• Clinical and Experimental Pediatrics
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• v.55 no.7
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• pp.238-248
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• 2012

Purpose: Hypoxic-ischemic encephalopathy is an important cause of neonatal mortality, as this brain injury disrupts normal mitochondrial respiratory activity. Carnitine plays an essential role in mitochondrial fatty acid transport and modulates excess acyl coenzyme A levels. In this study, we investigated whether treatment of primary cultures of rat cortical neurons with L-carnitine was able to prevent neurotoxicity resulting from oxygen-glucose deprivation (OGD). Methods: Cortical neurons were prepared from Sprague-Dawley rat embryos. L-Carnitine was applied to cultures just prior to OGD and subsequent reoxygenation. The numbers of cells that stained with acridine orange (AO) and propidium iodide (PI) were counted, and lactate dehydrogenase (LDH) activity and reactive oxygen species (ROS) levels were measured. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and the terminal uridine deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assay were performed to evaluate the effect of L-carnitine (1 ${\mu}M$, 10 ${\mu}M$, and 100 ${\mu}M$) on OGD-induced neurotoxicity. Results: Treatment of primary cultures of rat cortical neurons with L-carnitine significantly reduced cell necrosis and prevented apoptosis after OGD. L-Carnitine application significantly reduced the number of cells that died, as assessed by the PI/AO ratio, and also reduced ROS release in the OGD groups treated with 10 ${\mu}M$ and 100 ${\mu}M$ of L-carnitine compared with the untreated OGD group (P<0.05). The application of L-carnitine at 100 ${\mu}M$ significantly decreased cytotoxicity, LDH release, and inhibited apoptosis compared to the untreated OGD group (P<0.05). Conclusion: L-Carnitine has neuroprotective benefits against OGD in rat primary cortical neurons in vitro.

• Journal of Physiology & Pathology in Korean Medicine
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• v.21 no.4
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• pp.1010-1016
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• 2007

Sagunjatang (Sagunja), containing Radix Astragali, Radix Ginseng, Fructus Schizandrae, Radix Ophiopogonis and Radix Glycyrrhizae, has been used as a prescription for ischemic heart and brain diseases in Korean traditional medicine. This study was designed to investigate the protective mechanisms of Sagunja on $H_2O_2$-induced cytotoxicity of H9c2 cardiomyocytes. Treatment with $H_2O_2$ resulted in death of H9c2 cells, characterized by apparent apoptotic features, including the fragmentation of nucleus and increase in sub-GO/G1fraction of cell cycle. However, Sagunja markedly suppressed the apoptotic characteristics of H9c2 cells induced by $H_2O_2$ with decrease of intracellular peroxide level. In addition, Sagunja suppressed the features of mitochondrial dysfunction, including change of mitochondrial membrane potential, in $H_2O_2$- treated cells. Additionally, Sagunja induced the expression of HO-1 protein in both time-and dose-dependent manner. The role of HO-1 in ROS-scavenging activity of Sagunja is proposed.

• Journal of Microbiology and Biotechnology
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• v.31 no.6
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• pp.867-874
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• 2021

Epalrestat (EPS) is a brain penetrant aldose reductase inhibitor, an approved drug currently used for the treatment of diabetic neuropathy. At near-plasma concentration, EPS induces glutathione biosynthesis, which in turn reduces oxidative stress in the neuronal cells. In this study, we found that EPS reduces neurodegeneration by inhibiting reactive oxygen species (ROS)-induced oxidative injury, mitochondrial membrane damage, apoptosis and tauopathy. EPS treatment up to 50 µM did not show any toxic effect on SH-SY5Y cell line (neuroblastoma cells). However, we observed toxic effect at a concentration of 100 µM and above. At 50 µM concentration, EPS showed better antioxidant activity against H₂O₂ (100 µM)-induced cytotoxicity, ROS formation and mitochondrial membrane damage in retinoic acid-differentiated SH-SY5Y cell line. Furthermore, our study revealed that 50 µM of EPS concentration reduced the glycogen synthase kinase-3 β (GSK3-β) expression and total tau protein level in H₂O₂ (100 µM)-treated cells. Findings from this study confirms the therapeutic efficacy of EPS on regulating Alzheimer's disease (AD) by regulating GSK3-β and total tau proteins phosphorylation, which helped to restore the cellular viability. This process could also reduce toxic fibrillary tangle formation and disease progression of AD. Therefore, it is our view that an optimal concentration of EPS therapy could decrease AD pathology by reducing tau phosphorylation through regulating the expression level of GSK3-β.

