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World Journal of Gastroenterology Sep 2015To investigate the effects of salvianolic acid B (Sal B) on the morphological characteristics and functions of liver mitochondria of rats with nonalcoholic...
AIM
To investigate the effects of salvianolic acid B (Sal B) on the morphological characteristics and functions of liver mitochondria of rats with nonalcoholic steatohepatitis (NASH).
METHODS
A total of 60 male Sprague-Dawley rats were randomly divided into three groups: (1) a normal group fed a normal diet; (2) an NASH model group; and (3) a Sal B-treated group fed a high-fat diet. Two rats from each group were executed at the end of the 12th week to detect pathological changes. The rats in the Sal B-treated group were gavaged with 20 mL/kg Sal B (1 mg/mL) daily. The model group received an equal volume of distilled water as a control. At the end of the 24th weekend, the remaining rats were executed. Serum biochemical parameters and liver histological characteristics were observed. Malondialdehyde (MDA) and superoxide dismutase (SOD) in the liver were determined. Protein expression of CytC and caspase-3 was determined by immunohistochemistry. The mRNA transcripts of mitofusin-2 (Mfn2) and NF-κB in the liver tissue were detected by real-time PCR. Mitochondrial membrane potential was detected using a fluorescence spectrophotometer. Mitochondrial respiratory function was detected using a Clark oxygen electrode.
RESULTS
The model group showed significantly higher ALT, AST, TG, TC and MDA but significantly lower SOD than the normal group. In the model group, the histological characteristics of inflammation and steatosis were also evident; mitochondrial swelling and crest were shortened or even disappeared. CytC (18.46 ± 1.21 vs 60.01 ± 3.43, P < 0.01) and caspase-3 protein expression (30.26 ± 2.56 vs 83.31 ± 5.12, P < 0.01) increased significantly. The mRNA expression of NF-κB increased (0.81 ± 0.02 vs 0.91 ± 0.03, P < 0.05), whereas the mRNA expression of Mfn2 decreased (1.65 ± 0.31 vs 0.83 ± 0.16, P < 0.05). Mitochondrial membrane potential also decreased and breathing of rats was weakened. Steatosis and inflammation degrees in the treatment group were significantly alleviated compared with those of the model group. In the treatment group, mitochondrial swelling was alleviated. CytC (60.01 ± 3.43 vs 30.52 ± 2.01, P < 0.01) and caspase-3 protein expression (83.31 ± 5.12 vs 40.15 ± 3.26, P < 0.01) significantly decreased. The mRNA expression of NF-κB also decreased (0.91 ± 0.03 vs 0.74 ± 0.02, P < 0.01), whereas the mRNA expression of Mfn2 increased (0.83 ± 0.16 vs 1.35 ± 0.23, P < 0.01). Mitochondrial membrane potential increased and respiratory function was enhanced.
CONCLUSION
Sal B can treat NASH by protecting the morphological characteristics and functions of liver mitochondria, regulating lipid metabolism, controlling oxidative stress and lipid peroxidation and inhibiting apoptosis.
Topics: Animals; Apoptosis; Benzofurans; Biomarkers; Caspase 3; Cytochromes c; Diet, High-Fat; Disease Models, Animal; GTP Phosphohydrolases; Lipid Peroxidation; Liver; Male; Malondialdehyde; Membrane Potential, Mitochondrial; Membrane Proteins; Mitochondria, Liver; Mitochondrial Proteins; Mitochondrial Swelling; NF-kappa B; Non-alcoholic Fatty Liver Disease; Oxidative Stress; Rats, Sprague-Dawley; Superoxide Dismutase
PubMed: 26401075
DOI: 10.3748/wjg.v21.i35.10104 -
Biophysical Journal Sep 2008Mitochondrial swelling is a hallmark of mitochondrial dysfunction, and is an indicator of the opening of the mitochondrial permeability transition pore. We introduce...
Mitochondrial swelling is a hallmark of mitochondrial dysfunction, and is an indicator of the opening of the mitochondrial permeability transition pore. We introduce here a novel quantitative in situ single-cell assay of mitochondrial swelling based on standard wide-field or confocal fluorescence microscopy. This morphometric technique quantifies the relative diameter of mitochondria labeled by targeted fluorescent proteins. Fluorescence micrographs are spatial bandpass filtered transmitting either high or low spatial frequencies. Mitochondrial swelling is measured by the fluorescence intensity ratio of the high- to low-frequency filtered copy of the same image. We have termed this fraction the "thinness ratio". The filters are designed by numeric optimization for sensitivity. We characterized the thinness ratio technique by modeling microscopic image formation and by experimentation in cultured cortical neurons and astrocytes. The frequency domain image processing endows robustness and subresolution sensitivity to the thinness ratio technique, overcoming the limitations of shape measurement approaches. The thinness ratio proved to be highly sensitive to mitochondrial swelling, but insensitive to fission or fusion of mitochondria. We found that in situ astrocytic mitochondria swell upon short-term uncoupling or inhibition of oxidative phosphorylation, whereas such responses are absent in cultured cortical neurons.
