Disruption of mitochondrial bioenergetics and calcium homeostasis by phytanic acid in the heart: Potential relevance for the cardiomyopathy in Refsum disease.

Refsum disease is an inherited peroxisomal disorder caused by severe deficiency of phytanoyl-CoA hydroxylase activity. Affected patients develop severe cardiomyopathy of poorly known pathogenesis that may lead to a fatal outcome. Since phytanic acid (Phyt) concentrations are highly increased in tissues of individuals with this disease, it is conceivable that this branched-chain fatty acid is cardiotoxic. The present study investigated whether Phyt (10-30 µM) could disturb important mitochondrial functions in rat heart mitochondria. We also determined the influence of Phyt (50-100 µM) on cell viability (MTT reduction) in cardiac cells (H9C2). Phyt markedly increased mitochondrial state 4 (resting) and decreased state 3 (ADP-stimulated) and uncoupled (CCCP-stimulated) respirations, besides reducing the respiratory control ratio, ATP synthesis and the activities of the respiratory chain complexes I-III, II, and II-III. This fatty acid also reduced mitochondrial membrane potential and induced swelling in mitochondria supplemented by exogenous Ca2+, which were prevented by cyclosporin A alone or combined with ADP, suggesting the involvement of the mitochondrial permeability transition (MPT) pore opening. Mitochondrial NAD(P)H content and Ca2+ retention capacity were also decreased by Phyt in the presence of Ca2+. Finally, Phyt significantly reduced cellular viability (MTT reduction) in cultured cardiomyocytes. The present data indicate that Phyt, at concentrations found in the plasma of patients with Refsum disease, disrupts by multiple mechanisms mitochondrial bioenergetics and Ca2+ homeostasis, which could presumably be involved in the cardiomyopathy of this disease.

[Effect of ethanol extract of Gastrodiae Rhizoma on mitochondrial dysfunction in cerebral ischemia-reperfusion injury].

This study observed the effects of ethanol extract of Gastrodiae Rhizoma(GE) on multiple aspects of mitochondrial dysfunction by investigating the mitochondria isolated from rat brains subjected to focal middle cerebral artery occlusion/reperfusion(MCAO/R). SD rats were randomly divided into a sham operation group(Sham), a model group(MCAO/R), low-and high-dose ethanol extract of GE groups(262.3 and 524.6 mg·kg~(-1)), and a nimodipine group(Nim, 15 mg·kg~(-1)). After continuous intragastric administration for 7 days, the MCAO/R model was induced in rats by the suture method. The neurological function and percentage of cerebral infarction volume were measured 24 h after reperfusion, and mitochondrial ultrastructure was observed under an electron microscope. Mitochondria were separated by gradient centrifugation and detected for reactive oxygen species(ROS), malondialdehyde(MDA), respiratory chain enzyme complex Ⅰ-Ⅳ activity, mitochondrial permeability transition pore(mPTP), mitochondrial membrane potential(MMP), and mitochondrial adenosine triphosphate(ATP) content. Enzyme-linked immunosorbent assay(ELISA) was used to detect the expression of cytochrome C(Cyt C) in different sites. TUNEL staining was used to observe the apoptosis of brain tissues in each group, and Western blot was used to detect the expression of B-cell lymphoma 2(Bcl-2) and Bcl-2-associated X protein(Bax) in brain tissues. The experimental results revealed that compared with the Sham group, the MCAO/R group showed evident neurological dysfunction and cerebral infarction(P<0.01) accompanied by mitochondrial swelling and crest disappearance, increased ROS level and MDA content, inhibited activity of respiratory chain enzyme complex Ⅰ-Ⅳ, abnormal opening of mPTP, and reduced MMP and mitochondrial ATP(P<0.01). Besides, many Cyt C was released from mitochondria into the cytoplasm to induce apoptosis(P<0.01). The ethanol extract of GE positively affected the behavior deficit and mitochondrial health of MCAO/R rats, with the manifestations of decreased production of ROS and MDA(P<0.01), potentiated activity of mitochondrial respiratory chain enzyme complex Ⅰ-Ⅳ, and restored the level of mPTP(P<0.05). In addition, the ethanol extract of GE reduced the loss of MMP and the escape of Cyt C to the cytoplasm(P<0.05), reduced the number of TUNEL positive cells(P<0.01) accompanied by the decrease in Bax and the up-regulation of Bcl-2(P<0.01), and increased the level of ATP(P<0.01). In conclusion, GE ethanol extract has a protective effect against MCAO/R-induced mitochondrial dysfunction, and the mechanism may be related to the regulation of oxidative stress, maintenance of functional morphology of mitochondria, and inhibition of endogenous apoptosis.

Effects of dietary supplementation with selenium yeast and jujube powder on mitochondrial oxidative damage and apoptosis of chicken.

