myocardial ischemic preconditioning group(E1 group), 5hydroxydecanoate(5-HD) group (E2 group) and Diazoxide (Diaz) group(E3 group). Hemodynamics recovery rate, myocardial water content(MWC), the leakage rates of serum creatine kinase and lactate dehydrogenase, adenosine triphosphate content, superoxide dismutase activity, malondialdehyde content, myocardial cell Ca2+ content and myocardial mitochondrial Ca2+ content, myocardial mitochondrial Ca2+-ATPase activity, the adenosine triphosphate(ATP) synthesizing ability of myocardial mitochondria were tested, and myocardial ultrastructure was observed via electron microscopy. Results The hemodynamics recovery rate, myocardial water content(P＜0.05), adenosine triphosphate content, superoxide dismutase activity, myocardial mitochondrial Ca2+-adenosine triphosphyatase(ATPase) activity and the ATP synthesizing ability of myocardial mitochondria of the rabbits in E1 and E3 group were significantly better than that in I/R group and E2 group(P＜0.05). Malondialdehyde content, the leakage rates of serum creatine kinase and lactate dehydrogenase, myocardial cell Ca2+ content and myocardial mitochondrial Ca2+ content of the rabbits in E1 group and E3 group were significantly lower than that in I/R group and E2 group (P＜0.05). The myocardial ultrastructure injury in E1 and E3 group were significantly reduced compared with that in I/R and E2 group. Conclusion Myocardial ischemic preconditioning has significant protective effects on immature myocardium. Its mechanism may be related to the activation of mitoKATP.
ObjectiveTo investigate the expression of mitochondrial transcription factor A (TFAM) in colon cancer and the effect of its expression on proliferation of colon cancer cell. MethodsThirty cases of colon cancer in the First Affiliated Hospital of Sun Yat-sen University from March 2013 to April 2013 were studied. TFAM mRNA was detected both in colon cancer tissue and para-cancer tissue by real-time PCR. TFAM mRNA and protein were detected in normal colon cell strain and colon cancer strains SW480, HT-29, and HCT116 by real-time PCR and Western blot, respectively. The proliferation of SW480 cells was evaluated after up-regulating TFAM. ResultsThe expression of TFAM mRNA in the colon cancer tissue was significantly higher than that in the para-cancer tissue (P < 0.000 1). The expressions of TFAM mRNA were obviously increased in the SW480, HT-29, and HCT116 cells as compared with the normal colon cell strain (P value was 0.000 8, 0.002 3, and 0.000 6, respectively), among which the most notable increase was detected in the SW480 cells. The expressions of TFAM protein were obviously increased in the SW480, HT-29, and HCT116 cells as compared with the normal colon cell strain (P value was 0.000 2, 0.003 8, and 0.001 6, respectively), among which the most notable increase was detected in the SW480 cells. After up-regulating TFAM by plasmid transfection, the proliferation of the pcDNA3.1-TFAM-SW480 cell was increased significantly as compared with the pcDNA3.1-SW480 cell at 96 h and 120 h after transfection by the MTT test (P < 0.000 1). The proliferation of the pcDNA3.1-TFAM-SW480 cell was increased significantly as compared with the pcDNA3.1-SW480 cell at 48 h after transfection by the BrdU test (P < 0.001 0). ConclusionTFAM expression is high in colon cancer. Up-regulated TFAM could promote the proliferation of colon cancer cells.
Objective To explore the relationship between microsatellite instability （MSI） and gastric cancer. Methods The related literatures at home and abroad were consulted and reviewed. Results The MSI is the replication errors caused by mismatch repair system defects. Gastric cancer which exhibiting MSI has characteris clinicopathological feature and prognosis. Detection the MSI of precancerous lesions and gastric cancer tissues can evaluate the risk and prognosis of gastric cancer. MSI include nuclear microsatellite stability （nMSI） and mitochondrial microsatellite instability （mtMSI）. Conclusions MSI plays an important role in the occurrence and development of gastric cancer. MSI may become a important indicator to forecast precancerosis risks and clinical prognosis of gastric cancer.
