[논문]고초균에 의한 생강 발효 추출물의 신경세포 보호 효과

양희선; 김미진; 김민아; 최정숙

doi:10.4163/jnh.2021.54.6.618

고초균에 의한 생강 발효 추출물의 신경세포 보호 효과
Neuroprotective effect of fermented ginger extracts by Bacillus subtilis in SH-SY5Y cells 원문보기

Journal of nutrition and health, v.54 no.6, 2021년, pp.618 - 630

양희선 (농촌진흥청 국립농업과학원 농식품자원부) , 김미진 (농촌진흥청 국립농업과학원 농식품자원부) , 김민아 (농촌진흥청 국립농업과학원 농식품자원부) , 최정숙 (농촌진흥청 국립농업과학원 농식품자원부)

초록
AI-Helper

본 연구에서는 생강을 효소처리하여 수용화율을 높인 후 고초균을 이용하여 발효시킨 생강발효물 (FGEs)을 제조하고, 이를 SH-SY5Y에 6-OHDA와 함께 처리하여 세포 보호 효과 및 AChE 저해 활성을 평가하였다. 그 결과, 6-OHDA로 자극된 신경세포에서 FGE를 처리한 모든 실험군에서 세포 생존율이 증가하고 LDH 농도가 감소하였다. 6-OHDA로 유발된 세포자멸사를 억제할 수 있는지 확인하기 위해 핵의 형태학적인 변화 및 caspase-3 활성을 확인하였다. FGE를 처리한 모든 실험군에서 핵의 손상 및 apoptotic body의 감소와 caspase-3 억제 활성을 확인할 수 있었다. 또한 FGE는 AChE의 활성을 유의적으로 감소시켰다. 시료 간의 활성 차이를 비교하였을 때, 생강에 효소 처리 후 고초균으로 발효한 추출물군 (E/BKG와 E/BCG)의 신경세포 보호 활성이 효소 처리하지 않은 발효생강군 (BKG와 BCG) 보다 유의적으로 크게 나타났다. 그러나 발효에 사용된 고초균 2종 간의 활성은 유사하였으며 처리군 간 유의적인 차이를 보이지 않았다. 본 연구 결과로부터 효소처리하여 수용화율을 높여 고초균으로 발효한 FGE는 신경세포 보호 및 AChE 저해 효과를 나타내어 향후 신경질환 연구를 위한 기초자료 제공 및 고부가가치 식품소재 개발에 이용 가능할 것으로 판단된다.

Abstract ▼ AI-Helper

Purpose: The ginger rhizome (Zingiber officinale) is widely cultivated as a spice for its aromatic and pungent components. One of its constituents, 6-hydroxydopamine (6-OHDA) is usually thought to cross the cell membrane through dopamine uptake transporters, and induce inhibition of mitochondrial respiration and the generation of intracellular reactive oxygen species (ROS). This study examines the neuroprotective effect and acetylcholinesterase (AChE) inhibitory activity of fermented ginger extracts (FGEs) on 6-OHDA induced toxicity in SH-SY5Y human neuroblastoma cells. Methods: Ginger was fermented using 2 species of Bacillus subtilis, with or without enzyme pretreatment. Each sample was extracted with 70% ethanol. Neurotoxicity was assessed by applying the EZ-Cytox cell viability assay and by measuring lactic dehydrogenase (LDH) release. Morphological changes of apoptotic cell nuclei were observed by Hoechst staining. Cell growth and apoptosis of SH-SY5Y cells were determined by Western blotting and enzyme activity analysis of caspase-3, and AChE enzymatic activity was determined by the colorimetric assay. Results: In terms of cell viability and LDH release, exposure to FGE showed neuroprotective activities against 6-OHDA stimulated stress in SH-SY5Y cells. Furthermore, FGE reduced the 6-OHDA-induced apoptosis, as determined by Hoechst staining. The occurrence of apoptosis in 6-OHDA treated cells was confirmed by determining the caspase-3 activity. Exposure to 6-OHDA resulted in increased caspase-3 activity of SH-SY5Y cells, as compared to the unexposed group. However, pre-treatment with FGE inhibited the activity of caspase-3. The neuroprotective effects of FGE were also found to be caspase-dependent, based on reduction of caspase-3 activity. Exposure to FGE also inhibited the activity of AChE induced by 6-OHDA, in a dose-dependent manner. Conclusion: Taken together, our results show that FGE exhibits a neuroprotective effect in 6-OHDA treated SH-SY5Y cells, thereby making it a potential novel agent for the prevention or treatment of neurodegenerative disease.

주제어

표/그림 (5)