• Animal Bioscience
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• v.35 no.9
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• pp.1351-1359
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• 2022

Objective: The objective of this study was to analyse the differentially abundant proteins caused by freeze-thawing of ram sperm and explore candidate proteins of interest for their ability to improve ram sperm cryopreservation outcomes in vitro. Methods: Sperm were from three mature Dorper. Fresh and frozen sperm proteins were extracted, and the differentially abundant proteins were analysed by mass spectrometry. Among these proteins, lactoferrin (LTF) was selected to be added before cryopreservation. Next, sperm samples were diluted in Tris extender, with the addition of 0, 10, 100, 500, and 1,000 ㎍/mL of LTF. After thawing, sperm quality was evaluated by motility, plasma membrane integrity, mitochondrial activity and reactive oxygen species (ROS). Results: Cryopreservation significantly altered the abundance of 40 proteins; the abundance of 16 proteins was increased, while that of 24 proteins was decreased. Next, LTF was added to Tris extender applied to ram sperm. The results showed that sperm motility and plasma membrane integrity were significantly improved (p<0.05) by supplementation with 10 ㎍/mL LTF compared to those in the control group. There was no significant difference in mitochondrial activity between the 0 ㎍/mL group and other groups (p>0.05). Supplementation of the cryoprotective extender with 10 ㎍/mL LTF led to decreased ROS levels compared with those in the control and other groups (p<0.05). Conclusion: The LTF is an important protein during cryopreservation, and the addition of 10 ㎍/mL LTF to a cryoprotective extender can significantly improve the function of frozen ram sperm.

• Proceedings of the Plant Resources Society of Korea Conference
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• 2021.04a
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• pp.57-57
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• 2021

Oxygen glucose deprivation/re-oxygenation (OGD/R) induces neuronal injury via mechanisms that are believed to mimic the pathways associated with brain ischemia. Stachys sieboldii Miq. (Chinese artichoke), which has been extensively used in oriental traditional medicine to treat of ischemic stroke; however, the role of S. sieboldii Miq. (SSM) in OGD/R induced neuronal injury is not yet fully understood. The present research is aimed to investigate the protective effect and possible mechanisms of SSM extract treatment in an in vitro model of OGD/R to simulate ischemia/reperfusion Injury. Pretreatment of these cells with SSM significantly attenuated OGD/R-induced production of reactive oxygen species (ROS) by increasing GPx, SOD, and decreasing MDA. SSM decreased mitochondrial damage caused by OGD/R injury and inhibited the release of cyt-c from mitochondrion to cytoplasm in SH-SY5Y cells. Furthermore, neuronal cell apoptosis caused by OGD/R injury was inhibited by SSM, and SSM could decrease apoptosis by increasing ratio of Bcl-2/Bax and inhibiting caspase signaling pathway in SH-SY5Y cells. SSM demonstrated a neuroprotective effect on the simulated cerebral ischemia in vitro model, and this effect was the inhibition of mitochondria-mediated apoptosis pathway by scavenging of ROS generation. Therefore, SSM may be a promising neuroprotective strategy against ischemic stroke.

