Effect of the mitochondrial uncoupling agent BAM15 against the Toxoplasma gondii RH strain and Prugniaud strain.

BACKGROUND: Toxoplasmosis is a zoonotic disease caused by the infection of the protozoa Toxoplasma gondii (T. gondii), and safe and effective therapeutic drugs are lacking. Mitochondria, is an important organelle that maintains T. gondii survival, however, drugs targeting mitochondria are lacking. METHODS: The cytotoxicity of BAM15 was detected by CCK-8 and the in vitro effects of BAM15 was detected by qPCR, plaque assay and flow cytometry. Furthermore, the ultrastructural changes of T. gondii after BAM15 treatment were observed by transmission electron microscopy, and further the mitochondrial membrane potential (ΔΨm), ATP level and reactive oxygen species (ROS) of T. gondii after BAM15 treatment were detected. The pharmacokinetic experiments and in vivo infection assays were performed in mice to determine the in vivo effect of BAM15. RESULTS: BAM15 had excellent anti-T. gondii activity in vitro and in vivo with an EC50 value of 1.25 µM, while the IC50 of BAM15 in Vero cells was 27.07 µM. Notably, BAM15 significantly inhibited proliferation activity of T. gondii RH strain and Prugniaud strain (PRU), caused T. gondii death. Furthermore, BAM15 treatment induced T. gondii mitochondrial vacuolation and autolysis by TEM. Moreover, the decrease in ΔΨm and ATP level, as well as the increase in ROS production further confirmed the changes CONCLUSIONS: Our study identifies a useful T. gondii mitochondrial inhibitor, which may also serve as a leading molecule to develop therapeutic mitochondrial inhibitors in toxoplasmosis.'

Restricting bioenergetic efficiency enhances longevity and mitochondrial redox capacity in Drosophila melanogaster.

Mitochondria are essential for survival and as such, impairments in organelle homeostasis significantly accelerate age-related morbidity and mortality. Here, we determined the contribution of bioenergetic efficiency to life span and health span in Drosophila melanogaster utilizing the mitochondrial uncoupler BAM15. Life span was determined in flies fed a normal diet (ND) or high fat diet (HFD) supplemented with vehicle or BAM15. Locomotor function was determined by negative geotaxis assay in middle-aged flies fed vehicle or BAM15 under ND or HFD conditions. Redox capacity (high-resolution respirometry/fluorometry), citrate synthase (enzyme activity), mtDNA content (qPCR), gene expression (qPCR), and protein expression (western blot) were assessed in flight muscle homogenates of middle-aged flies fed vehicle or BAM15 ND. The molar ratio of H2O2 and O2 (H2O2:O2) in a defined respiratory state was calculated as a measure of redox balance. BAM15 extended life span by 9% on ND and 25% on HFD and improved locomotor activity by 125% on ND and 53% on HFD. Additionally, BAM15 enhanced oxidative phosphorylation capacity supported by pyruvate + malate, proline, and glycerol 3-phosphate. Concurrently, BAM15 enhanced the mitochondrial H2O2 production rate, reverse electron flow from mitochondrial glycerol-3-phosphate dehydrogenase (mGPDH) to Complex I, mGPDH, and Complex I without altering the H2O2:O2 ratio. BAM15 upregulated transcriptional signatures associated with mitochondrial function and fitness as well as antioxidant defense. BAM15-mediated restriction of bioenergetic efficiency prolongs life span and health span in Drosophila fed a ND or HFD. Improvements in life span and health span in ND were supported by synergistic enhancement of muscular redox capacity.

Polymeric Particle BAM15 Targeting Macrophages Attenuates the Severity of LPS-Induced Sepsis: A Proof of Concept for Specific Immune Cell-Targeted Therapy.

