Regulation of Mitochondria on Platelet Apoptosis and Activation / 中国实验血液学杂志

OBJECTIVE@#To explore the regulation of mitochondria on platelet apoptosis and activation, and the relationship between platelet apoptosis and activation.@*METHODS@#Platelets were isolated from peripheral venous blood of healthy volunteers. Cyclosporin A (CsA), which has a protective effect on the function of platelet mitochondria, BAPTA, which can chelate calcium ions across membranes in platelets, and NAC, an antioxidant that reduces the level of intracellular reactive oxygen species, were selected for coincubation with washed platelets, respectively. By flow cytometry, platelet aggregator was used to detect the changes of platelet mitochondrial function and platelet activation indexes after different interventions.@*RESULTS@#H89, staurosporine, and A23187 led to platelet mitochondrial abnormalities, while CsA could effectively reverse the decline of platelet mitochondrial membrane potential caused by them. Antioxidant NAC could reverse platelet mitochondrial damage correspondingly, and completely reverse platelet shrinkage and phosphatidylserine eversion induced by H89. BAPTA, prostaglandin E1, acetylsalicylic acid and other inhibitors could not reverse the decline of platelet mitochondrial membrane potential.@*CONCLUSION@#Mitochondrial function plays an important role in platelet apoptosis and activation. Abnormal mitochondrial function causes the imbalance of reduction/oxidation state in platelets, which leads to platelet apoptosis. Platelet apoptosis and activation are independent signal processes.

MicroRNA-132 promotes atherosclerosis by inducing mitochondrial oxidative stressmediated ferroptosis / 南方医科大学学报

OBJECTIVE@#To explore the expression of microRNA-132 (miR-132) and its potential role in the development of atherosclerosis (AS).@*METHODS@#Thirty AS samples and 30 samples of normal peripheral vessels were collected from atherosclerotic patients undergoing peripheral angiostomy in our hospital for detecting the expression level of miR-132 using RT-qPCR. The expression of miR-132 in human umbilical vein endothelial cells (HUVEC) was up-regulated by liposome transfection, and intracellular reactive oxygen species (ROS), localization relationship between ROS and mitochondria, functional changes of mitochondrial reactive oxygen superoxide species (mtROS), mitochondrial membrane potential (MMP) and opening of mitochondrial permeability transition pore (mPTP) were analyzed by flow cytometry and laser confocal microscopy. The activity of mitochondrial redox respiratory chain complex (type I, II, III, IV and V) in HUVECs was detected using ELISA, and the expression levels of key iron death proteins were detected with Western blotting.@*RESULTS@#RT-qPCR results showed that miR-132 was significantly up-regulated in atherosclerotic plaques compared with normal vascular samples (P < 0.001). Compared with control HUVECs, HUVECs overexpressing miR-132 showed a significantly increased level of intracellular ROS (P < 0.001), and most of ROS was colocalized with mitochondria. HUVECs overexpressing miR-132 also showed significantly decreased MMP (P < 0.001) and obviously increased mtROS (P < 0.001) and opening of mPTP (P < 0.001), which led to mitochondrial REDOX respiratory chain stress disorder. The key iron death protein GPX4 was significantly down-regulated and the oxidized protein NOX4 was significantly increased in miR-132-overexpressing HUVECs (P < 0.001).@*CONCLUSION@#MiR-132 promotes atherosclerosis by inducing mitochondrial oxidative stress-mediated ferroptosis, which may serve as a promising therapeutic target for AS.

The Effect of Glyphosate on Human Sperm: In Vitro Approximation / Efecto del glifosato sobre los espermatozoides humanos: una aproximación in vitro

Introduction Glyphosate is an herbicide used to eradicate illicit crops; however, its use is controversial due to different health problems associated with it. The present study aims to evaluate the effects of glyphosate on human sperm in vitro. Methods Twenty-two semen samples from healthy normozoospermic men were included; 11 semen samples were incubated with Panzer (INVESA S.A., Antiquia, Colombia) and 11 with Roundup (Monsanto Company, MO, USA). The changes in motility and viability were observed. Functional seminal parameters were evaluated as well. Results The samples exposed to glyphosate showed less motility and viability; a decrease in the potential of the mitochondrial membrane was observed, and an increase in the lipoperoxidation of the membrane was evidenced. Conclusion Based on the present results, we concluded that glyphosate has cytotoxic potential for exposed people and may affect their fertility.