The main objective of this study was to explore the effects of dietary selenium yeast and jujube powder on mitochondrial oxidative damage and cell apoptosis of broilers during postmortem aging, chicken breasts of broilers fed diets supplemented with different concentrations of selenium yeast and jujube powder were used as research subjects. With the prolongation of postmortem aging time, the levels of reactive oxygen species (ROS), carbonyl content, mitochondrial permeability transition pore (MPTP) openness, and mitochondrial membrane permeability increased significantly (P < 0.05). The contents of the sulfhydryl, mitochondrial membrane potential, shear force, and cytochrome C (Cyt-c) reduction level decreased significantly (P < 0.05). The activity of Caspase-3 and Caspase-9 increased from 0 to 24 h postmortem but fell from 24 to 72 h postmortem. Compared with the control group, dietary selenium yeast and jujube powder significantly reduced mitochondrial oxidative damage. They greatly increased the shear force, mitochondrial membrane potential, and Cyt-c reduction levels (P < 0.05). Among them, the combination group of high-dose selenium yeast and jujube powder had more significant effects on ROS scavenging, reducing cell membrane permeability, protecting cell membrane integrity, and increasing Cyt-c reduction level (P < 0.05). In conclusion, cell apoptosis intensifies during the chicken breast's aging time, and muscle tenderness continues. Still, different doses of dietary selenium yeast and jujube powder can inhibit mitochondrial oxidation to various degrees. The combined group of selenium yeast and jujube powder with 0.6 mg·kg-1 has the best effect. This study is of great significance for applying natural antioxidant ingredients such as selenium yeast and jujube powder in the development and utilization of poultry feed.

[Effect of Rehmanniae Radix Praeparata on energy metabolism in prefrontal cortex of rats with attention deficit hyperactivity disorder based on "static Yin and dynamic Yang" theory].

The present study investigated the effect of Rehmanniae Radix Praeparata(RRP) on the energy metabolism of prefrontal cortex(PFC) of spontaneously hypertensive rats with attention deficit hyperactivity disorder(ADHD) based on the "static Yin and dynamic Yang" theory.Thirty spontaneously hypertensive male rats aged 3 weeks were randomly divided into a model group, a methylphenidate(MPH) group(2 mg·kg~(-1)), and an RRP group(2.4 g·kg~(-1)).Wistar-Kyoto(WKY) male rats of the same age were assigned to the normal group.Rats were treated with corresponding drugs twice per day, and those in the model group and the normal group received the same volume of 0.9% sodium carboxymethyl cellulose(CMC-Na) solution by gavage.The open-field test was performed to evaluate the spontaneous and impulsive behaviors of rats before treatment and on the 4~(th) week after treatment.Four weeks after treatment, PFC was isolated and mitochondria were prepared.The content of adenosine triphosphate(ATP), adenosine diphosphate(ADP), and adenosine monophosphate(AMP) in the PFC was determined by high-performance liquid chromatography(HPLC), and energy charge(EC) was calculated.The parameters related to mitochondrial respiratory function were measured by the Clark oxygen electrode, specifically, state 3 respiration(ST3), state 4 respiration(ST4), and respiratory control rate(RCR).Enzymatic activities of succinate dehydrogenase(SDH), cytochrome C oxidase(COX), Na~+-K~+-ATPase, and Ca~(2+)-Mg~(2+)-ATPase were measured by chemical colorimetry.Mitochondrial permeability transition pore(mPTP) opening was measured by spectrophotometry.Protein expression of glucose transporter 1(GLUT1) and GLUT3 in PFC was tested by Western blot.Compared with the results in the model group, RRP could significantly reduce the total distance of movement, vertical times, and distance in the central area in the open field test(P<0.05 or P<0.01), increase the content of ATP and EC, decrease the content of AMP(P<0.05), elevate ST3 and RCR(P<0.05), potentiate activities of SDH, COX, Na~+-K~+-ATPase, and Ca~(2+)-Mg~(2+)-ATPase(P<0.05 or P<0.01), inhibit the opening of mPTP, and increase the expression levels of GLUT1 and GLUT3 proteins(P<0.05).It was inferred that RRP could inhibit hyperacti-vity and impulsivity by improving the energy metabolism disorder in PFC of ADHD rats, and its mechanism may be related to the improvement of mitochondrial respiratory function, potentiation of Na~+-K~+-ATPase, Ca~(2+)-Mg~(2+)-ATPase, and mitochondrial respiratory enzymes, inhibition of the opening of mPTP, and up-regulation of the expression of glucose transporter proteins.This study initially reveals the biological connotation of the "static Yin and dynamic Yang" theory in ADHD.

[Effect of Linggui Zhugan Decoction-containing serum on mitochondrial oxidative stress in cardiomyocytes: an exploration based on Nrf2/BNIP3 signaling pathway].

