• The Journal of Internal Korean Medicine
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• v.22 no.3
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• pp.383-391
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• 2001

목적 : 본 연구는 목향순기탕(木香順氣湯)이 인간의 장관상피세포 계열인 Caco-2 세포에서 항산화작용을 증진시키는 효과가 있는지 검증하기 위한 실험이다. 방법 : 배양된 인간장관 세포계열인 Caco-2 세포에서 세포의 사망은 trypan blue의 소실정도에 의해 평가했으며 $H_2O_2$는 표본산화제로 사용되었다. 결과 : $H_2O_2$에서 노출된 세포들은 용량에 비례하여 세포 사망하는 결과를 보였다. 목향순기탕(木香順氣湯)은 $H_2O_2$에 의해 유발된 세포사망을 방지하였고, 0.05-1%의 농도범위에 걸쳐서 효과가 극대화되었다. 목향순기탕(木香順氣湯)과 강력한 항산화제인 DPPD는 $H_2O_2$에 의해 억제된 SOD의 활성에는 영향을 주지는 못했다. 그러나 $H_2O_2$에 의해 유발된 catalase, glutathione peroxidase, hydroperoxide 탈취효소의 활성이 감소되는 것을 억제하였다. 또한 $H_2O_2$에 의해 유발된 glutathione의 감소는 목향순기탕(木香順氣湯)과 DPPD에 의해 억제되었다. 목향순기탕(木香順氣湯)은 $H_2O_2$에 의해 유발된 ATP의 소실을 회복시켰지만 DPPD는 ATP 소실을 회복시키지 못하였다. 결론 : 이러한 결과로 볼 때 Caco-2세포에서 목향순기탕이 세포사망을 억제하는 것은 다른 기전을 통하여 항산화작용을 하는 것으로 볼 수 있다. 따라서 본 연구는 목향순기탕(木香順氣湯)이 반응성산소기에 의해 유발된 인체 위장관질환의 치료에 사용할 수 있을 가능성을 제시하고 있다.

• Parasites, Hosts and Diseases
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• v.58 no.2
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• pp.173-179
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• 2020

Leishmaniasis is a prevalent cause of death and animal morbidity in underdeveloped countries of endemic area. However, there is few vaccine and effective drugs. Antimicrobial peptides are involved in the innate immune response in many organisms and are being developed as novel drugs against parasitic infections. In the present study, we synthesized a 5-amino acid peptide REDLK, which mutated the C-terminus of Pseudomonas exotoxin, to identify its effect on the Leishmania tarentolae. Promastigotes were incubated with different concentration of REDLK peptide, and the viability of parasite was assessed using MTT and Trypan blue dye. Morphologic damage of Leishmania was analyzed by light and electron microscopy. Cellular apoptosis was observed using the annexin V-FITC/PI apoptosis detection kit, mitochondrial membrane potential assay kit and flow cytometry. Our results showed that Leishmania tarentolae was susceptible to REDLK in a dose-dependent manner, disrupt the surface membrane integrity and caused parasite apoptosis. In our study, we demonstrated the leishmanicidal activity of an antimicrobial peptide REDLK from Pseudomonas aeruginosa against Leishmania tarentolae in vitro and present a foundation for further research of anti-leishmanial drugs.

• Toxicological Research
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• v.35 no.1
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• pp.83-91
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• 2019

Nanoparticles (NPs) have been recognized as both useful tools and potentially toxic materials in various industrial and medicinal fields. Previously, we found that zinc oxide (ZnO) NPs that are neurotoxic to human dopaminergic neuroblastoma SH-SY5Y cells are mediated by lipoxygenase (LOX), not cyclooxygenase-2 (COX-2). Here, we examined whether human bone marrow-derived mesenchymal stem cells (MSCs), which are different from neuroblastoma cells, might exhibit COX-2- and/or LOX-dependent cytotoxicity of ZnO NPs. Additionally, changes in annexin V expression, caspase-3/7 activity, and mitochondrial membrane potential (MMP) induced by ZnO NPs and ZnO were compared at 12 hr and 24 hr after exposure using flow cytometry. Cytotoxicity was measured based on lactate dehydrogenase activity and confirmed by trypan blue staining. Rescue studies were executed using zinc or iron chelators. ZnO NPs and ZnO showed similar dose-dependent and significant cytotoxic effects at concentrations ${\geq}15{\mu}g/mL$, in accordance with annexin V expression, caspase-3/7 activity, and MMP results. Human MSCs exhibited both COX-2 and LOX-mediated cytotoxicity after exposure to ZnO NPs, which was different from human neuroblastoma cells. Zinc and iron chelators significantly attenuated ZnO NPs-induced toxicity. Conclusively, these results suggest that ZnO NPs exhibit both COX-2- and LOX-mediated apoptosis by the participation of mitochondrial dysfunction in human MSC cultures.

