Permeability Transition Research Articles

Reactive Oxygen Species Damage Bovine Endometrial Epithelial Cells via the Cytochrome C-mPTP Pathway.

After parturition, bovine endometrial epithelial cells (BEECs) undergo serious inflammation and imbalance between oxidation and antioxidation, which is widely acknowledged as a primary contributor to the development of endometritis in dairy cows. Nevertheless, the mechanism of oxidative stress-mediated inflammation and damage in bovine endometrial epithelial cells remains inadequately defined, particularly the molecular pathways associated with mitochondria-dependent apoptosis. Hence, the present study was designed to explore the mechanism responsible for mitochondrial dysfunction-induced BEEC damage. In vivo, the expressions of proapoptotic protein caspase 3 and cytochrome C were increased significantly in dairy uteri with endometritis. Similarly, the levels of proapoptotic protein caspase 3, BAX, and cytochrome C were markedly increased in H2O2-treated BEECs. Our findings revealed pronounced BEEC damage in dairy cows with endometritis, accompanied by heightened expression of cyto-C and caspase-3 both in vivo and in vitro. The reduction in apoptosis-related protein of BEECs due to oxidant injury was notably mitigated following N-acetyl-L-cysteine (NAC) treatment. Furthermore, mitochondrial vacuolation was significantly alleviated, and mitochondrial membrane potential returned to normal levels after the removal of ROS. Excessive ROS may be the main cause of mitochondrial dysfunction. Mitochondrial permeability transition pore (mPTP) blockade by cyclophilin D (CypD) knockdown with CSA significantly blocked the flow of cytochrome C (cyto-C) and Ca2+ to the cytoplasm from the mitochondria. Our results indicate that elevated ROS and persistent opening of the mPTP are the main causes of oxidative damage in BEECs. Collectively our results reveal a new mechanism involving ROS-mPTP signaling in oxidative damage to BEECs, which may be a potential avenue for the clinical treatment of bovine endometritis.

Diverse mitochondrial effects, antiplasmodial and anti-inflammatory potentials of Costus afer (Ker Gawl), Nauclea latifolia (Sm) and Sphenocentrum jollyanum (Pierre) in mice infected with Plasmodium berghei

ObjectivesThis study reported the antiplasmodial, anti-inflammatory and mito-protrective effects of Costus afer (CA), Nauclea latifolia (NA) and Sphenocentrum jollyanum (SJ) methanol extracts in Plasmodium berghei-infected mice. MethodsAir-dried CA, NA and SJ were extracted with methanol. Antiplasmodial activity of these extracts were monitored using chloroquine-sensitive and resistant strains of Plasmodium berghei. Heme and hemozoin contents, interleukins and C-reactive protein as well as mitochondrial permeability transition (mPT) pore opening, lipid peroxidation (mLPO) and F0F1 ATPase activity were determined by spectrophotometry. Phytochemical constituents were determined using UPLC-QTOF-MS and NMR spectroscopy. Results and ConclusionsCA, NL and SJ decreased percentage parasitemia to 0.25 ± 0.07; 0.30 ± 0.14 and 0.25 ± 0.07% relative to control (8.60 ± 0.15%) in the chloroquine-sensitive model and to 0.40 ± 0.14; 0.30 ± 0.14 and 0.45 ± 0.07, respectively as against 10.88 ± 0.26% of the infected control in the chloroquine-resistant model. In chloroquine-resistant model, NL decreased mLPO (0.41 ± 0.04) F0F1 ATPase (0.15 ± 0.02 mmol pi/mg protein /min) while CA enhanced mPT pore opening at 100 mg/kg,and SJ (50 mg/kg) reversed parasite-induced mPT pore opening (1.66 vs 9.4 folds). The NL increased heme, decreased hemozoin, IL-6, CRP, TNF-α, while SJ dose-dependently increased IL-10. UPLC-QTOF-MS analysis showed that coumaric acid, divaricatinic acid, diocin and aferiosides A and C were present in CA, 3-caffeoylquinic acid, 18, 19-dihydroangustine, jatrorrhizine, 17-epinaucleidinal, strictosamide and quinovic acid 3-O-rhamnoside in NL and quinic acid, jatrorrhizine and mabioside B in SJ. While the three medicinal plants have varying antimalarial effects, their decoction will be better for a synergistic purpose.

Aging-induced mitochondrial dysfunction: two distinct populations of mitochondria versus a combined population.

