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Neuroprotective mechanisms of luteolin in glutamate-induced oxidative stress and autophagy-mediated neuronal cell death
Scientific Reports volume 14, Article number: 7707 (2024) Cite this article
Abstract
Neurodegenerative diseases, characterized by progressive neuronal dysfunction and loss, pose significant health challenges. Glutamate accumulation contributes to neuronal cell death in diseases such as Alzheimer's disease. This study investigates the neuroprotective potential of Albizia lebbeck leaf extract and its major constituent, luteolin, against glutamate-induced hippocampal neuronal cell death. Glutamate-treated HT-22 cells exhibited reduced viability, altered morphology, increased ROS, and apoptosis, which were attenuated by pre-treatment with A. lebbeck extract and luteolin. Luteolin also restored mitochondrial function, decreased mitochondrial superoxide, and preserved mitochondrial morphology. Notably, we first found that luteolin inhibited the excessive process of mitophagy via the inactivation of BNIP3L/NIX and inhibited lysosomal activity. Our study suggests that glutamate-induced autophagy-mediated cell death is attenuated by luteolin via activation of mTORC1. These findings highlight the potential of A. lebbeck as a neuroprotective agent, with luteolin inhibiting glutamate-induced neurotoxicity by regulating autophagy and mitochondrial dynamics.
점진적인 신경세포 기능 장애와 손실을 특징으로 하는 신경 퇴행성 질환은
건강에 심각한 문제를 일으킵니다.
글루타메이트 축적은
알츠하이머병과 같은 질환에서 신경세포 사멸의 원인이 됩니다.
이 연구는
글루타메이트에 의한 해마 신경세포 사멸에 대한
알비지아 레벡 잎 추출물과
그 주요 성분인 루테올린의 신경 보호 가능성을 조사합니다.
글루타메이트로 처리한 HT-22 세포는
생존력 감소, 형태 변화, ROS 증가 및 세포 사멸을 보였으며,
이는 알비지아 레벡 추출물과 루테올린으로 전처리하면 약화되었습니다.
또한
루테올린은
미토콘드리아 기능을 회복하고
미토콘드리아 슈퍼옥사이드를 감소시키며
미토콘드리아 형태를 보존하는 것으로 나타났습니다.
특히
루테올린이 BNIP3L/NIX의 비활성화를 통해
과도한 미토파지 과정을 억제하고
리소좀 활성을 억제한다는 사실을 처음으로 발견했습니다.
이 연구는
글루타메이트에 의한 자가포식 매개 세포 사멸이
루테올린에 의해 mTORC1의 활성화를 통해
약화된다는 것을 시사합니다.
이러한 연구 결과는
루테올린이 자가포식과 미토콘드리아 역학을 조절하여
글루타메이트에 의한 신경 독성을 억제하는 등
신경 보호제로서 A. 레벡의 잠재력을 강조합니다.
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Introduction
Neurodegenerative diseases are neurological disorders and age-related diseases that generally progress slowly and worsen over time. These diseases are characterized by the loss of neuronal structure and function in the central nervous system (CNS) and peripheral nervous system (PNS)1,2, which leads to clinical features such as movement disorder, cognitive impairment, and behavioral impairment3,4. Wilson et al. recently reported the eight hallmarks of neurodegenerative disease consist of pathological protein aggregation, synaptic and neuronal network dysfunction, aberrant proteostasis, cytoskeletal abnormalities, altered energy homeostasis, DNA and RNA defects, inflammation, and neuronal cell death5. Notably, many of these characteristic features play a role and work together to facilitate neuronal cell death.
Glutamate is the most abundant excitatory neurotransmitter in the central nervous system (CNS), which plays a crucial role in synaptic communication and neuronal signaling. In conditions such as Alzheimer's, Parkinson's, and Huntington's diseases, there is an abnormal accumulation of glutamate in the brain's extracellular space6,7,8. The glutamate accumulation is the primary factor responsible for excessive ROS generation via the overactivation of glutamate receptor N-methyl-D-aspartate (NMDA), resulting in an excess calcium influx to the cells. Moreover, high extracellular glutamate can lead to glutathione depletion through cystine/glutamate antiporter (Xc). Subsequently, the accumulation of ROS leads to the deterioration of neuronal cells and triggers various types of cell death, such as apoptosis, necrosis, ferroptosis, and autophagy9,10,11. Among those, autophagy is a cellular process involved in the degradation and recycling of cellular components, including damaged proteins and organelles. In neurons, autophagy helps to remove misfolded proteins and damaged organelles, thus protecting neurons from cellular stress12. However, under specific abnormal circumstances, such as prolonged nutrient deprivation and chronic stress, autophagy can become excessive or uncontrolled, crossing a critical threshold where it triggers irreversible neuronal cell death. Moreover, autophagic cell death leads to the degradation of essential cellular components, especially mitochondria via mitophagy receptor PINK1 and parkin-mediated mitophagy or BNIP3 and NIX-dependent mitophagy13,14. The overactivation of the mitochondria degradation also process leads to an alteration in energy homeostasis. Consequently, inhibition of glutamate toxicity by targeting the excessive degradation process is regarded as a promising strategy for alleviating neurodegenerative disease.
The mammalian target of rapamycin complex 1 (mTORC1) is a critical regulator of cell growth, metabolism, and autophagy. It plays a central role in coordinating cellular responses to various stress conditions, including nutrient deprivation, energy depletion, and other forms of cellular stress. Under normal physiological conditions, mTORC1 fosters cell growth and protein synthesis. Simultaneously, it hampers autophagy by phosphorylating ULK1 (Unc-51-like autophagy activating kinase 1)15. However, mTORC1 activity is suppressed under stress conditions16,17. Thus, the mTORC1 regulation is important for maintaining cellular homeostasis and preventing uncontrolled autophagy.
Dietary supplements from plants containing phytochemicals such as carotenoids, flavonoids, saponins, and vitamins tend to prevent ROS or oxidative stress-related chronic conditions18. Interestingly, some antioxidant agents such as acetylcholinesterase inhibitor can improve learning and memory19,20. It is noteworthy that some natural bioactive compounds exhibit anti-aging properties and can restore mTORC1 activity21. Albizia lebbeck, a medicinal plant native to part of Southeast Asia and the Indian subcontinent, and belonging to the Fabaceae family, is recognized for its notable antioxidant, anti-inflammatory, and neuroprotective activities22. A. lebbeck contains various phytochemical constituent, which shows promise as a potential therapeutic agent for neurodegenerative diseases23,24. However, the neuroprotective effect and molecular mechanism of A. lebbeck extract and luteolin against glutamate-induced hippocampal cell death have not been elucidated. In this study, we identified that the leaf extract of A. lebbeck and luteolin have neuroprotective activities against glutamate-induced hippocampal neuronal cell death. Moreover, we unveiled a new mechanism by which luteolin hinders excessive mitophagy and autophagy through mTOR signaling (Supplementay Information).
소개
신경 퇴행성 질환은
일반적으로 천천히 진행되며
시간이 지남에 따라 악화되는 신경 장애 및 노화 관련 질환입니다.
이러한 질환은
중추신경계(CNS)와 말초신경계(PNS)1,2의 신경세포 구조와 기능의 손실로 인해
운동 장애, 인지 장애, 행동 장애와 같은
윌슨 등은 최근
신경 퇴행성 질환의 8가지 특징으로
병적 단백질 응집,
시냅스 및 신경 네트워크 기능 장애,
비정상적인 단백질 항상성 protein homeostasis,
세포 골격 이상,
에너지 항상성 변화,
DNA 및 RNA 결함,
염증 및 신경 세포 사멸을 보고한 바 있습니다5.
pathological protein aggregation,
synaptic and neuronal network dysfunction,
aberrant proteostasis,
cytoskeletal abnormalities,
altered energy homeostasis,
DNA and RNA defects,
inflammation, and neuronal cell death
신경세포 기능장애 - 미토콘드리아, 리보솜, ER, 골지체, 리소좀 기능장애
--> ATP 생성장애, ROS 과다생성
--> Apoptosis 문제로 cellular senescence --> inflammation 만성화
--> 4차 단백질 구조 이상, 알파 시누클레인, 루이체 축적
--> Synapse 기능 이상, neuronal network 기능부전
----> neuronal cell death, brain atrophy
특히 이러한 특징적인 특징 중 다수는
신경 세포 사멸을 촉진하는 역할을 하며 함께 작용합니다.
글루타메이트는
중추신경계(CNS)에서 가장 풍부한 흥분성 신경전달물질로
시냅스 통신과 신경 신호 전달에 중요한 역할을 합니다.
알츠하이머병, 파킨슨병, 헌팅턴병과 같은 질환에서는
뇌의 세포 외 공간에 글루타메이트가
글루타메이트 축적은
글루타메이트 수용체 N-메틸-D-아스파르트산염(NMDA)의 과활성화를 통해
과도한 ROS를 생성하여
세포에 과도한 칼슘 유입을 초래하는 주요 요인입니다.
또한
세포 외 글루타메이트 농도가 높으면
시스틴/글루타메이트 항포터(Xc)를 통해
글루타티온이 고갈될 수 있습니다.
결과적으로
ROS의 축적은
신경세포의 기능 저하로 이어져
세포사멸, 괴사, 페로펩토시스, 자가포식 등
이 중 자가포식은
손상된 단백질과 세포 소기관을 포함한
세포 구성 요소의 분해 및 재활용에 관여하는 세포 과정입니다.
뉴런에서 자가포식은
잘못 접힌 단백질과 손상된 세포 소기관을 제거하여
세포 스트레스로부터 뉴런을 보호하는 데 도움을 줍니다12.
그러나
장기간의 영양 결핍이나
만성 스트레스와 같은
특정 비정상적인 상황에서는
자가포식이 과도하거나
통제되지 않아 임계점을 넘어
돌이킬 수 없는 신경 세포 사멸을 유발할 수 있습니다.
또한
자가포식 세포 사멸은
미토파지 수용체 PINK1과 파킨 매개 미토파지 또는
필수 세포 성분, 특히 미토콘드리아의 분해로 이어집니다.
미토콘드리아 분해 과정의 과활성화는
에너지 항상성의 변화로 이어집니다.
따라서
과도한 분해 과정을 표적으로 삼아
글루타메이트 독성을 억제하는 것이
신경 퇴행성 질환을 완화하는 유망한 전략으로 여겨지고 있습니다.
포유류의
라파마이신 복합체 1(mTORC1)은
세포 성장, 대사 및 자가포식의 중요한 조절 인자입니다.
영양소 부족,
에너지 고갈 및
기타 형태의 세포 스트레스를 포함한
다양한 스트레스 조건에 대한 세포 반응을 조정하는 데
중심적인 역할을 합니다.
정상적인 생리적 조건에서
mTORC1은
세포 성장과 단백질 합성을 촉진합니다.
동시에
ULK1(Unc-51 유사 오토파지 활성화 키나아제 1)을 인산화하여
오토파지를 방해합니다15.
