Mitochondrial Staining Research Articles

Curcumin and N-Acetylcysteine Nanocarriers Alone or Combined with Deferoxamine Target the Mitochondria and Protect against Neurotoxicity and Oxidative Stress in a Co-Culture Model of Parkinson's Disease.

As the blood-brain barrier (BBB) prevents most compounds from entering the brain, nanocarrier delivery systems are frequently being explored to potentially enhance the passage of drugs due to their nanometer sizes and functional characteristics. This study aims to investigate whether Pluronic® F68 (P68) and dequalinium (DQA) nanocarriers can improve the ability of curcumin, n-acetylcysteine (NAC) and/or deferoxamine (DFO), to access the brain, specifically target mitochondria and protect against rotenone by evaluating their effects in a combined Transwell® hCMEC/D3 BBB and SH-SY5Y based cellular Parkinson's disease (PD) model. P68 + DQA nanoformulations enhanced the mean passage across the BBB model of curcumin, NAC and DFO by 49%, 28% and 49%, respectively (p < 0.01, n = 6). Live cell mitochondrial staining analysis showed consistent co-location of the nanocarriers within the mitochondria. P68 + DQA nanocarriers also increased the ability of curcumin and NAC, alone or combined with DFO, to protect against rotenone induced cytotoxicity and oxidative stress by up to 19% and 14% (p < 0.01, n = 6), as measured by the MTT and mitochondrial hydroxyl radical assays respectively. These results indicate that the P68 + DQA nanocarriers were successful at enhancing the protective effects of curcumin, NAC and/or DFO by increasing the brain penetrance and targeted delivery of the associated bioactives to the mitochondria in this model. This study thus emphasises the potential effectiveness of this nanocarrier strategy in fully utilising the therapeutic benefit of these antioxidants and lays the foundation for further studies in more advanced models of PD.

Analysis of mitochondrial function in lymphocytes obtained from COVID-19 patients.

There is a significant decline in the lymphocyte subset counts in the peripheral blood of COVID-19 patients. However, the mitochondrial function of lymphocytes obtained from COVID-19 patients has rarely been studied. A case-control study was conducted in 115 COVID-19 patients and 50 healthy controls from December 2022 to February 2023. The extent of lymphocytic mitochondrial damage in these patients using mitochondrial fluorescence staining and flow cytometry. Clinical symptoms were evaluated using the SOFA and APACHE II scores. The mitochondrial function of lymphocytes was severely impaired in the peripheral blood of COVID-19 patients, compared to healthy controls, and was characterized by an increased single-cell mitochondrial mass (SCMM) and increased percentage of low mitochondrial membrane potential. The increase in the SCMM of T cells was more notable in patients with severe COVID-19 and was positively correlated with the SOFA and APACHE II scores. When the SCMM-CD8 cutoff value was 38.775, the AUC for distinguishing between severe and mild COVID-19 was 0.740, and the sensitivity, specificity, and Youden index were 65.8%, 82.1%, and 0.478, respectively. SCMM-CD8 could act as a diagnostic biomarker of COVID-19 progression. However, this needs to be verified in other multi-center studies with a larger sample size.

Chlorogenic acid regulates the proliferation and migration of high-grade serous ovarian cancer cells through modulating the miR199a5p/DDR1 axis

This study aimed to demonstrate that chlorogenic acid (CGA) has anticancer effects against ovarian cancer. The MTT assay was used to assess the optimum concentrations of CGA on the ovarian cancer cell lines OVCA433 and SKOV3, followed by the rate of apoptosis using Annexin V-FITC/PI. The mitochondrial membrane potential of ovarian tumour cells treated with CGA was evaluated using mitochondrial staining kits followed by Western blot analysis, immunofluorescence, and RT-PCR assays. The Trans-well migration assay conducted the percentage of cell migration, followed by wound healing and colony formation assays. CGA induces activation of mitochondria-mediated intrinsic apoptotic pathways in ovarian cancer cells. The discovery that miR-199a-5p is inversely correlated to DDR1, a receptor tyrosine kinase involved in collagen synthesis, was the major consequence of examining the various mechanisms involved in the development of ovarian cancer. After treatment with CGA, cells derived from ovarian cancer cells were deregulated partially via the miR199a5p/DDR1 axis, significantly affecting tumour suppression. DDR1 has been identified as a direct target of miR199a5p in these ovarian cancer cells. We found that CGA-induced loss of DDR1 caused the inactivation of NF-κB signalling downstream in the MMP, migration, and EMT pathways. The study results showed that CGA is a promising drug candidate for treating ovarian cancer, particularly because it exhibits anti-invasive and migrastatic properties.