Macrophage polarization requires different energy sources and metabolic processes. Therefore, cell energy interference to alter macrophage functions has been proposed as a treatment for severe inflammatory diseases, including sepsis. In this study, targeting cell energy using BAM15 (a mitochondrial uncoupling agent) in human THP-1 and mouse RAW264.7 macrophages prominently interfered with M1 but not M2 polarization. Free BAM15 (BAM15) and BAM15-loaded PLGA particles (BAM15 particles) reduced the inflammatory response of M1 macrophages and enhanced the expression of M2 signature genes with the restoration of mitochondrial activity (extracellular flux analysis) in RAW264.7 cells. Furthermore, BAM15 particles but not BAM15 showed specific effects on the inflammatory response of macrophages but not neutrophils, and the particles were actively captured by splenic and liver macrophages in vivo. Administration of BAM15 and BAM15 particles attenuated the severity of sepsis in LPS-induced sepsis mice. Interestingly, BAM15 particles but not BAM15 alleviated LPS-induced liver injury by reducing hepatic inflammation. Our findings substantiate the superior efficacy of macrophage-targeted therapy using a BAM15 particle-delivery system and provide further support for clinical development as a potential therapy for severe inflammatory diseases.

BAM15 Relieves Neurodegeneration in Aged Caenorhabditis elegans and Extends Lifespan.

BAM15 was recently screened as a protonophore uncoupler specifically for the mitochondrial membrane but not the plasma membrane. It is equally as potent as FCCP, but less toxic. Previously, mitochondrial uncoupling via DNP alleviates neurodegeneration in the nematode Caenorhabditis elegans during aging. Therefore, we investigated whether BAM15 uncouplers could phenotypically and functionally reduce neuronal defects in aged nematodes. We observed green fluorescence protein-tagged mechanosensory neurons and performed touch and chemotaxis assays during aging. Wild-type animals treated with both 50 µM BAM15 and 10 µM DNP showed reduced mechanosensory neuronal defects during aging, which correlates with the maintenance of touch responses and short-term memory during aging. Uncoupler mutant ucp-4 also responded the same way as the wild-type, reducing neurodegeneration in 50 µM BAM15 and 10 µM DNP-treated animals compared to the DMSO control. These results suggest that 50 µM BAM15 alleviates neurodegeneration phenotypically and functionally in C. elegans during aging, potentially through mitochondrial uncoupling. In accordance with the preserved neuronal shape and function in aged C. elegans, 50 µM BAM15 extended the mean lifespan of both wild-type and ucp-4 mutants.

Self-assembly drug-albumin nanocomposites for nonalcoholic fatty liver disease treatment.

Nonalcoholic fatty liver disease (NAFLD) is a chronic metabolic liver disease closely related to obesity, which has become a global health problem. However, current pharmacological therapies for NAFLD are limited by potential side effects, low effectiveness and poor aqueous solubility. Herein, we designed functionalized drug-albumin nanocomposites (BAM15@BSA NPs), which were prepared by self-assembly of the anti-obesity small-molecule drug (BAM15) and bovine serum albumin (BSA), for treatment of NAFLD. The proposed BAM15@BSA NPs not only improve aqueous solubility and half-life of BAM15 but also exhibit hepatic-targeted capacity and an increased therapeutic efficacy. In vitro experiments revealed that BAM15@BSA NPs possessed excellent biocompatibility, and improved resistance to adipogenesis and reduced lipid accumulation in human hepatocellular carcinoma cells. In vivo, BAM15@BSA NPs showed liver targeting ability and powerful anti-obesity effects without altering body temperature or affecting food intake, and could effectively alleviate hepatic steatosis and improve therapeutic efficacy for NAFLD treatment. The above findings demonstrated that BAM15@BSA NPs potentially served as a safe and effective drug for NAFLD treatment.

Sepsis Encephalopathy Is Partly Mediated by miR370-3p-Induced Mitochondrial Injury but Attenuated by BAM15 in Cecal Ligation and Puncture Sepsis Male Mice.

