Melatonin alleviates angiotensin-II-induced cardiac hypertrophy via activating MICU1 pathway.

Mitochondrial calcium uptake 1 (MICU1) is a pivotal molecule in maintaining mitochondrial homeostasis under stress conditions. However, it is unclear whether MICU1 attenuates mitochondrial stress in angiotensin II (Ang-II)-induced cardiac hypertrophy or if it has a role in the function of melatonin. Here, small-interfering RNAs against MICU1 or adenovirus-based plasmids encoding MICU1 were delivered into left ventricles of mice or incubated with neonatal murine ventricular myocytes (NMVMs) for 48 h. MICU1 expression was depressed in hypertrophic myocardia and MICU1 knockdown aggravated Ang-II-induced cardiac hypertrophy in vivo and in vitro. In contrast, MICU1 upregulation decreased cardiomyocyte susceptibility to hypertrophic stress. Ang-II administration, particularly in NMVMs with MICU1 knockdown, led to significantly increased reactive oxygen species (ROS) overload, altered mitochondrial morphology, and suppressed mitochondrial function, all of which were reversed by MICU1 supplementation. Moreover, peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC-1α)/MICU1 expression in hypertrophic myocardia increased with melatonin. Melatonin ameliorated excessive ROS generation, promoted mitochondrial function, and attenuated cardiac hypertrophy in control but not MICU1 knockdown NMVMs or mice. Collectively, our results demonstrate that MICU1 attenuates Ang-II-induced cardiac hypertrophy by inhibiting mitochondria-derived oxidative stress. MICU1 activation may be the mechanism underlying melatonin-induced protection against myocardial hypertrophy.

Opening of liver mitochondrial permeability transition pore in streptozotocin-induced diabetic rats and its inhibition by methanol fraction of Ficus mucoso (Welw) root bark.

OBJECTIVE: Several pathologies arise from the inappropriate opening of the mitochondrial permeability transition (mPT) pore. In this regard, inhibition of mPT pore represents a cytoprotective approach to preserve mitochondrial function for treatment of diseases characterized by excessive tissue wastage such as diabetes mellitus. The aim of this study, therefore, was to study the effects of fractions of Ficus mucoso, a medicinal plant used in the traditional treatment of diabetes, on mPT pore in normal and streptozotocin (STZ)-induced diabetic rat liver. METHODS: Different solvent fractions of the crude methanol extract of F. mucoso were obtained by vacuum liquid chromatography and were tested on the mPT pore. Of all the fractions tested, methanol fraction of F. mucoso (MFFM) was the most potent and was used for in vivo studies. Diabetes mellitus was induced by a single intraperitoneal injection of 60 mg/kg STZ, while treatment lasted for 14 d. In vivo, 30 male Wistar rats were divided into five groups: A, normo-glycemic control (distilled water); B, STZ (65 mg/kg; diabetic control); C, STZ + MFFM (25 mg/kg); D, STZ + MFFM (50 mg/kg); E, STZ + glibenclamide (5 mg/kg). The mPT, mitochondrial ATPase activity, lipid peroxidation and cytochrome c release were assessed spectrophotometrically while blood glucose levels were monitored using glucometer. RESULTS: In vitro, the solvent fractions of F. mucoso, at all concentrations tested, had no effect on the mPT pore, in the absence of calcium, with no significant release of cytochrome c. Interestingly, calcium-dependent pore opening was inhibited by all solvent fractions of F. mucoso, with the MFFM having the highest inhibitory effect of 83% at 3 mg/mL. Induction of opening of the mPT pore, significant (P < 0.001) enhancement of mitochondrial ATPase activity and elevated malondialdehyde (MDA) levels in STZ-induced diabetes were significantly (P < 0.001) reversed by MFFM and were comparable with the effects of glibenclamide, a standard antidiabetic drug. Also, treatment with MFFM at different doses significantly (P < 0.001) reduced high serum blood glucose compared to the diabetic control. CONCLUSION: F. mucoso could be useful in therapeutic management of diabetes mellitus given its ability to prevent excessive tissue wastage via inhibition of pore opening, and reduction in levels of MDA and serum blood glucose.

Cerebral protection of epigallocatechin gallate (EGCG) via preservation of mitochondrial function and ERK inhibition in a rat resuscitation model.

