Neuroprotective Effects of Egg White Protein Hydrolysates Produced with Naturally Derived Proteolytic Enzymes in Oxidatively Stressed Human Neuroblastoma Cells = 자연 유래 단백질 분해효소로 제조한 계란 흰자 단백질 가수분해물의 산화적 스트레스가 유도된 인간 모세포종 SH-SY5Y 세포에 대한 신경보호 효과

다국어 초록 (Multilingual Abstract)

Neurodegenerative diseases are closely related to aging and are caused by the loss of neuronal cells in the brain. To prevent neurodegenerative disorders, some bioactive materials, such as bioactive peptides, have been frequently studied for their preventive effects. Egg white protein is known as a valuable protein source for producing bioactive peptides and providing many biofunctional effective agents. Thus, this study was designed to confirm the neuroprotective effects of the egg white proteins, ovotransferrin and ovalbumin, and their hydrolysates against oxidatively stressed neuronal cells. In chapter 1, ovotransferrin was isolated from egg white protein and then hydrolyzed using Neutrase, pepsin, Protamex, and trypsin under optimum conditions. After that, the hydrolysates were used to validate neuroprotective effects. Hydrolyzed ovotransferrin showed higher antioxidative activities than pure ovotransferrin. Furthermore, 0.5 mg/mL of pepsin-derived ovotransferrin hydrolysate (OHPE) exhibited the highest cell viability (81.89%), and OHPE effectively reduced cellular reactive oxygen species (ROS) accumulation by 72% (as assessed by corrected total cell fluorescence) and decreased total apoptosis rate by 46.41%. These anti-apoptotic and antioxidative effects were demonstrated by the regulatory effects of OHPE on the mRNA and protein expression of Bax, Bcl-2, caspase-9, caspase-3, Nrf2, and HO-1, as well as on the mRNA expression of BDNF and TH. Additionally, nine peptides comprising OHPE were identified by LC-MS/MS and compared with the sequences that have antioxidant effects reported in Database of Food-derived bioactive peptides (DFBP) to explore their potential as antioxidants. In chapter 2, ovalbumin hydrolysates were produced using bromelain and papain at 37 ℃, pH 7.0, for 6 h and were then subjected to a cell viability test to investigate the neuroprotective effects of ovalbumin hydrolysates using bromelain (OAB) and papain (OAP). After optimal sample selection, morphological change observation, ROS production measurements, relative mRNA and protein expression analysis, apoptosis rates, and cell cycle analysis were conducted. According to the results, OAB showed the most effective protection at 1 mg/mL (96.40%) against oxidative stressed SH-SY5Y cells. OAB demonstrated the ability to regulate ROS production, apoptotic-related factor expressions, antioxidant production axis activation, and dopaminergic neuronal characteristics. In detail, OAB decreases Bax, caspase-9, and caspase-3 (pro-, active forms) expression and increases Bcl-2 expression. Additionally, OAB enhances the antioxidant production pathway by upregulating Nrf2 and HO-1 and promotes dopaminergic neuronal proliferation and function by enhancing BDNF and TH. Ultimately, this study suggested that OAB can be developed as a preventive agent against neurodegenerative diseases by inhibiting apoptotic neuronal cell death tendencies. These results indicate that selected the ovotransferrin hydrolysate, OHPE, and ovalbumin hydrolysate, OAB, can be used as nature-derived compounds to prevent neurodegenerative diseases by inhibiting ROS interactions and regulating protein expression in neuronal cells, ultimately inhibiting tendencies toward apoptotic neuronal cell death.

국문 초록 (Abstract)

신경퇴행성 질환은 노화와 밀접한 관련이 있으며, 뇌 신경세포 손실로 인해 발생한다. 신경퇴행성 질환을 예방하기 위해 생리활성 펩타이드와 같은 일부 생리활성 물질에 대한 연구가 활발히 진행되고 있다. 난백 단백질은 생리활성 펩타이드를 생성하고 다양한 생리기능성 유효 물질을 제공하는 귀중한 단백질 공급원으로 알려져 있다. 따라서 본 연구는 계란 흰자 단백질인 오보트랜스페린과 오브알부민 가수분해물의 신경 보호 효과를 확인하고, 산화 스트레스에 노출된 신경 세포에 대한 보호 효과를 규명하고자 설계되었다.
1장에서는 난백 단백질로부터 오보트랜스페린을 분리한 후, 뉴트라제, 펩신, 프로타멕스, 트립신을 이용하여 최적 조건에서 가수분해하여 신경 보호 효과 검증에 사용하였다. 가수분해된 오보트랜스페린은 순수 오보트랜스페린보다 높은 항산화 활성을 나타냈다. 또한, 0.5 mg/mL의 펩신 유래 오보트랜스페린 가수분해물(OHPE)은 가장 높은 세포 생존율(81.89%)을 나타냈으며, 세포 내 ROS 축적을 72% 감소시키고 총 세포 사멸율을 46.41% 감소시켰다. 이러한 항세포 사멸 및 항산화 효과는 Bax, Bcl-2, caspase-9, caspase-3, Nrf2, HO-1의 mRNA 및 단백질 발현 조절뿐만 아니라, BDNF와 TH의 mRNA 발현 조절을 통해서도 확인되었다. 또한, OHPE를 구성하는 9개의 펩타이드를 LC-MS/MS를 통해 동정되었으며, Database of Food-derived bioactive peptides (DFBP)에서 항산화 효과를 나타내는 것으로 보고된 서열과 비교하여 항산화제 및 신경세포 보호제로서의 가능성을 확인하였다.
2장에서는, 브로멜라인과 파파인을 이용하여 37 ℃, pH 7.0에서 6시간 동안 오브알부민 가수분해물을 생산하였다. 그 후, 브로멜라인 가수분해물(OAB)과 파파인 가수분해물(OAP)의 신경보호 효과를 조사하기 위해 세포 생존율 시험을 진행하였다. 최적의 시료 선택 후, 형태학적 변화 관찰, 활성산소(ROS) 생성 측정, 상대적 mRNA 및 단백질 발현 분석, 세포자멸사율, 세포 주기 분석을 수행하였다. 그 결과, OAB는 산화 스트레스를 받은 SH-SY5Y 세포에 대해 1 mg/mL에서 가장 효과적인 세포 보호(96.40%)를 나타내었다. OAB는 ROS 생성, 사멸 관련 인자 발현, 항산화제 생성 축 활성화, 그리고 도파민 신경 세포의 특성을 조절하는 것으로 나타났다. 구체적으로, OAB는 Bax, caspase-9 및 caspase-3(활성형) 발현을 감소시키고 Bcl-2 발현을 증가시켰다. 또한 OAB 처리는 Nrf2와 HO-1을 상향 조절하여 항산화 생산 경로를 강화하고, BDNF와 TH를 증가시켜 도파민 신경 세포의 증식과 기능을 촉진시켰다. 궁극적으로, 본 연구는 OAB는 신경 세포의 세포사멸 경향을 억제함으로써 신경퇴행성 질환의 예방제로 개발될 수 있는 가능성을 제시한다.
이러한 결과는 선별된 오보트랜스페린 가수분해물인 OHPE, 및 오브알부민 가수분해물인 OAB가 ROS 상호작용을 억제하고 신경 세포에서 단백질 발현을 조절하여 궁극적으로 사멸 경향을 억제함으로써 신경퇴행성 질환을 예방하는 천연 화합물로 사용될 수 있음을 나타낸다.