Preventing Lipid Peroxidation Stimulates Loss of Glutathione Reduction Potential Coincident with Increased Mitochondrial Staining in Articular Chondrocytes

Preventing Lipid Peroxidation Stimulates Loss of Glutathione Reduction Potential Coincident with Increased Mitochondrial Staining in Articular Chondrocytes

A Graphene Oxide-Angiogenin Theranostic Nanoplatform for the Therapeutic Targeting of Angiogenic Processes: The Effect of Copper-Supplemented Medium

Graphene oxide (GO) nanosheets with different content in the defective carbon species bound to oxygen sp3 were functionalized with the angiogenin (ANG) protein, to create a novel nanomedicine for modulating angiogenic processes in cancer therapies. The GO@ANG nanocomposite was scrutinized utilizing UV-visible and fluorescence spectroscopies. GO exhibits pro- or antiangiogenic effects, mostly attributed to the disturbance of ROS concentration, depending both on the total concentration (i.e., >100 ng/mL) as well as on the number of carbon species oxidized, that is, the C/O ratio. ANG is considered one of the most effective angiogenic factors that plays a vital role in the angiogenic process, often in a synergic role with copper ions. Based on these starting hypotheses, the GO@ANG nanotoxicity was assessed with the MTT colorimetric assay, both in the absence and in the presence of copper ions, by in vitro cellular experiments on human prostatic cancer cells (PC-3 line). Laser confocal microscopy (LSM) cell imaging evidenced an enhanced internationalization of GO@ANG than bare GO nanosheets, as well as significant changes in cell cytoskeleton organization and mitochondrial staining compared to the cell treatments with free ANG.

Placental mesenchymal stem cells restore glucose and energy homeostasis in obesogenic adipocytes.

Obesity (Ob) depicts a state of energy imbalance(s) being characterized by the accumulation of excessive fat and which predisposes to several metabolic diseases. Mesenchymal stem cells (MSCs) represent a promising option for addressing obesity and its associated metabolic co-morbidities. The present study aims at assessing the beneficial effects of human placental MSCs (P-MSCs) in mitigating Ob-associated insulin resistance (IR) and mitochondrial dysfunction both in vivo and in vitro. Under obesogenic milieu, adipocytes showed a significant reduction in glucose uptake, and impaired insulin signaling with decreased expression of UCP1 and PGC1α, suggestive of dysregulated non-shivering thermogenesis vis-a-vis mitochondrial biogenesis respectively. Furthermore, obesogenic adipocytes demonstrated impaired mitochondrial respiration and energy homeostasis evidenced by reduced oxygen consumption rate (OCR) and blunted ATP/NAD+/NADP+ production respectively. Interestingly, co-culturing adipocytes with P-MSCs activated PI3K-Akt signaling, improved glucose uptake, diminished ROS production, enhanced mitochondrial OCR, improved ATP/NAD+/NADP+ production, and promoted beiging of adipocytes evidenced by upregulated expression of PRDM16, UCP1, and PGC1α expression. In vivo, P-MSCs administration increased the peripheral blood glucose uptake and clearance, and improved insulin sensitivity and lipid profile with a coordinated increase in the ratio of ATP/ADP and NAD+ and NADP+ in the white adipose tissue (WAT), exemplified in WNIN/GR-Ob obese mutant rats. In line with in vitro findings, there was a significant reduction in adipocyte hypertrophy, increased mitochondrial staining, and thermogenesis. Our findings advocate for a therapeutic application of P-MSCs for improving glucose and energy homeostasis, i.e., probably restoring non-shivering thermogenesis towards obesity management.

FGFR2 Mutation p.Cys342Arg Enhances Mitochondrial Metabolism-Mediated Osteogenesis via FGF/FGFR-AMPK-Erk1/2 Axis in Crouzon Syndrome.