BACKGROUND: Various and opposite roles of epigallocatechin gallate (EGCG) have been reported in different studies. We aimed to investigate how EGCG affects the cerebral injury in a cardiac arrest/cardiopulmonary resuscitation (CA/CPR) model of rat. METHODS: The rats which were subjected to CA/CPR randomly received low dose of EGCG (3 mg/kg, Low-EGCG group, n=16), high dose of EGCG (9 mg/kg, High-EGCG group, n=16) and equal volume of 0.9% saline solution (NS group, n=16) at the first minute after return of spontaneous circulation (ROSC). The rats underwent anesthesia and intubation were defined as Sham group (n=16). Twenty-four hours after ROSC, neural defect score (NDS), ROS fluorescence intensity, degree of mitochondrial permeability transition pore (mPTP) opening, ATP contents and mitochondrial ATP synthase expression were evaluated in the four groups. The expression of extracellular signal-regulated kinase (ERK) activity and cleaved-caspase 3 were also detected by Western blot. RESULTS: CA/CPR induced severe ischemia-reperfusion injury (IRI), resulted in mitochondrial dysfunction and upregulated phosphorylation of ERK. EGCG dose-dependently alleviated the IRI after CA/CPR, inhibited ERK activity and restored mitochondrial function and, as indicated by improved NDS, reduced ROS level, decreased mPTP opening, elevated ATP content, increased ATPase expression and downregulated cleaved-caspase 3 level. CONCLUSION: EGCG alleviated global cerebral IRI by restoring mitochondrial dysfunction and ERK modulation in a rat CA/CPR model, which might make it a potential candidate agent against IRI after CA/CPR in the future. Further study is needed to determine whether higher dosage of EGCG might aggravate cerebral IRI post-CA/CPR.

Evaluation of (-)-epigallocatechin-3-gallate (EGCG)-induced cytotoxicity on astrocytes: A potential mechanism of calcium overloading-induced mitochondrial dysfunction.

(-)-epigallocatechin-3-gallate (EGCG), the main component of green tea, has long been explored in the treatment and/or prevention of central nervous system (CNS) disorders. However, EGCG has been recently shown to exhibit acute and subacute toxicity. Although a lot of work has been done, the mechanisms of EGCG-induced mitochondrial dysfunction has not been delineated in primary astrocyte. Here, the mitotoxic effect of EGCG on primary astrocytes was investigated by measuring Ca2+ overloading-induced mitochondrial dysfunction. As expected, EGCG dose-dependently inhibited astrocytes growth depending on Ca2+ overloading, especially at 50 µM EGCG group. It is interesting to note that Ca2+ influx from the extracellular space was responsible for an increase in the cytosolic Ca2+ level ([Ca2+]i) by opening voltage-gated calcium channels (VGCCs) and, consequently, mitochondrial Ca2+ ([Ca2+]m) overloaded via the mitochondrial Ca2+ uniporter (MCU). As a result, mitochondrial dysfunction was induced, including the opening of the mitochondrial permeability transition pore (mPTP), mitochondrial membrane depolarization, an increasing in reactive oxygen species (ROS), and cytochrosome c (cyt c) releasing. Therefore, more apoptotic cells were observed in 50 µM EGCG group than that of in 1 µM EGCG group. These findings suggested that a high dose of EGCG was toxic to astrocytes partly by targeting mitochondria via calcium pathway, which would extend our understanding of the toxicity of EGCG and the underlying mechanisms.

Qishen Yiqi Drop Pill, a novel compound Chinese traditional medicine protects against high glucose-induced injury in cardiomyocytes.