Mitochondrial function in aged hearts is impaired, and studies of isolated mitochondria are commonly used to assess their function. The two populations of cardiac mitochondria, subsarcolemmal mitochondria (SSM) and interfibrillar mitochondria (IFM), are affected by aging. However, the yield of these mitochondria, particularly SSM, is limited in the mouse heart because of the smaller heart size. To address this issue, the authors developed a method to isolate a mixed population (MIX) of SSM and IFM mitochondria from a single mouse heart. The aim of the study was to compare the mitochondrial function between SSM, IFM, and the MIX population from young and aged mouse hearts. The MIX population had a higher yield of total protein and citrate synthase activity from both young and aged hearts compared with the individual yields of SSM or IFM. Oxidative phosphorylation (OXPHOS) decreased in aged SSM and IFM compared with young SSM and IFM, as well as in the MIX population isolated from aged hearts compared with young hearts, when using complex I or IV substrates. Furthermore, aging barely affected the sensitivity to mitochondrial permeability transition pore (MPTP) opening in SSM, whereas the sensitivity was increased in IFM isolated from aged hearts and in the MIX population from aged hearts compared with the corresponding populations isolated from young hearts. These results suggest that mitochondrial dysfunction exists in aged hearts and the isolation of a MIX population of mitochondria from the mouse heart is a potential approach to studying mitochondrial function in the mouse heart.NEW & NOTEWORTHY We developed two methods to isolate mitochondria from a single mouse heart. We compared mitochondrial function in young and aged mice using mitochondria isolated with different methods. Both methods can be successfully used to isolate cardiac mitochondria from single mouse hearts. Our results provide the flexibility to isolate mitochondria from a single mouse heart based on the purpose of the study.

Elucidating the Influence of MPT-driven necrosis-linked LncRNAs on immunotherapy outcomes, sensitivity to chemotherapy, and mechanisms of cell death in clear cell renal carcinoma.

Clear cell renal carcinoma (ccRCC) stands as the prevailing subtype among kidney cancers, making it one of the most prevalent malignancies characterized by significant mortality rates. Notably,mitochondrial permeability transition drives necrosis (MPT-Driven Necrosis) emerges as a form of cell death triggered by alterations in the intracellular microenvironment. MPT-Driven Necrosis, recognized as a distinctive type of programmed cell death. Despite the association of MPT-Driven Necrosis programmed-cell-death-related lncRNAs (MPTDNLs) with ccRCC, their precise functions within the tumor microenvironment and prognostic implications remain poorly understood. Therefore, this study aimed to develop a novel prognostic model that enhances prognostic predictions for ccRCC. Employing both univariate Cox proportional hazards and Lasso regression methodologies, this investigation distinguished genes with differential expression that are intimately linked to prognosis.Furthermore, a comprehensive prognostic risk assessment model was established using multiple Cox proportional hazards regression. Additionally, a thorough evaluation was conducted to explore the associations between the characteristics of MPTDNLs and clinicopathological features, tumor microenvironment, and chemotherapy sensitivity, thereby providing insights into their interconnectedness.The model constructed based on the signatures of MPTDNLs was verified to exhibit excellent prediction performance by Cell Culture and Transient Transfection, Transwell and other experiments. By analyzing relevant studies, we identified risk scores derived from MPTDNLs as an independent prognostic determinant for ccRCC, and subsequently we developed a Nomogram prediction model that combines clinical features and associated risk assessment. Finally, the application of experimental techniques such as qRT-PCR helped to compare the expression of MPTDNLs in healthy tissues and tumor samples, as well as their role in the proliferation and migration of renal clear cell carcinoma cells. It was found that there was a significant correlation between CDK6-AS1 and ccRCC results, and CDK6-AS1 plays a key role in the proliferation and migration of ccRCC cells. Impressive predictive results were generated using marker constructs based on these MPTDNLs. In this research, we formulated a new prognostic framework for ccRCC, integrating mitochondrial permeability transition-induced necrosis. This model holds significant potential for enhancing prognostic predictions in ccRCC patients and establishing a foundation for optimizing therapeutic strategies.

Pharmaceutical Manipulation of Mitochondrial F0F1-ATP Synthase Enables Imaging and Protection of Myocardial Ischemia/Reperfusion Injury Through Stress-induced Selective Enrichment.