BAM15 (a mitochondrial uncoupling agent) was tested on cecal ligation and puncture (CLP) sepsis mice with in vitro experiments. BAM15 attenuated sepsis as indicated by survival, organ histology (kidneys and livers), spleen apoptosis (activated caspase 3), brain injury (SHIRPA score, serum s100ß, serum miR370-3p, brain miR370-3p, brain TNF-α, and apoptosis), systemic inflammation (cytokines, cell-free DNA, endotoxemia, and bacteremia), and blood-brain barrier (BBB) damage (Evan's blue dye and the presence of green fluorescent E. coli in brain after an oral administration). In parallel, brain miR arrays demonstrated miR370-3p at 24 h but not 120 h post-CLP, which was correlated with metabolic pathways. Either lipopolysaccharide (LPS) or TNF-α upregulated miR370-3p in PC12 (neuron cells). An activation by sepsis factors (LPS, TNF-α, or miR370-3p transfection) damaged mitochondria (fluorescent color staining) and reduced cell ATP, possibly through profound mitochondrial activity (extracellular flux analysis) that was attenuated by BAM15. In bone-marrow-derived macrophages, LPS caused mitochondrial injury, decreased cell ATP, enhanced glycolysis activity (extracellular flux analysis), and induced pro-inflammatory macrophages (iNOS and IL-1ß) which were neutralized by BAM15. In conclusion, BAM15 attenuated sepsis through decreased mitochondrial damage, reduced neuronal miR370-3p upregulation, and induced anti-inflammatory macrophages. BAM15 is proposed to be used as an adjuvant therapy against sepsis hyperinflammation.

Mitochondrial uncoupling attenuates sarcopenic obesity by enhancing skeletal muscle mitophagy and quality control.

BACKGROUND: Sarcopenic obesity is a highly prevalent disease with poor survival and ineffective medical interventions. Mitochondrial dysfunction is purported to be central in the pathogenesis of sarcopenic obesity by impairing both organelle biogenesis and quality control. We have previously identified that a mitochondrial-targeted furazano[3,4-b]pyrazine named BAM15 is orally available and selectively lowers respiratory coupling efficiency and protects against diet-induced obesity in mice. Here, we tested the hypothesis that mitochondrial uncoupling simultaneously attenuates loss of muscle function and weight gain in a mouse model of sarcopenic obesity. METHODS: Eighty-week-old male C57BL/6J mice with obesity were randomized to 10 weeks of high fat diet (CTRL) or BAM15 (BAM15; 0.1% w/w in high fat diet) treatment. Body weight and food intake were measured weekly. Body composition, muscle function, energy expenditure, locomotor activity, and glucose tolerance were determined after treatment. Skeletal muscle was harvested and evaluated for histology, gene expression, protein signalling, and mitochondrial structure and function. RESULTS: BAM15 decreased body weight (54.0 ± 2.0 vs. 42.3 ± 1.3 g, P < 0.001) which was attributable to increased energy expenditure (10.1 ± 0.1 vs. 11.3 ± 0.4 kcal/day, P < 0.001). BAM15 increased muscle mass (52.7 ± 0.4 vs. 59.4 ± 1.0%, P < 0.001), strength (91.1 ± 1.3 vs. 124.9 ± 1.2 g, P < 0.0001), and locomotor activity (347.0 ± 14.4 vs. 432.7 ± 32.0 m, P < 0.001). Improvements in physical function were mediated in part by reductions in skeletal muscle inflammation (interleukin 6 and gp130, both P < 0.05), enhanced mitochondrial function, and improved endoplasmic reticulum homeostasis. Specifically, BAM15 activated mitochondrial quality control (PINK1-ubiquitin binding and LC3II, P < 0.01), increased mitochondrial activity (citrate synthase and complex II activity, all P < 0.05), restricted endoplasmic reticulum (ER) misfolding (decreased oligomer A11 insoluble/soluble ratio, P < 0.0001) while limiting ER stress (decreased PERK signalling, P < 0.0001), apoptotic signalling (decreased cytochrome C release and Caspase-3/9 activation, all P < 0.001), and muscle protein degradation (decreased 14-kDa actin fragment insoluble/soluble ratio, P < 0.001). CONCLUSIONS: Mitochondrial uncoupling by agents such as BAM15 may mitigate age-related decline in muscle mass and function by molecular and cellular bioenergetic adaptations that confer protection against sarcopenic obesity.

The new mitochondrial uncoupler BAM15 induces ROS production for treatment of acute myeloid leukemia.

