• Journal of Life Science
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• v.17 no.12
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• pp.1709-1716
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• 2007

The health benefits of garlic (Allium sativum L.) are derived from a wide variety of components and from the different ways it is administered. The known health benefits of garlic include cardiovascular protective effects, stimulation of immune function, reduction of blood glucose level, protection against microbial, viral and fungal infections, as well as anticancer effects. In the present study, it was examined the effects of water extract of A. sativum (WEAS) on the growth of cultured human tumor cells in order to investigate its anti-proliferative mechanism. Treatment of WEAS to tumor cells resulted in the growth inhibition, especially in leukemia cells, which was associated with induction of G2/M arrest of the cell cycle and apoptosis. In order to further explore the critical events leading to apoptosis in WEAS-treated U937 human leukemia cells, the following effects of WEAS on components of the mitochondrial apoptotic pathway were examined: generation of reactive oxygen species (ROS), alteration of the mitochondrial membrane potential (MMP), and the expression changes of Bcl-2 and IAP family proteins. The cytotoxic effect of WEAS was mediated by its induction of apoptosis as characterized by the occurrence of DNA ladders, apoptotic bodies and chromosome condensation in U937 cells. The WEAS-induced apoptosis in U937 cells was correlated with the generation of intracellular ROS, collapse of MMP, activation of caspase-3 and down-regulation of anti-apoptotic proteins. The quenching of ROS generation with antioxidant N-acetyl-L-cysteine conferred significant protection against WEAS-elicited ROS generation, caspase-3 activation, G2/M arrest and apoptosis. In conclusion, the present study reveals that the cellular ROS generation plays a pivotal role in the initiation of WEAS-triggered apoptotic death in U937 cells.

• Experimental and Molecular Medicine
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• v.50 no.11
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• pp.13.1-13.15
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• 2018

Wound healing is delayed in diabetic patients. Increased apoptosis and endothelial progenitor cell (EPC) dysfunction are implicated in delayed diabetic wound healing. Melatonin, a major secretory product of the pineal gland, promotes diabetic wound healing; however, its mechanism of action remains unclear. Here, EPCs were isolated from the bone marrow of mice. Treatment of EPCs with melatonin alleviated advanced glycation end product (AGE)-induced apoptosis and cellular dysfunction. We further examined autophagy flux after melatonin treatment and found increased light chain 3 (LC3) and p62 protein levels in AGE-treated EPCs. However, lysosome-associated membrane protein 2 expression was decreased, indicating that autophagy flux was impaired in EPCs treated with AGEs. We then evaluated autophagy flux after melatonin treatment and found that melatonin increased the LC3 levels, but attenuated the accumulation of p62, suggesting a stimulatory effect of melatonin on autophagy flux. Blockage of autophagy flux by chloroquine partially abolished the protective effects of melatonin, indicating that autophagy flux is involved in the protective effects of melatonin. Furthermore, we found that the AMPK/mTOR signaling pathway is involved in autophagy flux stimulation by melatonin. An in vivo study also illustrated that melatonin treatment ameliorated impaired wound healing in a streptozotocin-induced diabetic wound healing model. Thus, our study shows that melatonin protects EPCs against apoptosis and dysfunction via autophagy flux stimulation and ameliorates impaired wound healing in vivo, providing insight into its mechanism of action in diabetic wound healing.

• Korean Journal of Clinical Laboratory Science
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• v.53 no.2
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• pp.158-164
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• 2021

Hypertriglyceridemia is the main risk factor for atherosclerosis. It is reported that triglyceride (TG) induces macrophage cell death, and is involved in the formation of plaques and development of atherosclerosis. We previously reported that TG-induced cell death of macrophages is mediated via pannexin-1 activation, which increases the extracellular ATP and subsequent increase in potassium efflux, thereby activating the caspase-2/caspase-1/apoptotic caspases, including the caspase-8 pathway. Contrarily, some studies have reported that caspase-8 is an upstream molecule of caspase-1 and caspase-2 in several cellular processes. Therefore, this study was undertaken to investigate whether caspase-8 influences its upstream molecules in TG-stimulated macrophage cell death. We first confirmed that caspase-8 induces caspase-3 activation and poly ADP-ribose polymerase (PARP) cleavage in TG-treated macrophages. Next, we determined that the inhibition of caspase-8 results in reduced caspase-1 and -2 activity, which are upstream molecules of caspase-8 in TG-induced cell death of macrophages. We also found that ATP treatment restores the caspase-8 inhibitor-induced caspase-2 activity, thereby implying that caspase-8 affects the upstream molecules responsible for increasing the extracellular ATP levels in TG-induced macrophage cell death. Taken together, these findings indicate that caspase-8 potentiates the TG-induced macrophage cell death by activating its upstream molecules.

