[논문]더덕순 에탄올 추출물의 신경세포 보호 효과

양희선; 황인국; 최애진; 최정숙

doi:10.4163/jnh.2023.56.2.140

더덕순 에탄올 추출물의 신경세포 보호 효과
Neuroprotective effect of Deodeok (Codonopsis lanceolata) bud extracts in H2O2-stimulated SH-SY5Y cells 원문보기

Journal of nutrition and health, v.56 no.2, 2023년, pp.140 - 154

양희선 (농촌진흥청 국립농업과학원 농식품자원부) , 황인국 (농촌진흥청 국립농업과학원 농식품자원부) , 최애진 (농촌진흥청 국립농업과학원 농식품자원부) , 최정숙 (농촌진흥청 국립농업과학원 농식품자원부)

초록
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본 연구에서는 15-20 cm 길이의 더덕순을 70% 에탄올로 추출하여 추출물 (CLBE)을 제조하고, H₂O₂로 산화적 스트레스를 유발한 SH-SY5Y세포에 전처리하여 신경세포 보호 효과를 평가하였다. 그 결과, CLBE는 H₂O₂로 자극된 세포에서 세포 손상 및 LDH 방출 억제, ROS 소거를 통하여 세포의 사멸을 막아주었다. 또한 CLBE는 Bcl-2와 Bax 단백질의 발현을 조절함으로써 caspase의 활성을 억제하여 신경세포를 보호하였다. 이상의 연구결과들을 종합할 때, CLBE는 산화적 스트레스에 대하여 신경세포 보호 효과가 있는 것으로 보이며, 향후 신경질환 연구를 위한 치료제 개발 및 고부가가치 식품 소재 개발에 유용하게 사용될 수 있을 것으로 판단된다.

Abstract ▼ AI-Helper

Purpose: Deodeok (Codonopsis lanceolata) is generally used in conventional medicines and is considered to have remedial properties to cure several diseases. However, application of the C. lanceolata bud as a novel food ingredient has not been fully explored. Hydrogen peroxide (H2O2) is associated with the production of oxidative damage that results in mutagenesis, carcinogenesis, and cell death. This study examines the neuroprotective effect of C. lanceolate bud extracts (CLBE) on H2O2-stimulated apoptosis in SH-SY5Y cells. Methods: C. lanceolata bud of length 10 to 15 cm was collected and extracted using 70% ethanol. Cytotoxicity was evaluated by the EZ-cytox reagent, measurement of lactic dehydrogenase (LDH) release and reactive oxygen species (ROS). The morphological changes of the nuclei were determined using the Hoechst 33258 dye. Enzyme activities were analyzed using the caspase activity assay kit. Related protein expressions were quantified by the Western blot immunoassay in H2O2-stimulated SH-SY5Y cells. Results: Cell viability, LDH release and ROS generation, demonstrated neuroprotective effects of CLBE in H2O2-stimulated SH-SY5Y cells. The occurrence of apoptosis in H2O2-stimulated cells was confirmed by caspase activity, which was increased in H2O2-stimulated SH-SY5Y cells compared to the unexposed group. Pretreatment of CLBE was observed to inhibit the H2O2-stimulated apoptosis. In addition, exposure to CLBE resulted in increased expression of the Bcl-2 (B cell lymphoma 2) protein and decreased expression of the Bax (Bcl2 associated X) protein. Conclusion: This study shows that exposure to CLBE alleviates the H2O2-stimulated neuronal damage in SH-SY5Y cells. Our results indicate the potential application of CLBE in neurodegenerative disease therapy or prevention.