To rescue ischemic myocardium from progressing to myocardial infarction, timely identification of the infarct size and reperfusion is crucial. However, fast and accurate identification, as well as the targeted protection of injured cardiomyocytes following ischemia/reperfusion (I/R)injury, remain significantly challenging. Here, a near infrared heptamethine dye IR-780 is shown that has the potential to quickly monitor the area at risk following I/R injury by selectively entering the cardiomyocytes of the at-risk heart tissues. Preconditioning with IR-780 or timely IR-780 administration before reperfusion significantly protects the heart from ischemia and oxidative stress-induced cell death, myocardial remodeling, and heart failure in both rat and pig models. Furthermore, IR-780 can directly bind to F0F1-ATP synthase of cardiomyocytes, rapidly decrease the mitochondrial membrane potential, and subsequently slow down the mitochondrial energy metabolism, which induces the mitochondria into a "quiescent state" and results in mitochondrial permeability transition pore inhibition by preventing mitochondrial calcium overload. Collectively, the findings show the feasibility of IR-780-based imaging and protection strategy for I/R injury in a preclinical context and indicate that moderate mitochondrial function depression is a mode of action that can be targeted in the development of cardioprotective reagents.

The mitochondrial ATP synthase is a negative regulator of the mitochondrial permeability transition pore.

The mitochondrial permeability transition pore (mPTP) is a channel in the inner mitochondrial membrane whose sustained opening in response to elevated mitochondrial matrix Ca2+ concentrations triggers necrotic cell death. The molecular identity of mPTP is unknown. One proposed candidate is the mitochondrial ATP synthase, whose canonical function is to generate most ATP in multicellular organisms. Here, we present mitochondrial, cellular, and in vivo evidence that, rather than serving as mPTP, the mitochondrial ATP synthase inhibits this pore. Our studies confirm previous work showing persistence of mPTP in HAP1 cell lines lacking an assembled mitochondrial ATP synthase. Unexpectedly, however, we observe that Ca2+-induced pore opening is markedly sensitized by loss of the mitochondrial ATP synthase. Further, mPTP opening in cells lacking the mitochondrial ATP synthase is desensitized by pharmacological inhibition and genetic depletion of the mitochondrial cis-trans prolyl isomerase cyclophilin D as in wild-type cells, indicating that cyclophilin D can modulate mPTP through substrates other than subunits in the assembled mitochondrial ATP synthase. Mitoplast patch clamping studies showed that mPTP channel conductance was unaffected by loss of the mitochondrial ATP synthase but still blocked by cyclophilin D inhibition. Cardiac mitochondria from mice whose heart muscle cells we engineered deficient in the mitochondrial ATP synthase also demonstrate sensitization of Ca2+-induced mPTP opening and desensitization by cyclophilin D inhibition. Further, these mice exhibit strikingly larger myocardial infarctions when challenged with ischemia/reperfusion in vivo. We conclude that the mitochondrial ATP synthase does not function as mPTP and instead negatively regulates this pore.

Neither too much nor too little: mitochondrial calcium concentration as a balance between physiological and pathological conditions.

Ca2+ ions serve as pleiotropic second messengers in the cell, regulating several cellular processes. Mitochondria play a fundamental role in Ca2+ homeostasis since mitochondrial Ca2+ (mitCa2+) is a key regulator of oxidative metabolism and cell death. MitCa2+ uptake is mediated by the mitochondrial Ca2+ uniporter complex (MCUc) localized in the inner mitochondrial membrane (IMM). MitCa2+ uptake stimulates the activity of three key enzymes of the Krebs cycle, thereby modulating ATP production and promoting oxidative metabolism. As Paracelsus stated, "Dosis sola facit venenum,"in pathological conditions, mitCa2+ overload triggers the opening of the mitochondrial permeability transition pore (mPTP), enabling the release of apoptotic factors and ultimately leading to cell death. Excessive mitCa2+ accumulation is also associated with a pathological increase of reactive oxygen species (ROS). In this article, we review the precise regulation and the effectors of mitCa2+ in physiopathological processes.

