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)
ObjectiveMitochondrial encephalomyopathy is a series of diseases that drag in central nervous system and generalized muscles. The pathogenesis of the disease is lack of ATP for the dysfunction of mitochondria. The misdiagnosis rate of the disease is high and the purpose of this study is to improve the recognition and diagnosis of mitochondrial encephalomyopathy and thus, clinicians could take rational treatment in time and improve patients' prognosis.
MethodsThe clinical data of 11 patients with mitochondrial encephalomyopathy were analyzed including the physical data, clinical presentations, laboratory data, neuroimaging findings, muscle biopsy, genetic testing, treatment and prognosis. Reviewing literature and summarizing the clinical characteristics of mitochondrial encephalomyopathy.
ResultsAmong the 11 patients with mitochondrial encephalomyopathy, the mean age was 17 years old. 1 case had family history. 7 cases were misdiagnosed in the first clinic visit. The onset of the 11 cases, 9 were paroxysmal and 2 were hidden. In the course, 10 cases had an epileptic seizure. Among the 9 cases who took the determination of serum lactate, 8 was in high level.9 cases had MRI examination and all found abnormality, 10 patients had EEG examination, and 9 cases found abnormality, 6 cases had muscle biopsy and all found the ragged red fiber(RRF). 6 cases had molecular genetic testing, and all found mutations in mitochondrial DNA. Among the 10 cases who had epileptic seizure, 3 cases can be controlled with single kind of antiepileptic drug. The other 7 cases had a recurrence of epilepsy with single kind of antiepileptic drugs, but can be cotrolled after drug adjusting or drug combination.
ConclusionMitochondrial encephalomyopathy is often accompanied by seizure, which is usually found in children, and also often accompanied by systemic muscle symptoms. The clinical manifestations of the disease is not typical, but is complex and varied symptoms, so the clinical misdiagnosis rate is high. Mitochondrial encephalomyopathy mainly involves the main intracranial artery distribution area (parietal lobe, temporal lobe, occipital lobe, etc.) in central nervous system, and can involve more than one part. Patients with mitochondrial myopathy brain are usually detected the elevation of serum lactate levels, but if the lactic acid level is normal, it does not rule out the possibility of the disease, the confirmation of the disease is mainly by muscle biopsy or genetic tests. There is no specific treatment for mitochondrial encephalomyopathy till now, and it still give priority to symptomatic treatment. And the prognosis is poorer.
Objective To review the research progress of mitochondrial dynamics mediated by optic atrophy 1 (OPA1) in skeletal system diseases. MethodsThe literatures about OPA1-mediated mitochondrial dynamics in recent years were reviewed, and the bioactive ingredients and drugs for the treatment of skeletal system diseases were summarized, which provided a new idea for the treatment of osteoarthritis. Results OPA1 is a key factor involved in mitochondrial dynamics and energetics and in maintaining the stability of the mitochondrial genome. Accumulating evidence indicates that OPA1-mediated mitochondrial dynamics plays an important role in the regulation of skeletal system diseases such as osteoarthritis, osteoporosis, and osteosarcoma. Conclusion OPA1-mediated mitochondrial dynamics provides an important theoretical basis for the prevention and treatment of skeletal system diseases.
Augmenter of liver regeneration (ALR) is a newly discovered cytokine that can promote liver regeneration and proliferation of damaged liver cells. In the renal tissue, ALR is mainly expressed in the cytoplasm of the medullary loops, collecting ducts and distal convoluted tubules in the renal medulla, and is low in the glomerular and cortical tubules. Various stimulation, such as ischemiacal, hypoxia, poisoning and inflammatory stimulation, can induce the expression of ALR in the epithelial cells of proximal tubule regeneration and the damaged areas of cortex, and participate in the repair process. Current studies have found that in acute kidney injury (AKI), exogenous ALR can protect renal tubular epithelial cells by inhibiting apoptosis of renal tubular epithelial cells, promoting proliferation of renal tubular epithelial cells, inhibiting the activities of inflammatory cells, and promoting the reduction of renal injury. This paper intends to review the basic characteristics of ALR and the pathogenesis of AKI, summarize the characteristics of the mechanism of ALR in AKI by combing the relevant literature on ALR and AKI in recent years, and provide knowledge reserve and direction reference for the in-depth study of ALR in kidney in the future.