Acute myeloid leukemia (AML) is a malignant proliferative disease of myeloid hematopoietic origin and cannot be treated appropriately at present. This is due to the fact that leukemia cells are not sensitive to some of the traditional chemotherapy drugs. Or some chemotherapeutic drugs are too toxic to normal cells, affecting their wide clinical application. In this study, we identified BAM15 as a novel mitochondrial uncoupling agent by screening a library of small molecule compounds that inhibit AML cell activity. BAM15 significantly inhibited proliferation and promoted apoptosis in AML cells while at the same time being less cytotoxic to normal cells. The mechanism may be related to the disturbance of the ROS production balance. In vivo investigations revealed that BAM15 effectively suppressed AML progression and prolonged the survival time of mice. In addition, we found that BAM15 can be used in combination with cytarabine to enhance its anti-cancer activity and inhibit the activity of primary cells in AML. Therefore, we identified BAM15 as a potential drug candidate for the treatment of AML.

Breast cancer growth and proliferation is suppressed by the mitochondrial targeted furazano[3,4-b]pyrazine BAM15.

BACKGROUND: Enhanced metabolic plasticity and diversification of energy production is a hallmark of highly proliferative breast cancers. This contributes to poor pharmacotherapy efficacy, recurrence, and metastases. We have previously identified a mitochondrial-targeted furazano[3,4-b]pyrazine named BAM15 that selectively reduces bioenergetic coupling efficiency and is orally available. Here, we evaluated the antineoplastic properties of uncoupling oxidative phosphorylation from ATP production in breast cancer using BAM15. METHODS: The anticancer effects of BAM15 were evaluated in human triple-negative MDA-MB-231 and murine luminal B, ERα-negative EO771 cells as well as in an orthotopic allograft model of highly proliferative mammary cancer in mice fed a standard or high fat diet (HFD). Untargeted transcriptomic profiling of MDA-MB-231 cells was conducted after 16-h exposure to BAM15. Additionally, oxidative phosphorylation and electron transfer capacity was determined in permeabilized cells and excised tumor homogenates after treatment with BAM15. RESULTS: BAM15 increased proton leak and over time, diminished cell proliferation, migration, and ATP production in both MDA-MB-231 and EO771 cells. Additionally, BAM15 decreased mitochondrial membrane potential, while inducing apoptosis and reactive oxygen species accumulation in MDA-MB-231 and EO771 cells. Untargeted transcriptomic profiling of MDA-MB-231 cells further revealed inhibition of signatures associated with cell survival and energy production by BAM15. In lean mice, BAM15 lowered body weight independent of food intake and slowed tumor progression compared to vehicle-treated controls. In HFD mice, BAM15 reduced tumor growth relative to vehicle and calorie-restricted weight-matched controls mediated in part by impaired cell proliferation, mitochondrial respiratory function, and ATP production. LC-MS/MS profiling of plasma and tissues from BAM15-treated animals revealed distribution of BAM15 in adipose, liver, and tumor tissue with low abundance in skeletal muscle. CONCLUSIONS: Collectively, these data indicate that mitochondrial uncoupling may be an effective strategy to limit proliferation of aggressive forms of breast cancer. More broadly, these findings highlight the metabolic vulnerabilities of highly proliferative breast cancers which may be leveraged in overcoming poor responsiveness to existing therapies.

BAM15, a Mitochondrial Uncoupling Agent, Attenuates Inflammation in the LPS Injection Mouse Model: An Adjunctive Anti-Inflammation on Macrophages and Hepatocytes.

Controlof immune responses through the immunometabolism interference is interesting for sepsis treatment. Then, expression of immunometabolism-associated genes and BAM15, a mitochondrial uncoupling agent, was explored in a proinflammatory model using lipopolysaccharide (LPS) injection. Accordingly, the decreased expression of mitochondrial uncoupling proteins was demonstrated by transcriptomic analysis on metabolism-associated genes in macrophages (RAW246.7) and by polymerase chain reaction in LPS-stimulated RAW246.7 and hepatocytes (Hepa 1-6). Pretreatment with BAM15 at 24 h prior to LPS in macrophages attenuated supernatant inflammatory cytokines (IL-6, TNF-α, and IL-10), downregulated genes of proinflammatory M1 polarization (iNOS and IL-1ß), upregulated anti-inflammatory M2 polarization (Arg1 and FIZZ), and decreased cell energy status (extracellular flux analysis and ATP production). Likewise, BAM15 decreased expression of proinflammatory genes (IL-6, TNF-α, IL-10, and iNOS) and reduced cell energy in hepatocytes. In LPS-administered mice, BAM15 attenuated serum cytokines, organ injury (liver enzymes and serum creatinine), and tissue cytokines (livers and kidneys), in part, through the enhanced phosphorylated αAMPK, a sensor of ATP depletion with anti-inflammatory property, in the liver, and reduced inflammatory monocytes/macrophages (Ly6C +ve, CD11b +ve) in the liver as detected by Western blot and flow cytometry, respectively. In conclusion, a proof of concept for inflammation attenuation of BAM15 through metabolic interference-induced anti-inflammation on macrophages and hepatocytes was demonstrated as a new strategy of anti-inflammation in sepsis.