• Journal of Pharmacopuncture
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• v.22 no.3
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• pp.154-159
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• 2019

Objectives: Toxoplasmosis is a worldwide health problem that caused by intracellular apicomplexan parasite, Toxoplasma gondii (T. gondii). Considering that the available drugs for toxoplasmosis have serious host toxicity, the aim of the current study was to survey the in vitro and in vivo anti-Toxoplasma activity of Zea mays (Z. mays) and Eryngium caucasicum (E. caucasicum) extracts. Methods: Four concentrations (5, 10, 25, and $50mg\;mL^{-1}$) of Z. mays and E. caucasicum methanolic extracts for 30, 60, 120, and 180 min were incubated with infected macrophages and then the viability of RH strain of T. gondii tachyzoites was evaluated by trypan blue staining method. Also, we evaluated the survival rate of acutely infected mice with the extracts (100 and $200mg\;kg^{-1}\;day^{-1}$) intraperitoneally for 5 days after infection with $2{\times}104$ tachyzoites of T. gondii. Results: The anti-Toxoplasma effect of the methanolic extracts were extremely significant compared to the negative control group in all exposure times (P < 0.05). The Z. mays (10, 25 and $50mg\;mL^{-1}$) killed 100% of the parasites after 180 and 120 min exposure, respectively. Also, high toxoplasmacidal activity was observed with E. caucasicum extract. Furthermore, treatment of experimentally infected mice with the Z. mays (100, $200mg\;kg^{-1}\;day^{-1}$) and E. caucasicum ($100mg\;kg^{-1}\;day^{-1}$) significantly increased their survival rate compared to untreated infected control (P < 0.05). Conclusion: These extracts are promising candidates for further medicine development on toxoplasmosis. However, further investigations are necessary to clarify effective fractions of the Z. mays and E. caucasicum extracts and the mechanisms of action.

• Herbal Formula Science
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• v.27 no.1
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• pp.45-52
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• 2019

Objectives : Hyperthermia is a widely used therapeutic tool for cancer therapy and a well-known inducer of apoptosis. Although the Cinnamomi cortex (CC) is a potent anticancer agent for several human carcinomas, it is less potent in the human U937 cell line. To explore any enhancing effects of CC with hyperthermia induced apoptosis, this study investigated the combined effects and apoptotic mechanisms of hyperthermia and CC in U937 cells. Methods : U937 cells were heat treated at $43^{\circ}C$ for 30 min with or without pre-treatment for 1h with CC and then incubated at $37^{\circ}C$ with 5% $CO_2$. Cell viability was analyzed by MTT assay and Trypan blue assay. Morphological changes reflecting apoptosis were visualized under microscope. Synergy effect of CC combined with hyperthermia were calculated by Compusyn software. The expression of proteins related to apoptosis and signaling pathways was determined by western blotting. Results : Hyperthermia with CC reduced cell viability and induced apoptosis. Combined hyperthermia and CC treatment markedly augmented apoptosis by upregulating proapoptotic proteins and suppressing antiapoptotic proteins, culminating in caspase-3 activation. Furthermore, the combined treatment, decreased the expression of in Bcl-2 family, cyclin D1, VEGF, MMP2 and MMP9 expression. Conclusion : This study provides compelling evidence that hyperthermia, in combination with CC, is a promising therapeutic strategy for enhancement of apoptosis and suggests a promising therapeutic approach for cancer.

