• Journal of Life Science
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• v.29 no.12
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• pp.1393-1400
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• 2019

In the present study, we examined the effect of mitochondrial K⁺ channel opener diazoxide on the mitochondria-dependent apoptotic signaling in endothelial cells exposed to high glucose (HG) media. Endothelial cells derived from human umbilical veins were exposed to HG media containing 30 mM glucose, and the degree of apoptotic cell death associated with activation of the mitochondria-dependent apoptotic signaling pathway was determined. Exposure to HG media was seen to enhance apoptotic cell death in a time-dependent manner. In these cells, activation of caspases 3, 8, and 9 was observed, and while caspase-3 and -9 inhibitors suppressed the HG-induced apoptotic cell death, a caspase-8 inhibitor did not. The HG-treated cells exhibited disruption of mitochondrial membrane potential, formation of permeability transition pores, and cytosolic release of cytochrome c. Subsequently, diazoxide was seen to attenuate the HG-induced apoptotic cell death; caspase-9 activation was suppressed but caspase 8 was not. Diazoxide also suppressed the depolarization of mitochondrial membrane potential, the formation of mitochondrial permeability transition pores, and the release of cytochrome c. These effects were significantly inhibited by 5-hydroxydecanoate, a selective blocker of ATP-sensitive K⁺ channels (K_ATP). The present results demonstrate that diazoxide exhibits a beneficial effect to ameliorate HG-induced endothelial cell apoptosis. Opening the K_ATP could help preserve the functional integrity of mitochondria and provide an underlying mechanism to suppress HG-triggered apoptotic signaling.

• Journal of Microbiology and Biotechnology
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• v.26 no.2
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• pp.287-294
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• 2016

The effect of kaempferol (3,5,7,4-tetrahydroxyflavone), a flavonoid compound that was identified in barnyard millet (Echinochloa crus-galli var. frumentacea) grains, on G₂-checkpoint and apoptotic pathways was investigated in human acute leukemia Jurkat T cell clones stably transfected with an empty vector (J/Neo) or a Bcl-xL expression vector (J/Bcl-xL). Exposure of J/Neo cells to kaempeferol caused cytotoxicity and activation of the ATM/ATR-Chk1/Chk2 pathway, activating the phosphorylation of p53 (Ser-15), inhibitory phosphorylation of Cdc25C (Ser-216), and inactivation of cyclin-dependent kinase 1 (Cdk1), with resultant G₂-arrest of the cell cycle. Under these conditions, apoptotic events, including upregulation of Bak and PUMA levels, Bak activation, mitochondrial membrane potential (Δψm) loss, activation of caspase-9, -8, and -3, anti-poly (ADP-ribose) polymerase (PARP) cleavage, and accumulation of apoptotic sub-G₁ cells, were induced without accompanying necrosis. However, these apoptotic events, except for upregulation of Bak and PUMA levels, were completely abrogated in J/Bcl-xL cells overexpressing Bcl-xL, suggesting that the G₂-arrest and the Bcl-xL-sensitive mitochondrial apoptotic events were induced, in parallel, as downstream events of the DNA-damage-mediated G₂-checkpoint activation. Together these results demonstrate that kaempferol-mediated antitumor activity toward Jurkat T cells was attributable to G₂-checkpoint activation, which caused not only G₂-arrest of the cell cycle but also activating phosphorylation of p53 (Ser-15) and subsequent induction of mitochondria-dependent apoptotic events, including Bak and PUMA upregulation, Bak activation, Δψm loss, and caspase cascade activation.

• Biomolecules & Therapeutics
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• v.27 no.1
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• pp.41-47
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• 2019

The apoptotic effects of shikonin (5,8-dihydroxy-2-[(1R)-1-hydroxy-4-methylpent-3-enyl]naphthalene-1,4-dione) on the human colon cancer cell line SNU-407 were investigated in this study. Shikonin showed dose-dependent cytotoxic activity against SNU-407 cells, with an estimated $IC_{50}$ value of $3{\mu}M$ after 48 h of treatment. Shikonin induced apoptosis, as evidenced by apoptotic body formation, sub-G_1$ phase cells, and DNA fragmentation. Shikonin induced apoptotic cell death by activating mitogen-activated protein kinase family members, and the apoptotic process was mediated by the activation of endoplasmic reticulum (ER) stress, leading to activation of the $PERK/elF2{\alpha}/CHOP$ apoptotic pathway, and mitochondrial $Ca^{2+}$ accumulation. Shikonin increased mitochondrial membrane depolarization and altered the levels of apoptosis-related proteins, with a decrease in B cell lymphoma (Bcl)-2 and an increase in Bcl-2-associated X protein, and subsequently, increased expression of cleaved forms of caspase-9 and -3. Taken together, we suggest that these mechanisms, including MAPK signaling and the ER- and mitochondria-mediated pathways, may underlie shikonin-induced apoptosis related to its anticancer effect.