Topics: Animals; Astrocytes; Cells, Cultured; Microscopy, Confocal; Microscopy, Fluorescence; Mitochondria; Mitochondrial Size; Mitochondrial Swelling; Models, Biological; Neurons; Oxidative Phosphorylation; Rats; Rats, Wistar
PubMed: 18424491
DOI: 10.1529/biophysj.107.118620 -
Biochimica Et Biophysica Acta Feb 2006Common deletion (CD) 4977 bp of mitochondrial DNA (mtDNA) disrupt specifically mitochondrial complex I, IV and V on the electron transport chain (ETC) and is closely...
Common deletion (CD) 4977 bp of mitochondrial DNA (mtDNA) disrupt specifically mitochondrial complex I, IV and V on the electron transport chain (ETC) and is closely associated with wide spectrums of clinical manifestations. To quantitatively investigate how CD-induced ETC defect alters mitochondrial reactive oxygen species (mROS) generation as well as down stream apoptotic signaling, we employed an established array of human CD cytoplasmic hybrids (cybrids) harboring 0%-80% of CD. Pathological effects of CD on the mitochondria were visualized at single cell level by the application of fluorescent probes coupled with conventional and multiphoton imaging microscopy. Intriguingly, we observed CD-augmented mROS generation omitted "threshold effect". CD-augmented mROS generation was associated with depolarized mitochondrial membrane potential (DeltaPsi(m)). Upon oxidative stress, the amount of CD-augmented mROS generation was greatly enhanced to cause pathological apoptotic deterioration including opening of the mitochondrial permeability transition, cytochrome c release, phosphatidylserine externalization and DNA fragmentation. In addition, heterogeneous mitochondrial dysfunctions were found in cybrids containing 80% of CD (D cybrids), i.e., low sensitive-D (LS-D, roughly 80%) and a super sensitive-D (SS-D, 20%). As compared to LS-D, SS-D had higher resting mROS level but slightly hyperpolarized DeltaPsi(m). Upon H2O2 treatment, much faster generation of mROS was observed which induced a faster depolarization of DeltaPsi(m) and later apoptotic deterioration in SS-D. We proposed a dose-dependent, feed-forward and self-accelerating vicious cycle of mROS production might be initiated in CD-induced ETC defect without threshold effect. As CD-augmented mROS generation is obligated to cause an enhanced pathological apoptosis, precise detection of CD-augmented mROS generation and their degree of heterogeneity in single cells may serve as sensitive pathological indexes for early diagnosis, prognosis and treatment of CD-associated diseases.
Topics: Apoptosis; Caspase 3; Caspases; Cells, Cultured; Cytochromes c; DNA, Mitochondrial; Enzyme Activation; Gene Deletion; Humans; Hydrogen Peroxide; Mitochondria; Mitochondrial Swelling; Oxidative Stress; Reactive Oxygen Species; Sequence Deletion
PubMed: 16368227
DOI: 10.1016/j.bbadis.2005.10.008 -
Biomedicine & Pharmacotherapy =... Mar 2021Trypanosoma cruzi is a protozoan parasite that causes Chagas disease, a neglected tropical disease that is endemic in Latin America and spreading worldwide due to...
Trypanosoma cruzi is a protozoan parasite that causes Chagas disease, a neglected tropical disease that is endemic in Latin America and spreading worldwide due to globalization. The current treatments are based on benznidazole and nifurtimox; however, these drugs have important limitations and limited efficacy during the chronic phase, reinforcing the necessity of an alternative chemotherapy. For the last 30 years, our group has been evaluating the biological activity of naphthoquinones and derivatives on T. cruzi, and of the compounds tested, N1, N2 and N3 were found to be the most active in vitro. Here, we show the synthesis of a novel β-lapachone-derived naphthoimidazolium named N4 and assess its activity on T. cruzi stages and the mechanism of action. The new compound was very active on all parasite stages (IC/24 h in the range of 0.8-7.9 μM) and had a selectivity index of 5.4. Mechanistic analyses reveal that mitochondrial ROS production begins after short treatment starts and primarily affects the activity of complexes II-III. After 24 h treatment, a partial restoration of mitochondrial physiology (normal complexes II-III and IV activities and controlled HO release) was observed; however, an extensive injury in its morphology was still detected. During treatment with N4, we also observed that trypanothione reductase activity increased in a time-dependent manner and concomitant with increased oxidative stress. Molecular docking calculations indicated the ubiquinone binding site of succinate dehydrogenase as an important interaction point with N4, as with the FMN binding site of dihydroorotate dehydrogenase. The results presented here may be a good starting point for the development of alternative treatments for Chagas disease and for understanding the mechanism of naphthoimidazoles in T. cruzi.