Introducción El glifosato es un herbicida utilizado ampliamente para la erradicación de cultivos ilícitos; sin embargo, su uso es polémico debido a diferentes problemas de salud asociados con él. El objetivo del presente estudio fue evaluar los efectos del glifosato sobre los espermatozoides humanos in vitro. Métodos Se incluyeron 22 muestras de semen de hombres sanos normozoospérmicos, de las cuales 11 se incubaron con Panzer y 11 con Roundup, y se evaluaron los cambios en la movilidad y la viabilidad espermática, además de valorar los parámetros seminales funcionales. Resultados Las muestras expuestas al glifosato presentaron una menor movilidad y viabilidad, una disminución en el potencial de la membrana mitocondrial, y un aumento en la lipoperoxidación de la membrana. Conclusiones El glifosato es potencialmente citotóxico para las personas que estén expuestas, y puede afectar su fertilidad.

MiR-484 Protects Rat Myocardial Cells from Ischemia-Reperfusion Injury by Inhibiting Caspase-3 and Caspase-9 during Apoptosis

BACKGROUND AND OBJECTIVES: To reveal the detail mechanism of miR-484 on myocardial ischemia-reperfusion (MI/R) injury.METHODS: Rats model of MI/R injury was established based on control (Con; sham operate) group, ischemia-reperfusion (I/R) group, miR-484 treatment (miR) group, and I/R-negative control (IR-C) group, followed by pathological and interleukin (IL)-6, tumor necrosis factor (TNF)-α, and IL-1β expression evaluation. Then the myocardial apoptosis, as well as the expression of miR-484, caspase-3, and caspase-9 in myocardium were examined. Finally, the regulatory relation between miR-484 and SMAD family member 7 (SMAD7) was predicated, followed by verification analysis.RESULTS: Compared with Con group, the expression of miR-484 in I/R and IR-C group was decreased. Compared with I/R and IR-C group, the expression of miR-484 was increased in miR group. Compared with Con group, the expression levels of IL-6, TNF-α, and IL-1β in cardiac myocytes of I/R group and IR-C group were increased. Compared with Con group, the apoptotic index, membrane potential of I/R, and the expression of caspase-3/9 were increased in IR-C group. Compared with the I/R and IR-C groups, the apoptotic index of myocardial cells in the ischemic region was decreased, the membrane potential was increased, and the expression of caspase-3/9 was decreased significantly in the miR group. SMAD7 was the target gene of miR-484.CONCLUSIONS: MiR-484 protected myocardial cells from I/R injury by suppressing caspase-3 and caspase-9 expression during cardiomyocyte apoptosis. MiR-484 reduced the expression of IL-6, TNF-α, and IL-1β in MI/R. MiR-484 might alleviate the decreasing of mitochondrial membrane potential in MI/R cells.

Effects of sulforaphane on brain mitochondria: mechanistic view and future directions / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

The organosulfur compound sulforaphane (SFN; C6H11NOS2) is a potent cytoprotective agent promoting antioxidant, anti-inflammatory, antiglycative, and antimicrobial effects in in vitro and in vivo experimental models. Mitochondria are the major site of adenosine triphosphate (ATP) production due to the work of the oxidative phosphorylation (OXPHOS) system. They are also the main site of reactive oxygen species (ROS) production in nucleated human cells. Mitochondrial impairment is central in several human diseases, including neurodegeneration and metabolic disorders. In this paper, we describe and discuss the effects and mechanisms of action by which SFN modulates mitochondrial function and dynamics in mammalian cells. Mitochondria-related pro-apoptotic effects promoted by SFN in tumor cells are also discussed. SFN may be considered a cytoprotective agent, at least in part, because of the effects this organosulfur agent induces in mitochondria. Nonetheless, there are certain points that should be addressed in further experiments, indicated here as future directions, which may help researchers in this field of research.

