• Title/Summary/Keyword: Western-blot

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Whitening Activity of Sambucus Sieboldiana Var. Pendula (Nakai) Extract (말오줌나무 추출물의 미백활성 검증)

  • Yoo, Dan-Hee;Kim, Jin-Tae;Oh, Min-Jeong;Yeom, Hyeon-Ji;Lee, Jin-Young
    • Journal of Life Science
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    • v.29 no.3
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    • pp.279-286
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    • 2019
  • This study evaluated the anti-oxidant and whitening effects of a 70% ethanol extract of the Sambucus sieboldiana var. pendula (Nakai) (SS). At $1,000{\mu}g/ml$ concentration, the electron donating ability of this SS extract was found to be 86.21% and the ABTS+ radical scavenging ability was 97.9%. In terms of whitening activity, the tyrosinase inhibitory effect of the extract was 37%, also at $1,000{\mu}g/ml$ concentration. To explore the extractefftoxicity to B16F10 melanoma cells, a 3-[4,5-dimethyl-thiazol-2-yl]-2,5-diphenyl-tetrazoliumbromide assay was performed. Results showed 90% or more cells remained viable at $100{\mu}g/ml$ concentration. A Western blot of the SS extract was used to measure microphthalmia-associated transcription factor (MITF), tyrosinase-related protein-1 (TRP-1), tyrosinase relate protein-2 (TRP-2), and the tyrosinase protein expression inhibitory effect at 25, 50, $100{\mu}g/ml$ concentrations; ${\beta}-actin$ was used as a positive control. Consequently, the MITF, TRP-1, TRP-2, and the tyrosinase protein expression inhibitory effect were seen to decrease by 34.5%, 45.6%, 58.4%, and 79.6%, respectively, at $100{\mu}g/ml$ concentration. These were also then measured by reverse transcription-polymerase chain reaction at 25, 50, $100{\mu}g/ml$ concentrations with GAPDH as a positive control. As a result, the SS extract was seen to decrease MITF, TRP-1, TRP-2, and the tyrosinase protein expression inhibitory effect by 85.4%, 67.5%, 85.2%, 67.1%, respectively at the $100{\mu}g/ml$ concentration. We therefore confirmed the possibility of Sambucus sieboldiana var. pendula (Nakai) extract as a whitening material.

Inhibitory Effects of Chrysanthemum boreale Makino on 3T3-L1 Preadipocyte Differentiation and Down-regulation of Adipogenesis and Lipogenesis (산국(Chrysanthemum boreale Makino) 꽃 유래 에센셜오일(Essential oil)이 지방세포 분화 및 지방생성에 미치는 영향)

  • Hwang, Dae Il;Choi, In-Ho;Kim, Do Yoon;Park, Soo Min;Kim, Ha Bin;Li, YaLi;Lee, Hwan Myung
    • Journal of Life Science
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    • v.29 no.3
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    • pp.332-336
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    • 2019
  • Obesity is associated with an increased risk of many diseases including type 2 diabetes mellitus, hypertension, and hyperlipidemia. The flowers of Chrysanthemum boreale have been used as traditional medicines for the treatment of diseases such as obesity and type 2 diabetes mellitus. This study aimed to evaluate the effect of C. boreale Makino flower essential oil (CFEO) on adipocyte differentiation using preadipocyte cell line 3T3-L1. CFEO at concentrations between 0.1 and $5{\mu}g/ml$ did not affect 3T3-L1 cell viability. A CFEO concentration of between 0.1 and $1{\mu}g/ml$ significantly inhibited lipid accumulation during MDI-induced differentiation in 3T3-L1 cells in a dose-dependent manner, reaching a maximal level at $1{\mu}g/ml$ ($28.94{\pm}2.01%$; approximately 30% of control treated with MDI alone). Western blot analysis revealed that CFEO concentrations between 0.1 and $1{\mu}g/ml$ suppressed the activations of three adipogenic transcription factors in the MDI-stimulated 3T3-L1 cells: peroxisome proliferator-activated receptor ${\gamma}$; CCATT/enhancer binding protein ${\alpha}$; and sterol regulatory element binding protein-1. Moreover, the expressions of lipogenic enzymes, acetyl-CoA carboxylase, and fatty acid synthase were also inhibited by treatment with CFEO between 0.1 and $1{\mu}g/ml$. CFEO may therefore be a promising functional material for obesity prevention.

