Enhanced Axon Guidance and Synaptic Markers in Rat Brains Using Ferric-Tannic Nanoparticles.

Ferric-tannic nanoparticles (FTs) are now considered to be new pharmaceuticals appropriate for the prevention of brain aging and related diseases. We have previously shown that FTs could activate axon guidance pathways and cellular clearance functioning in neuronal cell lines. Herein, we further investigated whether FTs could activate the two coordinated neuronal functions of axon guidance and synaptic function in rat brains and neuronal cell lines. A single intravenous injection of safe dose of FTs has been shown to activate a protein expression of axon attractant Netrin-1 and neurotransmitter receptor GABRA4 in the cerebral cortexes of male Wistar rats. According to RNA-seq with targeted analysis, axon guidance and synapses have been enriched and Ephrin membered genes have been identified as coordinating a network of genes for such processes. In vitro, as expected, FTs are also found to activate axon guidance markers and promote neuronal tubes in neuronal cell lines. At the same time, presynaptic markers (synaptophysin), post-synaptic markers (synapsin), and GABRA4 neurotransmitter receptors have been found to be activated by FTs. Interestingly, synaptophysin has been found to localize along the promoted neuronal tubes, suggesting that enhanced axon guidance is associated with the formation and transportation of pre-synaptic vesicles. Preliminarily, repeated injection of FTs into adult rats every 3 days for 10 times could enhance an expression of synaptophysin in cerebral cortex, as compared to control rat. This work demonstrates that FTs can be used for activating brain function associated with axon guidance and synaptic function.

Early Detection of Hepatocarcinogenicity in Rats Using MRI with Ferric-Tannic Nanoparticles.

Herein, we present molecular nanoparticles of ferric-tannic complexes (so called ferric-tannic nanoparticles, FT NPs) used to enhance the MRI signal in the early stage of hepatocarcinoma. FT NPs were found to accumulate in the hepatic parenchyma without tumor nodules of Wistar rats in which hepatocarcinogenicity had been induced using diethylnitrosamine (DEN). The MRI enhancement and accumulation of FT NPs were clearly observed in the early phase of hepatocarcinogenicity, which was possibly modulated by various solute carrier family members present in the entire hepatic parenchyma of the DEN-induced rats. These findings suggest that MRI with FT NPs is promising for the assessment of the early stage of hepatocarcinoma.

p38 MAPK Inhibitor (SB203580) and Metformin Reduces Aortic Protein Carbonyl and Inflammation in Non-obese Type 2 Diabetic Rats.

Microvascular and macrovascular diseases are the main causes of morbidity in type 2 diabetes patients through chronic hyperglycaemic condition via oxidative stress and inflammation. Reactive oxygen species (ROS) activate p38 MAPK phosphorylation and inflammation which enhances protein modification by carbonylation. The use of metformin and a p38 MAPK inhibitor is hypothesised to reduce ROS production and inflammation but effects of metformin and p38 MAPK inhibitor (SB203580) on ROS production and inflammation in vascular type 2 diabetes mellitus non-obese (T2DM) have not been investigated. The Goto-Kakizaki rat T2DM model was divided into three groups as T2DM, T2DM treated with 15 mg/kg bw metformin and T2DM treated with 2 mg/kg bw SB203580 for 4 weeks. Rat aortas were isolated and protein carbonyl (PC) contents were measured by spectrophotometric DNPH assay. Aortic IL-1ß level was determined by ELISA. Results showed that aortic PC contents in the T2DM group were significantly higher than in non-diabetic rats. Treatment with metformin or SB203580 significantly reduced PC contents while only metformin significantly reduced IL-1ß levels. Findings indicated that metformin reduced ROS production and inflammation in diabetic vessels and possibly reduce vascular complications in non-obese T2DM.

Combination of metformin and p38 MAPK inhibitor, SB203580, reduced myocardial ischemia/reperfusion injury in non-obese type 2 diabetic Goto-Kakizaki rats.

