Vascular smooth muscle cell (VSMC) apoptosis has been identified in various vascular diseases, including atherosclerosis and restenosis after angioplasty, and has been known to precipitate atherosclerotic plaque instability and rupture. Oxysterols are known as inducers of apoptosis in VSMC, and 7-ketocholesterol (7KC) is the major nonenzymically formed oxysterol in atherosclerotic lesions. The precise mechanism underlying VSMC apoptosis is still poorly understood. In this study, we investigated whether 7KC causes apoptosis, and characterized its apoptotic mechanisms in primary cultured rat aortic VSMC. Cell viability was assessed by MTT assay and trypan blue assay. Apoptosis was assessed by flow cytometry, immunofluorescence, immunoprecipitation, and Western blot analyses. 7KC markedly decreased the VSMC viability in a time- and concentration-dependent manner, and increased the production of 4-hydroxynonenal (HNE), a major end-product of lipid peroxidation, which also decreased the VSMC viability. Pretreatment with 2,4-dinitrophenylhydrazine, a well-known reagent of lipid peroxidation-derived aldehydes, significantly restored the 7KC-decreased viability of VSMC. Furthermore, HNE, as well as 7KC, reduced the level of total Akt, a major mediator of cell survival. The 7KC-decreased level of total Akt was significantly restored by pretreatments with 2,4-dinitrophenylhydrazine and N-acetylcysteine. Lactacystin, a proteasome inhibitor, protected VSMC against apoptosis and Akt degradation, but did not inhibit HNE production. In the immunoprecipitation assay, 7KC increased HNE-modified Akt. From the results, it seems that, in atherosclerotic lesions, 7KC induces HNE production in VSMC, and this HNE binds to Akt, proceeding to proteasomal degradation of Akt, through which mechanism the atherosclerotic plaque instability may be facilitated.
The objective of the present study was to investigate the chemical composition of anthocyanin-enriched extract of radiation-induced blackberry (Rubus fruticosus L.) mutant (${\gamma}$-B201) as well as the protective effect of ${\gamma}$-B201 against oxidative stress in vitro. The cytotoxicity, reactive oxygen species (ROS) scavenging capacity, and DNA damage were assessed by WST-1 assay, flow cytometry, and comet assay, respectively. Lactate dehydrogenase, superoxide dismutase, and catalase activities were determined by using a commercial kit. The in vitro results showed that ${\gamma}$-B201 increased the cell viability, reduction of lactate dehydrogenase release, and intracellular ROS scavenging capacity in hydrogen peroxide ($H_2O_2$)-treated HepG2 cells. Furthermore, treatment with ${\gamma}$-B201 attenuated DNA damage in $H_2O_2$-treated HepG2 cells and treatment with ${\gamma}$-B201 restored the activity of superoxide dismutase and catalase in $H_2O_2$-treated HepG2 cells. In conclusion, the present study suggests that ${\gamma}$-B201 blackberry extract can exert a significant cytoprotective effect against $H_2O_2$-induced cell damage.
Purpose: This study examined the immunological activity and optimized the mixture conditions of Sargassum horneri (S. horneri) extracts in vitro and in vivo models. Methods: S. horneri was extracted using three different methods: hot water extraction (HWE), 50% ethanol extraction (EE), and supercritical fluid extraction (SFE). Splenocyte proliferation and cytokine production (Interleukin-2 and Interferon-γ) were measured using a WST-1 assay and enzyme-linked immunosorbent assay, respectively. The levels of nitric oxide and T cell activation production were measured using a Griess assay and flow cytometry, respectively. The natural killer (NK) cell activity was determined using an EZ-LDH kit. Results: Among the three different types of extracts, HWE showed the highest levels of splenocyte proliferation and cytokine production in vitro. In the animal model, three different types of extracts were administrated for 14 days (once/day) at 50 and 100 mg/kg body weight. HWE and SFE showed a high level of splenocyte proliferation and cytokine production in the with and without mitogen-treated groups, whereas EE administration did not induce the splenocyte activation. When RAW264.7 macrophage cells were treated with different mixtures (HWE with 5, 10, 15, 20% of SFE) to determine the optimal mixture ratio of HWE and SFE, the levels of nitric oxide and cytokine production increased strongly in the HWE with 5% and 10% of SFE containing group. In the animal model, HWE with 5% and 10% of SFE mixture administration increased the levels of splenocyte proliferation, cytokine production, and activated CD4+ cell population significantly, with the highest level observed in the HWE with 5% of SFE group. Moreover, the NK cell activity was increased significantly in the HWE with 5% of SFE mixture-treated group compared to the control group. Conclusion: The optimal mixture condition of S. horneri with immune-enhancing activity is the HWE with 5% of SFE mixture. These results confirmed that the extracts of S. horneri and its mixtures are potential candidate materials for immune enhancement.