Immunomodulating activity of Sargassum horneri extracts in RAW264.7 macrophages (RAW264.7 대식세포에서 괭생이 모자반 추출물의 면역활성 증진 효과)

  • Kim, Dong-Sub;Sung, Nak-Yun;Park, Sang-Yun;Kim, Geon;Eom, Ji;Yoo, Jin-Gon;Seo, In-Ra;Han, In-Jun;Cho, Young-Baik;Kim, Kyung-Ah
    • Journal of Nutrition and Health
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    • v.51 no.6
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    • pp.507-514
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    • 2018
  • Purpose: Sargassum horneri (S. horneri) is a species of brown macroalgae that is common along the coast of Japan and Korea. The present study investigated the immuno-modulatory effects of different types of S. horneri extracts in RAW264.7 macrophages. Methods: S. horneri was extracted by three different methods, hot water extraction, 50% ethanol extraction, and supercritical fluid extraction. Cell viability was then measured by MTT assay, while the production levels of tumor necrosis factor-${\alpha}$ (TNF-${\alpha}$), interleukin-6 (IL-6), and nitric oxide (NO) were measured by enzyme-linked immunosorbent assay and Griess assay, respectively. The expression and activation levels of inducible NO synthase (iNOS), mitogen-activated protein kinase (MAPK) and nuclear factor ${\kappa}B$ ($NF-{\kappa}B$) were examined by western blot analysis. Results: The three different S. horneri extracts were nontoxic against RAW 264.7 cells up to $50{\mu}g/mL$, among which treatment with hot water extract (HWE) of S. horneri significantly enhanced the production of TNF-${\alpha}$, IL-6, and NO in a dose-dependent manner. Hot water extract of S. horneri also increased the expression level of iNOS, suggesting that up-regulation of iNOS expression by HWE of S. horneri was responsible for the induction of NO production. In addition, treatment of RAW 264.7 macrophages with HWE of S. horneri increased the phosphorylation levels of ERK, p38 and JNK. Furthermore, the activation and subsequent nuclear translocation of $NF-{\kappa}B$ was enhanced upon treatment with HWE of S. horneri, indicating that HWE of S. horneri activates macrophages to secrete TNF-${\alpha}$, IL-6 and NO and induces iNOS expression via activation of the $NF-{\kappa}B$ and MAPKs signaling pathways. Conclusion: Taken together, these findings suggest that HWE of S. horneri possesses potential as a functional food with immunomodulatory activity.

Protective effect of Gabjubaekmok (Diospyros kaki) extract against amyloid beta (Aβ)-induced cognitive impairment in a mouse model (아밀로이드 베타(amyloid beta)로 유도된 인지장애 마우스 모델에서 갑주백목(Diospyros kaki) 추출물의 인지기능 및 뇌 신경세포 보호 효과)

  • Yoo, Seul Ki;Kim, Jong Min;Park, Seon Kyeong;Kang, Jin Yong;Han, Hye Ju;Park, Hyo Won;Kim, Chul-Woo;Lee, Uk;Heo, Ho Jin
    • Korean Journal of Food Science and Technology
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    • v.51 no.4
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    • pp.379-392
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    • 2019
  • The current study investigated the effect of Gabjubaekmok (Diospyros kaki) ethanolic extract (GEE) on $H_2O_2$-induced human neuroblastoma MC-IXC cells and amyloid beta $(A{\beta})_{1-42}$-induced ICR (Institute of Cancer Research) mice. GEE showed significant antioxidant activity that was evaluated based on ABTS, DPPH scavenging activity, and inhibition of malondialdehyde (MDA) and acetylcholinesterase activity. Further, GEE inhibited ROS production and increased cell viability in $H_2O_2$-induced MC-IXC cells. Administration of GEE ameliorated the cognitive dysfunction on $A{\beta}$-induced ICR mice as evaluated using Y-maze, passive avoidance, and Morris water maze tests. Results of ex vivo test using brain tissues showed that, GEE protected the cholinergic system and mitochondrial functions by increasing the levels of antioxidants such as ROS, mitochondrial membrane potential (MMP), and adenosine triphosphate (ATP) against $A{\beta}$-induced cognitive dysfunction. Moreover, GEE decreasd the expression levels of apoptosis-related proteins such as $TNF-{\alpha}$, p-JNK, p-tau, BAX and caspase 3. While, expression levels of p-Akt and $p-GSK3{\beta}$ increased than $A{\beta}$ group. Finally, gallic acid was identified as the main compound of GEE using high performance liquid chromatography.

