The Effect of Betanin Containing Vegetables and Fruit in High Fat Diet on Mouse Induced Colon Inflammation and Cancer by Azoxymethane and Dextran Sulfate Sodium

根據行政院衛生署統計，2008年國人十大死亡原因之首為惡性腫瘤，而大腸癌為其中的第三位。飲食一直被認為是影響大腸癌發生率與致死率的重要因子，流行病學研究顯示廣泛攝取蔬菜與水果，對降低癌症的發生風險具相關性。蔬果除提供人體膳食纖維外，也提供具保健效果的植物二級代謝物。其中苷甜菜(betanin)雖在細胞培養研究中被證實具有抗癌的效果，但對於活體腸胃道的保健資訊，仍然缺乏。 本試驗目的在探討高油飲食中添加含betanin蔬果對受誘發小鼠大腸發炎及大腸癌發生之影響，採用的致癌模式為注射致癌劑Azoxymethane (AOM)與餵食致發炎劑Dextran sulfate sodium (DSS)誘發ICR小鼠大腸癌的動物試驗模式 。高油基礎配方是以AIN-93G為基本、提高其含油量至30% (HF)，再添加10% (w/w)含betanin蔬果，三種含beatain 蔬果配方為：紅龍果果肉組(HFPU)、紅龍果果皮組(HFPE)與菾菜組(HFB)，另一標準品組(HFS)是添加10% (w/w)結球萵苣作為膳食纖維來源並給予0.0025% betanin飲水。HF飲食為對照組。 高油飲食搭配大腸癌誘發模式下，AOM注射不會造成小鼠死亡或活動力降低，且體重持續上升；但DSS飲水導致腸道急性發炎而有部分小鼠死亡。小鼠存活率在添加紅龍果果肉、果皮或菾菜時會降低，但添加結球萵苣並飲用含betanin飲水則會升高。倖存的小鼠飼養15週犧牲分析腸道組織之免疫染色、病理評分、抗發炎與癌化相關基因表現，結果顯示高油飲食本身不會增加大腸組織氧化壓力及提高Nrf2、Ki-67、c-Myc與c-Myb表現量；但以AOM與DSS誘發後則會，且造成腸道細胞異常增生。高油飲食中再添加含betanin蔬果，會降低受AOM與DSS誘發大腸癌之小鼠腸道Nrf2、Ki-67與c-Myb表現量，但增加c-Myc與Cox2表現量。上述抗氧化與細胞異常增生基因之表現量改變，使腸道細胞異常增生與發炎情形加劇。腸道長期發炎下往往形成腫瘤，腫瘤相關分析指標也進一步證實，大腸癌誘發模式下，高油飲食中添加betanin會使大腸腫瘤發生情形更加嚴重。故腸道發炎期間不建議攝取含betanin之蔬果，但其對於未誘發健康小鼠腸道之保健效果則尚待進行。Cancer was the top cause leading to death in 2008, and colorectal cancer ranked the third among the cancers (Department of Health, Executive Yuan, Taiwan). Diet has been considered to affect the morbidity and mortality of colorectal cancer, and the results of epidemiological studies also suggest that intake of varieties of vegetables and fruits may reduce the risk of suffering cancers. In addition to dietary fiber, fruits and vegetables also provide secondary metabolites which may have the potential to promote human health. Among them, the anticarcinogen effect of betanin had been confirmed in cell culture study. However, the effects of health promoting on gastrointestinal system in living stocks has not yet been focused. The objective of this study was to study the effect of betanin containing vegetables and fruits in high fat diet on colon inflammation and cancer on ICR mouse. The animal experimental model of inducing colon cancer on ICR mouse we used was by injecting azoxymethane (AOM) as carcinogen and adding dextran sulfate sodium (DSS) in drinking water as acute inflammatory agent. The basal high fat medium (HF) was modified from AIN-93 by increasing the fat content to 30%. By adding 10% (w/w) of 3 betanin containing plant tissues, 3 feeds were provided as following: red pitaya fruit pulp containing feed (HFPU), red pitaya fruit peel containing feed (HFPE) and beetroot containing feed (HFB). Adding 10% (w/w) head lettuce to the HF basal medium and providing 0.0025% betanin in drinking water was designed as HFS. HF medium along was as control. AOM injection did not cause death or affect mouse’s activity and growing. Drinking DSS containing water induced acute intestinal inflammation and caused death. The survival rate decreased in the mouse feeding when pitaya (peel and plup) and beetroots were added to the HF medium; however, the survival rate increased when head lettuce and betanin containing drinking water was provided. The survived mouse were sacrificed after 15 weeks for the analyses of immunohistochemistry staining, pathological sections, expression levels of anti-inflammatory and cancer-associated gene in colorectal tissue. High fat diet along did not significantly affected the intestinal oxidative stress and the expression of Nrf2, Ki-67, c-Myc and c-Myb. However, all of them were increased after the AOM and DSS application, and intestinal proliferation was resulted. Adding betanin containing fruits and vegetables to high fat diet reduced Nrf2, Ki-67 and c-Myb expression but increased expression of Cox2 and c-Myc in the intestine of AOM-DSS treated mouse. The alteration in the expression of anti-inflammatory and intestinal proliferation genes might contribute to the severity of intestinal proliferation and inflammation. After long-term inflammation, tumors might appear in colon tissue. Tumor-related analysis further confirmed that adding betanin to high fat diet might increase the morbidity of colon tumor under the caner inducing model. Therefore, we concluded that betanin containing fruits and vegetables are not suggested while intestine is inflamming. However, the effects of health promoting on gastrointestinal system on normal mouse was not studied in this thesis