Membrane Permeability Transition Pore Research Articles

EFHD1 promotes osteosarcoma proliferation and drug resistance by inhibiting the opening of the mitochondrial membrane permeability transition pore (mPTP) by binding to ANT3

Chemoresistance is the main obstacle in the clinical treatment of osteosarcoma (OS). In this study, we investigated the role of EF-hand domain-containing protein 1 (EFHD1) in OS chemotherapy resistance. We found that the expression of EFHD1 was highly correlated with the clinical outcome after chemotherapy. We overexpressed EFHD1 in 143B cells and found that it increased their resistance to cell death after drug treatment. Conversely, knockdown of EFHD1 in 143BR cells (a cisplatin-less-sensitive OS cell line derived from 143B cells) increased their sensitivity to treatment. Mechanistically, EFHD1 bound to adenine nucleotide translocase-3 (ANT3) and inhibited its conformational change, thereby inhibiting the opening of the mitochondrial membrane permeability transition pore (mPTP). This effect could maintain mitochondrial function, thereby favoring OS cell survival. The ANT3 conformational inhibitor carboxyatractyloside (CATR), which can promote mPTP opening, enhanced the chemosensitivity of EFHD1-overexpressing cells when combined with cisplatin. The ANT3 conformational inhibitor bongkrekic acid (BKA), which can inhibit mPTP opening, restored the resistance of EFHD1 knockdown cells. In conclusion, our results suggest that EFHD1-ANT3-mPTP might be a promising target for OS therapy in the future.

Effects of Concomitant Use of N-acetylcysteine and Cyclosporine A on Acetaminophen-induced Acute Kidney Injury in Mice

Background: Acetaminophen (APAP), a commonly used analgesic, causes acute kidney injury (AKI) in overdose although it is rare. Mitochondrial dysfunction plays a major role in the pathophysiology of renal damage, although the exact molecular mechanism is unknown. This study aimed to evaluate the potential therapeutic effect of cyclosporin A (CsA), a mitochondrial membrane permeability transition pore (MPTP) inhibitor, with N-acetylcysteine (NAC) in APAP-induced AKI. Methods: Male BALB/c mice were divided into control, APAP, APAP+NAC, APAP+CsA and APAP+NAC+CsA groups (n=6). A single dose of APAP (400 mg/kg) was administered intraperitoneally. All other treatments (1200 mg/kg NAC, 50 mg/kg CsA) were performed intraperitoneally 3 h after APAP administration. All animals were decapitated and blood samples and kidney tissue samples were collected for evaluation. Serum creatinine (Cr) and blood urea nitrogen (BUN) levels were measured. The kidney tissue 8-hydroxy-deoxyguanosine (8-OHdG), cytochrome c (Cytc) and 3-nitrotyrosine (3-NT) levels and cytochrome c (Cytc) expressions were determined. Result: Increased Cr and BUN levels, histopathological examinations and expressions of 8-OHdG, 3-NT and Cytc were detected in the APAP group. Combined NAC+CsA treatment sufficiently reversed oxidative stress, serum Cr and BUN levels and histopathological alterations induced by APAP. Moreover, cytc levels and renal tubular injury were remarkably reduced by combined drug treatment compared to the APAP+NAC group. These data suggest that the therapeutic effect of combined NAC+CsA treatment in mice with APAP-induced nephrotoxicity can be related to the combination of the antioxidant effect of NAC and the mitochondrial MPTP inhibitor effect of CsA.

Research progress on the role of mitochondria in the process of hepatic ischemia-reperfusion injury.