Protonophoric action of BAM15 on planar bilayers, liposomes, mitochondria, bacteria and neurons.

Small molecules capable of uncoupling respiration and ATP synthesis in mitochondria are protective towards various cell malfunctions. Recently (2-fluorophenyl){6-[(2-fluorophenyl)amino](1,2,5-oxadiazolo[3,4-e]pyrazin-5-yl)}amine (BAM15), a new compound of this type, has become popular as a potent mitochondria-selective depolarizing agent producing minimal adverse effects. To validate protonophoric mechanism of BAM15 action, we examined its behavior in bilayer lipid membranes (BLM). BAM15 proved to be a typical anionic protonophore with the activity on planar membranes being suppressed upon decreasing membrane dipole potential. In both planar BLM and liposomes, BAM15 induced proton conductance with the potency close to that of the classical protonophoric uncoupler carbonyl cyanide m-chlorophenyl hydrazone (CCCP). In isolated rat liver mitochondria (RLM), BAM15 caused membrane potential collapse, increased respiration rate and induced Ca2+ efflux at concentrations slightly higher than those for CCCP. Surprisingly, the uncoupling action of BAM15 on isolated RLM, in contrast to that of CCCP, was partially reversed by carboxyatractyloside (CATR), an inhibitor of adenine nucleotide translocase, thereby indicating involvement of this protein in the BAM15-induced uncoupling. BAM15 inhibited growth of Bacillus subtilis at micromolar concentrations. In electrophysiological experiments on molluscan neurons, BAM15 caused plasma membrane depolarization and suppression of electrical activity, but the effect developed more slowly than that of CCCP.

BAM15-mediated mitochondrial uncoupling protects against obesity and improves glycemic control.

Obesity is a leading cause of preventable death worldwide. Despite this, current strategies for the treatment of obesity remain ineffective at achieving long-term weight control. This is due, in part, to difficulties in identifying tolerable and efficacious small molecules or biologics capable of regulating systemic nutrient homeostasis. Here, we demonstrate that BAM15, a mitochondrially targeted small molecule protonophore, stimulates energy expenditure and glucose and lipid metabolism to protect against diet-induced obesity. Exposure to BAM15 in vitro enhanced mitochondrial respiratory kinetics, improved insulin action, and stimulated nutrient uptake by sustained activation of AMPK. C57BL/6J mice treated with BAM15 were resistant to weight gain. Furthermore, BAM15-treated mice exhibited improved body composition and glycemic control independent of weight loss, effects attributable to drug targeting of lipid-rich tissues. We provide the first phenotypic characterization and demonstration of pre-clinical efficacy for BAM15 as a pharmacological approach for the treatment of obesity and related diseases.

In-vivo Imaging of Mitochondrial Depolarization of Myocardium With Positron Emission Tomography and a Proton Gradient Uncoupler.

BACKGROUND: We recently reported a method using positron emission tomography (PET) and the tracer 18F-labeled tetraphenylphosphonium (18F-TPP+) for mapping the tissue (i.e., cellular plus mitochondrial) membrane potential (ΔΨT) in the myocardium. The purpose of this work is to provide additional experimental evidence that our methods can be used to observe transient changes in the volume of distribution for 18F-TPP+ and mitochondrial membrane potential (ΔΨm). METHODS: We tested these hypotheses by measuring decreases of 18F-TPP+ concentration elicited when a proton gradient uncoupler, BAM15, is administered by intracoronary infusion during PET scanning. BAM15 is the first proton gradient uncoupler shown to affect the mitochondrial membrane without affecting the cellular membrane potential. Preliminary dose response experiments were conducted in two pigs to determine the concentration of BAM15 infusate necessary to perturb the 18F-TPP+ concentration. More definitive experiments were performed in two additional pigs, in which we administered an intravenous bolus plus infusion of 18F-TPP+ to reach secular equilibrium followed by an intracoronary infusion of BAM15. RESULTS: Intracoronary BAM15 infusion led to a clear decrease in 18F-TPP+ concentration, falling to a lower level, and then recovering. A second BAM15 infusion reduced the 18F-TPP+ level in a similar fashion. We observed a maximum depolarization of 10 mV as a result of the BAM15 infusion. SUMMARY: This work provides evidence that the total membrane potential measured with 18F-TPP+ PET is sensitive to temporal changes in mitochondrial membrane potential.