Background: Crouzon syndrome ([OMIM] #123500) caused by FGFR2 mutation is an autosomal dominant syndrome with craniosynostosis, the underlying mechanism of which remains obscure. Methods: First, whole exome sequencing was used to screen the possible pathogenic variant in two sporadic patients with Crouzon syndrome. The investigation of primary and secondary structures as well as the conservation analysis of FGFR2 mutation (p.Cys342Arg) was performed. Then, wild-type and mutant overexpression plasmids were constructed and transfected into pre-osteoblastic murine cell line MC3T3-E1 cells. Osteogenesis and mitochondrial metabolism were analyzed by CCK8, ALP staining and ALP activity, alizarin red staining, qRT-PCR, Western blot, seahorse assays and mitochondrial staining. The siRNA targeting FGFR2 and domain negative FGFR2 were designed for verification. Results: First, FGFR2 mutation (p.Cys342Arg) was detected in two sporadic Chinese Crouzon syndrome patients. FGFR2 p.Cys342Arg promoted the osteogenic differentiation of MC3T3-E1 cells through the upregulation of AMP-activated protein kinase (AMPK)-Erk1/2 signal pathway. Furthermore, FGFR2 p.Cys342Arg enhanced oxidative phosphorylation and converted mitochondrial fusion to the fission of MC3T3-E1, promoting osteogenic differentiation and craniosynostosis in Crouzon syndrome. Additionally, AMPK or Erk1/2 inhibitors delayed the cranial suture closure. Conclusion: FGFR2 mutation p.Cys342Arg promotes osteogenesis by enhancing mitochondrial metabolism-mediated via FGF/FGFR-AMPK-Erk1/2 axis, which indicates the potential of therapy targeting AMPK or Erk1/2 for syndromic craniosynostosis treatment.

Mitochondrial Damage of Lymphocytes in Patients with Acute Relapse of Schizophrenia: A Correlational Study with Efficacy and Clinical Symptoms.

Accumulating evidence has demonstrated that schizophrenia is associated with mitochondrial and immune abnormalities. In this pilot case-control study, we investigated the level of mitochondrial impairment in lymphocytes in patients with acute relapse of schizophrenia and explored the correlation between the level of mitochondrial damage and symptoms or treatment response. Lymphocytic mitochondrial damage was detected using mitochondrial fluorescence staining and flow cytometry in 37 patients (at admission and discharge) and 24 controls. Clinical symptoms were assessed using the Positive and Negative Syndrome Scale (PANSS) and Clinical Global Impression Scale (CGI-S). The levels of mitochondrial damage in CD3+ T, CD4+ T, and CD8+ T lymphocytes of the patients with schizophrenia at admission were significantly higher than those of the controls (p<0.05) and did not return to normal at discharge (p>0.05). The mitochondrial damage of T cells significantly improved at discharge for responsive patients only, as compared with that at admission (P<0.05). However, no significant difference was found in mitochondrial damage in CD19+ B cells between patients and healthy controls, or between admission and discharge (p>0.05). Furthermore, the reduction in mitochondrial damage of CD3, CD4, and CD8 lymphocytes was positively correlated with the reduction of the score of the PANSS positive scale at discharge (p<0.05), while no significant correlation was found between the level of mitochondrial damage in lymphocytes and the scores of PANSS and CGI-S. Acute relapse of schizophrenia might be associated with higher levels of mitochondrial damage in peripheral blood T lymphocytes. The degree of recovery of mitochondrial impairment in the T cells may be used as a predictor of treatment response in schizophrenia. As this is a pilot study, the conclusion still needs further verification in large-scale studies.

Melatonin ultrastructural localization in mitochondria of human salivary glands.

The hormone melatonin was initially believed to be synthesized exclusively by the pineal gland and the enterochromaffin cells, but nowadays its production and distribution were observed in several other tissues and organs. Among others, the ultrastructural localization of melatonin and its receptors has been reported in human salivary glands. In these glands, the fine localization of melatonin in intracellular organelles, above all in mitochondria, remains to be explored comprehensively. Bioptic samples of parotid and submandibular glands were treated to search for melatonin using the immunogold staining method by transmission electron microscopy. Morphometric analysis was applied to micrographs. The results indicated that, both in parotid and submandibular glands mitochondria, a certain melatonin positivity was present. Within glandular cells, melatonin was less retrieved in mitochondria than in secretory granules; however, its presence in this organelle was clearly evident. Inside striated duct cells, melatonin staining in mitochondria was more prominent than in glandular cells. Our data provide an ultrastructural report on the presence of melatonin in mitochondria of human major salivary glands and represent a fundamental prerequisite for a better understanding of the melatonin role in this organelle.