During liver ischemia-reperfusion injury, existing mechanisms involved oxidative stress, calcium overload, and the activation of inflammatory responses involve mitochondrial injury. Mitochondrial autophagy, a process that maintains the normal physiological activity of mitochondria, promotes cellular metabolism, improves cellular function, and facilitates organelle renewal. Mitochondrial autophagy is involved in oxidative stress and apoptosis, of which the PINK1-Parkin pathway is a major regulatory pathway, and the deletion of PINK1 and Parkin increases mitochondrial damage, reactive oxygen species production, and inflammatory response, playing an important role in mitochondrial quality regulation. In addition, proper mitochondrial permeability translational cycle regulation can help maintain mitochondrial stability and mitigate hepatocyte death during ischemia-reperfusion injury. This mechanism is also closely related to oxidative stress, calcium overload, and the aforementioned autophagy pathway, all of which leads to the augmentation of the mitochondrial membrane permeability transition pore opening and cause apoptosis. Moreover, the release of mitochondrial DNA (mtDNA) due to oxidative stress further aggravates mitochondrial function impairment. Mitochondrial fission and fusion are non-negligible processes required to maintain the dynamic renewal of mitochondria and are essential to the dynamic stability of these organelles. The Bcl-2 protein family also plays an important regulatory role in the mitochondrial apoptosis signaling pathway. A series of complex mechanisms work together to cause hepatic ischemia-reperfusion injury (HIRI). This article reviews the role of mitochondria in HIRI, hoping to provide new therapeutic clues for alleviating HIRI in clinical practice.

New cyclophilin D inhibitor rescues mitochondrial and cognitive function in Alzheimer's disease.

Mitochondrial dysfunction is an early pathological feature of Alzheimer disease and plays a crucial role in the development and progression of Alzheimer's disease. Strategies to rescue mitochondrial function and cognition remain to be explored. Cyclophilin D (CypD), the peptidylprolyl isomerase F (PPIase), is a key component in opening the mitochondrial membrane permeability transition pore, leading to mitochondrial dysfunction and cell death. Blocking membrane permeability transition pore opening by inhibiting CypD activity is a promising therapeutic approach for Alzheimer's disease. However, there is currently no effective CypD inhibitor for Alzheimer's disease, with previous candidates demonstrating high toxicity, poor ability to cross the blood-brain barrier, compromised biocompatibility and low selectivity. Here, we report a new class of non-toxic and biocompatible CypD inhibitor, ebselen, using a conventional PPIase assay to screen a library of ∼2000 FDA-approved drugs with crystallographic analysis of the CypD-ebselen crystal structure (PDB code: 8EJX). More importantly, we assessed the effects of genetic and pharmacological blockade of CypD on Alzheimer's disease mitochondrial and glycolytic bioenergetics in Alzheimer's disease-derived mitochondrial cybrid cells, an ex vivo human sporadic Alzheimer's disease mitochondrial model, and on synaptic function, inflammatory response and learning and memory in Alzheimer's disease mouse models. Inhibition of CypD by ebselen protects against sporadic Alzheimer's disease- and amyloid-β-induced mitochondrial and glycolytic perturbation, synaptic and cognitive dysfunction, together with suppressing neuroinflammation in the brain of Alzheimer's disease mouse models, which is linked to CypD-related membrane permeability transition pore formation. Thus, CypD inhibitors have the potential to slow the progression of neurodegenerative diseases, including Alzheimer's disease, by boosting mitochondrial bioenergetics and improving synaptic and cognitive function.

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.

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.

Jatonik polyherbal mixture induced rat liver MMPT pore opening in normal Wistar rat: In vitro and in vivo studies

