Natural Polyphenols Vanicoside B and Methylnissolin 3-O-β-glucoside Reverse Cellular Senescence in Human Dermal Fibroblasts

국문 초록 (Abstract)

세포 노화는 미토콘드리아 기능 저하, 산화적 스트레스, 그리고 비가역적인 증식 정지로 특징지어지며 피부 노화의 주요 원인으로 알려져 있다. 본 연구에서는 천연 폴리페놀 화합물인 Vanicoside B 와 Methylnissolin 3-O-β-glucoside (MNG)의 항노화 효과를 인간 진피섬유아세포에서 규명하였다. 두 화합물 모두 세포 독성을 유발하지 않으면서 노화세포의 증식능을 유의하게 향상시키고, 미토콘드리아 활성산소(ROS)를 감소시켰다. Vanicoside B는 미토콘드리아 호흡 기능을 회복시키고, 자가포식 활성 및 리소좀 균형을 정상화하였으며, 전사체 분석 결과 ADH1B 발현이 항노화 작용의 핵심 조절인자로 확인되었다. 또한 MNG 는 미토콘드리아 막전위를 회복시키고, 미토파지 활성화를 통해 미토콘드리아 질량을 감소시켰으며, 리소좀 분해 기능을 향상시켰다. 유전자 수준에서 두 화합물은 MMP1 발현을 억제하고 COL1A1 발현을 증가시켜 세포외기질(ECC) 구조 복원 및 콜라겐 합성을 촉진하였다. 중성 코멧 분석 결과, 두 화합물 모두 DNA 꼬리 길이를 유의하게 감소시켜 산화적 DNA 손상을 억제하는 것으로 나타났다. 종합적으로, Vanicoside B 와 MNG 는 미토콘드리아 및 리소좀 항상성을 회복시키고, 유전체 안정성을 유지하며, 세포 노화의 주요 특징을 역전시키는 다중 기능성 항노화 후보물질임을 확인하였다. 본 연구는 두 화합물이 피부 재생 및 노화 지연을 위한 천연 유래 항노화 치료제로서의 가능성을 제시한다.

다국어 초록 (Multilingual Abstract)

Cellular senescence is a key driver of skin aging, characterized by mitochondrial dysfunction, oxidative stress, and irreversible proliferation arrest. This study investigated the anti-senescence effects of two natural polyphenols, Vanicoside B and Methylnissolin 3-O-β-glucoside (MNG), in human dermal fibroblasts. Both compounds significantly enhanced cell proliferation and reduced mitochondrial reactive oxygen species (ROS) without cytotoxicity. Vanicoside B restored mitochondrial respiration, increased autophagy flux, and normalized lysosomal content, while transcriptional profiling revealed ADH1B upregulation as a key mediator of its rejuvenating activity. Similarly, MNG improved mitochondrial membrane potential, reduced mitochondrial mass through mitophagy activation, and enhanced lysosomal degradation. At the molecular level, both agents downregulated MMP1 and upregulated COL1A1, promoting extracellular matrix restoration and collagen synthesis. Neutral comet assays confirmed reduced DNA tail length, indicating protection against oxidative DNA damage. Collectively, Vanicoside B and MNG act as multifunctional senotherapeutic compounds that restore mitochondrial and lysosomal homeostasis, maintain genomic integrity, and reverse major hallmarks ii of fibroblast aging—highlighting their potential as natural anti-aging therapeutics for skin regeneration.

목차 (Table of Contents)

• General Abstract i
• Table of Contents iii
• List of Table vi
• List of Figure vii
• List of Supplementary Information ix
• General Introduction 1
• Chapter 1. Mitigation of oxidative stress via Vanicoside B-induced activation of ADH1B in senescent fibroblasts 3
• Abstract 4
• 1.1. Introduction 5
• 1.2. Materials and Methods 7
• 1.2.1 Preparation of compounds 7
• 1.2.2 Cell culture 7
• 1.2.3 Flow cytometric analysis of ROS 7
• 1.2.4 Cell proliferation assay 8
• 1.2.5 Cell viability assay 8
• 1.2.6 Neutral Comet assay 8
• 1.2.7 Analysis of the oxygen consumption rate (OCR) 9
• 1.2.8 Immunofluorescence 9
• 1.2.9 Analysis and quantification of autophagy flux 10
• 1.2.10 Flow cytometric analysis of mitochondrial mass, lysosomal mass 10
• 1.2.11 Real-time polymerase chain reaction (qPCR) 11
• 1.2.12 Transcriptome expression profiling 13
• 1.2.13 Plasmid engineering 13
• 1.2.14 Lenti–viral production and infection 15
• 1.2.15 Statistical analysis 15
• 1.3. Results 16
• 1.3.1 Vanicoside B reduces oxidative stress and promotes proliferation in senescent fibroblasts 16
• 1.3.2 Vanicoside B promotes mitochondrial oxygen respiration in senescent fibroblasts 21
• 1.3.3 Vanicoside B promotes autophagic clearance of dysfunctional mitochondria 23
• 1.3.4 Vanicoside B suppresses senescence-associated secretory phenotype (SASP) 27
• 1.3.5 Vanicoside B overexpresses alcohol dehydrogenase 1B (ADH1B) in senescent fibroblasts 32
• 1.3.6 ADH1B improves mitochondrial metabolism in senescent fibroblasts 34
• 1.3.7 ADH1B ameliorates SASP in senescent fibroblasts 36
• 1.4. Discussion 38
• 1.5. Conclusion 42
• Chapter 2. Methylnissolin 3-O-β-glucoside Restores Proliferation and Mitochondrial Function in Human Dermal Fibroblasts 43
• Abstract 44
• 2.1. Introduction 45
• 2.2. Materials and Methods 48
• 2.2.1 Cell culture 48
• 2.2.2 Cell proliferation assay 48
• 2.2.3 Flow cytometric analysis of reactive oxygen species (ROS) 49
• 2.2.4 Flow cytometric analysis of mitochondrial mass, lysosomal mass 49
• 2.2.5 Immunofluorescence 50
• 2.2.6 Analysis and quantification of autophagy flux 50
• 2.2.7 Senescent associated–β–galactosidase (SA–β–gal) staining 51
• 2.2.8 Real-time polymerase chain reaction (qPCR) 51
• 2.2.9 Neutral Comet assay 51
• 2.2.10 Statistical Analysis 52
• 2.3. Results 53
• 2.3.1 MNG enhances cell proliferation in senescent fibroblasts 53
• 2.3.2 MNG improves mitochondrial function and reduces oxidative stress in senescent fibroblasts 55
• 2.3.3 MNG enhances mitophagy and autophagic flux in senescent fibroblasts 57
• 2.3.4 MNG alleviates cellular senescence and lipofuscin accumulation 60
• 2.3.5 MNG reverses senescence-associated gene expression and protects genomic integrity. 62
• 2.4. Discussion 65
• 2.5. Conclusion 68
• Reference 69
• Supplementary Information 77
• 국문초록 99