Mitochondrial Permeability Transition Pore Opening Research Articles

PGC-1α attenuates the oxidative stress-induced impaired osteogenesis and angiogenesis regulation effects of mesenchymal stem cells in the presence of diabetic serum

Oxidative stress is believed to induce dysfunction of the bone remodeling process and be associated with progressive loss of bone mass. The peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α) is a master controller during mitochondrial biogenesis and the antioxidant response. We postulated that PGC-1α could function as a cyto-protective effector in mesenchymal stem cells (MSCs) under oxidative stress conditions. In this study, diabetic serum was firstly used to treat MSCs to induce oxidative damage. The anti-oxidative protective effects of PGC-1α overexpression on MSCs, as well as MSCs’ osteogenesis and angiogenic regulation effects were investigated in vitro. Results showed that diabetic conditions induced significantly increase of intracellular oxidative damage and mitochondrial permeability transition pore (mPTP) opening activity, decrease of cellular viability, and osteogenic differentiation and pro-angiogenic regulation effects of MSCs. However, the diabetic conditions induced oxidative impair on MSCs were significantly alleviated via PGC-1α overexpression under diabetic conditions. Taken together, this study indicates the anti-oxidative treatment potential of PGC-1α regulation as a promising strategy to promote coupling pro-osteogenesis and pro-angiogenesis effects of MSCs.

Abstract 2032: Vitamin B12 augments Ca2+ dependent cytotoxicity of diethyldithiocarbamate through excessive ER-vacuolization, oxidative stress and mitochondrial depolarization in MCF-7 cells in culture

Abstract Coordinated organization of intracellular organelles into functional networks is vital for cells. Mitochondrial and endoplasmic reticulum (ER) interaction in regulation of cell Ca2+ homeostasis is crucial for cell death and survival. Function of these intracellular organelles involved in two-way regulation of the status of major cell metabolites including protein, lipid exchange and intracellular Ca2+ homeostasis. Disulfiram and its derivatives recently attracted attention as a potential anticancer treatment (Kona et al., 2011). Combined use of hydroxycobalamin (vitamin B12b) and diethyldithiocarbamate (DDTC) demonstrated synergistic effect (combination index=0.25) and enhanced the cytotoxic effect of DDTC in number of transformed cell lines in culture (Solovieva et al., 2020). In the present work using MCF-7, human breast cancer cells in culture we demonstrate that combined action of DDTC and B12b resulted in induction of UPRER manifested by dilation of ER cisterns, increased level of polyubiquitinated proteins, and increased expression of CHOP. Confocal microscopy revealed disorganization of mitochondrial network and ER and treatment of these cells with B12b/DDTC and accompanied by mitochondrial depolarization (~50% after 6 h of exposure to drugs). Treatments also induced changes in morphology of mitochondrial network, and within 6 h of exposure, mitochondria demonstrated large amplitude swelling, characteristic feature of mitochondrial permeability transition pore opening. Drug exposure induced mitochondrial oxidative stress and increase in superoxide anion level (monitored with MitoSOX) and increased content of mitochondrial Ca2+ (xRhod-1) which almost doubled following treatment with B12b/DDTC. Short-time exposure of MCF-7 cells to mixture of drugs in Ca2+-free incubation media (2.5 mM EGTA) resulted in generation of significantly smaller size of vacuoles as compared with control. Similarly, pre-loading of cells with intracellular Ca2+ chelator (BAPTA, 5 µM, 30 min, 37oC) or inhibition of IP3 receptor(s) with 2-APB, delayed the drug-induced vacuolization. Taken together, our data indicate that the mechanism of B12b/DDTC-induced cytotoxicity in human MCF-7 cancer cell lines involves mitochondrial oxidative stress, activation of MPT pore and Ca2+ overload. Support of Funding Information: Russian Science Foundation № 19-75-20145 Citation Format: Marina E. Solovieva. Vitamin B12 augments Ca2+ dependent cytotoxicity of diethyldithiocarbamate through excessive ER-vacuolization, oxidative stress and mitochondrial depolarization in MCF-7 cells in culture [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2032.

Cyclosporine A-enhanced cardioplegia preserves mitochondrial basal respiration after ischemic arrest.

