Accumulated ROS Activates HIF-1α-Induced Glycolysis and Exerts a Protective Effect on Sensory Hair Cells Against Noise-Induced Damage

Noise exposure causes noise-induced hearing loss (NIHL). NIHL exhibits loss of inner ear sensory hair cells and is often irreparable. Although oxidative stress is involved in hearing loss, the complex mechanisms involved in NIHL are unclear. Hypoxia-inducible factor 1α (HIF-1α) has been suggested to be essential for protecting sensory hair cells. Additionally, it has been shown that ROS is involved in modulating the stability of HIF-1α. To investigate the NIHL pathogenesis, we established a tert-butyl hydroperoxide (t-BHP)-induced oxidative stress damage model in hair-like HEI-OC1 cells and an NIHL model in C57BL/6 mice. Protein and mRNA expression were determined, and biochemical parameters including reactive oxygen species (ROS) accumulation, glucose uptake, adenosine triphosphat (ATP) production, and mitochondrial content were evaluated. In HEI-OC1 cells, t-BHP induced ROS accumulation and reduced mitochondrial content and oxygen consumption, but the ATP level was unaffected. Additionally, there was increased glucose uptake and lactate release along with elevated expression of HIF-1α, glucose transporter 1, and several glycolytic enzymes. Consistently, noise trauma induced oxidative stress and the expression of HIF-1α and glycolytic enzymes in mice. Thus, we concluded that ROS induced HIF-1α expression, which promoted glycolysis, suggesting a metabolic shift maintained the ATP level to attenuate hair cell damage in NIHL.

Celastrol Protects against Cerebral Ischemia/Reperfusion Injury in Mice by Inhibiting Glycolysis through Targeting HIF-1α/PDK1 Axis

Cerebral ischemia/reperfusion (I/R) injury is closely related to dysfunctional glucose metabolism. Celastrol is a bioactive compound that has been found to exhibit neuroprotective effects in cerebral ischemia, while whether it can protect against cerebral I/R injury by regulating glycolysis remains unclear. The goal of this study is to investigate the role of celastrol on cerebral I/R injury and its underlying mechanisms in transient middle cerebral artery occlusion (tMCAO) mice. Methods. To observe the protective effect of celastrol and select its optimal dosage for further study, neurological score, TTC staining, and HE staining were used to evaluate neurological function, cerebral infarct volume, and cortical cell damage, respectively. QRT-PCR and Western blot were used to detect the mRNA and protein expression of hypoxia inducible factor-1α (HIF-1α), pyruvate dehydrogenasekinase1 (PDK1), lactate dehydrogenase A (LDHA), glucose transporter1 (GLUT1), and hexokinase2 (HK2), respectively. The lactate production, ATP level, and glucose content were assessed by assay kits. Results. Our results indicated that celastrol dose-dependently improved neurological function and reduced cerebral infarct volume and cortical cell death of tMCAO mice, and its optimal dosage was 4.5 mg/kg. In addition, celastrol significantly blocked I/R-induced increase of LDHA, GLUT1, HK2, and lactate production as well as decrease of ATP level and glucose content. Moreover, celastrol inhibited the I/R-induced upregulation of HIF-1α and PDK1. Overexpression of HIF-1α by DMOG reversed the protective effect of celastrol on cerebral I/R injury and blocked celastrol-induced suppression of glycolysis. Conclusions. Taken together, these results suggested that celastrol protected against cerebral I/R injury through inhibiting glycolysis via the HIF-1α/PDK1 axis.

MiR-21-5p inhibition attenuates Warburg effect and stemness maintenance in osteosarcoma cells via inactivation of Wnt/β-catenin signaling

MicroRNA (miR)-21 has been found to be overexpressed in osteosarcoma (OS). The aim of the present study was to investigate the effect of miR-21-5p on the Warburg effect and stemness maintenance in OS cells and its potential molecular mechanism. Herein, miR-21-5p was overexpressed or inhibited in MG-63 cells via transfection with mimics or inhibitors. The effect of miR-21-5p on cell viability, apoptosis, Warburg effect and stemness maintenance were explored in OS cells. The results demonstrated that miR-21-5p inhibition suppressed MG-63 cell viability and enhanced their apoptosis. Additionally, miR-21-5p inhibition attenuated the stemness maintenance of MG-63 cells, as demonstrated by the reduced proportion of CD133-positive MG-63 cells, the decrease in tumorsphere formation capacity, and the downregulation of Sox2, Oct4, and Nanog proteins. Moreover, miR-21-5p inhibition suppressed the Warburg effect in MG-63 cells, as indicated by the decrease in glucose uptake, lactic acid production, and ATP level and the downregulation of proteins involved in the Warburg effect (GLUT1, LDHA, HK2, and PKM2). Furthermore, the results suggested that the effect of miR-21-5p suppression on stemness and the Warburg effect may be associated with the decreased activity of the Wnt/β-catenin pathway in OS cells. Our findings suggest a novel potential biomarker for OS therapy.