This study aimed to explore the effect of Linggui Zhugan Decoction(LGZGD)-containing serum on mitochondrial oxidative stress in cardiomyocytes based on the NF-E2-related factor2(Nrf2)/Bcl-2/adenovirus E1 B 19 kDa interacting protein(BNIP3) signaling pathway. After the preparation of LGZGD-containing serum and blank serum, H9 c2 cardiomyocytes were exposed to H_2O_2 for inducing oxidative stress in vitro. The H9 c2 cells were divided into four groups, namely normal control group, H_2O_2 model group, 20% blank serum group, and 20% LGZGD-containing serum group. After the cells were pre-treated with different types of serum for 12 h and cultured with 100 µmol·L~(-1 )H_2O_2 for 6 h, the reactive oxygen species(ROS) level in each group was detected by DCFH-DA, and the openness of mitochondrial permeability transition pore(mPTP) was measured using a calcein AM fluorescent probe. The expression levels of cytoplasmic cytochrome C(CytC), mitochondrial CytC, cytoplasmic and nuclear Nrf2, and BNIP3 were detected by Western blot. Nrf2-silenced H9 c2 cells were prepared by transfecting them with siRNA-Nrf2 for investigating the efficacy of LGZGD-containing serum in regulating ROS, mPTP, cytoplasmic and mitochondrial CytC, and BNIP3. The results showed that compared with the normal control group, H_2O_2 significantly increased the ROS content and mPTP openness(P<0.01), and the expression of Nrf2, BNIP3, and cytoplasmic CytC(P<0.01), and decreased the expression of mitochondrial CytC(P<0.01), without causing obvious change in cytoplasmic Nrf2. LGZGD-containing serum significantly lowered ROS content(P<0.01), inhibited mPTP openness(P<0.01), down-regulated the expression of cytoplasmic CytC and BNIP3(P<0.01), up-regulated mitochondrial CytC expression(P<0.01), and promoted Nrf2 nuclear translocation(P<0.05). However, after Nrf2 silencing, the reduced ROS production, diminished BNIP3 expression, and inhibited mPTP openness and CytC release induced by LGZGD-containing serum were reversed(P<0.01). These results have suggested that LGZGD-containing serum is able to alleviate the mitochondrial oxidative stress injury of cardiomyocytes by regulating the Nrf2/BNIP3 signaling pathway.

Coix seed oil regulates mitochondrial functional damage to induce apoptosis of human pancreatic cancer cells via the PTEN/PI3K/AKT signaling pathway.

BACKGROUND: Coix seed oil (CSO) has a wide range of anticancer effects. However, the mechanism of action against pancreatic cancer (PC) and regulation of mitochondria in vitro is still unclear. MATERIALS AND RESULTS: This research investigated the possible mechanism of CSO induction of PC cell apoptosis and regulating mitochondrial functional damage. Proliferation of PC cells, mitochondrial membrane potential (MMP), qualitative and quantitative analysis of PC cell apoptosis, openness of mitochondrial permeability transition pore, related protein expression, generation of reactive oxygen species (ROS), and gene expression were determined by cell counting kit-8, JC-1 staining, acridine orange and ethidium bromide staining, flow cytometry, calcein-AM/cobalt staining, western blotting, dichlorofluorescein diacetate probe, and quantitative real-time reverse transcription-polymerase chain reaction, respectively. We confirmed that PTEN protein was involved in CSO-induced PANC-1 cell apoptosis and mitochondrial functional damage. CSO induced depolarization of MMP, increased opening of the mitochondrial permeability transition pore, increased ROS production, and further increased mitochondrial damage. Additionally, CSO downregulated expression of p-AKT and p-PI3K proteins; upregulated protein expression of cleaved caspase-9, Bax, cleaved caspase-3 and cytochrome c; and downregulated expression of Bcl-2 by upregulating the PTEN gene. The corresponding protein expression was consistent with the gene expression level. Furthermore, the loss of function of PTEN protein reduces the ability of CSO to induce apoptosis of PANC-1 cells and damage to mitochondrial function. CONCLUSIONS: CSO induces apoptosis of PANC-1 PC cells by modulating mitochondrial functional impairment and related apoptotic molecules via PTEN, which may be closely related to the PI3K/AKT signaling pathway.

Puerarin mitigates oxidative injuries, opening of mitochondrial permeability transition pores and pathological damage associated with liver and kidney in Xanthium strumarium-intoxicated rats.

In this study, the therapeutic effects of puerarin on Xanthium strumarium toxicity, which can develop in many species and does not have a specific antidote, were investigated. A single dose of 100 g/kg X. strumarium seeds was administered by gavage to female Sprague-Dawley rats, 6 h following which 200 mg/kg puerarin was administered by the same route, with puerarin administration being repeated daily at the same time. After completing the application, the blood, liver and kidney tissues of the rats were examined. Further, the biochemical parameters, glucose, MDA, GSH, SOD, mitochondrial Ca2+ and mitochondrial permeability transition pore (mPTP) opening levels, apoptotic factors (TUNEL, Bax and Bcl-2), ATP synthase and histopathological changes of the experimental rats were examined. The results revealed that while the administration of X. strumarium resulted in increased blood AST, ALT, ALP, LDH, CK, BUN and creatinine levels, it decreased glucose levels. In addition, it increased the MDA levels in the tissues and significantly increased the oxidative stress levels by decreasing the GSH levels and SOD activity. X. strumarium caused an increase in the mitochondrial Ca2+ and mPTP opening levels. Moreover, it increased the immunohistochemically determined ATP synthase expression and histopathologically identified necrotic liver cell death rates. Owing to its antioxidant properties and inhibitory effects on mPTP opening, puerarin administered for therapeutic purposes decreased the oxidative damage caused by X. strumarium toxicity, blood biochemical parameter levels, mitochondrial Ca2+ levels, mPTP opening, ATP synthase expression and the percentage of necrotic cells. Hence, the reduction in the liver and kidney damage in X. strumarium toxicity by puerarin indicates its potential use as an antidote for X. strumarium poisoning.