Topics: Chagas Disease; Dihydroorotate Dehydrogenase; Electron Transport Chain Complex Proteins; Energy Metabolism; Mitochondria; Mitochondrial Swelling; Naphthoquinones; Oxidoreductases Acting on CH-CH Group Donors; Protozoan Proteins; Reactive Oxygen Species; Succinate Dehydrogenase; Trypanocidal Agents; Trypanosoma cruzi
PubMed: 33395606
DOI: 10.1016/j.biopha.2020.111186 -
The Journal of Biological Chemistry Dec 1996The permeability transition process in rat liver mitochondria was studied by following the swelling consequent to external solute equilibration through the membrane...
The permeability transition process in rat liver mitochondria was studied by following the swelling consequent to external solute equilibration through the membrane pore. The kinetics of the transition was analyzed according to a model based on the assumptions that the transition rate follows a first-order process and that the solute diffusion rate strongly depends on the pore conformation. Three kinetic parameters, easily calculated from absorbance measurements during mitochondrial swelling, were used to determine whether changes of the swelling rate are due to changes of (i) the transition rate, (ii) the amount of permeabilized mitochondria, (iii) the mean pore dimension, or (iv) the number of pores per mitochondrion. The model was tested for transitions induced either by phenylarsine oxide (PhAsO) or by Ca2+ and Pi. Under both conditions, only a definite fraction of mitochondria was permeabilized, and the transition always followed a first-order reaction, indicating that mitochondria behaved as a homogeneous population. However, the equilibration of external solutes was rapid only in a fraction of permeabilized mitochondria and slow in the remaining fraction, due to restricted solute diffusion through narrower pores. With 0.2 mM PhAsO as the inducer, the fraction of permeabilized mitochondria was about 0.8. Sucrose diffusion was rapid only in 15% of this fraction (half-time less than 1 s) and restricted in the remaining 85% (half-time of about 60 s). Increasing PhAsO concentrations increased the number of pores per mitochondrion and the rate constant of the permeability transition, but not the mean pore diameter and the fraction of permeabilized mitochondria. With 0.28 microM free [Ca2+] and 1 mM Pi as the inducers, the fraction of permeabilized mitochondria was about 0.43. Sucrose diffusion was rapid in 60% of this fraction and restricted in the remaining 40% (half-time of about 9 s). The permeabilization process started above a threshold- free [Ca2+] of 0.15 microM, and the rate constant and the fraction of permeabilized mitochondria reached a maximum at about 0.2 microM, while both parameters were inhibited at higher free [Ca2+].
Topics: Animals; Arsenicals; Calcium; Carbohydrate Metabolism; Enzyme Inhibitors; Intracellular Membranes; Kinetics; Mitochondria, Liver; Mitochondrial Swelling; Permeability; Phosphates; Platelet Aggregation Inhibitors; Rats
PubMed: 8943240
DOI: No ID Found -
The Biochemical Journal Jun 1986The ability of alpha-adrenergic agonists and vasopressin to increase the mitochondrial volume in hepatocytes is dependent on the presence of extracellular Ca2+. Addition...
