Anti-inflammatory Effects of Fermented Lotus Root and Linoleic Acid in Lipopolysaccharide-induced RAW 264.7 Cells = Lipopolysaccharide로 유도된 RAW 264.7 세포에서 발효연근과 리놀레산의 항염증 효능 평가

다국어 초록 (Multilingual Abstract)

Lotus root, the edible rhizome of Nelumbo nucifera, is a popular traditional herbal medicine in East Asia. It has been known to several beneficial effects, including anti-oxidative effects and anti-inflammatory effects. However, the mechanisms underlying the anti-inflammatory effects of lotus root are still largely unknown. Furthermore, several recent studies have reported that physiological functions of plant-based foods were further enhanced through fermentation. Thus, the aim of this study is to examine the mechanisms underlying the anti-inflammatory effects of fermented lotus root (FLR) and its major constituent linoleic acid (LA) in lipopolysaccharide (LPS)-induced RAW 264.7 macrophages. First, cytotoxicity assay was carried out to investigate whether FLR and LA affect cell viability. When RAW 264.7 cells were treated with FLR or LA, there was no significant cytotoxicity, and cell proliferation increased by LPS was reduced. Nitric oxide (NO) production induced by LPS was decreased due to treatment with FLR in a dose-dependent manner, and treatment with LA significantly decreased NO production at the highest concentration. In addition to that, increased mRNA expression of immune genes including nitric oxide synthase 2 (Nos2), prostaglandin-endoperoxide synthase 2 (Ptgs2), tumor necrosis factor-alpha (Tnf-α), interleukin-1 beta (Il1b), and interleukin-6 (Il6) was reduced when cells were treated with FLR or LA. Phosphorylation of nuclear factor-kappa B (NF-κB) and inhibitor of kappa B alpha (IκBα) was the highest when cells were induced by LPS for 1 h, and treatment with FLR or LA inhibited phosphorylation of NF-κB and IκBα. Besides, nuclear translocation of NF-κB p65 was blocked by treatment with FLR or LA. These results suggest that FLR and LA attenuate pro-inflammatory response through inhibition of NF-κB activation in LPS-induced RAW 264.7 cells.

국문 초록 (Abstract)

Nelumbo nucifera의 식용 뿌리줄기인 연근은 동아시아에서 전통적인 한약재로서 사용되어왔다. 연근은 항산화 효과 및 항염증 효과를 비롯하여 몇 가지 유익한 효능을 가지는 것으로 알려져 있으나, 연근이 가지는 항염증 효과의 근본적인 메커니즘은 잘 알려져 있지 않다. 또한, 최근 여러 연구들에 의해 식물유래 식품들이 가지는 생리활성 기능이 발효를 통해 더욱 향상되었음이 밝혀졌다. 따라서, 이 연구의 목적은 lipopolysaccharide (LPS)로 유도된 RAW 264.7 세포에서 발효연근 (FLR)과 그 주요 성분인 리놀레산 (LA)의 항염증 효과와 함께 근본적인 메커니즘을 평가하는 것이다. 먼저 FLR과 LA가 세포 생존율에 미치는 영향을 조사하기 위해 세포독성시험을 수행하였다. RAW 264.7 세포가 FLR과 LA로 처리되었을 때 유의미한 세포 독성은 나타나지 않았으며 LPS에 의해 증가된 세포 증식은 감소되었다. LPS에 의해 유도된 nitric oxide (NO) 생성은 FLR 처리에 의해 농도의존적으로 감소하였으며, LA는 가장 고농도 (100 μM)에서만 유의미하게 NO 생성을 감소시켰다. 또한 면역관련 유전자인 nitric oxide synthase 2 (Nos2), prostaglandin-endoperoxide synthase 2 (Ptgs2), tumor necrosis factor-alpha (Tnf-α), interleukin-1 beta (Il1b) 및 interleukin 6 (Il6)의 mRNA 발현 증가는 FLR 또는 LA 처리에 의해 감소되었다. Nuclear factor-kappa B (NF-κB)와 inhibitor of κB alpha (IκBα)의 인산화 수준은 세포가 LPS에 의해 1시간동안 처리되었을 때 가장 높았으며, NF-κB와 IκBα의 인산화는 FLR 또는 LA 처리에 의해 억제되었다. FLR 또는 LA 처리는 NF-κB p65의 핵 전위 또한 억제하였다. 이러한 결과는 LPS로 유도된 RAW 264.7 세포에서 FLR과 LA가 NF-κB 활성화의 억제를 통해 전염증성 반응을 약화시킨다는 것을 시사한다.

목차 (Table of Contents)

• I. Introduction ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ 1
• 1. Nitric oxide (NO) in inflammation ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ 1
• 2. Pro-inflammatory cytokines ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ 2
• 3. Nuclear factor-kappa B (NF-κB) signaling pathway ∙∙∙∙∙∙∙∙∙∙ 4
• II. Materials and Methods ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ 8
• 1. Preparation of Fermented Lotus Root (FLR) and Linoleic Acid (LA) ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ 8
• 2. Cell Culture ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ 8
• 3. Cell Viability Assay ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ 9
• 4. Cell Morphology Observation ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ 9
• 5. Nitric Oxide (NO) Production Analysis ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ 9
• 6. RNA Extraction and complementary DNA (cDNA) Synthesis∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ 10
• 7. Real-time Reverse Transcription Polymerase Chain Reaction (RT-PCR) ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ 11
• 8. Luciferase Assay ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ 13
• 9. Western Blot Analysis ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ 13
• 10. Immunofluorescence Staining ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ 14
• 11. LA Detection by Gas Chromatography with Flame Ionization Detector (GC-FID) ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ 15
• 12. Statistical Analysis ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ 15
• III. Results ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ 16
• 1. Effects of FLR on Cell Viability and Morphologic Alteration in RAW 264.7 Cells ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ 16
• 2. Inhibitory Effects of FLR on NO Production and mRNA Expression of Pro-inflammatory Enzymes in Lipopoly-saccharide (LPS)-induced RAW 264.7 Cells ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ 18
• 3. Inhibitory Effects of FLR on mRNA Expression of Pro-inflammatory Cytokines in LPS-induced RAW 264.7 Cells ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ 20
• 4. Effects of FLR on NF-κB Activation in LPS-induced RAW 264.7 Cells ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ 22
• 5. Effects of FLR on Nuclear Translocation of NF-κB p65 in LPS-induced RAW 264.7 Cells ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ 24
• 6. Effects of LA on Cell Viability and Morphologic Alteration in RAW 264.7 Cells ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ 26
• 7. Inhibitory Effects of LA on NO Production and mRNA Expression of Pro-inflammatory Enzymes in LPS-induced RAW 264.7 Cells ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ 28
• 8. Inhibitory Effects of LA on mRNA Expression of Pro-inflammatory Cytokines in LPS-induced RAW 264.7 Cells ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ 30
• 9. Effects of LA on NF-κB Activation in LPS-induced RAW 264.7 Cells ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ 32
• 10. Effects of LA on Nuclear Translocation of NF-κB p65 in LPS-induced RAW 264.7 Cells ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ 34
• IV. Discussion ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ 36
• V. Conclusion ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ 39
• References ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ 40
• 국문 초록 ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ 49