• 한방재활의학과학회지
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• 제31권4호
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• pp.1-11
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• 2021

Objectives Hwanggeumjakyak-tang (HJT) has traditionally been used to treat gastrointestinal inflammatory diseases; however, its protective effects against neuronal inflammation are still undiscovered. Methods We investigated the anti-neuroinflammatory effects of HJT water extract on lipopolysaccharide (LPS)-stimulated BV2 mouse microglia cells. BV2 cells were treated with LPS (1 ㎍/mL) 1 hour prior to the addition of HJT. We measured cell viability using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay and nitrite production using the Griess assay. We performed a reverse transcription-polymerase chain reaction assay to measure messenger RNA expression of inflammatory cytokines including interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α. Western blot analysis was performed to determine protein expression of mitogen-activated protein kinases (MAPKs) and inhibitor of nuclear factor kappa B (NF-κB)α. Results HJT inhibited excessive nitrite release in LPS-stimulated BV2 cells and also significantly inhibited inflammatory cytokines such as IL-1β, IL-6, and TNF-α in LPS-stimulated BV2 cells. Moreover, HJT significantly suppressed LPS-induced MAPK and NF-κB activation and inhibited the elevation of IL-1β, IL-6, and TNF-α in the brain of LPS-injected mice. Conclusions Our study highlights the anti-neuroinflammatory effects of HJT via MAPK and NF-κB deactivation.

• 한국약용작물학회지
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• 제15권6호
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• pp.444-450
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• 2007

Dried leaves from Cedrela sinensis A. Juss. (CS), have been observed to possess various pharmacological activity and contain various antioxidant constituents. The protective effect of ethanol extract of CS on hydrogen peroxide $(H_2O_2)-induced$ neurotoxicity was examined using primary cultured rat cortical neurons in the present study. Exposure of cultured neurons to 100 ${\mu}M\;H_2O_2$ caused a significant neuronal death as assessed by a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay and Hoechst 33342 staining. The addition of CS, over a concentration range of 10 to $50{\mu}g/m{\ell}$, concentration-dependently prevented the $H_2O_2-induced$ neuronal apoptotic death. CS $(50{\mu}g/m{\ell})$ significantly inhibited $H_2O_2-induced$ elevation of the cytosolic $Ca^{2+}$ concentration $([Ca^{2+}]_c)$, which was measured by a fluorescent dye, Fluo-4 AM. CS (30 and $50{\mu}g/m{\ell})$ inhibited glutamate release and generation of reactive oxygen species (ROS) induced by $100{\mu}M\;H_2O_2$. These results suggest that CS may mitigate the $H_2O_2-induced$ neurotoxiciy by interfering with the increase of $[Ca^{2+}]_c$, and then inhibiting glutamate release and generation of ROS in cultured neurons.

• 동의생리병리학회지
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• 제23권6호
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• pp.1409-1415
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• 2009

Obesity is especially a serious health problem in industrialized countries, because it is considered to be a risk factor associated with the genesis or development of various metabolic diseases, including cardiovascular disease and type 2 diabetes mellitus. The purpose of this study was to investigate the effects of tanshinone IIA from Salvia miltiorrhiza Bunge on induction of apoptossis and inhibition of adipogenesis in in 3T3-L1 preadipocytes and adipocytes. The results demonstrated that tanshinone IIA decreased cell population growth of 3T3-L1 preadipocytes, assessed with the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] and LDH (lactate dehydrogenase) assay. Flow cytometric analysis of 3T3-L1 preadipocytes exposed to tanshinone IIA showed that apoptotic cells increased in a timeand dose-dependent manner. Treatment with tanshinone IIA decreased the number of normal cells and increased the number of apoptotic cells in a dose-dependent manner. The induction of apoptosis in 3T3-L1 preadipocytes by tanshinone IIA was mediated through the activation of caspase-3 and Bax, and then through the cleavage of PARP and the down-regulation of Bcl-2. Moreover, tanshinone IIA significantly decreased the amount of intracellular triglycerides and GPDH (glycerol-3-phosphate dehydrogenase) activity in 3T3-L1 adipocytes. Our results suggest that tanshinone IIA efficiently induces apoptosis and inhibits adipogenesis in 3T3-L1 preadipocytes and adipocytes.

• Food Science and Biotechnology
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• 제16권5호
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• pp.858-862
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• 2007

The present study was conducted to examine the antioxidant activities and neuroprotective effects of methanolic extracts from Schizonepeta tenuifolia Briquet (STE). STE ($100\;{\mu}g/mL$) showed $43.33\;{\mu}M$ of total phenolic content, 64.43% of radical scavenging activity, and 0.157 of reducing power. In addition, the effect of STE on $H_2O_2$-induced DNA damage in human leucocytes was evaluated by the comet assay, where STE was a dose dependent inhibitor of DNA damage induced by $200{\mu}M$ of $H_2O_2$. The protective effect of STE against $H_2O_2$-induced oxidative damage on PC12 cells was investigated by an 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) reduction assay and lactate dehydrogenase (LDH) release assays. After 2 hr of cell exposure to $H_2O_2\;(500\;{\mu}M)$, a marked reduction in cell survival was observed. However, this reduction was significantly prevented by $1-50\;{\mu}g/mL$ of STE. Therefore, these results suggest that STE could be a new antioxidant candidate against neuronal diseases.