Anti-obesity effects of the dual-active adenosine A2A/A3 receptor-ligand LJ-4378

Background and objectivesA2A adenosine receptor (A2AAR)-mediated signaling in adipose tissues has been investigated as a potential target for obesity-related metabolic diseases. LJ-4378 has been developed as a dual-acting ligand with A2AAR agonist and A3 adenosine receptor (A3AR) antagonist activity. The current study aimed to investigate the anti-obesity effects of LJ-4378 and its underlying molecular mechanisms.MethodsImmortalized brown adipocytes were used for in vitro analysis. A high-fat diet (HFD)-induced obesity and cell death-inducing DFFA-like effector A reporter mouse models were used for in vivo experiments. The effects of LJ-4378 on lipolysis and mitochondrial metabolism were evaluated using immunoblotting, mitochondrial staining, and oxygen consumption rate analyses. The in vivo anti-obesity effects of LJ-4378 were evaluated using indirect calorimetry, body composition analyses, glucose tolerance tests, and histochemical analyses.ResultsIn vitro LJ-4378 treatment increased the levels of brown adipocyte markers and mitochondrial proteins, including uncoupling protein 1. The effects of LJ-4378 on lipolysis of adipocytes were more potent than those of the A2AAR agonist or A3AR antagonist. In vivo, LJ-4378 treatment increased energy expenditure by 17.0% (P value < 0.0001) compared to vehicle controls. LJ-4378 (1 mg/kg, i.p.) treatment for 10 days reduced body weight and fat content by 8.24% (P value < 0.0001) and 24.2% (P value = 0.0044), respectively, and improved glucose tolerance in the HFD-fed mice. LJ-4378 increased the expression levels of brown adipocyte markers and mitochondrial proteins in interscapular brown and inguinal white adipose tissue.ConclusionThese findings support the in vivo anti-obesity effects of LJ-4378, and suggest a novel therapeutic approach to combat obesity and related metabolic diseases.

Metabolomic fingerprinting of renal disease progression in Bardet-Biedl syndrome reveals mitochondrial dysfunction in kidney tubular cells

SummaryChronic kidney disease (CKD) is a major clinical sign of patients with Bardet-Biedl syndrome (BBS), especially in those carrying BBS10 mutations. Twenty-nine patients with BBS and 30 controls underwent a serum-targeted metabolomic analysis. In vitro studies were conducted in two kidney-derived epithelial cell lines, where Bbs10 was stably deleted (IMCD3-Bbs10−/−cells) and over-expressed. The CKD status affected plasmatic metabolite fingerprinting in both patients with BBS and controls. Specific phosphatidylcholine and acylcarnitines discriminated eGFR decline only in patients with BBS. IMCD3-Bbs10−/ cells displayed intracellular lipidaccumulation, reduced mitochondrial potential membrane and citrate synthase staining. Mass-Spectrometry-based analysis revealed that human BBS10 interacted with six mitochondrial proteins, in vitro. In conclusion, renal dysfunction correlated with abnormal phosphatidylcholine and acylcarnitines plasma levels in patients with BBS; in vitro, Bbs10 depletion caused mitochondrial defects while human BBS10 interacted with several mitochondria-related proteins, suggesting an unexplored role of this protein.

Natural Xylooligosaccharides Exert Antitumor Activity via Modulation of Cellular Antioxidant State and TLR4.

