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.
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 investigate the preventive and therapeutic effects and the mechanisms of pyrrol idine dithiocarbamate (PDTC) on the atrophy of denervated skeletal muscle. Methods Thirty adult Wistar rats of either gender, weighing (200 ± 10) g were randomly divided into 3 groups: group A (n=6, control group), group B (n=12, denervation group), and group C (n=12, PDTC treatment group). The sciatic nerves of the rats were only exposed without cutting off in group A, and the rats were made denervated gastrocnemius models in groups B and C. PDTC of 100 mg/(kg?d) was injected peritoneally in group C and an intraperitoneal injection of the same amount normal sal ine was given in group B. After 14 and 28 days, the gastrocnemius was harvested to measure the ratio of muscle wet weight; the levels of nuclear factor of κB (NF-κB)p65 protein and the opening of the mitochondrial permeabil ity transition pore (MPTP) in the gastrocnemius were detectedrespectively by Western blot and laser confocal scanning microscope; and the apoptotic cells in atrophic muscle were measured with TUNEL. Results The ratio of muscle wet weight in group A was 1.039 ± 0.115, and it significantly decreased in groups B and C (P lt; 0.05); after 14 and 28 days of operation, the ratio of muscle wet weight in group C significantly increased when compared with those in group B (P lt; 0.05). The expression of NF-κB p65 protein in group A was 0.224 ± 0.041; the expressions of NF-κB p65 in groups B and C significantly increased when compared with that in group A (P lt; 0.05); however, the expression of NF-κB p65 in group C was significantly lower than that in group B (P lt; 0.05). The MPTP fluorescence intensity in group A was 31.582 ± 1.754; the MPTP fluorescence intensity was significantly lower in groups B and C than in group A (P lt; 0.05), and the MPTP fluorescence intensity in group C was significantly higher than that in group B (P lt; 0.05). The rate of apoptosis in group A was 4.542% ± 0.722%; after 14 and 28 days of operation, the rates of apoptosis significantly increased when compared groups B and C with group A, and signiticantly decreased when compared group C with group B (P lt; 0.05). Conclusion PDTC can retard denervated skeletal muscle atrophy, and the effect may have a relationship with its inhibition on NF-κB, the opening of the MPTP, and the ratio of apoptosis.
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.
Alzheimer’s disease (AD) is the most common degenerative disease of the nervous system. Studies have found that the 40 Hz pulsed magnetic field has the effect of improving cognitive ability in AD, but the mechanism of action is not clear. In this study, APP/PS1 double transgenic AD model mice were used as the research object, the water maze was used to group dementia, and 40 Hz/10 mT pulsed magnetic field stimulation was applied to AD model mice with different degrees of dementia. The behavioral indicators, mitochondrial samples of hippocampal CA1 region and electrocardiogram signals were collected from each group, and the effects of 40 Hz pulsed magnetic field on mouse behavior, mitochondrial kinetic indexes and heart rate variability (HRV) parameters were analyzed. The results showed that compared with the AD group, the loss of mitochondrial crest structure was alleviated and the mitochondrial dynamics related indexes were significantly improved in the AD + stimulated group (P < 0.001), sympathetic nerve excitation and parasympathetic nerve inhibition were improved, and the spatial cognitive memory ability of mice was significantly improved (P < 0.05). The preliminary results of this study show that 40 Hz pulsed magnetic field stimulation can improve the mitochondrial structure and mitochondrial kinetic homeostasis imbalance of AD mice, and significantly improve the autonomic neuromodulation ability and spatial cognition ability of AD mice, which lays a foundation for further exploring the mechanism of ultra-low frequency magnetic field in delaying the course of AD disease and realizing personalized neurofeedback therapy for AD.
The main function of mitochondrial fusion protein 1 (Mfn1) and mitochondrial fusion protein 2 (Mfn2) was originally thought to be just regulating the fusion of mitochondrial outer membrane. But in recent years,many studies on these two proteins show that they are involved in many important cellular physiological processes including proliferation,apoptosis,necrosis and regulation of respiratory function and oxidative metabolism. There are many aspects of the influenceof Mfn1 and Mfn2 on cardiomyocyte,which have not been thoroughly studied yet,sometimes with even contradictoryconclusions. But these two proteins definitely have significant impact on the growth,development and physiological functionof cardiomyocyte. To investigate the function and mechanism of Mfn1 and Mfn2 in various physiological processes of cardiomyocyte is of great significance for in vitro studies of physiological functions of cardiomyocyte and technological development of myocardial tissue engineering and transplantation in vivo. This article mainly focuses on recent research progress of the influence of Mfn1 and Mfn2 on various physiological functions of cardiomyocyte.
ObjectiveTo analyze the variation of perioperative concentration of mitochondrial DNA (mtDNA) in circulation system after cardiac surgery with cardiopulmonary bypass (CPB).
