Elevated Mitochondrial Membrane Potential Research Articles

Polyphenols from Maackia amurensis Heartwood Protect Neuronal Cells from Oxidative Stress and Prevent Herpetic Infection.

Here, we continued the investigation of anti-HSV-1 activity and neuroprotective potential of 14 polyphenolic compounds isolated from Maackia amurensis heartwood. We determined the absolute configurations of asymmetric centers in scirpusin A (13) and maackiazin (10) as 7R,8R and 1″S,2″S, respectively. We showed that dimeric stilbens maackin (9) and scirpusin A (13) possessed the highest anti-HSV-1 activity among polyphenols 1-14. We also studied the effect of polyphenols 9 and 13 on the early stages of HSV-1 infection. Direct interaction with the virus (virucidal activity) was the main mechanism of the antiviral activity of these compounds. The neuroprotective potential of polyphenolic compounds from M. amurensis was studied using models of 6-hydroxydopamine (6-OHDA)-and paraquat (PQ)-induced neurotoxicity. A dimeric stilbene scirpusin A (13) and a flavonoid liquiritigenin (6) were shown to be the most active compounds among the tested polyphenols. These compounds significantly increased the viability of 6-OHDA-and PQ-treated Neuro-2a cells, elevated mitochondrial membrane potential and reduced the intracellular ROS level. We also found that scirpusin A (13), liquiritigenin (6) and retusin (3) considerably increased the percentage of live Neuro-2a cells and decreased the number of early apoptotic cells. Scirpusin A (13) was the most promising compound possessing both anti-HSV-1 activity and neuroprotective potential.

Apelin‑13 reduces high glucose‑induced mitochondrial dysfunction in cochlear hair cells by inhibiting endoplasmic reticulum stress.

The complex manifestation of diabetic hearing loss and the relative inaccessibility of the inner ear contribute to the lack of research. The present study aimed to reveal the role of Apelin-13, a critical regulator of lipid metabolism, in diabetes-induced hearing loss. Cochlear hair cells treated with high glucose (HG) were adopted as an in vitro research model, and the impacts of Apelin-13 on cellular oxidative stress, apoptosis, mitochondrial dysfunction and endoplasmic reticulum (ER) stress were determined. In addition, cells were treated with the ER stress agonist tunicamycin to further explore its potential role in the regulatory effects of Apelin-13. Apelin-13 inhibited oxidative stress and apoptosis in the HG-induced cells. Additionally, Apelin-13 elevated mitochondrial membrane potential and ATP production, whereas it reduced mitochondrial reactive oxygen species levels. The levels of ER stress-related proteins exhibited a downward trend in response to Apelin-13. By contrast, tunicamycin reversed the effects of Apelin-13 on the aforementioned aspects, suggesting the role of ER stress in the regulatory effects of Apelin-13. In conclusion, the present study elucidated the protective role of Apelin-13 in ameliorating HG-induced mitochondrial functional impairment in cochlear hair cells by inhibiting ER stress.

Superior performance of biocomposite nanoparticles PLGA-RES in protecting oocytes against vitrification stimuli.

Irreversible cryogenic damage caused by oocyte vitrification limits its widespread use in female fertility preservation. In recent years, nanoparticles (NPs) have gained great attention as potential alternatives in protecting oocytes against cryoinjuries. In this paper, a novel composite nanoparticle, poly (lactic-co-glycolic acid)-resveratrol (PLGA-RES) was designed to improve the biocompatibility and sustained release properties by encapsulating natural antioxidant RES into PLGA NPs. Firstly, biotoxicity and oxidation resistance of PLGA-RES were determined, and the results showed that PLGA-RES had nontoxic effect on oocyte survival during in vitro maturation (IVM) (97.08% ± 0.24% vs. 98.89% ± 1.11%, p > 0.05). Notably, PLGA-RES even increased maturation (65.10% ± 4.11% vs. 52.85% ± 2.87%, p < 0.05) and blastocyst rate (56.13% ± 1.36% vs. 40.91% ± 5.85%, p < 0.05). Moreover, the reduced reactive oxygen species (ROS) level (13.49 ± 2.30 vs. 34.07 ± 3.30, p < 0.01), increased glutathione (GSH) (44.13 ± 1.57 vs. 37.62 ± 1.79, p < 0.01) and elevated mitochondrial membrane potential (MMP) levels (43.10 ± 1.81 vs. 28.52 ± 1.25, p < 0.01) were observed in oocytes treated with PLGA-RES when compared with that of the control group. Subsequently, the role of PLGA-RES played in oocytes during vitrification was systematically evaluated. The results showed that the addition of PLGA-RES during vitrification and thawing significantly improved the survival rate (80.42% ± 1.97% vs. 75.37% ± 1.3%, p < 0.05). Meanwhile, increased GSH (15.09 ± 0.86 vs. 14.51 ± 0.78, p < 0.01) and mitochondrial membrane potential (22.56 ± 3.15 vs. 6.79 ± 0.60, p < 0.01), decreased reactive oxygen species levels (52.11 ± 2.95 vs. 75.41 ± 7.23, p < 0.05) and reduced mitochondrial abnormality distribution rate (25.00% ± 0.29% vs. 33.33% ± 1.15%, p < 0.01) were assessed in vitrified MII oocytes treated with PLGA-RES. Furthermore, transcriptomic analyses demonstrated that PLGA-RES participated in endocytosis and PI3K/AKT/mTOR pathway regulation, which was verified by the rescued expression of ARRB2 and ULK3 protein after PLGA-RES treatment. In conclusion, PLGA-RES exhibited potent antioxidant activity, and could be used as an efficacious strategy to improve the quality of vitrified oocytes.