Activity-guided Purification of N-benzyl-N-methyldecan-1-amine from Garlic and Its Antitumor Activity against CT-26 Colorectal Carcinoma in BALB/C Mice (활성추적분리법에 의해서 순수분리한 마늘 N-benzyl-N-methyldecan-1-amine이 CT-26 세포주 이식 BALB/C mice의 항암효과)

  • Seetharaman, Rajasekar;Choi, Seong Mi;Guo, Lu;Cui, Zheng Wei;Otgonbayar, Duuriimaa;Park, Ju Ha;Kwon, Young-Seok;Kwak, Jung Ho;Kwon, Young Hee;Min, Ji Hyun;Kang, Jum Soon;Choi, Young Whan
    • Journal of Life Science
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    • v.29 no.10
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    • pp.1062-1070
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    • 2019
  • A components of garlic (Allium sativum) have anti-proliferative effects against various types of cancer. We aimed to investigate the capacity of garlic compounds to anti-tumor on a various cancer cell lines. Fractionation of garlic extract, guided by antiproliferative activity against human gastric cancer (AGS) cells, has resulted in the isolation of N-benzyl-N-methyldecan-1-amine (NBNMA). We investigated the effect of newly isolated NBNMA from garlic cloves on the inhibition of the growth of CT-26, AGS, HepG2, HCT-116, MCF7, B16F10, and Sarcoma-180 cells for in vitro and CT-26 colon carcinoma cells in vivo. NBNMA exhibited an antiproliferative effect in CT-26 cells by apoptotic cell death. NBNMA exhibited down-regulation of anti-apoptotic Bcl-2 proteins and up-regulation of apoptotic Bad protein expression in western blot analyses. In addition, NBNMA meagre activated caspase 3 and caspase 9, initiator caspases of the extrinsic and intrinsic pathways of apoptosis. NBNMA treatment at a dose of 10 mg/kg for 21 days in experimental mice implanted with tumors resulted in significant reduction of the tumor weight (43%). NBNMA exhibited both in vitro and in vivo anticancer activity. These results indicate that NBNMA has promising potential to become a novel anticancer agent from garlic cloves for the treatment of colon carcinoma cancer.

Effects of 2-methoxy-1,4-naphthoquinone (MQ) on MCP-1 Induced THP-1 Migration (MCP-1에 의해 유도된 THP-1 유주에 미치는 2-methoxy-1,4-naphthoquinone (MQ)의 영향)

  • Kim, Si Hyun;Park, Bo Bin;Hong, Sung Eun;Ryu, Sung Ryul;Lee, Jang Ho;Kim, Sa Hyun;Lee, Pyeongjae;Cho, Eun-Kyung;Moon, Cheol
    • Korean Journal of Clinical Laboratory Science
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    • v.51 no.2
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    • pp.245-251
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    • 2019
  • This study examined the effects of 2-methoxy-1,4-naphthoquinone (MQ) on the monocyte chemoattractant protein-1 (MCP-1)-induced migration of monocytes, which is an important phenomenon for the body defense and immune response. MQ is a major component extracted from Impatiens balsamina leaves, which have been used for many years in Asian medicine for the treatment of a range of diseases and pain. The cytotoxicity of MQ began to appear at a concentration of $10{\mu}M$, and approximately 50% cytotoxicity was confirmed at $100{\mu}M$. The MCP-1 induced migration of the THP-1 monocyte cell line increased after MQ treatment in a dose dependent manner and the largest increase was observed at $0.1{\mu}M$. The level of cAMP expression decreased after a treatment with $0.1{\mu}M$ MQ. The phosphorylation of extracellular signal-regulated kinases 1/2 (Erk1/2), a key signaling protein involved in the signaling pathway of C-C motif chemokine receptor 2 (CCR2), a receptor for MCP-1, was increased by the simultaneous treatment of $0.1{\mu}M$ MQ. These results show that MQ increases the MCP-1-induced migration of THP-1, decreases the level of cAMP expression, and increases the level of Erk1/2 phosphorylation.