Diabetic cardiomyopathy, especially myocardial ischemia reperfusion (I/R) injury, is a major cause of morbidity and mortality in type 2 diabetic patients. The increasing of basal p38 MAP Kinase (p38 MAPK) activation is a major factor that aggravates cardiac death on diabetic cardiomyopathy. In addition, metformin also shows cardio-protective effects on myocardial ischemia/reperfusion injury. In this study, we investigated the effect of the combination between metformin and p38 MAPK inhibitor (SB203580) in diabetic rats subjected to I/R injury. H9c2 cells were induced into a hyperglycemic condition and treated with metformin, SB203580 or the combination of metformin and SB203580. In addition, cells in both the presence and absence of drug treatment were subjected to simulated ischemia/reperfusion injury. Cell viability and cellular reactive oxygen species (ROS) were determined. Moreover, the Goto-Kakizaki (GK) rats were treated with metformin, SB203580, and the combination of metformin and SB203580 for 4 weeks. Diabetic parameters and cardiac functions were assessed. Finally, rat hearts were induced ischemia/reperfusion injury for the purpose of infarct size analysis and determination of signal transduction. A high-glucose condition did not reduce cell viability but significantly increased ROS production and significantly decreased cell viability after induced sI/R. Treatment using drugs was shown to reduce ROS generation and cardiac cell death. The GK rats displayed diabetic phenotype by increasing diabetic parameters and these parameters were significantly decreased when treated with drugs. Treatment with metformin or SB203580 could significantly reduce the infarct size. Interestingly, the combination of metformin and SB203580 could enhance cardio-protective ability. Myocardial I/R injury significantly increased p38 MAPK phosphorylation, Bax/Bcl-2 ratio and caspase-3 level. Treatment with drugs significantly decreased the p38 MAPK phosphorylation, Bax/Bcl-2 ratio, caspase-3 level and increased Akt phosphorylation. In conclusion, using the combination of metformin and SB203580 shows positive cardio-protective effects on diabetic ischemic cardiomyopathy.

The cardioprotective effects of secretory leukocyte protease inhibitor against myocardial ischemia/reperfusion injury.

Protease enzymes generated from injured cells and leukocytes are the primary cause of myocardial cell damage following ischemia/reperfusion (I/R). The inhibition of protease enzyme activity via the administration of particular drugs may reduce injury and potentially save patients' lives. The aim of the current study was to investigate the cardioprotective effects of treatment with recombinant human secretory leukocyte protease inhibitor (rhSLPI) on in vitro and ex vivo models of myocardial I/R injury. rhSLPI was applied to isolated adult rat ventricular myocytes (ARVMs) subjected to simulated I/R and to ex vivo murine hearts prior to I/R injury. Cellular injury, cell viability, reactive oxygen species (ROS) levels, and levels of associated proteins were assessed. The results demonstrated that administration of rhSLPI prior to or during sI/R significantly reduced the death and injury of ARVMs and significantly reduced intracellular ROS levels in ARVMs during H2O2 stimulation. In addition, treatment of ARVMs with rhSLPI significantly attenuated p38 mitogen-activated protein kinase (MAPK) activation and increased the activation of Akt. Furthermore, pretreatment of ex vivo murine hearts with rhSLPI prior to I/R significantly decreased infarct size, attenuated p38 MAPK activation and increased Akt phosphorylation. The results of the current study demonstrated that treatment with rhSLPI induced a cardioprotective effect and reduced ARVM injury and death, intracellular ROS levels and infarct size. rhSLPI also attenuated p38 MAPK phosphorylation and activated Akt phosphorylation. These results suggest that rhSLPI may be developed as a novel therapeutic strategy of treating ischemic heart disease.

Overexpression and pre-treatment of recombinant human Secretory Leukocyte Protease Inhibitor (rhSLPI) reduces an in vitro ischemia/reperfusion injury in rat cardiac myoblast (H9c2) cell.

One of the major causes of cardiac cell death during myocardial ischemia is the oversecretion of protease enzymes surrounding the ischemic tissue. Therefore, inhibition of the protease activity could be an alternative strategy for preventing the expansion of the injured area. In the present study, we investigated the effects of Secretory Leukocyte Protease Inhibitor (SLPI), by means of overexpression and treatment of recombinant human SLPI (rhSLPI) in an in vitro model. Rat cardiac myoblast (H9c2) cells overexpressing rhSLPI were generated by gene delivery using pCMV2-SLPI-HA plasmid. The rhSLPI-H9c2 cells, mock transfected cells, and wild-type (WT) control were subjected to simulated ischemia/reperfusion (sI/R). Moreover, the treatment of rhSLPI in H9c2 cells was also performed under sI/R conditions. The results showed that overexpression of rhSLPI in H9c2 cells significantly reduced sI/R-induced cell death and injury, intracellular ROS level, and increased Akt phosphorylation, when compared to WT and mock transfection (p <0.05). Treatment of rhSLPI prior to sI/R reduced cardiac cell death and injury, and intra-cellular ROS level. In addition, 400 ng/ml rhSLPI treatment, prior to sI, significantly inhibited p38 MAPK phosphorylation and rhSLPI at 400-1000 ng/ml could increase Akt phosphorylation.

