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.

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.

Vitexin induces apoptosis through mitochondrial pathway and PI3K/Akt/mTOR signaling in human non-small cell lung cancer A549 cells

BACKGROUND: Currently, the prognosis of patients with non-small cell lung cancer (NSCLC) remains dismal; hence, it is critical to identify effective anti-NSCLC agents with limited side effects. This study aimed to evaluate the therapeutic potential of flavonoid compound vitexin in human NSCLC cells and the underlying mechanisms. RESULTS: The experimental results indicated that vitexin reduced the viability of A549 cells in a dose-dependent manner with nearly no toxicity against normal human bronchial epithelial 16HBE cells. Vitexin also dose-dependently increased A549 cell apoptosis, accompanied by the decreased Bcl-2/Bax ratio and the increased expression of cleaved caspase-3. Moreover, the in vivo anticancer activity of vitexin was further determined in nude mice bearing A549 cells. In addition, vitexin induced the release of cytochrome c from the mitochondria to the cytosol and the loss of mitochondrial membrane potential. Vitexin also significantly reduced the levels of p-PI3K, p-Akt and p-mTOR, and the pro-apoptotic effect of vitexin on A549 cells was partly blocked by SC79, an Akt activator. CONCLUSIONS: Accordingly, we believed that vitexin could be used as a potential therapeutic agent for the treatment of NSCLC in the future.

Hydrogen sulfide protects H9c2 cardiomyoblasts against H2O2-induced apoptosis

Reactive oxygen species (ROS) are highly reactive chemical species that may cause irreversible tissue damage, and play a critical role in cardiovascular diseases. Hydrogen sulfide (H2S) is a gasotransmitter that acts as a ROS scavenger with cardio-protective effects. In this study, we investigated the cytoprotective effect of H2S against H2O2-induced apoptosis in cardiomyocytes. H9c2 rat cardiomyoblasts were treated with H2S (100 μM) 24 h before challenging with H2O2 (100 μM). Apoptosis was then assessed by annexin V and PI, and mitochondrial membrane potential was measured using a fluorescent probe, JC-1. Our results revealed that H2S improved cell viability, reduced the apoptotic rate, and preserved mitochondrial membrane potential. An increased Bcl-2 to Bax ratio was also seen in myocytes treated with H2S after H2O2-induced stress. Our findings indicated a therapeutic potential for H2S in preventing myocyte death following ischemia/reperfusion.

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.

MS-5, a Naphthalene Derivative, Induces the Apoptosis of an Ovarian Cancer Cell CAOV-3 by Interfering with the Reactive Oxygen Species Generation

Reactive oxygen species (ROS) are widely generated in biological processes such as normal metabolism and response to xenobiotic exposure. While ROS can be beneficial or harmful to cells and tissues, generation of ROS by diverse anti-cancer drugs or phytochemicals plays an important role in the induction of apoptosis. We recently identified a derivative of naphthalene, MS-5, that induces apoptosis of an ovarian cell, CAOV-3. Interestingly, MS-5 induced apoptosis by down-regulating the ROS. Cell viability was evaluated by water-soluble tetrazolium salt (WST-1) assay. Apoptosis was evaluated by flow cytometry analysis. Intracellular ROS (H₂O₂), mitochondrial superoxide, mitochondrial membrane potential (MMP) and effect on cycle were determined by flow cytometry. Protein expression was assessed by western blotting. The level of ATP was measured using ATP Colorimetric/Fluorometric Assay kit. MS-5 inhibited growth of ovarian cancer cell lines, CAOV-3, in a concentration- and time-dependent manner. MS-5 also induced G1 cell cycle arrest in CAOV-3 cells, while MS-5 decreased intracellular ROS generation. In addition, cells treated with MS-5 showed the decrease in MMP and ATP production. In this study, we found that treatment with MS-5 in CAOV-3 cells induced apoptosis but decreased ROS level. We suspect that MS-5 might interfere with the minimum requirements of ROS for survival. These perturbations appear to be concentration-dependent, suggesting that MS-5 may induce apoptosis by interfering with ROS generation. We propose that MS-5 may be a potent therapeutic agent for inducing apoptosis in ovarian cancer cell through regulation of ROS.

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.

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.

Induction of Apoptosis Scutellaria baicalensis Georgi Root Extract by Inactivation of the Phosphatidyl Inositol 3-kinase/Akt Signaling Pathway in Human Leukemia U937 Cells

BACKGROUND: The roots of Scutellaria baicalensis Georgi (Labiatae) have been widely used in traditional medicine for treatment of various diseases. In this study, we investigated the effects of ethanol extracts of S. baicalensis roots (EESB) on the growth ofn human leukemia U937 cells. METHODS: The effect of EESB on cell viability was measured by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay. Apoptosis was determined using 4,6-diamidino-2-phenyllindile staining and flow cytometry. The effects of EESB on the expression of regulatory proteins of apoptosis and phosphatidyl inositol 3-kinase (PI3K)/Akt signaling were determined by Western blotting. Caspase activity and mitochondrial membrane potential (MMP) were measured using flow cytometric analysis.

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.

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.

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.

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.

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.

Cyclosporin A aggravates hydrogen peroxide-induced cell death in kidney proximal tubule epithelial cells / 대한해부학회지

Cyclosporin A (CsA) does not only exert a toxic effect on kidney parenchymal cells, but also protects them against necrotic cell death by inhibiting opening of mitochondrial permeability transition pore. However, whether CsA plays a role in hydrogen peroxide-induced kidney proximal tubular cell death is currently unclear. In the present study, treatment with CsA further increased apoptosis and necrosis in HK-2 human kidney proximal tubule epithelial cells during exposure to hydrogen peroxide. In addition, hydrogen peroxide-induced p53 activation and BH3 interacting-domain death agonist (BID) expression were higher in CsA-treated cells than those in non-treated cells, whereas hydrogen peroxide-induced activation of mitogen-activated protein kinases including p38, c-Jun N-terminal kinase, and extracellular signal-regulated kinase and activation of protein kinase B were not significantly altered by treatment with CsA. In oxidant-antioxidant system, reactive oxygen species (ROS) production induced by hydrogen peroxide was further enhanced by treatment with CsA. However, expression levels of antioxidant enzymes including manganese superoxide dismutase, copper/zinc superoxide dismutase, and catalase were not altered by treatment with hydrogen peroxide or CsA. Treatment with CsA further enhanced mitochondrial membrane potential induced by exposure to hydrogen peroxide, although it did not alter endoplasmic reticulum stress based on expression of glucose-regulated protein 78 and 94. Taken together, these data suggest that CsA can aggravate hydrogen peroxide-induced cell death through p53 activation, BID expression, and ROS production.

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.