Nano-plastics and gastric health: Decoding the cytotoxic mechanisms of polystyrene nano-plastics size

Gastrointestinal diseases exert a profound impact on global health, leading to millions of healthcare interventions and a significant number of fatalities annually. This, coupled with escalating healthcare expenditures, underscores the need for identifying and addressing potential exacerbating factors. One emerging concern is the pervasive presence of microplastics and nano-plastics in the environment, largely attributed to the indiscriminate usage of disposable plastic items. These nano-plastics, having infiltrated our food chain, pose a potential threat to gastrointestinal health. To understand this better, we co-cultured human gastric fibroblasts (HGF) with polystyrene nano-plastics (PS-NPs) of diverse sizes (80, 500, 650 nm) and meticulously investigated their cellular responses over a 24-hour period. Our findings revealed PS particles were ingested by the cells, with a notable increase in ingestion as the particle size decreased. The cellular death induced by these PS particles, encompassing both apoptosis and necrosis, showcased a clear dependence on both the particle size and its concentration. Notably, the larger PS particles manifested more potent cytotoxic effects. Further analysis indicated a concerning reduction in cellular membrane potential, alongside a marked increase in ROS levels upon PS particles exposure. This suggests a significant disruption of mitochondrial function and heightened oxidative stress. The larger PS particles were especially detrimental in causing mitochondrial dysfunction. In-depth exploration into the PS particles impact on genes linked with the permeability transition pore (PTP) elucidated that these PS particles instigated an internal calcium rush. This surge led to a compromise in the mitochondrial membrane potential, which in tandem with raised ROS levels, further catalyzed DNA damage and initiated cell death pathways. In essence, this study unveils the intricate mechanisms underpinning cell death caused by PS particles in gastric epithelial cells and highlighting the implications of PS particles on gastrointestinal health. The revelations from this research bear significant potential to shape future healthcare strategies and inform pertinent environmental policies.

Activation of mitochondrial DNA-mediated cGAS-STING pathway contributes to chronic postsurgical pain by inducing type I interferons and A1 reactive astrocytes in the spinal cord

Chronic postsurgical pain (CPSP) is increasingly recognized as a public health issue. Recent studies indicated the innate immune pathway of cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS)-stimulator of interferon genes (STING) was involved in pain regulation. However, the detailed mechanisms remain unclear. Previous studies found A1 reactive astrocytes in the spinal cord contributed to CPSP. This study aimed to investigate the roles and mechanisms of the cGAS-STING pathway in regulating the generation of A1 reactive astrocytes during CPSP. First, CPSP model was established using skin/muscle incision and retraction (SMIR) in rats. We found that cGAS-STING pathway was activated accompanied with an increase in mitochondrial DNA in the cytosol in the spinal cord following SMIR. Second, a STING inhibitor C-176 was intrathecally administrated. We found that C-176 decreased the expression of type I interferons and A1 reactive astrocytes in the spinal cord, and alleviated mechanical allodynia in SMIR rats. Third, cyclosporin A as a mitochondrial permeability transition pore blocker was intrathecally administrated. We found that cyclosporin A decreased the leakage of mitochondrial DNA and inhibited the activation of cGAS-STING pathway. Compared with C-176, cyclosporin A exhibits similar analgesic effects. The expression of type I interferons and A1 reactive astrocytes in the spinal cord were also down-regulated after intervention with cyclosporin A. Moreover, simultaneous administration of cyclosporin A and C-176 did not show synergistic effects in SMIR rats. Therefore, our study demonstrated that the cGAS-STING pathway activated by the leakage of mitochondrial DNA contributed to chronic postsurgical pain by inducing type I interferons and A1 reactive astrocytes in the spinal cord.

Selenite ameliorates the ATP hydrolysis of mitochondrial F1FO-ATPase by changing the redox state of thiol groups and impairs the ADP phosphorylation

Selenite as an inorganic form of selenium can affect the redox state of mitochondria by modifying the thiol groups of cysteines. The F1FO-ATPase has been identified as a mitochondrial target of this compound. Indeed, the bifunctional mechanism of ATP turnover of F1FO-ATPase was differently modified by selenite. The activity of ATP hydrolysis was stimulated, whereas the ADP phosphorylation was inhibited. We ascertain that a possible new protein adduct identified as seleno-dithiol (-S-Se-S-) mercaptoethanol-sensitive caused the activation of F-ATPase activity and the oxidation of free –SH groups in mitochondria. Conversely, the inhibition of ATP synthesis by selenite might be irreversible. The kinetic analysis of the activation mechanism was an uncompetitive mixed type with respect to the ATP substrate. Selenite bound more selectively to the F1FO-ATPase loaded with the substrate by preferentially forming a tertiary (enzyme-ATP-selenite) complex. Otherwise, the selenite was a competitive mixed-type activator with respect to the Mg2+ cofactor. Thus, selenite more specifically bound to the free enzyme forming the complex enzyme-selenite. However, even if the selenite impaired the catalysis of F1FO-ATPase, the mitochondrial permeability transition pore phenomenon was unaffected. Therefore, the reversible energy transduction mechanism of F1FO-ATPase can be oppositely regulated by selenite.