Mitochondrial permeability transition pore (mPTP) opening plays a crucial role in cell death during ischemia-reperfusion injury (IRI). Cyclosporine A (CsA) inhibits mPTP opening. This study aimed to investigate the effects of CsA treatment during cardioplegia on the mitochondrial function and cardiac IRI. Landrace pigs (52.9 ± 3.7 kg) were subjected to midline sternotomy, cardiopulmonary bypass at 34°C and 90 minutes of cardiac arrest. They received either a single shot of standard 4°C cold histidine-tryptophan-α-ketoglutarate (HTK)-Bretschneider solution (n = 11) or HTK-Bretschneider plus 1.2 mg/L CsA (histidine-tryptophan-α-ketoglutarate plus cyclosporine A (HTK/CsA); n = 11). During reperfusion global left-ventricular function was assessed and myocardial biopsies were harvested at baseline, during ischemia and 45 minutes following reperfusion. High-resolution respirometry and hydrogen peroxide production were measured. Immunohistochemical stainings for apoptosis-inducing factor and hypoxia-inducible factor-1α as well as a flow cytometry-based JC-1 mitochondrial membrane potential assay were performed. Hemodynamic parameters were comparable between both groups. The cytochrome C release (HTK: 930.3 ± 804.4 pg/mg, HTK/CsA: 699.7 ± 394.0 pg/mg, p = 0.457) as well as PGC1α content (HTK: 66.7%, HTK/CsA: 33.3%, p = 0.284) was lower in the HTK/CsA group. Respiratory measurements revealed that the oxygen flux under basal respiration was higher in the HTK/CsA group (8.2 ± 1.3 pmol·O2·s-1·mg-1·ww) than in the HTK group (3.8 ± 1.4 pmol·O2·s-1·mg-1·ww, p = 0.045). There were no significant differences regarding histological surrogates of apoptosis and necrosis. Supplementing cardioplegic solutions with CsA enhances the basal mitochondrial respiration thereby exerting a cardioprotective effect and diminishing IRI-induced damage. CsA seems to preserve mitochondrial function via non-ROS related pathways.

The Alterations of Mitochondrial Function during NAFLD Progression-An Independent Effect of Mitochondrial ROS Production.

The progression of non-alcoholic fatty liver (NAFL) into non-alcoholic steatohepatitis implicates multiple mechanisms, chief of which is mitochondrial dysfunction. However, the sequence of events underlying mitochondrial failure are still poorly clarified. In this work, male C57BL/6J mice were fed with a high-fat plus high-sucrose diet for 16, 20, 22, and 24 weeks to induce NAFL. Up to the 20th week, an early mitochondrial remodeling with increased OXPHOS subunits levels and higher mitochondrial respiration occurred. Interestingly, a progressive loss of mitochondrial respiration along “Western diet” feeding was identified, accompanied by higher susceptibility to mitochondrial permeability transition pore opening. Importantly, our findings prove that mitochondrial alterations and subsequent impairment are independent of an excessive mitochondrial reactive oxygen species (ROS) generation, which was found to be progressively diminished along with disease progression. Instead, increased peroxisomal abundance and peroxisomal fatty acid oxidation-related pathway suggest that peroxisomes may contribute to hepatic ROS generation and oxidative damage, which may accelerate hepatic injury and disease progression. We show here for the first time the sequential events of mitochondrial alterations involved in non-alcoholic fatty liver disease (NAFLD) progression and demonstrate that mitochondrial ROS are not one of the first hits that cause NAFLD progression.

Insight into Crosstalk between Ferroptosis and Necroptosis: Novel Therapeutics in Ischemic Stroke.

Ferroptosis is a nonapoptotic form of cell death characterized by iron-dependent accumulation of lipid hydroperoxides to lethal levels. Necroptosis, an alternative form of programmed necrosis, is regulated by receptor-interacting protein (RIP) 1 activation and by RIP3 and mixed-lineage kinase domain-like (MLKL) phosphorylation. Ferroptosis and necroptosis both play important roles in the pathological progress in ischemic stroke, which is a complex brain disease regulated by several cell death pathways. In the past few years, increasing evidence has suggested that the crosstalk occurs between necroptosis and ferroptosis in ischemic stroke. However, the potential links between ferroptosis and necroptosis in ischemic stroke have not been elucidated yet. Hence, in this review, we overview and analyze the mechanism underlying the crosstalk between necroptosis and ferroptosis in ischemic stroke. And we find that iron overload, one mechanism of ferroptosis, leads to mitochondrial permeability transition pore (MPTP) opening, which aggravates RIP1 phosphorylation and contributes to necroptosis. In addition, heat shock protein 90 (HSP90) induces necroptosis and ferroptosis by promoting RIP1 phosphorylation and suppressing glutathione peroxidase 4 (GPX4) activation. In this work, we try to deliver a new perspective in the exploration of novel therapeutic targets for the treatment of ischemic stroke.