Phosphodiesterase-3 inhibitor (cilostazol) attenuates oxidative stress-induced mitochondrial dysfunction in the heart.

BACKGROUND: Cilostazol is a type 3 phosphodiesterase inhibitor which has been previously demonstrated to prevent the occurrence of tachyarrhythmia and improve defibrillation efficacy. However, the mechanism for this beneficial effect is still unclear. Since cardiac mitochondria have been shown to play a crucial role in fatal cardiac arrhythmias and that oxidative stress is one of the main contributors to arrhythmia generation, we tested the effects of cilostazol on cardiac mitochondria under severe oxidative stress. METHODS: Mitochondria were isolated from rat hearts and treated with H2O2 to induce oxidative stress. Cilostazol, at various concentrations, was used to study its protective effects. Pharmacological interventions, including a mitochondrial permeability transition pore (mPTP) blocker, cyclosporine A (CsA), and an inner membrane anion channel (IMAC) blocker, 4'-chlorodiazepam (CDP), were used to investigate the mechanistic role of cilostazol on cardiac mitochondria. Cardiac mitochondrial reactive oxygen species (ROS) production, mitochondrial membrane potential change and mitochondrial swelling were determined as indicators of cardiac mitochondrial function. RESULTS: Cilostazol preserved cardiac mitochondrial function when exposed to oxidative stress by preventing mitochondrial depolarization, mitochondrial swelling, and decreasing ROS production. CONCLUSIONS: Our findings suggest that cardioprotective effects of cilostazol reported previously could be due to its prevention of cardiac mitochondrial dysfunction caused by severe oxidative stress.

Dipeptidyl peptidase-4 inhibitor reduces infarct size and preserves cardiac function via mitochondrial protection in ischaemia-reperfusion rat heart.

AIM: We hypothesized that dipeptidyl peptidase (DPP)-4 inhibitor (vildagliptin) reduces fatal arrhythmias, cardiac dysfunction and infarct size caused by ischaemia-reperfusion (I/R) injury via its attenuation of cardiac mitochondrial dysfunction. METHODS: In total, 26 rats were randomized to receive either 1 mL normal saline solution or 2.0 mg/kg vildagliptin intravenously (n = 13/group) 30 min prior to a 30-min left anterior descending coronary artery occlusion, followed by a 120-min reperfusion. Arrhythmia scores, cardiac functions, infarct size and mitochondrial function were evaluated. RESULTS: Vildagliptin reduced the infarct size by 44% and mitigated cardiac dysfunction by preserving cardiac function without altering the incidence of cardiac arrhythmias. Vildagliptin increased expression of Bcl-2 and pro-caspase3 in the ischaemic area, whereas Bax and phosphorylated-connexin43/total-connexin43 were not altered. Vildagliptin attenuated cardiac mitochondrial dysfunction by reducing the reactive oxygen species level and mitochondrial swelling. CONCLUSIONS: DPP-4 inhibitor provides cardioprotection by reducing the infarct size and ameliorating cardiac dysfunction in I/R hearts by attenuating cardiac mitochondrial dysfunction and cardiomyocyte apoptosis.

Mitochondrial calcium uniporter blocker effectively prevents brain mitochondrial dysfunction caused by iron overload.

