Mitofusin-2 mediates cannabidiol-induced neuroprotection against cerebral ischemia in rats.

Cannabidiol (CBD) reportedly exerts protective effects against many psychiatric disorders and neurodegenerative diseases, but the mechanisms are poorly understood. In this study, we explored the molecular mechanism of CBD against cerebral ischemia. HT-22 cells or primary cortical neurons were subjected to oxygen-glucose deprivation insult followed by reoxygenation (OGD/R). In both HT-22 cells and primary cortical neurons, CBD pretreatment (0.1, 0.3, 1 µM) dose-dependently attenuated OGD/R-induced cell death and mitochondrial dysfunction, ameliorated OGD/R-induced endoplasmic reticulum (ER) stress, and increased the mitofusin-2 (MFN2) protein level in HT-22 cells and primary cortical neurons. Knockdown of MFN2 abolished the protective effects of CBD. CBD pretreatment also suppressed OGD/R-induced binding of Parkin to MFN2 and subsequent ubiquitination of MFN2. Overexpression of Parkin blocked the effects of CBD in reducing MFN2 ubiquitination and reduced cell viability, whereas overexpressing MFN2 abolished Parkin's detrimental effects. In vivo experiments were conducted on male rats subjected to middle cerebral artery occlusion (MCAO) insult, and administration of CBD (2.5, 5 mg · kg-1, i.p.) dose-dependently reduced the infarct volume and ER stress in the brains. Moreover, the level of MFN2 within the ischemic penumbra of rats was increased by CBD treatment, while the binding of Parkin to MFN2 and the ubiquitination of MFN2 was decreased. Finally, short hairpin RNA against MFN2 reversed CBD's protective effects. Together, these results demonstrate that CBD protects brain neurons against cerebral ischemia by reducing MFN2 degradation via disrupting Parkin's binding to MFN2, indicating that MFN2 is a potential target for the treatment of cerebral ischemia.

Ethylmalonic acid impairs bioenergetics by disturbing succinate and glutamate oxidation and induces mitochondrial permeability transition pore opening in rat cerebellum.

Tissue accumulation and high urinary excretion of ethylmalonic acid (EMA) are found in ethylmalonic encephalopathy (EE), an inherited disorder associated with cerebral and cerebellar atrophy whose pathogenesis is poorly established. The in vitro and in vivo effects of EMA on bioenergetics and redox homeostasis were investigated in rat cerebellum. For the in vitro studies, cerebellum preparations were exposed to EMA, whereas intracerebellar injection of EMA was used for the in vivo evaluation. EMA reduced state 3 and uncoupled respiration in vitro in succinate-, glutamate-, and malate-supported mitochondria, whereas decreased state 4 respiration was observed using glutamate and malate. Furthermore, mitochondria permeabilization and succinate supplementation diminished the decrease in state 3 with succinate. EMA also inhibited the activity of KGDH, an enzyme necessary for glutamate oxidation, in a mixed manner and augmented mitochondrial efflux of α-ketoglutarate. ATP levels were markedly reduced by EMA, reflecting a severe bioenergetic disruption. Docking simulations also indicated interactions between EMA and KGDH and a competition with glutamate and succinate for their mitochondrial transporters. In vitro findings also showed that EMA decreased mitochondrial membrane potential and Ca2+ retention capacity, and induced swelling in the presence of Ca2+ , which were prevented by cyclosporine A and ADP and ruthenium red, indicating mitochondrial permeability transition (MPT). Moreover, EMA, at high concentrations, mildly increased ROS levels and altered antioxidant defenses in vitro and in vivo. Our data indicate that EMA-induced impairment of glutamate and succinate oxidation and MPT may contribute to the pathogenesis of the cerebellum abnormalities in EE.

Enhancement of O-GlcNAcylation on Mitochondrial Proteins with 2-(4-Methoxyphenyl)ethyl-2-acetamido-2-deoxy-ß-d-pyranoside, Contributes to the Mitochondrial Network, Cellular Bioenergetics and Stress Response in Neuronal Cells under Ischemic-like Conditions.