Resveratrol Prevents Right Ventricle Dysfunction, Calcium Mishandling, and Energetic Failure via SIRT3 Stimulation in Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) is characterized by pulmonary vessel remodeling; however, its severity and impact on survival depend on right ventricular (RV) failure. Resveratrol (RES), a polyphenol found in red wine, exhibits cardioprotective effects on RV dysfunction in PAH. However, most literature has focused on RES protective effect on lung vasculature; recent finding indicates that RES has a cardioprotective effect independent of pulmonary arterial pressure on RV dysfunction, although the underlying mechanism in RV has not been determined. Therefore, this study is aimed at evaluating sirtuin-3 (SIRT3) modulation by RES in RV using a monocrotaline- (MC-) induced PAH rat model. Myocyte function was evaluated by confocal microscopy as cell contractility, calcium signaling, and mitochondrial membrane potential (ΔΨm); cell energetics was assessed by high-resolution respirometry, and western blot and immunoprecipitation evaluated posttranslational modifications. PAH significantly affects mitochondrial function in RV; PAH is prone to mitochondrial permeability transition pore (mPTP) opening, thus decreasing the mitochondrial membrane potential. The compromised cellular energetics affects cardiomyocyte function by decreasing sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) activity and delaying myofilament unbinding, disrupting cell relaxation. RES partially protects mitochondrial integrity by deacetylating cyclophilin-D, a critical component of the mPTP, increasing SIRT3 expression and activity and preventing mPTP opening. The preserved energetic capability rescues cell relaxation by maintaining SERCA activity. Avoiding Ca2+ transient and cell contractility mismatch by preserving mitochondrial function describes, for the first time, impairment in excitation-contraction-energetics coupling in RV failure. These results highlight the importance of mitochondrial energetics and mPTP in PAH.

Ischemia Injury induces mPTP opening by reducing Sirt3

Mitochondrial permeability transition pore (mPTP) opening is critical to mitochondrial apoptosis during ischemic injury. Sirtuin 3 (Sirt3) is a mitochondrial deacetylase known to play a major role in stress resistance and cell death. Our previous studies have shown that Sirt3 activates superoxide dismutase 2 and forkhead box O3a to reduce cellular reactive oxygen species. However, it is unclear the interaction between Sirt3 and mPTP and the roles they play in ischemic stroke. We used the middle cerebral artery occlusion (MCAO) model, a mouse model of stroke, to examine Sirt3 and mPTP-related protein levels. We then applied lentivirus packaged Sirt3 overexpression in HT22 cells, a mouse hippocampal neuronal cell line, to investigate the underlying mechanism. We found Sirt3 protein level was decreased in the penumbra area in MCAO mice, along with an increase in mPTP related proteins, namely voltage-dependent anion channel 1 (VDAC1) and adenine nucleotide translocator 1 (ANT1). Sirt3 overexpression suppressed the increase in VDAC1, ANT1 and cleaved caspase 3 that were induced by the serum and glucose deprivation (SGD) condition. Our studies suggest that ischemic injury induced mPTP opening and apoptosis by reducing Sirt3. It helps to identify new therapeutic targets for ischemic stroke.

Acylated Ghrelin Attenuates l-Thyroxin-induced Cardiac Damage in Rats by Antioxidant and Anti-inflammatory Effects and Downregulating Components of the Cardiac Renin-angiotensin System.