그러나
스트레스 조건에서
따라서
세포 항상성을 유지하고
통제되지 않는 자가포식을 방지하는 데
mTORC1 조절이 중요합니다.
카로티노이드,
플라보노이드,
사포닌,
비타민과 같은
식물성 화학 물질을 함유한 식이 보충제는
ROS 또는 산화 스트레스 관련 만성 질환을 예방하는 경향이 있습니다18.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7601865/
흥미롭게도
아세틸콜린에스테라아제 억제제와 같은
일부 항산화제는
일부
천연 생리 활성 화합물은
노화 방지 특성을 나타내며 mTORC1 활성을 회복할 수 있다는 점에
주목할 만합니다21.
동남아시아와 인도 아대륙 일부에 자생하며
파바세과에 속하는 약용식물인 알비지아 레벡은
주목할 만한 항산화, 항염증 및 신경 보호 활동으로 인정 받고 있습니다22.
알비지아 레벡에는
다양한 식물성 화학 성분이 함유되어 있어
신경 퇴행성 질환의 잠재적 치료제로서의 가능성 을 보여주고 있습니다23,24.
그러나
글루타메이트에 의한
해마 세포 사멸에 대한
A. 레 벡 추출물과 루테올린의 신경 보호 효과와 분자 기전은 밝혀지지 않았습니다.
본 연구에서는
A. 레 벡의 잎 추출물과 루테올린이
글루타메이트에 의한 해마 신경세포 사멸에 대한 신경보호 활성이 있음을 확인했습니다.
또한
루테올린이
mTOR 신호를 통해 과도한 미토파지와 자가포식을 저해하는
새로운 메커니즘을 밝혀냈습니다(보충설명).
Results
Glutamate-induced neuronal cell death
Glutamate toxicity was examined in various neuronal cell lines to determine the optimal dose of glutamate and the most appropriate model. HT-22 mouse hippocampal, SH-SY5Y human neuroblastoma, and Neuro-2A mouse neuroblastoma cell lines were tested in our study and exposed to varying concentrations of glutamate. Cell viability was evaluated using the MTS assay, revealing that 5 mM of glutamate is the most optimal concentration for HT-22 cells. This concentration resulted in approximately 25% cell viability (Fig. 1A). Moreover, SH-SY5Y human neuroblastoma cells were treated with glutamate at various concentrations including 40, 80, 160, and 200 mM for 18 h. Glutamate toxicity in SH-SY5Y cells was observed to begin at a concentration of 160 mM, resulting in approximately 50% cell viability (Fig. 1B). In addition, the Neuro-2A mouse neuroblastoma cell line underwent treatment with varying concentrations of glutamate, revealing that the onset of glutamate toxicity occurs at 40 mM, leading to an approximately 80% cell viability (Fig. 1C). Given that among the tested neuronal cell lines, HT-22 cells displayed the highest sensitivity to glutamate toxicity, they were selected for the subsequent experiments.
글루타메이트에 의한 신경세포 사멸
글루타메이트의 최적 용량과
가장 적합한 모델을 결정하기 위해
다양한 신경 세포주에서 글루타메이트 독성을 조사했습니다.
HT-22 마우스 해마, SH-SY5Y 인간 신경모세포종, Neuro-2A 마우스 신경모세포종 세포주를 다양한 농도의 글루타메이트에 노출시켜 테스트했습니다.
MTS 분석법을 사용하여 세포 생존력을 평가한 결과,
5mM의 글루타메이트가
HT-22 세포에 가장 적합한 농도라는 것이 밝혀졌습니다.
이 농도에서 약 25%의 세포 생존율을 보였습니다(그림 1A). 또한 SH-SY5Y 인간 신경모세포종 세포를 40, 80, 160, 200mM 등 다양한 농도의 글루타메이트로 18시간 동안 처리한 결과, SH-SY5Y 세포의 글루타메이트 독성은 160mM 농도에서 시작하여 약 50%의 세포 생존율을 보이는 것으로 관찰되었습니다(그림 1B). 또한 Neuro-2A 마우스 신경모세포종 세포주를 다양한 농도의 글루타메이트로 처리한 결과, 글루타메이트 독성이 40mM에서 시작되어 세포 생존율이 약 80%에 이르는 것으로 나타났습니다(그림 1C). 테스트한 신경세포주 중 HT-22 세포가 글루타메이트 독성에 가장 높은 민감도를 보였기 때문에 후속 실험을 위해 이 세포를 선택했습니다.
Figure 1
Glutamate-induced toxicity in different neuronal cells. The effect of glutamate-induced cytotoxicity in different types of neuronal cell lines was assessed by MTS assay (n = 3). HT-22 cells were treated with varying concentrations of glutamate, ranging from 5 to 20 mM. Similarly, SH-SY5Y mouse neuroblastoma cell line were treated with glutamate at 40–200 mM, and Neuro-2A mouse neuroblastoma cell line were treated with glutamate at 5–160 mM for 18 h. Bar graphs show the % cell viability of (A) HT-22 mouse hippocampal cell line, (B) SH-SY5Y mouse neuroblastoma cell line, and (C) Neuro-2A mouse neuroblastoma cell line. The data were collected from at least three independent experiments and the results were shown in mean ± SEM. **p-value < 0.01, ***p-value < 0.005, ****p-value < 0.001 compared with untreated control group.
다양한 신경 세포에서 글루타메이트에 의한 독성.
다양한 유형의 신경 세포주에서 글루타메이트에 의한 세포 독성의 영향은 MTS 분석으로 평가했습니다(n= 3). HT-22 세포를 5~20mM 범위의 다양한 농도의 글루타메이트로 처리했습니다. 마찬가지로, SH-SY5Y 마우스 신경모세포종 세포주를 40-200mM에서 글루타메이트로 처리하고 Neuro-2A 마우스 신경모세포종 세포주를 5-160mM에서 18시간 동안 글루타메이트로 처리했습니다. 막대 그래프는 (A) HT-22 마우스 해마 세포주, (B) SH-SY5Y 마우스 신경모세포종 세포주 및 (C) Neuro-2A 마우스 신경모세포종 세포주의 세포 생존율 %를 보여줍니다. 데이터는 최소 세 번의 독립적인 실험에서 수집되었으며 결과는 평균 ± SEM으로 표시되었습니다. 처리하지 않은 대조군과 비교하여 **p-값< 0.01, ***p-값< 0.005, ****p-값< 0.001.
A. lebbeck leaf ethanol extracts (ALE) inhibit glutamate-induced HT-22 hippocampal neuronal cell death
Previous research has shown that extracts of A. lebbeck leaves can inhibit neuronal cell death25. In this study, we aimed to assess the inhibitory potential of ALE against glutamate-induced hippocampal neuronal cell death. Therefore, HT-22 cells were pre-treated with ALE at concentrations ranging from 2.5 to 100 µg/ml, followed by exposure to 5 mM of glutamate. The results revealed that 5 mM of glutamate led to approximately 25% cell viability in HT-22 cells. However, when cells were pre-treated with ALE, there was a significant dose-dependent increase in cell viability (Fig. 2A). Furthermore, upon examination under a light microscope, the treatment of cells with glutamate exhibited nuclear condensation and cell shrinkage. In contrast, those that were pre-treated with ALE retained their original cell morphology (Fig. 2B). To further investigate the flavonoid constituents of ALE, we employed the HPLC analysis (Fig. 2C). We found that ALE possesses a high flavonoid content, especially quercetin and luteolin. Therefore, we directed our attention to these two compounds for further investigation.
A. 레벡 잎 에탄올 추출물(ALE)은 글루타메이트에 의한 HT-22 해마 신경세포 사멸을 억제합니다.
이전 연구에 따르면 A. 레벡 잎 추출물은 신경 세포 사멸을 억제할 수 있는 것으로 나타났습니다25. 이 연구에서는 글루타메이트에 의한 해마 신경세포 사멸에 대한 ALE의 억제 가능성을 평가하고자 했습니다. 따라서 HT-22 세포를 2.5 ~ 100 µg/ml 농도의 ALE로 전처리한 후 5mM의 글루타메이트에 노출시켰습니다. 그 결과 5mM의 글루타메이트는 HT-22 세포의 세포 생존율을 약 25% 감소시키는 것으로 나타났습니다. 그러나 세포를 ALE로 전처리했을 때는 세포 생존율이 용량 의존적으로 크게 증가했습니다(그림 2A). 또한, 광학 현미경으로 검사한 결과, 글루타메이트로 세포를 처리한 경우 핵 응축과 세포 수축이 나타났습니다. 반면, ALE로 사전 처리한 세포는 원래의 세포 형태를 유지했습니다(그림 2B). ALE의 플라보노이드 성분을 추가로 조사하기 위해 HPLC 분석을 사용했습니다(그림 2C).
우리는 ALE가 특히
퀘르세틴과 루테올린과 같은
플라보노이드 함량이 높다는 것을 발견했습니다.
따라서
추가 조사를 위해
이 두 가지 화합물에 주목했습니다.
Figure 2
ALE inhibits glutamate-induced cytotoxicity in mouse HT-22 hippocampal cells. (A) The effect of ALE on glutamate-induced cytotoxicity in HT-22 was assessed by MTS assay (n = 5). HT-22 cells were pre-treated with ALE at different concentrations (2.5–100 µg/ml) for 24 h, followed by 5 mM glutamate for 18 h. Bar graphs show the % cell viability. (B) The morphology of HT-22 cells was visualized under the inverted light microscope (scale bar is 1000 µm). (C) HPLC chromatograms of flavonoid standards (Top) and A. lebbeck (L.) Benth. leaf (Bottom). (1 = myricetin; 2 = quercetin; 3 = naringenin; 4 = luteolin; 5 = hesperidin; 6 = kaempferol; 7 = apigenin). The data were collected from at least three independent experiments and the results were shown in mean ± SEM. ***p-value < 0.005, ****p-value < 0.001 compared with glutamate group. #p-value < 0.05 compared with untreated control group.
Luteolin mitigates glutamate-induced cytotoxicity in HT-22 and reduces cellular stress
Considering our results, luteolin has been selected as the primary compound for investigating its neuroprotective effect against glutamate-induced HT-22 hippocampal neuronal death. To accomplish this, HT-22 cells were pre-treated with a range of luteolin concentrations (5–50 µM) for 24 h before exposing them to glutamate. We optimized the use of 10 µM quercetin (data not shown) as a positive control26. The cell viability assay result demonstrated that luteolin effectively restores HT-22 cell viability (Fig. 3A). Moreover, we used the LDH cytotoxicity assay to further support the cell viability data, showing that luteolin (5–50 µM) reduced glutamate toxicity in a dose-dependent manner (Fig. 3B). Glutamate is known to induce cytotoxicity in neuronal cells by promoting the production of ROS27. Therefore, we analyzed intracellular ROS levels using the H2DCF-DA probe to determine whether luteolin could alleviate glutamate-induced oxidative stress. Fluorescence intensity was measured in the CellInsight CX7 High-Content Screening (HCS) Platform and normalized with nuclear-specific staining (Hoechst 33342). Our findings revealed a notable elevation in intracellular ROS production due to glutamate exposure. Nevertheless, the pre-treatment of cells with luteolin and quercetin proficiently reinstated the levels of intracellular ROS accumulation (Fig. 3C). To assess the impact of luteolin on glutamate-induced cell death, we conducted an apoptosis assay using PE-Annexin V and 7AAD staining. The results demonstrated that treatment of HT-22 cells with 5 mM glutamate alone led to approximately 40% late apoptosis and 14% early apoptosis (Fig. 3D). However, pre-treatment with luteolin and quercetin prior to glutamate incubation significantly reduced glutamate-induced apoptosis, with the protective effect observed at a concentration of 5–25 µM and 10 µM respectively. Thus, cells pre-treated with luteolin showed a remarkable decrease in neuronal cell death induced by glutamate.