• Journal of Life Science
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• v.31 no.5
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• pp.488-495
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• 2021

The aim of this study was to investigate the possible antioxidant effect of Spirodela polyrhiza (SP) on rats fed a high fat and high cholesterol diet supplemented with either 5% (SPA group) or 10% (SPB group) SP for 4 weeks. The hepatic SOD activity of the HF group significantly decreased compared to that of the N group, but that of the SPA and SPB groups significantly increased. The GPx activity of the SPA and SPB groups in the liver was significantly greater than that of the HF group, and the hepatic catalase activity of the SPA and SPB groups significantly increased compared to the HF group. The hepatic superoxide radical content of the mitochondria and microsomes of the HF group significantly increased compared to that of the N group, but the contents were reduced in the group that took SP powder. The hepatic hydrogen peroxide content in the cytosol and mitochondria of the SP powder group was lower than in the HF group. The carbonyl content in the mitochondria and microsomes of the SPA and SPB groups was significantly lower than in the HF group. The TBARS values in the liver significantly decreased in the SPA and SPB groups. Spirodela polyrhiza was thus effective in reducing oxidative stress by regulating the hepatic antioxidant enzymes and the free radicals in rats fed high fat and high cholesterol diets.

• Applied Microscopy
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• v.28 no.4
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• pp.513-525
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• 1998

The spermatogenetic process in the edible giant snail is similar to those in the other snails, except for the axoneme formation process. In this study, the axoneme formation process in the giant snail was mainly examined by means of electron microscopy. The tail portion of a spermatozoon is about $160{\mu}m$ long, and extends straight to the rear, surrounded by two large and long mitochondria in spiral forms. A number of glycogen particles $(40\sim70nm)$ are found in the swollen matrix of the mitochodria. The axoneme which composes the tail of a spermatozoon is surrounded by $7\sim10$ lamella-form fibrous sheaths of about $0.2{\mu}m$ in thickness. Most of the mature spermatozoa are found to be clustered into a group of $5\sim7$ ea in syncytial bridges formed by cytoplasmic processes. Sertoli cells contain glycogen particles, endoplasmic reticulum, a lot of mitochondria, and lipids in their cytoplasm. They protrude their filiform pseudopodia and phagocytize abnormal spermatids or spermaozoa.

• The Journal of Internal Korean Medicine
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• v.41 no.1
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• pp.44-58
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• 2020

Objectives: This study was conducted to identify the protective effects of Chongmyunggongjin-dan (CMGJD) on Hydrogen peroxide (H₂O₂)-induced apoptosis mechanisms in C6 glial cells. Method: We used CMGJD after distilled water extraction, filtration, and lyophilization. The ROS scavenging effect was examined by fluorescence microscopy. Expression levels of proteins related to ROS generation were investigated by western blotting. Functional changes in organelles related to Reactive oxygen species (ROS) generation were investigated by immunoblotting and by verifying expression level of relevant enzymes. Results: The CMGJD extract protected the cells against H₂O₂-induced morphological changes and DNA fragmentation, inhibited the increase of Heme_oxygenase-1(HO^-1) and the decrease in catalase, protected against the loss of mitochondrial membrane potential, inhibited disturbances of lysosomal function, and induced an increase in peroxisomes. Conclusion: CMGJD was confirmed to have a protective effect on H₂O₂-induced C6 glial cell death possibly by blocking the pathways causing damage to subcellular organelles, such as mitochondria, lysosomes, and peroxisomes. We assume that CMGJD will be effective for the prevention and treatment of ischemic stroke in a clinical environment.

• The Journal of Internal Korean Medicine
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• v.42 no.6
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• pp.1269-1284
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• 2021

Objectives: Phragmitis Rhizoma is the fresh or dried rhizome of Phragmites communis Trin., which has been prescribed in traditional Korean medicine to relieve fever and vomiting and to nourish the body fluids. Recently, the protective effect of Phragmitis Rhizoma extract or its components on myelotoxicity and inflammatory responses have been reported, but no study has yet been conducted on oxidative stress. Methods: The present study investigated whether an ethanol extract of Phragmitis Rhizoma (PR) could protect against cellular damage induced by oxidative stress in Chang liver cells. Results: Pretreatment with PR significantly suppressed the hydrogen peroxide (H₂O₂)-induced reduction of Chang cell viability and generation of reactive oxygen species (ROS), thereby deferring apoptosis. PR also markedly inhibited H₂O₂-induced comet tail formation and phospho-γH2AX expression, suggesting that PR protected against oxidative stress-mediated DNA damage. PR also effectively prevented the inhibition of ATP synthesis in H₂O₂-treated Chang cells by inhibiting the loss of mitochondrial membrane potential, indicating that PR maintains energy metabolism through preservation of mitochondrial function while eliminating ROS generated by H₂O₂. Immunoblotting results indicated that PR attenuated the H₂O₂-induced downregulation of Bcl-2 and upregulation of Bax expression. Conclusions: PR protects against oxidative injury in Chang liver cells by regulating energy homeostasis via ROS generation blockade, which is at least partly mediated through inactivation of the mitochondria-mediated apoptosis pathway.