O-GlcNAcylation is a nutrient-driven post-translational modification known as a metabolic sensor that links metabolism to cellular function. Recent evidences indicate that the activation of O-GlcNAc pathway is a potential pro-survival pathway and that acute enhancement of this response is conducive to the survival of cells and tissues. 2-(4-Methoxyphenyl)ethyl-2-acetamido-2-deoxy-ß-d-pyranoside (SalA-4g), is a salidroside analogue synthesized in our laboratory by chemical structure-modification, with a phenyl ring containing a para-methoxy group and a sugar ring consisting of N-acetylglucosamine. We have previously shown that SalA-4g elevates levels of protein O-GlcNAc and improves neuronal tolerance to ischemia. However, the specific target of SalA-4g regulating O-GlcNAcylation remains unknown. To address these questions, in this study, we have focused on mitochondrial network homeostasis mediated by O-GlcNAcylation in SalA-4g's neuroprotection in primary cortical neurons under ischemic-like conditions. O-GlcNAc-modified mitochondria induced by SalA-4g demonstrated stronger neuroprotection under oxygen glucose deprivation and reoxygenation stress, including the improvement of mitochondrial homeostasis and bioenergy, and inhibition of mitochondrial apoptosis pathway. Blocking mitochondrial protein O-GlcNAcylation with OSMI-1 disrupted mitochondrial network homeostasis and antagonized the protective effects of SalA-4g. Collectively, these data demonstrate that mitochondrial homeostasis mediated by mitochondrial protein O-GlcNAcylation is critically involved in SalA-4g neuroprotection.

Ferroptosis mediated by the interaction between Mfn2 and IREα promotes arsenic-induced nonalcoholic steatohepatitis.

Exposure to arsenic is a risk factor for nonalcoholic steatohepatitis (NASH). Ferroptosis is a form of regulated cell death defined by the accumulation of lipid peroxidation. In the current study, we observed the occurrence of ferroptosis in arsenic-induced NASH by assessing ferroptosis related hallmarks. In vitro, we found that ferrostatin-1 effectively attenuated the executing of ferroptosis and NASH. Simultaneously, the expression of ACSL4 (acyl-CoA synthetase long-chain family member 4) was upregulated in rat's liver and L-02 cells exposed to arsenic. While, suppression of ACSL4 with rosiglitazone or ACSL4 siRNA remarkably alleviated arsenic-induced NASH and ferroptosis through diminishing 5-hydroxyeicosatetraenoic acid (5-HETE) content. Additionally, Mitofusin 2 (Mfn2), a physical tether between endoplasmic reticulum and mitochondria, has rarely been explored in the ferroptosis. Using Mfn2 siRNA or inositol-requiring enzyme 1 alpha (IRE1α) inhibitor, we found NASH and ferroptosis were obviously mitigated through reducing 5-HETE content. Importantly, Co-IP assay indicated that Mfn2 could interact with IRE1α and promoted the production of 5-HETE, ultimately led to ferroptosis and NASH. Collectively, our data showed that ferroptosis is involved in arsenic-induced NASH. These data provide insightful viewpoints into the mechanism of arsenic-induced NASH.

The expression of glucose transporters and mitochondrial division and fusion proteins in rats exposed to hypoxic preconditioning to attenuate propofol neurotoxicity.

Purpose: Evidence has shown that propofol may cause widespread apoptotic neurodegeneration. Hypoxic preconditioning has been demonstrated to provide neuroprotection and brain recovery from both acute and chronic neurodegeneration in several cellular and animal models. However, the mechanism has not been well elucidated. Therefore, the present study was designed to investigate the expression of glucose transporters (GLUT1 and GLUT3) and mitochondrial division and fusion (Drp1 and Mfn2) proteins in rats exposed to hypoxic preconditioning to attenuate propofol neurotoxicity.Methods: Propofol (100 mg/kg) was given to 7-day-old Sprague-Dawley rats; in some rats, hypoxic preconditioning was administered before intraperitoneal propofol injection by subjecting rats to five cycles of 10 min of hypoxia (8% O2) and 10 min of normoxia (21% O2). Then, the rats were allowed to breathe room air for 2 h. Neuronal mitochondrial morphology was observed by transmission electron microscopy. ATP content was detected using an ATP assay kit. The expression levels of GLUT1, GLUT3, pDrp1, Drp1 and Mfn2 were detected by Western blot, and the expression levels of GLUT1 and GLUT3 were further examined by immunohistochemistry.Results: Propofol damaged mitochondria, and decreased ATP content and GLUT3 and pDrp1 protein expression. However, our results suggested that hypoxic preconditioning could attenuate propofol neurotoxicity by reducing mitochondrial damage and increasing ATP content and pDrp1, GLUT1 and GLUT3 protein expression.Conclusion: Hypoxic preconditioning reduced propofol-induced damage in the hippocampus of neonatal rats by attenuating the increase in mitochondrial division and decrease in GLUT3 expression.