루테올린은 HT-22에서 글루타메이트에 의한 세포 독성을 완화하고 세포 스트레스를 감소시킵니다.
연구 결과를 고려할 때,
루테올린은 글루타메이트에 의한 HT-22 해마 신경세포 사멸에 대한 신경 보호 효과를 조사하기 위한 주요 화합물로 선택되었습니다. 이를 위해 HT-22 세포를 글루타메이트에 노출시키기 전에 24시간 동안 다양한 루테올린 농도(5-50 µM)로 사전 처리했습니다. 양성 대조군으로 10µM 케르세틴(데이터는 표시되지 않음)을 최적화하여 사용했습니다26. 세포 생존력 분석 결과 루테올린이 HT-22 세포 생존력을 효과적으로 회복시키는 것으로 나타났습니다(그림 3A).
또한 세포 생존력 데이터를 더욱 뒷받침하기 위해 LDH 세포 독성 분석법을 사용하여 루테올린(5-50 µM)이 용량 의존적으로 글루타메이트 독성을 감소시킨다는 것을 보여주었습니다(그림 3B). 글루타메이트는 ROS27의 생성을 촉진하여 신경세포에서 세포 독성을 유도하는 것으로 알려져 있습니다. 따라서 루테올린이 글루타메이트에 의한 산화 스트레스를 완화할 수 있는지 확인하기 위해 H2DCF-DA 프로브를 사용하여 세포 내 ROS 수준을 분석했습니다. 형광 강도는 CellInsight CX7 고함량 스크리닝(HCS) 플랫폼에서 측정하고 핵 특이 염색(Hoechst 33342)으로 정규화했습니다. 그 결과 글루타메이트 노출로 인해 세포 내 ROS 생성이 현저하게 증가한 것으로 나타났습니다. 그럼에도 불구하고 세포를 루테올린과 케르세틴으로 전처리하면 세포 내 ROS 축적 수준이 능숙하게 회복되었습니다(그림 3C). 글루타메이트에 의한 세포 사멸에 대한 루테올린의 영향을 평가하기 위해 PE-Annexin V 및 7AAD 염색을 사용하여 세포 사멸 분석을 수행했습니다. 그 결과, HT-22 세포를 5mM 글루타메이트 단독으로 처리하면 약 40%의 후기 세포 사멸과 14%의 조기 세포 사멸이 발생했습니다(그림 3D). 그러나 글루타메이트 배양 전에 루테올린과 케르세틴을 전처리하면 글루타메이트에 의한 세포 사멸이 크게 감소했으며, 각각 5-25 µM 및 10 µM의 농도에서 보호 효과가 관찰되었습니다. 따라서 루테올린으로 사전 처리된 세포는 글루타메이트에 의해 유도된 신경 세포 사멸이 현저하게 감소한 것으로 나타났습니다.
Figure 3
Luteolin inhibits glutamate-induced cytotoxicity in HT-22 cells. HT-22 cells were pre-treated with luteolin at different concentrations (5–50 µM) for 24 h and quercetin 10 µM was used as a positive control. Subsequently, the cells were exposed to 5 mM glutamate for 18 h. Bar graphs show (A) the % cell viability (n = 5) and (B) the % LDH release (n = 3). (C) The intracellular ROS was visualized under the CellInsight CX7 High-Content Screening (HCS) platform, the bottom bar graph shows the relative intracellular ROS level (n = 3). (D, left) The HT-22 cells were stained with PE-Annexin V/7-AAD probes, the numbers of cell deaths were analyzed via flow cytometry Q1: necrosis, Q2: late apoptosis, Q3: live, Q4: early apoptosis (n = 3). (D, right) The histogram represents the percentages of necrotic and apoptotic cells. The data were collected from at least three independent experiments and the results were shown in mean ± SEM. *p-value < 0.05, **p-value < 0.01, ****p-value < 0.001 compared with glutamate-treated group, #p-value < 0.05, ####p-value < 0.001 compared with untreated control group. L:luteolin, Q:quercetin.
Luteolin inhibits glutamate-induced mitochondrial dysfunction and restores mitochondrial content
Neurons have high energy demands to maintain their functions. Mitochondria are indeed the primary suppliers of adenosine triphosphate (ATP) in neurons and play a critical role in brain energy metabolism. Notably, glutamate excitotoxicity has been associated with mitochondria damage in neurons28. To assess the impact of luteolin on mitochondrial stress, the generation of mitochondria superoxide was evaluated utilizing the mitosox probe. The fluorescence intensity was then monitored using the CellInsight CX7 high content screening (HCS) Platform, with subsequent normalization with Hoechst 33342. Our result revealed that glutamate induced an increase in mitochondria superoxide production. However, pre-treatment of cells with luteolin (5–25 µM) and quercetin (10 µM) significantly restored the mitochondria superoxide level (Fig. 4A). Furthermore, the mitochondria membrane potential, which is susceptible to oxidative stress, was evaluated by staining with mitotracker orange CMTMRos (Fig. 4B). The control group (untreated group) showed a high fluorescent intensity, indicating a robust mitochondrial membrane potential. In contrast, the glutamate treatment group displayed a significant reduction in fluorescent intensity, demonstrating a loss of mitochondrial membrane potential. However, pre-treatment with luteolin and quercetin protected the mitochondrial membrane potential and restored it. Quantification of fluorescence intensity for mitotracker orange CMTMRos confirmed that glutamate significantly decreased the fluorescence intensity compared to the control group (Fig. 4B, bottom). Additionally, we used mitotracker orange CMTMRos staining to assess mitochondria morphology under the confocal microscope. The mitochondria network analysis plugin (MiNA) was used to analyze mitochondria morphology. Our results showed that glutamate could cause mitochondria fragmentation and decrease the branching network. However, pre-treatment of cells with luteolin and quercetin prior to glutamate exposure could maintain the mitochondria morphology and increase the branching network (Fig. 4C). Moreover, we investigated the mitochondrial content using the stable mtDNA fraction, 16S rRNA, comparing it with nuclear DNA (HKII). We found that the glutamate treatment group had a decrease in mtDNA/nDNA ratio, indicating the loss of mitochondria content (Fig. 4D). Conversely, the luteolin pre-treatment group had an increase in mtDNA/nDNA ratio, indicating an increase in mitochondria content. These findings suggest that luteolin has a positive impact on mitochondrial health and may protect against glutamate-induced mitochondrial stress.
루테올린은
글루타메이트에 의한 미토콘드리아 기능 장애를 억제하고
미토콘드리아 함량을 회복시킵니다.
신경세포는
기능을 유지하기 위해
많은 에너지를 필요로 합니다.
미토콘드리아는
실제로 뉴런에서 아데노신 삼인산(ATP)의 주요 공급원이며
뇌 에너지 대사에 중요한 역할을 합니다.
특히
글루타메이트 흥분 독성은
뉴런의 미토콘드리아 손상과 관련이 있습니다28.
루테올린이 미토콘드리아 스트레스에 미치는 영향을 평가하기 위해
미토옥스 프로브를 사용하여
미토콘드리아 슈퍼옥사이드의 생성을 평가했습니다.
그런 다음 형광 강도를 CellInsight CX7 고함량 스크리닝(HCS) 플랫폼을 사용하여 모니터링하고 이후 Hoechst 33342로 정규화했습니다. 그 결과 글루타메이트가 미토콘드리아 슈퍼옥사이드 생성의 증가를 유도한다는 사실이 밝혀졌습니다. 그러나 세포를 루테올린(5-25 µM)과 케르세틴(10 µM)으로 전처리하면 미토콘드리아 슈퍼옥사이드 수치가 현저히 회복되었습니다(그림 4A). 또한 산화 스트레스에 취약한 미토콘드리아 막 전위를 미토트래커 오렌지 CMTMRos로 염색하여 평가했습니다(그림 4B). 대조군(무처리 그룹)은 높은 형광 강도를 보여 미토콘드리아 막 전위가 강건함을 나타냅니다. 대조적으로 글루타메이트 처리 그룹은 형광 강도가 현저히 감소하여 미토콘드리아 막 전위가 손실되었음을 보여주었습니다. 그러나 루테올린과 케르세틴으로 전처리하면 미토콘드리아 막 전위가 보호되고 회복되었습니다. 미토트래커 오렌지 CMTMRos의 형광 강도를 정량화하여 글루타메이트가 대조군에 비해 형광 강도를 현저히 감소시키는 것을 확인했습니다(그림 4B, 하단). 또한 공초점 현미경으로 미토콘드리아 형태를 평가하기 위해 미토트래커 오렌지 CMTMRos 염색을 사용했습니다. 미토콘드리아 형태 분석을 위해 미토콘드리아 네트워크 분석 플러그인(MiNA)을 사용했습니다. 그 결과 글루타메이트가 미토콘드리아 분열을 유발하고 분지 네트워크를 감소시킬 수 있음을 보여주었습니다. 그러나 글루타메이트에 노출되기 전에 세포를 루테올린과 케르세틴으로 전처리하면 미토콘드리아 형태가 유지되고 분기 네트워크가 증가했습니다(그림 4C). 또한 안정된 mtDNA 분획인 16S rRNA를 사용하여 미토콘드리아 함량을 조사하고 이를 핵 DNA(HKII)와 비교했습니다. 그 결과 글루타메이트 처리 그룹은 미토콘드리아 함량이 감소했음을 나타내는 mtDNA/nDNA 비율이 감소한 것으로 나타났습니다(그림 4D). 반대로 루테올린 전처리 그룹은 미토콘드리아 함량이 증가했음을 나타내는 mtDNA/nDNA 비율이 증가했습니다.
이러한 결과는
루테올린이 미토콘드리아 건강에 긍정적인 영향을 미치며
글루타메이트로 인한 미토콘드리아 스트레스로부터
보호할 수 있음을 시사합니다.