Phosphate starvation signaling increases mitochondrial membrane potential through respiration-independent mechanisms.

Mitochondrial membrane potential directly powers many critical functions of mitochondria, including ATP production, mitochondrial protein import, and metabolite transport. Its loss is a cardinal feature of aging and mitochondrial diseases, and cells closely monitor membrane potential as an indicator of mitochondrial health. Given its central importance, it is logical that cells would modulate mitochondrial membrane potential in response to demand and environmental cues, but there has been little exploration of this question. We report that loss of the Sit4 protein phosphatase in yeast increases mitochondrial membrane potential, both by inducing the electron transport chain and the phosphate starvation response. Indeed, a similarly elevated mitochondrial membrane potential is also elicited simply by phosphate starvation or by abrogation of the Pho85-dependent phosphate sensing pathway. This enhanced membrane potential is primarily driven by an unexpected activity of the ADP/ATP carrier. We also demonstrate that this connection between phosphate limitation and enhancement of mitochondrial membrane potential is observed in primary and immortalized mammalian cells as well as in Drosophila. These data suggest that mitochondrial membrane potential is subject to environmental stimuli and intracellular signaling regulation and raise the possibility for therapeutic enhancement of mitochondrial function even in defective mitochondria.

Pulmonary Hypertension Induces Serotonin Hyperreactivity and Metabolic Reprogramming in Coronary Arteries via NOX1/4-TRPM2 Signaling Pathway.

Clinical evidence revealed abnormal prevalence of coronary artery (CA) disease in patients with pulmonary hypertension (PH). The mechanistic connection between PH and CA disease is unclear. Serotonin (5-hydroxytryptamine), reactive oxygen species, and Ca2+ signaling have been implicated in both PH and CA disease. Our recent study indicates that NOXs (NADPH [nicotinamide adenine dinucleotide phosphate] oxidases) and TRPM2 (transient receptor potential cation channel subfamily M member 2) are key components of their interplay. We hypothesize that activation of the NOX-TRPM2 pathway facilitates the remodeling of CA in PH. Left and right CAs from chronic hypoxia and monocrotaline-induced PH rats were collected to study vascular reactivity, gene expression, metabolism, and mitochondrial function. Inhibitors or specific siRNA were used to examine the pathological functions of NOX1/4-TRPM2 in CA smooth muscle cells. Significant CA remodeling and 5-hydroxytryptamine hyperreactivity in the right CA were observed in PH rats. NOX1/4-mediated reactive oxygen species production coupled with TRPM2-mediated Ca2+ influx contributed to 5-hydroxytryptamine hyperresponsiveness. CA smooth muscle cells from chronic hypoxia-PH rats exhibited increased proliferation, migration, apoptosis, and metabolic reprogramming in an NOX1/4-TRPM2-dependent manner. Furthermore, the NOX1/4-TRPM2 pathway participated in mitochondrial dysfunction, involving mitochondrial DNA damage, reactive oxygen species production, elevated mitochondrial membrane potential, mitochondrial Ca2+ accumulation, and mitochondrial fission. In vivo knockdown of NOX1/4 alleviated PH and suppressed CA remodeling in chronic hypoxia rats. PH triggers an increase in 5-hydroxytryptamine reactivity in the right CA and provokes metabolic reprogramming and mitochondrial disruption in CA smooth muscle cells via NOX1/4-TRPM2 activation. This signaling pathway may play an important role in CA remodeling and CA disease in PH.

EDA2R knockdown alleviates myocardial ischemia/reperfusion injury through inhibiting the activation of the NF-κB signaling pathway.