• Natural Product Sciences
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• 제10권6호
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• pp.289-295
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• 2004

Myristica fragrans seed from Myristica fragrans Houtt (Myristicaceae) has various pharmacological activities peripherally and centrally. The present study aims to investigate the effect of the methanol extract of Myristica fragrans seed (MF) on N-methyl-D-aspartate (NMDA)-induced neurotoxicity in primary cultured rat cerebellar granule neuron. MF, over a concentration range of 0.05 to $5\;{\mu}g/ml$, inhibited NMDA (1 mM)- induced neuronal cell death, which was measured by trypan blue exclusion test and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay. MF $(0.5\;{\mu}g/ml)$ inhibited glutamate release into medium induced by NMDA (1 mM), which was measured by HPLC. Pretreatrnent of MF $(0.5\;{\mu}g/ml)$ inhibited NMDA (1 mM)-induced elevation of cytosolic calcium concentration $([Ca^{2+}]_c)$, which was measured by a fluorescent dye, Fura 2-AM, and generation of reactive oxygen species (ROS). These results suggest that MF prevents NMDA-induced neuronal cell damage in vitro.

• International Journal of Oral Biology
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• 제46권2호
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• pp.85-93
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• 2021

Asarum sieboldii Miq. (Aristolochiaceae) is a perennial herbaceous plant and has been used as traditional medicine for treating diseases, cold, fever, phlegm, allergies, chronic gastritis, and acute toothaches. Also, it has various biological activities, such as antiallergic, antiinflammatory, antinociceptive, and antifungal. However, the anticancer effect of A. sieboldii have been rarely reported, except anticancer effect on lung cancer cell (A549) of water extracts of A. sieboldii. This study investigated the anticancer activity of methanol extracts of A. sieboldii (MeAS) and the underlying mechanism in human FaDu hypopharyngeal squamous carcinoma cells. MeAS inhibited FaDu cells grown dose-dependently without affecting normal cells (L929), as determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide and live and dead assay. In addition, concentration of MeAS without cytotoxicity (0.05 and 0.1 mg/mL) inhibited migration and colony formation. Moreover, MeAS treatment significantly induced apoptosis through the proteolytic cleavage of caspase-3, -7, -9, poly (ADP-ribose) polymerase, and downregulation of Bcl-2 and upregulation of Bax in FaDu cells, as determined by fluorescence-activated cell sorting analysis, 4'6-diamidino-2-phenylindole stain, and western blotting. Altogether, these results suggest that MeAS exhibits strong anticancer effects by suppressing the growth of oral cancer cells and the migration and colony formation via caspase- and mitochondrial-dependent apoptotic pathways in human FaDu hypopharyngeal squamous carcinoma cells. Therefore, MeAS can serve as a natural chemotherapeutic for human oral cancer.

• Preventive Nutrition and Food Science
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• 제20권1호
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• pp.73-77
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• 2015

As an approach to search for chemopreventive agents, we tested p-coumaric acid, 3-methoxy-p-coumaric acid (ferulic acid), and 3,5-dimethoxy-p-coumaric acid (sinapic acid) in B16/F10 melanoma cells. Intracellular melanin contents were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay and cytotoxicity of the compounds were examined by lactate dehydrogenase (LDH) release. p-Coumaric acid showed inhibitory effect on melanogenesis, but ferulic acid increased melanin content, and sinapic acid had almost no effect on melanogenesis. Treatment with ferulic acid resulted in a 2 to 3 fold elevation in the production of melanin. Correlatively, cell viability decreased in a dose-dependent manner when treated with ferulic acid. However, ferulic acid did not affect the LDH release from the cells. Treatment with sinapic acid resulted in a 50~60% elevation in the release of LDH when treated with a $200{\mu}g/mL$ concentration and showed neither cytostasis nor increase of melanin synthesis in a dose-dependent manner. Taken together, p-coumaric acid inhibits melanogenesis, ferulic acid induces melanogenesis, and sinapic acid exerts cytotoxic effects in B16/F10 murine melanoma cells. The results indicate that the addition of methoxy groups to p-coumaric acid shows the melanogenic or cytotoxic effects in melanoma cells compared to the original compound. Therefore, this study suggests the possibility that methoxylated p-coumaric acid, ferulic acid can be used as a chemopreventive agent.