OBJECTIVE: Qishen Yiqi Drop Pill (QSYQ) has been recognized as a potential protective agent for various cardiovascular diseases. However, the effect of QSYQ in cardiac complications associated with diabetes is not clear currently. In this study, we investigate whether QSYQ could exert cardiac protective effects against high glucose-induced injuries in cardiac H9c2 cells. METHODS: H9c2 cells were exposed to 24 hours of high glucose in presence or absence of QSYQ and LY294002. Cell cytotoxicity, apoptosis, reactive oxygen species (ROS) generation, mitochondrial membrane potential and mitochondrial permeability transition pore (mPTP) opening were determined. Levels of bax, bcl-2, p53, cleaved caspase-3, PI3K and Akt were evaluated by Western blot. RESULTS: Our data indicated that QSYQ significantly increased the cell viability and decreased cytotoxicity. By analysing the apoptotic rate as well as the expression levels of cytoapoptosis-related factors including cleaved caspase-3, bax, bcl-2, and p53, we found that QSYQ could remarkably suppress apoptosis of cardiomyoblasts caused by high glucose. In addition, it also showed that QSYQ reduced the generation of ROS. We further found that QSYQ treatment could inhibit the loss of mitochondrial membrane potential and mPTP opening. Moreover, Western blot analysis showed enhanced phosphorylation of PI3K/Akt. The specific inhibitor of PI3K, LY294002 not only inhibited QSYQ induced PI3K/Akt signalling pathway activation, but alleviated its protective effects. CONCLUSIONS: In summary, these findings demonstrated that QSYQ effectively protected H9c2 cells against the series injuries due to high glucose at least partially by activating the PI3K/Akt signalling pathway.

Melatonin as a master regulator of cell death and inflammation: molecular mechanisms and clinical implications for newborn care.

Melatonin, more commonly known as the sleep hormone, is mainly secreted by the pineal gland in dark conditions and regulates the circadian rhythm of the organism. Its intrinsic properties, including high cell permeability, the ability to easily cross both the blood-brain and placenta barriers, and its role as an endogenous reservoir of free radical scavengers (with indirect extra activities), confer it beneficial uses as an adjuvant in the biomedical field. Melatonin can exert its effects by acting through specific cellular receptors on the plasma membrane, similar to other hormones, or through receptor-independent mechanisms that involve complex molecular cross talk with other players. There is increasing evidence regarding the extraordinary beneficial effects of melatonin, also via exogenous administration. Here, we summarize molecular pathways in which melatonin is considered a master regulator, with attention to cell death and inflammation mechanisms from basic, translational and clinical points of view in the context of newborn care.

Epigallocatechin gallate prevents mitochondrial impairment and cell apoptosis by regulating miR-30a/p53 axis.

PURPOSE: This study was designed to investigate whether EGCG prevents cardiac I/R mitochondrial impairment and cell apoptosis by regulating miR-30a/p53 axis. METHODS: The H9c2 cardiomyocytes hypoxia/reoxygenation (H/R) model in vitro and myocardial ischemia /reperfusion (I/R) model in vivo were made, with or without EGCG treatment. The levels of I/R-induced creatine kinase-MB (CK-MB) and the release of lactate dehydrogenase (LDH), as well as the adenosine triphosphate (ATP) and cardiac functional impairment were examined. Stablely transfecting miR-30a mimic or inhibitor in H9c2 cardiomyocytes was built. The expression of miR-30a, p53 and related proteins in cells was measured by western blotting and qRT-PCR. Cell viability and apoptosis were examined using CCK-8 assay and flow cytometry. The content of reactive oxygen species (ROS), mitochondrial permeability transition pores (MPTP) opening and mitochondrial transmembrane potential (ΔΨm) in cells was measured by fluorescent probes. The levels of miR-30a and p53, some related proteins expression and apoptosis in the cardiac muscle tissues were determined by quantitative real-time PCR (qRT-PCR), H&E staining, western blotting and TUNEL assays. RESULTS: We found that EGCG preconditioning significantly decreased the levels of CK-MB and LDH, increased the activity of ATP, reduced the apoptotic rate and partially preserved heart function. Furthermore, EGCG decreased ROS levels, MPTP opening and depolarization of ΔΨm, and improved the activity of post-I/R cardiomyocyte. The beneficial effect of EGCG was associated with restored levels of miR-30a expression in the I/R injury that correspond to p53 mRNA downregulation. The regulatory effect of EGCG was greatly enhanced by miR-30a mimic and suppressed by miR-30a inhibitor. More importantly, EGCG pretreatment inhibited the expression of mitochondrial apoptotic related proteins downstream of the miR-30a/p53 pathway. CONCLUSION: This study demonstrated that EGCG pretreatment may attenuate mitochondrial impairment and myocardial apoptosis by regulation of miR-30a/p53 axis.