It has been recently proven that xylooligosaccharides (XOS) with prebiotic properties have diverse beneficial biological effects including immunomodulatory and antitumor activities. The present article focused on the chemical and biological evaluation of corn-derived commercially available XOS and aimed to elucidate their cytotoxicity and inhibitory potential against tumor cells. Spectrophotometric chemical analyses, Fourier transform infrared spectroscopy, and high-performance liquid chromatography analyses were performed. Antioxidant activity was determined by measuring the oxygen radical absorbance capacity and hydroxyl radical averting capacity. In vitro cytotoxicity assays with human cell lines derived from normal and tumor tissues, assessments of ATP production, mitochondrial membrane potential specific staining, cytokine assays, and molecular docking were used to evaluate the biological activity of XOS. The sample showed significant antioxidant activity, and it was determined that most xylose oligomers in it are composed of six units. XOS exhibited antitumor activity with pronounced inhibitory effect on lysosomes, but mitochondrial functionality was also affected. The production of proinflammatory cytokines by lipopolysaccharide-stimulated U-937 cells was reduced by XOS treatment, which suggested the involvement of Toll-like receptor 4 (TLR4)-mediated signaling in the mechanism of XOS action. Molecular docking analyses confirmed the potential inhibitory interaction between the sample and TLR4. In addition, XOS treatment had significant tumor-cell-specific influence on the glutathione antioxidant system, affecting its balance and thus contributing to the inhibition of cellular viability. The present study elucidated the tumor-inhibitory potential of commercially available XOS that could be utilized in pharmaceutical and food industry providing disease-preventive and therapeutic benefits.

Imidazobenzothiadiazole: A new multifunction dyes for mechanochromic luminescence, acid responsing and mitochondrial staining

To develop new luminophores, three imidazobenzothiadiazole (ImBTD) fluorescent dyes (4a, 4b, and 4c) have been successfully synthesized and characterized. Interestingly, the introduction of imidazole groups at the 5- and 6-positions of benzothiadiazole can effectively enhance the fluorescence in both solid and aqueous solution and lead to a fluorescence response to acid. Modification of different aromatic groups at position 4 and 8 of ImBTD can produce tunable mechanochromic luminescence (MCL), which has been studied by theoretical calculations, single crystal X-ray diffraction (XRD), powder XRD analysis, and differential scanning calorimeter (DSC). More importantly, the neutral dyes 4a-c show mitochondrial staining comparable to commercially positively charged dyes. As a novel multifunctional fluorescent dye, it has application value in the fields of solid-state fluorescence and biological imaging.

P1400: BOOSTING MITOCHONDRIAL RECYCLING REJUVENATES HEMATOPOIETIC AND IMMUNE SYSTEMS

Background: Hematopoietic stem cells (HSCs) generate all blood lineages and ensure the correct homeostasis between myeloid and lymphoid lineages during the entire life of an organism. However, HSCs decrease their regenerative potential and undergo toward a myeloid-biased fate skewing with age. This phenomenon leads to a reduction of B and T cell compartments and immune dysfunction. Consequently, elderly patients are more susceptible to severe viral and fungal infections. It was reported that HSC aging is associated with defective autophagy and accumulation of damaged mitochondria. And emerging studies showed that dysregulation of mitochondria metabolism correlates closely with hematopoietic aging process. However, the clear causal relationship between mitochondrial metabolism and HSC aging remains unknown. Aims: We were wondering if metabolic modulator can rejuvenate aged HSCs, rescue loss-of-function of aged HSCs and improve overall immune response. Methods: We used mito-QC reporter mice and mitochondrial stain to analyze mitochondrial network upon in vitro treatment of metabolic modulator. In vivo long-term blood reconstitution assays and LCMV infection model were used to estimate the effect of metabolic modulator on HSC functionality. Results: Here we reported that interventions aimed to modulate mitochondria recycling and metabolism can rejuvenate aged HSCs and thus preserving the lymphoid compartments and immune function. In vitro administration of the mitophagy inducer Urolithin A on aged HSCs restore their blood and immune reconstitution capability. Moreover, Urolithin A supplemented in the mouse diet, improves bone marrow functionality, expands the lymphoid compartment and boost immune response against viral infection in old mice. Finally, we discovered that HSCs rejuvenation is associated with mitophagy induction and re-establishment of mitochondria homeostasis and fitness in aged HSCs. Summary/Conclusion: In summary, we identified mitophagy as potent regulator of aging process and as valuable target for rejuvenating approaches in HSCs.

Sindbis Virus Replication Reduces Dependence on Mitochondrial Metabolism During Infection.