Objective
To analyze the glucolipotoxicity effects of glucose combined with free fatty acid (FFA) on ketone production and ultrastructure of skeletal muscle, by exogenous elevating circulating glucose and FFA concentration.
Methods
Fifty Wistar rats were divided into high-fat-feed induced obesity group (OB group, n=40) and ordinary feed as normal control group (NC group, n=10). Circulating glucose and FFA levels were increased by infusion in high-fat-fed obese rats. The levels of serum lipid, plasma FFA and beta-hydroxybutyric acid were detected by the horizontal colorimetry, and the microstructure of skeletal muscle was observed by transmission electron microscopy, especially the changes of the mitochondrial structure. Euglycemic-hyperinsulinemic clamp with tracer infusion was performed to assess peripheral insulin sensitivity.
Results
The average weight and body fat ratio in the OB group was higher than that in the NC group (P<0.05). Insulin clamp test to assess peripheral insulin sensitivity showed that the steady-state glucose Infusion rate in the OB group during clamp test was significantly lower than that in the NC group [OB: (19.26±1.84) mg/(kg·min)vs. NC: (28.82±1.69) mg/(kg·min), P<0.05]. The mitochondrial denaturation of skeletal muscle in the OB group of rats was observed, and the swelling and crest permutation, the accumulation of lipid droplets and cavitation were formed, and hypertrophy of mitochondria were also seen after intralipid and glucose infusion, which was obvious in the combined infusion group.
Conclusions
By exogenous elevating circulating glucose and FFA concentration, the products of ketone body increases. The mitochondrial damage of skeletal muscle suggests that mitochondrial may be the potential target of glucoxicity and lipotocicity.
ObjectiveTo observe the effects of p21 activated kinase 4 (PAK4) on the mitochondrial function and biological behavior in retinal vascular endothelial cells. MethodsThe experimental study was divided into two parts: in vivo animal experiment and in vitro cell experiment. In vivo animal experiments: 12 healthy C57BL/6J male mice were randomly divided into normal control group and diabetes group, with 6 mice in each group. Diabetes mice were induced by streptozotocin to establish diabetes model. Eight weeks after modeling, quantitative real-time polymerase chain reaction and Western blots were performed to detect the expression of PAK4 in diabetic retinas. In vitro cell experiments: the human retinal microvascular endothelial cells (hRMEC) were divided into three groups: conventional cultured cells group (N group), empty vector transfected (Vector group); pcDNA-PAK4 eukaryotic expression plasmid transfected group (PAK4 group). WB and qPCR were used to detect transfection efficiency, while scratching assay, cell scratch test was used to detect cell migration in hRMEC of each group. In vitro white blood cell adhesion experiment combined with 4 ', 6-diamino-2-phenylindole staining was used to detect the number of white blood cells adhering to hRMEC in each group. The Seahorse XFe96 cell energy metabolism analyzer measures intracellular mitochondrial basal respiration, adenosine triphosphate (ATP) production, maximum respiration, and reserve respiration capacity. The t-test was used for comparison between the two groups. Single factor analysis of variance was used for comparison among the three groups. ResultsIn vivo animal experiments: compared with normal control group, the relative expression levels of PAK4 mRNA and protein in retina of diabetic mice were significantly increased, with statistical significance (t=25.372, 22.419, 25.372; P＜0.05). In vitro cell experiment: compared with the N group and Vector group, the PAK4 protein, mRNA relative expression and cell mobility in the hRMEC of PAK4 group were significantly increased, with statistical significance (F=36.821, 38.692, 29.421; P＜0.05). Flow cytometry showed that the adhesion number of leukocytes on hRMEC in PAK4 group was significantly increased, and the difference was statistically significant (F=39.649, P＜0.01). Mitochondrial pressure measurement results showed that the capacity of mitochondrial basic respiration, ATP production, maximum respiration and reserve respiration in hRMEC in PAK4 group was significantly decreased, with statistical significance (F=27.472, 22.315, 31.147, 27.472; P＜0.05). ConclusionOver-expression of PAK4 impairs mitochondrial function and significantly promotes leukocyte adhesion and migration in retinal vascular endothelial cells.