Salvianolic Acid A Protects Neonatal Cardiomyocytes Against Hypoxia/Reoxygenation-Induced Injury by Preserving Mitochondrial Function and Activating Akt/GSK-3ß Signals.

OBJECTIVE: To investigate the effects of salvianolic acid A (SAA) on cardiomyocyte apoptosis and mitochondrial dysfunction in response to hypoxia/reoxygenation (H/R) injury and to determine whether the Akt signaling pathway might play a role. METHODS: An in vitro model of H/R injury was used to study outcomes on primary cultured neonatal rat cardiomyocytes. The cardiomyocytes were treated with 12.5, 25, 50 µg/mL SAA at the beginning of hypoxia and reoxygenation, respectively. Adenosine triphospate (ATP) and reactive oxygen species (ROS) levels were assayed. Cell apoptosis was evaluated by flow cytometry and the expression of cleaved-caspase 3, Bax and Bcl-2 were detected by Western blotting. The effects of SAA on mitochondrial dysfunction were examined by determining the mitochondrial membrane potential (△Ψm) and mitochondrial permeability transition pore (mPTP), followed by the phosphorylation of Akt (p-Akt) and GSK-3ß (p-GSK-3ß), which were measured by Western blotting. RESULTS: SAA significantly preserved ATP levels and reduced ROS production. Importantly, SAA markedly reduced the number of apoptotic cells and decreased cleaved-caspase 3 expression levels, while also reducing the ratio of Bax/Bcl-2. Furthermore, SAA prevented the loss of △Ψm and inhibited the activation of mPTP. Western blotting experiments further revealed that SAA significantly increased the expression of p-Akt and p-GSK-3ß, and the increase in p-GSK-3ß expression was attenuated after inhibition of the Akt signaling pathway with LY294002. CONCLUSION: SAA has a protective effect on cardiomyocyte H/R injury; the underlying mechanism may be related to the preservation of mitochondrial function and the activation of the Akt/GSK-3ß signaling pathway.

Rhein reverses doxorubicin resistance in SMMC-7721 liver cancer cells by inhibiting energy metabolism and inducing mitochondrial permeability transition pore opening.

Rhein, a monomeric anthraquinone obtained from the plant herb species Polygonum multiflorum and P. cuspidatum, has been proposed to have anticancer activity. This activity has been suggested to be associated with mitochondrial injury due to the induction of mitochondrial permeability transition pore (mPTP) opening. In this study, the effects of 5-80 µM rhein on cell viability, half-maximal inhibitory concentration (IC50 value), resistance index, and apoptosis were assessed in the liver cancer cell lines SMMC-7721 and SMMC-7721/DOX (doxorubicin-resistant cells). Rhein (10-80 µM) significantly reduced the viability of both cell lines; 20 µM rhein significantly increased sensitivity to DOX and increased apoptosis in SMMC-7721 cells, but reversed resistance to DOX by 7.24-fold in SMMC-7721/DOX cells. Treatment with rhein increased accumulation of DOX in SMMC-7721/DOX cells, inhibited mitochondrial energy metabolism, decreased cellular ATP, and ADP levels, and altered the ratio of ATP to ADP. These effects may result from the binding of rhein with voltage-dependent ion channels (VDACs), adenine nucleotide translocase (ANT), and cyclophilin D, affecting their function and leading to the inhibition of ATP transport by VDACs and ANT. ATP synthesis was greatly reduced and mitochondrial inner membrane potential decreased. Together, these results indicate that rhein could reverse drug resistance in SMMC-7721/DOX cells by inhibiting energy metabolism and inducing mPTP opening. © 2018 BioFactors, 45(1):85-96, 2019.

Mitochondrial bioenergetics and cardiolipin alterations in myocardial ischemia-reperfusion injury: implications for pharmacological cardioprotection.

Mitochondrial dysfunction plays a central role in myocardial ischemia-reperfusion (I/R) injury. Increased reactive oxygen species production, impaired electron transport chain activity, aberrant mitochondrial dynamics, Ca2+ overload, and opening of the mitochondrial permeability transition pore have been proposed as major contributory factors to mitochondrial dysfunction during myocardial I/R injury. Cardiolipin (CL), a mitochondria-specific phospholipid, plays a pivotal role in multiple mitochondrial bioenergetic processes, including respiration and energy conversion, in mitochondrial morphology and dynamics as well as in several steps of the apoptotic process. Changes in CL levels, species composition, and degree of oxidation may have deleterious consequences for mitochondrial function with important implications in a variety of pathophysiological conditions, including myocardial I/R injury. In this review, we focus on the role played by CL alterations in mitochondrial dysfunction in myocardial I/R injury. Pharmacological strategies to prevent myocardial injury during I/R targeting mitochondrial CL are also examined.