Ischemia/reperfusion (I/R) is a pathological process that occurs in numerous organs and is often associated with severe cellular damage and death. Ectodysplasin-A2 receptor (EDA2R) is a member of the TNF receptor family that has anti-inflammatory and antioxidant effects. However, to the best of our knowledge, its role in the progression of myocardial I/R injury remains unclear. The present study aimed to investigate the role of EDA2R during myocardial I/R injury and the molecular mechanisms involved. In vitro, dexmedetomidine (DEX) exhibited a protective effect on hypoxia/reoxygenation (H/R)-induced cardiomyocyte injury and downregulated EDA2R expression. Subsequently, EDA2R silencing enhanced cell viability and reduced the apoptosis of cardiomyocytes. Furthermore, knockdown of EDA2R led to an elevated mitochondrial membrane potential (MMP), repressed the release of Cytochrome C and upregulated Bcl-2 expression. EDA2R knockdown also resulted in downregulated expression of Bax, and decreased activity of Caspase-3 and Caspase-9 in cardiomyocytes, reversing the effects of H/R on mitochondria-mediated apoptosis. In addition, knockdown of EDA2R suppressed H/R-induced oxidative stress. Mechanistically, EDA2R knockdown inactivated the NF-κB signaling pathway. Additionally, downregulation of EDA2R weakened myocardial I/R injury in mice, as reflected by improved left ventricular function and reduced infarct size, as well as suppressed apoptosis and oxidative stress. Additionally, EDA2R knockdown repressed the activation of NF-κB signal in vivo. Collectively, knockdown of EDA2R exerted anti-apoptotic and antioxidant effects against I/R injury in vivo and in vitro by suppressing the NF-κB signaling pathway.

Mutations in DNAJC19 cause altered mitochondrial structure and increased mitochondrial respiration in human iPSC-derived cardiomyocytes

BackgroundDilated cardiomyopathy with ataxia (DCMA) is an autosomal recessive disorder arising from truncating mutations in DNAJC19, which encodes an inner mitochondrial membrane protein. Clinical features include an early onset, often life-threatening, cardiomyopathy associated with other metabolic features. Here, we aim to understand the metabolic and pathophysiological mechanisms of mutant DNAJC19 for the development of cardiomyopathy. MethodsWe generated induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) of two affected siblings with DCMA and a gene-edited truncation variant (tv) of DNAJC19 which all lack the conserved DnaJ interaction domain. The mutant iPSC-CMs and their respective control cells were subjected to various analyses, including assessments of morphology, metabolic function, and physiological consequences such as Ca2+ kinetics, contractility, and arrhythmic potential. Validation of respiration analysis was done in a gene-edited HeLa cell line (DNAJC19tvHeLa). ResultsStructural analyses revealed mitochondrial fragmentation and abnormal cristae formation associated with an overall reduced mitochondrial protein expression in mutant iPSC-CMs. Morphological alterations were associated with higher oxygen consumption rates (OCRs) in all three mutant iPSC-CMs, indicating higher electron transport chain activity to meet cellular ATP demands. Additionally, increased extracellular acidification rates suggested an increase in overall metabolic flux, while radioactive tracer uptake studies revealed decreased fatty acid uptake and utilization of glucose. Mutant iPSC-CMs also showed increased reactive oxygen species (ROS) and an elevated mitochondrial membrane potential. Increased mitochondrial respiration with pyruvate and malate as substrates was observed in mutant DNAJC19tv HeLa cells in addition to an upregulation of respiratory chain complexes, while cellular ATP-levels remain the same. Moreover, mitochondrial alterations were associated with increased beating frequencies, elevated diastolic Ca2+ concentrations, reduced sarcomere shortening and an increased beat-to-beat rate variability in mutant cell lines in response to β-adrenergic stimulation. ConclusionsLoss of the DnaJ domain disturbs cardiac mitochondrial structure with abnormal cristae formation and leads to mitochondrial dysfunction, suggesting that DNAJC19 plays an essential role in mitochondrial morphogenesis and biogenesis. Moreover, increased mitochondrial respiration, altered substrate utilization, increased ROS production and abnormal Ca2+ kinetics provide insights into the pathogenesis of DCMA-related cardiomyopathy.

Rapid degeneration of iPSC-derived motor neurons lacking Gdap1 engages a mitochondrial-sustained innate immune response

Charcot-Marie-Tooth disease is a chronic hereditary motor and sensory polyneuropathy targeting Schwann cells and/or motor neurons. Its multifactorial and polygenic origin portrays a complex clinical phenotype of the disease with a wide range of genetic inheritance patterns. The disease-associated gene GDAP1 encodes for a mitochondrial outer membrane protein. Mouse and insect models with mutations in Gdap1 have reproduced several traits of the human disease. However, the precise function in the cell types affected by the disease remains unknown. Here, we use induced-pluripotent stem cells derived from a Gdap1 knockout mouse model to better understand the molecular and cellular phenotypes of the disease caused by the loss-of-function of this gene. Gdap1-null motor neurons display a fragile cell phenotype prone to early degeneration showing (1) altered mitochondrial morphology, with an increase in the fragmentation of these organelles, (2) activation of autophagy and mitophagy, (3) abnormal metabolism, characterized by a downregulation of Hexokinase 2 and ATP5b proteins, (4) increased reactive oxygen species and elevated mitochondrial membrane potential, and (5) increased innate immune response and p38 MAP kinase activation. Our data reveals the existence of an underlying Redox-inflammatory axis fueled by altered mitochondrial metabolism in the absence of Gdap1. As this biochemical axis encompasses a wide variety of druggable targets, our results may have implications for developing therapies using combinatorial pharmacological approaches and improving therefore human welfare.A Redox-immune axis underlying motor neuron degeneration caused by the absence ofGdap1. Our results show that Gdap1–/– motor neurons have a fragile cellular phenotype that is prone to degeneration. Gdap1–/– iPSCs differentiated into motor neurons showed an altered metabolic state: decreased glycolysis and increased OXPHOS. These alterations may lead to hyperpolarization of mitochondria and increased ROS levels. Excessive amounts of ROS might be the cause of increased mitophagy, p38 activation and inflammation as a cellular response to oxidative stress. The p38 MAPK pathway and the immune response may, in turn, have feedback mechanisms, leading to the induction of apoptosis and senescence, respectively. CAC, citric acid cycle; ETC, electronic transport chain; Glc, glucose; Lac, lactate; Pyr, pyruvate.