Jung, Gi Hwan;Kim, Sung Hyun;Woo, Joong Seok;Yoo, Eun Seon;Lee, Jae Han;Han, So Hee;Jung, Soo Hyun;Kim, Sae Man;Kim, Eun Gee;Shin, Man Jae;Cho, Ho Min;Jung, Ji Youn
Journal of Life Science
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v.31
no.9
/
pp.827-833
/
2021
Dendropanax morbiferus leaves (DPL) has been used as a medicine since ancient times in various diseases such as inflammation, diabetes, and cancer. In particular, it has been found to have anticancer effects on several types of cancer cells, but the anticancer effect on breast cancer cells SK-BR-3 has not yet been revealed. Therefore, in this study, DPL caused proliferation inhibition in breast cancer cells SK-BR-3 and the anticancer effect by inducing apoptosis was confirmed, through an in vitro experiment. In order to examine the effect of DPL on cell viability, MTT assay was performed to confirm a significant decrease in the concentration of cell viability. DAPI staining was performed to examine the effect of DPL on cellular morphological changes and increase of apoptotic bodies. To supplement this, an increase in the apoptosis rate was also confirmed through flow cytometry after staining with annexin V/PI. Western blot was performed to confirm apoptosis-related proteins. DPL increased the expression of Cleaved-PARP, Bax whereas decreased the expression of Bcl-2. Changes in the expression levels of MAPK pathway proteins p-ERK1/2, p-JNK, and p-p38 were also confirmed, and a significant increase in p-p38 was observed. These results indicated that DPL induced apoptosis, through p-p38 MAPK signal pathway in SK-BR-3 breast cancer cells.
Kim, Min-Jeong;Kim, Jihee;Lee, Jin-Ho;Kim, Minah;Woo, Keunjung;Kim, Han Sung;Kim, Tack-Joong
Journal of Life Science
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v.31
no.9
/
pp.787-795
/
2021
Eupatorium chinensis var. simplicifolium (EUC) has anti-inflammatory and antioxidant effects. Young sprouts of EUC have been used as food for a long time, and the whole EUC plant has been used as an herbal remedy in oriental medicine. Arteriosclerosis, or chronic inflammation in arterial vessels, is a cardiovascular disease and is involved in various disorders. Cardiovascular diseases such as restenosis and neuropathic hyperplasia are mainly caused by abnormal growth and movement due to multiple growth factors in vascular smooth muscle cells (VSMCs). Platelet-derived growth factor (PDGF) is a mitogen released from damaged vessel walls and is involved in the proliferation and migration of VSMCs. To determine the effects of EUC on the abnormal proliferation and migration of VSMCs, the present study investigated intracellular signaling pathways in PDGF-BB-induced VSMCs treated with and without EUC. Pretreating PDGF-BB-induced VSMCs with EUC tended to effectively decrease cell proliferation and migration. Subsequently, the intracellular growth-related signaling pathways of AKT, phospholipase C gamma (PLC-γ), and mitogen-activated protein kinase (MAPK) were investigated using western blotting to confirm inhibited phosphorylation. Furthermore, flow cytometry data showed that EUC blocked the cell cycle of VSMCs. These results suggest that EUC can inhibit the proliferation and migration of VSMCs by controlling the cell cycle and growth factor receptors. Furthermore, this indicates that EUC can be used as a preventative against cardiovascular disease resulting from abnormal proliferation and migration of VSMCs.