Exogenous H2S alleviates senescence of glomerular mesangial cells through up-regulating mitophagy by activation of AMPK-ULK1-PINK1-parkin pathway in mice.

Hydrogen sulfide (H2S) is the third gas signaling molecule that has been shown to be involved in the regulating vital activities in the body, including inhibition of aging. However, it is unknown whether H2S alleviates aging in the kidney and glomerular mesangial cells (GMCs) by modulating their mitophagy. Here, results of experiments in vivo and in vitro showed that compared with control group, the renal function of mice and GMCs viability were decreased in D-gal (D-galactose) group, while the activity of SA-β-gal and p21 expression were increased, Cyclin D1 and Klotho expressions were decreased; H2S content and CSE expression were lower; ROS and MDA contents and mitochondrial permeability transition pore (mPTP) opening were risedose; ATP production and mitochondrial membrane potential (Δψm) were reduced; Apoptotic rate, the expression of Cleaved caspase-9 and -3, Cyt c, p62 and Drp1 were enhanced and the expression of Bcl-2, Mfn2, Beclin-1, LC3 II/I, PINK1 and parkin were decreased. In addition, phospho-AMPK/AMPK and phospho-ULK1/ULK1 were also decreased significantly. Compared with the D-gal group, the changes of above indexes were reversed in the D-gal + NaHS (Sodium hydrosulfide, an exogenous H2S donor) group. The reverse effects of NaHS were similar to that of AICAR (an AMPK agonist) and kinetin (a PINK1 agonist), respectively. Taken together, these results suggest that exogenous H2S increases mitophagy and inhibits apoptosis as well as oxidative stress through up-regulation of AMPK-ULK1-PINK1-parkin pathway, which delays kidney senescence in mice.

Investigating the role of mitochondrial calcium uniporter beta in Alzheimer’s disease

AbstractBackgroundWe have recently shown that mitochondrial calcium (mCa2+) overload significantly contributes to the development of Alzheimer’s disease (AD). We found that either genetic inhibition of the neuronal mitochondrial calcium uniporter (MCU, the primary route for mCa2+ influx)1 or enhancement of mitochondrial Na+/Ca2+ exchanger (NCLX, the primary route for mCa2+ efflux)2 in the AD mouse model reduces AD‐associated phenotype and mitochondrial permeability transition pore (MPTP)‐induced cell death. However, blocking either the mCa2+ influx or enhancing mCa2+ efflux can also shut down the bioenergetics. Thus, it is vital to find a regulator of mCa2+ homeostasis that will positively impact Ca2+‐dependent physiological functions.MethodsTo define the new molecular regulatory mechanism to limit mCa2+ overload in AD that will maintain bioenergetics and prevent cell death, we have examined the role of Mitochondrial Calcium Uniporter Beta (MCUB), a paralog of MCU that acts as a negative regulator of mCa2+ uptake3. This study used AD patient samples, mice, and cell lines to examine MCUB expression. A neuroblastoma cell line stably expressing the human Swedish mutant amyloid precursor protein (N2a/APPswe) with adenovirus encoding MCUB was examined for mitochondrial alterations in: Ca2+ handling, ROS generation, permeability transition pore activation, oxidative phosphorylation (OxPHOS) and cell death.Results5xFAD mice and human AD brain samples displayed significant reductions in the expression of MCUB. Studies performed in N2a/APPswe neurons discovered that mCa2 handling, redox, and bioenergetics was severely impaired in AD cells. Rescue of MCUB via adenoviral expression enhanced the clearance of pathogenic mCa2+, enhanced OxPHOS, and prevented ROS and cell death.ConclusionsOur data suggest that MCUB modulation has the therapeutic potential to preserve bioenergetics and limit cell death in AD. This project will further utilize neuronal‐specific knockouts and overexpression of MCUB to increase or decrease mCa2+ uptake, respectively, in 5xFAD mouse models and will explore whether and how MCUB‐mediated changes in uptake are able to preserve metabolism, mCa2+ dynamics, and AD‐phenotype in vivo.

Role of Oxidative Stress and Reactive Metabolites in Cytotoxicity & Mitotoxicity of Clozapine, Diclofenac and Nifedipine in CHO-K1 Cells In Vitro.

