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Yoon, Han, Fang, Gundeti, Lee, Song, Hwang, Kim, Sung, and Park: Inhibitory Activity of Cordyceps bassiana Extract on LPS-induced Inflammation in RAW 264.7 Cells by Suppressing NF-κB Activation
Original Article

Natural Product Sciences 2017;23(3):162-168.

Published online: 20 January 2017

DOI: https://doi.org/10.20307/nps.2017.23.3.162

Inhibitory Activity of Cordyceps bassiana Extract on LPS-induced Inflammation in RAW 264.7 Cells by Suppressing NF-κB Activation

Deok Hyo Yoon1, †, Changwoo Han2, †, Yuanying Fang2, Shankariah Gundeti2, In-Sook Han Lee3, Won O Song4, Ki-Chul Hwang5, Tae Woong Kim1, Gi-Ho Sung5, ∗, Haeil Park2, ∗

1Department of Biochemistry, Kangwon National University, Chunchon 200–701, Republic of Korea

2College of Pharmacy, Kangwon National University, Chunchon 200–701, Republic of Korea

3Department of Science Education, Kangwon National University, Chunchon 200–701, Republic of Korea

4Department of Food Science and Human Nutrition, Michigan State University, East Lansing, MI48824, USA

5Institute for Bio-Medical Convergence, Catholic Kwandong University, Incheon 404–834, Republic of Korea

∗Author for correspondence Haeil Park, College of Pharmacy, Kangwon National University, Chunchon 200-701, Republic of Korea. Tel: +82-33-250-6920 E-mail: haeilp@kangwon.ac.kr Gi-Ho Sung, Institute for Bio-Medical Convergence, Catholic Kwandong University, Incheon 404-834, Republic of Korea. Tel: +82-32-290-2772 E-mail: ung97330@gmail.com

† These authors contributed equally to this work

Received 1 April 2017       Revised 5 May 2017       Accepted 5 May 2017

Copyright © 2017 The Korean Society of Pharmacognosy

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Cordyceps bassiana has long been used as an oriental medicine and reported to possess diverse biological activities. The fruiting bodies of Cordyceps bassiana was extracted with ethanol and then further fractionated with n-hexane, ethyl acetate, n-butanol and water. The butanol fraction from Cordyceps bassiana (CBBF) exhibited the most effective in anti-inflammatory activity in RAW 264.7 macrophages and the roles of CBBF on the anti-inflammation cascade in LPS-stimulated RAW 264.7 cells were studied. To investigate the mechanism by which CBBF inhibits NO, iNOS and COX-2, the activation of IκB and MAPKs in LPS-activated macrophage were examined. Our present results demonstrated that CBBF inhibits NO production and iNOS expression in LPS-stimulated RAW 264.7 macrophage cells, and these effects were mediated through the inhibition of IκB-α, JNK and p38 phosphorylation. Also, CBBF suppressed activation of MAPKs including p38 and SAPK/JNK. Furthermore, CBBF significantly suppressed LPS-induced intracellular ROS generation. Its inhibition on iNOS expression, together with its antioxidant activity, may support its anti-inflammatory activity. Thus Cordyceps bassiana can be used as a useful medicinal food or drug for further studies.

Keywords: Cordyceps bassiana, Oriental ethnopharmacology, n-butanol fraction, Anti-inflammatory activity

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Fig. 1.
Effects of CBEE on NO production, iNOS and COX-2 protein expression in LPS-induced macrophages: (A) RAW 264.7 cells (4 × 106 cells/well) were incubated for 16 h with 1 μg/ml LPS in the presence or absence of CBEE. RAW 264.7 cells were pretreated with the indicated concentrations of CBEE for 30 min before incubation with LPS for 16 h. The culture supernatants were subsequently isolated and analyzed for nitrite levels. Data are expressed as mean ± S.D.(n = 3). Asterisks indicate a significant difference from LPS alone (∗ P < 0.05, ∗∗ P < 0.01 vs. LPS alone). (B) The cells were lysed, and the lysates were analyzed by immunoblotting used anti-iNOS and anti-COX-2. The blot was stripped from the bound antibody and reprobed with anti-β-actin to confirm equal loading. (C∼D) The intensity of the bands was scanned and quantified by Scion Image software.
nps-23-162f1.tif
Fig. 2.
Effect of Cordyceps bassiana solvent fractions on NO production in LPS-induced macrophages. RAW 264.7 cells (4 × 106 cells/well) were incubated for 16 h with 1 μg/ml LPS in the presence or absence of CBEE. RAW 264.7 cells were pretreated with 150 μg/ml of various solvent fractions for 30 min before incubation with LPS for 16 h. HF, n-hexane fraction; BF, n-butanol fraction; EAF, ethyl acetate fraction; AF, aqueous fraction. The culture supernatants were subsequently isolated and analyzed for nitrite levels. Data are expressed as mean ± S.D. (n = 3). Asterisks indicate a significant difference from LPS alone (∗ P < 0.05, ∗∗ P < 0.01 vs. LPS alone).
nps-23-162f2.tif
Fig. 3.
Effects of CBBF on NO production, iNOS protein and mRNA expression in LPS-induced macrophages. RAW 264.7 cells (4 × 106 cells/well) were incubated with 1 μg/ml LPS in the presence or absence of CBBF. (A) RAW 264.7 cells were pretreated with indicated concentrations of CBBF for 30 min before incubation with LPS for 16 h. The culture supernatants were subsequently isolated and analyzed for nitrite levels. Data are expressed as mean ± S.D. (n = 3). Asterisks indicate a significant difference from LPS alone (∗P < 0.05, ∗∗P < 0.01 vs. LPS alone). (B) The cells were lysed, and the lysates were analyzed by immunoblotting used anti-iNOS. The blot was stripped from the bound antibody and reprobed with anti-β-actin to confirm equal loading. (C) After 8 h stimulation, total RNAs were obtained from RAW 264.7 cells using a Trizol reagent kit. The mRNA levels of iNOS was determined by RT-PCR analysis. Actin was used as an internal control.
nps-23-162f3.tif
Fig. 4.
Effect of CBBF on LPS-induced phosphorylation of IκB-α. Raw 264 Macrophages were incubated with LPS (1 μg/ml) without or with different concentration of CBBF for 2 h. Cytosolic extracts of the cells were subjected to Western blot analysis with anti-phospho-IκBα (Ser-32/36) antibody and anti-IκBα.
nps-23-162f4.tif
Fig. 5.
Effects of CBBF on Akt and MAPKs activation in RAW 264.7 macrophage cells: (4 × 106 cells/well) were treated with or without LPS (1 μg/ml), or with LPS plus different concentrations of CBBF. Control values were obtained in case of LPS and CBBF. CBBF was pretreated for 30 min and then treated with LPS for 30 min. Western blot analysis was performed using a specific antibody raised against Akt (A) and MAPKs (B). (C) The intensity of the bands was scanned and quantified.
nps-23-162f5.tif
Fig. 6.
Effect of CBBF on LPS-activated ROS production. RAW 264.7 cells were treated with LPS in the presence or absence of CBBF (150 μg/ml). After 30 min incubation, cells were incubated with DCFH2-DA (5 μM) for an additional 30 min. Cells were washed twice with PBS, and the intracellular levels of ROS were analyzed by confocal microscope. DCF fluorescence intensities were determined from the same numbers of cells in randomly selected area. DPI, NADPH oxidase inhibitor, 10 μM.
nps-23-162f6.tif
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