Objective To investigate the protective effects of ischemic postconditioning (IPo) on ischemiareperfusion (I/R) myocardium and the relationship with mitochondrial adenosine triphosphate (ATP) sensitive K+ channels (mitoKATP) and provide evidences to the development of druginduced postconditioning. Methods Langendorff models were established in 40 Wistar rats which were divided into 5 groups by random number table with 8 rats in each group. Normal control group(NC group): the rat hearts were continuously reperfused by KrebsHenseleit bicarbonate buffer (K-HB) for 100 min without any other treatment; I/R group: the rat hearts underwent a 40-min global ischemia followed by a 60-min reperfusion; IPo group: after a 40-min global ischemia, the process of 10-second reperfusion followed by a 10-second ischemia was repeated 6 times, then there was a continuous 58min reperfusion; 5-hydroxydecanoic acid(5-HD) group: after a 40min global ischemia, hearts with 5HD(100 μmol/L) K-HB were reperfused for 15min and then perfused without 5HD for 45min；IPo+5-HD group: after a 40-min global ischemia, the process that the isolated hearts with 5-HD(100 μmol/L) KHB were reperfused for 10second followed by a 10second ischemia was repeated 6 times, then the hearts with 5-HD(100 μmol/L) KHB were continuously [CM(159mm]perfused for 13-min followed by reperfusion without 5-HD(100 μmol/L) K-HB for 45-min. The cardiac function,coronary flow(CF), cardiac troponin I(cTnI) content in coronary effluent, the area of acute myocardial infarction (AMI) and myocardial ultrastructure were observed. Results Left ventricular developed pressure(74.3±3.3 mm Hg vs. 57.1±3.3 mm Hg,t=1300, P=0.000),+dp/dtmax(1 706.6±135.6 mm Hg/s vs. 1 313.3±96.2 mm Hg/s,t=6.28,P=0.000),－dp/dtmax(1 132.8±112.1 mm Hg/s vs. 575.7±67.7 mm Hg/s,t=13.48, P=0.000) and CF(6.49±0.30 ml/min vs. 3.70±0.24 ml/min,t=28.6,P=0.000) in IPo group were higher than those in I/R group. Left ventricular enddiastolic pressure(10.9±1.7mm Hg vs. 26.2±1.5 mm Hg,t=－19.21, P=0000)and cTnI content in coronary effluent (0.62±0.01 ng/ml vs. 0.71±0.01 ng/ml, t=-12.00,P=0.000) were lower than those in I/R group; the area of AMI decreased 20.8％ compared with that in I/R group (Plt;0.05). The myocardial protective effect in IPo+5HD group was similar with that in IPo group, but lower than that in IPo group. The electron microscope showed that IPo and IPo+5HD could reduce myocardial fiber damage and mitochondrial damage caused by I/R. Conclusion IPo can protect I/R myocardium, which is achieved mainly by activating mitoK-ATP channels.
ObjectiveTo investigate the feasibility of immoribund skin fibroblast cell line derived from Leber′s hereditary optic neuropathy (LHON) patients as a cell model. MethodsA basic research. Two LHON patients and 2 healthy volunteers were recruited from Department of Ophthalmology of Genetic Clinic of Henan Provincial Eye Hospital. The skin tissue of participants was obtained, and the 4 immortalized skin fibroblasts were constructed by SV40 virus infection, including 2 LHON patient cells (LHON-1 and LHON-2 cells) and 2 healthy volunteers cells (NC-1 and NC-2 cells). Mitochondrial morphology in cells was observed by electron microscope. The levels of reactive oxygen species (ROS), nicotinamide adenine dinucleotide-oxidation state (NAD+), nicotinamide adenine dinucleotide-reduction state (NADH) and adenosine triphosphate (ATP) in fibroblasts were detected. Cellular oxygen consumption was measured by seahorse mitochondrial pressure assay. Cell viability was detected using cell counting kit-8 (CCK8). One-way ANOVA was performed to compare the levels of ROS, NAD+, NADH and ATP in LHON and NC cells, as well as basal oxygen consumption, maximal oxygen consumption, ATP-coupled oxygen consumption, and cell viability. ResultsCompared with NC-1 and NC-2, the number of mitochondrial crest in LHON fibroblasts was significantly reduced, indicating abnormal mitochondrial morphology. Biochemical analysis showed that ROS levels in LHON cells increased, but NAD+/NADH and ATP levels decreased, and the oxygen consumption was significantly inhibited, indicating the presence of mitochondrial damage and respiratory dysfunction. The results of CCK-8 detection showed that the survival ability of LHON-1 and LHON-2 cells was worse under stress conditions. ConclusionImmortalized skin fibroblast cell lines from LHON patients presented mitochondrial dysfunction.
【Abstract】 Objective To analyze the correlations between the mt5351G and mt6680C genotypes in mitochondrial DNA ( mtDNA) haplogroup M and susceptibility to high altitude pulmonary edema ( HAPE)among the Hans. Methods Specimens from206 Hans cases of HAPE and 144 matched Hans controls were collected. Then PCR-RFLP method was used to determine haplogroup M and N of mtDNA, and PCR-LDR was used to genotype mt5351G and mt6680C in the haplogroup M in these samples. Results The frequencies of haplogroup Mand N were 49. 0% and 51.0% in the HAPE patients, and 47. 2% and 52. 8% in the controls, respectively, with no significant difference between the HAPE patients and the controls. In the haplogroup M, the genotype of mt6680C and mt5351G frequencies in the HAPE patients were both significantly higher than the controls ( both 12. 0% vs. 1. 5% , P = 0. 016) . Conclusion The existence of mt5351G and mt6680C genotypes in the haplogroup Mis a risk factor for HAPE among the Hans.