Tanshinone IIA Pretreatment Protects H9c2 Cells against Anoxia/Reoxygenation Injury: Involvement of the Translocation of Bcl-2 to Mitochondria Mediated by 14-3-3η.

Tanshinone IIA is an important component that is isolated from danshen (Salvia miltiorrhiza), which is known to be beneficial for cardiovascular health. In this study, we determined the effects of Tanshinone IIA and its underlying mechanisms of action in an anoxia/reoxygenation (A/R) cell line model. Prior to inducing A/R injury, rat cardiomyocyte-derived cell line H9c2 was stimulated with 8 µM of Tanshinone IIA for 48 hours. When compared with the A/R group, the Tanshinone IIA treatment significantly increased cell viability and decreased lactate dehydrogenase activity. Tanshinone IIA upregulated 14-3-3η expression and facilitated Bcl-2 translocation to the mitochondrial outer membrane, which bound with voltage-dependent anion channel 1. In addition, pretreatment with Tanshinone IIA reduced the generation of reactive oxygen species and cytochrome c release, inactivated caspase-3, prevented mitochondrial permeability transition pore opening, and reduced the percentage of apoptotic cells. Moreover, treatment with Tanshinone IIA reduced the level of malondialdehyde, thereby increasing the activity of superoxide dismutase and glutathione peroxidase. Silencing the expression of 14-3-3η by adenovirus blocked the above-mentioned results. These novel findings showed that pretreatment with Tanshinone IIA alleviated H9c2 cell damage against A/R injury and was associated with upregulation of 14-3-3η, thereby facilitating Bcl-2 translocation to the mitochondrial outer membrane and preventing mitochondrial permeability transition pore opening, decreasing cytochrome c release, preventing caspase-3 activation, and restraining apoptosis.

Cardioprotective Effect of Hydroxysafflor Yellow A via the Cardiac Permeability Transition Pore.

Myocardial ischemia damages cardiac myocytes in part via opening of the mitochondrial permeability transition pore. Preventing this pore's opening is therefore a useful therapeutic goal in treating cardiovascular disease. Hydroxysafflor yellow A has been proposed as a nontoxic alternative to other agents that modulate mitochondrial permeability transition pore opening. In this study, we proposed that hydroxysafflor yellow A prevents mitochondrial permeability transition pore formation in anoxic cardiac myocytes, and thus protects the cell from damage seen during reoxygenation of the cardiac myocytes. Experiments with hydroxysafflor yellow A transport in aerobic myocytes show that roughly 50% of the extracellular dye concentration crosses the cell membrane in a 2-h incubation. In our anoxia/reoxygenation protocol, hydroxysafflor yellow A modulated both the reduction of viability and the loss of rod-shaped cells that attend anoxia and reoxygenation. Hydroxysafflor yellow A's protective effect was similar to that of cyclosporin A, an agent known to inhibit mitochondrial permeability transition pore opening. In additional experiments, plated myocytes were loaded with calcein/MitoTracker Red, then examined for intracellular dye distribution/morphology after anoxia/reoxygenation. Hydroxysafflor yellow A-containing cells showed a cardioprotective pattern similar to that of cyclosporin A (an agent known to close the mitochondrial permeability transition pore). We conclude that hydroxysafflor yellow A can enter the cardiac myocyte and is able to modulate anoxia/reoxygenation-induced damage by interacting with the mitochondrial permeability transition pore.

Sasanquasaponin-induced cardioprotection involves inhibition of mPTP opening via attenuating intracellular chloride accumulation.

