Anti-Inflammatory and Apoptosis-Inducing Potential of Lactic Acid Bacteria Isolated from Kimchi in Macrophages and Colorectal Epithelial Cells = 김치 유래 프로바이오틱 유산균의 염증 완화, 장 환경 개선 및 대장암 예방 효과

다국어 초록 (Multilingual Abstract)

Probiotics, defined as beneficial live microorganisms that interact with the host to promote physiological balance, have been increasingly recognized for their role in managing chronic inflammatory conditions. These microorganisms help regulate intestinal homeostasis, reinforce barrier integrity, and produce bioactive compounds that support immune and metabolic health. This study aimed to evaluate the anti-inflammatory, gut-protective, and anticancer effects of Levilactobacillus brevis 20080 and G1 strains, isolated from kimchi. Their biological activities were assessed in RAW 264.7 murine macrophages and HT-29 human colorectal epithelial cells, which were used as experimental models for intestinal inflammation and colorectal cancer.
Firstly, to assess probiotic potential, L. brevis 20080 and G1 strains were evaluated for their tolerance to acidic and bile salt conditions, as well as their adhesion to intestinal epithelial cells. Both strains exhibited high survivability under simulated gastrointestinal conditions and adhered effectively to HT-29 cells, as confirmed by scanning electron microscopy (SEM). Safety evaluation showed that neither strain exhibited hemolytic activity and both displayed acceptable antibiotic susceptibility profiles, supporting their suitability as probiotics. In lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages, both strains significantly reduced the production of nitric oxide (NO), prostaglandin E₂ (PGE₂), and leukotriene B4 (LTB4), as well as the mRNA expression of pro-inflammatory cytokines including tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-6, indicating a strong anti-inflammatory response. These effects were accompanied by the inhibition of phosphorylation in inflammation-related signaling pathways, including nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), mitogen-activated protein kinase (MAPK), and activator protein-1 (AP-1). In HT-29 cells, the strains also suppressed the production of NO and the expression of pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-8 under sodium nitroprusside (SNP) or LPS stimulation. Furthermore, both strains enhanced the expression of tight junction-related genes—zonula occludens-1 (ZO-1) and occludin—as well as the mucin gene, mucin 2 (MUC2), thereby contributing to intestinal barrier integrity under inflammatory conditions.
Secondly, the anticancer effects of L. brevis 20080 and G1 strains were investigated using HT-29 cells. Treatment with both strains led to a significant decrease in cell viability and increased intracellular reactive oxygen species (ROS) levels, indicating oxidative stress-induced cytotoxicity. Apoptotic morphological changes, including nuclear condensation and fragmentation, were clearly observed through confocal laser scanning microscopy (CLSM) following 4′,6-diamidino-2-phenylindole (DAPI) staining. Transmission electron microscopy (TEM) further revealed characteristic apoptotic structures such as mitochondrial swelling, cytoplasmic vacuolization, and apoptotic bodies. Gene expression analysis demonstrated that both strains upregulated pro-apoptotic markers such as Bcl-2-associated X protein (Bax), caspase-9, and caspase-3, while downregulating the anti-apoptotic gene B-cell lymphoma 2 (Bcl-2), thereby increasing the Bax/Bcl-2 ratio. Flow cytometric analysis confirmed these findings by showing sub-G1 phase cell cycle arrest and a higher proportion of apoptotic cells in treated groups. These results suggest that L. brevis 20080 and G1 induce apoptosis in HT-29 cells via the intrinsic mitochondrial pathway, highlighting their potential as functional food components for colorectal cancer prevention.
Collectively, these findings demonstrate that L. brevis 20080 and G1 strains possess multifunctional bioactivities, including anti-inflammatory, gut-protective, and anticancer effects. By attenuating inflammatory responses in both immune and intestinal epithelial cells, enhancing gut barrier integrity, and inducing apoptosis in colorectal cancer cells via mitochondrial pathways, these strains exhibit promising potential as probiotic candidates. Their incorporation into functional food products may contribute to the prevention or management of chronic inflammatory conditions and colorectal cancer.

국문 초록 (Abstract)

프로바이오틱스는 숙주와 상호작용하여 생리적 균형을 유지하는 데 기여하는 유익한 미생물로, 최근 만성 염증 질환, 항비만, 항암 등과 관련된 예방 및 치료적 효과가 주목받고 있다. 본 연구에서는 김치에서 분리한 Levilactobacillus brevis 20080 및 L. brevis G1균주의 항염증, 장 건강 증진, 항암 효능을 확인하고, 기능성 식품소재로서의 가능성을 평가하고자 하였다.
첫째, 본 연구에서는 L. brevis 20080 및 L. brevis G1의 프로바이오틱스 특성과 lipopolysaccharide (LPS)로 염증 유도된 RAW 264.7 대식세포와 LPS 및SNP로 염증 유도된 HT-29 장 상피세포에서의 항염증 및 장 환경 개선 효능을 평가하였다. 모든 균주는 인공 위장관 환경에서 높은 생존율과 강한 장 부착 능력을 나타내어 잠재적 프로바이오틱스로 확인되었다. L. brevis균주는 LPS로 자극한 RAW 264.7 대식세포에서는 NO, PGE₂, LTB4 생성과 TNF-α, IL-1β, IL-6 등 염증성 사이토카인의 발현을 유의적으로 억제하였으며, NF-κB, MAPK, AP-1 경로의 인산화를 억제하여 염증 반응을 효과적으로 완화하였다. HT-29 장 상피세포 모델에서도 sodium nitroprusside (SNP) 또는 LPS로 유도된 염증 반응을 억제하고, ZO-1, Occludin, MUC2 등의 발현을 증가시켜 장 장벽 강화 효과를 나타내었다.
둘째, L. brevis 20080 및 L. brevis G1의 항암 효과를 HT-29 인간 유래 대장암 세포에서 평가하였다. 모든 균주는 세포 생존율 저하, LDH 방출 증가 및 ROS생성을 유도하였으며 Bax, caspase-9, caspase-3 유전자 및 단백질 발현 증가, Bcl-2 발현 감소, Bax/Bcl-2 비율 증가가 나타나 내재성 미토콘드리아 자멸사 경로 활성화가 확인되었다. 또한, 유세포 분석 결과 L. brevis 균주는 세포 자멸사 비율을 유의하게 증가시켰으며, sub-G1기 축적을 유도하여 세포 주기 정지와 자멸사를 동시에 유발하였다. 아울러, DAPI 염색 및 CLSM 관찰을 통해 세포핵 응축 및 단편화, TEM 분석을 통해 자멸소체 형성, 미토콘드리아 팽창, 세포질 공포화 등 자멸사 특이적 형태 변화를 확인하였다.
본 연구는 L. brevis 균주의 프로바이오틱 특성과 안전성을 확인하였으며, RAW 264.7 대식세포와 HT-29 세포에서 항염증 효능, HT-29 세포에서 장 환경 개선 효능을 검증하였다. 또한, L. brevis 균주의 HT-29 인간 대장 선암세포에서의 대장암 예방 및 완화 효능을 확인하였다. 따라서, L. brevis균주는 프로바이오틱 특성을 가지며 염증, 장환경 개선 및 대장암 예방을 위한 기능성 식품 소재로 활용될 가능성이 있음을 시사한다.