This study investigated the protective effect of acylated ghrelin (AG) against l-thyroxin (l-Thy)-induced cardiac damage in rats and examined possible mechanisms. Male rats were divided into five intervention groups of 12 rats/group: control, control + AG, l-Thy, l-Thy + AG, and l-Thy + AG + [D-Lys3]-GHRP-6 (AG antagonist). l-Thy significantly reduced the levels of AG and des-acyl ghrelin and the AG to des-acyl ghrelin ratio. Administration of AG to l-Thy-treated rats reduced cardiac weights and levels of reactive oxygen species and preserved the function and structure of the left ventricle. In addition, AG also reduced the protein levels of cleaved caspase-3 and cytochrome c and prevented mitochondrial permeability transition pore opening. In the left ventricle of both control + AG-treated and l-Thy + AG-treated rats, AG significantly increased left ventricular levels of manganese superoxide dismutase (SOD2), total glutathione (GSH), and Bcl2. It also reduced the levels of malondialdehyde, tumor necrosis factor-α (TNF-α), interleukin-6, and Bax and the nuclear activity of nuclear factor-kappa B. Concomitantly, in both treated groups, AG reduced the mRNA and protein levels of NADPH oxidase 1, angiotensin (Ang) II type 1 receptor, and Ang-converting enzyme 2. All the beneficial effects of AG in l-Thy-treated rats were prevented by the coadministration of [D-Lys3]-GHRP-6, a selective growth hormone secretagogue receptor subtype 1a antagonist. In conclusion, AG protects against hyperthyroidism-induced cardiac hypertrophy and damage, which is mainly due to its antioxidant and anti-inflammatory potentials and requires the activation of GHS-R1a.

Metformin induces apoptosis via uterus mitochondrial permeability transition pore opening and protects against estradiol benzoate-induced uterine defect and associated pathophysiological disorder in female Wistar rats

BackgroundSome antitumor or anticancer agents have been shown to execute cell death by induction of mitochondrial permeability transition (mPT) pore opening in order to elicit their chemotherapeutic effect. Therefore, this study investigated the effect of metformin on cell death via rat uterus mPT pore and estradiol benzoate-induced uterine defect and associated pathophysiological disorder in female rat. Mitochondria were isolated using differential centrifugation. The mPT pore opening, cytochrome c release and mitochondrial ATPase activity were determined spectrophotometrically. Caspases 9 and 3 activities, MDA and estradiol levels and SOD, GSH activities, were determined using ELISA technique. Histological and histochemical assessments of the uterine section were carried out using standard methods.ResultsMetformin at concentrations 10–90 μg/mL, showed no significant effect on mPT pore opening, mATPase activity and release of cytochrome c. However, oral administration of metformin caused mPT pore opening, enhancement of mATPase activity and activation of caspases 9 and 3 significantly at 300 and 400 mg/kg. Metformin protected against estradiol benzoate (EB)-induced uterine defect and other associated pathophysiological disorder. It also improved the antioxidant defense system. The histological evaluation revealed the protective effect of metformin on the cellular architecture of the uterus while the histochemical examination showed severe hyperplasia in the uterine section of EB-treated rats, remarkably reversed by metformin co-treatment.ConclusionThis study suggests that metformin at high doses induces apoptosis via rat uterus mPT pore opening and protects against EB-induced uterine defect (hyperplasia) and associated pathophysiological disorder.

Cerebral and myocardial mitochondrial injury differ in a rat model of cardiac arrest and cardiopulmonary resuscitation

Brain mitochondria are more sensitive to global ischemia compared to heart mitochondria. Complex I in the electron transport chain (ETC) is sensitive to ischemic injury and is a major control point of the rate of ADP stimulated oxygen consumption. The purpose of this study was to explore whether changes in cerebral and myocardial mitochondria differ after cardiac arrest. Animals were randomized into 4 groups (n = 6): 1) Sham 2) VF 3) VF+CPR 4) ROSC 1hr. Ventricular Fibrillation (VF) was induced through a guide wire advanced from the right jugular vein into the ventricle and untreated for 8 min. Resuscitation was attempted with a 4J defibrillation after 8 min of cardiopulmonary resuscitation (CPR). Brain mitochondria and cardiac mitochondrial subpopulations were isolated. Calcium retention capacity was measured to assess susceptibility to mitochondrial permeability transition pore opening. ADP stimulated oxygen consumption and ETC activity assays were performed. Brain mitochondria are far more sensitive to injury during cardiac arrest and resuscitation compared to cardiac mitochondria. Complex I is highly sensitive to injury in brain mitochondria. With markedly decreased calcium retention capacity, mitochondria contribute to cerebral reperfusion injury. Therapeutic preservation of cerebral mitochondrial activity and mitochondrial function during cardiac arrest may improve post-resuscitation neurologic function.