Aqua-soluble DDQ reduces the levels of Drp1 and Aß and inhibits abnormal interactions between Aß and Drp1 and protects Alzheimer's disease neurons from Aß- and Drp1-induced mitochondrial and synaptic toxicities.

The purpose of our study was to develop a therapeutic target that can reduce Aß and Drp1 levels, and also can inhibit abnormal interactions between Aß and Drp1 in AD neurons. To achieve this objective, we designed various compounds and their 3-dimensional molecular structures were introduced into Aß and Drp1 complex and identified their inhibitory properties against Aß-Drp1 interaction. Among all, DDQ was selected for further investigation because of 1) its best docking score and 2) its binding capability at interacting sites of Drp1 and Aß complex. We synthesized DDQ using retro-synthesis and analyzed its structure spectrally. Using biochemical, molecular biology, immunostaining and transmission electron microscopy (TEM) methods, we studied DDQ's beneficial effects in AD neurons. We measured the levels of Aß and Drp1, Aß and Drp1 interaction, mRNA and protein levels of mitochondrial dynamics, biogenesis and synaptic genes, mitochondrial function and cell viability and mitochondrial number in DDQ-treated and untreated AD neurons. Our qRT-PCR and immunoblotting analysis revealed that reduced levels of mitochondrial fission and increased fusion, biogenesis and synaptic genes in DDQ-treated AD neurons. Our immunoblotting and immunostaining analyses revealed that Aß and Drp1 levels were reduced in DDQ-treated AD neurons. Interaction between Aß and Drp1 is reduced in DDQ-treated AD neurons. Aß42 levels were significantly reduced in DDQ-treated mutant APPSwe/Ind cells. Mitochondrial number is significantly reduced and mitochondrial length is significantly increased. Mitochondrial function and cell viability were maintained in AD neurons treated with DDQ. These observations indicate that DDQ reduces excessive mitochondrial fragmentation, enhances fusion, biogenesis and synaptic activity and reduces Aß42 levels and protects AD neurons against Aß-induced mitochondrial and synaptic toxicities.

Oxaliplatin induces muscle loss and muscle-specific molecular changes in Mice.

INTRODUCTION: Muscle wasting is a frequent, debilitating complication of cancer. The impact of colorectal cancer chemotherapeutic oxaliplatin on the development of muscle loss and associated molecular changes is of clinical importance. METHODS: C57BL/6J male mice were treated with oxaliplatin. Total body weights were measured and behavioral studies performed. Hindlimb muscle weights (gastrocnemius and soleus) were recorded in conjunction with gene and protein expression analysis. RESULTS: Oxaliplatin-treated mice displayed reduced weight gain and behavioral deficits. Mice treated over a shorter course had significantly increased STAT3 phosphorylation in gastrocnemius muscles. Mice receiving extended oxaliplatin treatment demonstrated reduced hindlimb muscle mass with upregulation of myopathy-associated genes Foxo3, MAFbx, and Bnip3. DISCUSSION: The findings suggest that oxaliplatin treatment can directly disrupt skeletal muscle homeostasis and promote muscle loss, which may be clinically relevant in the context of targeting fatigue and weakness in cancer patients. Muscle Nerve 57: 650-658, 2018.

The trypanosome alternative oxidase: a potential drug target?