Objective To investigate the potential mechanism of cellular senescence-related mitochondrial autophagy genes in diabetic retinopathy (DR). MethodsThe DR gene datasets GSE53257 and GSE60436 from the GEO database and screened the differentially expressed genes (DEG) were downloaded. Cellular senescence-related genes and mitochondrial autophagy-related genes from the GeneCards database, and the intersection of the two to obtain the DR-related differentially expressed genes (CSRMRDEG) were collected. The obtained CSRMRDEG was subjected to Gene Ontology (GO) functional enrichment analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway enrichment analysis, protein-protein interaction network (PPI) analysis, and hub gene identification using Maximal Clique Centrality (MCC), Degree, Maximum Neighborhood Component (MNC)、Edge Percolated Component (EPC) and Closeness algorithms. Gene Set Enrichment Analysis (GSEA) was conducted to obtain the enriched pathways of DEG, and ssGSEA immune infiltration analysis was performed to screen the correlation between immune cells and DR. The diagnostic efficacy of hub genes for DR was evaluated by drawing the receiver operating characteristic (ROC) curve and calculating the area under the curve (AUC). Meanwhile, the Wilcoxon rank sum test was used to compare the differences in the infiltration level of immune cells between the DR Group and the control group. Results23 DR-related CSRMRDEG were obtained. GO analysis showed that they were mainly enriched in the pathways of dicarboxylic acid, biosynthetic process of folate-containing compounds, tetrahydrofolate conversion, mitochondrial matrix, mitochondrial endomembrane, structural components of ribosomes, and glutamate transmembrane transporter protein activity. The results of KEGG pathway enrichment analysis showed that CSRMRDEG was highly enriched in pathways such as the folate carbon pool, biosynthesis of cofactors, and pyruvate metabolism. The PPI analysis results show that there are 16 related CSRMRDEG. Five algorithms (MCC, Degree, MNC, EPC, Closeness) obtained the nine Hub genes. The results of ROC curve analysis showed that the AUC of the expression levels of 9 hub genes for diagnosing DR ranged from 0.7-0.9. The ssGSEA results showed that there were statistically significant differences in Wilcoxon of central memory CD4+ T cells, macrophages, natural killer cells, and helper T cell 1 between the DR group and the control group (Z=−2.85, −2.23, −2.10, −2.52; P＜0.05). ConclusionMitochondrial autophagy genes related to cellular senescence are potential diagnostic targets for DR.
Abstract： Objective To study the changes of the cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) expression of isolated rat hearts after diazoxide preconditioning (DPC), and to explore the possible mechanism of cAMP signaling pathway in myocardial protection by DPC. Methods Isolated working heart Langendorff perfusion models of 40 Wistar rats were set up and were divided randomly into four groups. For the ischemia reperfusion injury(I/R) group (n=10), 30 min of equilibrium perfusion was followed by a 60 min reperfusion of KrebsHenseleit (K-H) fluid. The DPC group (n=10) had a 10 min equilibrium perfusion and two cycles of 5 min of 100 μmol/L diazoxide perfusion followed by a 5 min diazoxidefree period before the 30 min ischemia and the 60 min reperfusion of K-H fluid. The blank control group (control group, n=10) and the Dimethyl Sulphoxide(DMSO) group (n=10) were perfused with the same treatment as in the DPC group except that diazoxide was replaced by natriichloridum and DMSO respectively. The activity of creatine kinase (CK) in coronary outflow, the activity of malonyldialdehyde (MDA) and superoxide dismutase (SOD) in myocardium were detected. And the scope of myocardial infarction and the concentrations of myocardial cAMP and PKA were also assessed. Results Compared with the I/R group, the level of MDA for the DPC group decreased significantly (8.28±2.04 nmol/mg vs. 15.52±2.18 nmol/mg, q=11.761,Plt;0.05), the level of SOD increased significantly (621.39±86.23 U/mg vs. 477.48±65.20 U/mg, q=5.598,Plt;0.05). After a 30 min reperfusion, compared with the I/R group, the content of CK decreased significantly (82.55±10.08 U/L vs. 101.64±19.24 U/L, q=5.598, Plt;0.05) and the infarct size reduced significantly (5.63%±9.23% vs.17.58%±5.76%, q=6.176,Plt;0.05) in the DPC group. The cAMP concentration in the DPC group was much higher than that in the I/R group (0.64±0.07 pmol/g vs. 0.34±0.05 pmol/g, q=14.738,Plt;0.05), and PKA concentration was also much higher than that in the I/R group ［17.13±1.57 pmol/(L·min·mg) vs. 12.85±2.01 pmol/(L·min·mg), Plt;0.05］. However, there were no significant differences between the I/R group, DMSO group and the control group in the above indexs (Pgt;0.05). Conclusion DPC significantly improves the releasing of cAMP and PKA, decreases oxygen free radicals, and relieves myocardial ischemia reperfusion injury. The cAMP signaling pathway may be involved in triggering the process of myocardial protection mechanisms of DPC.