Alphaviruses are single stranded, positive sense RNA viruses that are often transmitted through mosquito vectors. With the increasing spread of mosquito populations throughout the world, these arboviruses represent a significant global health concern. Viruses such as Sindbis Virus (SINV), Chikungunya Virus (CHIKV) and Equine Encephalitis Viruses (EEV) are all alphaviruses. As viruses, these pathogens are dependent on the host cell environment for successful viral replication. It has been observed that viruses manipulate cellular metabolism and mitochondrial shape, activity, and dynamics to favor viral infection. This report looked to understand the metabolic changes present during Sindbis virus infection of hamster and human kidney cells. Cells were infected with increasing levels of SINV and at 24 hours post infection the mitochondria morphology was assessed with staining and mitochondrial activity was measured with a real-time Seahorse Bioanalyzer. The relative amount of mitochondrial staining intensity decreased with Sindbis virus infected cells. Both oxygen consumption rate and ATP production were decreased during SINV infection while non-mitochondrial respiration and extracellular acidification rate increased during infection. Collectively, the data indicates that SINV primarily utilizes non-mitochondrial metabolism to support viral infection within the first 24 hours. This understanding of viral preference for host cell metabolism may provide critical targets for antiviral therapies and help further define the nature of alphavirus infection.

Fluorophore dyes based on 9-thienylanthracene: AIEE behaviors, performance in solid state, and bioimaging in living cells

Four new 9-thienylanthracene fluorescent dyes (TAs) bearing 1-methylindole/triphenylamine moieties were successfully synthesized. They all displayed typical aggregation-induced emission enhancement (AIEE) features with bright and varying aggregative-state emission involving multiple fluorescence colors containing cyan, green, and jacinth. Meanwhile, their solid state fluorescence could be effectively switched by mechanical grinding and dichloromethane fuming except for TPATA-Py12. Specifically, ITA, TPATA and ITA-Py12 showed reversible high contrast mechanofluorochromic (MFC) behaviors involving distinct fluorescent color changes from cyan/yellow to green/red. Notably, these TA dyes displayed excellent biocompatibility and have been successfully realized in living cell imaging. Particularly, ITA-Py12 and TPATA-Py12 can steadily immobilize in the mitochondria of HeLa cells by strong hydrophobic interaction between the C12-alkyl chain and lipid bilayer, resulting in high specificity mitochondrial staining. These good properties of them could provide a reference for the design and development of multi-functional AIEE dyes based on TA-skeleton.

A versatile AIE fluorogen with selective reactivity to primary amines for monitoring amination, protein labeling, and mitochondrial staining

AbstractSpecific bioconjugation for native primary amines is highly valuable for both chemistry and biomedical research. Despite all the efforts, scientists lack a proper strategy to achieve high selectivity for primary amines, not to mention the requirement of fast response in real applications. Herein, we report a chromone‐based aggregation‐induced emission (AIE) fluorogen called CMVMN as a self‐reporting bioconjugation reagent for selective primary amine identification, and its applications for monitoring bioprocesses of amination and protein labeling. CMVMN is AIE‐active and capable of solid‐state sensing. Thus, its electrospun films are manufactured for visualization of amine diffusion and leakage process. CMVMN also shows good biocompatibility and potential mitochondria‐staining ability, which provides new insight for organelle‐staining probe design. Combined with its facile synthesis and good reversibility, CMVMN would not only show wide potential applications in biology, but also offer new possibilities for molecular engineering.

A method for isolating highly purified and active mitochondria from insects

So far, methods that yield the high purity and activity of the isolated mitochondria from insects have not been reported and determined. Here, we develop methods that combine differential centrifugation and discontinuous Nycodenz density gradient centrifugation to isolate highly purified mitochondria from the thorax muscle of insects, and the methods were widely validated across three orders (Coleoptera, Hymenoptera, and Blattaria) covering four insect species using Western blot and transmission electron microscopy (TEM) analysis. The results showed the removal of the residual contamination with nonmitochondrial components such as nucleus, sarcolemma, cytosol, and endoplasmic reticulum. Furthermore, TEM, mitochondria staining, fluorescence detection, and flow cytometry analyses were employed to assess membrane integrity and activity of the isolated mitochondria. The results showed no loss of mitochondria activity/integrity after isolation. In addition, temporal dynamics in activity of the isolated mitochondria under commonly used laboratory temperature (-20 °C, 4 °C, and 25 °C) were respectively detected using a fluorescence microplate reader. The results showed that it should be avoided to store the isolated mitochondria at room temperature, and the mitochondria can meet the requirements of the most downstream experiments when they were stored at −20 °C. Overall, the study presented a method for isolating highly purified and active mitochondria from insects. This study firstly described a high-speed discontinuous density gradient centrifugation-based method that could be widely applied for mitochondria isolation in insects. The present study also provided an example to assess purity and integrity/activity of the isolated mitochondria.