• Journal of Life Science
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• v.31 no.5
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• pp.464-474
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• 2021

Inflammation induced by metabolic syndromes, cancers, injuries, and sepsis can alter cellular metabolism by reducing mitochondrial function via oxidative stress, thereby resulting in neuropathy and muscle atrophy. In this study, we investigated whether butyrate, a short chain fatty acid produced by gut microbiota, could prevent mitochondrial dysfunction and muscle atrophy induced by lipopolysaccharide (LPS) in the C2C12 cell line. LPS-activated MAPK signaling pathways increased the levels of the mitochondrial fission signal, p-DRP1 (Ser616), and the muscle atrophy marker, atrogin 1. Interestingly, butyrate significantly inhibited the phosphorylation of JNK and p38 and reduced the atrogin 1 level in LPS-treated C2C12 cells while increasing the phosphorylation of DRP1 (Ser637) and levels of mitofusin2, which are both mitochondrial fusion markers. Next, we investigated the effect of MAPK inhibitors, finding that butyrate had the same effect as JNK inhibition in C2C12 cells. Also, butyrate inhibited the LPS-induced expression of pyruvate dehydrogenase kinase 4 (PDK4), resulting in decreased PDHE1α phosphorylation and lactate production, suggesting that butyrate shifted glucose metabolism from aerobic glycolysis to oxidative phosphorylation. Finally, we found that these effects of butyrate on LPS-induced mitochondrial dysfunction were caused by its antioxidant effects. Thus, our findings demonstrate that butyrate prevents LPS-induced muscle atrophy by improving mitochondrial dynamics and metabolic stress via the inhibition of JNK phosphorylation. Consequently, butyrate could be used to improve LPS-induced mitochondrial dysfunction and myopathy in sepsis.

• Journal of Ginseng Research
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• v.45 no.1
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• pp.134-148
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• 2021

Background: Lung cancer has a high incidence worldwide, and most lung cancer-associated deaths are attributable to cancer metastasis. Although several medicinal properties of Panax ginseng Meyer have been reported, the effect of ginsenosides Rk1 and Rg5 on epithelial-mesenchymal transition (EMT) stimulated by transforming growth factor beta 1 (TGF-β1) and self-renewal in A549 cells is relatively unknown. Methods: We treated TGF-β1 or alternatively Rk1 and Rg5 in A549 cells. We used western blot analysis, real-time polymerase chain reaction (qPCR), wound healing assay, Matrigel invasion assay, and anoikis assays to determine the effect of Rk1 and Rg5 on TGF-mediated EMT in lung cancer cell. In addition, we performed tumorsphere formation assays and real-time PCR to evaluate the stem-like properties. Results: EMT is induced by TGF-β1 in A549 cells causing the development of cancer stem-like features. Expression of E-cadherin, an epithelial marker, decreased and an increase in vimentin expression was noted. Cell mobility, invasiveness, and anoikis resistance were enhanced with TGF-β1 treatment. In addition, the expression of stem cell markers, CD44, and CD133, was also increased. Treatment with Rk1 and Rg5 suppressed EMT by TGF-β1 and the development of stemness in a dose-dependent manner. Additionally, Rk1 and Rg5 markedly suppressed TGF-β1-induced metalloproteinase-2/9 (MMP2/9) activity, and activation of Smad2/3 and nuclear factor kappa B/extra-cellular signal regulated kinases (NF-kB/ERK) pathways in lung cancer cells. Conclusions: Rk1 and Rg5 regulate the EMT inducing TGF-β1 by suppressing the Smad and NF-κB/ERK pathways (non-Smad pathway).

• Advances in nano research
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• v.12 no.5
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• pp.501-514
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• 2022