Figure 4
Luteolin reduces mitochondrial oxidative stress and restores mitochondrial function. (A) Representative the mitochondrial superoxide generation stained with mitosox indicator. HT-22 cells were pre-treated with luteolin at different concentrations (5–50 µM) for 24 h and quercetin 10 µM was used as a positive control, followed by 5 mM glutamate for 18 h. The bottom bar graph shows the relative mitochondria superoxide production (n = 3). (B) The mitochondria membrane potential was stained with mitotracker orange CMTMRos. The relative mean fluorescence staining was compared with control group (n = 3). (C) The mitochondria morphology was analyzed under a LSM 800 confocal microscope with 40X objective magnification (scale bar is 10 µm). The bottom bar graph shows the mitochondrial network branches mean analyzed with ImageJ Mitochondria Network Analysis plugin (MiNA) (n = 3). (D) The bar graph shows the relative mtDNA content (16S rRNA) / nuclear DNA ratio (n = 3). The data were collected from at least three independent experiments and the results were shown in mean ± SEM. *p-value < 0.05, **p-value < 0.01 compared with glutamate-treated group, #p-value < 0.001 compared with untreated control group. L:luteolin, Q:quercetin.
루테올린은 미토콘드리아의 산화 스트레스를 줄이고 미토콘드리아 기능을 회복시킵니다.
(A) 미토옥스 지표로 염색한 미토콘드리아 슈퍼옥사이드 발생을 나타냅니다. HT-22 세포에 루테올린을 다양한 농도(5-50 µM)로 24시간 동안 전처리하고, 양성 대조군으로 케르세틴 10 µM을 사용한 후 5mM 글루타메이트를 18시간 동안 처리한 후 하단 막대 그래프는 상대적인 미토콘드리아 슈퍼옥사이드 생성량을 나타낸다(n= 3).
(B) 미토콘드리아 막 전위를 미토트래커 오렌지 CMTMRos로 염색했습니다. 상대적 평균 형광 염색을 대조군과 비교했습니다(n= 3).
(C) 미토콘드리아 형태를 40배 대물 배율의 LSM 800 공초점 현미경으로 분석했습니다(눈금 막대는 10µm). 하단 막대 그래프는 ImageJ 미토콘드리아 네트워크 분석 플러그인(MiNA)으로 분석한 미토콘드리아 네트워크 가지의 평균을 보여줍니다(n= 3).
(D) 막대 그래프는 상대적인 mtDNA 함량(16S rRNA) / 핵 DNA 비율을 보여줍니다(n= 3). 데이터는 최소 세 번의 독립적인 실험에서 수집되었으며 결과는 평균 ± SEM으로 표시되었습니다.
글루타메이트 처리군과 비교하여 *p-값< 0.05, **p-값< 0.01, 무처리 대조군과 비교하여 #p-값< 0.001. L:루테올린, Q:케르세틴.
Luteolin inhibits the excessive mitochondria degradation process and autophagy-mediated neuronal cell death
To gain insight into the mechanism underlying the glutamate-induced reduction in mitochondria content, we investigated cellular degradation processes. This involved examining protein expression levels of autophagy markers, namely LC3B and Beclin-1 using Western blot analysis. In line with a previous study29, we employed chloroquine at a concentration of 50 µM to stimulate autophagy in HT-22 cells, serving as a positive control for verifying autophagy activation. Our findings demonstrate that both the glutamate-treated group and the chloroquine-treated group exhibited elevation in protein levels of LC3B-II (autophagosome marker) and Beclin-1, compared to the untreated group. Interestingly, both luteolin and quercetin exhibited inhibitory effects on LC3B conversion and led to a decrease in Beclin-1 protein expression levels as compared to the glutamate-treated group (Fig. 5A,B). In addition, we investigated the protein expression of the stress-sensitive mitophagy receptor BNIP3L/NIX. Notably, the glutamate treatment group showed an increase in BNIP3L/NIX protein expression, suggesting an over-activation of the mitochondria degradation process by glutamate. In contrast, both luteolin and quercetin treatments significantly decrease the level of BNIP3L/NIX protein expression compared to the glutamate-treated group (Fig. 5A,B). The co-localization of lysosomes and mitochondria was used to clarify the glutamate-stimulated excessive mitophagy. The results revealed that the chloroquine and glutamate-treated group exhibited an increase in lysosomal fluorescence intensity, suggesting lysosomal activation. In contrast, the cells treated with luteolin and quercetin showed a decrease in lysosomal fluorescence intensity, suggesting a potential suppression of lysosomal activity (Fig. 5C). Moreover, we computed the Pearson's correlation coefficient to evaluate co-localization. Co-localization was observed in the group receiving glutamate treatment, while it decreased in the groups treated with luteolin and quercetin (Fig. 5D). These results suggest that glutamate can lead to the overactivation of the mitophagy process, but this effect can be inhibited by luteolin. Finally, we investigated whether overactivation of the mitophagy process results in HT-22 cell death by inhibiting autophagolysosomal fusion with ammonium chloride (NH4Cl) and assessing cell viability with the MTS assay. Our findings indicated that NH4Cl increased the cell viability compared to the glutamate treatment group, suggesting that the overactivation of the mitophagy process is associated with HT-22 cell death (Fig. 5E).
루테올린은 과도한 미토콘드리아 분해 과정과 오토파지 매개 신경세포 사멸을 억제합니다.
글루타메이트에 의한
미토콘드리아 함량 감소의 근본적인 메커니즘에 대한 통찰력을 얻기 위해
세포 분해 과정을 조사했습니다.
여기에는 웨스턴 블롯 분석을 통해 자가포식 마커, 즉 LC3B와 Beclin-1의 단백질 발현 수준을 조사하는 것이 포함되었습니다. 이전 연구29에 따라 HT-22 세포의 자가포식을 자극하기 위해 50 µM 농도의 클로로퀸을 사용하여 자가포식 활성화를 확인하기 위한 양성 대조군으로 사용했습니다. 연구 결과, 글루타메이트를 처리한 그룹과 클로로퀸을 처리한 그룹 모두 무처리 그룹에 비해 LC3B-II(오토파지 마커)와 베클린-1의 단백질 수치가 상승한 것으로 나타났습니다. 흥미롭게도 루테올린과 케르세틴 모두 글루타메이트 처리 그룹에 비해 LC3B 전환에 대한 억제 효과를 보였으며 Beclin-1 단백질 발현 수준이 감소했습니다(그림 5A,B). 또한 스트레스에 민감한 미토파지 수용체 BNIP3L/NIX의 단백질 발현을 조사했습니다. 특히 글루타메이트 처리 그룹은 BNIP3L/NIX 단백질 발현이 증가하여 글루타메이트에 의한 미토콘드리아 분해 과정이 과도하게 활성화되었음을 시사합니다. 반면, 루테올린과 케르세틴을 처리한 그룹은 글루타메이트 처리 그룹에 비해 BNIP3L/NIX 단백질 발현 수준이 현저히 감소했습니다(그림 5A,B). 리소좀과 미토콘드리아의 공동 국소화를 사용하여 글루타메이트에 의해 자극된 과도한 미토파지를 명확히 했습니다. 그 결과 클로로퀸과 글루타메이트로 처리한 그룹은 리소좀 형광 강도가 증가하여 리소좀이 활성화되었음을 시사하는 것으로 나타났습니다. 반면, 루테올린과 케르세틴으로 처리한 세포는 리소좀 형광 강도가 감소하여 리소좀 활동이 억제될 가능성이 있음을 시사했습니다(그림 5C). 또한 피어슨 상관 계수를 계산하여 공동 국소화를 평가했습니다. 글루타메이트 처리를 받은 그룹에서는 공동 국소화가 관찰된 반면, 루테올린과 케르세틴 처리를 받은 그룹에서는 감소했습니다(그림 5D). 이러한 결과는 글루타메이트가 미토파지 과정의 과활성화를 유발할 수 있지만 루테올린에 의해 이 효과가 억제될 수 있음을 시사합니다. 마지막으로, 우리는 염화암모늄(NH4Cl)으로 자가포식소체 융합을 억제하고 MTS 분석법으로 세포 생존력을 평가함으로써 미토파지 과정의 과활성화가 HT-22 세포 사멸을 초래하는지 여부를 조사했습니다. 연구 결과, NH4Cl은 글루타메이트 처리 그룹에 비해 세포 생존력을 증가시켜 미토파지 과정의 과활성화가 HT-22 세포 사멸과 관련이 있음을 시사했습니다(그림 5E).
Figure 5
Luteolin inhibits glutamate-induced excessive autophagy and mitophagy activation. HT-22 cells were pre-treated with luteolin at different concentrations (5–50 µM) for 24 h and 10 µM quercetin was used as a positive control, followed by 5 mM glutamate for 18 h. Chloroquine (CQ, 50 µM) served as the positive control for autophagy induction. (A) The protein expression level of LC3B, Beclin-1 (autophagy) and BNIP3L/NIX (mitophagy) were analyzed by Western blot, and β-actin served as the loading control. (B) Relative protein levels of LC3B, Beclin-1 (autophagy) and BNIP3L/NIX (mitophagy) were quantified by densitometry and the mean data from at least three independent experiments were normalized to the results (n = 3). (C) The co-localization of lysosome and mitochondria. HT-22 cells were stained with the mitochondria (Mitotracker: red) Lysosome (Lysotracker: green) and nucleus (Hoechst: blue). They were observed under the confocal laser scanning microscope with 40X objective magnification (scalebar is 10 µm). (D) The bar graph of co-localization was considered with Pearson’s correlation coefficient analyzed using ImageJ JACoP plugin. Data represent the means ± SEM and represent averages of results from at least 50 cells (n = 3). (E) Autophagy inhibitor, ammonium chloride (NH4Cl), inhibits glutamate-induced HT-22 cell death. The morphology of HT-22 cells was visualized under the inverted light microscope. A bar graph shows the MTS cell viability results (n = 3). The data represent the means ± SEM collected from at least three independent experiments and. *p value < 0.05, **p value < 0.01, ****p value < 0.001 compared with only glutamate-treated group, #p value < 0.05 compared with untreated control group. CQ:chloroquine, L:luteolin, Q:quercetin.
Luteolin triggers the activation of mTORC1 to prevent glutamate-induced autophagy-mediated cell death.