Nitrogen-obtaining and -conserving strategies in xylotrophic basidiomycetes.

Nitrogen in sufficient quantities is strictly necessary for all living organisms. In this study, the ability of some xylotrophic basidiomycetes to grow extremely long on a solid growth medium full of carbon nutrition but lacking a nitrogen source in its composition was discovered. The nitrogen oligotrophy of wood-decaying fungi is associated with their adaptation to live in a wood substrate, which is also deficient in nitrogen content. This nitrogen-depleted cultural growth is called "pseudo-foraging" and can be used as a simplified model of wood-decaying growth. Four main nitrogen-obtaining and -conserving strategies (nitrogen concentration, using alternative sources of nitrogen, economy of growth, and nutritional recycling), which are attributed to wood-colonizing xylotrophs in the literature, were revised studying the pseudo-foraging model. Based on the results, some aspects of the behavior of xylotrophs deep in undecomposed wood were predicted. For example, one of the results is that for pseudo-foraging xylotrophs, the main way to obtain nitrogen is its concentration in their mycelium from the nutrient medium in which nitrogen is contained in the impurities of the components of the medium. The result suggests that in bulk solid wood, the nitrogen concentration strategy also dominates the strategy of using diazotrophic and other alternative nitrogen. In addition, three individual unprecedented mechanisms, which supposedly help the xylotrophic fungi to colonize wood in nature (generation of fine mycelium, macrovesicular endocytosis, formation and conversion of super-elongated mitochondria), were investigated in the laboratory.

BAM15 attenuates transportation-induced apoptosis in iPS-differentiated retinal tissue.

BACKGROUND: BAM15 is a novel mitochondrial protonophore uncoupler capable of protecting mammals from acute renal ischemic-reperfusion injury and cold-induced microtubule damage. The purpose of our study was to investigate the effect of BAM15 on apoptosis during 5-day transportation of human-induced pluripotent stem (hiPS)-differentiated retinal tissue. METHODS: Retinal tissues of 30 days and 60 days were transported with or without BAM15 for 5 days in the laboratory or by real express. Immunofluorescence staining of apoptosis marker cleaved caspase3, proliferation marker Ki67, and neural axon marker NEFL was performed. And expression of apoptotic-related factors p53, NFkappaB, and TNF-a was detected by real-time PCR. Also, location of ganglion cells, photoreceptor cells, amacrine cells, and precursors of neuronal cell types in retinal tissue was stained by immunofluorescence after transportation. Furthermore, cell viability was assessed by CCK8 assay. RESULTS: Results showed transportation remarkably intensified expression of apoptotic factor cleaved caspase3, p53, NFkappaB, and TNF-a, which could be reduced by supplement of BAM15. In addition, neurons were severely injured after transportation, with axons manifesting disrupted and tortuous by staining NEFL. And the addition of BAM15 in transportation was able to protect neuronal structure and increase cell viability without affecting subtypes cells location of retinal tissue. CONCLUSIONS: BAM15 might be used as a protective reagent on apoptosis during transporting retinal tissues, holding great potential in research and clinical applications.