The role of mitochondria in metabolic disease: a special emphasis on heart dysfunction.

Metabolic diseases (MetDs) embrace a series of pathologies characterized by abnormal body glucose usage. The known diseases included in this group are metabolic syndrome, prediabetes and diabetes mellitus types 1 and 2. All of them are chronic pathologies that present metabolic disturbances and are classified as multi-organ diseases. Cardiomyopathy has been extensively described in diabetic patients without overt macrovascular complications. The heart is severely damaged during the progression of the disease; in fact, diabetic cardiomyopathies are the main cause of death in MetDs. Insulin resistance, hyperglycaemia and increased free fatty acid metabolism promote cardiac damage through mitochondria. These organelles supply most of the energy that the heart needs to beat and to control essential cellular functions, including Ca2+ signalling modulation, reactive oxygen species production and apoptotic cell death regulation. Several aspects of common mitochondrial functions have been described as being altered in diabetic cardiomyopathies, including impaired energy metabolism, compromised mitochondrial dynamics, deficiencies in Ca2+ handling, increases in reactive oxygen species production, and a higher probability of mitochondrial permeability transition pore opening. Therefore, the mitochondrial role in MetD-mediated heart dysfunction has been studied extensively to identify potential therapeutic targets for improving cardiac performance. Herein we review the cardiac pathology in metabolic syndrome, prediabetes and diabetes mellitus, focusing on the role of mitochondrial dysfunctions.

Influence of Microbial Metabolites on the Nonspecific Permeability of Mitochondrial Membranes under Conditions of Acidosis and Loading with Calcium and Iron Ions.

Mitochondrial dysfunction is currently considered one of the main causes of multiple organ failure in chronic inflammation and sepsis. The participation of microbial metabolites in disorders of bioenergetic processes in mitochondria has been revealed, but their influence on the mitochondrial membrane permeability has not yet been studied. We tested the influence of various groups of microbial metabolites, including indolic and phenolic acids, trimethylamine-N-oxide (TMAO) and acetyl phosphate (AcP), on the nonspecific permeability of mitochondrial membranes under conditions of acidosis, imbalance of calcium ions and excess free iron, which are inherent in sepsis. Changes in the parameters of the calcium-induced opening of the mitochondrial permeability transition pore (MPTP) and iron-activated swelling of rat liver mitochondria were evaluated. The most active metabolites were indole-3-carboxylic acid (ICA) and benzoic acid (BA), which activated MPTP opening and swelling under all conditions. AcP showed the opposite effect on the induction of MPTP opening, increasing the threshold concentration of calcium by 1.5 times, while TMAO activated swelling only under acidification. All the redox-dependent effects of metabolites were suppressed by the lipid radical scavenger butyl-hydroxytoluene (BHT), which indicates the participation of these microbial metabolites in the activation of membrane lipid peroxidation. Thus, microbial metabolites can directly affect the nonspecific permeability of mitochondrial membranes, if conditions of acidosis, an imbalance of calcium ions and an excess of free iron are created in the pathological state.

Stigmasterol isolated from the chloroform fraction of Adenopus breviflorus Benth fruit induces mitochondrial-dependent apoptosis in rat liver.