This study aimed to investigate the effects and potential mechanisms of Chikusetsusaponin V (CsV) on endothelial nitric oxide synthase (eNOS) and vascular endothelial cell functions. Different concentrations of CsV were added to animal models, bovine aorta endothelial cells (BAECs) and human umbilical vein endothelial cells (HUVECs) cultured in vitro. qPCR, Western blotting (WB), and B ultrasound were performed to explore the effects of CsV on mouse endothelial cell functions, vascular stiffness and cellular eNOS mRNA, protein expression and NO release. Bioinformatics analysis, network pharmacology, molecular docking and protein mass spectrometry analysis were conducted to jointly predict the upstream transcription factors of eNOS. Furthermore, pulldown and ChIP and dual luciferase assays were employed for subsequent verification. At the presence or absence of CsV stimulation, either overexpression or knockdown of purine rich element binding protein A (PURA) was conducted, and PCR assay was employed to detect PURA and eNOS mRNA expressions, Western blot was used to detect PURA and eNOS protein expressions, cell NO release and serum NO levels. Tube formation experiment was conducted to detect the tube forming capability of HUVECs cells. The animal vasodilation function test detected the vasodilation functions. Ultrasonic detection was performed to determine the mouse aortic arch pulse wave velocity to identify aortic stiffness. CsV stimulus on bovine aortic cells revealed that CsV could upregulate eNOS protein levels in vascular endothelial cells in a concentration and time dependent manner. The expression levels of eNOS mRNA and phosphorylation sites Ser1177, Ser633 and Thr495 increased significantly after CsV stimulation. Meanwhile, CsV could also enhance the tube forming capability of HUVECs cells. Following the mice were gavaged using CsV, the eNOS protein level of mouse aortic endothelial cells was upregulated in a concentration- and time-dependent manner, and serum NO release and vasodilation ability were simultaneously elevated whereas arterial stiffness was alleviated. The pulldown, ChIP and dual luciferase assays demonstrated that PURA could bind to the eNOS promoter and facilitate the transcription of eNOS. Under the conditions of presence or absence of CsV stimulation, overexpression or knockdown of PURA indicated that the effect of CsV on vascular endothelial function and eNOS was weakened following PURA gene silence, whereas overexpression of PURA gene could enhance the effect of CsV upregulating eNOS expression. CsV could promote NO release from endothelial cells by upregulating the expression of PURA/eNOS pathway, improve endothelial cell functions, enhance vasodilation capability, and alleviate vessel stiffness. The present study plays a role in offering a theoretical basis for the development and application of CsV in vascular function improvement, and it also provides a more comprehensive understanding of the pharmacodynamics of CsV.

• Journal of Life Science
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• v.32 no.1
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• pp.23-28
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• 2022

CopA3 (LLCIALRKK), an antimicrobial peptide isolated from the Korean dung beetle, has been shown to suppress apoptosis in various cell types. CopA3 inhibits not only bacterial toxin-induced colonocyte apoptosis but also 6-hydroxy dopamine-induced neural cell apoptosis. Our recent study revealed that CopA3 directly binds to caspases (key regulators of apoptosis) and inhibits the proteolytic cleavage required for their activation. But molecular mechanisms underlying CopA3-mediated inhibition of apoptosis in multiple cell types remain unknown. Here we assessed possible effects of CopA3 on expression of survivin, which is known to inhibit apoptosis. In HT29 human colonocytes, CopA3 exposure markedly upregulated survivin expression in a concentration- and time-dependent manner. RT-PCR revealed that CopA3-mediated upregulation of survivin was attributable to increased gene transcription, and further showed that CopA3 also increased expression of Sp1, one of many transcription factors known to be involved in transcription of the survivin gene. Notably, blocking Sp1 by treatment with the Sp1 inhibitor, tolfenamic acid, significantly reduced CopA3-mediated upregulation of survivin. These results collectively suggest that CopA3 induces Sp1 expression, which in turn is involved in upregulation of survivin in human colonocytes. These novel findings establish another pathway for explaining the anti-apoptotic effects of CopA3 against various cellular apoptosis systems.

• Journal of Ginseng Research
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• v.47 no.1
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• pp.97-105
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• 2023

Background: Hyperactivated airway mucosa cells overproduce mucin and cause severe breathing complications. Here, we aimed to identify the effects of saponins derived from Panax ginseng on inflammation and mucin overproduction. Methods: NCI-H292 cells were pre-incubated with 16 saponins derived from P. ginseng, and mucin overproduction was induced by treatment with phorbol 12-myristate 13-acetate (PMA). Mucin protein MUC5AC was quantified by enzyme-linked immunosorbent assay, and mRNA levels were analyzed using quantitative polymerase chain reaction (qPCR). Moreover, we performed a transcriptome analysis of PMA-treated NCI-H292 cells in the absence or presence of Rg5, and differential gene expression was confirmed using qPCR. Phosphorylation levels of signaling molecules, and the abundance of lipid droplets, were measured by western blotting, flow cytometry, and confocal microscopy. Results: Ginsenoside Rg5 effectively reduced MUC5AC secretion and decreased MUC5AC mRNA levels. A systematic functional network analysis revealed that Rg5 upregulated cholesterol and glycerolipid metabolism, resulting in the production of lipid droplets to clear reactive oxygen species (ROS), and modulated the mitogen-activated protein kinase and nuclear factor (NF)-kB signaling pathways to regulate inflammatory responses. Rg5 induced the accumulation of lipid droplets and decreased cellular ROS levels, and N-acetyl-ⳑ-cysteine, a ROS inhibitor, reduced MUC5AC secretion via Rg5. Furthermore, Rg5 hampered the phosphorylation of extracellular signal-regulated kinase and p38 proteins, affecting the NF-kB signaling pathway and pro-inflammatory responses. Conclusion: Rg5 alleviated inflammatory responses by reducing mucin secretion and promoting lipid droplet-mediated ROS clearance. Therefore, Rg5 may have potential as a therapeutic agent to alleviate respiratory disorders caused by hyperactivation of mucosa cells.

