Effects of Astaxanthin on Inhibition of Inflammation and BACE1 in LPS induced Microglia BV-2 Cells

국문 초록 (Abstract)

연구목적
이 연구는 Lipopolysaccharide（LPS）로 유도된 BV-2 미세아교세포 모델에서 항산화 및 항염 작용을 지닌 것으로 알려진 Astaxanthin（AX）의 경도인지장애 관련 인자 발현 및 염증에 미치는 영향을 살펴보고자 하였다.

방법
BV-2 미세아교세포 주를 사용하였으며, LPS 처리를 통해 신경염증 및 경도인지장 애 관련 인자의 발현을 유도하였다. BV-2 미세아교세포를 RPMI 1640 또는 MEM 배지 에서 배양하였으며, 1 μg/㎖의 LPS를 처리하여 염증 반응을 유도하였다. 실험군은 무 처치군（Normal）, 대조군（Control, LPS 처리）, AX 처리군（5, 10, 20 μM）으로 설정하였 다. CCK-8 assay를 통해 세포 생존율을 측정하였고, griess reagent system을 이용하 여 Nitric oxide（NO） 생성량을 정량하였다. β-secretase activity 측정을 위해 FRET 기 술 기반의 분석 키트를 사용하였으며, western blot을 통해 APP, BACE1, iNOS, COX-2, IBA-1 단백질의 발현량을 분석하였다. qRT-PCR을 통해 전염증성 사이토카인 （TNF-α, IL-1β, IL-6） 및 케모카인（MCP-1, MIP-1α, MIP-1β）의 mRNA 발현 수준을 측정하였다.

결과
1. AX20 처리군에서 대조군에 비하여 β-secretase activity가 유의미하게 감소하였다. 2. AX5, AX10, AX20 처리군에서 대조군에 비하여 APP expression이 유의미하게 감소 하였다. 3. AX5, AX10, AX20 처리군에서 대조군에 비하여 BACE1 expression이 유의미하게 감 소하였다. 4. AX10, AX20 처리군에서 대조군에 비하여 iNOS와 COX-2 expression이 유의미하게 감소하였다. 5. AX5, AX10, AX20 처리군에서 대조군에 비하여 IBA-1 expression이 유의미하게 감 소하였다. 6. AX5, AX10, AX20 처리군에서 대조군에 비하여 TNF-α, IL-1β, IL-6의 mRNA expression이 유의미하게 감소하였다. 7. AX5, AX10, AX20 처리군에서 대조군에 비하여 MCP-1, MIP-1α, MIP-1β의 mRNA expression이 유의미하게 감소하였다. 8. AX5, AX10, AX20 처리군에서 대조군에 비하여 NO generation이 유의미하게 감소 하였다.

결론
아스타잔틴은 LPS로 유도된 BV-2 미세아교세포 모델에서 베타아밀로이드 생성 관 련 인자와 염증 억제로 경도인지장애 치료에 응용될 수 있는 가능성을 보여준다. 그 러나 앞으로 이를 위한 추가적인 심화 연구가 필요할 것으로 사료된다.

다국어 초록 (Multilingual Abstract)

Research Objective
This study aimed to investigate the effects of Astaxanthin（AX）, known for its antioxidant and anti-inflammatory properties, on the expression of factors related to mild cognitive impairment and inflammation in a Lipopolysaccharide（LPS） induced microglia BV-2 model.

Methods
Microglia BV-2 cells were used, and the expression of neuroinflammation and mild cognitive impairment related factors was induced through LPS treatment. Microglia BV-2 cells were cultured in RPMI 1640 or MEM medium, and an inflammatory response was induced by treatment with 1 μg/㎖ of LPS. The experimental groups were established as a non treated group （Normal）, a control group （Control, LPS treated）, and AX treated groups （5, 10, 20 μM）. Cell viability was measured using the CCK-8 assay, and nitric oxide （NO） production was quantified using the griess reagent system. A FRET-based assay kit was used to measure β-secretase activity, and the expression levels of APP, BACE1, iNOS, COX-2 and IBA-1 proteins were analyzed via western blot. mRNA expression levels of pro-inflammatory cytokines （TNF-α, IL-1β, IL-6） and chemokines （MCP-1, MIP-1α, MIP-1β） were measured using qRT-PCR.

Results
1. β-secretase activity was significantly reduced in the AX20 treated group compared with the control group.
2. APP expression was significantly reduced in the AX5, AX10 and AX20 treated groups compared with the control group.
3. BACE1 expression was significantly reduced in the AX5, AX10 and AX20 treated groups compared with the control group.
4. iNOS and COX-2 expression were significantly reduced in the AX10 and AX20 treated groups compared with the control group.
5. IBA-1 expression was significantly reduced in the AX5, AX10 and AX20 treated groups compared with the control group.
6. mRNA expression of TNF-α, IL-1β and IL-6 were significantly reduced in the AX5, AX10 and AX20 treated groups compared with the control group.
7. mRNA expression of MCP-1, MIP-1α and MIP-1β were significantly reduced in the AX5, AX10 and AX20 treated groups compared with the control group.
8. NO generation was significantly reduced in the AX5, AX10 and AX20 treated groups compared with the control group.

Conclusion
AX demonstrates potential for the treatment of mild cognitive impairment by inhibiting Aβ production related factors and suppressing inflammation in an LPS induced microglia BV-2 model. However, further in-depth research is deemed necessary for this purpose.

목차 (Table of Contents)

• I. Introduction 1
• II. Materials and Methods 4
• 1. Materials and Cell Culture 4
• 2. Experimental Groups 6
• 3. Cell Viability Assay 8
• 4. NO Assay 10
• 5. β-Secretase Activity Assay 13
• 6. Western Blot Analysis 15
• 7. Quantitative Real-time PCR (qRT-PCR) Analysis 16
• 8. Statistical Analysis 18
• III. Results 19
• 1. The Effects of AX on Cell Viability in the Microglia BV-2 Cells 19
• 2. The Effects of AX on β-Secretase Activity in the Microglia BV-2 Cells 21
• 3. The Effects of AX on APP Expression in the Microglia BV-2 Cells 23
• 4. The Effects of AX on BACE1 Expression in the Microglia BV-2 Cells 25
• 5. The Effects of AX on iNOS Expression in the Microglia BV-2 Cells 27
• 6. The Effects of AX on COX-2 Expression in the Microglia BV-2 Cells 29
• 7. The Effects of AX on IBA-1 Expression in the Microglia BV-2 Cells 31
• 8. The Effects of AX on mRNA Expression Level of TNF-α in the Microglia BV-2 Cells 33
• 9. The Effects of AX on mRNA Expression Level of IL-1β in the Microglia BV-2 Cells 35
• 10. The Effects of AX on mRNA Expression Level of IL-6 in the Microglia BV-2 Cells 37
• 11. The Effects of AX on mRNA Expression Level of MCP-1 in the Microglia BV-2 Cells 39
• 12. The Effects of AX on mRNA Expression Level of MIP-1α in the Microglia BV-2 Cells 41
• 13. The Effects of AX on mRNA Expression Level of MIP-1β in the Microglia BV-2 Cells 43
• 14. The Effects of AX on NO generation in the Microglia BV-2 Cells 45
• IV. Discussion 47
• References 51