To provide in vitro data on toxicity mechanisms of clozapine, diclofenac and nifedipine. CHO-K1 cells were used as in vitro model to explore mechanisms of cytotoxicity of the test drugs. Cytotoxic mechanisms of clozapine (CLZ), diclofenac (DIC) and nifedipine (NIF) were studied in CHO-K1 cells in vitro. All three drugs induce adverse reactions in some patients with partially unknown mechanisms. Following the determination of time- and dose-dependency of cytotoxicity by the MTT test, cytoplasmic membrane integrity was explored by the LDH leakage test. Both end-points were further examined in the presence of soft and hard nucleophilic agents, glutathione (GSH) and potassium cyanide (KCN), respectively, and either individual or general cytochrome P450 (CYP) inhibitors, whether CYP-catalysed formation of electrophilic metabolites play a role in the observed cytotoxicity and membrane damage. The generation of reactive metabolites during the incubations was also explored. Formation of malondialdehyde (MDA) and oxidation of dihydrofluorescein (DCFH) were monitored whether peroxidative membrane damage and oxidative stress take place in cytotoxicity. Incubations were also conducted in the presence of chelating agents of EDTA or DTPA to explore any possible role of metals in cytotoxicity by facilitating electron transfer in redox reactions. Finally, mitochondrial membrane oxidative degradation and permeability transition pore (mPTP) induction by the drugs were tested as markers of mitochondrial damage. The presence of an individual or combined nucleophilic agents significantly diminished CLZ- and NIF-induced cytotoxicities, while the presence of both agents paradoxically increased DIC-induced cytotoxicity by a factor of three with the reason remaining unknown. The presence of GSH significantly increased DIC-induced membrane damage too. Prevention of membrane damage by the hard nucleophile KCN suggests the generation of a hard electrophile upon DIC and GSH interaction. The presence of CYP2C9 inhibitor sulfaphenazol significantly diminished DIC-induced cytotoxicity, probably by preventing the formation of 4-hydroxylated metabolite of DIC, which further converts to an electrophilic reactive intermediate. Among the chelating agents, EDTA caused a marginal decrease in CLZ-induced cytotoxicity, while DIC-induced cytotoxicity was amplified by a factor of five. Both reactive and stable metabolites of CLZ could be detected in the incubation medium of CLZ with CHO-K1 cells, which are known to have low metabolic capacity. All three drugs caused a significant increase in cytoplasmic oxidative stress by means of DCFH oxidation, which was confirmed by increased MDA from cytoplasmic as well as mitochondrial membranes. The addition of GSH paradoxically and significantly increased DIC-induced MDA formation, in parallel with the increase in membrane damage when DIC and GSH combined. Our results suggested that the soft electrophilic nitrenium ion of CLZ is not responsible for the observed in vitro toxicities, and this may originate from a relatively low amount of the metabolite due to the low metabolic capacity of CHO-K1. A hard electrophilic intermediate may contribute to cellular membrane damage incubated with DIC, while a soft electrophilic intermediate seems to exacerbate cell death by a mechanism other than membrane damage. A significant decrease in cytotoxicity of NIF by GSH and KCN suggested that both soft and hard electrophiles contribute to NIF-induced cytotoxicity. All three drugs induced peroxidative cytoplasmic membrane damage, while only DIC and NIF induced peroxidative mitochondrial membrane damage, which suggested mitochondrial processes may contribute to adverse effects of these drugs in vivo.

Mild excitotoxic effect on mitochondrial membrane potential, Ca2+ homeostasis and energy production in a mouse model of Alzheimer’s disease