Sasanquasaponin (SQS) has been reported to elicit cardioprotection by suppressing hypoxia/reoxygenation (H/R)-induced elevation of intracellular chloride ion concentration ([Cl-]i). Given that the increased [Cl-]i is involved to modulate the mitochondrial permeability transition pore (mPTP), we herein sought to further investigate the role of mPTP in the cardioprotective effect of SQS on H/R injury. H9c2 cells were incubated for 24h with or without 10µM SQS followed by H/R. The involvement of mPTP was determined with a specific mPTP agonist atractyloside (ATR). The results showed that SQS attenuated H/R-induced the elevation of [Cl-]i, accompanied by reduction of lactate dehydrogenase release and increase of cell viability. Moreover, SQS suppressed mPTP opening, and protected mitochondria, as indicated by preserved mitochondrial membrane potential and respiratory chain complex activities, decreased mitochondrial reactive oxygen species generation, and increased ATP content. Interestingly, extracellular Cl--free condition created by replacing Cl- with equimolar gluconate resulted in a decrease in [Cl-]i and induced protective effects similar to SQS preconditioning, whereas pharmacologically opening of the mPTP with ATR abolished all the protective effects induced by SQS or Cl--free, including suppression of mPTP opening, maintenance of mitochondrial membrane potential, and subsequent improvement of mitochondrial function. The above results allow us to conclude that SQS-induced cardioprotection may be mediated by preserving the mitochondrial function through preventing mPTP opening via inhibition of H/R-induced elevation of [Cl-]i.

Ex Vivo Treatment with a Polyphenol-Enriched Cocoa Extract Ameliorates Myocardial Infarct and Postischemic Mitochondrial Injury in Normotensive and Hypertensive Rats.

Our objective was to determine the effects of a polyphenol-enriched cocoa extract (PCE) on myocardial postischemic alterations in normotensive (Wistar rats, W) and spontaneously hypertensive rats (SHR). Isolated hearts were submitted to 110 min of perfusion or 20 min stabilization, 30 min global ischemia, and 60 min reperfusion (R). Other hearts were treated with PCE at the onset of R. Infarct size, the reduced glutathione (GSH), and the expression of phospho-Akt, P-GSK-3ß, and P-eNOS were assessed. In isolated mitochondria, the Ca(2+)-mediated response of mitochondrial permeability transition pore (mPTP), membrane potential (Δψm), and superoxide production were determined. PCE decreased infarct size, partly preserved GSH, increased the P-Akt, P-GSK-3ß, and P-eNOS contents, improved mPTP response to Ca(2+), decreased the superoxide production, and restored Δψm. These data show that PCE decreases the cardiac postischemic damage in W rats and SHR and suggest that Akt/GSK-3ß/eNOS dependent pathways are involved.

Gypenosides alleviate myocardial ischemia-reperfusion injury via attenuation of oxidative stress and preservation of mitochondrial function in rat heart.

Gypenosides (GP) are the predominant components of Gynostemma pentaphyllum, a Chinese herb medicine that has been widely used for the treatment of chronic inflammation, hyperlipidemia, and cardiovascular disease. GP has been demonstrated to exert protective effects on the liver and brain against ischemia-reperfusion (I/R) injury, yet whether it is beneficial to the heart during myocardial I/R is unclear. In this study, we demonstrate that pre-treatment with GP dose-dependently limits infarct size, alleviates I/R-induced pathological changes in the myocardium, and preserves left ventricular function in a rat model of cardiac I/R injury. In addition, GP pre-treatment reduces oxidative stress and protects the intracellular antioxidant machinery in the myocardium. Further, we show that the cardioprotective effect of GP is associated with the preservation of mitochondrial function in the cardiomyocytes, as indicated by ATP level, enzymatic activities of complex I, II, and IV on the mitochondrial respiration chain, and the activity of citrate synthase in the citric acid cycle for energy generation. Moreover, GP maintains mitochondrial membrane integrity and inhibits the release of cytochrome c from the mitochondria to the cytosol. The cytoprotective effect of GP is further confirmed in vitro in H9c2 cardiomyoblast cell line with oxygen-glucose deprivation and reperfusion (OGD/R), and the results indicate that GP protects cell viability, reduces oxidative stress, and preserves mitochondrial function. In conclusion, our study suggests that GP may be of clinical value in cytoprotection during acute myocardial infarction and reperfusion.

5-diphenylacetamido-indirubin-3'-oxime as a novel mitochondria-targeting agent with anti-leukemic activities.