Study on the mechanism of Wuzi-Yanzong-Wan-medicated serum interfering with the mitochondrial permeability transition pore in the GC-2 cell induced by atractyloside.

Wuzi-Yanzong-Wan (WZYZW) is a classic prescription for male infertility. Our previous investigation has demonstrated that it can inhibit sperm apoptosis via affecting mitochondria, but the underlying mechanisms are unclear. The purpose of the present study was to explore the actions of WZYZW on mitochondrial permeability transition pore (mPTP) in mouse spermatocyte cell line (GC-2 cells) opened by atractyloside (ATR). At first, WZYZW-medicated serum was prepared from rats following oral administration of WZYZW for 7 days. GC-2 cells were divided into control group, model group, positive group, as well as 5%, 10%, 15% WZYZW-medicated serum group. Cyclosporine A (CsA) was used as a positive control. 50 µmol·L-1 ATR was added after drugs incubation. Cell viability was assessed using CCK-8. Apoptosis was detected using flow cytometry and TUNEL method. The opening of mPTP and mitochondrial membrane potential (MMP) were detected by Calcein AM and JC-1 fluorescent probe respectively. The mRNA and protein levels of voltage-dependent anion channel 1 (VDAC1), cyclophilin D (CypD), adenine nucleotide translocator (ANT), cytochrome C (Cyt C), caspase 3, 9 were detected by RT-PCR (real time quantity PCR) and Western blotting respectively. The results demonstrated that mPTP of GC-2 cells was opened after 24 hours of ATR treatment, resulting in decreased MMP and increased apoptosis. Pre-protection with WZYZ-medicated serum and CsA inhibited the opening of mPTP of GC-2 cells induced by ATR associated with increased MMP and decreased apoptosis. Moreover, the results of RT-qPCR and WB suggested that WZYZW-medicated serum could significantly reduce the mRNA and protein levels of VDAC1 and CypD, Caspase-3, 9 and CytC, as well as a increased ratio of Bcl/Bax. However, ANT was not significantly affected. Therefore, these findings indicated that WZYZW inhibited mitochondrial mediated apoptosis by attenuating the opening of mPTP in GC-2 cells. WZYZW-medicated serum inhibited the expressions of VDAC1 and CypD and increased the expression of Bcl-2, which affected the opening of mPTP and exerted protective and anti-apoptotic effects on GC-2 cell induced by ATR.

Modulatory Potential of Citrus sinensis and Moringa oleifera Extracts and Epiphytes on Rat Liver Mitochondrial Permeability Transition Pore.

BACKGROUND: Bioactive agents from medicinal and dietary plants have been reported to modulate the mitochondrial membrane permeability transition pores. OBJECTIVE: This study investigated the in vitro effects of C. sinensis (CSE) and M. oleifera (MOE) methanol leaf extracts and their epiphytes (CEP and MEP) on mitochondria permeability transition pores. METHODS: In vitro antioxidant activities of the extracts were determined using standard procedures and quantification of polyphenolic compounds in the extract was done using HPLC-DAD. Opening of the mitochondrial permeability transition pores was assessed as mitochondrial swelling and observed spectrophotometrically as changes in absorbance under succinate-energized conditions. Cytochrome c release was also assessed spectrophotometrically. RESULTS: From the results, CSE, MOE, CEP, and MEP inhibited lipid peroxidation and scavenged nitric oxide and DPPH radicals in a concentration-dependent manner. All extracts exhibited greater ferric reducing antioxidant potential. More so, the results showed that CSE, MOE, CEP, and MEP possess the substantive amount of total flavonoids and total phenolics. CSE and MOE had higher total flavonoids and total phenolic content when compared with the epiphytes. HPLC-DAD results revealed Tangeretin as the most abundant in CSE; Eriocitrin in citrus epiphytes; Moringine in MOE and Flavones in moringa epiphytes. All extracts inhibited calcium-induced opening of the pores in a concentration- dependent manner, with C. sinensis leaf extract (CSE) and moringa epiphyte (MEP) being the most potent in this regard with no significant release of cytochrome c at all concentrations. CONCLUSION: The results suggest that CSE and MEP have bioactive agents, which could be useful in the management of diseases where too much apoptosis occurs characterized by excessive tissue wastage, such as neurodegenerative conditions.

Chronic magnesium deficiency causes reversible mitochondrial permeability transition pore opening and impairs hypoxia tolerance in the rat heart.