ObjectiveTo assess acute toxicity, the in vitro and in vivo effects of methanol and ethyl acetate extracts (JME and JEE) of Jatonik polyherbal mixture on some mitochondria-related parameters and their effect on the activity of some liver enzymes. MethodsAcute toxicity of JME and JEE was determined using Lorke’s method. In vitro and in vivo opening of the mitochondrial membrane permeability transition pore (MMPT pore) was spectrophotometrically assayed. Production of malondialdehyde (MDA) as an index of lipid peroxidation and the activity of mitochondrial ATPase was evaluated in vitro and in vivo and the effect of JME and JEE on the activity of liver enzymes such as alkaline phosphatase (ALP), aspartate and alanine aminotransferase (AST and ALT) and gamma-glutamyl transferase (GGT) was also investigated. ResultsJME had an LD50 of 3 808 mg/kg b.w whereas JEE had an LD50 greater than 5 000 mg/kg b.w. of rats. After the rats have been fed with both extracts, a photomicrograph of a piece of liver tissue showed no apparent symptoms of toxicity. From the in vitro and in vivo studies, both extracts prompted intact mitochondria to open their MMPT pores. When compared to the control, lipid peroxide product release and ATPase activity were significantly increased (P < 0.05) in vitro and in vivo. The activities of AST, ALT, and GGT were all reduced at 50 mg/kg when treated with JME, but the activity of AST was considerably enhanced when treated with JEE (P < 0.05). The results revealed that both JME and JEE of the Jatonik polyherbal mixture had low toxicity, profound MMPTpore induction, and enhanced ATPase activity, but an increased MDA production. ConclusionJatonik extracts may be a promising target for drug development in diseases where there is dysregulation of apoptosis, however, further studies are needed to better clarify the molecular mechanism involved in these phenomena.

In silico and In vivo Studies of Calotropis procera leaf and root extracts on Mitochondrial-Related parameters in Wistar Rats

Drugs targeting Mitochondrial Membrane Permeability Transition (MMPT) pore opening are of great interest for conditions arising from apoptosis dysregulation. This study investigate MMPT pore inducing effect of Calotropis procera leaves and root extracts on mitochondrial-related parameters in the liver of healthy male Wistar rats. Extraction was done by standard methods, using Ethylacetate and Butanol solvent. Fifty–two rats weighing between 130 g - 150 g were divided into thirteen groups (n=4). Group 1 received distilled water, other groups were administered ethylacetate leaf extract (ELE), butanol leaf extract (BLE), ethylacetate root extract (ERE) and butanol root extracts (BRE). Liver Mitochondrial pore opening, mitochondrial ATPase activity and lipid peroxidation were assessed spectrophotometrically, while the interaction between human Cyclophilin D (a pore activating protein with PDB ID: 2BIT) and identified phytochemicals of Calotropis procera leaves and roots extracts were studied In-silico. Varying concentrations of the extracts induced MMPT pore opening by 6.6, 7.4 and 5.9 folds for BLE and 10.2, 7.3 and 6.4 folds for ELE at 40, 50 and 60 mg/100g bw respectively when compared with control group. Root extracts showed significant MMPT pore opening with a fold increase of 12.2, 10.1 and 20.4 for BRE and 11.37, 11.84, for ERE at at 40 , 50 and 60 mg/100g bw respectively. BLE, BRE, ELE and ERE showed increase in ATPase activities with respect to the control group. Malondialdehyde (MDA) levels as an indication of lipid peroxidation increased significantly when compared with control. Molecular docking and simulation showed the existence of stable interactions between human Cyclophilin D with phytochemicals of Calotropis procera with 2‶-Oxovoruscharin having highest binding affinity of -7.9 kcal/mol. C. procera has potential for therapeutic use for the treatment of disorders related to derangement of

Effect of Potato Glycoside Alkaloids on Energy Metabolism of Fusarium solani.

Fusarium solani is one of the primary pathogens causing root rot of wolfberry. The aims of this study were to investigate the inhibitory effect of potato glycoside alkaloids (PGA) on F. solani and its energy metabolism. In this study, the effects of PGA treatment on the growth and development of F. solani were investigated and the changes in the glycolytic pathway (EMP), ATPase activity, mitochondrial complex activity, mitochondrial structure, and energy charge level were analyzed to elucidate the possible antifungal mechanism of PGA on F. solani. The results showed that PGA treatment inhibited the colony growth, biomass, and spore germination of F. solani. PGA treatment reduced the glucose content and Hexokinase (HK) activity of F. solani, but increased the activity of Fructose-6-Phosphate Kinase (PFK) and Pyruvate Kinase (PK) and promoted the accumulation of pyruvic acid. In addition, PGA treatment inhibited the activities of H+-ATPase, Ca2+-ATPase, and mitochondrial complex IV, increased the mitochondrial inner membrane Ca2+ content and mitochondrial membrane permeability transition pore, and decreased the contents of ATP, ADP, and AMP as well as the energy charge. These results indicate that PGA treatment inhibits the growth and development of F. solani, activates the glycolysis pathway, inhibits ATPase activity and mitochondrial complex activity, and destroys the structure and function of mitochondrial membrane, resulting in a lower energy charge level.