Polyphenolic extracts of walnut (Juglans regia) green husk containing juglone inhibit the growth of HL-60 cells and induce apoptosis

BACKGROUND: Juglone is a naphthoquinone currently obtained by chemical synthesis with biological activities including antitumor activity. Additionally, juglone is present in the green husk of walnut, which suggests evaluating the effect of GH extracts on carcinogenic cell lines. RESULTS: Walnut green husk ethanolic extract was obtained as 169.1 mg juglone/100 g Green Husk and antioxidant activity (ORAC) of 44,920 µmol Trolox Equivalent/100 g DW Green Husk. At 1 µM juglone in HL-60 cell culture, green husk extract showed an antiproliferative effect, but pure juglone did not; under these conditions, normal fibroblast cells were not affected. A dose-dependent effect on mitochondrial membrane potential loss was observed. Apoptosis of HL-60 was detected at 10 µM juglone. Despite high ORAC values, neither purified juglone nor the extract showed protective effects on HL-60 cells under oxidative conditions. CONCLUSIONS: Green husk extract generates an antiproliferative effect in HL-60 cells, which is related to an induction of the early stages of apoptosis and a loss of mitochondrial membrane potential. The normal cells were not affected when juglone is present at concentrations of 1 µM, while at higher concentrations, there is loss of viability of both cancerous and healthy cells.

Myricetin Protects Against High Glucose-Induced β-Cell Apoptosis by Attenuating Endoplasmic Reticulum Stress via Inactivation of Cyclin-Dependent Kinase 5

BACKGROUND: Chronic hyperglycemia has deleterious effects on pancreatic β-cell function and turnover. Recent studies support the view that cyclin-dependent kinase 5 (CDK5) plays a role in β-cell failure under hyperglycemic conditions. However, little is known about how CDK5 impair β-cell function. Myricetin, a natural flavonoid, has therapeutic potential for the treatment of type 2 diabetes mellitus. In this study, we examined the effect of myricetin on high glucose (HG)-induced β-cell apoptosis and explored the relationship between myricetin and CDK5. METHODS: To address this question, we subjected INS-1 cells and isolated rat islets to HG conditions (30 mM) in the presence or absence of myricetin. Docking studies were conducted to validate the interaction between myricetin and CDK5. Gene expression and protein levels of endoplasmic reticulum (ER) stress markers were measured by real-time reverse transcription polymerase chain reaction and Western blot analysis. RESULTS: Activation of CDK5 in response to HG coupled with the induction of ER stress via the down regulation of sarcoendoplasmic reticulum calcium ATPase 2b (SERCA2b) gene expression and reduced the nuclear accumulation of pancreatic duodenal homeobox 1 (PDX1) leads to β-cell apoptosis. Docking study predicts that myricetin inhibit CDK5 activation by direct binding in the ATP-binding pocket. Myricetin counteracted the decrease in the levels of PDX1 and SERCA2b by HG. Moreover, myricetin attenuated HG-induced apoptosis in INS-1 cells and rat islets and reduce the mitochondrial dysfunction by decreasing reactive oxygen species production and mitochondrial membrane potential (Δψm) loss. CONCLUSION: Myricetin protects the β-cells against HG-induced apoptosis by inhibiting ER stress, possibly through inactivation of CDK5 and consequent upregulation of PDX1 and SERCA2b.