Decoction of Adenopus breviflorus fruit is used in folkloric medicine for treating dysmenorrhea and gonorrhea. Phytochemicals from A.breviflorus may be potent in inducing mitochondrial-dependent apoptosis via the opening of the mitochondrial permeability transition (MPT) pore. Therefore, this study investigated the in vitro effects of stigmasterol isolated from the chloroform fraction of A.breviflorus (CFAB) and also the increasing concentration of CFAB on the opening of rat liver mitochondrial permeability transition (MPT) pore. Fractionation of CFAB on column chromatography yielded a needle-like crystal which structure was elucidated by standard spectroscopic techniques. The effects of stigmasterol and CFAB on MPT pore opening were assayed spectrophotometrically. Also, the effect of CFAB on mitochondrial ATPase (mATPase) activity and cytochrome c (Cyt c) release were determined. Stigmasterol isolated from CFAB induced MPT pore opening significantly (p<0.05) when compared withthe control. Similarly, CFAB significantly (p<0.05) induced MPT pore opening in rat liver mitochondria in a concentration-dependent manner in the presence and absence of the triggering agent- calcium ion. Furthermore, the increasing concentration of CFAB significantly (p<0.05) stimulated mitochondrial ATPase (mATPase) activity and Cyt c release in a concentration-dependent manner. The study showed that stigmasterol isolated from the chloroform fraction of A.breviflorus is a potent inducer of mitochondrial-dependent apoptosis. Also, the study further revealed that CFAB possesses potent bioactive compounds which can induce the mitochondrial-dependent apoptosis through the opening of the mitochondrial permeability transition pore, activation of mitochondrial ATPase (mATPase) activity and cytochrome c release.

Fractions of Ageratum conyzoides L. (Compositae) induce mitochondrial-mediated apoptosis in rats: Possible option in monosodium glutamate-induced hepatic and uterine pathological disorder

Ethnopharmacological relevanceSeveral pathological disorders have been attributed to either oxidative stress or defect in apoptotic signaling pathway. Some bioactive compounds elicit their antiproliferative properties by induction of apoptosis via mitochondrial permeability transition (mPT) pore opening. Aim of studyThe present study therefore investigated the effects of various fractions of methanol extract of Ageratum conyzoides L. (MEAC) on mitochondrial-mediated apoptosis and the possible protective potential of the most potent against monosodium glutamate (MSG)-induced hepatic damage and uterine pathological disorder. The plant is folklorically used in the treatment of cancer and gynecological disorder. Materials and methodsThe MEAC was partitioned in succession and concentrated at 40 °C to obtain chloroform(CFAC), ethylacetate(EFAC) and methanol(MFAC) fractions. Mitochondria were isolated by differential centrifugation. The opening of mPT pore, mATPase activity and hepatic DNA fragmentation were assessed spectrophotometrically. Caspases 9 and 3, SOD and GSH-Px activities and MDA level were determined using ELISA technique. Histological assessment of the liver and uterine sections and GC-MS analysis of the most potent fraction were carried out. ResultsThe investigation showed that oral administration of the fractions caused induction of mPT pore opening, enhanced mATPase activity, upregulated the activities of caspases 9 and 3 and also, caused hepatic DNA fragmentation with CFAC being the most potent. The CFAC reversed severe MSG-induced hepatic damage and uterine hyperplasia. The MSG-induced oxidative stress was normalized by CFAC. The GC-MS analysis of CFAC revealed the presence of some pharmacologically relevant phytochemicals. ConclusionThese findings therefore suggest that fractions of Ageratum conyzoides induce mitochondrial-mediated apoptosis. Moreover, CFAC, which is the most potent has a promising antioxidant and antiproliferative potential against MSG-induced hepatic and uterine pathological disorder.