Current treatment for leukemia largely depends on chemotherapy. Despite the progress in treatment efficacy of chemotherapy, a poor outcome consequent upon chemoresistance against conventional anti-cancer drugs still remains to be solved. In this study, we report 5-diphenylacetamido-indirubin-3'-oxime (LDD398) as a novel mitochondria-targeting anti-leukemic agent, which is a derivative of indirubin used in traditional medicine. Treatment with LDD398 resulted in caspase activation, cell death, and growth arrest at G2/M phases in leukemia cells. Interestingly, LDD398 quickly collapsed mitochondrial membrane potential (MMP) within 1 h, accompanied by cytochrome c release into cytosol and severe depletion of cellular ATP. However, the LDD398-induced cellular events was significantly mitigated by blockage of mitochondrial permeability transition pore (MPTP) opening with chemical and genetic modifications, strongly supporting that LDD398 executes its anti-leukemic activity via an inappropriate opening of MPTP and a consequent depletion of ATP. The most meaningful finding was the prominent effectiveness of LDD398 on primary leukemia cells and also on malignant leukemia cells resistant to anticancer drugs. Our results demonstrate that, among a series of indirubin derivatives, LDD398 induces leukemia cell death via a different mode from indirubin or conventional chemotherapeutics, and can be employed as a potent anti-cancer agent in the treatment for newly diagnosed and relapsed leukemia.

Protection effect of piperine and piperlonguminine from Piper longum L. alkaloids against rotenone-induced neuronal injury.

Currently available treatment approaches for Parkinson׳s disease (PD) are limited in terms of variety and efficacy. Piper longum L. (PLL; Piperaceae) is used in traditional medicine in Asia and the Pacific Islands, with demonstrated anti-inflammatory and antioxidant activities in preclinical studies, and alkaloid extracts of PLL have shown protective effects in PD models. The present study investigated the mechanistic basis for the observed protective effects of PLL. Rats treated with PLL-derived alkaloids showed improvement in rotenone-induced motor deficits, while reactive oxygen species (ROS) production was decreased, mitochondrial membrane potential was stabilized, and the opening of the mitochondrial permeability transition pore (mPTP)-which is involved in ROS production-was inhibited. In addition, rotenone-induced apoptosis was abrogated in the presence of these alkaloids, while a pretreatment stimulated autophagy, likely mitigating neuronal injury by the removal of damaged mitochondria. These findings provide novel insight into the neuroprotective function of PLL as well as evidence in favor of its use in PD treatment. This article is part of a Special Issue entitled SI: Neuroprotection.

Saffron extracts alleviate cardiomyocytes injury induced by doxorubicin and ischemia-reperfusion in vitro.

Doxorubicin (DOX), a highly active chemotherapeutic drug, faces limitations in clinical application due to severe cardiotoxic effects (mainly through increased oxidative stress). Therefore, its effect is exacerbated in subjects with ischemic heart disease. We have recently reported that saffron extract (SAF), a natural compound mainly consisting of safranal and corcins, exerts a protective effect against DOX oxidative cytotoxicity in isolated rabbit hearts. Here, we aimed to investigate whether SAF exerts cardioprotection against combined ischemia-reperfusion (I/R) and DOX toxicity in H9c2 cardiomyocytes. H9c2 were subjected to simulated I/R, with or without DOX treatment at reperfusion, in the presence or absence of SAF prior to ischemia or at reperfusion. We evaluated the effects of these treatments by MTT, LDH and western blot analysis. Apoptosis was assessed by Hoechst 33258 staining, tetramethyl rhodamine methyl ester fluorescence and caspase activity. The results showed that I/R and DOX significantly decreased cardiomyocytes viability, inhibited reperfusion injury salvage kinase cardioprotective pathway, reduced contractile proteins (α-Actinine, Troponine C and MLC), increased caspase-3 expression and induced loss of mitochondrial membrane potential. These effects were remarkably inhibited by treatment with SAF (10 µg/mL) at reperfusion. SAF activated AKT/P70S6K and ERK1/2, restored contractile proteins expression, inhibited mitochondrial permeability transition pore and decreased caspase-3 activity. In conclusion, our findings indicate that SAF treatment exerted cardioprotection against I/R and DOX toxicity by reducing oxidative stress (LDH assay). Thereby, SAF offers a potential novel antioxidant therapeutic strategy to counteract I/R and DOX cardiotoxicity, paving the way for future clinical trials.