Restorative Mechanism of Neural Progenitor Cells Overexpressing Arginine Decarboxylase Genes Following Ischemic Injury

Cell replacement therapy using neural progenitor cells (NPCs) following ischemic stroke is a promising potential therapeutic strategy, but lacks efficacy for human central nervous system (CNS) therapeutics. In a previous in vitro study, we reported that the overexpression of human arginine decarboxylase (ADC) genes by a retroviral plasmid vector promoted the neuronal differentiation of mouse NPCs. In the present study, we focused on the cellular mechanism underlying cell proliferation and differentiation following ischemic injury, and the therapeutic feasibility of NPCs overexpressing ADC genes (ADC-NPCs) following ischemic stroke. To mimic cerebral ischemia in vitro , we subjected the NPCs to oxygen-glucose deprivation (OGD). The overexpressing ADC-NPCs were differentiated by neural lineage, which was related to excessive intracellular calcium-mediated cell cycle arrest and phosphorylation in the ERK1/2, CREB, and STAT1 signaling cascade following ischemic injury. Moreover, the ADC-NPCs were able to resist mitochondrial membrane potential collapse in the increasingly excessive intracellular calcium environment. Subsequently, transplanted ADC-NPCs suppressed infarct volume, and promoted neural differentiation, synapse formation, and motor behavior performance in an in vivo tMCAO rat model. The results suggest that ADC-NPCs are potentially useful for cell replacement therapy following ischemic stroke.

Asiatic Acid Protects Dopaminergic Neurons from Neuroinflammation by Suppressing Mitochondrial ROS Production

This study sought to evaluate the effects of Asiatic acid in LPS-induced BV2 microglia cells and 1-methyl-4-phenyl-pyridine (MPP⁺)-induced SH-SY5Y cells, to investigate the potential anti-inflammatory mechanisms of Asiatic acid in Parkinson’s disease (PD). SH-SY5Y cells were induced using MPP⁺ to establish as an in vitro model of PD, so that the effects of Asiatic acid on dopaminergic neurons could be examined. The NLRP3 inflammasome was activated in BV2 microglia cells to explore potential mechanisms for the neuroprotective effects of Asiatic acid. We showed that Asiatic acid reduced intracellular production of mitochondrial reactive oxygen species and altered the mitochondrial membrane potential to regulate mitochondrial dysfunction, and suppressed the NLRP3 inflammasome in microglia cells. We additionally found that treatment with Asiatic acid directly improved SH-SY5Y cell viability and mitochondrial dysfunction induced by MPP⁺. These data demonstrate that Asiatic acid both inhibits the activation of the NLRP3 inflammasome by downregulating mitochondrial reactive oxygen species directly to protect dopaminergic neurons from, and improves mitochondrial dysfunction in SH-SY5Y cells, which were established as a model of Parkinson’s disease. Our finding reveals that Asiatic acid protects dopaminergic neurons from neuroinflammation by suppressing NLRP3 inflammasome activation in microglia cells as well as protecting dopaminergic neurons directly. This suggests a promising clinical use of Asiatic acid for PD therapy.

Etoposide Induces Mitochondrial Dysfunction and Cellular Senescence in Primary Cultured Rat Astrocytes

Brain aging is an inevitable process characterized by structural and functional changes and is a major risk factor for neurodegenerative diseases. Most brain aging studies are focused on neurons and less on astrocytes which are the most abundant cells in the brain known to be in charge of various functions including the maintenance of brain physical formation, ion homeostasis, and secretion of various extracellular matrix proteins. Altered mitochondrial dynamics, defective mitophagy or mitochondrial damages are causative factors of mitochondrial dysfunction, which is linked to age-related disorders. Etoposide is an anti-cancer reagent which can induce DNA stress and cellular senescence of cancer cell lines. In this study, we investigated whether etoposide induces senescence and functional alterations in cultured rat astrocytes. Senescence-associated β-galactosidase (SA-β-gal) activity was used as a cellular senescence marker. The results indicated that etoposide-treated astrocytes showed cellular senescence phenotypes including increased SA-β-gal-positive cells number, increased nuclear size and increased senescence-associated secretory phenotypes (SASP) such as IL-6. We also observed a decreased expression of cell cycle markers, including Phospho-Histone H3/Histone H3 and CDK2, and dysregulation of cellular functions based on wound-healing, neuronal protection, and phagocytosis assays. Finally, mitochondrial dysfunction was noted through the determination of mitochondrial membrane potential using tetramethylrhodamine methyl ester (TMRM) and the measurement of mitochondrial oxygen consumption rate (OCR). These data suggest that etoposide can induce cellular senescence and mitochondrial dysfunction in astrocytes which may have implications in brain aging and neurodegenerative conditions.