Sex differences in renal mitochondrial function of young Sprague Dawley rats

Introduction Sex differences are implicated in many cardiovascular and renal pathologies; for instance, premenopausal females are typically less prone to renovascular damage than males. However, although mitochondrial bioenergetics per se is a well-known factor that can mediate the progression of the renal diseases, it is not known if there are sex-related dissimilarities in the performance of renal mitochondria prior to the development of any disease. The goal of this study was to test the differences in renal cortical and medullary mitochondrial function in healthy male versus female rats. Methods . Mitochondria were isolated from the kidneys collected from male and female Sprague Dawley (SD) rats procured from Charles River labs at 11 weeks of age. Mitochondrial membrane potential, superoxide and H2O2 levels were measured with luminescent or fluorescent dyes (TMRM, MCLA and Amplex Red) in mitochondria isolated from renal cortex and medulla. To measure the oxygen consumption rate (OCR) and mitochondrial calcium uptake, seahorse assay and spectrofluorimetry with CaGreen dye, respectively, were performed on isolated mitochondria. Lipid peroxide radical formation was detected using electron spin resonance spectroscopy (ESR) with in vivo spin trapping. The SOD activity and total antioxidant capacity were measured using commercially available kits. In order to analyze the expression levels of various mitochondria-related proteins, Western blot analysis was performed on snap-frozen isolated renal mitochondria. Results . Kidneys from SD male (SDM) and female rats (SDF) were divided into cortex (SDMC, FC) and medulla (SDMM, FM). We report higher membrane potential in SDFM compared to SDMM (p<0.001). H2O2 levels were elevated in both the SDFC and SDFM mitochondria compared to SDM (p<0.01), and GPX4 level was significantly increased the SDFM samples. Interestingly, superoxide production was increased in the medulla compared to the cortex for both SDM and SDF, while SOD2 expression was similar. Total SOD activity was increased in SDFC compared to all other groups (p<0.01). ESR showed similar lipid peroxide radical levels in all groups. Antioxidant capacity was reduced in SDFM tissues compared to other groups (p<0.05). Female mitochondria exhibited decreased OCR compared to males, and all OCR parameters were lower in medullary vs cortical mitochondria, independent of sex. Interestingly, calcium uptake was more active in the medulla vs cortex for both males and females, whereas the mitochondrial permeability transition pore (mPTP) opening was recorded earlier in females than males. Conclusions . We report differences in mitochondrial function in the cortex and medulla of young healthy male and female rats, primarily, in their ability to handle reactive oxygen species. The observed sex-related dissimilarities open new avenues of research aimed at establishing the mechanisms that may affect the predisposition of males and females to kidney disease development later in life.

The role of PGC-1α and metabolic signaling pathway in kidney injury following chronic administration with 3-MCPD as a food processing contaminant.

3-Monochloropropane-1,2-diol (3-MCPD) as a byproduct of food processing and a carcinogenic agent has attracted much attention in the last decades. Kidney is the main target organ that is sensitive to the toxicity of 3-MCPD. Due to limited evidence about possible 3-MCPD toxicity, we design an investigation to determine the role of mitochondrial biogenesis following chronic oral administration of 3-MCPD (2, 4, 8 and 32mg/kg) for 2months in male C57 mice. The present study evaluated the affects of 3-MCPD in modulating metabolic signalling which is associated with Il-18, PGC-1α, Nrf-2 and Sir3 which are the major transcription factors. Our data confirms controversial behaviors after chronic exposure with 3-MCPD. Over expression of the PGC-1α and Sir3 and IL-18 were observed after exposure with 2,4 & 8mgkg-1 day-1 of 3-MCPD. In front, PGC-1α down-regulation occurs at the highest dose (32mg/kg) resulted in kidney injury. Based on the findings, PGC-1α plays an important role in the restoration of the mitochondrial function during the recovery from chronic kidney injury. We suggest that the PGC-1α can be consider as a therapeutic target in prevention and treatment of kidney injury after chronic exposure of 3-MCPD. PRACTICAL APPLICATIONS: 3-Monochloropropane-1, 2-diol (3-MCPD) existed in several foods, can induce nephrotoxicity, progressive nephropathy and renal tubule dilation following acute and chronic exposure. It revealed that 3-MCPD toxicity is related to metabolites which can cause oxidative stress and activation of cell death signaling. It seems that cytotoxicity of 3-MCPD has disruptive effect on kidney cells due to rise in ROS production and decrease in mitochondrial membrane permeability. These effects can lead to MPT pore opening, cytochrome c release and activation of programed cell death signaling pathway. Therefore, present study was investigated the role of PGC-1a and the metabolic signaling involved in 3-MCPD-induced nephrotoxicity for the first time. Our data revealed that up-regulation of mitochondrial biogenesis following chronic exposure with 3-MCPD accelerates recovery of mitochondrial and cellular function in kidney by deacetylation of histones, overexpression of transcription factors (PGC-1α, Nrf-2, and Sir3) and maintaining cellular homeostasis.

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

Tissue accumulationand 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.

Capsaicin protects cardiomyocytes against lipopolysaccharide-induced damage via 14-3-3γ-mediated autophagy augmentation.