AbstractBackgroundNeurons are bound to maintain a tight control over their cellular homeostasis in order to preserve their many functions, and specifically to precisely regulate ion fluxes for the generation of action potential and neurotransmission. In particular, the energetic balance has to be finely adjusted since its alteration may hamper cellular functionality with consequent cell suffering and death. Mitochondria play a central role in the homeostatic regulation within the cells due to the many vital tasks they perform in terms of energy production, participation in the regulation of Ca2+ concentration, modulation of intracellular signaling that allow the cell to respond to external solicitationMethodPrimary hippocampal neuronal cultures of 14‐17 DIV from WT and AD mouse model were challenged with μM concentration of glutamate to induce a mild excitotoxic effect; mitochondrial membrane potential, mitochondrial an cytosolic Ca2+ transients and cytosolic ATP levels were evaluated with fluorescent probes. Cells were treated also with inhibitors of the permeability transition pore to evaluate its effect on cell functionalityResultWe observed that in a model of Familial Alzheimer’s disease, hippocampal neurons are sensitive to a modest excitotoxic signal that produces a transient cytosolic and mitochondrial matrix Ca2+ with consequent partial loss of mitochondrial membrane potential and delayed recovery of mitochondrial matrix [Ca2+] which might contribute in the activation of the mitochondria permeability transition pore and consequent impairment of mitochondrial and cellular functions. This is accompanied by a reduced cytosolic ATP content that can result in an energy deficit for the cell. A brief treatment with inhibitors of the permeability transition pore is able to reduce these effects and might have protective roles against exogenous insults to the cells and help mitigate the neuronal loss characteristic of Alzheimer’s disease.ConclusionAD hippocampal neurons are more sensitive to a mild excitotoxic insult that affect mitochondrial functionality and in particular the activation of the permeability transition pore (PTP). Treatment with μM concentrations of two different inhibitors of the PTP prevent mitochondrial impairment.

Involvement of the Mitochondrial Ca2+-Independent Phospholipase iPLA2 in the Induction of Mitochondrial Permeability Transition Pore by Long-Chain Acylcarnitines

Involvement of the Mitochondrial Ca2+-Independent Phospholipase iPLA2 in the Induction of Mitochondrial Permeability Transition Pore by Long-Chain Acylcarnitines

Rational design peptide inhibitors of Cyclophilin D as a potential treatment for acute pancreatitis.

Cyclophilin D (CypD) is a mitochondrial matrix peptidyl prolidase that regulates the mitochondrial permeability transition pore. Inhibition of CypD was suggested as a therapeutic strategy for acute pancreatitis. Peptide inhibitors emerged as novel binding ligand for blocking receptor activity. In this study, we present our computational approach for designing peptide inhibitors of CypD. The 3-D structure of random peptides were built, and docked into the active center of CypD using Rosetta script integrated FlexPepDock module. The peptide displayed the lowest binding energy against CypD was further selected for virtual iterative mutation based on virtual mutagenesis and molecular docking. Finally, the top 5 peptides with the lowest binding energy was selected for validating their affinity against CypD using inhibitory assay. We showed 4 out of the selected 5 peptides were capable for blocking the activity of CypD, while WACLQ display the strongest affinity against CypD, which reached 0.28 mM. The binding mechanism between WACLQ and CypD was characterized using molecular dynamics simulation. Here, we proved our approach can be a robust method for screening peptide inhibitors.

T-2 toxin induces mitochondrial dysfunction in chondrocytes via the p53-cyclophilin D pathway

Kashin–Beck disease is an endemic joint disease characterized by deep chondrocyte necrosis, and T-2 toxin exposure has been confirmed its etiology. This study investigated mechanism of T-2 toxin inducing mitochondrial dysfunction of chondrocytes through p53-cyclophilin D (CypD) pathway. The p53 signaling pathway was significantly enriched in T-2 toxin response genes from GeneCards. We demonstrated the upregulation of the p53 protein and p53-CypD complex in rat articular cartilage and ATDC5 cells induced by T-2 toxin. Transmission electron microscopy showed the damaged mitochondrial structure of ATDC5 cells induced by T-2 toxin. Furthermore, it can lead to overopening of the mitochondrial permeability transition pore (mPTP), decreased mitochondrial membrane potential, and increased reactive oxygen species generation in ATDC5 cells. Pifithrin-α, the p53 inhibitor, alleviated the increased p53-CypD complex and mitochondrial dysfunction of chondrocytes induced by T-2 toxin, suggesting that p53 played an important role in T-2 toxin-induced mitochondrial dysfunction. Mechanistically, T-2 toxin can activate the p53 protein, which can be transferred to the mitochondrial membrane and form a complex with CypD. The increased binding of p53 and CypD mediated the excessive opening of mPTP, changed mitochondrial membrane permeability, and ultimately induced mitochondrial dysfunction and apoptosis of chondrocytes.