• Herbal Formula Science
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• v.31 no.4
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• pp.265-281
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• 2023

Objectives : Ukgan-san plus Citri Pericarpium and Pinelliae Rhizoma (UCP) is used as a traditional herbal formula in Korea and Japan for treatment of fever, fever-induced convulsions, and liver dysfunction and so on. In this study, we investigated the cytoprotective effect and underlying mechanism of UCP against oxidative stress induced by cotreatment of arachidonic acid (AA) and iron. Methods : To evaluate the hepatoprotective effects of UCP against AA + iron-induced oxidative stress in HepG2 cell, cell viability and changes on apoptosis-related proteins were assessed by MTT and immunoblot analyses. The changes in intracellular reactive oxygen species (ROS), glutathione (GSH), and mitochondrial membrane permeability (MMP) were investigated against to the oxidative stress. Furthermore, to verify underlying molecular mechanism, NF-E2-related factor 2 (Nrf2) and its downstream target genes were examined by immunoblot analysis. Results : Treatment of UCP increased the cell viability and altered the expression levels of apoptosis-related proteins such as PARP, caspase-9, caspase-3, Bcl-2. UCP also inhibited the GSH depletion, excessive ROS production and mitochondrial dysfunction induced by AA + iron. In addition, the Nrf2 and the Nrf2 target genes activation were increased by UCP. Conclusions : These results indicated that UCP has the ability to protect against oxidative stress-induced hepatocyte damage, which may be mediated with Nrf2 pathway.

• Animal Bioscience
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• v.37 no.5
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• pp.852-861
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• 2024

Objective: The present study aimed to investigate the effect of β-nicotinamide mononucleotide (NMN) supplementation on ram sperm quality during storage at 4℃ in vitro. Methods: Tris-citric acid-glucose solution containing different doses of NMN (0, 30, 60, 90, and 120 µM) was used to dilute semen collected from rams and it was stored at 4℃. Sperm motility, plasma membrane integrity as well as acrosome integrity were evaluated at 0, 24, and 48 h time points after storage at 4℃. In addition, sperm mitochondrial activity, lipid peroxidation (LPO), malondialdehyde (MDA) content, reactive oxygen species (ROS) content, glutathione (GSH) content, superoxide dismutase (SOD) activity, and apoptosis were measured at 48 h time point after storage at 4℃. Results: Results demonstrate that the values obtained for sperm motility, acrosome integrity, and plasma membrane integrity in the NMN treatments were significantly higher than control (p<0.05). The addition of 60 µM NMN significantly improved ram sperm mitochondrial activity and reduced LPO, MDA content, and ROS content compared to control (p<0.05). Interestingly, sperm GSH content and SOD activity for the 60 µM NMN treatment were much higher than those observed for control. NMN treatment also decreased the level of Cleaved-Caspase 3, Cleaved-Caspase 9, and Bax while increasing Bcl-2 level in sperm at 48 h time point after storage at 4℃. Conclusion: Ram sperm quality can be maintained during storage at 4℃ with the addition of NMN at 60 µM to the semen extender. NMN also reduces oxidative stress and apoptosis. Overall, these findings suggest that NMN is efficient in improving the viability of ram sperm during storage at 4℃ in vitro.

• Journal of Ginseng Research
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• v.47 no.2
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• pp.199-209
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• 2023

Background: Due to the interrupted blood supply in cerebral ischemic stroke (CIS), ischemic and hypoxia results in neuronal depolarization, insufficient NAD+, excessive levels of ROS, mitochondrial damages, and energy metabolism disorders, which triggers the ischemic cascades. Currently, improvement of mitochondrial functions and energy metabolism is as a vital therapeutic target and clinical strategy. Hence, it is greatly crucial to look for neuroprotective natural agents with mitochondria protection actions and explore the mediated targets for treating CIS. In the previous study, notoginseng leaf triterpenes (PNGL) from Panax notoginseng stems and leaves was demonstrated to have neuroprotective effects against cerebral ischemia/reperfusion injury. However, the potential mechanisms have been not completely elaborate. Methods: The model of middle cerebral artery occlusion and reperfusion (MCAO/R) was adopted to verify the neuroprotective effects and potential pharmacology mechanisms of PNGL in vivo. Antioxidant markers were evaluated by kit detection. Mitochondrial function was evaluated by ATP content measurement, ATPase, NAD and NADH kits. And the transmission electron microscopy (TEM) and pathological staining (H&E and Nissl) were used to detect cerebral morphological changes and mitochondrial structural damages. Western blotting, ELISA and immunofluorescence assay were utilized to explore the mitochondrial protection effects and its related mechanisms in vivo. Results: In vivo, treatment with PNGL markedly reduced excessive oxidative stress, inhibited mitochondrial injury, alleviated energy metabolism dysfunction, decreased neuronal loss and apoptosis, and thus notedly raised neuronal survival under ischemia and hypoxia. Meanwhile, PNGL significantly increased the expression of nicotinamide phosphoribosyltransferase (NAMPT) in the ischemic regions, and regulated its related downstream SIRT1/2/3-MnSOD/PGC-1α pathways. Conclusion: The study finds that the mitochondrial protective effects of PNGL are associated with the NAMPT-SIRT1/2/3-MnSOD/PGC-1α signal pathways. PNGL, as a novel candidate drug, has great application prospects for preventing and treating ischemic stroke.