PER2 Promotes Odontoblastic/Osteogenic Differentiation of Dental Pulp Stem Cells by Modulating Mitochondrial Metabolism.

Human dental pulp stem cells (hDPSCs) possess remarkable self-renewal and multilineage differentiation ability. PER2, an essential circadian molecule, regulates various physiological processes. Evidence suggests that circadian rhythm and PER2 participate in physiological functions of DPSCs. However, the influence of PER2 on DPSCs' differentiation remains largely unknown. This study aimed to explore the effect and potential mechanism of PER2 on hDPSCs' differentiation. Dental pulp tissues were extracted, and hDPSCs were cultured for in vitro and in vivo experiments. Dorsal subcutaneous transplantation was performed in 6-week-old male BALB/c mice. The hDPSCs' odontoblastic/osteogenic differentiation was assessed, and mitochondrial metabolism was evaluated. The results indicated PER2 expression increasing during hDPSCs' odontoblastic/osteogenic differentiation. Gain- and loss-of function studies confirmed that PER2 promoted alkaline phosphatase (ALP) activity, mineralized nodules deposition, mRNA expression of DSPP, DMP1, COL1A1 and protein expression of DSPP and DMP1 in hDPSCs. Furthermore, PER2 enhanced collagen deposition, osteodentine-like tissue formation and DSPP expression in vivo. Mitochondrial metabolic evaluation aimed to investigate the mechanism of PER2-mediated hDPSC odontoblastic/osteogenic differentiation, which showed that PER2 increased ATP synthesis, elevated mitochondrial membrane potential and changed expression of proteins regulating mitochondrial dynamics. This study demonstrated that PER2 promoted hDPSCs' odontoblastic/osteogenic differentiation, which involved mitochondrial metabolic change.

Mdivi-1 Rescues Memory Decline in Scopolamine-Induced Amnesic Male Mice by Ameliorating Mitochondrial Dynamics and Hippocampal Plasticity.

Memory loss, often known as amnesia, is common in the elderly population and refers to forgetting facts and experiences. It is associated with increased mitochondrial fragmentation, though the contribution of mitochondrial dynamics in amnesia is poorly understood. Therefore, the present study is aimed at elucidating the role of Mdivi-1 in mitochondrial dynamics, hippocampal plasticity, and memory during scopolamine(SC)-induced amnesia. The findings imply that Mdivi-1 significantly increased the expression of Arc and BDNF proteins in the hippocampus of SC-induced amnesic mice, validating improved recognition and spatial memory. Moreover, an improved mitochondrial ultrastructure was attributed to a decline in the percentage of fragmented and spherical-shaped mitochondria after Mdivi-1 treatment in SC-induced mice. The significant downregulation of p-Drp1 (S616) protein and upregulation of Mfn2, LC3BI, and LC3BII proteins in Mdivi-1-treated SC-induced mice indicated a decline in fragmented mitochondrial number and healthy mitochondrial dynamics. Mdivi-1 treatment alleviated ROS production and Caspase-3 activity and elevated mitochondrial membrane potential, Vdac1 expression, ATP production, and myelination, resulting in reduced neurodegeneration in SC mice. Furthermore, the decline of pro-apoptotic protein cytochrome-c and increase of anti-apoptotic proteins Procaspase-9 and Bcl-2 in Mdivi-1-treated SC-induced mice suggested improved neuronal health. Mdivi-1 also increased the dendritic arborization and spine density, which was further corroborated by increased expression of synaptophysin and PSD95. In conclusion, the current study suggests that Mdivi-1 treatment improves mitochondrial ultrastructure and function through the regulation of mitochondrial dynamics. These changes further improve neuronal cell density, myelination, dendritic arborization, and spine density, decrease neurodegeneration, and improve recognition and spatial memory. Schematic presentation depicts that Mdivi-1 rescues memory decline in scopolamine-induced amnesic male mice by ameliorating mitochondrial dynamics and hippocampal plasticity.