Experimental study of compound BAM15 alleviating cryopreservation-induced primary hepatocyte injury in rats / 器官移植

Objective To investigate the effect of compound BAM15 on the primary hepatocyte injury induced by cold storage in rats. Methods The primary rat hepatocytes were extracted by collagenase perfusion method. According to different cell culture conditions, the cells were divided into 4 groups: group A (Hibernate cell culture solution containing 250 nmol/L BAM15), group B (Hibernate cell culture solution containing 500 nmol/L BAM15), group C (Hibernate cell culture solution containing 1 000 nmol/ L BAM15), control group (Hibernate cell culture solution). The cells of each group were cryopreserved for 12 h. The purity of primary hepatocytes was observed under fluorescence microscope. The changes in the cell proliferation ability, cell apoptosis rate and mitochondrial reactive oxygen species (ROS) were measured in each group. Results The cell proliferation ability in groups B and C was significantly higher than that in the control group (both P < 0.05). The apoptosis rates in groups A, B and C were (33.7±2.2)%, (19.7±1.1)% and (28.7±1.2)%, which were significantly lower than (82.7±4.2)% in the control group (all P < 0.05). The positive rates of intracellular ROS in groups A, B and C were (11.8±4.0)%, (7.6±1.3)% and (8.9±1.6)%, remarkably lower than (27.4±4.5)% in the control group (all P < 0.05). Conclusions Compound BAM15 can effectively mitigate the primary hepatocyte injury in rats induced by cryopreservation. The underlying mechanism is probably associated with the role of BAM15 in reducing ROS generation during cold ischemia.

Mitochondrial uncoupler BAM15 inhibits artery constriction and potently activates AMPK in vascular smooth muscle cells.

Our previous studies found that mitochondrial uncouplers CCCP and niclosamide inhibited artery constriction and the mechanism involved AMPK activation in vascular smooth muscle cells. BAM15 is a novel type of mitochondrial uncoupler. The aim of the present study is to identify the vasoactivity of BAM15 and characterize the BAM15-induced AMPK activation in vascular smooth muscle cells (A10 cells). BAM15 relaxed phenylephrine (PE)-induced constricted rat mesenteric arteries with intact and denuded endothelium. Pretreatment with BAM15 inhibited PE-induced constriction of rat mesenteric arteries with intact and denuded endothelium. BAM15, CCCP, and niclosamide had the comparable IC50 value of vasorelaxation in PE-induced constriction of rat mesenteric arteries. BAM15 was less cytotoxic in A10 cells compared with CCCP and niclosamide. BAM15 depolarized mitochondrial membrane potential, induced mitochondrial fission, increased mitochondrial ROS production, and increased mitochondrial oxygen consumption rate in A10 cells. BAM15 potently activated AMPK in A10 cells and the efficacy of BAM15 was stronger than that of CCCP, niclosamide, and AMPK positive activators metformin and AICAR. In conclusion, BAM15 activates AMPK in vascular smooth muscle cells with higher potency than that of CCCP, niclosamide and the known AMPK activators metformin and AICAR. The present work indicates that BAM15 is a potent AMPK activator.

Mitochondrial uncoupler BAM15 inhibits artery constriction and potently activates AMPK in vascular smooth muscle cells

Our previous studies found that mitochondrial uncouplers CCCP and niclosamide inhibited artery constriction and the mechanism involved AMPK activation in vascular smooth muscle cells. BAM15 is a novel type of mitochondrial uncoupler. The aim of the present study is to identify the vasoactivity of BAM15 and characterize the BAM15-induced AMPK activation in vascular smooth muscle cells (A10 cells). BAM15 relaxed phenylephrine (PE)-induced constricted rat mesenteric arteries with intact and denuded endothelium. Pretreatment with BAM15 inhibited PE-induced constriction of rat mesenteric arteries with intact and denuded endothelium. BAM15, CCCP, and niclosamide had the comparable IC value of vasorelaxation in PE-induced constriction of rat mesenteric arteries. BAM15 was less cytotoxic in A10 cells compared with CCCP and niclosamide. BAM15 depolarized mitochondrial membrane potential, induced mitochondrial fission, increased mitochondrial ROS production, and increased mitochondrial oxygen consumption rate in A10 cells. BAM15 potently activated AMPK in A10 cells and the efficacy of BAM15 was stronger than that of CCCP, niclosamide, and AMPK positive activators metformin and AICAR. In conclusion, BAM15 activates AMPK in vascular smooth muscle cells with higher potency than that of CCCP, niclosamide and the known AMPK activators metformin and AICAR. The present work indicates that BAM15 is a potent AMPK activator.