New drugs against Trypanosoma brucei, the causative agent of Human African Trypanosomiasis, are urgently needed to replace the highly toxic and largely ineffective therapies currently used. The trypanosome alternative oxidase (TAO) is an essential and unique mitochondrial protein in these parasites and is absent from mammalian mitochondria, making it an attractive drug target. The structure and function of the protein are now well characterized, with several inhibitors reported in the literature, which show potential as clinical drug candidates. In this review, we provide an update on the functional activity and structural aspects of TAO. We then discuss TAO inhibitors reported to date, problems encountered with in vivo testing of these compounds, and discuss the future of TAO as a therapeutic target.

Exogenous H2S regulates endoplasmic reticulum-mitochondria cross-talk to inhibit apoptotic pathways in STZ-induced type I diabetes.

The upregulation of reactive oxygen species (ROS) is a primary cause of cardiomyocyte apoptosis in diabetes cardiomyopathy (DCM). Mitofusin-2 (Mfn-2) is a key protein that bridges the mitochondria and endoplasmic reticulum (ER). Hydrogen sulfide (H2S)-mediated cardioprotection is related to antioxidant effects. The present study demonstrated that H2S inhibited the interaction between the ER and mitochondrial apoptotic pathway. This study investigated cardiac function, ultrastructural changes in the ER and mitochondria, apoptotic rate using TUNEL, and the expression of ER stress-associated proteins and mitochondrial apoptotic proteins in cardiac tissues in STZ-induced type I diabetic rats treated with or without NaHS (donor of H2S). Mitochondria of cardiac tissues were isolated, and MPTP opening and cytochrome c (cyt C) and Mfn-2 expression were also detected. Our data showed that hyperglycemia decreased the cardiac function by ultrasound cardiogram, and the administration of exogenous H2S ameliorated these changes. We demonstrated that the expression of ER stress sensors and apoptotic rates were elevated in cardiac tissue of DCM and cultured H9C2 cells, but the expression of these proteins was reduced following exogenous H2S treatment. The expression of mitochondrial apoptotic proteins, cyt C, and mPTP opening was decreased following treatment with exogenous H2S. In our experiment, the expression and immunofluorescence of Mfn-2 were both decreased after transfection with Mfn-2-siRNA. Hyperglycemia stimulated ER interactions and mitochondrial apoptotic pathways, which were inhibited by exogenous H2S treatment through the regulation of Mfn-2 expression.

Melatonin suppresses platelet activation and function against cardiac ischemia/reperfusion injury via PPARγ/FUNDC1/mitophagy pathways.

Platelet activation is a major (patho-) physiological mechanism that underlies ischemia/reperfusion (I/R) injury. In this study, we explored the molecular signals for platelet hyperactivity and investigated the beneficial effects of melatonin on platelet reactivity in response to I/R injury. After reperfusion, peroxisome proliferator-activated receptor γ (PPARγ) was progressively downregulated in patients with acute myocardial infarction undergoing coronary artery bypass grafting (CABG) surgery and in mice with I/R injury model. Loss of PPARγ was closely associated with FUN14 domain containing 1 (FUNDC1) dephosphorylation and mitophagy activation, leading to increased mitochondrial electron transport chain complex (ETC.) activity, enhanced mitochondrial respiratory function, and elevated ATP production. The improved mitochondrial function strongly contributed to platelet aggregation, spreading, expression of P-selectin, and final formation of micro-thromboses, eventually resulting in myocardial dysfunction and microvascular structural destruction. However, melatonin powerfully suppressed platelet activation via restoration of the PPARγ content in platelets, which subsequently blocked FUNDC1-required mitophagy, mitochondrial energy production, platelet hyperactivity, and cardiac I/R injury. In contrast, genetic ablation of PPARγ in platelet abolished the beneficial effects of melatonin on mitophagy, mitochondrial ATP supply, and platelet activation. Our results lay the foundation for the molecular mechanism of platelet activation in response to I/R injury and highlight that the manipulation of the PPARγ/FUNDC1/mitophagy pathway by melatonin could be a novel strategy for cardioprotection in the setting of cardiac I/R injury.