• Herbal Formula Science
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• v.27 no.1
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• pp.53-63
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• 2019

Objectives : In this study, we investigated the combination effects of Cinnamomi cortex Ethanol Extract (CcEE) and hyperthermia in the human AGS gastric cancer cell line. Methods : AGS cells were treated with the indicated concentrations of CcEE (0, 50 or $60{\mu}g/mL$) for 1h prior to hyperthermia. And then incubated for a further 30 min at the indicated temperatures (37, 42 or $43^{\circ}C$) in a humidified incubator containing 5% $CO_2$ or a thermostatically controlled water bath for hyperthermia. The cell viability was measured by MTT assay, Morphology assay and Trypan blue assay. To investigate the possible molecular signaling pathways, the activation of mitogen-activated protein kinase (MAPK) proteins (ERK, p38 and JNK) and expression of various anti-apoptotic proteins such as Caspase-3, Caspase-9, p53, Cyclin D1 and MMP-2 were assessed by Western blot analysis. In addition, Annexin V and 7-amino-actinomycin D (7-AAD) staining was performed to examine the apoptotic mechanism. Results : Combination of CcEE with hyperthermia effectively suppressed the cell viability and changed cellmorphology compared with CcEE or hyperthermia treatment alone. Combined treatment also abated the expression of Caspase-3, Caspase-9, Cyclin D1 and MMP-2. Whereas, the expression level of p53 was up-regulated by co-treatment. Moreover, combination treatment enhanced phosphorylation of ERK, p38 and JNK. In addition, this combination increased anti-cancer effect by inducing cell death through the apoptosis. Conclusions : Taken together, all these findings suggest that the combination treatment with CcEE and hyperthermia may have therapeutic potential as a promising approach to patients with stomach cancer.

• The Korean Journal of Pain
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• v.34 no.1
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• pp.19-26
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• 2021

Background: Prolotherapy is a proliferation therapy as an alternative medicine. A combination of dextrose solution and lidocaine is usually used in prolotherapy. The concentrations of dextrose and lidocaine used in the clinical field are very high (dextrose 10%-25%, lidocaine 0.075%-1%). Several studies show about 1% dextrose and more than 0.2% lidocaine induced cell death in various cell types. We investigated the effects of low concentrations of dextrose and lidocaine in fibroblasts and suggest the optimal range of concentrations of dextrose and lidocaine in prolotherapy. Methods: Various concentrations of dextrose and lidocaine were treated in NIH-3T3. Viability was examined with trypan blue exclusion assay and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Migration assay was performed for measuring the motile activity. Extracellular signal-regulated kinase (Erk) activation and protein expression of collagen I and α-smooth muscle actin (α-SMA) were determined with western blot analysis. Results: The cell viability was decreased in concentrations of more than 5% dextrose and 0.1% lidocaine. However, in the concentrations 1% dextrose (D1) and 0.01% lidocaine (L0.01), fibroblasts proliferated mildly. The ability of migration in fibroblast was increased in the D1, L0.01, and D1 + L0.01 groups sequentially. D1 and L0.01 increased Erk activation and the expression of collagen I and α-SMA and D1 + L0.01 further increased. The inhibition of Erk activation suppressed fibroblast proliferation and the synthesis of collagen I. Conclusions: D1, L0.01, and the combination of D1 and L0.01 induced fibroblast proliferation and increased collagen I synthesis via Erk activation.

• Journal of Physiology & Pathology in Korean Medicine
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• v.35 no.4
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• pp.117-123
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• 2021