Chronic magnesium (Mg) deficiency induces and exacerbates various cardiovascular diseases. We previously investigated the mechanisms underlying decline in cardiac function caused by chronic Mg deficiency and the effectiveness of Mg supplementation on this decline using the Langendorff-perfused isolated mouse heart model. Herein, we used the Langendorff-perfused isolated rat heart model to demonstrate the chronic Mg-deficient rats (Mg-deficient group) had lower the heart rate (HR) and left ventricular pressure (LVDP) than rats with normal Mg levels (normal group). Furthermore, decline in cardiac function due to hypoxia/reoxygenation injury was significantly greater in the Mg-deficient group than in the normal group. Experiments on mitochondrial permeability transition pore (mPTP) using isolated mitochondria revealed that mitochondrial membrane was fragile in the Mg-deficient group, implying that cardiac function decline through hypoxia/reoxygenation injury is associated with mitochondrial function. Mg supplementation for chronic Mg-deficient rats not only improved hypomagnesemia but also almost completely restored cardiac and mitochondrial functions. Therefore, proactive Mg supplementation in pathological conditions induced by Mg deficiency or for those at risk of developing hypomagnesemia may suppress the development and exacerbation of certain disease states.

Cajanus cajan ameliorated CCl4-induced oxidative stress in Wistar rats via the combined mechanisms of anti-inflammation and mitochondrial-membrane transition pore inhibition.

ETHNOPHARMACOLOGICAL RELEVANCE: Liver diseases is a public health issue in sub-saharan Africa and has been reported to be the major cause of many hospital admissions. Oxidative stress, mitochondrial dysfunction and inflammation play important roles in several diseases including liver injury. Cajanus cajan is an indigenous medicinal plant useful in the traditional treatment of jaundice, inflammation and liver injury. AIM OF STUDY: This study assessed the effects of methanol extract Cajanus cajan (MECC) on mitochondrial permeability transition (mPT) pore opening, biomarkers of oxidative stress and inflammation in CCl4-induced liver injury in rats. METHODS: Wistar albino rats (200-210g) were completely randomized into five (5) groups of six animals each. Group I (control) was given distilled water orally once daily. Animals in group II were administered CCl4 in parafin (1:1) at a dose of 0.5 mL/kg i.p on the seventh day. Animals in groups III, IV and V were administered methanol extract of Cajanus cajan (MECC) at doses of 100, 200 mg/kg and silymarin (100 mg/kg) respectively for 7 days prior to a single intraperitoneal dose of CCl4. After 24 h of CCl4 treatment, serum and liver tissues were collected. Mitochondrial permeability transition (mPT) pore opening, mitochondrial ATPase activities and biomarkers of oxidative stress were determined spectrophotometrically. Tumor necrosis factor (TNFα), NF-κB and COX-2 were determined by immunohistochemistry and the phytochemicals present in the extract were determined by GC-MS. RESULTS: Liver enzyme (AST, ALP, ALT and γGT) activities and MDA levels were significantly decreased in rats pretreated with MECC at the dose of 100, 200 and silymarin (100 mg/kg) when compared to the rats administered CCl4 alone (p < 0.05). GSH, GST, CAT and SOD increased and the expressions of TNFα, NF-κB and COX- 2 were also reduced when compared to the CCl4- treated animals. In addition, the liver histopathological analyses revealed that MECC markedly alleviated inflammatory cell infiltration, hepatic fibrosis, hepatocyte ballooning, necrosis and severe apoptosis of hepatocytes induced by CCl4. GC-MS analysis yielded 23 compounds including flavonoids, terpenoids and fatty acids. CONCLUSION: Cajanus cajan leaf extract elicited hepatoprotective action on CCl4-induced liver injury via inhibition of mPT pore opening, prevention of CCl4-induced hepatic oxidative stress and suppression of inflammatory response thus it may become useful for chemoprevention of liver injury. This supports its traditional use.

Kigelia africana (Lam.) Benth leaf extract inhibits rat brain and liver mitochondrial membrane permeability transition pore opening.

The effect of Kigelia africana on mitochondrial membrane permeability transition has not been explored. In this study, the effect of a solvent fraction of Kigelia africana leaf extract on mitochondrial membrane permeability transition of rat brain and liver was evaluated. A methanol extract of K. africana leaves was fractionated into different solvents by vacuum liquid chromatography and following preliminary screening, the dichloromethane:ethylacetate (1:1) fraction was selected for further assays. Constituent phytochemicals in the fraction were revealed by thin-layer chromatography and further purification was carried out to characterize the compounds. Brain and liver mitochondria were isolated and used for mitochondrial membrane permeability transition and adenosine triphosphatase assays. Exogenous Ca2+ and Al3+ were used to trigger the mitochondrial membrane permeability transition opening. Physicochemical properties revealed by thin-layer chromatography showed that the isolated compounds were flavonoids. The extract inhibited mitochondrial membrane permeability transition opening in the presence and absence of triggering agents in brain and liver mitochondria. It also inhibited mitochondrial lipid peroxidation and adenosine triphosphatase activity. These results suggest that the extract can limit the rate of apoptosis via inhibition of mitochondrial membrane permeability transition which is pivotal to the mitochondrial apoptotic pathway and is an important therapeutic target in some pathological conditions.