• Journal of Acupuncture Research
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• 제35권4호
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• pp.182-186
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• 2018

Background: The purpose of this study was to investigate the effects of wild ginseng pharmacopuncture on melanin production in B16/F10 murine melanoma cells. Methods: To determine the effect of wild ginseng pharmacopuncture solution on B16/F10 cells, cytotoxicity was evaluated using the 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyl-tetrazolium bromide (MTT) method. To observe B16/F10 cell growth, death, and morphological changes, Trypan blue solution was used. The Hosoi method was used to investigate the effect of wild ginseng pharmacopuncture solution on melanin production. The Martinez-Esparza method was used to investigate the effect of wild ginseng pharmacopuncture solution on tyrosinase activity. To determine the pathway involved in the melanogenesis in cells exposed to wild ginseng pharmacopuncture solution, a cell-free tyrosinase was used. Results: Following treatment with $200{\mu}L$ of wild ginseng solution, the cell survival rate was $76.32{\pm}2.45%$ which significantly decreased with higher concentrations (${\mu}L$) of wild ginseng (up to $200{\mu}L$). When $100{\mu}L$ of wild ginseng was used, the cell survival rate was $89.95{\pm}2.07%$. No morphological changes or abnormalities were observed in the B16/F10 murine melanoma cells as observed in the Trypan blue test. Melanin production was significantly reduced to $72.17{\pm}3.74%$ at $100{\mu}L$. Using $100{\mu}L$ of wild ginseng solution, tyrosinase activity was significantly decreased to $80.15{\pm}1.05%$. Wild ginseng pharmacopuncture solution reduced melanin production both directly and indirectly. Conclusion: This study suggests that wild ginseng pharmacopuncture solution may be effective in inhibiting melanin production. Further studies are needed to determine safe and effective clinical applications.

• Asian Pacific Journal of Cancer Prevention
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• 제14권6호
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• pp.3897-3901
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• 2013

Background: Optimal treatment for prostate cancer remains a challenge worldwide. Recently, T cell immunoglobulin mucin-3 (TIM-3) has been implicated in tumor biology but its contribution prostate cancer remains unclear. The aim of this study was to investigate the role of TIM-3 as a prognostic marker in patients with prostate cancer. Methods: TIM-3 protein expression was determined by immunohistochemistry and Western blotting in 137 prostate cancer tumor samples and paired adjacent benign tissue. We also performed cell proliferation assays using 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl- 2H tetrazolium bromide (MTT) and cell invasion assays. The effects of small interfering RNA (siRNA)-mediated knockdown of TIM-3 (TIM-3 siRNA) in two human prostate cancer cell lines were also evaluated. Results: TIM-3 expression was higher in prostate cancer tissue than in the adjacent benign tissue (P<0.001). High TIM-3 expression was an independent predictor of both recurrence-free survival and progression-free survival. TIM-3 protein was expressed in both prostate cancer cell lines and knockdown suppressed their proliferation and invasion capacity. Conclusions: TIM-3 expression is associated with a poor prognosis in prostate cancer. Taken together, our resutlts indicate that TIM-3 is a potential prognostic marker in prostate cancer.

• Journal of Applied Biological Chemistry
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• 제62권4호
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• pp.347-353
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• 2019

Stings of jellyfish, which frequently occur in a warm season, cause severe pain, inflammation and sometimes irreversible results such as the death. Harmful venoms from jellyfish, therefore, have been studied for finding the therapeutic agents to relieve pain or to neutralize toxic components. However, it is still unclear if and how jellyfish venom reveal neuronal toxicity even though pain induction seems to result from the activation of nociceptors such as nerve endings. In this study, using HT-22 cell line, we investigated neurotoxic effects of the venom of Chrysaora pacifica (CpV) which appears in South-East ocean of Korea. In 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay, CpV significantly reduced the viability of HT-22 cells in a dose-dependent manner. Additionally, in 2',7'-Dichlorofluorescin diacetate fluorescence test under the culture condition lacking dominant inflammatory factors, CpV remarkably increased the production of intracellular reactive oxygen species (ROS). Reduced responsive fluorescence to Rhodamine123 and increased expression of intracellular cytochrome c were also observed in HT-22 cells treated with CpV. These indicate that CpV-reduced viability of HT-22 cells may be due to the activation of apoptotic signalings mediated with oxidative stress and mitochondrial dysfunction. Furthermore, removing Ca²⁺ ion or adding N-acetyl-Lcystein remarkably blocked the CpV effect to reduce the viability of HT-22 cells. The findings in this study clearly demonstrate that CpV may activate Ca²⁺-mediated ROS signalings and mitochondrial dysfunction resulting in neuronal damage or death, and suggest that blocking Ca²⁺ pathway is a therapeutic approach to possibly block toxic effects of jellyfish venoms.