Oleic acid attenuates asthma pathogenesis via Th1/Th2 immune cell modulation, TLR3/4-NF-κB-related inflammation suppression, and intrinsic apoptotic pathway induction

다국어 초록 (Multilingual Abstract)

Asthma, a chronic inflammatory respiratory disease without a known cure, disproportionately affects children and the elderly, presenting a growing health concern in an aging population. This underscores the urgent need for novel therapeutics with minimal side effects. Previous research has highlighted the anti-asthmatic properties of camellia and its active component, oleic acid; however, the underlying mechanisms remain underexplored. Asthma’s etiology is linked to an imbalance of Th1 and Th2 cells, inflammation within lung airways, and the proliferation of cells within the respiratory system. This study delineates the fundamental anti-asthmatic mechanisms of oleic acid by examining its regulatory effects on Th1/Th2 immune cells, its anti-inflammatory action, and its role in apoptosis induction along the asthma pathway. Inflammation was experimentally induced in RAW 264.7 cells using LPS to investigate oleic acid’s inhibitory effects. Furthermore, an asthma model was established in BALB/c mice with ovalbumin to study immune cell modulation and apoptosis. The efficacy and mechanisms were assessed using a suite of analytical methods, including MTT, qPCR, ELISA, Western blotting, immunofluorescence, gene transfection, immunohistochemistry, and various staining techniques (Diff Quik, H&E, PAS). In vitro findings demonstrated oleic acid’s capability to diminish the LPS-induced elevation of inflammatory cytokines (TNF-α, IL-6, IL-1β). Molecular docking analyses revealed oleic acid’s potential to interact with TLR3 and TLR4 proteins, forming a ligand-protein complex, which was further substantiated by TLR4 knockdown experiments. Additionally, oleic acid attenuated the expression of MAPK pathway components (JNK, p38 MAPK) and NF-κB pathway constituents (IκB, NF-κB, COX-2, PGE2). In vivo results indicated that oleic acid mitigated the levels of inflammatory cells (WBC, eosinophils, neutrophils, lymphocytes) and IgE activity, reduced the expression of the Th2 cell transcription factor GATA-3, and decreased Th2/Th17-related cytokines (IL-4, TNF-α, IL-6). Oleic acid also alleviated OVA-induced pathological changes in the lung, such as epithelial cell proliferation, inflammatory cell infiltration, and mucus hypersecretion. It restored apoptosis in lung epithelial cells, which was suppressed by OVA, by modulating the expression of Bcl-2 and Bax. In summary, oleic acid exhibits potential as a novel candidate for asthma treatment through its regulation of immune cells, anti-inflammatory effects, and promotion of apoptosis, thereby ameliorating asthma manifestations.

목차 (Table of Contents)

• LIST OF TABLES ⅳ
• LIST OF FIGURES ⅴ
• ABSTRACT ⅵ
• ABBREVIATION ⅷ
• Ⅰ. INTRODUCTION 01
• 1. Disease 01
• 1) Statistics 01
• 2) Pathogenic mechanism 01
• 2. Drug 07
• 1) Classification 07
• 2) Adverse effect 07
• 3. Candidate 08
• 1) Research result 08
• 2) Study objectives 09
• Ⅱ. MATERIALS AND METHODS 10
• 1. In vitro study 10
• 1) Cell culture 10
• 2) Cell viability assay 10
• 3) Nitric oxide assay 11
• 4) Real Time-polymerase chain reaction (RT-PCR) analysis 11
• 5) Enzyme-linked immunosorbent assay (ELISA) analysis 12
• 6) Western blot analysis 13
• 7) Immunofluorescence (IF) analysis 13
• 8) Gene transfection 14
• 2. In vivo study 15
• 1) Ethics statement 15
• 2) Animal experiments 15
• 3) Bronchoalveolar liquid fluid (BALF) analysis 15
• 4) Immunoglobulin E (IgE) in serum analysis 16
• 5) Histopathological observation 16
• 6) RT-PCR analysis 17
• 7) ELISA analysis 17
• 8) IF analysis 18
• 9) Immunohistochemistry (IHC) analysis 18
• 3. In silico study 19
• 4. Statistics 19
• Ⅲ. RESULTS 20
• 1. In vitro study 20
• 1) Safety of oleic acid in RAW 264.7 cells 20
• 2) Oleic acid reduces the release of pro-inflammatory cytokines (such as TNF-α, IL-6, and IL-1β) and nitric oxide 22
• 3) Oleic acid docks into TLR3 and TLR4/MD-2 protein complexes 24
• 4) Oleic acid suppresses the expressions of pro-inflammatory cytokines, such as IL-1β, IL-6, and TNF-α, via TLR3 and TLR4 27
• 5) Oleic acid inactivates the MAPK signaling pathway 29
• 6) Oleic acid inactivates the NF-κB signaling pathway 31
• 2. In vivo study 34
• 1) Oleic acid reduces inflammatory cells in BALF in a dose-dependent manner 34
• 2) Oleic acid reduces serum IgE levels 36
• 3) Oleic acid prevents OVA-induced morphological changes in the pulmonary system 37
• 4) Oleic acid modulates the balance between helper T cells via downregulation of Th2/Th17 cell activation 39
• 5) Oleic acid suppresses inflammation via NF-κB/COX-2/PGE2 pathway inactivation 42
• 6) Oleic acid induces apoptosis of pulmonary epithelial cells 45
• 7) Oleic acid modulates pulmonary epithelial cell death via downregulation of Bcl-2 expression and upregulation of Bax expression 47
• Ⅳ. DISCUSSION 49
• V. CONCLUSION 53
• REFERENCES 55
• KOREAN ABSTRACT 62