그림 Fig. 1. Effect of FGE and 6-OHDA on viability of SH-SY5Y cells. (A) SH-SY5Y cells were treated with FGE at various concentrations for 24 hours. (B) SH-SY5Y cells were treated with 6-OHDA at various concentrations for 24 hours. Cell viability was tested using EZ-Cytox cell viability assay. Data are presented as the mean ± SD of 3 independent experiments. Statistical significance of the differences was evaluated by Student's t-test. 6-OHDA, 6-hydroxydopamine; KACC 10854, B. subtilus standard strain; CMIN-4, B. subtilus isolated from Cheonggukjang; BKG, fermented ginger extract by B. subtilus KACC 10854; E/BKG, fermented ginger extract by B. subtilus KACC 10854 after enzyme treatment filtrate; BCG, fermented ginger extract by B. subtilus CMIN-4; E/BCG, fermented ginger extract by B. subtilus CMIN-4 after enzyme treatment filtrate. *p < 0.05 compared with the control (none) group.
그림 Fig. 2. Protective effect of FGE against 6-OHDA-stimulated cytotoxicity in SH-SY5Y cells. (A) SH-SY5Y cells were treated with 100 μM 6-OHDA for 24 hours after pre-treatment with or without FGE for 1 hour. Cell viability was tested using EZ-Cytox cell viability assay. Data are presented as the mean ± SD of 3 independent experiments. Statistical significance of the differences was evaluated by Student's t-test. (B) SH-SY5Y cells were treated with 100 μM 6-OHDA for 24 hours after pre-treatment with or without 100 μg/mL E/BKG and E/BCG for 1 hour. The morphological images were obtained by a phase-contrast microscope (magnification 200×, scale bar 50 μm). These images are representative of at least 3 independent experiments. Donepezil used as a positive control at a concentration of 1 μM. Samples are the same as Fig. 1. 6-OHDA, 6-hydroxydopamine; FGE, fermented ginger extract; KACC 10854, B. subtilus standard strain; CMIN-4, B. subtilus isolated from Cheonggukjang; BKG, fermented ginger extract by B. subtilus KACC 10854; E/BKG, fermented ginger extract by B. subtilus KACC 10854 after enzyme treatment filtrate; BCG, fermented ginger extract by B. subtilus CMIN-4; E/BCG, fermented ginger extract by B. subtilus CMIN-4 after enzyme treatment filtrate. *p < 0.05 compared with the 6-OHDA treated group; ^†p < 0.01 compared with the control (none) group; ^‡p < 0.05 compared with the enzyme treated group at the same concentration.
그림 Fig. 3. Protective effect of FGE against 6-OHDA-stimulated LDH release in SH-SY5Y cells. SH-SY5Y cells were treated with 100 μM 6-OHDA for 24 hours after pre-treatment with or without FGE for 1 hour. LDH concentration was assessed by LDH cytotoxicity assay kit. Data are presented as the mean ± SD of 3 independent experiments. Statistical significance of the differences was evaluated by Student's t-test. Donepezil used as a positive control at a concentration of 1 μM. Samples are the same as Fig. 1. 6-OHDA, 6-hydroxydopamine; FGE, fermented ginger extract; LDH, lactic dehydrogenase; KACC 10854, B. subtilus standard strain; CMIN-4, B. subtilus isolated from Cheonggukjang; BKG, fermented ginger extract by B. subtilus KACC 10854; E/BKG, fermented ginger extract by B. subtilus KACC 10854 after enzyme treatment filtrate; BCG, fermented ginger extract by B. subtilus CMIN-4; E/BCG, fermented ginger extract by B. subtilus CMIN-4 after enzyme treatment filtrate. *p < 0.05 compared with the 6-OHDA treated group; ^†p < 0.01 compared with the control (none) group; ^‡p < 0.05 compared with the enzyme treated group at the same concentration.
그림 Fig. 4. Inhibitory effects of FGE against 6-OHDA-stimulated apoptosis in SH-SY5Y cells. SH-SY5Y cells were treated with 100 μM 6-OHDA for 24 hours after pre-treatment with or without 100 μg/mL FGE for 1 hour. (A) The morphological images were obtained by a fluorescence microscope after hoechst staining (magnification 400×, scale bar 50 μm). These images are representative of at least 3 independent experiments. (B) Activation of FGE was confirmed using z-VAD-fmk. SH-SY5Y cells were treated with 100 μM 6-OHDA for 24 hours after pre-treatment with FGE or 20 μM z-VAD-fmk for 1 hour. Cell viability was tested using EZ-Cytox cell viability assay. (C) Caspase-3 activation was tested using caspase-3 activity assay kit. (D) Western blot analysis of the caspase-3 activation protein expression. β-actin was used as a control. Data are presented as the mean ± SD of 3 independent experiments. Statistical significance of the differences was evaluated by Student's t-test. Donepezil used as a positive control at a concentration of 1 μM. Samples are the same as Fig. 1. 6-OHDA, 6-hydroxydopamine; FGE, fermented ginger extract; KACC 10854, B. subtilus standard strain; CMIN-4, B. subtilus isolated from Cheonggukjang; BKG, fermented ginger extract by B. subtilus KACC 10854; E/BKG, fermented ginger extract by B. subtilus KACC 10854 after enzyme treatment filtrate; BCG, fermented ginger extract by B. subtilus CMIN-4; E/BCG, fermented ginger extract by B. subtilus CMIN-4 after enzyme treatment filtrate. *p < 0.05 compared with the 6-OHDA treated group; ^†p < 0.01 compared with the control (none) group; ^‡p < 0.05 compared with the enzyme treated group at the same concentration.
그림 Fig. 5. AChE inhibitory effects of FGE. SH-SY5Y cells were treated with 100 μM 6-OHDA for 24 hours after pre-treatment with or without FGE for 1 hour. AChE activity was tested using AChE assay kit. Data are presented as the mean ± SD of 3 independent experiments. Statistical significance of the differences was evaluated by Student's t-test. Donepezil used as a positive control at a concentration of 1 μM. Samples are the same as Fig. 1. 6-OHDA, 6-hydroxydopamine; FGE, fermented ginger extract; AChE, acetylcholinesterase; KACC 10854, B. subtilus standard strain; CMIN-4, B. subtilus isolated from Cheonggukjang; E/BKG, fermented ginger extract by B. subtilus KACC 10854 after enzyme treatment filtrate; E/BCG, fermented ginger extract by B. subtilus CMIN-4 after enzyme treatment filtrate. *p < 0.05 compared with the 6-OHDA treated group; ^†p < 0.01 compared with the control (none) group; ^‡p < 0.05 compared with the enzyme treated group at the same concentration.

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