AIMS: Although iron overload induces oxidative stress and brain mitochondrial dysfunction, and is associated with neurodegenerative diseases, brain mitochondrial iron uptake has not been investigated. We determined the role of mitochondrial calcium uniporter (MCU) in brain mitochondria as a major route for iron entry. We hypothesized that iron overload causes brain mitochondrial dysfunction, and that the MCU blocker prevents iron entry into mitochondria, thus attenuating mitochondrial dysfunction. MAIN METHODS: Isolated brain mitochondria from male Wistar rats were used. Iron (Fe(2+) and Fe(3+)) at 0-286 µM were applied onto mitochondria at various incubation times (5-30 min), and the mitochondrial function was determined. Effects of MCU blocker (Ru-360) and iron chelator were studied. KEY FINDINGS: Both Fe(2+) and Fe(3+) entered brain mitochondria and caused mitochondrial swelling in a dose- and time-dependent manner, and caused mitochondrial depolarization and increased ROS production. However, Fe(2+) caused more severe mitochondrial dysfunction than Fe(3+). Although all drugs attenuated mitochondrial dysfunction caused by iron overload, only an MCU blocker could completely prevent ROS production and mitochondrial depolarization. SIGNIFICANCE: Our findings indicated that iron overload caused brain mitochondrial dysfunction, and that an MCU blocker effectively prevented this impairment, suggesting that MCU could be the major portal for brain mitochondrial iron uptake.

Calcium-induced cardiac mitochondrial dysfunction is predominantly mediated by cyclosporine A-dependent mitochondrial permeability transition pore.

BACKGROUND AND AIMS: Cardiac mitochondrial Ca(2+) overload plays a critical role in mechanical and electrical dysfunction leading to cardiac cell death and fatal arrhythmia. Because Ca(2+) overload is related to mitochondrial permeability transition, reactive oxygen species (ROS) production and membrane potential (ΔΨm) dissipation, we probed the mechanistic association between Ca(2+) overload, oxidative stress, mitochondrial permeability transition pore (mPTP) and mitochondrial calcium uniporter (MCU) in isolated cardiac mitochondria. METHODS: Various concentrations of Ca(2+) (5-200 µM) were used to induce mitochondrial dysfunction. Cyclosporin A (CsA, an mPTP blocker) and Ru360 (an MCU blocker) were used to test its protective effects on Ca(2+)-induced mitochondrial dysfunction. RESULTS: High concentrations of Ca(2+) (≥100 µM) caused overt mitochondrial swelling and ΔΨm collapse. However, only slight increases in ROS production were detected. Blocking the MCU by Ru360 is less effective in protecting mitochondrial dysfunction. CONCLUSIONS: A dominant cause of Ca(2+)-induced cardiac mitochondrial dysfunction was mediated through the mPTP rather than MCU. Therefore, CsA could be more effective than Ru360 in preventing Ca(2+)-induced cardiac mitochondrial dysfunction.

Synaptic and nonsynaptic mitochondria demonstrate a different degree of calcium-induced mitochondrial dysfunction.

AIMS: Since variety in response to Ca(2+)-induced mitochondrial dysfunction in different neuronal mitochondrial populations is associated with the pathogenesis of several neurological diseases, we investigated the effects of Ca(2+) overload on synaptic (SM) and nonsynaptic mitochondrial (NM) dysfunction and probed the effects of cyclosporin A (CsA), 4'-chlorodiazepam (CDP) and Ru360 on relieving mitochondrial damage. MAIN METHODS: SM and NM mitochondria were isolated from rats' brains (n=5/group) and treated with various concentrations (5, 10, 100, and 200 µM) of Ca(2+), with and without CsA (mPTP blocker), CDP (PBR/TSPO blocker) and Ru360 (MCU blocker) pretreatments. Mitochondrial function was determined by mitochondrial swelling, ROS production and mitochondrial membrane potential changes (ΔΨm). KEY FINDINGS: At 200-µM Ca(2+), SM presented mitochondrial swelling to a greater extent than NM. At 100 and 200-µM Ca(2+), the ROS production of SM was higher than that of NM and ΔΨm dissipation of SM was also larger. CsA, CDP and Ru360 could reduce ROS production of SM and NM with exposure to 200-µM Ca(2+). However, only Ru360 could completely inhibit ROS generation in both SM and NM, whereas CsA and CDP could only partially reduce the ROS level in SM. Moreover, CsA and CDP pretreatments were not able to restore ΔΨm. However, Ru360 pretreatment could protect ΔΨm dissipation in both SM and NM, with complete protection observed only in NM. SIGNIFICANCE: Our findings suggested that mitochondrial calcium uniporter is a possible major pathway for calcium uptake in both mitochondrial populations. However, SM might have additional pathways involved in the calcium uptake.