• Journal of The Korean Society of Integrative Medicine
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• v.11 no.4
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• pp.73-82
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• 2023

Purpose : Cymbopogon citratus, also known as lemongrass, has widely spread around the world and its essential oil is usually applied in food, perfume, and other industrial purposes. In addition, C. citratus has also been used for the treatment of inflammation, digestive disorders, and diabetes in traditional medicine. In this study, the antioxidative activity of C. citratus ethanol extract (CCEE) was analyzed in RAW 264.7 cells through the induction of one of phase II enzymes, heme oxygenase (HO)-1 by nuclear factor-erythroid 2 p45-related factor (Nrf)2, mitogen-activated protein kinase (MAPK), and phosphoinositide 3-kinase (PI3K)/Akt. Methods : The antioxidative activity of CCEE against oxidative stress and its underlying molecular mechanisms were analyzed by the cell viability assay, intracellular reactive oxygen species (ROS) formation assay, and Western blot analysis in RAW 264.7 cells. Results : The results exhibited that CCEE potently attenuated tert-butyl hydroperoxide (t-BHP) induced intracellular ROS levels in a dose-dependent manner without any cytotoxicity. CCEE treatment significantly induced the expression of HO-1 which is known for its antioxidative capacity. In addition, CCEE treatment significantly upregulated the expression of Nrf2, a corresponding transcription factor for the regulation of antioxidative enzymes, which was in accordance with the HO-1 overexpression. MAPK and PI3K/Akt were also evaluated for their important roles in the regulation of cellular redox homeostasis against oxidative damage. As a result, the potent HO-1 expression was mediated by not extracellular regulated kinase (ERK), c-Jun NH2 terminal kinase (JNK), p38, but phosphoinositide 3-kinase (PI3K) phosphorylation. To confirm the antioxidative activity of CCEE-induced HO-1 expression, oxidative damage was initiated by t-BHP and attenuated by CCEE treatment, which was identified by HO-1 selective inhibitor and inducer. Conclusion : Consequently, CCEE potently induced the HO-1-mediated antioxidative potential through the modulation of Nrf2 and PI3K/Akt signaling pathways in RAW 264.7 cells. These results suggest that CCEE could be a promising strategy for the mitigation against cellular oxidative damage.

• The Korean Journal of Physiology and Pharmacology
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• v.27 no.6
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• pp.521-531
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• 2023

Transmembrane protein TMEM16A, which encodes calcium-activated chloride channel has been implicated in tumorigenesis. Overexpression of TMEM16A is associated with poor prognosis and low overall survival in multiple cancers including lung adenocarcinoma, making it a promising biomarker and therapeutic target. In this study, three structure-related sesquiterpene lactones (mecheliolide, costunolide and dehydrocostus lactone) were extracted from the traditional Chinese medicine Aucklandiae Radix and identified as novel TMEM16A inhibitors with comparable inhibitory effects. Their effects on the proliferation and migration of lung adenocarcinoma cells were examined. Whole-cell patch clamp experiments showed that these sesquiterpene lactones potently inhibited recombinant TMEM16A currents in a concentration-dependent manner. The half-maximal concentration (IC50) values for three tested sesquiterpene lactones were 29.9 ± 1.1 µM, 19.7 ± 0.4 µM, and 24.5 ± 2.1 µM, while the maximal effect (E_max) values were 100.0% ± 2.8%, 85.8% ± 0.9%, and 88.3% ± 4.6%, respectively. These sesquiterpene lactones also significantly inhibited the endogenous TMEM16A currents and proliferation, and migration of LA795 lung cancer cells. These results demonstrate that mecheliolide, costunolide and dehydrocostus lactone are novel TMEM16A inhibitors and potential candidates for lung adenocarcinoma therapy.

• Animal Bioscience
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• v.36 no.12
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• pp.1775-1784
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• 2023