Maresin1 alleviates liver ischemia/reperfusion injury by reducing liver macrophage pyroptosis

BackgroundCell pyroptosis has a strong proinflammatory effect, but it is unclear whether pyroptosis of liver macrophages exacerbates liver tissue damage during liver ischemia‒reperfusion (I/R) injury. Maresin1 (MaR1) has a strong anti-inflammatory effect, and whether it can suppress liver macrophage pyroptosis needs further study.MethodsThis study aimed to investigate whether MaR1 can alleviate liver I/R injury by inhibiting macrophage pyroptosis. The effects of MaR1 on cell pyroptosis and mitochondrial damage were studied by dividing cells into control, hypoxia/reoxygenation, and hypoxia/reoxygenation + MaR1 groups. Knocking out RORa was used to study the mechanism by which MaR1 exert its protective effects. Transcriptome analysis, qRT‒PCR and Western blotting were used to analyze gene expression. Untargeted metabolomics techniques were used to analyze metabolite profiles in mice. Flow cytometry was used to assess cell death and mitochondrial damage.ResultsWe first found that MaR1 significantly reduced liver I/R injury. We observed that MaR1 decreased liver I/R injury by inhibiting liver macrophage pyroptosis. Then, we discovered that MaR1 promotes mitochondrial oxidative phosphorylation, increases the synthesis of ATP, reduces the generation of ROS, decreases the impairment of mitochondrial membrane potential and inhibits the opening of mitochondrial membrane permeability transition pores. MaR1 inhibits liver macrophage pyroptosis by protecting mitochondria. Finally, we found that MaR1 exerts mitochondrial protective effects through activation of its nuclear receptor RORa and the PI3K/AKT signaling pathway.ConclusionsDuring liver I/R injury, MaR1 can reduce liver macrophage pyroptosis by reducing mitochondrial damage, thereby reducing liver damage.

Modulation of mitochondrial membrane permeability transition pore by Annonaceous acetogenins (ACGs)-Loaded Transferrin Conjugated Nanoparticles

Making the optimal cancer treatment choice can be challenging and more challenging is harnessing the therapeutic potential of bioactive molecules isolated from plants. The mitochondrion is the main organelle of oxidative stress in cells. Increased permeability of the inner mitochondrial membrane is a key phenomenon in cell death. The protein transferrin (Tf) was conjugated to the NPs with the role to actively targeting them to the cancerous cells. Peg-GNPs, Tf-GNPs, Peg-G-ACGs and Tf-Peg-G-ACGs were successfully synthesized by probe sonication method and yielded NPs with size about 100 nm, with polydispersity index around 0.20 and a negative zeta potential of about − 30 mV. Mitochondria, isolated from rat liver were exposed to different concentrations of Tf-peg-G-ACGs. Their ability to induce mitochondrial permeability transition (mPT) pore opening were assessed spectrophotometrically. In the absence of CaCl2, Tf-peg-G-ACGs caused a concentration-dependent induction of mPT pore opening by 13, 11.3, 8.7, and 2.7 folds, at concentrations 10, 20,50 and 100 μg/mL, respectively, when compared with the control with no attendant inhibitory activity which was intensified in the presence of Calcium to 22, 19.7, 15 and 8.3 folds at concentrations 10, 20,50 and 100 μg/mL, respectively. Tf-peg-G-ACGs showed improved ability to induced mitochondrial membrane pore opening which can be explored for the treatment of cancer.