• Animal Bioscience
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• v.36 no.7
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• pp.1022-1033
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• 2023

Objective: p66Shc, a 66 kDa protein isoform encoded by the proto-oncogene SHC, is an essential intracellular redox homeostasis regulatory enzyme that is involved in the regulation of cellular oxidative stress, apoptosis induction and the occurrence of multiple age-related diseases. This study investigated the expression profile and functional characteristics of p66Shc during preimplantation embryo development in sheep. Methods: The expression pattern of p66Shc during preimplantation embryo development in sheep at the mRNA and protein levels were studied by quantitative real-time polymerase chain reaction (RT-qPCR) and immunofluorescence staining. The effect of p66Shc knockdown on the developmental potential were evaluated by cleavage rate, morula rate and blastocyst rate. The effect of p66Shc deficiency on reactive oxygen species (ROS) production, DNA oxidative damage and the expression of antioxidant enzymes (e.g., catalase and manganese superoxide dismutase [MnSOD]) were also investigated by immunofluorescence staining. Results: Our results showed that p66Shc mRNA and protein were expressed in all stages of sheep early embryos and that p66Shc mRNA was significantly downregulated in the 4-to 8-cell stage (p<0.05) and significantly upregulated in the morula and blastocyst stages after embryonic genome activation (EGA) (p<0.05). Immunofluorescence staining showed that the p66Shc protein was mainly located in the peripheral region of the blastomere cytoplasm at different stages of preimplantation embryonic development. Notably, serine (Ser36)-phosphorylated p66Shc localized only in the cytoplasm during the 2- to 8-cell stage prior to EGA, while phosphorylated (Ser36) p66Shc localized not only in the cytoplasm but also predominantly in the nucleus after EGA. RNAi-mediated silencing of p66Shc via microinjection of p66Shc siRNA into sheep zygotes resulted in significant decreases in p66Shc mRNA and protein levels (p<0.05). Knockdown of p66Shc resulted in significant declines in the levels of intracellular ROS (p<0.05) and the DNA damage marker 8-hydroxy2'-deoxyguanosine (p<0.05), markedly increased MnSOD levels (p<0.05) and resulted in a tendency to develop to the morula stage. Conclusion: These results indicate that p66Shc is involved in the metabolic regulation of ROS production and DNA oxidative damage during sheep early embryonic development.

• Animal Bioscience
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• v.36 no.9
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• pp.1336-1349
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• 2023

Objective: The study was conducted to screen differentially expressed miRNAs in sows at early pregnancy by high-throughput sequencing and explore its mechanism of action on embryo implantation. Methods: The blood serum of pregnant and non-pregnant Landrace×Yorkshire sows were collected 14 days after artificial insemination, and exosomal miRNAs were purified for high throughput miRNA sequencing. The expression patterns of 10 differentially expressed (DE) miRNAs were validated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The qRT-PCR quantified the abundance of serum exosomal miR-192 in pregnant and control sows, and the diagnostic power was assessed by receiver operating characteristic (ROC) analysis. The target genes of DE miRNAs were predicted with bioinformatics software, and the functional and pathway enrichment analysis was performed on gene ontology and the Kyoto encyclopedia of genes and genomes terms. Furthermore, a luciferase reporter system was used to identify the target relation between miR-192 and integrin alpha 4 (ITGA4), a gene influencing embryo implantation in pigs. Finally, the expression levels of miRNAs and the target gene ITGA4 were analyzed by qRT-PCR, and western blot, with the proliferation of BeWo cells detected by cell counting kit-8 (CCK-8). Results: A total of 221 known miRNAs were detected in the libraries of the pregnant and non-pregnant sows, of which 55 were up-regulated and 67 were down-regulated in the pregnant individuals compared with the non-pregnant controls. From these, the expression patterns of 10 DE miRNAs were validated. The qRT-PCR analysis further confirmed a significantly higher expression of miR-192 in the serum exosomes extracted from pregnant sows, when compared to controls. The ROC analysis revealed that miR-192 provided excellent diagnostic accuracy for pregnancy (area under the ROC curve [AUC]=0.843; p>0.001). The dual-luciferase reporter assay indicated that miR-192 directly targeted ITGA4. The protein expression of ITGA4 was reduced in cells that overexpressed miR-192. Overexpression of miR-192 resulted in the decreased proliferation of BeWo cells and regulated the expression of cell cycle-related genes. Conclusion: Serum exosomal miR-192 could serve as a potential biomarker for early pregnancy in pigs. miR-192 targeted ITGA4 gene directly, and miR-192 can regulate cellular proliferation.