Background: The myocardium is susceptible to lipopolysaccharide (LPS)-induced damage in sepsis, and cardiac dysfunction is a leading cause of mortality in patients with sepsis. The changes in cardiomyocyte autophagy in sepsis and the effects and mechanism of action of capsaicin (Cap) remain unclear. Methods and Results: The potential pathway of 14-3-3γ-dependent autophagy and the effects and mechanisms of Cap were studied in LPS-induced injury to primary cultured neonatal rat cardiomyocytes. The results showed that cardiomyocyte viability decreased, lactate dehydrogenase and creatine kinase activities increased, 14-3-3γ expression was downregulated, and autophagy was inhibited after LPS challenge. Cap pretreatment augmented autophagy by upregulating 14-3-3γ expression and activating AMP-activated protein kinase (AMPK) and unc-51 like autophagy-activating kinase 1 (ULK1), suppressing mammalian target of rapamycin (mTOR), alleviating cardiac dysfunction and improving the inflammation response, whereas pAD/14-3-3γ-shRNA nullified the above effects. Cap pretreatment also decreased the levels of IL-1β, TNF-α, IL-6, and IL-10; suppressed intracellular oxidative stress; reduced the intracellular/mitochondrial reactive oxygen species (ROS); balanced GSH/GSSG; increased GSH-Px, catalase, and SOD activities; and decreased MDA contents. It also increased ATP content, activated complex Ⅰ and complex Ⅲ, stabilized the mitochondrial membrane potential, and decreased the mitochondrial permeability transition pore opening, thereby improving mitochondrial function. Conclusion: Pretreatment with Cap can regulate autophagy by upregulating 14-3-3γ expression, inhibiting oxidative stress and inflammation, maintaining mitochondrial function, and protecting cardiomyocytes against LPS-induced injury.

Polydatin Attenuates OGD/R-Induced Neuronal Injury and Spinal Cord Ischemia/Reperfusion Injury by Protecting Mitochondrial Function via Nrf2/ARE Signaling Pathway.

Spinal cord ischemia/reperfusion injury (SCII) is a devastating complication of spinal or thoracic surgical procedures and can lead to paraplegia or quadriplegia. Neuronal cell damage involving mitochondrial dysfunction plays an important role in the pathogenesis of SCII. Despite the availability of various treatment options, there are currently no mitochondria-targeting drugs that have proven effective against SCII. Polydatin (PD), a glucoside of resveratrol, is known to preserve mitochondrial function in central nervous system (CNS) diseases. The aim of the present study was to explore the neuro- and mito-protective functions of PD and its underlying mechanisms. An in vitro model of SCII was established by exposing spinal cord motor neurons (SMNs) to oxygen–glucose-deprivation/reperfusion (OGD/R), and the cells were treated with different dosages of PD for varying durations. PD improved neuronal viability and protected against OGD/R-induced apoptosis and mitochondrial injury in a dose-dependent manner. In addition, PD restored the activity of neuronal mitochondria in terms of mitochondrial membrane potential (MMP), intracellular calcium levels, mitochondrial permeability transition pore (mPTP) opening, generation of reactive oxygen species (ROS), and adenosine triphosphate (ATP) levels. Mechanistically, PD downregulated Keap1 and upregulated Nrf2, NQO-1, and HO-1 in the OGD/R-treated SMNs. Likewise, PD treatment also reversed the neuronal and mitochondrial damage induced by SCII in a mouse model. Furthermore, the protective effects of PD were partially blocked by the Nrf2 inhibitor. Taken together, PD relieves mitochondrial dysfunction-induced neuronal cell damage by activating the Nrf2/ARE pathway and is a suitable therapeutic option for SCII.

Polyamine treatment ameliorates pericarp browning in cold-stored ‘Nanguo’ pears by protecting mitochondrial structure and function

Refrigeration is the most common way to store ‘Nanguo’ pears. However, their pericarps are prone to browning during the shelf life after refrigeration. In this study, we examined the effects of polyamines (PAs) on pericarp browning and analyzed their impact on mitochondrial morphology and function. Exogenous PAs substantially alleviated the onset and development of pericarp browning in pear fruit treated with them. PA content and the expression and activity of anabolic enzymes related to PAs increased, whereas the reactive oxygen species levels decreased. Moreover, the PA treatment preserved pericarp mitochondrial structure. Exogenous PA diminished the rate of increase and amplitude of mitochondrial membrane permeability transition pore opening and mitochondrial membrane permeability. To a certain extent, the PA treatment maintained mitochondrial energy levels. These results imply that exogenous PA treatment may be a strategy for ameliorating the browning of cold stored ‘Nanguo’ pears.