Characterization of Collagen/Beta Glucan Hydrogels Crosslinked with Tannic Acid

Hydrogels based on collagen/β-glucan crosslinked with tannic acid were obtained by neutralization using dialysis. The presence of tannic acid allowed obtaining stable hydrogel materials with better mechanical properties. Tannic acid was released from matrices gradually and not rapidly. The antioxidant properties of the obtained hydrogels increased over the course of their incubation in culture media and were dependent on the concentration of tannic acid in the matrices. The obtained materials influenced dehydrogenase activity and the ATP level of pathogens. Additionally, the materials’ extracts improved the HaCaT cells’ viability. Therefore, the obtained hydrogels seem to be promising biocompatible materials which display antimicrobial properties.

Hsp22 Inhibits Oxidative Stress-Induced Endplate Chondrocyte Apoptosis by Regulating Mitochondrial Pathway

Background: Facet joint degeneration (FJD), which is also called facet joint syndrome (FJS), has become one of the most commonly seen etiological factors for lumbago. Cartilage lesion triggered by lumbar facet joint (LFJ) degeneration might be related to mitochondrial impairment, but the its underlying mechanism remains unclear. Materials and methods: The endplate chondrocytes were induced by hydrogen peroxide (H2O2) to mimic the pathological conditions of oxidative stress. Enzyme linked immunosorbent assay (ELISA) were used for the evaluation of reactive oxygen species (ROS). Adenosine-triphosphate (ATP) level was assessed using ATP detection, along with the detection the expression of cytochrome C in mitochondria (mito-cyt c) and in cell cytoplasm (cyto-cyt c) and cleaved caspase 3 by Western blot analysis. TUNEL assay was conducted for the measurement of cell apoptosis in endplate chondrocytes. Reverse transcription-polymerase chain reaction (RT-qPCR) was used to verify the expression of heat shock protein 22 (HSP22) and the transfection efficiency of HSP22 interference plasmid. Results: It was found that H2O2 promoted the mitochondrial dysfunction, ROS generation and cell apoptosis in endplate chondrocytes. Moreover, HSP22 was down-regulated in H2O2-induced endplate chondrocytes, and interference of HSP22 decreased the ROS production, increased the ATP level and promoted the cell apoptosis, resulting in the enhanced impairment of endplate chondrocytes. Additionally, mitochondrial ROS inhibitor (Mito-TEMPO) ameliorated the injury effects of HSP22 silencing in the H2O2-induced endplate chondrocytes. Conclusion: In conclusion, HSP22 inhibits oxidative stress-induced endplate chondrocyte apoptosis by regulating mitochondrial pathway, possibly providing novel guidance direction for the treatment of LFJ degeneration.

Melatonin Protects TEGDMA induced Pre-odontoblasts Mitochondrial Apoptosis via JNK/MAPK Signaling Pathway

Background: Resin monomer induced dental pulp injury presents a mitochondrial dysfunction related pathology. Melatonin has been regarded as a strong mitochondrial protective bioactive compound from pineal gland. However, it remains unknown whether melatonin can prevent dental pulp from resin monomer induced injury. The aim of the study is to investigate the effects of melatonin on TEGDMA, a major component in dental resin, induced mouse pre-odontoblast cell lines (mDPC6T) mitochondrial apoptosis and to verify whether JNK/MAPK signaling pathway mediate the protective effect of melatonin. Methods: We adopted a well-established TEGDMA-induced mDPC6T apoptosis model to investigate the preventive effect of melatonin by detecting cell viability, apoptosis rate, expression of apoptosis related protein, mitochondrial ROS (mtROS) production, mitochondrial membrane potential (MMP) and adenosine triphosphate (ATP) level. Inhibitors of MAPKs signaling were used to explore which pathway was participated in TEGDMA induced apoptosis. Finally, we verified the role of JNK/MAPK pathway during the protective effects of melatonin above by the agonist and antagonists of JNK.Results: Melatonin attenuated TEGDMA induced mDPC6T apoptosis via reducing mtROS production, rescuing MMP and ATP level. Meanwhile, the mitochondrial dysfunction and apoptosis was alleviated by the JNK/MAPK inhibitor SP600125 but not the other MAPKs signaling inhibitors. Furthermore, melatonin down-regulated the expression of phosphorylated-JNK, and eliminated the active effects of Anisomycin on JNK/MAPK pathway, which mimicked the effects of the SP600125.Conclusion: Our findings demonstrated that melatonin protected mDPC6T against TEGDMA induced apoptosis via JNK/MAPK signaling and maintenance of mitochondrial function, which presented a novel therapeutic strategy for prevention against resin monomer-induced dental pulp injury.