Protective effects of Shen Yuan Dan on myocardial ischemia-reperfusion injury via the regulation of mitochondrial quality control.

Myocardial cell death resulting from ischemia-reperfusion (I/R) injury has been a predominant contributor to morbidity and mortality globally. The mitochondria-centered mechanism plays an important role in the formation of I/R injury. This study intended to discuss the protective mechanism of Shen Yuan Dan (SYD) on cardiomyocytes hypoxia-reoxygenation (H/R) injury via the regulation of mitochondrial quality control (MQC). Additionally, this study clarified the mechanism by which SYD suppressed mitophagy activity through the suppression of the PTEN-induced kinase 1 (PINK1)/Parkin pathway. To induce cellular injury, H9c2 cardiomyocytes were exposed to H/R stimulation. Following the pretreatment with SYD, cardiomyocytes were subjected to H/R stimulation. Mitochondrial membrane potential (MMP), adenosine triphosphate (ATP), superoxide dismutase (SOD), and methane dicarboxylic aldehyde (MDA) were detected to evaluate the degree of cardiomyocyte mitochondrial damage. Laser confocal microscopy was applied to observe the mitochondrial quality, and the messenger (mRNA) levels of mitofusin 1 (Mfn1), mitofusin 2 (Mfn2), optic atrophy protein 1 (Opa1), dynamin-related protein 1 (Drp1), fission 1 (Fis1), and peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) in cardiomyocytes were assessed using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Western blotting was employed for the estimation of light chain 3 (LC3)-I, LC3-II, PINK1, and Parkin in cardiomyocytes. It was discovered that SYD pretreatment elevated MMP in H/R injury cardiomyocytes, enhanced ATP content, activated SOD activity, and reduced MDA level. SYD treatment increased the mRNA levels of Mfn1, Mfn2, Opa1 and PGC-1α decreased the mRNA levels of Drp1 and Fis1, and reduced the protein levels of LC3, PINK1, and Parkin. SYD plays a protective role in H/R injury to cardiomyocytes by regulating mitochondrial quality. Meanwhile, SYD may inhibit mitophagy activity through inhibiting the PINK1/Parkin pathway. This study provides insights into the underlying mechanism of SYD in alleviating myocardial I/R injury.

Elevated ROS levels during the early development of Angelman syndrome alter the apoptotic capacity of the developing neural precursor cells

Angelman syndrome (AS) is a rare genetic neurodevelopmental disorder caused by the maternally inherited loss of function of the UBE3A gene. AS is characterized by a developmental delay, lack of speech, motor dysfunction, epilepsy, autistic features, happy demeanor, and intellectual disability. While the cellular roles of UBE3A are not fully understood, studies suggest that the lack of UBE3A function is associated with elevated levels of reactive oxygen species (ROS). Despite the accumulating evidence emphasizing the importance of ROS during early brain development and its involvement in different neurodevelopmental disorders, up to date, the levels of ROS in AS neural precursor cells (NPCs) and the consequences on AS embryonic neural development have not been elucidated. In this study we show multifaceted mitochondrial aberration in AS brain-derived embryonic NPCs, which exhibit elevated mitochondrial membrane potential (ΔΨm), lower levels of endogenous reduced glutathione, excessive mitochondrial ROS (mROS) levels, and increased apoptosis compared to wild-type (WT) littermates. In addition, we report that glutathione replenishment by glutathione-reduced ethyl ester (GSH-EE) corrects the excessive mROS levels and attenuates the enhanced apoptosis in AS NPCs. Studying the glutathione redox imbalance and mitochondrial abnormalities in embryonic AS NPCs provides an essential insight into the involvement of UBE3A in early neural development, information that can serve as a powerful avenue towards a broader view of AS pathogenesis. Moreover, since mitochondrial dysfunction and elevated ROS levels were associated with other neurodevelopmental disorders, the findings herein suggest some potential shared underlying mechanisms for these disorders as well.

A Novel Three-Dimensional Follicle Culture System Decreases Oxidative Stress and Promotes the Prolonged Culture of Human Granulosa Cells.

Tissue engineering advancements have made it possible to modify biomaterials to reconstruct a similar three-dimensional structure of the extracellular matrix (ECM) for follicle development and to supply the required biological signals. We postulated that an artificial polysaccharide hydrogel modified with an ECM mimetic peptide may produce efficient irritation signals by binding to specific integrins providing a suitable environment for follicular development and influencing the behavior of human granulosa cells (hGCs). Laminin, an important component of the extracellular matrix, can modulate hGCs and oocyte growth. Specifically, follicles of mice were randomly divided into two-dimensional (2D) and three-dimensional (3D) culture systems established by a hydrogel modified with RGD or laminin mimetic peptides (IKVAV and YIGSR) and RGD (IYR). Our results showed that 3D cultured systems significantly improved follicle survival, growth, and viability. IYR peptides enhanced the oocyte meiosis competence. Additionally, we explored the effect of 3D culture on hGCs, which improved hGCs viability, increased the proportion of S- and G2/M-phase cells, and inhibited cell apoptosis of hGCs. On days 1 and 2, the secretion of progesterone was reduced in 3D-cultured hGCs. Notably, 3D-cultured hGCs exhibited delayed senescence, decreased oxidative stress, and elevated mitochondrial membrane potential. Moreover, the expression levels of cumulus expansion-related genes (COX2, HAS2, and PTX3) and integrin α6β1 were upregulated in 3D-cultured hGCs. In conclusion, a 3D culture utilizing hydrogels modified with Laminin-mimetic peptides can provide a durable physical environment suitable for follicular development. The laminin-mimetic peptides may regulate the biological activity of hGCs by attaching to the integrin α6β1.