Evaluation of bioenergetic and mitochondrial function in liver transplantation

BACKGROUND/AIMS: We measured changes in mitochondrial function and bioenergetics that occur during ischemia/reperfusion in fresh liver samples of patients undergoing liver transplantation. These variations correlated with markers of liver function and clinical outcome. Ischemia/reperfusion injury related to liver transplantation affects mitochondrial function and bioenergetics. Experimental studies were conducted to identify the role of bioenergetics and mitochondrial dysfunction. To the best of our knowledge, no investigation of these two factors’ impacts on liver transplantation has been performed. METHODS: This was a prospective study of 28 patients who underwent liver transplantation. We measured parameters of mitochondrial function and bioenergetics in biopsies performed during the procedure. RESULTS: We observed a statistically significant reduction in mitochondrial membrane potential, an increase in lag phase, and decreases in mitochondrial respiration and adenosine triphosphate content (P<0.010). Higher postoperative aminotransferase peaks correlated with worse mitochondrial function; mitochondrial respiration correlated with arterial lactate (P<0.010). CONCLUSIONS: There is a relationship between mitochondrial function and ischemia/reperfusion injury. The future use of these clinical markers as prognostic factors may allow early identification of post-transplant liver failure and may indicate the need to perform a new transplant.

Enhanced Anti-Cancer Effects of Conditioned Medium from Hypoxic Human Umbilical Cord–Derived Mesenchymal Stem Cells

BACKGROUND AND OBJECTIVES: There have been contradictory reports on the pro-cancer or anti-cancer effects of mesenchymal stem cells. In this study, we investigated whether conditioned medium (CM) from hypoxic human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) (H-CM) showed enhanced anti-cancer effects compared with CM from normoxic hUC-MSCs (N-CM). METHODS AND RESULTS: Compared with N-CM, H-CM not only strongly reduced cell viability and increased apoptosis of human cervical cancer cells (HeLa cells), but also increased caspase-3/7 activity, decreased mitochondrial membrane potential (MMP), and induced cell cycle arrest. In contrast, cell viability, apoptosis, MMP, and cell cycle of human dermal fibroblast (hDFs) were not significantly changed by either CM whereas caspase-3/7 activity was decreased by H-CM. Protein antibody array showed that activin A, Beta IG-H3, TIMP-2, RET, and IGFBP-3 were upregulated in H-CM compared with N-CM. Intracellular proteins that were upregulated by H-CM in HeLa cells were represented by apoptosis and cell cycle arrest terms of biological processes of Gene Ontology (GO), and by cell cycle of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. In hDFs, negative regulation of apoptosis in biological process of GO and PI3K-Akt signaling pathway of KEGG pathways were represented. CONCLUSIONS: H-CM showed enhanced anti-cancer effects on HeLa cells but did not influence cell viability or apoptosis of hDFs and these different effects were supported by profiling of secretory proteins in both kinds of CM and intracellular signaling of HeLa cells and hDFs.

Modulatory action of enrofloxacin in lipopolysaccharide-induced hyper-activated mouse spleen cells / 대한수의학회지

Enrofloxacin, a fluoroquinolone, is a broad-spectrum antibiotic widely used in veterinary medicine that inhibits the action of bacterial DNA gyrase, resulting in anti-bacterial effects. This study was performed to examine whether enrofloxacin has modulatory and anti-inflammatory activity on immune cells. A few studies have reported the anti-inflammatory effects of enrofloxacin. In this study, we used mouse spleen cells treated with lipopolysaccharide (LPS) and examined the effects of enrofloxacin. Several assays were performed in LPS-treated spleen cells after the enrofloxacin treatment. Enrofloxacin inhibited the metabolic activity and mitochondrial membrane potential of LPS-treated spleen cells significantly. On the other hand, enrofloxacin did not alter the proportion of the subsets in spleen cells, and did not induce cell death. The production of tumor necrosis factor-alpha in LPS-treated spleen cells was inhibited by enrofloxacin. Overall, enrofloxacin had modulatory activity in spleen cells treated with LPS. These data may broaden the use of enrofloxacin as an antibiotic with anti-inflammatory activity in veterinary clinics.