MethodsBetween July and December 2014, 40 continuous patients underwent aortic valve replacement (AVR) and mitral valve replacement (MVR) in Department of Cardiovascular Surgery, West China Hospital, Sichuan University, including 16 males and 14 females with their mean age of 48.7±11.0 years and mean body weight of 59.0±6.9 kg. Perioperative mtDNA concentrations of circulatory blood were tested at different time points:before general anesthesia (T1), 1 min before CPB (T2), reperfusion of the ascending aorta (T3), 6 h after operation (T4), 24 h after operation (T5), 48 h after operation (T6).
ResultsAll the surgeries were successfully performed without early death. Postoperative complications were low cardiac output syndrome in 3 cases and acute kidney failure in 1 cases. The concentration of mtDNA in circulation system rising gradually after CPB. The mtDNA concentration of T3, T4 and T5 were significantly higher than T1 (P < 0.05). The peak level was observed at T5 and the mtDNA concentration of T6 was still significantly higher than that of T1 (P < 0.05).
ConclusionThe concentration of mtDNA in circulation system was rising after CPB and peak level appeared at 24 h after CPB.
Objective To investigate the cell growth inhibition and apoptosis induced by rapamycin on human hepatocellular carcinoma Bel-7402 cells and to study the role of mitochondrium membrane potential in the process of apoptosis. Methods Bel-7402 cells in vitro were given 5, 10, 20, 30, 40 and 50 nmol/L different concentrations of rapamycin, and the cell growth inhibiting ratio of Bel-7402 was assessed by MTT assay. The changes of morphology of Bel-7402 were observed by Hoechst 33258 staining and flow cytometry (FCM), respectively; The cell mitochondrial membrane potential was detected by using JC-1 staining method. Results Rapamycin could inhibit the growth of Bel-7402 cells significantly by inducing apoptosis, and the growth suppression and the cell apoptosis both presented time-effect relationship and were also dose-dependent. The rates of inhibiting and cell apoptosis after 72 h exposure to 50 nmol/L rapamycin were significantly higher that those of other groups (P<0.01). Typical morphological changes of cell apoptosis were observed very clearly after the Bel-7402 cells had been exposed to rapamycin for 48 hours using Hoechst 33258 staining method, and it was also observed that the mitochondrial membrane potential decreased when apoptosis occured (P<0.01). Conclusion Rapamycin could inhibit the growth of Bel-7402 cells by inducing cell apoptosis, and the descent of mitochondrial membrane potential may play an important role in the process of cell apoptosis.
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 observe the retinal manifestations and classification of mitochondrial encephalomyopathy,and explore the relationship between retinopathy and systemic manifestations.Method The clinical data of 88 inpatients with mitochondrial encephalomyopathy were retrospectively analyzed,in whom 12 patients(24 eyes)with retinal manifestations who diagnosed by ophthalmology consultation and complete medical records were collected. There were nine males and three females aged from 14 to 33 years with the mean age of(20.1±7.0)years. The disease duration ranged from 2.5 to 20 years,with the mean of(9.5±6.8)years. All the patients had the eye symptoms of the different degree,such as limbs weakness,hearing decline and central nervous system symptoms. Ophthalmologic examination including best corrected visual acuity,slit lampa microscope,indirect ophthalmoscopy,noncontact Tonometer,ptosis,ocular movement,pupillary reflex and color fundus photography. Among the patients,three,one,two and five patients had undergone fundus fluorescein angiography(FFA),optical coherence tomography(OCT),lectroretinogram(ERG)and visual field examination respectively. Diabetic retinopathy were divided into “salt and pepper”, retinitis pigmentosa(RP),retinal pigment epithelium(RPE),choroidal capillary atrophy and simplex optic atrophy according to the inspection results.Results All the patients′ both eyes were involved,the disease degree of bilateral eyes was accordant. The ptosis and(or)eye movement limitation were found in nine patients(75.0%),and decreased visual acuity was in six patients(50.0%).“Salt and pepper” was found in six patients(12 eyes),presenting retinal granular pigmentation and depigmentation;the visual acuity was 0.4-1.2;no central nervous system symptoms were found in patients,such as hearing decline,twitch,ataxia and hypophrenia. RP was found in one patient(two eyes),presenting retinal cells sample pigmentation,retinal vessel shrink,optic atrophy;the vision were light perception in both eyes;hypophrenia,hearing decline,bilateral lower limbs pain and onset twitch were also found in them. RPE and choroidal capillary atrophy were found in three patients(six eyes),the choroidal great vessels and flake pigment accumulation surrounding the retina were observed;the visual acuity was hand movement0.7;limbs weakness was found in two patients;hearing decline was found in three patients;barylalia and hypophrenia were found in two patients;somnolence was found in one patient. Simplex optic atrophy was found in two patients(four eyes);the vision was 0.1-0.7;central nervous system symptoms were found in patients,such as limbs weakness,twitch,hypophrenia and headache.Conclusion Retinopathy types is concerned with visual prognosis and central nervous system symptoms.