Objective To analyze the thickness of peripapillary retinal nerve fiber layer (pRNFL) and photoreceptor (PR) sublayer in Leber hereditary optic neuropathy (LHON) and G11778A mutation carriers. MethodsA cross sectional study. From September 2020 to October 2021, 68 LHON patients (136 eyes) (patient group) and 40 G11778A mutation carriers (80 eyes) of LHON patients' families (carrier group) were included in the study. All patients were found to have G11778A mutation by Genetic testing. Forty healthy volunteers with 80 eyes matched to the age and gender of the patient group were recruited as a normal control group. All eyes were examined by optical coherence tomography (OCT). The pRNFL thickness was automatically measured by the built-in software of the OCT device. The total retinal thickness (MT) and the thickness of the outer bundle layer (OPL), outer nuclear layer (ONL), external limiting membrane to retinal pigment epithelium (ELM-RPE) in macular OCT images were measured by Image J software. Linear mixed model was used to analyze and compare the thickness of pRNFL, macular fovea and four layers above the nasal and temporal paracentral retina in patients, carriers and normal controls. The correlation between pRNFL and macular retinal sublayer thickness and the course of disease was also analyzed. ResultsThe thickness of the upper and lower pRNFL, temporal pRNFL and average pRNFL of the patients were smaller than those of the carriers and the normal control group (P＜0.01), and the nasal pRNFL thickness of the patients was smaller than that of the carriers (P＜0.01). Fovea: compared with the normal control group, the thickness of MT and ONT in the patient group was decreased, ONL thickness decreased in carrier group, with the significant different (P＜0.05). Parafovea: compared with normal control group, the thickness of MT and temporal ONL decreased and temporal OPL increased in the patients group, with the significant different (P＜0.05). In the carrier group, the thickness of MT and temporal, nasal ONL decreased, and the thickness of nasal OPL increased, with the significant different (P＜0.05). Compared with the carrier group, the MT thickness of the patient group was decreased, and the nasal ONL and nasal ELM-RPE thickness were increased, with the significant different (P＜0.05). Correlation analysis results showed that the thinning of pRNFL in the superior, nasal, temporal and average (r=－0.22, －0.21, －0.25, －0.22), and the thickening of ELM-RPE in foveo-temporal (r=0.19) were correlated with the course of disease (P＜0.05). ConclusionsThe pRNFL of LHON patients with G11778A mutation becomes thinner and is related to the course of the disease. There were significant differences in the thickness of MT and PR sublayers between patients and carriers compared to the normal control group.
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.
RCBTB1 gene associated hereditary retinopathy is an extremely rare inherited retinal disease (IRD) discovered recently. The mutation of RCBTB1 gene can lead to a variety of IRD clinical phenotypes, such as early retinitis pigmentosa and delayed chorioretinal atrophy. The hereditary mode of RCBTB1 gene associated retinopathy is autosomal recessive. RCBTB1 gene plays an important role in maintaining mitochondrial function and anti-oxidative stress defense mechanism of retinal pigment epithelium cells. In the future, it is necessary to further determine whether there is a genotypic and phenotypic correlation in the age of onset of RCBTB1 gene associated retinopathy or multi-organ involvement, and evaluate the safety and efficacy of adeno-associated virus-mediated RCBTB1 gene replacement therapy in animal models, to explore the feasibility of gene replacement therapy and stem cell therapy.
Leber’s hereditary optic neuropathy (LHON) is a paradigm maternal hereditary eye disease, mainly involving the retinal and macular fibers of the optic disc in the anterior ethmoid plate of the sclera. LHON has the characteristics of sex bias among males and incomplete penetrance. Primary mitochondrial DNA mutations m.11778G>A, m. 14484T>C, m.3460G>A are the molecular basis of LHON. However, other risk factors, such as secondary mitochondrial DNA mutations, mitochondrial haplotypes, nuclear modification genes, estrogen, vitamin B12 and environmental factors, work together to affect its phenotypic expression. The clinical diagnosis of LHON mainly limited to the detection of the primary mutation site of mitochondrial DNA. Therefore, comprehensive analysis of multiple risk factors of LHON will facilitate to construct multi-dimensional model of prevention, diagnosis and treatment system, which provide accurate and individualized medical services for patients. These may alleviate the incidence in LHON families. It also provides new ideas and different angles for the in-depth study of the pathogenesis of LHON.