The mammalian target of rapamycin complex 1 (mTORC1) regulates autophagy but is often inhibited during stress conditions16,30. To explore this further, we conducted an experiment on HT-22 cells. The cells were pre-treated with luteolin at various concentrations (5–25 µM) and then exposed to glutamate for 18 h. We evaluated the mTOR phosphorylation level at S2448. Phosphorylation at this particular site is thought to regulate the function of mTORC1 and its ability to inhibit autophagy31. Our result revealed that the mTOR phosphorylation at S2448 decreased in the glutamate treatment group. In contrast, luteolin increases mTOR phosphorylation at S2448 in a dose-dependent manner (Fig. 6A,B). However, quercetin did not restore the mTOR phosphorylation levels, suggesting that quercetin may inhibit autophagy activation through other pathways. We also measured the protein level of a biomarker of the mTORC1 complex (Raptor); however, no significant change in the Raptor protein was observed after 18 h of glutamate induction (Fig. 6A). To assess the impact of luteolin on mTORC1 activation, HT-22 cells were pre-treated with 25 µM of luteolin and then exposed them to glutamate for 3 and 6 h. Our result revealed that the mTOR phosphorylation at S2448 significantly increased in the luteolin-treated group at 3 and 6 h. Interestingly, the increase in Raptor protein level was observed at 6 h of glutamate exposure (Fig. 6C,D). Furthermore, to confirm that luteolin triggers the activation of mTORC1 under glutamate-induced neuronal cell death, we used rapamycin, which decreases mTOR activity without affecting its abundance. Rapamycin was pre-treated for 1 h before glutamate induction for 6 h. The results showed that luteolin increases the protein expression of p-mTOR (S2448), Raptor, and the levels of mTOR downstream targets, such as p-S6 (S235/236), p-4E-BP1 (Thr37/46), and p-ULK1 (S757). Moreover, p-S6 (S235/236) was decreased in the glutamate-treated group, indicating the inhibition of mTORC1 activity (Fig. 6E,F). Whereas rapamycin treatment inhibited the mTORC1 activation in the luteolin treatment group. Thus, these data demonstrated that glutamate suppressed mTORC1 activity, while luteolin stimulated the mTORC1 activity.
Figure 6
Luteolin increases the mTORC1 activation. HT-22 cells were pre-treated with luteolin at 5–25 µM for 24 h, followed by 5 mM glutamate for 18 h. (A) The dose-dependent mTORC1 protein expression. The protein expression level of p-mTOR S2448 (mTOR activation) and Raptor (mTORC1 complex) were analyzed by Western blot, and β-actin served as the loading control. (B) Relative protein levels of p-mTOR S2448 (mTOR activation) and Raptor (mTORC1 complex) were quantified by densitometry and the mean data from at least three independent experiments were normalized to the results (n = 3). (C) The time dependent mTORC1 protein expression. The protein expression level of p-mTOR S2448 (mTOR activation) and Raptor (mTORC1 complex) were analyzed at 3 h or 6 h of glutamate induction, and β-actin served as the loading control. (D) Relative protein levels of p-mTOR S2448 (mTOR activation) and Raptor (mTORC1 complex) (n = 3). (E) Representative immunoblotting of mTORC1 downstream target p-S6 (S235/236), p-4E-BP1 (Thr37/46), and p-ULK1 (S757). (F) Relative protein levels of p-mTOR S2448, Raptor, p-S6 (S235/236), p-4E-BP1 (Thr37/46), and p-ULK1 (S757) after rapamycin treatment (n = 3). The Western blot was quantified using NIH Imaging J. The data represent the means ± SEM collected from at least three independent experiments and. *p value < 0.05, **p value < 0.01, ****p value < 0.001 compared with the glutamate treatment group. C:control, Glu:glutamate, L:luteolin, Q:quercetin.
Luteolin decreases the mitophagy receptor and mTORC1 downstream target UVRAG mRNA expression
To investigate the impact of luteolin on mRNA expression related to autophagy, we conducted a comparative analysis of 84 autophagy-related genes between the glutamate/vehicle treatment group and the glutamate/luteolin treatment group using the RT2 profiler mouse autophagy PCR array. As shown in Fig. 7A, luteolin was found to increase the mRNA expression of the autophagy substrate p62. Furthermore, luteolin induced a reduction in the mRNA expression of BNIP3, a factor implicated in promoting mitophagy via BNIP3L/NIX. Interestingly, luteolin also contributed to a reduction exceeding twofold in the mRNA expression of the Ultraviolet irradiation resistance-associated gene (UVRAG), a crucial protein in autophagosome formation (Fig. 7B). Since the previous study reported the negative regulation between mTORC1 and UVRAG32, PCR array results are supportive evidence that luteolin inhibits autophagy activation through increasing mTORC1 activity.
Figure 7
Luteolin inhibits autophagy-related mRNA expression. Luteolin decreases the Bnip3 (Mitophagy receptor) and UVRAG (mTORC1 downstream target) mRNA expression and increases p62 (Autophagy substrate) mRNA expression. HT-22 cells were pre-treated with luteolin at 25 µM for 24 h, followed by 5 mM glutamate for 12 h. (A) The heatmap results of mouse autophagy PCR array of glutamate-treated vs luteolin + glutamate treated. Upregulation: red, Down regulation: blue. (B) The scatter plot of mRNA expression with fold regulation = 2, upregulation: red, down regulation: green.
Discussion
Neuronal cell death is a complex and multifactorial process that plays a critical role in various neurological disorders and neurodegenerative diseases. One significant factor in this cell death is the oxidative stress caused by excessive glutamate8,33,34,35,36. Glutamate is an excitatory neurotransmitter that plays a crucial role in normal brain function; however, excessive release or impaired clearance of glutamate can lead to excitotoxicity, triggering a cascade of events that ultimately results in neuronal cell death. Glutamate toxicity has been reported in many cases of neurodegenerative disorders and accelerates the pathophysiology of neurodegenerative diseases37,38,39.
In this study, we present the protective effects of A. lebbeck leaf extract (ALE) and its primary active component luteolin, against oxidative stress caused by glutamate in mouse hippocampal neuronal cells. To start, we evaluated three distinct brain cell lines (mouse hippocampus, mouse neuroblastoma, and human neuroblastoma) and ultimately chose HT-22 mouse hippocampal cells because of their increased susceptibility to glutamate-induced toxicity (Fig. 1). This sensitivity is probably due to the lack of N-methyl-D-aspartate (NMDA) receptor and the mechanism is undergoing through cysteine glutamate antiporter, which consequently triggers a reduction in glutathione level11. Of importance, HT-22 cells have shown the presence of Alzheimer's disease-specific markers under glutamate toxicity conditions40 and it is worth noting that glutamate-induced oxidative toxicity contributes to 50% of neuronal cell fatality41. Thus, HT-22 cells can serve as a valuable model system for assessing and testing novel agents with potential anti-neurodegenerative disease properties.
Our study demonstrates that ALE enhances the survival of neuronal cells against glutamate-induced neuronal cell death. ALE contains a substantial number of flavonoids, with quercetin and luteolin as major phytochemical components (Fig. 2C). Notably, quercetin is the predominant compound within ALE and has previously reported neuroprotective effects against glutamate oxidative toxicity in HT-22 cells26,42. On the other hand, luteolin is the second major phytochemical which is a natural flavonoid compound found in various fruits, vegetables, and herbs. It is also reported in several studies that luteolin exhibits neuroprotective effects by acting as an antioxidant and anti-inflammatory agent24,43. Despite this, the specific neuroprotective mechanisms of luteolin against glutamate toxicity have not been fully understood. Our findings reveal that luteolin effectively prevents glutamate-induced neuronal apoptosis and various other forms of cell death, while also diminishing the accumulation of intracellular ROS (Fig. 3).
Mitochondria are the most abundant in neurons and essential for neuronal function. During glutamate excitotoxicity, mitochondria are involved in the intrinsic apoptotic pathway by releasing pro-apoptotic factors such as cytochrome c and apoptosis-inducing factor (AIF) into the cytoplasm, triggering a cascade of events that ultimately lead to cell death44. This situation compromises vital aspects like maintaining the potential of mitochondrial membranes and the characteristic branching of mitochondria, culminating in the accumulation of impaired mitochondria within neuronal cells45,46. Apparently, luteolin intervenes in this process, effectively curbing mitochondrial dysfunction and even restoring the count of functional mitochondria, as shown in Fig. 4. These outcomes might be attributed to luteolin's antioxidant activity, which recent studies have linked to hindering elevated calcium levels and addressing mitochondrial dysfunction47. Interestingly, previous in vivo studies revealed that luteolin ameliorates Alzheimer’s disease mice via inhibited endoplasmic reticulum stress and inhibited Aβ-induced mitochondrial dysfunction and neuronal apoptosis48,49. Additionally, in a stroke model, luteolin mitigated CA1 hippocampal damage by reducing glial cell activation and suppressing autophagy in MCAO/R-treated rats. Furthermore, it decreased mitochondrial vacuolization50. These results suggest that luteolin serves as a neuroprotective compound in conditions characterized by mitochondrial dysfunction and oxidative stress. In parallel, damaged mitochondria and other cellular components can be eliminated and renewed through the autophagy and mitophagy processes51. Intriguingly, prolonged periods of stress can provoke an excessive autophagy and mitophagy response, eventually leading to the demise of neuronal cells. These occurrences have been documented in a range of instances involving neuronal cell death52,53,54.
BNIP3L (BCL2/adenovirus E1B 19kDa interacting protein 3-like), also known as NIX (Nip3-like protein X) localizes at mitochondria outer membrane which serves as a stress sensing protein and induction of cell death. Moreover, BNIP3L/NIX acts as a mitophagy receptor that interacts with LC3 through its LIR (LC3-interacting region) domain. This interaction serves to attract LC3 family proteins to impaired mitochondria55. Our findings firstly demonstrate that there is an increased expression of BNIP3L/NIX and the presence of the autophagy marker LC3 following exposure to glutamate. Furthermore, we observed the activation of lysosomes along with their colocalization with mitochondria, suggesting the initiation of cellular degradation and mitophagy processes (Fig. 5). These observations align with an earlier study, indicating that glutamate prompts the activation of autophagy56.
Regarding the impact of glutamate-induced toxicity on HT-22 cells, we also observed increased levels of mitophagy. Damaged mitochondria are identified with the aid of diverse mitophagy-related proteins and PINK1 augments the mitophagy signaling by generating phosphorylated ubiquitin, facilitating the connection of autophagosomal components like P62 and LC3 to envelop the targeted mitochondria57. Alternatively, both BNIP3L/NIX and BNIP3 respond to cellular stress signals, orchestrating the recruitment of autophagosomes. Moreover, BNIP3L/NIX has been documented to trigger the disruption of mitochondrial transmembrane potential58. As a result, these pathways synergistically contribute to the excessive degradation of mitochondria. Luteolin intervenes in these processes, reinstating the integrity of mitochondria and counteracting mitochondrial stress. This intervention leads to a decline in the protein expression of mitophagy receptors (BNIP3L/NIX) and autophagy markers (LC3). Notably, luteolin has been found to inhibit autophagy activation induced by ovalbumin (OVA), acting via the PI3K/Akt/mTOR pathway, and suppressing the Beclin-1 complex59.