Effect of Scytosiphon lomentaria Ethanol Extracts on Myostatin Activity and Zebrafish Obesity Induced by High Feeding (고리매(Scytosiphon lomentaria) 에탄올 추출물이 마이오스타틴 활성과 고 급식으로 유도된 비만 제브라피쉬에 미치는 영향)

  • Jung, Jun Gyo;Kim, Jae Hong;Kim, Jeong Hwan;Kim, Yong Soo;Jin, Deuk-Hee;Jin, Hyung-Joo
    • Journal of Life Science
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    • v.31 no.8
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    • pp.699-709
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    • 2021
  • Muscle mass improvement through lifestyle modification has been shown to reduce the risk of metabolic syndrome. This study examined the capacity of ethanol extracts of Scytosiphon lomentaria (SLE) to suppress the bioactivity of myostatin, a potent negative regulator of skeletal muscle mass, as well as the effect of SLE treatment on metabolic homeostasis in obese zebrafish induced by high feeding. A total of 10 ㎍/ml SLE completely blocked myostatin (1 nM/ml) signaling in the pGL3-(CAGA)12 luciferase assay and suppressed myostatin-induced Smad2 phosphorylation in the Western blot analysis. In the zebrafish larvae analysis, the whole body glucose concentration of the high feeding control (HFC) group was significantly higher than that of the normal feeding control (NFC) group. However, the glucose levels of the high feeding group treated with 12.5 ug SLE and of the high feeding group treated with 18.75 ug SLE were similar to those of the NFC group. The mRNA expression level of the GLUT2 gene of the HFC group was significantly lower than that of the NFC group. SLE treatment restored the expression of the GLUT2 gene to a level that was close to that of the NFC group, indicating that SLE is capable of regulating glucose levels in zebrafish larvae. The current results highlight the potential of SLE as a natural MSTN inhibitor and supplement that can be used to facilitate the treatment of metabolic syndrome.

Resveratrol Ameliorates NMDA-induced Mitochondrial Injury by Enhanced Expression of Heme Oxygenase-1 in HT-22 Neuronal Cells (NMDA를 처리한 HT-22 신경세포에서 미토콘드리아 손상을 완화하는 레스베라트롤의 보호 효과와 헴 산화효소-1의 역할)

  • Kang, Jae Hoon;Woo, Jae Suk
    • Journal of Life Science
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    • v.32 no.1
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    • pp.11-22
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    • 2022
  • N-methyl-D-aspartate (NMDA) receptors have received considerable attention regarding their involvement in glutamate-induced neuronal excitotoxicity. Resveratrol has been shown to exhibit neuroprotective effects against this kind of overactivation, but the underlying cellular mechanisms are not yet clearly understood. In this study, HT-22 neuronal cells were treated with NMDA in Mg2+-free buffer and subsequently used as an experimental model of glutamate excitotoxicity to elucidate the mechanisms of resveratrol-induced neuroprotection. We found that NMDA treatment causes a drop in MTT reduction ability, disrupts inside-negative transmembrane potential of mitochondria, depletes cellular ATP levels, and stimulates intracellular ROS production. Double fluorescence imaging studies demonstrated an increased formation of mitochondrial permeability transition (MPT) pores accompanied by apoptotic cell death, while cobalt protoporphyrin and bilirubin showed protective effects against NMDA-induced mitochondrial injury. On the other hand, zinc protoporphyrin IX significantly attenuated the protective effects of resveratrol which was itself shown to enhance heme oxygenase-1 (HO-1) mRNA and protein expression levels. In cells transfected with HO-1 small interfering RNA, resveratrol failed to suppress the NMDA-induced effects on MTT reduction ability and MPT pore formation. The present study suggests that resveratrol may prevent mitochondrial injury in NMDA- treated HT-22 cells and that enhanced expression of HO-1 is involved in the underlying cellular mechanism.