The ability of alpha-adrenergic agonists and vasopressin to increase the mitochondrial volume in hepatocytes is dependent on the presence of extracellular Ca2+. Addition of Ca2+ to hormone-treated cells incubated in the absence of Ca2+ initiates mitochondrial swelling. In the presence of extracellular Ca2+, A23187 (7.5 microM) induces mitochondrial swelling and stimulates gluconeogenesis from L-lactate. Isolated liver mitochondria incubated in KCl medium in the presence of 2.5 mM-phosphate undergo energy-dependent swelling, which is associated with electrogenic K+ uptake and reaches an equilibrium when the volume has increased to about 1.3-1.5 microliter/mg of protein. This K+-dependent swelling is stimulated by the presence of 0.3-1.0 microM-Ca2+, leading to an increase in matrix volume at equilibrium that is dependent on [Ca2+]. Ca2+-activated K+-dependent swelling requires phosphate and shows a strong preference for K+ over Na+, Li+ or choline. It is not associated with either uncoupling of mitochondria or any non-specific permeability changes and cannot be produced by Ba2+, Mn2+ or Sr2+. Ca2+-activated K+-dependent swelling is not prevented by any known inhibitors of plasma-membrane ion-transport systems, nor by inhibitors of mitochondrial phospholipase A2. Swelling is inhibited by 65% and 35% by 1 mM-ATP and 100 microM-quinine respectively. The effect of Ca2+ is blocked by Ruthenium Red (5 micrograms/ml) at low [Ca2+]. Spermine (0.25 mM) enhanced the swelling seen on addition of Ca2+, correlating with its ability to increase Ca2+ uptake into the mitochondria as measured by using Arsenazo-III. Mitochondria derived from rats treated with glucagon showed less swelling than did control mitochondria. In the presence of Ruthenium Red and higher [Ca2+], the mitochondria from hormone-treated animals showed greater swelling than did control mitochondria. These data imply that an increase in intramitochondrial [Ca2+] can increase the electrogenic flux of K+ into mitochondria by an unknown mechanism and thereby cause swelling. It is proposed that this is the mechanism by which alpha-agonists and vasopressin cause an increase in mitochondrial volume in situ.
Topics: Animals; Calcium; Female; Liver; Mitochondria, Liver; Mitochondrial Swelling; Potassium; Rats; Rats, Inbred Strains; Ruthenium Red; Spermine; Valinomycin
PubMed: 2431681
DOI: 10.1042/bj2360779 -
Journal of Orthopaedic Surgery and... Jul 2020Ischemia-reperfusion injury of the spinal cord (SCII) often leads to unalterable neurological deficits, which may be associated with apoptosis induced by oxidative...
BACKGROUND
Ischemia-reperfusion injury of the spinal cord (SCII) often leads to unalterable neurological deficits, which may be associated with apoptosis induced by oxidative stress and inflammation. Astaxanthin (AST) is a strong antioxidant and anti-inflammatory agent with multitarget neuroprotective effects. This study aimed to investigate the potential therapeutic effects of AST for SCII and the molecular mechanism.
METHODS
Rat models of SCII with abdominal aortic occlusion for 40 min were carried out to investigate the effects of AST on the recovery of SCII. Tarlov's scores were used to assess the neuronal function; HE and TUNEL staining were used to observe the pathological morphology of lesions. Neuron oxidative stress and inflammation were measured using commercial detection kits. Flow cytometry was conducted to assess the mitochondrial swelling degree. Besides, Western blot assay was used to detect the expression of PI3K/Akt/GSK-3β pathway-related proteins, as well as NOX2 and NLRP3 proteins.
RESULTS
The results demonstrated that AST pretreatment promoted the hind limb motor function recovery and alleviated the pathological damage induced by SCII. Moreover, AST significantly enhanced the antioxidative stress response and attenuated mitochondrial swelling. However, AST pretreatment hardly inhibited the levels of proinflammatory cytokines after SCII. Most importantly, AST activated p-Akt and p-GSK-3β expression levels. Meanwhile, cotreatment with LY294002 (a PI3K inhibitor) was found to abolish the above protective effects observed with the AST pretreatment.
CONCLUSION
Overall, these results suggest that AST pretreatment not only mitigates pathological tissue damage but also effectively improves neural functional recovery following SCII, primarily by alleviating oxidative stress but not inhibiting inflammation. A possible underlying molecular mechanism of AST may be mainly attributed to the activation of PI3K/Akt/GSK-3β pathway.
Topics: Animals; Anti-Inflammatory Agents; Antioxidants; Apoptosis; Glycogen Synthase Kinase 3 beta; Male; Mitochondrial Swelling; Neuroprotective Agents; Oxidative Stress; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Rats, Sprague-Dawley; Reperfusion Injury; Signal Transduction; Spinal Cord Ischemia; Xanthophylls
PubMed: 32703256
DOI: 10.1186/s13018-020-01790-8 -
Respiratory Research Feb 2015Denatonium, a widely used bitter agonist, activates bitter taste receptors on many cell types and plays important roles in chemical release, ciliary beating and smooth...
BACKGROUND
Denatonium, a widely used bitter agonist, activates bitter taste receptors on many cell types and plays important roles in chemical release, ciliary beating and smooth muscle relaxation through intracellular Ca(2+)-dependent pathways. However, the effects of denatonium on the proliferation of airway epithelial cells and on the integrity of cellular components such as mitochondria have not been studied. In this study, we hypothesize that denatonium might induce airway epithelial cell injury by damaging mitochondria.