A pivotal regulatory protein mTOR governs an array of cellular processes encompassing cell growth, proliferation, and autophagy. Particularly in neurons, mTOR exerts control over neuronal development, function, and survival. Activation of mTORC1 yields the ability to temper excessive autophagy, while also influencing protein synthesis and remodeling of the cytoskeleton. This orchestration facilitates neuronal expansion, dendritic and axonal branching60,61. Perturbation of mTOR signaling has been linked to neurodegenerative pathways30,62. Our study reveals that prolonged exposure to glutamate diminishes the phosphorylation of mTOR at the s2448 site, a key regulator of mTOR activity (Fig. 6A). During instances of cellular stress, the functionality of mTORC1 diminishes due to a lack of energy and inadequate energy availability adversely impacts mTORC1's operational capacity, given its dependence on ample ATP levels. Intriguingly, our findings indicate that luteolin restores mitochondrial function, while concurrently upregulating mTOR phosphorylation. This phosphorylation occurs at the S2448 position of mTOR and is present within both mTORC1 and mTORC2 complexes.
The impact of luteolin on mTORC1 activation was confirmed using rapamycin treatment, as shown in Fig. 6. Luteolin prompts an increase in the protein expression of key downstream targets of mTORC1, such as p-S6 (S235/236), p-4E-BP1 (Thr37/46), and p-ULK1 (S757). In contrast, rapamycin treatment suppresses mTORC1 activation in the luteolin-treated group. A noteworthy protein, ULK1, plays a crucial role in initiating both autophagy and mitophagy processes. It collaborates with other autophagy-related proteins like ATG13, FIP200, and ATG101 to initiate the assembly of autophagosomes61. Furthermore, ULK1's activation at the mitochondria signals the start of engulfing damaged mitochondria by autophagosomes. Our data indicated that luteolin increased ULK1 phosphorylation at the S757 site (inactive form), thereby hindering the initiation of autophagy. Interestingly, luteolin also increases the expression of UVRAG mRNA, as shown in Fig. 7. UVRAG interacts with Beclin-1 and class III phosphatidylinositol 3-kinase (PI3K) complexes, which support the formation of precursors for autophagosomes. Based on array data, luteolin steps into reducing excessive autophagy and mitophagy triggered by glutamate through mTORC1 activation. This subsequently inhibits the ULK1-mediated assembly of autophagosomes and suppresses the expression of UVRAG, thereby curbing the initiation of autophagosome formation. In this investigation, luteolin demonstrated the ability to inhibit autophagy and mitophagy through mechanisms involving the suppression of reactive oxygen species (ROS) accumulation, restoration of mitochondrial function, and activation of mTORC1. Nevertheless, it is noted that luteolin exhibits divergent effects across various cellular contexts. While luteolin presents potential in cancer therapy through its modulation of autophagy, its impact on autophagic processes is contingent upon the specific autophagic activity within each cell type63,64. Moreover, the effect depends on the concentration of luteolin.
토론
신경 세포 사멸은
다양한 신경 장애와 신경 퇴행성 질환에서 중요한 역할을 하는 복잡하고 다인자적인 과정입니다.
이러한
세포 사멸의 중요한 요인 중 하나는
산화 스트레스입니다.
글루타메이트는
정상적인 뇌 기능에 중요한 역할을 하는
흥분성 신경전달물질이지만,
글루타메이트의 과도한 방출 또는 제거 장애는
흥분 독성을 유발하여
궁극적으로 신경 세포 사멸을 초래하는 일련의 사건을 촉발할 수 있습니다.
글루타메이트 독성은
신경 퇴행성 질환의 많은 사례에서 보고되었으며
신경 퇴행성 질환의 병리 생리학을 가속화합니다37,38,39.
이 연구에서는
마우스 해마 신경세포에서
글루타메이트에 의한 산화 스트레스에 대한
A. 레벡 잎 추출물(ALE)과
그 주요 활성 성분인 루테올린의 보호 효과를 제시합니다.
우선 세 가지 뇌 세포주(마우스 해마, 마우스 신경모세포종, 인간 신경모세포종)를 평가한 결과
글루타메이트에 의한 독성에 대한 감수성이 높은
HT-22 마우스 해마 세포를 최종적으로 선택했습니다(그림 1).
이러한 민감성은
아마도 N-메틸-D-아스파르트산염(NMDA) 수용체가 부족하고 시
스테인 글루타메이트 항포터가 글루타치온 수치를 감소시키는
메커니즘을 통해 발생하기 때문일 것입니다11.
중요한 것은 HT-22 세포가
글루타메이트 독성 조건에서
알츠하이머병 특이적 마커의 존재를 보여주었으며40,
글루타메이트에 의한 산화 독성이
신경세포 사망률의 50%에 기여한다는 사실41에 주목할 필요가 있다는 점입니다.
it is worth noting that glutamate-induced oxidative toxicity contributes to 50% of neuronal cell fatality
따라서
HT-22 세포는
잠재적인 항퇴행성 질환 특성을 가진
새로운 약제를 평가하고 테스트하는 데 유용한 모델 시스템으로 사용될 수 있습니다.
우리의 연구는 ALE가
글루타메이트에 의한 신경세포 사멸에 대항하여
신경세포의 생존을 향상시킨다는 것을 보여줍니다.
ALE에는
퀘르세틴과 루테올린을 주요 파이토케미컬 성분으로 하는
상당수의 플라보노이드가 함유되어 있습니다(그림 2C).
특히
케르세틴은
ALE의 주요 화합물이며
이전에 HT-22 세포에서 글루타메이트 산화 독성에 대한
반면에
루테올린은 다양한 과일, 채소 및 허브에서 발견되는
천연 플라보노이드 화합물인
두 번째 주요 파이토케미컬입니다.
또한 여러 연구에서
루테올린이 항산화 및 항염증제로 작용하여
그럼에도 불구하고
글루타메이트 독성에 대한 루테올린의 구체적인 신경 보호 메커니즘은
완전히 이해되지 않았습니다.
우리의 연구 결과에 따르면
루테올린은
글루타메이트에 의한 신경 세포 사멸과
다양한 다른 형태의 세포 사멸을 효과적으로 예방하는 동시에
세포 내 ROS 축적을 감소시키는 것으로 나타났습니다(그림 3).
미토콘드리아는
신경세포에 가장 많이 존재하며
신경세포 기능에 필수적입니다.
글루타메이트 흥분 독성 동안
미토콘드리아는 세포질 내로 사이토크롬 c 및
세포 사멸 유도 인자(AIF)와 같은
세포 사멸 촉진 인자를 방출하여
내재적 세포 사멸 경로에 관여하여
궁극적으로 세포 사멸로 이어지는 일련의 사건을 촉발 합니다44.
이러한 상황은
미토콘드리아 막의 잠재력 유지 및
미토콘드리아의 특징적인 분지와 같은
중요한 측면을 손상시켜 신경 세포 내에 손상된 미토콘드리아가 축적되어45,46 절정에 이르게 됩니다.
루테올린은
이 과정에 개입하여 미토콘드리아 기능 장애를 효과적으로 억제하고
심지어 그림 4에서 볼 수 있듯이
기능성 미토콘드리아의 수를 회복시키는 것으로 나타났습니다.
이러한 결과는
루테올린의 항산화 작용 때문일 수 있으며,
최근 연구에 따르면 루테올린은 칼슘 수치 상승을 억제하고
미토콘드리아 기능 장애를 해결하는 것과 관련이 있는 것으로 밝혀졌습니다47.
흥미롭게도
이전의 생체 내 연구에 따르면
루테올린은 소포체 스트레스를 억제하고
Aβ로 인한 미토콘드리아 기능 장애와 신경 세포 사멸을 억제하여
알츠하이머병 마우스를 개선하는 것으로 나타났습니다48,49.
또한 뇌졸중 모델에서 루테올린은 MCAO/R로 치료한 쥐의 신경교세포 활성화를 줄이고 오토파지를 억제하여 CA1 해마 손상을 완화했습니다. 또한 미토콘드리아 공포증도 감소시켰습니다50. 이러한 결과는 루테올린이 미토콘드리아 기능 장애와 산화 스트레스가 특징인 조건에서 신경 보호 화합물로서 작용한다는 것을 시사합니다. 이와 동시에 손상된 미토콘드리아 및 기타 세포 구성 요소는 자가포식 및 미토파지 과정을 통해 제거되고 재생될 수 있습니다51. 흥미롭게도 장기간의 스트레스는 과도한 자가포식 및 미토파지 반응을 유발하여 결국 신경 세포의 사멸로 이어질 수 있습니다. 이러한 현상은 신경세포 사멸과 관련된 다양한 사례에서 문서화되었습니다52,53,54.
BNIP3L(BCL2/아데노바이러스 E1B 19kDa 상호작용 단백질 3-like)은 NIX(Nip3 유사 단백질 X)로도 알려져 있으며 미토콘드리아 외막에 위치하여 스트레스 감지 및 세포 사멸 유도 단백질로 작용합니다. 또한, BNIP3L/NIX는 LIR(LC3 상호작용 영역) 도메인을 통해 LC3와 상호 작용하는 미토파지 수용체 역할을 합니다. 이러한 상호 작용은 LC3 계열 단백질을 손상된 미토콘드리아로 끌어당기는 역할을 합니다55. 우리의 연구 결과는 먼저 글루타메이트에 노출된 후 BNIP3L/NIX의 발현이 증가하고 자가포식 마커 LC3의 존재가 있음을 보여줍니다. 또한 리소좀의 활성화와 미토콘드리아와의 공동화 현상을 관찰하여 세포 분해 및 미토파지 과정이 시작되었음을 시사했습니다(그림 5). 이러한 관찰은 글루타메이트가 오토파지의 활성화를 촉진한다는 이전 연구와 일치합니다56.
HT-22 세포에 대한 글루타메이트 유발 독성의 영향과 관련하여, 우리는 또한 미토파지의 증가된 수준을 관찰했습니다. 손상된 미토콘드리아는 다양한 미토파지 관련 단백질의 도움으로 확인되며, PINK1은 인산화 유비퀴틴을 생성하여 미토파지 신호를 강화함으로써 P62 및 LC3와 같은 자가포식체 구성 요소의 연결을 촉진하여 표적 미토콘드리아를 감싸는 것을 촉진합니다57. 또는 BNIP3L/NIX와 BNIP3는 모두 세포 스트레스 신호에 반응하여 오토파지솜의 모집을 조율합니다. 또한, BNIP3L/NIX는 미토콘드리아 막전위 파괴를 유발하는 것으로 기록되어 있습니다58. 결과적으로 이러한 경로는 미토콘드리아의 과도한 분해에 상승적으로 기여합니다. 루테올린은 이러한 과정에 개입하여 미토콘드리아의 완전성을 회복하고 미토콘드리아의 스트레스에 대응합니다. 이러한 개입은 미토파지 수용체(BNIP3L/NIX)와 오토파지 마커(LC3)의 단백질 발현 감소로 이어집니다. 특히 루테올린은 PI3K/Akt/mTOR 경로를 통해 작용하고 베클린-1 복합체59를 억제하여 오발부민(OVA)에 의해 유도되는 오토파지 활성화를 억제하는 것으로 밝혀졌습니다.