Park, Kyung Woo;Yoo, Man Ho;Oh, Hyun Ju;Youn, Seok Hyun;Kwon, Kee Young;Moon, Chang Ho
Korean Journal of Environmental Biology
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v.37
no.1
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pp.93-108
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2019
To investigate the temporal-spatial distribution of picophytoplankton in relation to different water masses in the northern East China Sea (ECS), picophytoplankton abundance were investigated using flow cytometry with environmental factors in 2016-2017. The results from the analysis of flow cytometer data showed that Synechococcus appeared across all seasons, exhibiting its minimum abundance in winter and maximum abundance in summer. Furthermore, high abundance was detected in the surface mixed layer during spring and summer when vertical stratification occurs; in particular, Synechococcus exhibited maximum abundance in thermocline layer, indicating a close correlation to water temperature and thermocline formation. In addition, the abundance of Synechococcus indicated a decrease in the western seas in 2017 compared to 2016 under the strong influence of the Changjiang Diluted Water (CDW). This was determined by the significant influence of the CDW on the abundance of Synechococcus during summer in the northern waters of the ECS. In contrast, Prochlorococcus did not appear during winter and spring, and its distribution was limited during summer and autumn in the eastern seas under the influence of the Kuroshio current. The largest range of Prochlorococcus distribution was confirmed during autumn without the influence of the CDW. Thus, the distribution pattern of each picophytoplankton genus was found to be changing in accordance to the extension and reduction of sea current in different seasons and periods of time. This is anticipated to be a useful biological marker in understanding the distribution of sea currents and their influence in the northern waters of the ECS.
Journal of the Society of Cosmetic Scientists of Korea
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v.49
no.3
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pp.193-201
/
2023
Hydrogen peroxide (H2O2) is a type of active oxygen species (ROS) that causes oxidative stress in cells and affects cell growth, proliferation, senescence, and death. The purpose of this study is to find active peptides that attenuate cytotoxicity of H2O2. A positional scanning synthetic tetrapeptide combinatorial library was screened to predict the sequence of potentially active peptides. As a result of comparing the effect of peptide pools on H2O2-induced death of human keratinocytes (HaCaT cells), various active peptide sequences were predicted. Especially, peptides containing cysteine (C) residue were predicted to be active. In follow-up experiments, the cytotoxicity and activity of cysteine-containing peptides of different lengths, such as C-NH2, CC-NH2, CCC-NH2, and CCCC-NH2 were examined. C-NH2 and CC-NH2 showed no significant cytotoxicity up to 1.0 mM, but CCC-NH2, and CCCC-NH2 showed relatively strong cytotoxicity. C-NH2 and CC-NH2 alleviated H2O2-induced cytotoxicity. CC-NH2 was more cytoprotective compared to C-NH2, C, N-acetyl cysteine (NAC), and glutathione (GSH). When intracellular ROS was measured by flow cytometry, H2O2 increased ROS production, and CC-NH2 suppressed ROS production more effectively than C-NH2, and it was as effective as C, NAC, and GSH. This study suggests that CC-NH2 of the cysteine-containing peptides of different lengths has an antioxidant property that safely and effectively alleviates H2O2-induced cytotoxicity and ROS production.
Da Jung Ha;Seohwi Kim;Byunwoo Son;Myungho Jin;Sungwoo Cho;Sang Hoon Hong;Yung Hyun Choi;Sang Eun Park
Journal of Life Science
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v.33
no.12
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pp.1002-1014
/
2023
The root of Cirsium japonicum var. maackii (Maxim.) has long been used in traditional medicine to prevent the onset and progression of various diseases and has been reported to exert a wide range of physiological effects, including antioxidant activity. However, research on its effects on hepatocytes remains scarce. This study used the human hepatocellular carcinoma HepG2 cell line to investigate the antioxidant activity of ethanol extract of C. japonicum root (EECJ) on hepatocytes. Hydrogen peroxide (H2O2) was used to mimic oxidative stress. The results showed that EECJ significantly reverted the decrease in cell viability and suppressed the release of lactate dehydrogenase in HepG2 cells treated with H2O2. Moreover, an analysis of changes in cell morphology, flow cytometry, and microtubule-associated protein light chain 3 (LC3) expression showed that EECJ significantly inhibited HepG2 cell autophagy induced by H2O2. Furthermore, it attenuated H2O2-induced apoptosis and cell cycle disruption by blocking intracellular reactive oxygen species and mitochondrial superoxide production, indicating strong antioxidant activity. EECJ also restored the decreased levels of intracellular glutathione (GSH) and enhanced the expression and activity of superoxide dismutase and GSH peroxidase in H2O2-treated HepG2 cells. Although an analysis of the components contained in EECJ and in vivo validation using animal models are needed, these findings indicate that EECJ is a promising candidate for the prevention and treatment of oxidative stress-induced liver cell damage.