METHODS
Bright-field microscopy, cell counting kit-8 (CCK-8) assay and flow cytometry analysis were used to examine cellular morphology, proliferation and cell cycle, respectively. Transmission electron microscopy (TEM) was used to examine mitochondrial integrity. JC-1 dye and western blotting techniques were used to measure mitochondrial membrane potential and protein expression, respectively.
RESULTS
For airway epithelial cells, we observed that denatonium significantly effects cellular morphology, decreases cell proliferation and reduces the number of cells in S phase in a dose-dependent manner. TEM analysis demonstrated that denatonium causes large amplitude swelling of mitochondria, which was confirmed by the loss of mitochondrial membrane potential, the down-regulation of Bcl-2 protein and the subsequent enhancement of the mitochondrial release of cytochrome c and Smac/DIABLO after denatonium treatment.
CONCLUSIONS
In this study, we demonstrated for the first time that denatonium damages mitochondria and thus induces apoptosis in airway epithelial cells.
Topics: Animals; Apoptosis; Apoptosis Regulatory Proteins; Cell Line, Tumor; Cell Proliferation; Cell Shape; Dose-Response Relationship, Drug; Epithelial Cells; Humans; Lung; Membrane Potential, Mitochondrial; Mice, Inbred C57BL; Mitochondria; Mitochondrial Swelling; Quaternary Ammonium Compounds; Receptors, G-Protein-Coupled; S Phase Cell Cycle Checkpoints; Signal Transduction
PubMed: 25652218
DOI: 10.1186/s12931-015-0183-9 -
Biochimica Et Biophysica Acta Feb 2015The permeability transition pore (PTP) is a large channel of the mitochondrial inner membrane, the opening of which is the central event in many types of stress-induced...
The permeability transition pore (PTP) is a large channel of the mitochondrial inner membrane, the opening of which is the central event in many types of stress-induced cell death. PTP opening is induced by elevated concentrations of mitochondrial calcium. It has been demonstrated that spermine and other polyamines can delay calcium-induced swelling of isolated mitochondria, suggesting their role as inhibitors of the mitochondrial PTP. Here we further investigated the mechanism by which spermine inhibits the calcium-induced, cyclosporine A (CSA) -sensitive PTP by using three indicators: 1) calcium release from the mitochondria detected with calcium green, 2) mitochondrial membrane depolarization using TMRM, and 3) mitochondrial swelling by measuring light absorbance. We found that despite calcium release and membrane depolarization, indicative of PTP activation, mitochondria underwent only partial swelling in the presence of spermine. This was in striking contrast to the high-amplitude swelling detected in control mitochondria and in mitochondria treated with the PTP inhibitor CSA. We conclude that spermine selectively prevents opening of the high-conductance state, while allowing activation of the lower conductance state of the PTP. We propose that the existence of lower conductance, stress-induced PTP might play an important physiological role, as it is expected to allow the release of toxic levels of calcium, while keeping important molecules (e.g., NAD) within the mitochondrial matrix.
Topics: Animals; Calcium; Mitochondria; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Mitochondrial Swelling; Osmolar Concentration; Rats; Rats, Sprague-Dawley; Spermine
PubMed: 25448536
DOI: 10.1016/j.bbabio.2014.10.007 -
American Journal of Physiology. Renal... Dec 2003Isolated kidney mitochondria swell when incubated in hyposmotic solutions containing K+ salts in a manner inhibited by ATP, ADP, 5-hydroxydecanoate, and glibenclamide...
Isolated kidney mitochondria swell when incubated in hyposmotic solutions containing K+ salts in a manner inhibited by ATP, ADP, 5-hydroxydecanoate, and glibenclamide and stimulated by GTP and diazoxide. These results suggest the existence of ATP-sensitive K+ channels in these mitochondria, similar to those previously described in heart, liver, and brain. Renal mitochondrial ATP-sensitive K+ uptake rates are approximately 140 nmol.min-1.mg protein-1. This K+ transport results in a slight increase in respiration and decrease in the inner membrane potential. In addition, the activation of ATP-inhibited K+ uptake using diazoxide leads to a decrease of ATP hydrolysis through the reverse activity of the F0F1 ATP synthase when respiration is inhibited. In conclusion, we characterize an ATP-sensitive K+ transport pathway in kidney mitochondria that affects volume, respiration, and membrane potential and may have a role in the prevention of mitochondrial ATP hydrolysis.
Topics: Adenosine Triphosphate; Animals; Biological Transport; Cell Respiration; Ischemic Preconditioning; Kidney; Male; Membrane Potentials; Mitochondria; Mitochondrial Swelling; Potassium; Potassium Channels; Proton-Translocating ATPases; Rats; Rats, Wistar
PubMed: 12952853
DOI: 10.1152/ajprenal.00103.2003