[Protective effect of Wuziyanzong Pills on rats with experimental oligoasthenospermia and its action mechanism].

OBJECTIVE: To investigate the protective effect of Wuziyanzong Pills (WYP) in the rat model of oligoasthenospermia (OAS) and its action mechanism. METHODS: Sixty male SD rats were equally randomized into six groups: normal control, OAS model, Shengjing Capsules (1.6 g per kg of the body weight), low-dose WYP (1 g per kg of the body weight), medium-dose WYP (2 g per kg of the body weight), and high-dose WYP (4 g per kg of the body weight). The OAS model was established by intragastric administration of Tripterygium glucoside at 30 mg per g per d for 6 weeks. From the 3rd week of modeling, the rats of the medication groups were treated intragastrically with corresponding drugs for 4 weeks. Then all the rats were sacrificed for measurement of the testicular and epididymal organ coefficients, examination of epididymal sperm quality and apoptosis, and detection of the openness of the sperm mitochondrial permeability transition pore (MPTP). Histopathological changes in the testis were observed by HE staining and the apoptosis of spermatogenic cells determined by Hochest staining. RESULTS: WYP obviously improved the organ coefficients of the testis and epididymis, increased sperm concentration, motility and viability, decreased the apoptosis of spermatogenic cells, and inhibited the abnormal openness of MPTP in the OAS model rats. HE staining showed that the number and levels of spermatogenic cells were significantly increased while Hochest staining manifested that the apoptosis of spermatogenic cells was remarkably inhibited in the seminiferous tubules of the testis in the WYP-treated rats. CONCLUSIONS: WYP can improve sperm quality and reduce the apoptosis of spermatogenic cells (including sperm) in OAS model rats, which may be related with its inhibitory effect on the abnormal openness of MPTP.

Danhong injection protects cardiomyocytes against hypoxia/reoxygenation- and H2O2-induced injury by inhibiting mitochondrial permeability transition pore opening.

ETHNOPHARMACOLOGICAL RELEVANCE: Danhong injection (DHI), a Chinese medical product extracted from Radix et Rhizoma Salviae Miltiorrhizae (Salvia miltiorrhiza Bge., Labiatae, Danshen in Chinese) and Flos Carthami (Carthamus tinctorius L., Compositae, Honghua in Chinese), has been widely used for the treatment of ischemic heart disease, and clinical and experimental studies have demonstrated the protective effects against myocardial ischemia/reperfusion injury. Nevertheless, the underlying cellular mechanisms responsible for this protective effect are poorly understood. AIM OF THE STUDY: The present study aimed to examine the mechanism of DHI in regulating hypoxia/reoxygenation- and H2O2-induced cardiomyocytes injury. MATERIALS AND METHODS: Neonatal rat cardiomyocytes were subjected to hypoxia (9h)-reoxygenation (2h) or H2O2 (100 µM) in the presence or absence of DHI (2.5, 5, 10 µg/mL). Intracellular reactive oxygen species (ROS), cytosolic and mitochondrial Ca(2+) concentrations, mitochondrial membrane potential (ΔΨm) and mitochondrial permeability transition pore (mPTP) opening were monitored using CMH2DCFDA, Fluo-4 and rhod-2, JC-1 and calcein, respectively. Cell survival was evaluated using the 2-(4,5-dimethylthiazol-2-yl)-2,5 -diphenyltetrazolium bromide (MTT) assay and apoptosis was detected by Annexin V/propidium iodide (PI) staining. RESULTS: DHI improved cell survival following H/R and H2O2 injury and reduced H/R-induced cytochrome c release and apoptosis when compared with non-DHI treated cells. In addition, DHI attenuated H/R-induced ROS generation, H2O2-induced cytosolic and mitochondrial Ca(2+) overload, and cellular ROS generation when compared with H/R- and H2O2-only groups. Moreover, DHI significantly inhibited both mPTP opening and ΔΨm depolarization. CONCLUSION: These data demonstrate that the protective mechanism of DHI against H/R- and H2O2-induced injury is mediated by the inhibition of mPTP opening via mitigating Ca(2+) overload and ROS generation.