Alisporivir Treatment Alleviates Mitochondrial Dysfunction in the Skeletal Muscles of C57BL/6NCrl Mice with High-Fat Diet/Streptozotocin-Induced Diabetes Mellitus.

Diabetes mellitus is a systemic metabolic disorder associated with mitochondrial dysfunction, with mitochondrial permeability transition (MPT) pore opening being recognized as one of its pathogenic mechanisms. Alisporivir has been recently identified as a non-immunosuppressive analogue of the MPT pore blocker cyclosporin A and has broad therapeutic potential. The purpose of the present work was to study the effect of alisporivir (2.5 mg/kg/day i.p.) on the ultrastructure and functions of the skeletal muscle mitochondria of mice with diabetes mellitus induced by a high-fat diet combined with streptozotocin injections. The glucose tolerance tests indicated that alisporivir increased the rate of glucose utilization in diabetic mice. An electron microscopy analysis showed that alisporivir prevented diabetes-induced changes in the ultrastructure and content of the mitochondria in myocytes. In diabetes, the ADP-stimulated respiration, respiratory control, and ADP/O ratios and the level of ATP synthase in the mitochondria decreased, whereas alisporivir treatment restored these indicators. Alisporivir eliminated diabetes-induced increases in mitochondrial lipid peroxidation products. Diabetic mice showed decreased mRNA levels of Atp5f1a, Ant1, and Ppif and increased levels of Ant2 in the skeletal muscles. The skeletal muscle mitochondria of diabetic animals were sensitized to the MPT pore opening. Alisporivir normalized the expression level of Ant2 and mitochondrial susceptibility to the MPT pore opening. In parallel, the levels of Mfn2 and Drp1 also returned to control values, suggesting a normalization of mitochondrial dynamics. These findings suggest that the targeting of the MPT pore opening by alisporivir is a therapeutic approach to prevent the development of mitochondrial dysfunction and associated oxidative stress in the skeletal muscles in diabetes.

[Macrophage migration inhibitory factor meditates MPP+/MPTP-induced NLRP3 inflammasome activation in microglia cells].

OBJECTIVE: To explore the mechanisms of macrophage migration inhibitory factor (MIF)/nucleus factor-κB (NF-κB) in mediating 1-methyl-4-phenylpyridinium iodide (MPP +)/1-Methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP)-induced activation of Nod-like receptor protein 3 (NLRP3) inflammasome in microglials and the its effects on neurons. METHODS: Murine microglial cell line Bv-2 was infected with a lentivirus carrying MIF shRNA for MIF knockdown and then treated with MPP+. The total protein levels of NLRP3, caspase-1, p65 and p65 in the cell nuclei and cytoplasm were detected. ELISA was used to detect the levels of IL-1ß and IL-18 in the culture supernatant, which served as the conditioned culture medium for MN9D cells, whose TH expression level was detected using Western blotting. The effect of stereotactic injection of an adeno-associated virus (AAV) carrying MIF shRNA on behaviors was assessed in a C57BL/6 mouse model of Parkinson disease (PD) induced by intraperitoneal MPTP injection. TH and Iba-1 immunohistochemistry was used to evaluate the number of substantia nigra neurons and the activation of microglia cells, and the protein expressions of MIF, NLRP3 and TH in the substantia nigra were detected with Western blotting. RESULTS: MPP+ significantly increased NLRP3 and MIF expressions in Bv-2 cells (P < 0.05). MIF knockdown in Bv-2 cells significantly lowered NLRP3 and caspase-1 protein expressions and IL-1ß and IL-18 levels in the culture supernatant (P < 0.05) without affecting total protein level of p65. Bv-2 cells with MIF knockdown showed significantly lowered p65 protein expression in the nuclei but increased p65 expression in the cytoplasm (P < 0.05). The conditioned medium derived from Bv-2 cells with MIF knockdown, as compared with that from than MPP +-treated Bv-2 cells, significantly increased TH expression in MN9D cells (P=0.01). Compared with those in MPTP group, the mice receiving injections of AAV-MIF-shRNA had higher scores in pole test and open field test with lower scores in traction test, and showed increased TH-positive neurons, decreased Iba-1 microglia cell activation, reduced expressions of MIF and NLRP3, and increased expression of TH in he substantia nigra (all P < 0.05). CONCLUSION: Inhibition of MIF can reduce the expression of NLRP3 inflammasomes and inflammatory factor caused by MPP+ in microglia cells to relieve the damage of dopaminergic neurons and alleviate microglia cell activation, thus offering protection against neuroinflammation in Parkinson's disease.

Selenium-containing protein from selenium-enriched Spirulina platensis antagonizes oxygen glucose deprivation-induced neurotoxicity by inhibiting ROS-mediated oxidative damage through regulating MPTP opening.