중추적인 조절 단백질인 mTOR는 세포 성장, 증식, 자가포식을 아우르는 일련의 세포 과정을 관장합니다. 특히 신경세포에서 mTOR는 신경세포의 발달, 기능 및 생존을 제어합니다. mTORC1이 활성화되면 과도한 자가포식을 억제하는 동시에 단백질 합성과 세포 골격의 리모델링에도 영향을 미칩니다. 이러한 조율은 신경세포 확장, 수상돌기 및 축삭 분지를 촉진합니다60,61. mTOR 신호의 교란은 신경 퇴행성 경로와 관련이 있습니다30,62. 우리의 연구에 따르면 글루타메이트에 장기간 노출되면 mTOR 활성의 핵심 조절자인 s2448 부위에서 mTOR의 인산화가 감소하는 것으로 나타났습니다(그림 6A). 세포 스트레스가 발생하면 에너지 부족으로 인해 mTORC1의 기능이 저하되고, 충분한 ATP 수준에 의존하기 때문에 부적절한 에너지 가용성은 mTORC1의 작동 능력에 악영향을 미칩니다. 흥미롭게도 루테올린은 미토콘드리아 기능을 회복하는 동시에 mTOR 인산화를 상향 조절한다는 사실을 발견했습니다. 이 인산화는 mTOR의 S2448 위치에서 발생하며 mTORC1 및 mTORC2 복합체 내에 모두 존재합니다.
그림 6과 같이 라파마이신 처리를 통해 루테올린이 mTORC1 활성화에 미치는 영향을 확인했습니다. 루테올린은 p-S6(S235/236), p-4E-BP1(Thr37/46), p-ULK1(S757) 등 mTORC1의 주요 다운스트림 표적의 단백질 발현을 증가시켰습니다. 대조적으로 라파마이신 치료는 루테올린 치료 그룹에서 mTORC1 활성화를 억제합니다. 주목할 만한 단백질인 ULK1은 오토파지와 미토파지 과정을 모두 개시하는 데 중요한 역할을 합니다. 이 단백질은 ATG13, FIP200, ATG101과 같은 다른 오토파지 관련 단백질과 협력하여 오토파지솜61의 조립을 시작합니다. 또한 미토콘드리아에서 ULK1이 활성화되면 오토파지좀이 손상된 미토콘드리아를 포식하기 시작한다는 신호가 됩니다. 우리의 데이터에 따르면 루테올린은 S757 부위(비활성 형태)에서 ULK1 인산화를 증가시켜 오토파지의 시작을 방해하는 것으로 나타났습니다. 흥미롭게도 루테올린은 그림 7에서 볼 수 있듯이 UVRAG mRNA의 발현도 증가시킵니다. UVRAG는 베클린-1 및 클래스 III 포스파티딜이노시톨 3-키나아제(PI3K) 복합체와 상호 작용하여 오토파지좀의 전구체 형성을 지원합니다. 어레이 데이터에 따르면 루테올린은 mTORC1 활성화를 통해 글루타메이트에 의해 유발되는 과도한 자가포식과 미토파지를 감소시키는 단계에 들어갑니다. 이후 루테올린은 ULK1을 매개로 한 오토파지솜의 조립을 억제하고 UVRAG의 발현을 억제하여 오토파지솜 형성의 시작을 억제합니다. 이번 연구에서 루테올린은 활성산소종(ROS) 축적 억제, 미토콘드리아 기능 회복, mTORC1 활성화 등의 기전을 통해 자가포식 및 미토파지를 억제하는 능력을 입증했습니다. 그럼에도 불구하고 루테올린은 다양한 세포 상황에서 다양한 효과를 나타낸다는 사실이 밝혀졌습니다. 루테올린은 자가포식 조절을 통해 암 치료의 잠재력을 제시하지만, 자가포식 과정에 미치는 영향은 각 세포 유형 내 특정 자가포식 활성에 따라 달라집니다63,64. 또한 효과는 루테올린의 농도에 따라 달라집니다.
Conclusion
In this study, we unveil luteolin's ability to prevent glutamate-induced neuronal apoptosis and reduce ROS accumulation. Remarkably, luteolin restores mitochondrial function, mitigates mitochondrial dysfunction, and curtails excessive autophagy and mitophagy as shown in Fig. 8. This protective role might stem from its antioxidant attributes. However, further investigations are required to examine both animal and clinical studies for more understanding and clarifying the neuroprotective effects and deep mechanisms of this A. lebbeck leaf. Overall, our findings provide valuable insights into luteolin's potential as a therapeutic agent against neurodegenerative diseases.
결론
이 연구에서는 루테올린이 글루타메이트에 의한 신경 세포 사멸을 방지하고 ROS 축적을 줄이는 능력을 밝혀냈습니다. 놀랍게도 루테올린은 그림 8과 같이 미토콘드리아 기능을 회복하고 미토콘드리아 기능 장애를 완화하며 과도한 자가포식 및 미토파지를 억제하는 것으로 나타났습니다. 이러한 보호 역할은 항산화 특성에서 비롯된 것일 수 있습니다.
그러나
A. 레벡 잎의 신경 보호 효과와 깊은 메커니즘을 더 많이 이해하고 명확히하기 위해
동물 및 임상 연구를 모두 조사하기위한 추가 조사가 필요합니다.
전반적으로 이번 연구 결과는
신경 퇴행성 질환에 대한 치료제로서
루테올린의 잠재력에 대한 귀중한 통찰력을 제공합니다.
Figure 8
A proposed mechanism of luteolin against glutamate-induced neuronal death through autophagy-mediated neuronal cell death. Elevated glutamate levels within neurons cause an increase in mitochondrial ROS. These heightened ROS levels can damage mitochondria and trigger an excessive and specific process of removing damaged mitochondria, known as mitophagy, facilitated by BNIP3L/NIX. This sequence of events ultimately leads to the death of neurons. On the other hand, luteolin directly counteracts ROS, rescues mitochondrial health, and enhances the expression of mTORC1. Moreover, luteolin decreases autophagy-related gene expression (BNIP3 and UVRAG) and increases p62 gene expression. Furthermore, luteolin can reduce the initiation of mitophagy, resulting in a reduction of neuronal cell death. This figure was created with BioRender.com.
Materials and methods
Plant collection and extraction
The A. lebbeck (L.) Benth. leaves were collected during February to March from the Srinakarind Dam area in Kanchanaburi province, Thailand. The permission for plant sample collecting was obtained from the Plant Genetic Conservation Project initiated by the Royal Patronage of Her Royal Highness Princess Maha Chakri Sirindhorn. All protocols concerning plant access and collecting were in accordance with Thai national regulations, notably the Plant Variety Protection Act (1999), obtaining appropriate permits and maintaining ethical academic standards throughout the process. This plant was identified and confirmed as a scientific name by Assistant Professor Dr. Thaya Jenjittikul, Department of Plant Science, Faculty of Science, Mahidol University, Thailand. A voucher specimen was deposited at Suan Luang Rama IX Herbarium, Bangkok, Thailand (No.9429).
The edible part of the A. lebbeck leaves was separated and washed with deionized water according to the procedure described by Phoraksa and colleagues25. Briefly, A. lebbeck leaves were boiled for 2 min, followed by homogenization using an electric blender. Subsequently, the homogenized leaf mixture underwent lyophilization using a freeze dryer. The resultant A. lebbeck powder was then subjected to ethanol extraction. For the extraction process, 2 g of the sample was dissolved in 30 ml of each solvent. The mixture underwent sonication in a sonicator bath for 10 min, followed by centrifugation at 3,000 rpm. for 10 min. The resulting supernatant was collected in amber glass bottles. Subsequently, the extract was dried using a rotary evaporator and nitrogen blower. The stock solution was prepared by dissolving in DMSO and kept at − 20 °C. The study was conducted in accordance with relevant guidelines and legislation.
Determination of flavonoid contents
The modified method from Dawilai et al. was used to assess the flavonoid contents65. First, the ALE sample was subjected to acid methanol hydrolysis to obtain the aglycone form. Subsequently, the sample was boiled with a mixture of 62.5% (v/v) methanol, t-butyl hydroquinone (0.5 g/L), and 6 N hydrochloric acid for 2 h. Afterward, the sample was cooled on ice for 5 min, followed by the addition of 0.1% (w/v) ascorbic acid. Next, the sample was sonicated for 5 min and then filtered using a 0.2 µm PTFE syringe filter. Samples were analyzed with HPLC (Agilent 1260 Series liquid chromatograph, USA) using a ZORBAX Eclipse XDB-C18 column (4.6 × 150 mm). The mobile phase contains 0.1% trifluoroacetic acid (TFA) in water and 0.1% TFA in methanol. Quantification of flavonoid contents was accomplished by comparing retention times and spectral absorption with standard compounds (namely myricetin, quercetin, kaempferol, luteolin, apigenin, naringenin, and hesperidin). The results are presented as µg/g FW.
Antibodies and reagents
Antibody: LC3B (D11 Cell Signaling Technology, Danvers, MA, USA), BNIP3L/NIX (D4R4B Cell Signaling Technology, Danvers, MA, USA), Beclin-1 (D40C5 Cell Signaling Technology, Danvers, MA, USA), Phospho-mTOR (ser2448) (D9C2 Cell Signaling Technology, Danvers, MA, USA), mTOR (7C10 Cell Signaling Technology, Danvers, MA, USA), Raptor (24C12 Cell Signaling Technology, Danvers, MA, USA), Phospho-S6 Ribosomal Protein (Ser235/236 Cell Signaling Technology, Danvers, MA, USA), Phospho-4E-BP1 (Thr37/46) (236B4 Cell Signaling Technology, Danvers, MA, USA) mouse anti-β-actin (C4) HRP (#SC47778 Santa Cruz Biotechnology, Dallas, TX, USA) and goat anti-rabbit IgG (H + L) secondary antibody, HRP conjugate (#31,460 Invitrogen, Carlsbad, CA, USA). Dimethyl sulfoxide (DMSO) (DR1022) was purchased from Biosesang (Gyeonggi, South Korea). Fetal bovine serum (FBS), Dulbecco's Modified Eagle Medium (DMEM), phosphate-buffered saline (PBS), penicillin–streptomycin, SuperSignal™ West Femto maximum sensitivity substrate, SuperSignal™ West Pico PLUS chemiluminescent substrate, MitoTracker™ Orange CM-H2TMRos (Cat. #M7510), LysoTracker™ Deep Red (Cat. #L12492), MitoSOX™ mitochondrial superoxide indicators (Cat. #M36008) and Pierce™ BCA protein assay kit were purchased from Thermo Scientific (Waltham, MA, USA). The CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS) and CytoTox 96® Non-Radioactive Cytotoxicity Assay were purchased from Promega (Madison, WI, USA). 2′7’-dichlorodihydrofluorescein diacetate (H2DCFDA) was purchased from Molecular Probes (Eugene, OR, USA). RIPA lysis buffer was purchased from Biomax (Gyeonggi, South Korea). Dako Fluorescence mounting medium was purchased from Agilent (Santa Clara, CA, USA). Chloroquine diphosphate (Cat. #C6628) and luteolin (Cat #L9283) were purchased from Sigma (USA). Ammonium chloride 99 + % pure (Cat #123,340,250) was purchased from Acros Organics (Geel, Belgium). Quercetin dihydrate was purchased from MP Biomedicals (Santa Ana, CA, USA). Rapamycin (Cat #HY-10219) was purchased from MedChemExpress (NJ, USA).