• Molecules and Cells
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• v.43 no.7
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• pp.632-644
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• 2020

The molecular mechanism underlying autophagy impairment in the retinal pigment epithelium (RPE) in dry age-related macular degeneration (AMD) is not yet clear. Based on the causative role of poly(ADP-ribose) polymerase 1 (PARP1) in RPE necrosis, this study examined whether PARP1 is involved in the autophagy impairment observed during dry AMD pathogenesis. We found that autophagy was downregulated following H₂O₂-induced PARP1 activation in ARPE-19 cells and olaparib, PARP1 inhibitor, preserved the autophagy process upon H₂O₂ exposure in ARPE-19 cells. These findings imply that PARP1 participates in the autophagy impairment upon oxidative stress in ARPE-19 cells. Furthermore, PARP1 inhibited autolysosome formation but did not affect autophagosome formation in H₂O₂-exposed ARPE-19 cells, demonstrating that PARP1 is responsible for impairment of late-stage autophagy in particular. Because PARP1 consumes NAD⁺ while exerting its catalytic activity, we investigated whether PARP1 impedes autophagy mediated by sirtuin1 (SIRT1), which uses NAD⁺ as its cofactor. A NAD⁺ precursor restored autophagy and protected mitochondria in ARPE-19 cells by preserving SIRT1 activity upon H₂O₂. Moreover, olaparib failed to restore autophagy in SIRT1-depleted ARPE-19 cells, indicating that PARP1 inhibits autophagy through SIRT1 inhibition. Next, we further examined whether PARP1-induced autophagy impairment occurs in the retinas of dry AMD model mice. Histological analyses revealed that olaparib treatment protected mouse retinas against sodium iodate (SI) insult, but not in retinas cotreated with SI and wortmannin, an autophagy inhibitor. Collectively, our data demonstrate that PARP1-dependent inhibition of SIRT1 activity impedes autophagic survival of RPE cells, leading to retinal degeneration during dry AMD pathogenesis.

• Clinical and Experimental Reproductive Medicine
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• v.50 no.1
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• pp.26-33
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• 2023

Objective: Human exposure to multiple xenobiotics, over various developmental windows, results in adverse health effects arising from these concomitant exposures. Humans are widely exposed to bisphenol A, and acetaminophen is the most commonly used over-the-counter drug worldwide. Bisphenol A is a well-recognized male reproductive toxicant, and increasing evidence suggests that acetaminophen is also detrimental to the male reproductive system. The recent recognition of male reproductive system dysfunction in conditions of suboptimal reproductive outcomes makes it crucial to investigate the contributions of toxicant exposures to infertility and sub-fertility. We aimed to identify toxicity in the male reproductive system at the mitochondrial level in response to co-exposure to bisphenol A and acetaminophen, and we investigated whether melatonin ameliorated this toxicity. Methods: Male Wistar rats were divided into six groups (n=10 each): a control group and groups that received melatonin, bisphenol A, acetaminophen, bisphenol A and acetaminophen, and bisphenol A and acetaminophen with melatonin treatment. Results: Significantly higher lipid peroxidation was observed in the testicular mitochondria and sperm in the treatment groups than in the control group. Levels of glutathione and the activities of catalase, glutathione peroxidase, glutathione reductase, and manganese superoxide dismutase decreased significantly in response to the toxicant treatments. Likewise, the toxicant treatments significantly decreased the sperm count and motility, while significantly increasing sperm mortality. Melatonin mitigated the adverse effects of bisphenol A and acetaminophen. Conclusion: Co-exposure to bisphenol A and acetaminophen elevated oxidative stress in the testicular mitochondria, and this effect was alleviated by melatonin.

• Applied Microscopy
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• v.17 no.2
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• pp.55-71
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• 1987

To investigate the effects of lead on the electron transport system and ultrastructure of mouse kidney mitochondria, various lead acetate concentrations were treated in vitro and respiration rate, enzyme activities were measured. Ultrastructural changes at state IV respiration were also observed. To compare with in vivo experiments, mouse were injected intraperitoneally of 100 mg lead acetate per kg body weight and state IV respiration rate and enzyme activities were measured. Ultrastructure of renal proximal tubular cells were also observed. In in vitro treatement, decreased state IV respiration, decreased enzyme activities, ruptured membranes and inhibition of condensed to orthodox transformation were observed. In in vivo treatment, decreased state IV respiration and decreased enzyme activities were observed after 24 hrs of i.p. injection. Cytochrome c oxidase activity showed twice the inhibition compared to NADH-CoQ reductase activity at 24 hrs. Continuous decreased state IV respiration was observed after 48 and 72 hrs of injection, however, the enzyme activities were increased to control level. Lead-protein complex which probably inhibits the toxic effects of lead appeared. To conclude, dominant effect of lead on the electron transport system appeared at cytochrome c oxidase activity, and the increased enzyme activities may be a result of appearance of lead-protein complex.