CONTEXT: Selenium-containing protein from selenium-enriched Spirulina platensis (Se-SP) (syn. Arthrospira platensis [Microcoleaceae]) showed novel antioxidant activity. However, the protective effect of Se-SP against oxygen glucose deprivation (OGD)-induced neural apoptosis has not been reported yet. OBJECTIVE: To verify whether Se-SP can inhibit OGD-induced neural apoptosis and explore the underlying mechanism. MATERIALS AND METHODS: Primary hippocampal neurons were separated from Sprague-Dawley (SD) rats. 95% N2 + 5% CO2 were employed to establish OGD model. Neurons were treated with 5 and 10 µg/mL Se-SP under OGD condition for 6 h. Neurons without treatment were the control group. Neural viability and apoptosis were detected by MTT, immunofluorescence and western blotting methods. RESULTS: Se-SP significantly improved neuronal viability (from 57.2% to 94.5%) and inhibited apoptosis in OGD-treated primary neurons (from 45.6% to 6.3%), followed by improved neuronal morphology and caspases activation. Se-SP co-treatment also effectively suppressed OGD-induced DNA damage by inhibiting ROS accumulation in neurons (from 225.6% to 106.3%). Additionally, mitochondrial dysfunction was also markedly improved by Se-SP co-treatment via balancing Bcl-2 family expression. Moreover, inhibition of mitochondrial permeability transition pore (MPTP) by CsA (an MPTP inhibitor) dramatically attenuated OGD-induced ROS generation (from 100% to 56.2%), oxidative damage, mitochondrial membrane potential (MPP) loss (from 7.5% to 44.3%), and eventually reversed the neuronal toxicity and apoptosis (from 57.4% to 79.6%). DISCUSSION AND CONCLUSIONS: Se-SP showed enhanced potential to inhibit OGD-induced neurotoxicity and apoptosis by inhibiting ROS-mediated oxidative damage through regulating MPTP opening, indicating that selenium-containing protein showed broad application in the chemoprevention and chemotherapy against human ischaemic brain injury.

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

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

Modulatory effect of methanol extract of Annona muricata stem bark on mitochondrial membrane permeability transition pore in normal rat liver and monosodium glutamate-induced uterine hyperplasia.

OBJECTIVES: Uterine fibroids are benign tumors that develop in many women of reproductive age. Surgery is the main approach to treatment while other options are also associated with adverse effects. Studies have shown that certain bioactive agents present in medicinal plants elicit their anti-tumor activity by induction of mitochondrial permeability transition (mPT) opening. This research therefore aimed at investigating the effect of methanol extract of Annona muricata (MEAM) on mPT pore opening in normal and monosodium glutamate-induced uterine hyperplasia using female Wistar rats. METHODS: Mitochondria, isolated from rat liver were exposed to different concentrations (20, 60, 100, 140 and 180 µg/mL) of MEAM. The mPT pore opening, cytochrome c release, mitochondrial ATPase (mATPase) activity and the percentage lipid peroxidation were assessed spectrophotometrically. Histological effects of MEAM on the liver, brain and uterus of normal and MSG-treated rats were investigated. RESULTS: The in vitro results showed a significant induction of mPT pore opening by 2.4, 4.2 and 6.4 folds, release of cytochrome c and enhancement of mATPase activity at 100,140 and 180 µg/mL, respectively. However, oral administration of MEAM did not induce mPT pore opening, neither any significant release of cytochrome c nor enhancement of mATPase activity at all the dosages used. However, histological assay revealed the presence of MSG-induced cellular damage and uterine hyperplasia which was ameliorated by MEAM co-administration. CONCLUSIONS: These findings suggest that MEAM contains phytochemicals that can ameliorate MSG-induced damage and uterine hyperplasia in rats; however, the mechanism might not be via upregulation of mitochondrial-mediated apoptosis.

Hydroxysafflor yellow A alleviates cerebral ischemia reperfusion injury by suppressing apoptosis via mitochondrial permeability transition pore.

BACKGROUND: Mitochondria are key cellular organelles that are essential for cell fate decisions. Hydroxysafflor yellow A (HSYA) has displayed an impressively essential role in protection of cerebral ischemia/reperfusion (I/R). However, the mitochondrial effect of HSYA on Brain Microvascular Endothelial Cells (BMECs) under I/R remains to be largely unclear. PURPOSE: To evaluate the protective effects of HSYA-mediated mitochondrial permeability transition pore (mPTP) on cerebral I/R injury and its mechanism. METHODS: Cerebral I/R injury was established by the model of Middle cerebral artery occlusion (MCAO) in rats. Furthermore, to further clarify the relevant mechanism of HSYA's effects on mPTP, inhibition of extracellular regulated protein kinases (ERK) with U0126 and transfect with Cyclophilin D (CypD) SiRNA to reversely verified whether the protective effects of HSYA were exerted by regulating the Mitogen-activated protein kinase kinase (MEK)/ERK/CypD pathway. RESULTS: HSYA treatment significantly increased BMECs viability, decreased the generation of ROS, opening of mPTP and translocation of cytochrome c after OGD/R. In addition to inhibited CypD, HSYA potentiated MEK and increased phosphorylation of ERK expression in BMECs, inhibited apoptosis mediated by mitochondrial. Notably, HSYA also significantly ameliorated neurological deficits and decreased the infarct volume in rats. CONCLUSION: HSYA reduced the CytC export from mitochondrial by inhibited the open of mPTP via MEK/ERK/CypD pathway, contributing to the protection of I/R. Thus, our study not only revealed novel mechanisms of HSYA for its anti-I/R function, but also provided a template for the design of novel mPTP inhibitor for the treatment of various mPTP-related diseases.