Neuroprotective mechanisms of dieckol against glutamate toxicity through reactive oxygen species scavenging and nuclear factor-like 2/heme oxygenase-1 pathway

Glutamate toxicity-mediated mitochondrial dysfunction and neuronal cell death are involved in the pathogenesis of several neurodegenerative diseases as well as acute brain ischemia/stroke. In this study, we investigated the neuroprotective mechanism of dieckol (DEK), one of the phlorotannins isolated from the marine brown alga Ecklonia cava, against glutamate toxicity. Primary cortical neurons (100 µM, 24 h) and HT22 neurons (5 mM, 12 h) were stimulated with glutamate to induce glutamate toxic condition. The results demonstrated that DEK treatment significantly increased cell viability in a dose-dependent manner (1–50 µM) and recovered morphological deterioration in glutamate-stimulated neurons. In addition, DEK strongly attenuated intracellular reactive oxygen species (ROS) levels, mitochondrial overload of Ca²⁺ and ROS, mitochondrial membrane potential (ΔΨ(m)) disruption, adenine triphosphate depletion. DEK showed free radical scavenging activity in the cell-free system. Furthermore, DEK enhanced protein expression of heme oxygenase-1 (HO-1), an important anti-oxidant enzyme, via the nuclear translocation of nuclear factor-like 2 (Nrf2). Taken together, we conclude that DEK exerts neuroprotective activities against glutamate toxicity through its direct free radical scavenging property and the Nrf-2/HO-1 pathway activation.

The effects of Sestrin2 on apoptosis of heat-exposed lung epithelial cells and its mechanism / 中国应用生理学杂志

OBJECTIVE@#To investigate the protective effects of Sestrin2 protein on lung epithelial Beas-2B cells in the heat-exposure environment and its mechanism.@*METHODS@#Lung epithelial Beas-2B cells were cultured at 37℃, 39℃, 40℃ and 41℃ respectively. Cells were harvested at different times (0, 3, 6 and 12 h) after pancreatin digestion. The expressions of Sestrin2, superoxide dismutase(SOD), reactive oxygen species(ROS), cell mitochondrial membrane potential and apoptosis rate of cells were detected by Western blot, fluorescence spectrophotometer and flow cytometry, respectively. Gene expression sequence was cloned into high expression plasmid pcDNA3.1. Beas-2B cells were transfected by Lipfectamine 2000 to construct Sestrin2 and SOD high expression cells. The changes of mitochondrial membrane potential and cell apoptosis were observed in the Sestrin2 and SOD high expression cells.@*RESULTS@#With the increase of temperature, the expression level of Sestrin2 protein in heat treatment group was decreased compared with the control group. When Beas-2B cells were exposed to 41℃, the ROS level was increased, mitochondrial membrane potential was decreased significantly and apoptosis rate was increased at different time points. After high expression of Sestrin2 and SOD in the Beas-2B cells, the expression level of ROS was decreased and the change tendency of mitochondrial membrane potential was decreased, and the apoptosis rate was reduced at 41℃ exposure.@*CONCLUSION@#Sestrin2 can alleviate the apoptosis of lung epithelial cells induced by heat exposure through mitochondrial membrane potential and SOD, which has protective effect on lung epithelial Beas-2B cells.