Cyclophilin D as a potential therapeutic target of liver ischemia/reperfusion injury by mediating crosstalk between apoptosis and autophagy.

Liver ischemia/reperfusion (I/R) injury is a complex and multifactorial pathophysiological process. It is well recognized that the membrane permeability transition pore (mPTP) opening of mitochondria plays a crucial role in cell death after I/R injury. Cyclophilin D (CypD) is a critical positive regulator of mPTP. However, the effect of CypD on the pathogenesis of liver I/R injury and whether CypD is a potential therapeutic target are still unclear. We constructed liver-specific CypD knockout and AAV8-peptidyl prolyl isomerase F (PPIF) overexpression mice. Then, a 70% liver I/R injury model was established in mice, with 90 min of ischemia and 6 h of reperfusion. The liver function was detected by the level of serum glutamic pyruvic transaminase (alanine transaminase) and glutamic oxaloacetic transaminase (aspartate aminotransferase), the liver damage score and degree of necrosis were measured by hematoxylin and eosin (H&E) staining of liver tissues. Reactive oxygen species (ROS) staining, apoptosis, and autophagy-related molecules were used to detect apoptosis and autophagy during liver I/R. The liver-specific knockout of CypD alleviated necrosis and dysfunction in liver I/R injury, by reducing the excessive production of ROS, and inhibiting cell apoptosis and autophagy. On the contrary, overexpression of CypD exacerbated I/R-induced liver damage. We found that the downregulation of CypD expression alleviated liver I/R injury by reducing apoptosis and autophagy through caspase-3/Beclin1 crosstalk; in contrast, the upregulation of CypD expression aggravated liver I/R injury. Therefore, interfering with the expression of CypD seems to be a promising treatment for liver I/R injury.

APELIN AS A POTENTIAL DOWNSTREAM MEDIATOR OF THE IGF-1 CARDIOPROTECTIVE PATHWAY IN THE HYPERTROPHIED MYOCARDIUM OF SPONTANEOUSLY HYPERTENSIVE RATS

Objective: Hypertension is a complex cardiovascular disease and contributes to worldwide morbidity and mortality associated to the development of hypertensive cardiac hypertrophy and the progression of heart failure. Hypertensive myocardium is characterized by mitochondrial dysfunction and redox disbalance. On the other hand, aerobic exercise upregulates several humoral factors such as insulin like growth factor-1 (IGF-1) and apelin, involved in cardioprotective effects. Particularly, whether apelin mediates IGF-1 cardioprotective effects by an autocrine paracrine mechanism is not clear yet. Objective. To determine the acute effects of IGF-1/apelin on oxidative stress and mitochondrial status, as well as the possible crosstalk between the signaling pathways triggered by them in spontaneously hypertensive rats (SHR) myocardium. Design and method: Adult SHR isolated hearts or cardiomyocytes were perfused during 10 min in the presence or absence of IGF-1 or apelin, and the specific antagonist of their receptors (AG1024 and ML221 for IGF1R and APJ, respectively). We explored reactive oxygen species (ROS) production, SOD activity, APJ cellular distribution, mitochondrial membrane potential (↖ ⇘) and permeability transition pore opening through the calcium retention capacity (CRC). Results: IGF-1 exerted an antioxidant effect evidenced by an increase in SOD activity (U/mg, IGF-1: 59.8±5.2, vs Control: 41.6±3) and decrease in ROS production (%, IGF-1: 66±12, vs Control: 100±8). In mitochondria, IGF-1 prevented loss induced by H2O2 (IGF-1: 0.934±0.016, vs Control: 0.827±0.027) and increased CRC (IGF-1: 251±34, vs Control: 135±23). Interestingly, IGF-1 upregulated APJ expression (IGF-1: 175.8±27.7 vs Control: 100±16 % mRNA) while favoring its translocation to the sarcolemma (Pearson coefficient: IGF-1: 0.880±0.011 vs Control: 0.758±0.031) in isolated cardiomyocytes. Moreover, exogenous apelin was able to emulate IGF-1 antioxidant and mitochondrial actions (SOD: 64±5.3 U/mg; ROS: 64± 7 %; ↖ ⇘ loss induced by H2O2: 0.981±0.025 and CRC 264±31). These effects were prevented by inhibition of APJ (Apelin+ML221: SOD: 33±4.4, CRC: 84±17, 0.808±0.042) but not by AG1024 (Apelin+AG: 0.985±0.068, CRC: 245±47). Conclusions: In conclusion, our data suggest that IGF-1 by sequentially activating its own receptor and the apelin/APJ pathway is responsible for cardioprotection. Therefore, apelin emerges as a downstream effector of the cardiac IGF-1 signaling pathway.