• Journal of Life Science
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• v.25 no.11
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• pp.1235-1243
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• 2015

Hwangheuk-san (HHS) is a Korean multi-herb formula comprising four medicinal herbs. HHS, which was recorded in “Dongeuibogam,” has been used to treat patients with inflammation syndromes and digestive tract cancer for hundreds of years. However, little is known about its anti-tumor efficacy. The present study investigated the pro-apoptotic effect and mode of action of HHS against AGS human gastric carcinoma cells. HHS inhibited the cell growth of AGS cells in a dose-dependent manner, which was associated with the induction of apoptotic cell death, as evidenced by the formation of apoptotic bodies, chromatin condensation, and an accumulation of cells in the sub-G1 phase. HHS-induced apoptotic cell death was associated with the up-regulation of pro-apoptotic Bax protein expression, down-regulation of antiapoptotic Bcl-2 protein, and the release of cytochrome c from mitochondria to the cytosol. The treatment of AGS cells with HHS significantly elevated the generation of reactive oxygen species (ROS). Additionally, apoptosis-inducing concentrations of HHS induced the activation of both caspase-9 and -8, initiator caspases of the mitochondrial-mediated intrinsic and death receptor-mediated extrinsic pathways, respectively, and caspase-3, accompanied by proteolytic degradation of poly (ADP-ribose)-polymerase. However, ROS scavenger and pan-caspases inhibitor significantly blocked HHS-induced growth inhibition and apoptosis. Taken together, these findings suggest that HHS induces apoptosis through ROS- and caspase-dependent mechanisms and that HHS may be a potential chemotherapeutic agent for the control of human gastric cancer.

• Journal of Physiology & Pathology in Korean Medicine
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• v.27 no.6
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• pp.764-770
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• 2013

Stevia rebaudiana is a traditional herb used as a sweetener in Brazil and Paraguay as well as Korea and China. This study investigated the efficacy of Stevia rebaudiana methanol extract (SRE) to protect cells against the mitochondrial dysfunction and apoptosis in hepatocyte. To determine the effects of SRE on oxidative stress, we used the human derived hepatocyte cell line, HepG2 cell. Treatment of arachidonic acid (AA)+iron in HepG2 cells synergistically amplified cytotoxicity, as indicated by the excess reactive oxygen species (ROS) and mitochondrial permeability transition by fluorescence activated cell sorter (FACS) and immunoblot analysis. Treatment with SRE protected hepatocytes from AA+iron-induced cellular toxicity, as shown by alterations in the protein levels related with cell viability such as procaspase-3. SRE also prevented the mitochondrial dysfunction induced by AA+iron, and showed anti-oxidant effects as inhibition of $H_2O_2$ production and GSH depletion. Moreover, we measured the effects of SRE on AMP-activated protein kinase (AMPK), a key regulator in determining cell survival or death. Acetyl-CoA Carboxylase (ACC), a direct downstream target of AMPK. SRE increased phosphorylation of ACC, and prevented the inhibition of ACC phosphorylation by AA+iron. These results indicated that SRE has the ability to protect cells against AA+iron-induced $H_2O_2$ production and mitochondrial impairment, which may be mediated with AMPK-ACC pathway.