Gefitinib, a first generation epidermal growth factor receptor tyrosine kinase inhibitor (EGFR TKI), provides obvious clinical benefit in patients with EGFR-mutant non-small cell lung cancer (NSCLC). However, patients ultimately develop gefitinib resistance which mainly caused by EGFR T790M secondary mutation. In the current study, we investigated whether the root extract of Scutellaria baicalensis (SB) overcomes gefitinib resistance. Gefitinib-resistant H1975 human NSCLC cells (EGFR L858R/T790M double mutant) were treated with gefitinib and/or ethanol extract of SB (ESB) to evaluate the effect of ESB on the gefitinib sensitivity. The cell viability was measured by MTT assay and trypan blue exclusion assay. The colony-forming ability was evaluated by anchorage-dependent colony formation assay. Combined treatment with gefitinib and ESB markedly decreased the cell viability and colony formation than single treatment with gefitinib or ESB in H1975 cells. In addition, cells treated with both gefitinib and ESB exhibited a significant increase of sub-G1 DNA content which indicates apoptotic cells compared with those treated with gefitinib or ESB alone. As a molecular mechanism, combined treatment with gefitinib and ESB strongly downregulated the phosphorylation of ERK and JNK than single treatment with gefitinib or ESB. Taken together, our results demonstrate that ESB sensitizes H1975 cells to gefitinib treatment. We cautiously propose that ESB can be used in combination with gefitinib for the advanced NSCLC patients with acquired resistance to EGFR TKIs.

• Journal of Ginseng Research
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• v.46 no.1
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• pp.167-174
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• 2022

Background: 20(S)-protopanaxadiol (20(S)-PPD), one of the main active metabolites of ginseng, performs a broad spectrum of anti-tumor effects. Our aims are to search out new strategies to enhance anti-tumor effects of natural products, including 20(S)-PPD. In recent years, fasting has been shown to be multi-functional on tumor progression. Here, the effects of fasting combined with 20(S)-PPD on hepatocellular carcinoma growth, apoptosis, migration, invasion and cell cycle were explored. Methods: CCK-8 assay, trypan blue dye exclusion test, imagings photographed by HoloMonitorTM M4, transwell assay and flow cytometry assay were performed for functional analyses on cell proliferation, morphology, migration, invasion, apoptosis, necrosis and cell cycle. The expressions of genes on protein levels were tested by western blot. Tumor-bearing mice were used to evaluate the effects of intermittent fasting combined with 20(S)-PPD. Results: We firstly confirmed that fasting-mimicking increased the anti-proliferation effect of 20(S)-PPD in human HepG2 cells in vitro. In fasting-mimicking culturing medium, the apoptosis and necrosis induced by 20(S)-PPD increased and more cells were arrested at G0-G1 phase. Meanwhile, invasion and migration of cells were decreased by down-regulating the expressions of matrix metalloproteinase (MMP)-2 and MMP-9 in fasting-mimicking medium. Furthermore, the in vivo study confirmed that intermittent fasting enhanced the tumor growth inhibition of 20(S)-PPD in H22 tumor-bearing mice without obvious side effects. Conclusion: Fasting significantly sensitized HCC cells to 20(S)-PPD in vivo and in vitro. These data indicated that dietary restriction can be one of the potential strategies of chinese medicine or its active metabolites against hepatocellular carcinoma.

• The Korea Journal of Herbology
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• v.24 no.2
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• pp.87-92
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• 2009

Objectives : This study was performed to evaluate the neuroprotective effect of water extracts from Nelumbinis semen (NSW) in dopaminergic cells. Methods : We performed 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay, 2,2-azinobis3-ethyl-benzothiazoline-6-sulfonic acid (ABTS) cation scavenging assay, and determination of total polyphenolic content to examine the antioxidant effects of NSW. We also evaluated the neuroprotective effects against 6-hydroxydopamine (6-OHDA)-induced toxicity using 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyl-tetrazolium bromide assay (MIT) assay, trypan blue cytotoxicity assay, and nitric oxide assay in SH-SY5Y cells and tyrosine hydroxylase (TH) immunohistochemistry in primary rat dopaminergic neurons. Results : NSW showed $IC_{50}$ values of 184.80 and 92.90 ${\mu}$g/mL in DPPH and in ABTS assays, respectively. NSW showed 1.05% of total polyphenol contents. NSW showed protective effect against 6-0HDA-induced neurotoxicity whereas no influence on cell viability at the concentration of 1${\sim}$50 ${\mu}$g/mL. NSW reduced NO generation while 6-OHDA produced it. Moreover, it protected rat dopaminergic neurons against 6-0HDA at a dose of 1 ${\mu}$g/mL. Conclusions : These results indicated that NSW has neuroprotective effect against 6-0HDA-induced neurotoxicity through antioxidant activity in dopaminergic cell culture.