Baicalin mitigated IL-1β-Induced osteoarthritis chondrocytes damage through activating mitophagy.

Mitophagy is related to chondrocyte homeostasis and plays a key role in the progress of osteoarthritis (OA). Baicalin has a protective effect on OA chondrocytes, the aim of this study was to explore whether the effect of Baicalin on IL-1β-induced chondrocyte injury is related to the regulation of mitophagy. The expression of collagen II in chondrocytes was detected to identify chondrocytes. The effects of different concentrations of Baicalin (10, 20 and 40 μM), autophagy inhibitor (3-Methyladenine), autophagy activator (rapamycin) and Baicalin combined with PI3K agonist (740Y-P) on the viability (cell counting kit 8), apoptosis (flow cytometry), autophagy activation (Monodansylcadaverine staining) and mitochondrial membrane potential (JC-1 kit) of IL-1β-induced chondrocytes were evaluated. The co-localization of autophagosome and mitochondria was determined by immunofluorescence. Apoptosis-, autophagy-, PI3K/AKT/mTOR pathway- and mitophagy-related proteins were detected by western blot. Our result revealed that Baicalin and rapamycin facilitated cell viability, autophagy and mitophagy, elevated mitochondrial membrane potential and suppressed apoptosis of IL-1β-induced rat chondrocytes. In addition, Baicalin and rapamycin upregulated the levels of Bcl-2, Beclin 1, LC3-II/LC3-I, p-Drp1, PINK1 and Parkin as well as downregulated the levels of Bax, cleaved caspase-3, P62, p-PI3K/PI3K, p-mTOR/mTOR and Drp1 in IL-1β-induced rat chondrocytes. However, 3-Methyladenine did the opposite effects of Baicalin and 740Y-P reversed the effects of Baicalin on IL-1β-induced rat chondrocytes. In conclusion, Baicalin activated mitophagy in IL-1β-induced chondrocytes by inhibiting PI3K/AKT/mTOR pathway and activating PINK1/Parkin and PINK1/Drp-1 pathway, thereby reducing the chondrocyte injury.

The effects of purslane (Portulaca oleracea) and fennel (Foeniculum vulgare Mill) hydroalcoholic extracts on the functional parameters of human spermatozoa after vitrification.

Reactive oxygen species (ROS) are produced during cryopreservation of human sperm and impair sperm function. Antioxidant compounds, such as fennel and purslane, reduce the damaging effects of ROS. This study aimed to evaluate motility parameters, plasma membrane integrity (PMI), mitochondrial membrane potential (MMP), intracellular ROS, and DNA damage to determine the optimum concentrations of hydroalcoholic extracts of fennel and purslane for human spermatozoa cryopreservation. Twenty human sperm samples were used and divided into seven equal groups consisting of fennel hydroalcoholic extract (5, 10, and 15 mg/L), purslane hydroalcoholic extract (25, 50, and 100 mg/L), and no additive. Supplementation of 25 mg/L and 50 mg/L purslane extract and 10 mg/L fennel extract in cryopreservation extender significantly increased the motility and PMI of sperm with a significant reduction in intracellular ROS compared to control groups (p<0.05). A 50 mg/L concentration of purslane extract elevated progressive motility and MMP compared to the control group (p<0.05). No significant differences were seen for motion patterns and DNA damage of frozen-thawed human sperm in extender containing these extracts. The results showed that supplementation of 50 mg/L purslane extract and 10 mg/L fennel extract in semen cryopreservation extender has the potential to decrease intracellular ROS and subsequently elevate the motility and PMI of human sperm.

Jian Pi Sheng Sui Gao (JPSSG) alleviation of skeletal myoblast cell apoptosis, oxidative stress, and mitochondrial dysfunction to improve cancer-related fatigue in an AMPK-SIRT1- and HIF-1-dependent manner.