Dynamic changes in postmortem quality of mirror carp (Cyprinus carpio L.): Based on oxidation reaction and mitochondrial function properties

The dynamic changes in the postmortem quality of mirror carp (Cyprinus carpio L.) were investigated. With extended postmortem time, conductivity, redness, lipid oxidation, and protein oxidation all increased, while lightness, whiteness, and freshness decreased. At 4 h postmortem, the pH value reached a minimum (6.58), while the centrifugal loss and hardness reached a maximum (17.13% and 2539 g). Additionally, variations in mitochondria-related parameters during apoptosis were studied. Within 72 h postmortem, the content of reactive oxygen species initially decreased and subsequently increased; furthermore, there was a significant increase in the mitochondrial membrane permeability transition pore, membrane fluidity, and swelling (P < 0.05). Meanwhile, the cytosolic cytochrome c level decreased from 0.71 to 0.23, which indicated potential mitochondrial damage. Thus, mitochondrial dysfunction during postmortem aging can give rise to oxidation and the production of ammonia and amine compounds, which leads to flesh quality deterioration.

Mannose delays senescence in broccoli florets via maintaining mitochondrial morphological structure and function

The influence of mannose treatment on mitochondrial physiological parameters was investigated to postpone broccoli senescence. The results showed that mannose treatment could reduce the level of mitochondrial reactive oxygen species (ROS) to slow down the damage to its ultrastructure. Mannose inhibited malondialdehyde (MDA) accumulation and the decline of cytochrome c/a (Cyt c/a), and delayed the increase of mitochondrial membrane permeability. It also alleviated the over-opening of the mitochondrial membrane permeability transition pore (MPTP) by regulating expressions of MPTP component genes such as BoVDAC, BoHXK and BoANT. Moreover, mannose treatment improved the activities of mitochondrial antioxidant enzymes, including ascorbate oxidase (APX), superoxide dismutase (SOD), catalase (CAT), and ascorbic acid (AsA) content. Also, it increased the activities of succinate dehydrogenase (SDH), malate dehydrogenase (MDH), and Ca2+-ATPase, which were crucial for the mitochondrial energy metabolism. Taken together, our data indicated that mannose treatment inhibited the senescence of broccoli by maintaining the mitochondrial morphological structure and function.

Cytotoxicity and Autophagy Induced by Ivermectin via AMPK/mTOR Signaling Pathway in RAW264.7 Cells.

