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Protective effect of furosin isolated from Euphorbia helioscopia against glutamate-induced HT22 cell death

Article information

J Korean Med. 2018;39(1):35-43
Publication date (electronic) : 2018 March 31
doi : https://doi.org/10.13048/jkm.18004
Ji Yun Baek1,2orcid_icon, Ji Hoon Song2orcid_icon, Sung Youl Choi3orcid_icon
1Kyeongnam National University of Science and Technology
2College of Korean Medicine, Gachon University
3Department of Oriental Neuropsychiatry, College of Korean Medicine, Gachon University
Correspondence to: 최성열 (Sung Youl Choi), 가천대학교 한의과대학 한방신경정신과, 1342 Seongnam-Daero, Sujung-Gu, Seongnam 13120, South Korea Tel: 82-32-770-1343, e-mail: pc1075@gachon.ac.kr
Received 2017 February 13; Revised 2018 March 13; Accepted 2018 March 14.

Abstract

Objectives

In the brain, glutamate is the most important excitable neurotransmitter in physiological and pathological conditions. However, the high level of glutamate induces neuronal cell death due to exitotoxicity and oxidative stress. The present study investigated to evaluate a possible neuroprotective effect of furosin isolated from Euphorbia helioscopia against glutamate-induced HT22 cell death.

Methods

Furosin was isolated from methanol extract of Euphorbia helioscopia and examined whether it protects glutamate-induced neuronal cell death. The cell viability was determined using Ez-Cytox assay. Anti-oxidative effect of furosin was determined by DPPH scavenging activities, and the levels of intracellular reactive oxygen species (ROS) were determined by the fluorescent intensity after staining the cells with H2DCFDA. To evaluate apoptotic cell death, we performed nuclear staining and image-based cytometeric analysis.

Results

The cell viability was significantly increased by treatement with furosin compared with the treatment with glutamate. Furosin showed a strong DPPH radical scavenging activity (EC50=1.83 μM) and prevented the accumulation of intra cellular ROS. Finally, the presence of 50 and 100 μM furosin significantly the percentage of apoptotic cells compared with glutamate treatment.

Conclusion

The present study found that furosin is a potent neuroprotectant against glutamate-induced oxidative stress through inhibition of apoptotic cell death induced by glutamate. Therefore, the present study suggests that furosin as a bioactive compound of E. helioscopia can be a useful source to develop a drug for the treatment of neurodegenerative diseases and acute brain injuries.

Keywords: Furosin; Euphorbia helioscopia; Glutamate; Apoptotic cell death; HT22 cells
Fig. 1

Chemical structure of furosin (Fur).

Fig. 2

Furosin inhibited glutamate-induced HT22 cell death. HT22 cells were treated with the indicated concentrations of furosin in the presence or absence of 5 mM glutamate. (A and B) To determine the self toxicity and protective effect of furosin, the cell viability was determined using Ez-Cytox assay kit after the exposure to glutamate for 24 h. Bars denote the percentage of cell viability compared with non-treated groups (mean±S.E.M., *p<0.05 compared with glutamate-treated group). (C)Morphological changes were observed under the phase contrast microscope.

Fig. 3

Furosin prevented intracellular ROS accumulation through their antioxidant capacity. (A)The indicated concentrations of furosin was incubated with DPPH radical reagent for 30 min and obtained using microplate reader at 550 nm. (B)HT22 cells were treated with 50 or 100 uM furosin in the presence (dark gray) or absence (gray) of 5 mM glutamate for 8 h and stained with H2DCFDA, a fluorescent indicator of ROS. The fluorescent intensity of DCF was measured using a fluorescent microplate reader. Bars denote the fold increases of intracellular ROS level compared with non-treated groups (mean ±S.E.M., *<0.05 compared with glutamate-treated group).

Fig. 4

Furosin prevented glutamate-induced apoptotic HT22 cell death. HT22 cells were incubated with glutamate in the presence or absence of furosin for 12 h. (A) Chromatin condensation was determined using Hoechst33342 and observed under a fluorescent microscope. (B) The number of apoptotic cells were analyzed using Tali-image based cytometric analysis. The proportion of apoptotic cells were represented by the percentage of Annexin V-positive cells (mean±S.E.M., * <0.05 compared with glutamate-treated group).

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Article information Continued

Copyright © 2018 The Society of Korean Medicine all rights reserved

Fig. 1

Fig. 1

Chemical structure of furosin (Fur).

Fig. 2

Fig. 2

Furosin inhibited glutamate-induced HT22 cell death. HT22 cells were treated with the indicated concentrations of furosin in the presence or absence of 5 mM glutamate. (A and B) To determine the self toxicity and protective effect of furosin, the cell viability was determined using Ez-Cytox assay kit after the exposure to glutamate for 24 h. Bars denote the percentage of cell viability compared with non-treated groups (mean±S.E.M., *p<0.05 compared with glutamate-treated group). (C)Morphological changes were observed under the phase contrast microscope.

Fig. 3

Fig. 3

Furosin prevented intracellular ROS accumulation through their antioxidant capacity. (A)The indicated concentrations of furosin was incubated with DPPH radical reagent for 30 min and obtained using microplate reader at 550 nm. (B)HT22 cells were treated with 50 or 100 uM furosin in the presence (dark gray) or absence (gray) of 5 mM glutamate for 8 h and stained with H2DCFDA, a fluorescent indicator of ROS. The fluorescent intensity of DCF was measured using a fluorescent microplate reader. Bars denote the fold increases of intracellular ROS level compared with non-treated groups (mean ±S.E.M., *<0.05 compared with glutamate-treated group).

Fig. 4

Fig. 4

Furosin prevented glutamate-induced apoptotic HT22 cell death. HT22 cells were incubated with glutamate in the presence or absence of furosin for 12 h. (A) Chromatin condensation was determined using Hoechst33342 and observed under a fluorescent microscope. (B) The number of apoptotic cells were analyzed using Tali-image based cytometric analysis. The proportion of apoptotic cells were represented by the percentage of Annexin V-positive cells (mean±S.E.M., * <0.05 compared with glutamate-treated group).