Insights into the Response of Soybean Mitochondrial Proteins to Various Sizes of Aluminum Oxide Nanoparticles under Flooding Stress.

Rapid developments in nanotechnology have led to the increasing use of nanoparticles (NPs) in the agricultural sector. For possible interactions between NPs and crops under flooding stress to be investigated, the molecular mechanisms in soybeans affected by exposure to various sizes of Al2O3 NPs were analyzed using a proteomic technique. In plants exposed to 30-60 nm Al2O3 NPs, the length of the root including hypocotyl was increased, and proteins related to glycolysis were suppressed. Exposure to 30-60 nm Al2O3 NPs mediated the scavenging activity of cells by regulating the ascorbate/glutathione pathway. Hierarchical clustering analysis indicated that ribosomal proteins were also increased upon exposure to flooding-stressed plants with 30-60 nm Al2O3 NPs. Mitochondrion was the target organelle of Al2O3 NPs under flooding-stress conditions. Mitochondrial proteomic analysis revealed that the abundance of voltage-dependent anion channel protein was increased upon exposure to flooding-stressed soybeans with 135 nm Al2O3 NPs, indicating the permeability of the mitochondrial membrane was increased. Furthermore, isocitrate dehydrogenase was increased upon exposure of plants to 5 nm Al2O3 NPs under flooding conditions. These results suggest that Al2O3 NPs of various sizes affect mitochondrial proteins under flooding stress by regulating membrane permeability and tricarboxylic acid cycle activity.

Effects of dietary phosphate on glucose and lipid metabolism.

Recent epidemiological and animal studies have suggested that excess intake of phosphate (Pi) is a risk factor for the progression of chronic kidney disease and its cardiovascular complications. However, little is known about the impact of dietary high Pi intake on the development of metabolic disorders such as obesity and type 2 diabetes. In this study, we investigated the effects of dietary Pi on glucose and lipid metabolism in healthy rats. Male 8-wk-old Sprague-Dawley rats were divided into three groups and given experimental diets containing varying amounts of Pi, i.e., 0.2 [low Pi(LP)], 0.6 [control Pi(CP)], and 1.2% [high Pi(HP)]. After 4 wk, the HP group showed lower visceral fat accumulation compared with other groups, accompanied by a low respiratory exchange ratio (V̇CO2/V̇O2) without alteration of locomotive activity. The HP group had lower levels of plasma insulin and nonesterified fatty acids. In addition, the HP group also showed suppressed expression of hepatic lipogenic genes, including sterol regulatory element-binding protein-1c, fatty acid synthase, and acetyl-CoA carboxylase, whereas there was no difference in hepatic fat oxidation among the groups. On the other hand, uncoupling protein (UCP) 1 and peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) expression were significantly increased in the brown adipose tissue (BAT) of the HP group. Our data demonstrated that a high-Pi diet can negatively regulate lipid synthesis in the liver and increase mRNA expression related to lipid oxidation and UCP1 in BAT, thereby preventing visceral fat accumulation. Thus, dietary Pi is a novel metabolic regulator.

Protein coingestion with alcohol following strenuous exercise attenuates alcohol-induced intramyocellular apoptosis and inhibition of autophagy.