Jian Pi Sheng Sui Gao (JPSSG), a Chinese traditional herbal paste, possesses certain efficacy in patients with cancer-related fatigue (CRF); however, its related mechanism remains unclear. Hence, network pharmacology analysis, followed by in vivo and in vitro experiments were conducted in this study with the aim to evaluate the effect of JPSSG on CRF and clarify its potential mechanism. Network pharmacology analysis was performed. Subsequently, 12 mice were injected with CT26 cells to establish CRF mouse models and randomly divided into a model group (n=6) and JPSSG group (n=6); meanwhile, another 6 normal mice served as a control group. Then, 3.0 g/kg JPSSG was given to mice in JPSSG group for 15 days, while mice in the n control and model groups received phosphate-buffered saline (PBS) of the same volume for 15 days. For the in vitro experiment, CT26 conditioned medium (CM) was established; meanwhile, the mitochondrial damage model was constructed through C2C12 myotubes stimulated with H2O2. C2C12 myotubes were divided into 5 groups: control group (without treatment), CM group, CM + JPSSG group, H2O2 group, and H2O2 + JGSSP group. Network pharmacology analysis identified 87 bioactive compounds and 132 JPSSG-CRF interaction targets. Moreover, according to the Kyoto Encyclopedia of Genes and Genomes enrichment analysis and the subsequent in vivo and in vitro experiments, JPSSG activated adenosine 5'-monophosphate-activated protein kinase-silent-information-regulator factor 2-related-enzyme 1 (AMPK-SIRT1) and hypoxia-inducible factor-1 (HIF-1) signaling pathways during CRF. Moreover, the in vivo experiment showed that JPSSG attenuated CRF in mice, reflected by increased distance traveled, mobile time in open field test, and swimming time in exhaustive swimming test, and decreased absolute rest time and tail suspension test in the JPSSG group (vs. model group). Furthermore, JPSSG upregulated gastrocnemius weight, adenosine triphosphate (ATP), superoxide dismutase (SOD), and the cross-sectional area of the gastrocnemius. With regard to in vitro study, JPSSG elevated cell viability, B-cell lymphoma-2, ATP, SOD, and mitochondrial membrane potential, while it decreased apoptosis rate, cleaved-caspase3, malondialdehyde, and reactive oxygen species in C2C12 myotubes. JPSSG ameliorates CRF via alleviating skeletal myoblast cell apoptosis, oxidative stress, and mitochondrial dysfunction in an AMPK-SIRT1- and HIF-1-dependent manner.

Melatonin promoted osteogenesis of human periodontal ligament cells by regulating mitochondrial functions through the translocase of the outer mitochondrial membrane 20.

Melatonin plays an important role in various beneficial functions, including promoting differentiation. However, effects on osteogenic differentiation, especially in human periodontal cells (hPDLCs), still remain inconclusive. Mitochondria are highly dynamic organelles that play an important role in various biological processes in cells, including energy metabolism and oxidative stress reaction. Furthermore, the translocase of the outer mitochondrial membrane 20 (TOM20) is responsible for recognizing and transporting precursor proteins. Thus, the objective of this study was to evaluate the functionality of melatonin on osteogenesis in human periodontal cells and to explore the involved mechanism of mitochondria. The hPDLCs were extracted and identified by flow cytometry and multilineage differentiation. We divided hPDLCs into control group, osteogenic induction group, and osteogenesis with melatonin treatment group (100, 10, and 1μM). Then we used a specific siRNA to achieve interference of TOM20. Alizarin red and Alkaline phosphatase staining and activity assays were performed to evaluate osteogenic differentiation. Osteogenesis-related genes and proteins were measured by qPCR and western blot. Mitochondrial functions were tested using ATP, NAD+/NADH, JC-1, and Seahorse Mito Stress Test kits. Finally, TOM20 and mitochondrial dynamics-related molecules expression were also assessed by qPCR and western blot. Our results showed that melatonin-treated hPDLCs had higher calcification and ALP activity as well as upregulated OCN and Runx2 expression at mRNA and protein levels, which was the most obvious in 1μM melatonin-treated group. Meanwhile, melatonin supplement elevated intracellular ATP production and mitochondrial membrane potential by increasing mitochondrial oxidative metabolism, hence causing a lower NAD+ /NADH ratio. In addition, we also found that melatonin treatment raised TOM20 level and osteogenesis and mitochondrial functions were both suppressed after knocking down TOM20. We found that melatonin promoted osteogenesis of hPDLCs and 1μM melatonin had the most remarkable effect. Melatonin treatment can reinforce mitochondrial functions by upregulating TOM20.