Jung Ah Kim;Sung-Hee Kim;Jeong Jin Kim;Hyuna Noh;Su-bin Lee;Haengdueng Jeong;Jiseon Kim;Donghun Jeon;Jung Seon Seo;Dain On;Suhyeon Yoon;Sang Gyu Lee;Youn Woo Lee;Hui Jeong Jang;In Ho Park;Jooyeon Oh;Sang-Hyuk Seok;Yu Jin Lee;Seung-Min Hong;Se-Hee An;Joon-Yong Bae;Jung-ah Choi;Seo Yeon Kim;Young Been Kim;Ji-Yeon Hwang;Hyo-Jung Lee;Hong Bin Kim;Dae Gwin Jeong;Daesub Song;Manki Song;Man-Seong Park;Kang-Seuk Choi;Jun Won Park;Jun-Won Yun;Jeon-Soo Shin;Ho-Young Lee;Ho-Keun Kwon;Jun-Young Seo;Ki Taek Nam;Heon Yung Gee;Je Kyung Seong
IMMUNE NETWORK
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v.24
no.2
/
pp.7.1-7.19
/
2024
Viral load and the duration of viral shedding of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are important determinants of the transmission of coronavirus disease 2019. In this study, we examined the effects of viral doses on the lung and spleen of K18-hACE2 transgenic mice by temporal histological and transcriptional analyses. Approximately, 1×105 plaque-forming units (PFU) of SARS-CoV-2 induced strong host responses in the lungs from 2 days post inoculation (dpi) which did not recover until the mice died, whereas responses to the virus were obvious at 5 days, recovering to the basal state by 14 dpi at 1×102 PFU. Further, flow cytometry showed that number of CD8+ T cells continuously increased in 1×102 PFU-virus-infected lungs from 2 dpi, but not in 1×105 PFU-virus-infected lungs. In spleens, responses to the virus were prominent from 2 dpi, and number of B cells was significantly decreased at 1×105 PFU; however, 1×12 PFU of virus induced very weak responses from 2 dpi which recovered by 10 dpi. Although the defense responses returned to normal and the mice survived, lung histology showed evidence of fibrosis, suggesting sequelae of SARS-CoV-2 infection. Our findings indicate that specific effectors of the immune response in the lung and spleen were either increased or depleted in response to doses of SARS-CoV-2. This study demonstrated that the response of local and systemic immune effectors to a viral infection varies with viral dose, which either exacerbates the severity of the infection or accelerates its elimination.
Dongju Seo;Se-Hui Lee;Sun Park;Hyeyun Kim;Jin-Young Yang
Journal of Life Science
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v.34
no.1
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pp.48-58
/
2024
Salmonella is a common food-borne intracellular bacterial pathogen that has triggered significant public health concerns. Salmonella hosts' genetic factors play a pivotal role in determining their susceptibility to the pathogen. Cysteine-rich intestinal protein 1 (CRIP1), a member of LIM/double zinc finger protein family, is widely expressed in humans, such as in the lungs, spleen, and especially the gut. Recently, CRIP1 has been reported as a key marker of several immune disorders; however, the effect of CRIP1 on bacterial infection remains unknown. We aimed to elucidate the relationship between Salmonella infection and CRIP1 gene deficiency, as Salmonella spp. is known to invade the Peyer's patches of the small intestine, where CRIP1 is highly expressed. We found that CRIP1-deficient conditions could not alter the characteristics of bone marrow-derived myeloid cells in terms of phagocytosis on macrophages and the activation of costimulatory molecules on dendritic cells using ex vivo differentiation. Moreover, flow cytometry data showed comparable levels of MHCII+CD11b+CD11c+ dendritic cells and MHCII+F4/80+CD11b+ macrophages between WT and CRIP1 knockout (KO) mice. Interestingly, the basal population of monocytes in the spleen and neutrophils in MLNs is more abundant in a steady state of CRIP1 KO mice than WT mice. Here, we demonstrated that the CRIP1 genetic factor plays dispensable roles in host susceptibility to Salmonella Typhimurium infections and the activation of myeloid cells. In addition, differential immune cell populations without antigen exposure in CRIP1 KO mice suggest that the regulation of CRIP1 expression may be a novel immunotherapeutic approach to various infectious diseases.
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