Alcohol ingestion decreases postexercise rates of muscle protein synthesis, but the mechanism(s) (e.g., increased protein breakdown) underlying this observation is unknown. Autophagy is an intracellular "recycling" system required for homeostatic substrate and organelle turnover; its dysregulation may provoke apoptosis and lead to muscle atrophy. We investigated the acute effects of alcohol ingestion on autophagic cell signaling responses to a bout of concurrent (combined resistance- and endurance-based) exercise. In a randomized crossover design, eight physically active males completed three experimental trials of concurrent exercise with either postexercise ingestion of alcohol and carbohydrate (12 ± 2 standard drinks; ALC-CHO), energy-matched alcohol and protein (ALC-PRO), or protein (PRO) only. Muscle biopsies were taken at rest and 2 and 8 h postexercise. Select autophagy-related gene (Atg) proteins decreased compared with rest with ALC-CHO (P < 0.05) but not ALC-PRO. There were parallel increases (P < 0.05) in p62 and PINK1 commensurate with a reduction in BNIP3 content, indicating a diminished capacity for mitochondria-specific autophagy (mitophagy) when alcohol and carbohydrate were coingested. DNA fragmentation increased in both alcohol conditions (P < 0.05); however, nuclear AIF accumulation preceded this apoptotic response with ALC-CHO only (P < 0.05). In contrast, increases in the nuclear content of p53, TFEB, and PGC-1α in ALC-PRO were accompanied by markers of mitochondrial biogenesis at the transcriptional (Tfam, SCO2, and NRF-1) and translational (COX-IV, ATPAF1, and VDAC1) level (P < 0.05). We conclude that alcohol ingestion following exercise triggers apoptosis, whereas the anabolic properties of protein coingestion may stimulate mitochondrial biogenesis to protect cellular homeostasis.

Activation of IFN/STAT1 signalling predicts response to chemotherapy in oestrogen receptor-negative breast cancer.

BACKGROUND: Oestrogen receptor-negative (ER-) breast cancer is intrinsically sensitive to chemotherapy. However, tumour response is often incomplete, and relapse occurs with high frequency. The aim of this work was to analyse the molecular characteristics of residual tumours and early response to chemotherapy in patient-derived xenografts (PDXs) of breast cancer. METHODS: Gene and protein expression profiles were analysed in a panel of ER- breast cancer PDXs before and after chemotherapy treatment. Tumour and stromal interferon-gamma expression was measured in xenografts lysates by human and mouse cytokine arrays, respectively. RESULTS: The analysis of residual tumour cells in chemo-responder PDX revealed a strong overexpression of IFN-inducible genes, induced early after AC treatment and associated with increased STAT1 phosphorylation, DNA-damage and apoptosis. No increase in IFN-inducible gene expression was observed in chemo-resistant PDXs upon chemotherapy. Overexpression of IFN-related genes was associated with human IFN-γ secretion by tumour cells. CONCLUSIONS: Treatment-induced activation of the IFN/STAT1 pathway in tumour cells is associated with chemotherapy response in ER- breast cancer. Further validations in prospective clinical trials will aim to evaluate the usefulness of this signature to assist therapeutic strategies in the clinical setting.

Valproate Attenuates Nitroglycerin-Induced Trigeminovascular Activation by Preserving Mitochondrial Function in a Rat Model of Migraine.

BACKGROUND Migraine is a chronic disease that interferes with life quality and work productivity. Valproate shows protective effects against migraine, yet the underlying mechanisms are unclear. This study aimed to evaluate the potential effect of valproate on migraine using a rat model of nitroglycerin-induced trigeminovascular activation, as well as to explore the underlying mechanism. MATERIAL AND METHODS Intraperitoneal injection of nitroglycerin was conducted to induce trigeminovascular activation in rats. To explore the protective effect of valproate, a low dose (100 mg/kg) or a high dose (200 mg/kg) of valproate was intraperitoneally injected into rats, and then the levels of 5-hydroxytryptamine and nitric oxide in the peripheral blood were examined. The mtDNA copy number and the protein levels of peroxisome proliferator-activated receptor-γ coactivator 1α, mitochondrial transcription factor A, and peroxisome proliferator-activated receptor-γ in the spinal trigeminal nucleus were detected to evaluate the biogenesis of mitochondria. The mitochondrial energy metabolism was determined by the mitochondrial membrane potential and the levels of adenosine triphosphate, cytochrome C oxidase, and reactive oxygen species. RESULTS Valproate attenuated nitroglycerin-induced trigeminovascular activation in rats, with reduced scratching behavior and restored 5-hydroxytryptamine and nitric oxide levels. Moreover, the mitochondrial energy metabolism and the biogenesis of mitochondria were preserved by valproate in nitroglycerin-treated rats. CONCLUSIONS The protective effect of valproate against migraine may be achieved through the modulation of mitochondrial biogenesis and function. Our study provides evidence for the potential use of valproate in the treatment of migraine.

