식이섬유의 대사 산물인 단쇄지방산이 인체 면역 반응에 미치는 영향 : 2015년 국민건강영양조사자료와 극성화된 인체 장 상피 세포를 이용하여

다국어 초록 (Multilingual Abstract)

CRP(C - reactive protein) is an commonly used inflammatory biomarker in the clinical studies. Dietary fiber has been well known to be fermented to SCFAs(Short Chain Fatty Acids) by microbiome in the colon and absorbed to the intestinal epithelial cells and used as energy source for intestinal cells. However, recently, there are many studies looking for the protective effect of SCFA on innate immunity as well as adaptive immunity in the gut.
In this study, we analyzed the dietary factors associated with inflammation using human serum CRP data from the 2015 Korea National Health and Nutrition Examination Survey(KNHANES) and suggested the mechanism SCFAs regulate the level of inflammation.
In first part, we used the high-sensitivity C-reactive protein (hsCRP) measured by the 2015 KNHANES to identify the distribution of CRP levels according to sex, age, and obesity in healthy Korean adults. Our analysis showed that hsCRP level is closely associated with several lifestyle variables (obesity, smoking, physical activity) and nutrients intake (dietary fiber, fat, saturated fatty acid, omega-3 fatty acid, cholesterol) in healthy Korean adults. The average hsCRP level of healthy Korean adults was 0.95±0.03 mg/L (0.97±0.04 mg/L in men, 0.92±0.05 mg/L in women). Obese subjects had significantly higher hsCRP than non-obese subjects in both sexes. The hsCRP level was positively associated with current smoking, physical inactivity, BMI, waist circumference, fasting blood glucose, triglycerides, total cholesterol, LDL-cholesterol, and blood pressure and inversely associated with HDL-cholesterol. Low CRP Group(LCRPG) had significantly higher intake of dietary fiber compared to High CRP Group(HCRPG) in women. High hsCRP level was associated with more dietary cholesterol intake but less omega-3 fatty acid intake among subjects aged ≥ 50y. HCRPG of the obese subjects had higher intakes of fat and saturated fatty acid than LCRPG.
In second part, we investigated the effects of SCFAs, a product of probiotics and prebiotics, and its mechanisms involved in inflammatory response and endoplasmic reticulum stress pathway. SCFAs has a protective effect on intestinal epithelium barrier in the normal condition represented by intestinal epithelial cells(Caco-2). Also it has anti-inflammatory effects by decreasing cytokine-induced nitric oxide production and cytokine-induced IL8 mRNA expression on Caco-2.
In addition, we confirmed that SCFAs reduced the ratio of Thapsigargin-induced mIL6 mRNA expression significantly by the IRE1α-independent, XBP1-dependent pathway using Mouse Embryonic Fibroblast cells(MEF) knock out cell system. Also we confirmed this result by examining the expression of Thapsigargin-induced mXBP1s mRNA on wt, IRE1α KO and PERK KO MEF cell line showing XBP1s induction by Thapsigargin is PERK and IRE1α independent.
Overall, the differences in dietary fiber intake among CRP groups from the results of Part 1 is presumably due to the anti-inflammatory effect and protective effect of the tight junction of the intestinal epithelial cells by SCFAs shown on Part 2. Based on these results, we suggest that healthy diets (dietary fiber, omega-3, etc.) are necessary to maintain proper inflammation level in the normal condition and presumably also in the pathological inflammation condition.

목차 (Table of Contents)

• 1부 - 한국 성인의 혈중 hsCRP와 영양소 섭취의 관련성 1
• Ⅰ. 서 론 1
• Ⅱ. 연구내용 및 방법 3
• 1. 연구 자료 3
• 2. 연구 대상 3
• 3. 연구 방법 5
• 1) 식사 변수 5
• 2) 인구사회학 및 생활습관 변수 5
• 3) 신체 계측 및 생화학 지표 6
• 4. 통계처리 6
• Ⅲ. 결과 및 고찰 8
• 1. 대상자의 일반적 특성 8
• 2. 한국인의 CRP 수준 10
• 3. 대상자의 그룹별 CRP 수준 11
• 4. CRP 그룹에 따른 집단별 일반적 특성 13
• 5. 남녀별 CRP 그룹에 따른 영양소 섭취량 비교 17
• 6. 연령별 CRP 그룹에 따른 영양소 섭취량 비교 20
• 7. 비만여부별 CRP 그룹에 따른 영양소 섭취량 비교 22
• 8. 연구의 제한점과 후속 연구방향 25
• 2부 – 장상피세포에서 단쇄지방산의 항염증 효과 26
• Ⅰ. 서 론 26
• Ⅱ. 연구재료 및 방법 31
• 1. 시료 31
• 2. 세포주 및 세포 배양 32
• 3. zebra fish 사육 및 알 생산 32
• 1) 사육 조건 32
• 2) 알 생산 및 처리 방법 33
• 4. 단쇄지방산의 독성 평가 33
• 1) Caco-2 세포의 독성 평가 (WST assay) 33
• 2) zebra fish 수정란의 독성 평가 34
• 5. Paracellular permeability assay 34
• 6. Nitric Oxide assay 35
• 7. mRNA 발현 측정 35
• 1) Total RNA 추출 및 cDNA 합성 방법 35
• 2) mRNA 발현 측정 36
• 8. 통계 처리 36
• Ⅲ. 결과 및 고찰 38
• 1. 단쇄지방산의 세포 독성 평가 38
• 2. 단쇄지방산의 zebra fish 수정란 독성 평가 40
• 3. 단쇄지방산을 처리한 Caco-2 세포의 투과성 평가 42
• 4. 단쇄지방산을 처리한 Caco-2 세포에서의 항염증 효과 평가 44
• 5. 단쇄지방산을 처리한 세포에서의 염증인자와 소포체 스트레스 인자 변화 47
• 1) Caco-2 세포에서의 IL8 mRNA 발현 47
• 2) Caco-2 세포에서의 BiP mRNA 발현 51
• 6. 단쇄지방산이 세포의 소포체 스트레스 경로에 미치는 영향 54
• 1) MEF knockout 세포에서의 IL6 mRNA 발현 54
• 2) MEF knockout 세포에서의 XBP1s mRNA 발현 57
• Ⅳ. 요약 및 결론 60
• Ⅴ. 참고문헌 63