Potential biomarkers and antagonists for fluoranthene-induced cellular toxicity of bone marrow derived mesenchymal stem cells

BACKGROUND: Fluoranthene (FR) is a common environmental pollutant that exists in a complex mixture with other polycyclic aromatic hydrocarbons (PAHs). We identified biomarkers for monitoring FR exposure and investigated the rescue effect of FR-induced cellular toxicity via aryl hydrocarbon receptor (AHR) antagonist activity in bone marrow derived mesenchymal stem cells (BM-MSCs).METHODS: Morphological changes, viability, and rescue effects of an AHR antagonist (CH223191) were examined in BM-MSCs after exposure to FR. Cytotoxic effects were assayed using the tetrazolium-based colorimetric assay. Apoptosis was measured by annexin V and propidium iodide dye-based flowcytometry assay, mitochondrial membrane potential assay, and nuclear DNA fragmentation assay. Molecular signaling pathways of apoptosis and autophagy were investigated using immunoblotting. Proteomics were performed in order to reveal the spectra of cellular damage and identify biomarkers for FR exposure.RESULTS: Exposing BM-MSCs to FR (IC₅₀=50 µM) induced cell death and morphological changes, while the AHR antagonist showed rescue effects. Autophagy was activated and mitochondrial membrane potential was decreased. Proteomic analysis identified 48 deregulated proteins (26 upregulated and 22 downregulated). Among them, annexin A6, pyruvate kinase, UDP-glucose dehydrogenase, and phospholipase A2 could be potential biomarkers for FR exposure.CONCLUSION: The exposure of BM-MSCs to FR induced remarkable alterations in cellular biology and the proteome, allowing for identification of novel biomarkers for FR exposure. Furthermore, AHR antagonists might be able to prevent cellular damage due to FR exposure.

Tetrabromobisphenol A Promotes the Osteoclastogenesis of RAW264.7 Cells Induced by Receptor Activator of NF-kappa B Ligand In Vitro

BACKGROUND: Tetrabromobisphenol A (TBBPA), one of the most widely used brominated flame-retardants, is a representative persistent organic pollutants group. Studies on TBBPA toxicity have been conducted using various target cells; however, few studies have investigated TBBPA toxicity in bone cells. Therefore, this study investigated the in vitro effects of TBBPA on osteoclasts, a cell type involved in bone metabolism. METHODS: RAW264.7 cells were cultured in medium containing 50 ng/mL receptor activator of nuclear factor kappa B ligand (RANKL) and varying concentrations of TBBPA. To evaluate the effects of TBBPA on the differentiation and function of osteoclasts, osteoclast-specific gene expression, tartrate-resistant acid phosphatase (TRAP) activity, bone resorbing activity, mitochondrial membrane potential (MMP) and mitochondrial superoxide were measured. RESULTS: The presence of 20 μM TBBPA significantly increased TRAP activity in RANKL-stimulated RAW264.7 cells, the bone resorbing activity of osteoclasts, and the gene expression of Akt2, nuclear factor of activated T-cells cytoplasmic 1, and chloride channel voltage-sensitive 7. However, TBBPA treatment caused no change in the expression of carbonic anhydrase II, cathepsin K, osteopetrosis-associated transmembrane protein 1, Src, extracellular signal-related kinase, GAB2, c-Fos, or matrix metalloproteinase 9. Furthermore, 20 μM TBBPA caused a significant decrease in MMP and a significant increase in mitochondrial superoxide production. CONCLUSION: This study suggests that TBBPA promotes osteoclast differentiation and activity. The mechanism of TBBPA-stimulated osteoclastogenesis might include increased expression of several genes involved in osteoclast differentiation and reactive oxygen species production.

Interaction between necroptosis and apoptosis in MC3T3-E1 cell death induced by dexamethasone / 南方医科大学学报