Chemical Conditioning as an Approach to Ischemic Stroke Tolerance: Mitochondria as the Target.

It is well established that the brain can be prepared to resist or tolerate ischemic stroke injury, and mitochondrion is a major target for this tolerance. The preparation of ischemic stroke tolerance can be achieved by three major approaches: ischemic conditioning, hypoxic conditioning and chemical conditioning. In each conditioning approach, there are often two strategies that can be used to achieve the conditioning effects, namely preconditioning (Pre-C) and postconditioning (Post-C). In this review, we focus on chemical conditioning of mitochondrial proteins as targets for neuroprotection against ischemic stroke injury. Mitochondrial targets covered include complexes I, II, IV, the ATP-sensitive potassium channel (mitoKATP), adenine dinucleotide translocase (ANT) and the mitochondrial permeability transition pore (mPTP). While numerous mitochondrial proteins have not been evaluated in the context of chemical conditioning and ischemic stroke tolerance, the paradigms and approaches reviewed in this article should provide general guidelines on testing those mitochondrial components that have not been investigated. A deep understanding of mitochondria as the target of chemical conditioning for ischemic stroke tolerance should provide valuable insights into strategies for fighting ischemic stroke, a leading cause of death in the world.

UCP2-related mitochondrial pathway participates in oroxylin A-induced apoptosis in human colon cancer cells.

Oroxylin A is a flavonoid extracted from the root of Scutellaria baicalensis Georgi. Our previous research demonstrated that oroxylin A have various anti-tumor effects including apoptosis, cell cycle arrest, drug-resistant reversion, and others. This paper explores the mechanism how oroxylin A induce apoptosis by regulating uncoupling protein 2 (UCP2) in human colon cancer cells. We found that the inhibition of UCP2 by UCP2 siRNA significantly increased the sensitivity of cells to drugs, reactive oxygen species (ROS) generation and the opening of mitochondrial permeability transition pore (MPTP) of CaCo-2 cells. We also found that UCP2 inhibition could lead to ROS-mediated MPTP activation. Furthermore, we demonstrated that oroxylin A triggered MPTP-dependent pro-apoptotic protein release from mitochondria to matrix and then induced apoptotic cascade by inhibiting UCP2. Intriguingly, the inhibition of UCP2 by oroxylin A was able to block Bcl-2 translocation to the mitochondria, keeping MPTP at open-state. In conclusion, we have demonstrated that UCP2 plays a key role in mitochondrial apoptotic pathway; UCP2s inhibition by oroxylin A triggers the MPTP opening, and promotes the apoptosis in CaCo-2 cells.

Increased activity of mitochondrial uncoupling protein 2 improves stress resistance in cultured endothelial cells exposed in vitro to high glucose levels.

The endothelium is relatively independent of the mitochondrial energy supply, but mitochondria-derived ROS may play an important role in the development of many cardiovascular diseases. Energy-dissipating uncoupling proteins (UCPs) mediate free fatty acid-activated, purine nucleotide-inhibited proton conductance (uncoupling) in the inner mitochondrial membrane. We have described a functional characteristic and an antioxidative role for UCP2 in endothelial cells and isolated mitochondria and how this function is altered by long-term growth in high concentrations of glucose. Human umbilical vein endothelial cells (EA.hy926 line) were grown in media with either high (25 mM) or normal (5.5 mM) glucose concentrations. Under nonphosphorylating and phosphorylating conditions, UCP activity was significantly higher in mitochondria isolated from high glucose-treated cells. More pronounced control of the respiratory rate, membrane potential, and ROS by UCP2 was observed in these mitochondria. A greater UCP2-mediated decrease in ROS generation indicates an improved antioxidative role for UCP2 under high glucose conditions. Mitochondrial and nonmitochondrial ROS generations were significantly higher in high glucose-treated cells independent of UCP2 expression. UCP2 gene silencing led to elevated mitochondrial ROS formation and ICAM1 expression, especially in high glucose-cultured cells. UCP2 influenced endothelial cell viability and resistance to oxidative stress. Endothelial cells exposed to high glucose concentrations were significantly more resistant to peroxide. In these cells, the increased activity of UCP2 led to improved stress resistance and protection against acute oxidative stress. Our results indicate that endothelial UCP2 may function as a sensor and negative regulator of mitochondrial ROS production in response to hyperglycemia.