A glycyrrhetinic acid-iridium(III) conjugate as a theranostic NIR probe for hepatocellular carcinoma with mitochondrial-targeting ability

Hepatocellular carcinoma (HCC) is a major contributor to global mortality rates, but current treatment options have limitations. Advanced theranostics are needed to effectively integrate diagnosis and therapeutic of HCC. Glycyrrhetinic acid (GA) has abundant binding sites with glycyrrhetinic acid receptors (GA-Rs) on the surface of HCC cells and has also been reported to possess ligands with mitochondrial-targeting capability but with limited efficacy. Herein, we report a near-infrared (NIR) luminescent theranostic complex 1 through conjugating an iridium(III) complex to GA, which exhibits the desired photophysical properties and promotes mitochondrial-targeting capability. Complex 1 was selectively taken up by HepG2 liver cancer cells and was imaged within mitochondria with NIR emission. Complex 1 targeted mitochondria and opened mitochondrial permeability transition pores (MPTPs), resulting in ROS accumulation, mitochondrial damage, disruption of Bax/Bcl-2 equilibrium, and tumor cell apoptosis, resulting in significantly improved anticancer activity compared to GA. This work offers a methodology for developing multifunctional theranostic probes with amplified specificity and efficacy.

Euphorbia factor L1 inhibited transport channel and energy metabolism in human colon adenocarcinoma cell line Caco-2

Euphorbia factor L1 (EFL1) is a kind of lathyrane-type diterpenoid and is isolated from the medical herb Euphorbia lathyris L. (Euphorbiaceae); it has been reported with the toxicity that causes intestinal irritation, but the underlying mechanisms are still obscure. The objective of this study was to assess the EFL1-induced intestinal cytotoxicity in human colon adenocarcinoma Caco-2 cells. The Caco-2 cells were treated with EFL1, and the intracellular calcium ion concentration, mitochondrial membrane potential (MMP), mitochondrial permeability transition pore (mPTP), adenosine 5′-triphosphate (ATP) content, ATPase activities, TGF-β1 concentration, and transepithelial electrical resistance (TEER) were detected. The interaction between EFL1 and the tight junction proteins Occludin, Claudin-4, Tricellulin, ZO-1, JAM-1, and E-cadherin was simulated by molecular docking. The expression of proteins involved in the energy metabolism, the ion transporters and aquaporins, the tight junction, and the F-actin cytoskeleton were detected by Western blotting and cell immunofluorescence. As a result, EFL1 decreased the intracellular Ca2+, MMP, mPTP, ATP content, and ATPase activities in the Caco-2 cells. The AMPK/SIRT1/PGC-1α signaling pathway, which regulates the energy metabolism, was inhibited. The ion transporters NEH and CFTR, as well as the aquaporins in the Caco-2 cells, were decreased. The tight junction proteins were down-regulated, and the integrity of the intestinal barrier was injured; TGF-β1 was compensatively increased; so, the intestinal permeability was increased and was characterized by decreased TEER. The morphology of the F-actin cytoskeleton was destroyed. These findings indicated that EFL1 caused cytotoxicity in the human intestinal Caco-2 cells through mitochondrial damage, inhibition of the energy metabolism, and suppression of the ion and water molecule transporters, as well as the down-regulation tight junction and cytoskeleton protiens.

Arvanil induces ferroptosis of hepatocellular carcinoma by binding to MICU1.

Hepatocellular carcinoma (HCC) is a primary liver cancer with a high mortality rate that requires research and improved treatment strategies. Chemotherapy is still one of the main methods of HCC treatment, but it may lead to drug resistance and damage to normal organs. Capsaicin, a naturally occurring active ingredient in chili peppers, has demonstrated anticancer properties in a variety of malignant tumor cell lines. However, the anti-cancer mechanism of capsaicin needs to be further explored in HCC. In this study, we utilized Arvanil, a non-stimulating synthetic capsaicin analog, in place of capsaicin. We found that Arvanil induced high mitochondrial calcium flow, which contributed to a decrease in mitochondrial membrane permeability transition pore (mPTP) opening and oxidative phosphorylation levels, ultimately triggering cellular ferroptosis by live cells in real time with a high content screening (HCS) platform and confocal microscopy. It was further confirmed by vina molecular docking and point mutation experiments that Arvanil directly binds to two amino acid sites of mitochondrial calcium uptake protein 1 (MICU1), namely Ser47 and Phe128, to trigger this process, which in turn inhibits the growth of HCC cells. In addition, it was confirmed that Arvanil enhances cisplatin chemosensitivity by inducing HCC cellular ferroptosis in vivo. In conclusion, our study suggests that Arvanil induces ferroptosis in HCC cells and is a candidate drug for the treatment of HCC.