OBJECTIVE@#To investigate the relationship between necroptosis and apoptosis in MCET3-E1 cell death induced by glucocorticoids.@*METHODS@#MC3T3-E1 cells were incubated with 10-6 mol/L dexamethasone followed by treatment with the apoptosis inhibitor z-VAD-fmk (40 μmol/L) or the necroptosis inhibitor necrostatin-1 (40 μmol/L) for 2 h. At 72 h after incubation with dexamethasone, the cells were harvested to determine the cell viability using WST-1 assay and the rate of necrotic cells using annexin V/PI double staining; the percentage of apoptotic cells was determined using Hoechst staining. The mitochondrial membrane potential and the level of ATP in the cells were also evaluated. Transmission electron microscopy was used to observe the microstructural changes of the cells. The expressions of RIP-1 and RIP-3 in the cells were detected by Western blotting.@*RESULTS@#At a concentration of 10-6 mol/L, dexamethasone induced both apoptosis and necroptosis in MC3T3- E1 cells. Annexin V/PI double staining showed that inhibition of cell apoptosis caused an increase in cell necrosis manifested by such changes as mitochondrial swelling and plasma membrane disruption, as shown by electron microscopy; Hoechst staining showed that the percentage of apoptotic cells was significantly reduced. When necroptosis was inhibited by necrostatin-1, MC3T3-E1 cells showed significantly increased apoptosis as shown by both AV/PI and Hoechst staining, and such changes were accompanied by changes in mitochondrial membrane potential and ATP level in the cells.@*CONCLUSIONS@#In the process of dexamethasone-induced cell death, necroptosis and apoptosis can transform reciprocally accompanied by functional changes of the mitochondria.

High-content analysis of in vitro hepatocyte injury induced by various hepatotoxicants

In vitro prediction of hepatotoxicity can enhance the performance of non-clinical animal testing for identifying chemical hazards. In this study, we assessed high-content analysis (HCA) using multi-parameter cell-based assays as an in vitro hepatotoxicity testing model using various hepatotoxicants and human hepatocytes such as HepG2 cells and human primary hepatocytes (hPHs). Both hepatocyte types were exposed separately to multiple doses of ten hepatotoxicants associated with liver injury whose mechanisms of action have been described. HCA data were obtained using fluorescence probes for nuclear size (Hoechst), mitochondrial membrane potential (TMRM), cytosolic free calcium (Fluo-4AM), and lipid peroxidation (BODIPY). Cellular alterations were observed in response to all hepatotoxicants tested. The most sensitive parameter was TMRM, with high sensitivity at a low dose, next was BODIPY, followed by Fluo-4AM. HCA data from HepG2 cells and hPHs were generally concordant, although some inconsistencies were noted. Both hepatocyte types showed mild or severe mitochondrial impairment and lipid peroxidation in response to several hepatotoxicants. The results demonstrate that the application of HCA to in vitro hepatotoxicity testing enables more efficient hazard identification, and further, they suggest that certain parameters could serve as sensitive endpoints for predicting the hepatotoxic potential of chemical compounds.

Cis-3-O-p-hydroxycinnamoyl Ursolic Acid Induced ROS-Dependent p53-Mediated Mitochondrial Apoptosis in Oral Cancer Cells

Cis-3-O-p-hydroxycinnamoyl ursolic acid (HCUA), a triterpenoid compound, was purified from Elaeagnus oldhamii Maxim. This traditional medicinal plant has been used for treating rheumatoid arthritis and lung disorders as well as for its anti-inflammation and anticancer activities. This study aimed to investigate the anti-proliferative and apoptotic-inducing activities of HCUA in oral cancer cells. HCUA exhibited anti-proliferative activity in oral cancer cell lines (Ca9-22 and SAS cells), but not in normal oral fibroblasts. The inhibitory concentration of HCUA that resulted in 50% viability was 24.0 µM and 17.8 µM for Ca9-22 and SAS cells, respectively. Moreover, HCUA increased the number of cells in the sub-G1 arrest phase and apoptosis in a concentration-dependent manner in both oral cancer cell lines, but not in normal oral fibroblasts. Importantly, HCUA induced p53-mediated transcriptional regulation of pro-apoptotic proteins (Bax, Bak, Bim, Noxa, and PUMA), which are associated with mitochondrial apoptosis in oral cancer cells via the loss of mitochondrial membrane potential. HCUA triggered the production of intracellular reactive oxygen species (ROS) that was ascertained to be involved in HCUA-induced apoptosis by the ROS inhibitors YCG063 and N-acetyl-L-cysteine. As a result, HCUA had potential antitumor activity to oral cancer cells through eliciting ROS-dependent and p53-mediated mitochondrial apoptosis. Overall, HCUA could be applicable for the development of anticancer agents against human oral cancer.