The widespread and excessive use of ivermectin (IVM) will not only cause serious environmental pollution, but will also affect metabolism of humans and other mammals that are exposed. IVM has the characteristics of being widely distributed and slowly metabolized, which will cause potential toxicity to the body. We focused on the metabolic pathway and mechanism of toxicity of IVM on RAW264.7 cells. Colony formation and LDH detection assay showed that IVM significantly inhibited the proliferation of and induced cytotoxicity in RAW264.7 cells. Intracellular biochemical analysis using Western blotting assay showed that LC3-B and Beclin-1 were upregulated and p62 was down-regulated. The combination of confocal fluorescence, calcein-AM/CoCl2, and fluorescence probe results showed that IVM could induce the opening of the mitochondrial membrane permeability transition pore, reduce mitochondrial content, and increase lysosome content. In addition, we focused on induction of IVM in the autophagy signal pathway. The Western blotting results showed that IVM increased expression of p-AMPK and decreased p-mTOR and p-S6K expression in protein levels, indicating that IVM activated the AMPK/mTOR signaling pathway. Therefore, IVM may inhibit cell proliferation by inducing cell cycle arrest and autophagy.

Molecular Mechanisms of Mitochondrial Quality Control in Ischemic Cardiomyopathy.

Ischemic cardiomyopathy (ICM) is a special type of coronary heart disease or an advanced stage of the disease, which is related to the pathological mechanism of primary dilated cardiomyopathy. Ischemic cardiomyopathy mainly occurs in the long-term myocardial ischemia, resulting in diffuse myocardial fibrosis. This in turn affects the cardiac ejection function, resulting in a significant impact on myocardial systolic and diastolic function, resulting in a decrease in the cardiac ejection fraction. The pathogenesis of ICM is closely related to coronary heart disease. Mainly due to coronary atherosclerosis caused by coronary stenosis or vascular occlusion, causing vascular inflammatory lesions and thrombosis. As the disease progresses, it leads to long-term myocardial ischemia and eventually ICM. The pathological mechanism is mainly related to the mechanisms of inflammation, myocardial hypertrophy, fibrosis and vascular remodeling. Mitochondria are organelles with a double-membrane structure, so the composition of the mitochondrial outer compartment is basically similar to that of the cytoplasm. When ischemia-reperfusion induces a large influx of calcium into the cell, the concentration of calcium ions in the mitochondrial outer compartment also increases. The subsequent opening of the membrane permeability transition pore in the inner mitochondrial membrane and the resulting calcium overload induces the homeostasis of cardiomyocytes and activates the mitochondrial pathway of apoptosis. Mitochondrial Quality Control (MQC), as an important mechanism for regulating mitochondrial function in cardiomyocytes, affects the morphological structure/function and lifespan of mitochondria. In this review, we discuss the role of MQC (including mitophagy, mitochondrial dynamics, and mitochondrial biosynthesis) in the pathogenesis of ICM and provide important evidence for targeting MQC for ICM.

In vitro effects of 2-methyl-3-propylbutane-1,4-diol purified from Alstonia boonei on erythrocyte membrane stabilization and mitochondrial membrane permeabilization.

A recent review on the ethnomedicinal, chemical, pharmacological, and toxicological properties of Alstonia boonei revealed the plant's potential in the treatment and management of a range of diseases. However, most of these pharmacological effects are only traceable to the crude form of the plant extract and not specific natural products. Phytochemical investigation of the methanol fraction of the methanol extract of the stem-bark of Alstonia boonei led to the isolation and identification of 2-methyl-3-propylbutane-1,4-diol. The structures were elucidated by the application of 1D-, and 2D-NMR spectroscopic analyses and by comparison with literature data. In this study, the membrane stabilizing activity, mitochondrial membrane permeability transition pore opening, cytochrome c release, mitochondrial ATPase activity, and prevention of mitochondrial lipid peroxidation activity of 2-methyl-3-propylbutane-1,4-diol (MPBD) isolated from A. boonei were determined. The results showed that MPBD significantly (p < .05) prevented peroxidation of mitochondrial membrane lipids and hemolysis using both the heat-induced and hypotonic solution-induced membrane stabilization assays. On the contrary, the compound caused large amplitude swelling of rat liver mitochondria in the absence of calcium, significant (p < .05) cytochrome c release and enhancement of mitochondrial ATPase activity in vitro. Our findings suggest that MPBD showed characteristic biological properties useful in modulating cell death.