Aconitine attenuates mitochondrial dysfunction of cardiomyocytes via promoting deacetylation of cyclophilin-D mediated by sirtuin-3.

ETHNOPHARMACOLOGICAL RELEVANCE: Aconite is a processed product of seminal root of perennial herbaceous plant Aconitum Carmichaclii Debx. of Ranunculaceae. It has the effects of warming and tonifying heart yang and restoring yang to save from collapse. Aconitine is the main effective constituent of aconite and used to prevent and treat heart disease. However, how aconitine exerts myocardial protection is still poorly understood. AIM OF THE STUDY: The present study aimed to investigate the effects of aconitine on mitochondrial dysfunction and explore its mechanism of action. MATERIALS AND METHODS: The model of myocardial injury was induced by Angiotensin II (Ang II) (1 × 10-6 mol L-1), and H9c2 cells were incubated with different concentrations of aconitine. The effect of aconitine on mitochondrial was determined by flow cytometry, transmission electron microscopy, luciferase, Seahorse technique and Western blot. The effects of aconitine on sirtuin-3 (Sirt3) activity and Cyclophilin D (CypD) acetylation were detected by immunofluorescence, RT-PCR and co-immunoprecipitation. RESULTS: We demonstrate that aconitine alleviates the energy metabolic dysfunction of H9c2 cells by activating Sirt3 to deacetylate CypD and inhibiting mitochondrial permeability transition pore (mPTP) opening. In cardiomyocytes, aconitine significantly reduced mitochondrial fragmentation, inhibited acetylation of CypD, suppressed the mPTP opening, mitigated mitochondrial OXPHOS disorders, and improved the synthesis ability of ATP. In contrast, Sirt3 deficiency abolished the effects of aconitine on mPTP and OXPHOS, indicating that aconitine improves mitochondrial function by activating Sirt3. CONCLUSIONS: These results showed that aconitine attenuated the energy metabolism disorder by promoting Sirt3 expression and reducing CypD-mediated mPTP excess openness, rescuing mitochondrial function. Improve mitochondrial function may be a therapeutic approach for treating heart disease, which will generate fresh insight into the cardioprotective of aconitine.

Study on the protective effects of danshen-honghua herb pair (DHHP) on myocardial ischaemia/reperfusion injury (MIRI) and potential mechanisms based on apoptosis and mitochondria.

CONTEXT: Danshen, the dried root and rhizome of Salvia miltiorrhiza Bunge (Labiatae) and honghua, the dried flower of Carthamus tinctorius L. (Compositae) as the herb pair was used to treat cardiovascular diseases (CVD). OBJECTIVE: To study the effects of DHHP on MIRI and mechanisms based on apoptosis and mitochondria. MATERIALS AND METHODS: 36 SD rats (n = 6) were randomly divided into control group (Con), the ischaemia-reperfusion group (IR), positive control (Xinning tablets, XNT, 1 g/kg/d) and DHHP (1.2, 2.4, and 4.8 g/kg/d). Except for Con, the other groups were intragastrically administrated for 5 d, the rat hearts were isolated to establish the MIRI model in vitro for evaluating the effects of DHHP on MIRI. 24 SD rats (n = 6) were randomly divided into Con, IR, DPPH2.4 (2.4 g/kg/d) and DPPH 2.4 + Atractyloside (ATR) (2.4 + 5 mg/kg/d), administered intragastrically for 5 d, then treated with ATR (5 mg/kg/d) by intraperitoneal injection in DPPH2.4 + ATR group, took rat hearts to establish MIRI model in vitro for revealing mechanism. RESULTS: Myocardial infarct sizes were, respectively, 0.35%, 40.09%, 15.84%, 30.13%, concentrations of NAD+ (nmol/gw/w) were 144, 83, 119, and 88, respectively, in Con, IR, DHHP2.4, DHHP2.4 + ATR group. Cleaved caspase-3 were 0.3, 1.6, 0.5 and 1.3% and cleaved caspase-9 were 0.2, 1.1, 0.4 and 0.8%, respectively, in Con, IR, DHHP2.4 and DHHP2.4 + ATR group. The beneficial effects of DHHP on MIRI were reversed by ATR. CONCLUSIONS: The improvement of MIRI by DHHP may be involved in inhibiting MPTP opening, decreasing oxidative damage, alleviating ischaemic injury and inhibiting cardiomyocyte apoptosis.