Cell culture
The mouse hippocampal neuronal HT-22 cells were a generous gift from Prof. David Schubert (The Salk Institute, San Diego, CA, USA). SH-SY5Y neuroblastoma cells were purchased from a cell line service (Heidelberg, Germany; Catalogue number 300154). Neuro-2A cells were obtained from the Health Science Research Resources Bank (Osaka, Japan). HT-22 and Neuro-2A cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin. SH-SY5Y cells were cultured in F12/DMEM (F12/DMEM 1:1 mixture) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin. The cells were incubated at 37 °C in a humidified atmosphere with 5% CO2. The culture medium was changed every 3 days, and the cells were grown until they reached 80–85% confluence for the experiments. The cells passage between 10 and 25 were used.
Cell viability assay
To evaluate the cell viability, we employed the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay. HT-22, Neuro-2A, and SH-SY5Y cells were seeded in 96-well plates at a density of 3,000 cells per well and allowed to adhere for 18–24 h. Subsequently, the cells were pre-treated with A. lebbeck leaf extract (2.5 µg/ml-100 µg/ml), quercetin (10 µM), and luteolin (5 µM-50 µM) for 24 h, followed by exposure to glutamate (5 mM) in complete medium. Incubation was carried out at 37 °C in a 5% CO2 incubator with a humidified atmosphere for 18 h. Next, 20 µl of MTS CellTiter 96® Aqueous one solution reagent (Promega, Madison, WI, USA) was added, and the cells were further incubated for 1 h. The absorbance was measured by a microplate reader (Multiskan™ FC Microplate Photometer, Thermo Scientific, Waltham, MA, USA) at 490 nm. The percentage of cell viability was calculated by comparing the results to the control group. Additionally, for qualitative assessment of cell morphology, the Nikon Eclipse Ti-U inverted microscope was employed.
Lactate dehydrogenase assay
To assess cytotoxicity, the release of lactate dehydrogenase (LDH) enzyme from damaged cells was measured. HT-22 cells were seeded overnight in 96-well plates at a density of 3,000 cells per well. The cells were then pre-treated with quercetin (10 µM) and luteolin (5 µM-50 µM) for 24 h, after which they were exposed to glutamate (5 mM) in a complete medium. The incubation was carried out at 37 °C in a 5% CO2 incubator with a humidified atmosphere for 18 h. After the incubation period, 50 µl of supernatant was transferred to a new 96-well plate. The CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, WI, USA) was employed for analyzing cell cytotoxicity. Briefly, a lysis buffer was added and incubated for 45 min to serve as a cell lysis control. For LDH enzyme measurement, 50 µl of cytotoxic reagent was added to the aforementioned 96-well plate, and the mixture was incubated for 30 min at room temperature. The reaction was halted by adding 50 µl of stop solution. Subsequently, absorbance was measured at 490 nm using a microplate reader (Thermo Fisher Scientific, Waltham, MA, USA). The percentages of LDH release were calculated and compared to the control group.
Intracellular ROS
H2DCF-DA was utilized to measure intracellular ROS levels66. HT-22 cells were seeded in 48-well plates at a density of 8,000 cells per well. Following a 24 h pre-treatment with quercetin (10 µM) and luteolin (5 µM-25 µM), the cells were exposed to glutamate (5 mM) in a complete medium. The treated cells were incubated at 37 °C in a 5% CO2 incubator with a humidified atmosphere for 18 h. After the incubation period, 10 µM H2DCF-DA and 1 µM Hoechst were added to the cells and incubated at 37 °C for 30 min. The stained cells were washed twice with cold Hank’s balanced salt solution (HBSS). The fluorescence of dihydroethidium (DHE) was analyzed using a CX7 LZR high content screening (HCS) platform (Thermo Fisher Scientific, Waltham, MA, USA).
Mitochondria ROS level (Mitosox)
HT-22 cells were seeded in a 48-well plate at a density of 8,000 cells per well. The cells were treated with quercetin (10 µM) and luteolin (5 µM-25 µM) for 24 h, and subsequently exposed to 5 mM glutamate in a complete medium for 18 h. The cells were stained with a specific mitochondria superoxide indicator (MitoSOX-red) (Molecular Probes, Eugene, OR, USA) at a concentration of 5 µM for 10 min. Subsequently, the stained cells were washed twice with cold HBSS. The fluorescence of mitochondria superoxide was analyzed using a CX7 LZR high-content screening (HCS) platform.
Apoptosis assay
PE-Annexin V/7-AAD staining was performed on HT-22 cells that were seeded in 6-well plates at a density of 100,000 cells per well and allowed to adhere for 18–24 h. The cells were pre-treated with luteolin (5 µM-25 µM) for 24 h. Following this pre-treatment, the cells were exposed to glutamate for 18 h. Subsequently, the cells were harvested from the plate, washed with PBS, and resuspended in 100 μL of 1X Annexin V binding buffer. They were then incubated in the dark at room temperature with 5 µL of PE Annexin V and 5 µL of 7-AAD viability staining solution for 15 min. Then, the cells were diluted with 400 μL of binding buffer. For each experiment, unstained and single-channel controls were used to calculate compensation. Flow cytometry analysis was performed using a Cell sorter SH800S (Sony Biotechnology, San Jose, CA, USA).
Western blotting
Cells were washed with cold PBS and then lysed on ice using pre-cooled RIPA lysis buffer containing protease inhibitors (Biomax, Gyeonggi, South Korea). The proteins (25 µg) were loaded into each lane of an 8–12% acrylamide gel (Biosesang, Gyeonggi, South Korea). After the separation step, the proteins were transferred to PVDF membranes (GVS, Bologna, Italy) and blocked with 5% nonfat dry milk in Tris-Buffered saline with 0.1% Tween 20 detergent (1X TBS-T) for 1 h. Primary antibodies LC3B 1:5000, BNIP3L/NIX (D4R4B 1:1000), Beclin-1 (D40C5 1:1000), Phospho-mTOR (ser2448) (D9C2 1:1000), mTOR (7C10 1:1000), Raptor (24C12 1:1000), Phospho-S6 Ribosomal Protein (Ser235/236 1:1000), Phospho-4E-BP1 (Thr37/46) (236B4 1:1000) were used to probe the target proteins at 4 °C overnight. Next, the proteins were probed with an HRP-conjugated secondary antibody (1:5000, Invitrogen, USA) at room temperature for 1 h. Finally, the proteins were detected using a SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific, Waltham, MA, USA), and the protein analysis was performed using NIH ImageJ.
mtDNA analysis
The mtDNA copy number was analyzed according to previously reported67. To assess the mtDNA copy number, total genomic DNA was extracted by AccuPrep® Genomic DNA extraction kit (Bioneer, Daejeon, South Korea) according to the manufacturer’s instructions with the mouse mitochondrial genome. Since 16S rRNA represents a stable fraction (less prone to deletions)68, the 16S rRNA was selected to examine the relative copy number of mitochondrial DNA (mtDNA) and nuclear DNA (nDNA). Hexokinase 2 (HK2), a gene encoded in the nucleus, was specifically selected as a nuclear DNA (nDNA) for normalization (Table 1). The mtDNA expression was calculated by comparing the expression of 16S rRNA DNA to HK2 DNA expression. The mtDNA/nDNA ratio was calculated using the ΔΔCt method by calculating the number of mtDNA per nDNA. The Ct values, obtained from the qPCR machine software, represent the mean of triplicate Ct values for each DNA sample.
Table 1 Primer for mtDNA copynumber analysis.
Mitophagy and mitochondrial morphology analysis
HT-22 cells were placed on coverslips in a 6-well plate. The cells underwent the treatment as outlined previously. Each well was stained with 50 nM lysotracker deep red and 150 nM mitotracker orange CMTMRos. After rinsing with PBS, the cells were fixed with 4% paraformaldehyde at room temperature for 15 min. Subsequently, cells were stained with 1 µM Hoechst for 10 min. After rinsing with PBS, stained cells were mounted with Dako fluorescence mounting medium. Finally, the slides were examined using a confocal laser scanning microscope (LSM 800) from Carl Zeiss, Germany. The colocalization was analyzed by Image J software, the JACoP plugin. In addition, mitochondrial morphology and branching were analyzed by Image J software, the MiNA plugin.
Mitochondria membrane potential
The cells were seeded in a 48-well plate at a density of 8,000 cells per well and cultured under the same conditions as mentioned previously. After glutamate treatment for 12 h, the cells were subjected to staining with MitoTracker® Orange CMTMRos (25 nM) and incubated at 37 °C for 30 min. Subsequently, the cells were washed three times with PBS and fixed using 4% paraformaldehyde (PFA) for 10 min at room temperature (RT). For nucleus staining, the cells were stained with DAPI (10 μg/ml) for 10 min at RT and washed with PBS. The fluorescence intensity was quantified and visualized using the Thermo Scientific™ CellInsight CX7 high-content screening platform.
PCR array assay
Total RNA was extracted from the cells using Trizol reagent (Thermo Scientific, Waltham, MA, USA), and the RNA concentration was measured using a Nabi- UV/Vis Nano Spectrophotometer from Microdigital, Gyeonggi, South Korea. The RNA was then converted into complementary DNA (cDNA) through reverse transcription using the Verso cDNA synthesis kit from Thermo Scientific, USA. The real-time RT2 profiler mouse autophagy PCR array (QIAGEN, Cat. no. PAMM-084Z) was used to analyze the autophagy-related gene expression. The 96-array plate consists of controls for genomic DNA contamination, reverse transcription and positive PCR controls. Glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) served as the reference gene for the assay. CT values were recorded in an Excel file to generate a table of CT values, which was then uploaded to the data analysis web portal at http://www.qiagen.com/geneglobe. The samples included both control and test groups, and the CT values were normalized using a manual selection from the full panel of reference genes.
Data analysis
Each experiment was performed at least three independent experiments, and the values are expressed as the mean ± standard error of the mean (SEM). One-way analysis of variance (ANOVA) was used for the evaluation of the statistical significance with post hoc Dunnett’s test and Bonferroni. A p-value of less than 0.05 was considered statistically significant.
Data availability
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.
Abbreviations
ALE:
Albizia lebbeck leaf extracts
ROS:
Reactive oxygen species
H2DCF-DA:
2,7-Dichlorodihydrofluorescein diacetate
BNIP3L/NIX:
BCL2/adenovirus E1B interacting protein 3-like
mtDNA:
Mitochondrial DNA
nDNA:
Nuclear DNA
mTORC1:
Mammalian target of rapamycin complex 1
CQ:
Chloroquine
UVRAG:
UV radiation resistance associated
References
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