Enforcing ATP hydrolysis enhanced anaerobic glycolysis and promoted solvent production in Clostridium acetobutylicum

Background The intracellular ATP level is an indicator of cellular energy state and plays a critical role in regulating cellular metabolism. Depletion of intracellular ATP in (facultative) aerobes can enhance glycolysis, thereby promoting end product formation. In the present study, we examined this s trategy in anaerobic ABE (acetone-butanol-ethanol) fermentation using Clostridium acetobutylicum DSM 1731. Results Following overexpression of atpAGD encoding the subunits of water-soluble, ATP-hydrolyzing F1-ATPase, the intracellular ATP level of 1731(pITF1) was significantly reduced compared to control 1731(pIMP1) over the entire batch fermentation. The glucose uptake was markedly enhanced, achieving a 78.8% increase of volumetric glucose utilization rate during the first 18 h. In addition, an early onset of acid re-assimilation and solventogenesis in concomitant with the decreased intracellular ATP level was evident. Consequently, the total solvent production was significantly improved with remarkable increases in yield (14.5%), titer (9.9%) and productivity (5.3%). Further genome-scale metabolic modeling revealed that many metabolic fluxes in 1731(pITF1) were significantly elevated compared to 1731(pIMP1) in acidogenic phase, including those from glycolysis, tricarboxylic cycle, and pyruvate metabolism; this indicates significant metabolic changes in response to intracellular ATP depletion. Conclusions In C. acetobutylicum DSM 1731, depletion of intracellular ATP significantly increased glycolytic rate, enhanced solvent production, and resulted in a wide range of metabolic changes. Our findings provide a novel strategy for engineering solvent-producing C. acetobutylicum, and many other anaerobic microbial cell factories.

The Energy Status of Astrocytes Is the Achilles’ Heel of eIF2B-Leukodystrophy

Translation initiation factor 2B (eIF2B) is a master regulator of global protein synthesis in all cell types. The mild genetic Eif2b5(R132H) mutation causes a slight reduction in eIF2B enzymatic activity which leads to abnormal composition of mitochondrial electron transfer chain complexes and impaired oxidative phosphorylation. Previous work using primary fibroblasts isolated from Eif2b5(R132H/R132H) mice revealed that owing to increased mitochondrial biogenesis they exhibit normal cellular ATP level. In contrast to fibroblasts, here we show that primary astrocytes isolated from Eif2b5(R132H/R132H) mice are unable to compensate for their metabolic impairment and exhibit chronic state of low ATP level regardless of extensive adaptation efforts. Mutant astrocytes are hypersensitive to oxidative stress and to further energy stress. Moreover, they show migration deficit upon exposure to glucose starvation. The mutation in Eif2b5 prompts reactive oxygen species (ROS)-mediated inferior ability to stimulate the AMP-activated protein kinase (AMPK) axis, due to a requirement to increase the mammalian target of rapamycin complex-1 (mTORC1) signalling in order to enable oxidative glycolysis and generation of specific subclass of ROS-regulating proteins, similar to cancer cells. The data disclose the robust impact of eIF2B on metabolic and redox homeostasis programs in astrocytes and point at their hyper-sensitivity to mutated eIF2B. Thereby, it illuminates the central involvement of astrocytes in Vanishing White Matter Disease (VWMD), a genetic neurodegenerative leukodystrophy caused by homozygous hypomorphic mutations in genes encoding any of the 5 subunits of eIF2B.

O-039 Mitochondrial ATP generation in mammalian eggs and Ca2+ signaling

text In metaphase II arrested mammalian oocytes (eggs) and cleavage stage embryos the mitochondria are responsible for nearly all ATP production because glycolysis is inactivated. Luciferase assays show that ATP levels in eggs are strictly dependent upon pyruvate and fatty acid oxidation. The level of ATP in eggs appears to be maximal in conventional medium because the addition of extra mitochondrial substrates to eggs does not increase cytosolic ATP. The only clear elevation of ATP is seen at fertilization and is associated with sperm induced Ca2+ oscillations. Our recent findings suggest that the level of ATP modulates events at fertilization. At fertilization, the egg is activated by sperm derived PLCzeta which triggers a series of Ca2+ oscillations, with each Ca2+ release event causes by inositol trisphosphate (InsP3). Previous studies have shown that mouse eggs are more sensitive to PLCzeta, and generate higher frequency Ca2+ oscillations, than human eggs. Mouse eggs also generate Ca2+ oscillations and activate in response to Sr2+ that directly stimulates InsP3 receptors. In contrast, human eggs that contain the same type of InsP3 receptors do not generate Ca2+ oscillations in response to Sr2+. The difference in sensitivity of Ca2+ release between species can be explained by the fact that mouse eggs are about ten times more sensitive to InsP3 than human eggs. The reason for this difference appears to be due to ATP. The ATP level in unfertilized mouse eggs is about twice that in human eggs. Furthermore, the ability of mouse eggs to Sr2+ medium can be abolished by removing the mitochondrial substrate pyruvate, which reduces the ATP level. Adding back pyruvate to such eggs restores ATP levels promotes Sr2+ induced Ca2+ levels in mouse eggs. These data suggest that the level of ATP, possibly as ATP4-, modulates the sensitivity of the InsP3 receptor and the ability of eggs to generate Ca2+ oscillations. The level of cytosolic ATP may represent a significant ‘egg factor’ in determining the success of fertilization in humans. Enhancing mitochondrial ATP production could be useful in improving activation and embryo development after fertilization, or after artificial egg activation. References: Dumollard et al. (2009) Seminar in Cell and Developmental Biology 20, 346-353 Campbell and Swann (2006) Developmental Biology 298, 225-233 Storey et al. (2021) Molecular Human Reproduction 27, gaaa086