Anti-inflammatory and Antioxidative Effects of Lotus Root Extract in LPS-PG-Stimulated Human Gingival Fibroblast-1 Cells (치주염 원인균 LPS-PG로 유도된 인체 치은섬유아세포에서 연뿌리 추출물에 대한 항염증 및 항산화 효과)

  • Lee, Young-Kyung;Kim, Chul Hwan;Jeong, Dae Won;Lee, Ki Won;Oh, Young Taek;Kim, Jeong Il;Jeong, Jin-Woo
    • Korean Journal of Plant Resources
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    • v.35 no.5
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    • pp.565-573
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    • 2022
  • Gingival inflammation is one of the main causes that can be related to various periodontal diseases. Human gingival fibroblast (HGF) is the major constituent in periodontal connective tissue and secretes various inflammatory mediators, such as nitric oxide (NO) and prostaglandin E2 (PGE2), upon lipopolysaccharide stimulation. This study is aimed at investigating the anti-inflammatory and antioxidative activities of Lotus Root extract (LRE) in Porphyromonas gingivalis derived lipopolysaccharide (LPS-PG)-stimulated HGF-1 cells. The concentration of NO and PGE2, as well as their responsible enzymes, inducible NO synthase (iNOS), and cyclooxygenase-2 (COX-2), was analyzed by Griess reaction, ELISA, and western blot analysis. LPS-PG sharply elevated the production and protein expression of inflammatory mediators, which were significantly attenuated by LRE treatment in a dose-dependent manner. LRE treatment also suppressed activation of Toll-like receptor 4 (TLR4)/myeloid differentiation primary response gene 88 (MyD88) and nuclear factor-κB (NF-κB) in LPS-PG-stimulated HGF-1 cells. In addition, one of phase II enzyme, NAD(P)H quinone dehydrogenase (NQO)-1, and its transcription factor, Nuclear factor erythroid 2-related factor 2 (Nrf2), were significantly induced by LRE treatment. Consequently, these results suggest that LRE ameliorates LPS-PG-induced inflammatory responses by attenuating TLR4/MyD88-mediated NF-κB, and activating NQO-1/Nrf2 antioxidant response element signaling pathways in HGF-1 cells.

Cell Migration and Wound Healing Activities of Recombinant Thymosin β-4 Expressed in Escherichia coli (재조합 Thymosin β-4의 세포이동능과 상처치유능)

  • Hong, Kyo-Chang;Choi, Yung Hyun;Kim, Gun-Do;Cha, Hee-Jae;Jeon, Sung-Jong;Nam, Soo-Wan
    • Journal of Life Science
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    • v.32 no.2
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    • pp.135-141
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    • 2022
  • Thymosin β-4 (TB4) is a small peptide composed of 43 amino acids. To obtain sufficient biologically active mouse TB4 economically, we cloned and overexpressed this gene in an Escherichia coli system. With the isopropyl β-D-1-thiogalactopyranoside induction of the E. coli transformant, TB4 fusion protein with intein- and chitin-binding domain was successfully expressed in the soluble fraction within the E. coli cell. The TB4-intein - chitin-binding domain fusion protein was purified from the soluble fraction of E. coli cell lysate. The affinity chromatography with chitin beads and dithiothreitol-mediated intein self-cleavage reaction releases the TB4 peptide into the stripping solution. Sodium dodecyl sulphate - polyacrylamide gel electrophoresis and Western blot analyses were used to confirm that the recombinant TB4 peptide was produced with the expected size of 5 kDa. We found that the recombinant TB4 stimulated cell migration in the transwell plate chamber assay. After 18 hr of the treatment of the recombinant TB4 with 1 ng/ml concentration, the migration of the HT1080 cell was increased by 20% compared with that of the chemically synthesized TB4. The recombinant TB4 was also observed to promote the healing of a wound area in C57BL/6 mice by as high as 35% compared with that of the chemically synthesized TB4. These results suggest that the recombinant TB4 has better biological activity for cell migration and wound healing than that of the chemically synthesized TB4 peptide.