Objective: The aim of this study was to reveal the role and regulatory mechanism of miR-188-5p in the proliferation and differentiation of goat muscle satellite cells. Methods: Goat skeletal muscle satellite cells isolated in the pre-laboratory were used as the test material. First, the expression of miR-188-5p in goat muscle tissues at different developmental stages was detected by quantitative reverse transcription polymerase chain reaction (qRT-PCR). In addition, miR-188-5p was transfected into goat skeletal muscle satellite cells by constructing mimics and inhibitors of miR-188-5p, respectively. The changes of differentiation marker gene expression were detected by qPCR method. Results: It was highly expressed in adult goat latissimus dorsi and leg muscles, goat fetal skeletal muscle, and at the differentiation stage of muscle satellite cells. Overexpression and interference of miR-188-5p showed that miR-188-5p inhibited the proliferation and promoted the differentiation of goat muscle satellite cells. Target gene prediction and dual luciferase assays showed that miR-188-5p could target the 3'untranslated region of the calcium/calmodulin dependent protein kinase II beta (CAMK2B) gene and inhibit luciferase activity. Further functional studies revealed that CAMK2B promoted the proliferation and inhibited the differentiation of goat muscle satellite cells, whereas si-CAMK2B restored the function of miR-188-5p inhibitor. Conclusion: These results suggest that miR-188-5p inhibits the proliferation and promotes the differentiation of goat muscle satellite cells by targeting CAMK2B. This study will provide a theoretical reference for future studies on the molecular mechanisms of skeletal muscle development in goats.

• Journal of Ginseng Research
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• v.48 no.3
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• pp.298-309
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• 2024

Background: 20(S)-ginsenoside Rh2(GRh2), an effective natural histone deacetylase inhibitor, can inhibit acute myeloid leukemia (AML) cell proliferation. Lactate regulated histone lactylation, which has different temporal dynamics from acetylation. However, whether the high level of lactylation modification that we first detected in acute promyelocytic leukemia (APL) is associated with all-trans retinoic acid (ATRA) resistance has not been reported. Furthermore, Whether GRh2 can regulate lactylation modification in ATRA-resistant APL remains unknown. Methods: Lactylation and METTL3 expression levels in ATRA-sensitive and ATRA-resistant APL cells were detected by Western blot analysis, qRT-PCR and CO-IP. Flow cytometry (FCM) and APL xenograft mouse models were used to determine the effect of METTL3 and GRh2 on ATRA-resistance. Results: Histone lactylation and METTL3 expression levels were considerably upregulated in ATRA-resistant APL cells. METTL3 was regulated by histone lactylation and direct lactylation modification. Overexpression of METTL3 promoted ATRA-resistance. GRh2 ameliorated ATRA-resistance by downregulated lactylation level and directly inhibiting METTL3. Conclusions: This study suggests that lactylation-modified METTL3 could provide a promising strategy for ameliorating ATRA-resistance in APL, and GRh2 could act as a potential lactylation-modified METTL3 inhibitor to ameliorate ATRA-resistance in APL.

• Journal of Life Science
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• v.34 no.1
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• pp.37-47
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• 2024

FUN14 domain-containing protein 1 (FUNDC1), an outer mitochondrial membrane protein, contributes to removal of damaged mitochondria through mitophagy. In this study, to elucidate the role of the FUNDC1 in the amyloid beta peptide (A_β)-induced neuropathy, changes in the degree of mitochondrial dysfunction and cell injury caused by A_β treatment were examined in the HT-22 neuronal cells in which the FUNDC1 expression was transiently silenced or overexpressed. We found that A_β treatment causes a time-dependent decrease of the FUNDC1 expression. In the A_β-treated cells, there were a drop in MTT reduction ability, depletion of cellular ATP, disruption of mitochondrial membrane potential, stimulation of cellular ROS production, and increased mitochondrial Ca²⁺ load. Activation of caspase-3 and induction of apoptotic cell death were also observed. Transient silencing of the FUNDC1 expression by transfection with the FUNDC1 small interfering RNA per se caused mitochondrial dysfunction and apoptotic cell death like the effect of A_β treatment. Conversely, in cells in which the FUNDC1 was transiently overexpressed by FUNDC1-Myc transfection, overexpression itself had no effect on the mitochondrial functional integrity and cell survival but showed a significant prevention effect against mitochondrial and cell injury caused by A_β treatment. Overall, these results suggest that the FUNDC1 is importantly involved in the A_β-induced mitochondrial dysfunction and cell injury in the HT-22 neuronal cells.

• IMMUNE NETWORK
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• v.22 no.5
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• pp.40.1-40.24
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• 2022