Ghrelin protects rats against traumatic brain injury and hemorrhagic shock through upregulation of UCP2.

OBJECTIVE: To determine the mechanism responsible for ghrelin's neuroprotective effects after traumatic brain injury (TBI) and hemorrhagic shock. BACKGROUND: Ghrelin, a gastrointestinal hormone, has been demonstrated to possess multiple functions, including upregulation of uncoupling protein 2 (UCP2) and stimulation of the vagus nerve. Recent evidence has indicated that ghrelin is neuroprotective. We, therefore, hypothesized that ghrelin protects rats against TBI and hemorrhagic shock through upregulation of UCP2, involving stimulation of the vagus nerve. METHODS: Brain injury was induced by dropping a 450 g of weight from 1.5 m onto a steel helmet attached to the skull of male adult rats. Immediately after TBI, a midline laparotomy was performed, and both lumbar veins were isolated and severed at the junction with the vena cava. The abdomen was kept open for 20 minutes. At 45 minutes after TBI and uncontrolled hemorrhage (UH), ghrelin (4, 8, or 16 nmol/rat) or 1 mL of normal saline (vehicle) was intravenously administered. The Neurological Severity Scale (NSS), morphological alterations and ß-amyloid precursor protein expression in the brain, systemic organ injury markers (ie, alanine aminotransferase, aspartate aminotransferase, and lactate), and UCP2 expression in the cortex were measured. To determine whether the protective effect of ghrelin is mediated through upregulation of UCP2, genipin, a specific UCP2 antagonist, was administered intravenously before the injection of ghrelin in animals with TBI and UH. The role of the vagus nerve was assessed by performing vagotomy immediately before ghrelin administration. RESULTS: Ghrelin attenuated brain injury and facilitated functional recovery after TBI and UH. Ghrelin increased UCP2 expression in the cortex, and administration of genipin abolished ghrelin's protection after TBI and UH. Furthermore, vagotomy prevented the beneficial effects of ghrelin and eliminated ghrelin-induced UCP2 upregulation after TBI and UH. CONCLUSIONS: The protective effects of ghrelin after TBI and UH seem to be related to upregulation of UCP2 expression in the brain and requiring the intact vagus nerve.

Imatinib inhibits the expression of SCO2 and FRATAXIN genes that encode mitochondrial proteins in human Bcr-Abl⁺ leukemia cells.

Imatinib mesylate (IM/Gleevec®), a selective inhibitor of chimeric Bcr-Abl tyrosine kinase, was developed as a first line drug to treat CML and ALL Ph(+) patients. Earlier studies have shown that hemin counteracts the IM-induced cell killing in human K-562 CML cells. In this study, we investigated whether IM disrupts the heme-dependent Cytochrome c Oxidase (COX) Biosynthesis and Assembly Pathway (HDCBAP) in Bcr-Abl(+) and Bcr-Abl(-) cells by affecting the expression of key-genes. Cells were exposed to IM and evaluated at time intervals for cell growth, cell death, expression of various genes by RT-PCR analysis as well as Sco2 mature protein levels by western blot analysis and COX enzymatic activity. IM at 1 µM induced extensive cell growth inhibition and cell death as well as marked suppression of the expression of SCO2 and FRATAXIN (FXN) genes in human K-562 and KU-812 Bcr-Abl(+) CML cells. IM also reduced the protein level of mature Sco2 mitochondrial protein as well as COX activity in these cell lines. However, treatment of human MOLT-4 Bcr-Abl(-) cells with 1µM and even with higher concentrations (4×10(-5)M) of IM neither reduced the expression of SCO2 and FXN genes nor suppressed the protein level of mature Sco2 protein and COX activity. Our findings indicate that SCO2 and FXN genes, involved in HDCBAP, are repressed by IM in human Bcr-Abl(+) CML cells and may represent novel target sites in leukemia therapy.