목차 (Table of Contents)

• List of Tables ⅳ
• List of Figures ⅴ
• Abstract vii
• Chapter 1. Inflammation-modulating and gut-protective roles of Levilactobacillus brevis strains in macrophage and intestinal cell models 1
• 1.1. Introduction 1
• 1.2. Materials and methods 4
• 1.2.1. Bacterial strains and sample preparation 4
• 1.2.2. Cell line conditions 4
• 1.2.3. Evaluation of probiotic characteristics 5
• 1.2.3.1. Tolerance to artificial gastric juice and bile salts 5
• 1.2.3.2. Adhesion ability to HT-29 cells 5
• 1.2.3.3. Visualization of bacterial adhesion via scanning electron microscopy (SEM) 6
• 1.2.4. Safety evaluation 7
• 1.2.4.1. Antibiotic susceptibility 7
• 1.2.4.2. Hemolytic and bile salt hydrolase activity 7
• 1.2.5. Cell viability 8
• 1.2.6. Quantification of nitric oxide production 8
• 1.2.7. Enzyme-linked immunosorbent assay (ELISA) 9
• 1.2.8. Quantification of gene expression by qRT-PCR 9
• 1.2.9. Western blot analysis 11
• 1.2.10. Statistical analysis 12
• 1.3. Results and discussion 13
• 1.3.1. Probiotic characteristics of L. brevis strains 13
• 1.3.2. Safety evaluation of L. brevis strains 17
• 1.3.3. Effects of L. brevis strains on cell viability and NO production 20
• 1.3.4. Effects of L. brevis strains on the protein levels of PGE2 and LTB4 22
• 1.3.5. Effects of L. brevis strains on mRNA expression of iNOS, COX-2, pro-inflammatory cytokines, and anti-inflammatory cytokines in LPS-stimulated RAW 264.7 cells 24
• 1.3.6. Effects of L. brevis strains on mRNA expression of COX-2, pro-inflammatory cytokines, tight junction proteins, and mucins in LPS-stimulated HT-29 cells 27
• 1.3.7. Effects of L. brevis strains on the NF-κB and MAPK pathway 30
• 1.3.8. Effects of L. brevis strains on iNOS, COX-2, and AP-1 expression 34
• 1.4. Conclusion 38
• Chapter 2. Apoptosis-inducing and anti-proliferative effects of Levilactobacillus brevis strains in HT-29 colorectal cancer cells 39
• 2.1. Introduction 39
• 2.2. Materials and methods 42
• 2.2.1. Sample preparation 42
• 2.2.2. Cell culture 42
• 2.2.3. Cytotoxicity and morphological changes 42
• 2.2.3.1. Evaluation of anti-proliferative and cytotoxic effects 42
• 2.2.3.2. Microscopic analysis 44
• 2.2.4. Quantitative real-time polymerase chain reaction (qRT-PCR) 44
• 2.2.5. Caspase colorimetric assay 46
• 2.2.6. Western blotting 46
• 2.2.7. Flow cytometry analysis of apoptosis and cell cycle progression 47
• 2.2.7.1. Flow cytometry analysis of apoptosis 47
• 2.2.7.2. Flow cytometry analysis of cell cycle distribution 48
• 2.2.8. Cell morphology by confocal laser scanning microscope 48
• 2.2.9. Cell morphology by transmission electron microscopy (TEM) 49
• 2.2.10. ROS measurement 49
• 2.2.11. Statistical analysis 50
• 2.3. Results and discussion 51
• 2.3.1. Cytotoxicity and morphological changes induced by L. brevis strains 51
• 2.3.2. Effects of L. brevis strains on the relative mRNA expression of apoptosis-related genes 54
• 2.3.3. Activation of caspases involved in apoptosis 57
• 2.3.4. Effects of L. brevis strains on apoptosis-related protein expression 59
• 2.3.5. Effects of L. brevis strains on apoptosis and cell cycle progression 62
• 2.3.6. Effects of L. brevis strains on nuclear morphological alterations 66
• 2.3.7. Ultrastructural evidence of apoptosis by TEM 68
• 2.3.8. Effects of ROS generation by L. brevis strains 70
• 2.4. Conclusion 72
• References 73
• Abstract (in Korean) 87