Mesenchymal stem cells (MSCs) are attractive alternatives to conventional anti-asthmatic drugs for severe asthma. Mechanisms underlying the anti-asthmatic effects of MSCs have not yet been elucidated. This study evaluated the anti-asthmatic effects of intravenously administered MSCs, focusing on macrophages and monocytes. Seven-week-old transgenic (Tg) mice with lung-specific overexpression of IL-13 were used to simulate chronic asthma. MSCs were intravenously administered four days before sampling. We examined changes in immune cell subpopulations, gene expression, and histological phenotypes. IL-13 Tg mice exhibited diverse features of chronic asthma, including severe type 2 inflammation, airway fibrosis, and mucus metaplasia. Intravenous administration of MSCs attenuated these asthmatic features just four days after a single treatment. MSC treatment significantly reduced SiglecF^-CD11c^-CD11b⁺ monocyte-derived macrophages (MoMs) and inhibited the polarization of MoMs into M2 macrophages, especially M2a and M2c. Furthermore, MSCs downregulated the excessive accumulation of Ly6c^- monocytes in the lungs. While an intravenous adoptive transfer of Ly6c^- monocytes promoted the infiltration of MoM and Th2 inflammation, that of MSC-exposed Ly6c^- monocytes did not. Ex vivo Ly6c^- MoMs upregulated M2-related genes, which were reduced by MSC treatment. Molecules secreted by Ly6c^- MoMs from IL-13 Tg mice lungs upregulated the expression of fibrosis-related genes in fibroblasts, which were also suppressed by MSC treatment. In conclusion, intravenously administered MSCs attenuate asthma phenotypes of chronic asthma by modulating macrophages. Identifying M2 macrophage subtypes revealed that exposure to MSCs transforms the phenotype and function of macrophages. We suggest that Ly6c^- monocytes could be a therapeutic target for asthma management.

• International Journal of Stem Cells
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• v.16 no.1
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• pp.52-65
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• 2023

Background and Objectives: Epithelial-Mesenchymal transition (EMT) is one of the origins of myofibroblasts in renal interstitial fibrosis. Mesenchymal stem cells (MSCs) alleviating EMT has been proved, but the concrete mechanism is unclear. To explore the mechanism, serum-free MSCs conditioned medium (SF-MSCs-CM) was used to treat rat renal tubular epithelial cells (NRK-52E) fibrosis induced by transforming growth factor-β1 (TGF-β1) which ameliorated EMT. Methods and Results: Galectin-3 knockdown (Gal-3 KD) and overexpression (Gal-3 OE) lentiviral vectors were established and transfected into NRK-52E. NRK-52E fibrosis model was induced by TGF-β1 and treated with the SF-MSCs-CM for 24 h after modelling. Fibrosis and autophagy related indexes were detected by western blot and immunocytochemistry. In model group, the expressions of α-smooth muscle actin (α-SMA), fibronectin (FN), Galectin-3, Snail, Kim-1, and the ratios of P-Akt/Akt, P-GSK3β/GSK3β, P-PI3K/PI3K, P-mTOR/mTOR, TIMP1/MMP9, and LC3B-II/I were obviously increased, and E-Cadherin (E-cad) and P62 decreased significantly compared with control group. SF-MSCs-CM showed an opposite trend after treatment compared with model group. Whether in Gal-3 KD or Gal-3 OE NRK-52E cells, SF-MSCs-CM also showed similar trends. However, the effects of anti-fibrosis and enhanced autophagy in Gal-3 KD cells were more obvious than those in Gal-3 OE cells. Conclusions: SF-MSCs-CM probably alleviated the EMT via inhibiting Galectin-3/Akt/GSK3β/Snail pathway. Meanwhile, Gal-3 KD possibly enhanced autophagy via inhibiting Galectin-3/Akt/mTOR pathway, which synergistically ameliorated renal fibrosis. Targeting galectin-3 may be a potential target for the treatment of renal fibrosis.

• Journal of Chest Surgery
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• v.38 no.12 s.257
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• pp.835-843
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• 2005

Background: Cancer of the esophagus is one of the most malignant tumors with poor prognosis. The p53 gene alteration, over expression of Cyclin D1, and Ki67 index were thought to be the prognostic factors. However, their clinical significances in esophageal squamous cell carcinoma are controversial and p53 accumulation may not correlate with genetic mutation. The current study investigates their prognostic significance in squamous cell carcinoma of the esophagus. Material and Method: The Subjects studied were 124 esophageal squamous cell carcinoma patients who underwent esophagectomy. The mutation of p53, over expression of Cyclin D1, Ki67 labelling index, mitotic index were examined by using an immunohistochemical staining. We compared the results and investigated the correlation with the mutation of p53, overexpression of Cyclin D1, Ki67 labelling index, mitotic index and tumor size, and duration of survival. Result: There was no correlation between the results in immunohistochemical staining according to age, sex, tumor size, Iymph node status, and clinical stage of the disease. Mutant p53 protein was found in 69 cases (55.6$\%$). Median survival time was 21 months in cases with negative for mutant p53 protein and 22 months in positive cases. There was no significant difference in survival (p=0.46). Median survival time was 22 months in cases with negative for Cyclin D1 and 16 months in positive cases (p=0.18). Median and mean survival time was 22 months and 36 months when Ki67 labeling index was 40 or less (102 cases). Median and mean survival was 16 months and 23 months, when Ki67 labeling index was more than 40 (22 cases). There was significant difference in survival rate (p=0.011). Conciusion: Positivity of p53 and cyclin D1 was not useful in predicting the prognosis in our study. There was no significant correlation among mutant p53 protein accumulation, Cyclin D1 over expression, and Ki67 labeling index. However, in several studies, PCR single strand conformational polymorphism analysis of p53 showed a correlation to the prognosis. We thought that there was a significant discordance between p53 gene mutation and mutant p53 protein accumulation. When Ki67 labeling index was more than 40, prognosis was poorer, Ki67 seems to be a prognostic factor in our study. Therefore, we confirmed the possibility of using molecular markers as prognostic factors.