Downhill running induced DNA damage enhances mitochondrial membrane permeability by facilitating ER-mitochondria signaling.

To observe whether downhill running can lead to DNA damage in skeletal muscle cells and changes in mitochondrial membrane permeability and to explore whether the DNA damage caused by downhill running can lead to changes in mitochondrial membrane permeability by regulating the components of the endoplasmic reticulum mitochondrial coupling structure (MAM). A total of 48 male adult Sprague-Dawley rats were randomly divided into a control group (C, n = 8) and a motor group (E, n = 40). Rats in Group E were further divided into 0h (E0), 12h (E12), 24h (E24), 48h (E48) and 72h (E72) after prescribed exercise, with 8 rats in each group. At each time point, flounder muscle was collected under general anaesthesia. The DNA oxidative damage marker 8-hydroxydeoxyguanosine (8-OHdG) was detected by immunofluorescence. The expression levels of the DNA damage-related protein p53 in the nucleus and the EI24 protein and reep1 protein in whole cells were detected by Western blot. The colocalization coefficients of the endoplasmic reticulum protein EI24 and the mitochondrial protein Vdac2 were determined by immunofluorescence double staining, and the concentration of Ca2+ in skeletal muscle mitochondria was detected by a fluorescent probe. Finally, the opening of the mitochondrial membrane permeability transition pore (mPTP) was detected by immunofluorescence. Twelve hours after downhill running, the mitochondrial membrane permeability of the mPTP opened the most (P < 0.05), the content of 8-OHdG in skeletal muscle peaked (P < 0.05), and the levels of the regulatory protein p53, mitochondrial Ca2+, and the EI24 and reep1 proteins peaked (P < 0.01). Moreover, the colocalization coefficients of EI24 and Vdac2 and the Mandes coefficients of the two proteins increased first and then recovered 72h after exercise (P < 0.05). (1) Downhill running can lead to DNA damage in skeletal muscle cells, overload of mitochondrial Ca2+ and large opening of membrane permeability transformation pores. (2) The DNA damage caused by downhill running may result in p53 promoting the transcriptional activation of reep1 and EI24, enhancing the interaction between EI24 and Vdac2, and then leading to an increase in Ca2+ in skeletal muscle mitochondria and the opening of membrane permeability transition pores.

Modulation of mitochondrial permeability transition pore opening by Myricetin and prediction of its-drug-like potential using in silico approach

Myricetin has been demonstrated to have multiple biological functions with promising research and development prospects. This study investigated the effect of myricetin on liver mitochondrial membrane permeability transition pores and its inhibitory potential on proteins that are important in the apoptotic process in silico. Mitochondrial swelling was assessed as changes in absorbance under succinate-energized conditions. Cytochrome c release, mitochondrial-lipid peroxidation, caspase 3 and 9 expressions, as well as calcium ATPase, were assessed. Pharmacokinetic properties of myricetin were predicted through the SwissADME server while the binding affinity of myricetin toward the proteins was computed using the AutodockVina Screening tool. The conformational stability of protein-ligand interactions was evaluated using molecular dynamics simulations analysis through the iMODS server. Myricetin inhibited the opening of the mitochondrial permeability transition pore and also reversed the increase in mitochondrial lipid peroxidation caused by calcium and other toxicants. Myricetin also caused a reduction in the expression of caspase 3 and 9 as well as calcium ATPase activity. The molecular docking results revealed that myricetin had a considerable binding affinity to the pocket site of caspase 3 and 9 as well as calcium ATPase. Myricetin showed a good drug-likeness based on the predicted pharmacokinetic properties as revealed by low CYP 450 inhibitory promiscuity and relatively low toxicity. It could therefore be suggested that myricetin could be useful in the management of diseases where too many apoptosis occur characterized by excessive tissue wastage such as neurodegenerative conditions and could as well play a role in protecting the physicochemical properties of membrane bilayers from free radical-induced severe cellular damage.