목차 (Table of Contents)

• List of Tables ⅳ
• List of Figures ⅴ
• Abstract ⅵ
• Chapter 1. Neuroprotective effects of pepsin-hydrolyzed ovotransferrin against H2O2-induced oxidative damage in SH-SY5Y cells via anti-apoptotic and antioxidant mechanisms 1
• Ⅰ. Introduction 1
• Ⅱ. Materials and methods 4
• 1.2.1. Preparation of OTF and its hydrolysates 4
• 1.2.2. Molecular weight distribution change analysis 5
• 1.2.3. Antioxidant activity tests 5
• 1.2.3.1. ABTS radical scavenging assay 5
• 1.2.3.2. Reducing power assay 6
• 1.2.4. Cell culture 7
• 1.2.5. Cell viability 7
• 1.2.6. Cell morphology observation 8
• 1.2.7. Reactive oxygen species (ROS) accumulation analysis 8
• 1.2.8. qRT-PCR analysis 9
• 1.2.9. Western blot analysis 12
• 1.2.10. Flow cytometry analysis 13
• 1.2.11. Peptide identification using LC-MS/MS 13
• 1.2.12. Statistical analysis 14
• Ⅲ. Results and discussion 15
• 1.3.1. Molecular weight distribution pattern changes of OTHs 15
• 1.3.2. Antioxidant activity evaluations of OTF and OTHs 17
• 1.3.3. Cell viability evaluation of OTF and OTHs against SH-SY5Y cells 19
• 1.3.4. Cell morphological changes effect of OHPE on H2O2 induced SH-SY5Y cells 23
• 1.3.5. Inhibition effect of OHPE on intracellular ROS generation 25
• 1.3.6. Gene expression effects of OHPE in H2O2-treated SH-SY5Y cells 27
• 1.3.7. Protein expression effects of OHPE in H2O2-treated SH-SY5Y cells .. 30
• 1.3.8. Effects of OHPE on apoptosis induced with H2O2 in SH-SY5Y cells . 34
• 1.3.9. Identification of OHPE fragments with neuroprotective potential 36
• Ⅳ. Conclusion 38
• Chapter 2. Cytoprotective effects of ovalbumin hydrolysates against H2O2-induced oxidative stress in SH-SY5Y cells through anti-apoptotic and antioxidant defense-enhancing mechanisms 39
• Ⅰ. Introduction 39
• Ⅱ. Materials and methods 42
• 2.2.1. Preparation of OVA and its hydrolysates using bromelain and papain 42
• 2.2.2. Molecular weight distribution changes 43
• 2.2.3. Cell culture 43
• 2.2.4. Cell viability 43
• 2.2.5. Morphological changes observation 45
• 2.2.6. Intracellular ROS generation measurement 45
• 2.2.7. Relative intracellular mRNA expression measurement 46
• 2.2.8. Relative intracellular protein expression measurement 48
• 2.2.9. Degree of apoptotic rates 49
• 2.2.10. Cell cycle analysis 49
• 2.2.11. Statistical analysis 50
• Ⅲ. Results and discussion 51
• 2.3.1. Molecular weight composition changes of OVA and OAHs 51
• 2.3.2. Effects of OVA and OAHs on cell viability with or without H2O2 treatment 53
• 2.3.3. Effects of OAB and H2O2 on morphological change of SH-SY5Y cells 56
• 2.3.4. Effects of OAB and H2O2 on ROS accumulation in SH-SY5Y cells 58
• 2.3.5. Effects of OAB and H2O2 on gene expression in SH-SY5Y cells 60
• 2.3.6. Effects of OAB and H2O2 on protein expression in SH-SY5Y cells 64
• 2.3.7. Effects of OAB and H2O2 on apoptotic rates in SH-SY5Y cells 68
• 2.3.8. Effects of OAB and H2O2 on DNA fragmentation in SH-SY5Y cells . 70
• Ⅳ. Conclusion 72
• References 73
• Abstract (in Korean) 87