• Journal of Life Science
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• v.28 no.7
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• pp.835-840
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• 2018

Quantitative determination of the protein expression levels is one of the most important parts in assessment of the safety of foods derived from genetically modified (GM) crops. Overexpression of AtCYP78A7, a gene encoding cytochrome P450 protein, has been reported to improve tolerance to abiotic stress, such as drought and salt stress, in transgenic rice (Oryza sativa L.). In the present study, an enzyme-linked immunosorbent assay (ELISA) kit for diagnosing AtCYP78A7 protein including AtCYP78A7-specific monoclonal antibody was developed. GST-AtCYP78A7 recombinant protein was induced and purified by affinity column. Four monoclonal antibodies (mAb 6A7, mAb 4C2, mAb 11H6, and mAb 7E8) against recombinant protein were also produced and biotinylated with avidin-HRP. After pairing test using GST-AtCYP78A7 protein and lysate of rice samples, mAb 4C2 and mAb 7E8 were selected as a capture antibody and a detecting antibody, respectively, for ELISA kit. Product test using rice samples indicated that percentages of detected protein in total protein were greater than 0.1% in AtCYP78A7-overexpressing transgenic rice (Line 10B-5 and 18A-4), whereas those in negative control non-transgenic rice (Ilpum and Hwayoung) were less than 0.1%. The ELISA kit developed in this study can be useful for the rapid detection and safety assessment of transgenic rice overexpressing AtCYP78A7.

• Journal of Life Science
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• v.16 no.3 s.76
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• pp.546-554
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• 2006

To understand antitumor activity of Zanthoxylum schinfolium, which has been used as an aromatic and medicinal plant in Korea, the cytotoxic effect of various organic solvent extracts of its leaves on human tumor cells were investigated. Among these extracts such as methanol extract (SL-13), methylene chloride extract (SL-14), ethyl acetate extract (SL-15), n-butanol extract (SL-16), and residual fraction (SL-17), SL-14 appeared to contain the most cytotoxic activity against leukemia and breast cancer cells tested. The methylene chloride extra.1 (SL-14) possessed an apoptogenic activity causing apoptotic DNA fragmentation of human acute leukemia Jurkat T cells via mitochondrial cytochrome c release into cytoplasm, subsequent activation of caspase-9 and caspase-3, and cleavage of PARP, which could be negatively regulated by antiapoptotic protein Bcl-xL. The GC-MS analysis of SL-14 revealed that the twenty-two ingredients of SL-14 were 9,19-cyclolanost-24-en-3-ol (15.1%), 2-a-methyl-17, b-hop-21-ene (15.1%), 15-methyl-2,3-dihydro-1H benzazepin (11.95%), phytol (10.38%), lupeol (9.92%), 12-methylbenzofuran (8.23%), hexadecanoic acid (5.96%), cis,cis,cis-9,12,15-octadecatrienoic acid-methyl-ester (5.49%), 9,12,15-octadecatrienoic acid-methylester (3.59%), 15-methyl-4-(1-methylethylidene)-2-(4-nitrophenyl) (3.36%), hexadecanoic acid methyl ester (1.93%), vitamine E (1.88%), beta-amyrin (0.96%), and auraptene (0.89%). These results demonstrate that the cytotoxicity of the methylene chloride extract of the leaves of Z. schinifolium toward Jurkat T cells is mainly attributable to apoptosis mediated by mitochondria-dependent caspase cascade regulated by Bcl-xL, and provide an insight into the mechanism underlying antitumor activity of the edible plant Z. schinifolium.