Zeb1 sustains hematopoietic stem cell functions by suppressing mitofusin-2-mediated mitochondrial fusion

Metabolic status is essential in maintaining normal functions of hematopoietic stem cells (HSCs). However, how the dynamic of the mitochondrion, as a central organelle in metabolism, is molecularly regulated to orchestrate metabolism and HSC stemness remains to be elucidated. Here, we focus on the role of Zeb1, a well-characterized epithelial-to-mesenchymal transition (EMT) inducer which has been demonstrated to confer stem-cell-like characteristics in multiple cancer types in stemness regulation of HSCs. Using a Zeb1-tdTomato reporter mouse model, we find that Zeb1+Lin−Sca-1+c-Kit+ cells (Zeb1+-LSKs) represent a subset of functional long-term HSCs. Zeb1+LSKs exhibit a reduced reactive oxygen species (ROS) level, low mitochondrial mass, low mitochondrial membrane potential (MMP), and particularly small, round fragmented mitochondria. Of note, ectopic expression of Zeb1 leads to a fragmented mitochondrial morphology with a low mitochondrial metabolic status in EML cells. In addition, Zeb1-knockout (Zeb1-KO) LSKs from fetal liver display an exhausted stem-cell activity. Zeb1 deficiency results in elongated and tubulated mitochondria with increased mitochondrial mass, elevated MMP, and higher ROS production. Mechanistically, Zeb1 acts as a transcriptional suppressor on the key mitochondrial-fusion protein Mitofusin-2 (encoded by Mfn2). We highlight an important role of Zeb1 in the regulation of mitochondrial morphology in HSC and the metabolic control of HSC stemness by repressing Mfn2-mediated mitochondrial fusion.

Superoxide dismutase 2 ameliorates mitochondrial dysfunction in skin fibroblasts of Leber's hereditary optic neuropathy patients.

BackgroundIn Leber’s hereditary optic neuropathy (LHON), mtDNA mutations mediate mitochondrial dysfunction and apoptosis of retinal ganglion cells. Mitochondrial superoxide dismutase 2 (SOD2) is a crucial antioxidase against reactive oxygen species (ROS). This study aims to investigate whether SOD2 could ameliorate mtDNA mutation mediated mitochondrial dysfunction in skin fibroblasts of LHON patients and explore the underlying mechanisms.MethodsThe skin of normal healthy subjects and severe LHON patients harboring m.11778G > A mutation was taken to prepare immortalized skin fibroblast cell lines (control-iFB and LHON-iFB). LHON-iFB cells were transfected with SOD2 plasmid or negative control plasmid, respectively. In addition, human neuroblastoma SH-SY5Y cells and human primary retinal pigmental epithelium (hRPE) cells were stimulated by H2O2 after gene transfection. The oxygen consumption rate (OCR) was measured with a Seahorse extracellular flux analyzer. The level of ATP production, mitochondrial membrane potential, ROS and malondialdehyde (MDA) were measured separately with the corresponding assay kits. The expression level of SOD2, inflammatory cytokines and p-IκBα/IκBα was evaluated by western-blot. Assessment of apoptosis was performed by TUNEL assay.ResultsLHON-iFB exhibited lower OCR, ATP production, mitochondrial membrane potential but higher level of ROS and MDA than control-iFB. Western-blot revealed a significantly increased expression of IL-6 and p-IκBα/IκBα in LHON-iFB. Compared with the negative control, SOD2 overexpression increased OCR, ATP production and elevated mitochondrial membrane potential, but impaired ROS and MDA production. Besides, western-blot demonstrated exogenous SOD2 reduced the protein level of IL-6 and p-IκBα/IκBα. TUNEL assays suggested SOD2 inhibited cells apoptosis. Analogously, in SH-SY5Y and hRPE cells, SOD2 overexpression increased ATP production and mitochondrial membrane potential, but decreased ROS, MDA levels and suppressed apoptosis.ConclusionSOD2 upregulation inhibited cells apoptosis through ameliorating mitochondrial dysfunction and reducing NF-κB associated inflammatory response. This study further support exogenous SOD2 may be a promising therapy for the treatment of LHON.

ATF5 Attenuates Apoptosis in Hippocampal Neurons with Seizures Evoked by Mg2+-Free Medium via Regulating Mitochondrial Unfolded Protein Response.

The mitochondrial unfolded protein response (mtUPR)-a stress response pathway for maintaining protein homeostasis-is critical in seizures-induced neuronal injury. The activating transcription factor 5 (ATF5) regulates mtUPR; however, whether ATF5-regulated mtUPR has a role in neuronal injury in epilepsy remains uncertain. Here, we investigated the effects of ATF5-regulated mtUPR on neuronal injury in hippocampal neurons with seizures evoked by Mg2+-free medium. HSP60 and ClpP, key proteins of mtUPR, were upregulated, indicating mtUPR activation. ATF5 overexpression by lentiviral vector infection potentiated mtUPR, whereas ATF5 downregulation by lentiviral vector infection attenuated this response. Moreover, ATF5 overexpression elevated mitochondrial membrane potential and reduced reactive oxygen species (ROS) generation, suggesting that ATF5 overexpression protected mitochondrial homeostasis, while ATF5 downregulation had the opposite effect. ATF5 overexpression also reversed Bcl2 downregulation and Bax upregulation and attenuated seizures-induced neuronal apoptosis, while ATF5 downregulation aggravated the injury. Our study demonstrates that ATF5 attenuates seizures-induced neuronal injury, possibly by regulating mtUPR pathways, to prevent mitochondrial dysfunction.