Mitochonic Acid-5 Inhibits Reactive Oxygen Species Production and Improves Human Chondrocyte Survival by Upregulating SIRT3-Mediated, Parkin-dependent Mitophagy.

Mitochondrial dysfunction is related to the pathogenesis of osteoarthritis (OA); however, there are no effective drugs to treat OA for maintaining mitochondrial homeostasis. Studies have shown that mitochonic acid-5 (MA-5) has a protective effect against mitochondrial damage and plays a role in mitophagy. However, it is not clear whether MA-5 has a beneficial effect on inflammatory articular cartilage. Here, human OA cartilage was obtained from patients undergoing total joint replacement. Interleukin-1β (IL-1β) was used to stimulate chondrocytes and induce inflammatory injury. Cell Counting Kit-8, TUNEL, and flow cytometry assays were used to assess apoptosis. Gene expression was examined using quantitative reverse transcription-polymerase chain reaction. Mitochondrial function was evaluated using immunoblotting, mitochondrial membrane potential assay, JC-1 staining, and immunofluorescence analysis. Mitophagy was detected using immunoblotting and immunofluorescence. 3-(1H-1,2,3-triazol-4-yl) pyridine (3-TYP), a specific inhibitor of Sirtuin 3 (SIRT3), was used to block the SIRT3/Parkin pathway. Mitophagy in the cartilage sections was evaluated via immunohistochemistry. IL-1β was found to induce chondrocyte apoptosis by inhibiting SIRT3 expression and mitophagy. In addition, inflammatory damage reduced the mitochondrial membrane potential and promoted the production of intracellular reactive oxygen species (ROS), leading to increased mitochondrial division, mitochondrial fusion inhibition, and the consequent mitochondrial damage. In contrast, the MA-5 treatment inhibited excessive ROS production by upregulating mitophagy, maintaining the mitochondrial membrane potential, and reducing mitochondrial apoptosis. After chemically blocking SIRT3 with 3-TYP, Parkin-related mitophagy was also inhibited, an effect that was prevented by pretreatment of the chondrocytes with MA-5, thereby suggesting that SIRT3 is upstream of Parkin. Overall, MA-5 was found to enhance the activity of SIRT3, promote Parkin-dependent mitophagy, eliminate depolarized/damaged mitochondria in chondrocytes, and protect cartilage cells. In conclusion, MA-5 inhibits IL-1β-induced oxidative stress and protects chondrocytes by upregulating the SIRT3/Parkin-related autophagy signaling pathway.

A Novel Fluorescent Dye Extracted from Buddleja officinalis for Labeling Mitochondria after Fixation.

Mitochondria are versatile organelles and function by communicating with cellular ecosystems. The fluorescent colocalization analysis after fixation is a highly intuitive method to understand the role of mitochondria. However, there are few fluorescent dyes available for mitochondrial staining after fixation. In this study, a novel fluorescent dye (BO-dye), extracted from Buddleja officinalis, was applied for mitochondrial staining in fixed immortalized human oral keratinocytes. The BO-dye (excitation: 414 nm, emission: 677 nm) is a small fluorescent molecular dye, which can cross the cytomembrane without permeabilization. We assume that the BO-dye could aggregate and bind to the mitochondria stably. BO-dye exhibited a mega-Stokes shift (>250 nm), which is an important feature that could reduce self-quenching and enhance the signal-to-noise ratio. Analysis of photophysical properties revealed that the BO-dye is temperature and pH insensitive, and it exhibits superior photostability. These results indicate that BO-dye can be considered an alternative fluorescent dye for labeling mitochondria after fixation.