주제어

표/그림 (6)

그림 Fig. 1. Effect of CLBE and H₂O₂ on viability of SH-SY5Y cells. (A) SH-SY5Y cells were treated with CLBE at various concentrations for 24 hours. (B) SH-SY5Y cells were treated with H₂O₂ at various concentrations for 24 hours. Cell viability was tested using EZ-cytox cell viability assay. Data are presented as the mean ± SD of three independent experiments. Statistical significance of the differences was evaluated by Student's t-test. CLBE, Codonopsisa lanceolata bud extracts. ^#p < 0.05 compared with the control (none) group.
그림 Fig. 2. Protective effect of CLBE against H₂O₂ -stimulated cytotoxicity in SH-SY5Y cells. SH-SY5Y cells were treated with 200 μM H₂O₂ for 24 hours after pretreatment with or without CLBE for 1 hour. (A) Cell viability was tested using EZ-cytox cell viability assay. Data are presented as the mean ± SD of three independent experiments. Statistical significance of the differences was evaluated by Student's t-test. (B) The morphological images were obtained by a phase-contrast microscope (magnification 200×, scale bar 50 μm). These images are representative of three independent experiments. CLBE, Codonopsisa lanceolata bud extracts. ^#p < 0.05 compared with the control (none) group, *p < 0.05 compared with the H₂O₂ alone treated group.
그림 Fig. 3. Inhibitory effect of CLBE against H₂O₂-stimulated ROS generation in SH-SY5Y cells. SH-SY5Y cells were treated with 200 μM H₂O₂ for 24 hours after pretreatment with or without CLBE for 1 hour. After staining with DCFH-DA florescent dye, (A) DCF fluorescence intensity was measured using a fluorescence microplate reader. Data are presented as the mean ± SD of three independent experiments. (B) The morphological images were obtained by a fluorescence microscope (magnification 200×, scale bar 50 μm). These images are representative of three independent experiments. CLBE, Codonopsisa lanceolata bud extracts. ^#p < 0.05 compared with the control (none) group, *p < 0.05 compared with the H₂O₂ alone treated group.
그림 Fig. 4. Inhibitory effects of CLBE against H₂O₂-stimulated LDH release and nuclear damage in SH-SY5Y cells. SH-SY5Y cells were treated with 200 μM H₂O₂ for 24 hours after pretreatment with or without CLBE for 1 hour. (A) LDH concentration was assessed by LDH cytotoxicity assay kit. Data are presented as the mean ± SD of three independent experiments. Statistical significance of the differences was evaluated by Student's t-test. (B) The morphological images were obtained by a fluorescence microscope after Hoechst staining (magnification 400×, scale bar 50 μm). These images are representative of three independent experiments. CLBE, Codonopsisa lanceolata bud extracts. ^#p < 0.01 compared with the control (none) group, *p < 0.05 compared with the H₂O₂ alone treated group.
그림 Fig. 5. Inhibitory effects of CLBE against H₂O₂-stimulated caspase activity in SH-SY5Y cells. SH-SY5Y cells were treated with 200 μM H₂O₂ for 24 hour after pretreatment with or without CLBE for 1 hour. (A) Caspase-9 and (B) caspase-3 activation were tested using caspase activity assay kit. (C) Caspase proteins expression were assayed by western blot analysis using an ECL detection system. Expression level of β-actin was used as an internal control. (D) Caspase activation of CLBE was confirmed using z-VAD-fmk. SH-SY5Y cells were treated with 200 μM H₂O₂ for 24 hours after pretreatment with CLBE or 20 μM z-VAD-fmk for 1 hour. Cell viability was tested using EZ-cytox cell viability assay. Data are presented as the mean ± SD of three independent experiments. Statistical significance of the differences was evaluated by Student's t-test. CLBE, Codonopsisa lanceolata bud extracts. ^#p < 0.01 compared with the control (none) group, *p < 0.05 compared with the H₂O₂ alone treated group.
그림 Fig. 6. Effects of CLBE on protein expression of Bcl-2 and Bax in H₂O₂-stimulated SH-SY5Y cells. SH-SY5Y cells were treated with 200 μM H₂O₂ for 24 hours after pretreatment with or without CLBE for 1 hour. (A) Bcl-2 and Bax protein expressions were assayed by western blot analysis using an ECL detection system. Expression level of β-actin was used as an internal control. (B and C) Bands were quantified using Image J, normalized to actin and ratios were determined. Data are presented as the mean ± SD of three independent experiments. Statistical significance of the differences was evaluated by Student's t-test. CLBE, Codonopsisa lanceolata bud extracts. ^#p < 0.01 compared with the control (none) group, *p < 0.05 compared with the H₂O₂ alone treated group.

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